Physiol Rev
82: 701–734, 2002; 10.1152/physrev.00006.2002.
Polarized Calcium and Calmodulin Signaling
in Secretory Epithelia
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
Medical Research Council Secretory Control Research Group, The Physiological Laboratory,
University of Liverpool, Liverpool, United Kingdom
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
I. Introduction
II. Techniques Used in Studies of Polarized Calcium and Calmodulin Signaling
A. Overview of techniques and methods
B. Calcium indicators
C. Instruments
D. Visualization of the distribution of organelles and molecules involved in calcium signaling
E. Measurements of calmodulin distribution and calcium-calmodulin binding
III. Polarity of Intracellular Calcium Signals
A. Patterns of calcium signaling in secretory epithelial cells
B. Subcellular heterogeneity of calcium signals
C. Distribution of calcium-releasing channels and polarity of calcium responses
D. Distribution of cellular organelles and polarized calcium signaling
E. Calcium propagation through the ER lumen: signaling by “tunneling”
F. Polarization in distribution of calcium pumps and other cytoplasmic calcium
removal mechanisms
IV. Transcellular Calcium Transport
A. Transcellular transport process
B. Calcium entry and its role in maintenance of calcium stores and transcellular calcium transport
C. Extrusion of calcium at the apical membrane
D. Movement of calcium across the cell
E. Pathophysiology and physiology of calcium secretion
V. Polarity of Intercellular Calcium Signaling
A. Mechanisms underlying the polarity of intercellular calcium waves
B. Propagation of intercellular calcium waves
VI. Polarized Calmodulin Signaling
A. General properties of CaM
B. General functions of CaM
C. CaM signaling in exocrine secretory cells
D. CaM translocation and oscillations of CaM distribution
VII. Summary
702
702
702
703
704
705
705
706
706
707
709
712
713
713
715
715
715
717
717
718
719
719
721
722
722
722
723
724
725
Ashby, Michael C., and Alexei V. Tepikin. Polarized Calcium and Calmodulin Signaling in Secretory Epithelia.
Physiol Rev 82: 701–734, 2002; 10.1152/physrev.00006.2002.—This review examines polarized calcium and calmodulin signaling in exocrine epithelial cells. The calcium ion is a simple, evolutionarily ancient, and universal second
messenger. In exocrine epithelial cells, it regulates essential functions such as exocytosis, fluid secretion, and gene
expression. Exocrine cells are structurally polarized, with the apical region usually dedicated to secretion. Recent
advances in technology, in particular the development of videoimaging and confocal microscopy, have led to the
discovery of polarized, subcellular calcium signals in these cell types. The properties of a rich variety of local and
global calcium signals have now been described in secretory epithelial cells. Secretagogues stimulate apical-to-basal
waves of calcium in many exocrine cell types, but there are some interesting exceptions to this rule. The shapes of
intracellular calcium signals are determined by the distribution of calcium-releasing channels and mechanisms that
limit calcium elevation. Polarized distribution of calcium-handling mechanisms also leads to transcellular calcium
transport in exocrine epithelial cells. This transport can deliver considerable amounts of calcium into secreted fluids.
Multicellular polarized calcium signals can coordinate the activity of many individual cells in epithelial secretory
tissue. Certain particularly sensitive cells serve as pacemakers for initiation of intercellular calcium waves. Many
www.prv.org
0031-9333/02 $15.00 Copyright © 2002 the American Physiological Society
701
702
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
calcium signaling pathways involve activation of calmodulin. This ubiquitous protein regulates secretion in exocrine
cells and also activates interesting feedback interactions with calcium channels and transporters. Very recently it
became possible to directly study polarized calcium-calmodulin reactions and to visualize the process of hormoneinduced redistribution of calmodulin in live cells. The structural and functional polarity of secretory epithelia
alongside the polarity of its calcium and calmodulin signaling present an interesting lesson in tissue organization.
I. INTRODUCTION
Physiol Rev • VOL
II. TECHNIQUES USED IN STUDIES
OF POLARIZED CALCIUM AND
CALMODULIN SIGNALING
A. Overview of Techniques and Methods
Whether it be in control of granule/vesicle secretion,
transcellular fluid movement, or driving cell growth and
differentiation, the magnitude and location of calcium
increases are extremely important. Understanding of this
importance has necessarily evolved hand-in-hand with
technology that has allowed measurement of cellular calcium and the principle elements of calcium-dependent
signaling cascades. This section highlights techniques
that have been employed in this research field and looks
forward to how recent advances in technology should
allow further insight into the role of calcium in epithelial
biology.
Before the advent of cellular calcium measurements
with fluorescent probes, 45Ca2⫹ was extensively used to
investigate calcium fluxes. 45Ca2⫹ measurements are not
suitable for monitoring intracellular Ca2⫹ responses, particularly at the subcellular level. 45Ca2⫹ measurements
did provide, however, a considerable amount of useful
information on calcium fluxes from exocrine glands or
suspensions of isolated exocrine secretory cells (68, 205,
305, 307).
The correlation between 45Ca2⫹ fluxes and the functional consequences of calcium signaling were assessed
by monitoring of amylase secretion (307). Amylase secretion from isolated, intact pancreas has also been measured in other studies using various assays (140, 181, 213).
Functional tests of secretion are also available for other
exocrine tissues. For example, Canaff et al. (35) measured
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
Calcium is an essential second messenger (18, 24, 56).
This ion mediates the major functions of exocrine epithelial
cells. Most importantly, intracellular calcium regulates both
fluid and exocytotic secretion. Exocrine epithelial cells display structural polarity, functional polarity, and polarity of
calcium signals. Although the polarity of the cells can often
be seen using conventional microscopy, the complex spatiotemporal organization of calcium signals was only revealed following relatively recent developments in technology. These findings led to a rapid expansion of the
epithelial research field, and there is now a large group of
epithelial cell types in which many different forms of
polarized calcium signals have been described. Information on these signals has come from very many sources
and, although there is an obvious similarity between such
signals, comparison is not easily made when the data are
so widely spread. This review brings together information
about different types of polarized calcium signals, the
mechanisms underlying them, and their functional consequences for secretory epithelial cells.
Section II of the review describes the techniques that
have molded the polarized signaling research field. We
highlight those technological advances that have been
most important and discuss in detail some specific applications of that technology. We also outline the latest
technical developments and try to assess their possible
impact on the field.
Section III discusses the polarity of intracellular calcium signals. Secretagogues induce release of calcium
from internal stores resulting in very specific patterns of
calcium oscillations and/or waves. How these signals are
generated and controlled in various types of secretory
epithelial cells is reviewed. In particular, we consider the
correlation between the distribution of calcium-handling
mechanisms (pumps and channels) and the shape of the
calcium signals.
Many of the mechanisms underlying intracellular calcium signals also have roles in the movement of substantial amounts of calcium from the interstitial fluid to the
lumen of epithelial tissue. We discuss the different stages
of this transcellular transport in section IV. At the end of
this section, we consider the implications of the transport
process for the pathology and physiology of exocrine glands.
Cells within epithelial tissue tend to be highly connected, forming multicellular secretory units. Calcium signals are able to pass between cells and across the tissue
as a whole. These intercellular calcium waves are described in section V. We discuss the polarity of the waves
and, importantly, we note the implications of intercellular
communication for the sensitivity of the tissue to secretagogues.
In section VI, we move one step downstream of the
calcium signaling cascade. Calmodulin is one of the major
targets for calcium signals, and important work on the
activation of calmodulin and calmodulin-dependent pathways has been carried out in secretory epithelial cells.
The translocation of calmodulin and functional polarity of
calmodulin signaling are considered.
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
B. Calcium Indicators
The early direct intracellular Ca2⫹ measurements in
secretory epithelia used luminescent proteins (67). The
Physiol Rev • VOL
discovery of calcium oscillations was made in secretory
epithelial cells (hepatocytes), also using a luminescent
probe aequorin (309). Since then, however, the fluorescent dyes have become the standard probes used in secretory epithelial investigations. This has happened because secretory epithelial cells are relatively small and are
difficult to microinject. The other reason is that most
primary isolated secretory epithelial cells are difficult to
transfect and maintain in culture. Microinjection and
transfection are two of the most frequently used techniques for intracellular protein loading. These techniques
of loading are difficult to implement on secretory epithelial cells.
Fluorescent dyes can be introduced into cells directly using microinjection or diffusion from a pipette in
whole cell, patch-clamp configuration. Most commonly
and simply, however, cells are loaded by incubation with
the acetoxymethyl ester (AM) form of the dye, which is
membrane permeant. Once inside the cell, the dye becomes trapped following cleavage of the AM group by the
cell’s own esterase enzymes (276). AM loading can, however, present some problems as dyes may become sequestered into intracellular compartments. Such compartmentalization can lead to difficulty in interpretation of the
Ca2⫹ signals measured. However, as described later, the
tendency of some dyes to specifically compartmentalize
during AM loading can be used to the experimenter’s
advantage (118, 120).
Fluorescent dyes can be placed into three categories
based on their spectral properties: 1) dual-wavelength
excitation (e.g., fura 2), 2) dual emission (e.g., indo 1), and
3) single wavelength (e.g., fluo 3, CaGreen, fura red).
The dual-wavelength indicators display spectral shift
(along the wavelength axis) upon calcium binding. The
major advantage of dual-wavelength dyes is that they
allow ratiometric analysis of fluorescence, which can minimize inconsistencies produced by dye loading, leakage,
bleaching, and cell movement artifacts (154, 276).
An interesting property of fura 2 and indo 1 is that
Mn2⫹ will quench their fluorescence. This has been put to
good use in studies of store-operated calcium entry (32,
166, 262). The calcium channels in internal stores are also
permeable to Mn2⫹, so measurement of the quench effect
allowed Thomas and collaborators (237) to study opening
of intracellular calcium channels.
Probes have been developed that can be used to
measure Ca2⫹ in different cellular compartments, such as
the ER and mitochondria. Some of the probes used for ER
calcium measurements were originally designed as Mg2⫹
indicators: mag-fura 2, mag-indo 1, mag-fura red, and magfluo 4. They have low affinity for Ca2⫹ and should not
usually react to calcium changes in the cytosol. However, in
the calcium stores (most importantly the ER) where the
calcium concentration is relatively high, these indicators,
although designed as Mg2⫹ probes, behave as calcium indi-
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
the level of bile flow to assess the effects of in situ
stimulation of a calcium-sensing protein in hepatocytes.
Microelectrode techniques yielded important early
information on calcium-dependent conductance changes
in exocrine cells (230). The further development of electrophysiological techniques, including patch-clamp techniques (110), had a profound effect on epithelial biology
and, in particular, on our understanding of calcium handling in secretory epithelial cells. These techniques have
permitted detailed electrophysiological analysis of calcium-dependent ionic currents (13, 58, 77, 137, 170, 228).
Also, the relationship between intracellular calcium level
and activation of some channels (e.g., Ca2⫹-dependent
Cl⫺ channel in pancreatic acinar cells) has permitted
indirect monitoring of the calcium level by the whole cell,
voltage-clamp technique (228).
Patch-clamp techniques have been extensively used to
characterize the channels that are responsible for Ca2⫹ influx in nonexcitable cells (215). The functional consequences of calcium signals were also assessed using electrophysiological techniques. As vesicles fuse with the
plasma membrane during exocytosis, the area, and hence
the capacitance, of the membrane is increased. The link
between exocytosis and calcium transients was characterized using capacitance measurements (124, 168, 169, 236).
Measurements of the activity of Cl⫺ channels have provided
important information about calcium-dependent activation
and regulation of fluid secretion in exocrine cells (137, 228).
Electrophysiology has played an important role in
gaining a general understanding of the mechanisms of
calcium release. Detailed characterization of the inositol
1,4,5-trisphosphate (IP3) receptors, channels that mediate
calcium release from the endoplasmic reticulum (ER),
and their calcium sensitivity was made in electrophysiological studies of the proteins in reconstituted lipid bilayers (20). Single-channel recordings on the surface of ER
have also allowed characterization of release channels
from internal stores (162).
Optical techniques developed during the last 15 years
have allowed direct visualization of calcium signaling at
the cellular and subcellular level. The optical probes used
for calcium measurements fall into one of three categories: luminescent photoproteins (e.g., aequorin), fluorescent proteins (e.g., cameleons), and fluorescent dyes (e.g.,
fura 2). Each of the three approaches has advantages and
drawbacks (for review, see Refs. 27, 276). It is the development of optical techniques that has been the basis of
our understanding of polarized calcium and calmodulin
signaling. Therefore, the remainder of this section focuses
on such techniques.
703
704
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
C. Instruments
Single cell measurements using fluorescent calcium
probes require a fluorescent microscope with appropriate
light detector (usually photomultiplier). The distribution
of fluorescence in different regions of the cell can be
imaged using fluorescent microscopes equipped with a
charge-coupled device (CCD) camera (154). The increase
of sensitivity of the cameras can be achieved by cooling or
by using optical intensifiers. Deconvolution techniques
can be applied to decrease contamination of images by
out-of-focus fluorescence (256). Smaller and discrete calcium signals (for example, small cytosolic signals or signals in mitochondria) become difficult to assess accurately due to interference from so-called “out-of-focus”
light. This is the light emitted from above and below the
focal plane that adds an inherent noise that may mask the
fluorescence signal coming from the structures in the
focal plane. It is in this respect that confocal microscopy
has distinct advantages over conventional imaging techniques. Confocal microscopy can increase vertical resolution (sometimes termed “Z” resolution) of fluorescent
images more than 10 times. The principles of confocal
microscopy are well described in several reviews (241,
270) and will not be discussed here. Most importantly,
confocal microscopy allows higher signal-to-noise ratios
of recordings of small subcellular Ca2⫹ signals and more
accurate localization of organelles involved in calcium
signaling. Most modern confocals are of the laser scanning variety; that is, the laser excitation light is scanned
across the sample to produce the image. Recent technological developments in control of this scanning have
dramatically increased the speed with which images can
be acquired. This means that it is possible to follow events
at the same rate as conventional imaging while maintainPhysiol Rev • VOL
ing the high spatial resolution (154). The improved control of the excitation light has also been effectively put to
use in studies based around rapid and localized changes
in excitation. These include fluorescence recovery after
photobleaching (FRAP) experiments in which it is possible to monitor the movement of a fluorescent molecule
into a region that has previously been photobleached by
transient application of very strong excitation light. This
allows insights into the speed of translocation of molecules and the connectivity of different regions of the cell.
An extension of the same process is used for uncaging of
caged compounds. These compounds are specialized derivatives of molecules (e.g., cell signaling messengers)
that are attached to an ultraviolet (UV)-sensitive group
which effectively “inactivates” the bioactive molecule.
When the caged compound is exposed to UV light, the
caging group is removed and the signaling molecule is
free to complete its function. The most common of the
caged compounds is caged Ca2⫹, which has been used to
elevate Ca2⫹ inside the cell in a controlled manner that is
independent of the usual calcium release pathways (1,
72). IP3, cAMP, cADP-ribose, and nicotinic acid adenine
dinucleotide phosphate (NAADP) are also available in
caged form (112). The combination of carefully controlled
UV exposure and simultaneous confocal monitoring of
the effects of uncaging allows the experimenter to dissect
intracellular signaling pathways and localize them within
the cell.
Multiphoton (MP) microscopy is the latest innovation in imaging technology to have a significant impact on
this field (64). MP uses long (infrared; IR) wavelength
laser light to excite the fluorophore. Excitation is
achieved when two (or more) photons hit the fluorophore
and become absorbed at almost exactly the same time.
The two-photon reaction depends nonlinearly on the intensity of excitation light; it occurs effectively only when
the density of photons is very high. Therefore, excitation
is only achieved in that very small volume where the laser
is focused. Emitted light will only come from this region
and so can be collected without a pinhole (since there is
no out-of-focus light). Another important advantage of
two-photon microscopy is that its depth of penetration is
more than double that of confocal. Because IR excitation
light is absorbed or scattered by tissues less than visible
light, it is possible to scan areas ⬎100 ␮m deep into the
sample. This has been used in other tissues (for review,
see Ref. 142) but could be particularly well-suited to
secretory epithelia since it offers the possibility of studying intact secretory units or even tissue in situ (54).
Principles, advantages, and limitations (photobleaching,
phototoxicity) of two-photon microscopy have been considered in detail in a number of recent publications (141,
202) and are not discussed here.
As yet, there are few examples of this technique
applied to signaling in secretory epithelia. However, those
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
cators. There is also now a range of indicators with intermediate affinity to calcium and low affinity to magnesium
(e.g., fura 2-FF, fluo 3-FF) which should be even more suitable for measurements of calcium in internal stores (118).
Indicators can be loaded into ER by incubation with
appropriate AM forms. Normally, to reduce cross-compartment signal interference, it is necessary to wash out
the dye that has remained in the cytoplasm. Various approaches have been reported to achieve this including
permeabilization (120, 301, 325) and dialysis via patch
pipette in whole cell patch-clamp configuration (218).
Rhod 2-AM, by virtue of its positively charged nature
(in the AM form), is compartmentalized into negatively
charged organelles. This leads to the dye being trapped
predominantly in the mitochondria after AM loading. By
careful adjustment of loading protocols, it has proven
possible to measure mitochondrial calcium transients using rhod 2 (69, 216).
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
D. Visualization of the Distribution of Organelles
and Molecules Involved in Calcium Signaling
It is noteworthy that there are many fluorescent dyes
that can be used for labeling of cellular organelles. Fluorescent indicators are available for labeling ER, secretory
vesicles and other acidic organelles, mitochondria, and
nucleus (112). These dyes have proven useful for the
localization of organelles in live cells and for correlation
of the position of organelles and calcium signals. The
accurate localization of (often small) organellar structures has relied on confocal detection of specialized dyes
(9, 95, 216). The number of these dyes is now very large,
most intracellular organelles can be targeted, and various
wavelengths of excitation and emission light can be used.
Although more traditional microscopy methods are
applicable, confocal microscopy has been particularly
useful in the immunofluorescent analysis of protein distribution in fixed sections. Particularly important have
been studies to determine the distribution patterns of the
major proteins involved in calcium signaling pathways.
The only limitations here are the antibodies available,
their specificity, and the condition of the cells after the
necessary permeabilization and fixing procedures. This
approach has been widely used in many secretory epithelial cell types to localize calcium release channels (113,
148, 150, 191, 197, 278, 314, 318), calcium pumps (147, 150,
302), and other proteins involved in calcium signal transduction (35, 50, 59, 135).
An interesting extension of this is the use of bioactive
fluorescently labeled compounds to target proteins in live
cells. A recent example is the use of BIODIPY-conjugated
ryanodine by Straub et al. (268) to localize ryanodine
receptors in live pancreatic acinar cells. The advantages
Physiol Rev • VOL
of this approach are that the cell disruption is minimal
and the integrity of the cells can be determined by functional assays.
Fluorescent dyes that label lipid bilayers (e.g., FM1–
43) were used to monitor granular dynamics while simultaneously measuring intracellular calcium using another
dye (95). Optical measurement of exocytosis from exocrine epithelium was extended by Kasai’s group (200) to
multiphoton microscopy (described above). Another
study of exocytosis in pancreatic acinar cells used nonfluorescent microscopy to visualize granule movements
(34). This technique is based on the fact that the zymogen
granules are very absorptive of visible light and thus
appear dark on transmitted images. By continuously subtracting one bright-field image from the previous one, the
authors of this study were able to see the changes in
granule darkness which occur upon exocytosis. The technique seems to correlate well with fluorescent dye analysis of secretion and is applicable to any cell type with
large, optically dense vesicles (254).
Particularly high resolution in studies of localization
of specific organelles and molecules responsible for calcium signaling is produced by immunoelectron microscopy. This usually involves detection of electron-dense
gold particles conjugated via antibodies to the protein
under investigation. This technique can only be applied to
fixed cells, but the spatial resolution is unrivalled. Khoo et
al. (135) have used this technique to detect the presence
of CD38 (an enzyme suggested to have a role in production of cADPr) to the inner nuclear membrane in hepatocytes. Also, IP3 receptors (IP3Rs) were localized to vesicles located in the apical region of submandibular gland
duct cells using this technique (314).
E. Measurements of Calmodulin Distribution
and Calcium-Calmodulin Binding
A range of fluorescently labeled calmodulins that are
suitable for live cell studies was recently developed (297,
298). 5-(4,6-Dichlorotriazinyl)-amino-fluorescein (DTAF)calmodulin does not change its fluorescent intensity upon
calcium binding, whereas 2-chloro-(⑀-amino-Lys75)-[6-(4N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl (TA)-calmodulin increases its fluorescence when it binds calcium. The
development of TA-calmodulin gave experimenters the
fascinating opportunity to monitor the calcium-calmodulin reaction in real time at a cellular and subcellular level
(298, 327). Importantly, DTAF- and TA-calmodulins could
be used simultaneously, due to differences in their optical
properties. This allows the distinction between changes
of fluorescence due to redistribution of the calmodulin
and changes due to calcium binding (298). Studies by
Craske et al. (59) used these two forms of calmodulin in
pancreatic acinar cells. They were able to see transloca-
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
studies that have been published are significant and highlight the possibilities of the technique. The molecular
structures of the cytoskeleton (microtubules/actin) and
ER strands that underlie the polarity of pancreatic acinar
cells were visualized by Fogarty et al. (82), and the importance of these structures to normal calcium signaling
was demonstrated. Very recently, in the same cell type,
Kasai’s group has used MP microscopy to simultaneously
visualize the exocytosis of individual granules and measure intracellular Ca2⫹ concentration ([Ca2⫹]i). This led
them to important insights into the link between different
calcium signals and secretion and into to mechanism of
secretion itself (200). Both these studies were dependent
on the accurate detection of small fluorescent structures
achieved by MP microscopy. Furthermore, some common
calcium indicators (e.g., fura 2) appear to be well suited to
two-photon microscopy (144). Recently developed calcium-sensitive fluorescent proteins, called cameleons, can
also be used for two-photon calcium measurements (75).
705
706
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
III. POLARITY OF INTRACELLULAR CALCIUM
SIGNALS
A. Patterns of Calcium Signaling in Secretory
Epithelial Cells
Calcium oscillations are the major form of intracellular calcium signaling in secretory epithelial cells stimulated by hormones or neurotransmitters. Below is a brief
overview of studies that have described calcium oscillations in exocrine cell types.
Oscillations of cytoplasmic Ca2⫹ were first directly
demonstrated in hepatocytes using the bioluminescent
protein aequorin (309). Since then, calcium oscillations
have been recorded in most exocrine cell types studied.
The calcium oscillations seen with aequorin in hepatocytes have subsequently been confirmed using fluorescent
calcium dyes (243, 244). Several different types of calcium
oscillations are triggered by vasopressin, phenylephrine,
angiotensin II, ATP, ADP, and bile acid in this cell type
(42, 65, 243, 244, 250, 309). Such oscillations are normally
dependent on cyclic release from and uptake into intracellular calcium stores (usually ER).
Physiol Rev • VOL
Different agonists produced specific calcium signaling
patterns in hepatocytes. For example, the duration of calcium spikes was shortest for phenylephrine, whereas ADPinduced spikes were more prolonged. The duration of spikes
was even longer when stimulated by vasopressin, angiotensin II, or endothelin-1 (57, 287). The rate of calcium rise and
peak calcium level were similar for transients induced by
different agonists, whereas the rate of decline showed considerable variability and was agonist specific. Therefore, it is
the differences in the rate of decline which underlies the
agonist-dependent differences in transient duration (57,
287). ATP triggered complex Ca2⫹ responses that could be
comprised of short (⬃9 s in duration) spikes and much
longer (⬃50 s) transients. It is interesting to note that increasing the concentration of some agonists (e.g., phenylephrine) resulted in an increase of frequency of oscillations
while the shape and duration of transients remained unchanged. This led to the suggestion that the intensity of the
stimulus could be encoded by frequency modulation of calcium signals rather than by amplitude modulation (57, 240,
243, 287, 309).
Oscillatory calcium behavior was also described in
gastric parietal cells stimulated by histamine, carbachol
(156), or gastrin (63). Spontaneous calcium oscillations,
calcium oscillations triggered by mechanical stimulation,
and oscillations induced by ATP were found in experiments on mammary epithelial cells (87). Electrophysiological and optical measurements identified calcium oscillations in lacrimal acinar cells stimulated by muscarinic
agonists (21, 167). In parotid acinar cells, stimulation of
cell surface muscarinic and ␣-adrenergic receptors induces intracellular calcium oscillations (101).
It was in parotid acinar cells that a very interesting
and unusual type of oscillation was discovered. These
oscillations are triggered by thapsigargin, an inhibitor of
the sarcoplasmic/endoplasmic reticulum calcium pumps
(SERCA), which normally replenish intracellular calcium
stores. The inhibition of store uptake initiates oscillations
that are based on variations of calcium exchange between
the cell and the external media. This exchange is mediated
by calcium influx channels and by the plasma membrane
pumps (83, 84).
Calcium oscillations in exocrine pancreas were described in both duct cells (266) and acinar cells (226, 319).
Pancreatic acinar cells display complex patterns of oscillatory behavior, which are specific for particular hormones and neurotransmitters. The circulating hormone,
cholecystokinin (CCK), produces one type of pattern. At
submaximal doses of this hormone, the oscillations are
usually composed of individual calcium elevations from a
baseline that is similar to the resting calcium level (226,
272, 321). Bombesin produces a similar pattern of calcium
oscillations (172). However, the neurotransmitter acetylcholine (ACh), in the same cell type, produces faster
calcium oscillations that are superimposed on a plateau
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
tion of DTAF-calmodulin upon stimulation with Ca2⫹releasing agonist and monitor the interaction of Ca2⫹ and
calmodulin using TA-calmodulin (59).
Persechini and colleagues (242) developed an alternative calcium-calmodulin (Ca-CaM) sensor based on fluorescent proteins (FPs) (242). This sensor used a short
Ca-CaM binding protein to link two FP molecules (CFP
and YFP), which allows the fluorescence resonance energy transfer (FRET) to occur between the two fluorophores. The spectral relationship between the two FPs
meant that when Ca-CaM bound to the link protein, inducing a steric change, there was a change in the fluorescent emission of the molecule. This change was used to
monitor activity and position of CaM (285).
The expression of targeted fluorescent (e.g., GFP)
and bioluminescent (e.g., aequorin) proteins is being used
more and more to investigate specific organelles or proteins within the cell (52, 323). As mentioned above, the
expression of such proteins in secretory epithelia is not
easy, and hence, there is little use of the approach in this
field. There are, however, some studies in other cell types
which tackle general phenomena, and it may be possible
to extrapolate these results to problems in epithelial cells.
For example, the interaction of ER and mitochondria
described using targeted GFPs in HeLa cells (239) offers
a tempting explanation for results found in pancreatic
(216, 268, 291) and salivary acinar cells (325). Also, recent
work using GFP constructs, which highlighted the role
of gap junctions in the propagation of calcium signals
(214), may be very relevant to intercellular signaling in
secretory glands.
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
Physiol Rev • VOL
of these polarized calcium signals. The way in which
these mechanisms are activated determines the characteristics of the transients and thus forms the basis for
agonist-specific oscillation patterns. The properties of
these mechanisms are discussed in the following sections.
B. Subcellular Heterogeneity of Calcium Signals
Soon after the discovery of calcium oscillations, imaging techniques became widely available for studies of
cellular calcium distribution. Results from the last 10
years of videoimaging experiments have demonstrated
that a number of secretory epithelial cell types, hepatocytes, pancreatic acinar cells, lacrimal acinar cells, submandibular gland acinar cells, and submandibular gland
duct cells, exhibit a common property. In all these cell
types, calcium transients are initiated in the apical region.
This initiation is usually followed by the formation of
calcium waves, which propagate into the basal part of the
cells (131, 148, 195, 290, 293). However, some types of
smaller calcium signals can have an entire life cycle (initiation, local spreading, and decline) that is confined to
only a small, subcellular compartment, the apical region
of the cells (132, 290, 314) (example shown in Fig. 1A).
This form of localized signaling is probably very energy efficient. Calcium signals simply occur where they
are needed. The apical membrane of exocrine epithelial
cells is the place where secretion, both fluid and exocytotic secretion, takes place. The primary purpose of the
calcium signals is to initiate the secretion process, and
therefore, it is a most economical strategy to produce the
signals in the region where the cell’s business is taking
place.
Interestingly, in pancreatic acinar cells, short applications of ACh at very high concentration produce calcium responses localized to the apical (secretory granule)
region. ACh is a neurotransmitter and is expected to have
a short lifetime after its release from the synaptic terminals of peripheral neurons, due to activity of ACh esterase
present in pancreas. It is therefore conceivable that this
localized apical calcium response is the main calcium
signaling mode of this important neurotransmitter in exocrine pancreas (90).
There is, however, a necessity to inform the rest of
the cellular organelles about the apical secretion so that
all the functions of the cell can be coordinated. For example, to replenish the store of proteins lost as a result of
secretion, mRNA transcription and translation should be
stimulated. Propagating calcium waves could play the
role of long-range, coordinating messenger by delivering
the signals to the nucleus, rough ER, mitochondria, and
other organelles outside apical region. It would seem
most reasonable, evolutionarily, for initiation of these
waves to use the same mechanism that is involved in
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
that is considerably elevated compared with the resting
calcium level (321). At low doses, ACh and CCK can
generate oscillating calcium signals that are confined to a
small region of the cell (132, 210, 290).
The role of individual second messengers in generating oscillations was examined in a number of studies. It
was found that calcium oscillations can occur at constant
concentration of the calcium-releasing messenger IP3
(306). The bell-shaped sensitivity to Ca2⫹ of the IP3R
suggests that Ca2⫹ itself, by action on the IP3R, could
provide the negative feedback necessary to generate oscillations (20). Some recent studies on nonepithelial cells
indicate that IP3 oscillations after agonist stimulation can
occur and underlie oscillatory calcium responses (114,
192, 193, 208).
An interesting hypothesis on the mechanisms determining calcium signaling patterns was put forward in
studies from Muallem’s laboratory (159, 311, 324). The
hypothesis suggests that regulators of G protein signaling
(RGS) proteins specifically interact with different Gq-coupled receptors for calcium-releasing secretagogues. The
effect of RGS proteins was to inhibit phospholipase C
(PLC)-␤ activity and consequently to suppress IP3 generation and downstream calcium signaling. Thus it is proposed that RGS proteins may be responsible for the control and formation of oscillations in pancreatic acinar
cells via the interaction with the G protein-receptor complex. It is suggested that cyclic binding of Ca-CaM to RGS
proteins is essential for generating oscillations. Although
this hypothesis provides a conceptually novel mechanism
for control of oscillations, important questions concerning the dynamics of IP3 production and Ca-CaM binding to
RGS proteins remain to be tested by direct experiments.
An interesting aspect of these studies is that the
effect of the RGS proteins was found to be determined by
the nature of the receptors and was very different for
different secretagogues (311, 324). For example, both responses to carbachol and to CCK were inhibited by RGS4,
but responses to carbachol were 33 times more sensitive
to RGS4 than responses to CCK (311); the difference was
even more dramatic (⬃1,000-fold) for RGS1. It was suggested that the interaction of amino terminus of RGS
proteins with receptors determined the specificity of the
RGS protein action (311, 324). The specificity of this
interaction may determine the individuality of agonistinduced oscillation patterns.
In addition to IP3, there is strong evidence that “novel” calcium-releasing messengers (cADP-ribose and
NAADP) may have a role in determining patterns and
receptor specificity of oscillations in pancreatic acinar
cells (40, 55, 289).
During the oscillatory phase, the rise of each individual calcium transient inside the cell is heterogeneous in
both space and time. There are some interesting general
mechanisms that underlie the formation and propagation
707
708
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
generation of localized apical calcium responses. Therefore, the waves should start in the apical part of the cells
(example in Fig. 1B).
It is important to note that to organize localized
calcium signals, small cells need to have a high calcium
buffering capability. Without buffering, calcium will rapidly spread through the cytoplasm by diffusion, limiting
the possibility of localized signaling. The numerical measure of buffering is the calcium binding capacity (for
definition, see Ref. 199). Pancreatic acinar cells are the
only exocrine secretory cell type in which calcium binding capacity was estimated (182). The buffering was
found to be very high, with a binding capacity of ⬃2,000.
This means that out of 2,000 calcium ions added to the
cytoplasm of the cell, only 1 will remain free and the rest
will be bound by buffer (182). The high binding capacity is
probably important for localized apical calcium signaling
and must be overwhelmed to allow propagation of calcium waves.
Physiol Rev • VOL
The nuclei of these cells appear to be particularly
well protected against calcium rises induced by short
applications of ACh. Large calcium gradients of many
hundreds of nanomolar per micrometer can form along a
line connecting the secretory region and the nucleus.
Despite their size, these gradients eventually dissipate
without spreading into the nucleus (90). It is not yet clear
whether this nuclear protection is mediated by the nuclear envelope acting as a physical barrier to diffusion or
by elevated nuclear calcium buffering capacity.
The discovery of apical-to-basal calcium waves and
their correlation with agonist-stimulated ion currents in
pancreatic acinar cells led Kasai and Augustine (131) to
propose the “push-pull” model for unidirectional Cl⫺ secretion (131). This model is based on early activation of
apically located Ca2⫹-dependent Cl⫺ channels leading to
secretion of Cl⫺ into the lumen, the “push” phase. Cl⫺
uptake through the basal membrane channels only begins
later when the calcium wave reaches that area of the cell;
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
FIG. 1. Examples of the subcellular dynamics of secretagogue-induced calcium responses in secretory epithelia. All
images show pseudocolor representation of fluo 3 fluorescence obtained using confocal mocroscopy. A: an isolated
pancreatic acinar cell, shown in transmitted image (top left image), produces a calcium spike that is localized wholly in
the apical (secretory granule) region when stimulated with low doses of ACh (10 nM). Scale bar ⫽ 10 ␮m. B: the same
cell stimulated later with a higher concentration of ACh (50 nM) undergoes a global calcium transient that spreads as
a wave in an apical-to-basal direction. C: in blowfly salivary gland epithelial cells, stimulation with 3 nM 5-hydroxytryptamine triggers calcium waves that travel in the opposite direction to those in pancreatic acinar cells. The calcium
response begins in the basal area of the cell (cell delineated by white box) and spreads as a wave toward the apical
region. Scale bar ⫽ 20 ␮m. [C from Zimmermann (326), copyright Harcourt Publishers.]
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
C. Distribution of Calcium-Releasing Channels and
Polarity of Calcium Responses
The basis of the apical-to-basal pattern of calcium
signaling is polarized distribution of calcium-releasing
channels on the internal calcium stores. The IP3Rs are
frequently considered to be the channels responsible for
the activation of calcium signals, whereas the propagation
of the signal was thought to involve ryanodine receptors
(RyRs). Recent studies both enriched and complicated
this general notion.
1. Initiation of calcium signals
One of the earliest descriptions of polarized calcium
signals came from a study on hepatocytes in which
Rooney et al. (244) reported that intracellular calcium
oscillations originate repeatedly in the same region of the
cell, adjacent to the plasma membrane. The initiation site
remained the same during sequential stimulation with
different agonists (244, 287). Polarized calcium signaling
in hepatocytes was later confirmed in experiments on
intact, perfused liver. In the periportal hepatocytes, the
initiation of calcium signals occurred repeatedly in particular intracellular regions, the sinusoidal domains (240).
As noted above, the calcium waves in hepatocytes propagate in an apical-to-basal direction (195). Importantly,
IP3Rs are found in apical region of hepatocytes (but also
in the nucleus) (195). Initiation is therefore suggested to
be IP3 dependent. The propagation speed and the amplitude of the propagating wave are, however, reduced by
Physiol Rev • VOL
substances that inhibit RyRs (dantrolene, ryanodine).
This suggests the involvement of RyRs in propagating the
calcium signal into the basal part of the cell (195).
The geography of calcium signal initiation was advanced in work by Kasai et al. (132). It was found that
calcium signals are initiated in a small subregion of the
apical part of pancreatic acinar cells termed the “trigger
zone” (132). After initiation in the trigger zone, the signals
spread over the granular area. Injection of IP3 from the
patch pipette resulted in calcium signals originating in the
trigger zone, indicating the presence of high-affinity IP3Rs
in this part of the cell. These IP3Rs are essential in the
initiation of local and global calcium signals in pancreatic
acinar cells (132). A more recent study, which probed the
cytosol of pancreatic acinar cells with local uncaging of
IP3, revealed that apical uncaging systematically produced calcium signals, whereas uncaging in basal regions
produced calcium responses in only 50% of the experiments. The amount of uncaged IP3 necessary to generate
responses in basal part of the cells was also larger than in
the apical region (81). These data suggest that high-affinity IP3Rs concentrate in the apical part of pancreatic
acinar cells, whereas the basal part contains IP3Rs of
lower affinity that are distributed nonuniformly (present
only at a number of discrete sites) (81). The existence of
IP3-induced release in the basal region suggests that these
receptors could contribute to propagation of calcium
waves.
The distribution of release channels underlying polarized calcium signaling was directly elucidated using
immunostaining. IP3Rs (type 3) were found at the extreme apex of pancreatic acinar cells (197).
The suitability of different types of IP3Rs for initiating calcium signals recently became a topic of lively discussion. Hagar et al. (107) considered that the absence of
inhibition of IP3R3 by high levels of calcium makes it a
particularly suitable channel for initiation sites. Later
work, however, indicated that IP3R3 has a bell-shaped
dependency on calcium (274). In the presence of IP3, it is
activated by low concentrations of calcium and inhibited
by higher concentrations (274). In other words, it behaves
similarly to other types of IP3Rs. This does not preclude,
however, that IP3R3 could play an important role in initiating calcium signals. Indeed, studies using electrophysiological analysis of recombinant IP3Rs in oocytes have
suggested that, although many of the general properties of
the different subtypes are similar, there are sufficient
differences to suggest specific roles for certain isoforms
(163, 164). The relatively high sensitivity to IP3 of subtype
3 implicates this isoform in initiation of calcium signals
while the highly Ca2⫹-sensitive IP3R1 is ideally suited to
amplification and propagation of the signal (163).
In another study on pancreatic acinar cells, IP3R1,
IP3R2, and IP3R3 were resolved in the apical part of the
cells close to luminal and lateral membranes. In this work,
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
this is the “pull” phase. The sequential activation of the
two phases would be necessary because the depolarization of the cell membrane potential caused by the push
phase is prerequisite to the pulling of Cl⫺ into the cell.
This was the first description of how a subcellular calcium
gradient triggered by agonists could be vital in the control
of cell function. The model obviously relies on the existence of both basal and apical Ca2⫹-dependent Cl⫺ channels and has been thrown into some doubt by work from
Greger’s laboratory in which the basal channels could not
be found (265). Recent experiments using local uncaging
of caged Ca2⫹ to activate the Cl⫺ current have also demonstrated the absence of Cl⫺ channels in the basal and
lateral regions of the plasma membrane while confirming
the presence of the channels on the apical membrane
(217). Furthermore, in the fluid-secreting blowfly salivary
gland, agonist-induced Ca2⫹ waves initiate in the basal
region and spread to the apical part of the cell (326). This
leaves the situation somewhat unresolved. Although Cl⫺
secretion appears to be activated by the Ca2⫹ transient
and will be dependent on cell membrane potential as
Kasai and Augustine showed, the exact mechanism of
transcellular Cl⫺ movements remains elusive.
709
710
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
Physiol Rev • VOL
hibition; short exposure to a high concentration of
NAADP inhibits further NAADP responses for a long period of time. Remarkably, high autoinhibitory concentrations of NAADP also block CCK-induced calcium signaling. These data invite the suggestion that NAADP could
serve as a second messenger linking CCK receptors with
the calcium signaling cascade (36).
At the moment, it is not clear whether different agonists produce signals that have absolutely identical initiation sites. One study found no difference in initiation
sites of signals induced by CCK and by ACh (37), whereas
another work reported that the ACh analog carbachol and
CCK initiate signals at different sites (260). However, both
studies agree that for all agonists the signals originate in
some part of the apical region.
In pancreatic acinar cells, then, IP3, cADP-ribose, and
NAADP can all release calcium and all seem to be involved in the initiation of agonist-evoked signals. This has
led to the idea of cooperativity between the release mechanisms being necessary for initiation of the calcium signals in the apical region of the cell (37). This postulate
obviously requires that calcium stores available to each
messenger should be present in this highly sensitive region. Experiments performed on cultured pancreatic acinar cells permeabilized by streptolysin-O (SLO) indicate
the existence of three types of calcium stores. The first
type possesses receptors to cADP-ribose and IP3, the
second type has only IP3Rs, and the third type contains
Ca2⫹ that cannot be mobilized by IP3 or cADP-ribose but
can be released with ionomycin (97). This finding is in
contradiction with a previous study (also performed on
SLO-permeabilized pancreatic acinar cells) which reports
uniformity of IP3 sensitivity in different parts of the cell
(301). Recent experiments with photobleaching of indicators trapped in the lumen of ER and uncaging of caged
calcium loaded into the ER lumen showed considerable
connectivity of this major calcium store in pancreatic
acinar cells (218). The intracellular stores of hepatocytes
are also luminally connected, allowing full access to all
the available stored calcium after opening of any release
channel (109). Luminal connectivity of internal calcium
stores means that the localization of signal initiation is
very dependent on the positioning of release channels on
the store membrane rather than on the positioning of the
stores themselves.
Recent work from Muallem’s group (260) reported
that apical initiation of calcium signals may be aided by
preferential localization of ACh and CCK receptors to
apical and lateral membranes. This potentially interesting
new finding is, however, in contradiction with classical
work from the Jamieson laboratory (247) that describes
the presence of CCK receptors on the basal membrane
and explicitly states the absence of CCK receptors on the
apical membrane (247). The results of the Muallem group
are also in apparent contradiction to biophysical studies
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
the location of initiation sites was compared with the
distribution of immunofluorescent staining obtained with
different types of antibodies for IP3Rs. In some pancreatic
acinar cells, calcium initiation sites correlated with the
position of IP3R2 (148), although there is a substantial
overlap in distribution of all the subtypes in the apical
region (148, 197).
The position and the density of receptors can apparently be influenced by secretagogues. Treatment with
secretagogues causes redistribution and downregulation
of IP3Rs in the pancreas. The downregulation utilizes the
ubiquitin/proteasome pathway but is not limited by this
pathway (308). Calcium release by IP3Rs is also subject to
regulation by other intracellular pathways. IP3R3 is rapidly phosphorylated by cAMP-dependent kinase (PKA)
leading to inhibition. This phosphorylation occurs after
stimulation with physiological concentrations of CCK but
not with ACh. PKA and IP3R3 are colocalized in the apical
region of pancreatic acinar cells. Treatment with dibutyryl-cAMP or forskolin (activators of PKA) inhibits local
apical calcium transients induced by uncaging of small
doses of caged IP3. Brought together, these data suggest
that PKA-mediated phosphorylation of IP3R3 receptors
may be important in shaping the pattern of hormoneinduced calcium signaling in these cells (94).
The notion that IP3Rs are the only type of receptors
responsible for the activation of calcium signals in the
apical region of pancreatic acinar cells was shaken by
experiments with recently discovered calcium-releasing
substances. cADP-ribose, a potential activator of RyRs,
induces repetitive calcium transients localized to the secretory granule region of pancreatic acinar cells. This
suggests that both RyRs and IP3Rs could be involved in
generation of local calcium signals in the apical region of
pancreatic acinar cells (289). The calcium-releasing channels involved in activation of calcium signals could be
agonist specific. The cADP-ribose antagonist 8-NH2cADP-ribose inhibits local calcium responses induced by
cADP-ribose but not responses triggered by IP3. These
results were not unexpected but, more surprisingly,
8-NH2-cADP-ribose also blocks apical calcium oscillations
triggered by small, physiological doses of CCK and bombesin (29, 38, 39). In contrast, ACh-induced calcium signals
were unaffected by 8-NH2-cADPr (29). It was mentioned
previously that agonists produce different patterns of calcium signals, and these results suggest that it may be the
recruitment of different calcium release channels following agonist-stimulated messenger production that underlies this specificity.
Another recently discovered (89, 146) calcium-releasing compound, NAADP, also induces calcium responses
in pancreatic acinar cells. Calcium transients can be local
(confined to the apical region) or global. The shape of
NAADP-triggered responses is similar to those triggered
by CCK. An interesting property of NAADP is its autoin-
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
Physiol Rev • VOL
and IP3R3 were detected close to luminal and lateral
membranes (148).
Lacrimal acinar cells also conform to the general rule
of apical initiation of calcium signals. ACh and ATP induce calcium signals that are initiated in the secretory
(apical) pole of the lacrimal acinar cells and rapidly
spread toward the basal pole (293).
An interesting exception from the rule of apical-tobasal propagation of calcium waves is found in acidsecreting parietal cells of the stomach. The polarity of
calcium signaling seems to be reversed in these cells.
Histamine stimulation triggers calcium signals that are
initiated in the basal part of the cells (although closer to
the central part of the cell rather to the extreme basal
membrane, judging by the traces on Fig. 3 from this
paper) (9).
2. Propagation of calcium signals
The mechanism underlying the propagation of hormone-induced calcium signals into the basal part of polarized epithelial cells was touched upon above, and the
suggestion was made that propagation could involve both
IP3Rs and RyRs. There are some arguments that the propagation of the calcium signals is mainly dependent on
IP3Rs. Of particular interest in this respect is a study in
which different types of calcium responses (local and
global calcium spikes), which can normally be triggered
by ACh, were mimicked by uncaging of IP3. Rapid and
transient uncaging of IP3 produces local calcium signals
similar to that triggered by ACh. To reproduce the shape
of global calcium transients, gradual slow IP3 uncaging
was necessary, indicating that global calcium transients
may depend on continuous IP3 production (125).
One study compared the importance of RyRs for
calcium signaling in pancreatic and SMG acinar cells
(148). This paper described that RyRs were found in SMG
acinar and duct cells but not in pancreatic acinar cells
(148). Caffeine, which increases the Ca2⫹ sensitivity of
RyRs, elevated calcium in SMG acinar and duct cells but
not in pancreatic acinar cells. cADP-ribose was very effective in mobilizing calcium from the stores in SMG
acinar and duct cells. Although cADP-ribose can activate
calcium release from the stores of pancreatic acinar cells,
it does so less effectively. Furthermore, the cADP-riboseinduced calcium response was blocked by the IP3R antagonist heparin (148). These data argue that, in pancreatic
acinar cells, RyRs are not important for calcium signaling, so
propagation should be IP3 mediated. Intriguingly, though,
infusion of IP3 through the patch pipette mainly produces
localized nonpropagating calcium signals (227). Furthermore, there are data supporting the central role of RyRs in
propagating the calcium signals into the basal part of pancreatic acinar cells. The existence of RyRs in pancreatic
acinar cells and their localization to the basal part of the
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
in which stimulation of apical calcium release was induced by basal application of ACh (290). The clarification
of agonist receptor localization is important for our understanding of the process of calcium signal initiation in
pancreatic acinar cells.
Duct cells of the submandibular gland (SMG) present
an interesting example of polarized calcium signals which
appears to rely on unconnected intracellular calcium
stores. As with pancreatic acinar cells, calcium waves
initiate in the apical region of SMG duct cells and propagate toward the basal part of the cells (314). The calcium
elevation in the basal region is small and not statistically
distinguishable from noise, which indicates that calcium
waves are confined to the apical and middle regions of the
cells while the basal part remains inert with respect to
calcium signaling. Immunofluorescence imaging localized
IP3R types 2 and 3 to the apical region of SMG duct cells,
whereas IP3R1 was not detected. The amount of IP3R2 in
the duct cells was higher than in any other tissue examined by the authors. Immunoelectron microscopy identified the IP3R2 on small apical vesicles of some SMG duct
cells (314). This study is unique since it actually ascribes
IP3Rs to particular separate organelles, namely, the tiny
vesicles concentrated in subplasmalemmal region in the
apical part of the cell. The implication of these findings is
that these discrete stores provide the calcium for signals
that follow IP3-mediated agonist stimulation.
There was considerable heterogeneity of the amount of
IP3R2 between different cells. The calcium signals in the
submandibular gland duct cells were also nonuniform from
cell to cell. Some showed rapid calcium mobilization,
whereas others responded slowly. These findings led to the
concept of “pioneer cells” that possess IP3R2 in considerable
numbers. As a result of their higher release capability, these
cells are the first to initiate calcium responses and therefore
coordinate calcium signaling throughout the duct by transmitting their signals to neighboring cells (314) (see sect. V).
Somewhat different results regarding IP3Rs in this cell type
were reported by Lee et al. (148). In SMG ducts, they resolved IP3R1 and IP3R2 close to luminal and lateral membranes. Furthermore, one IP3R3 antibody used in this study
detected receptors around the nucleus of SMG duct cells,
whereas another type did not stain the duct at all (148). Both
papers report the presence of more than one type of IP3R in
the apical region of SMG duct cells.
RyRs confined to the luminal pole and lateral membrane were reported in SMG duct cells (148). Both caffeine and cADP-ribose release calcium from internal
stores in these cells (148). The RyR stimulators, caffeine
and cADP-ribose, both release calcium in SMG acinar and
duct cells, and this release was not inhibited by the IP3R
antagonist heparin (148).
Acinar cells of the SMG give another example of
apical-to-basal pattern of propagation of calcium waves
triggered by secretagogues. In these cells, IP3R1, IP3R2,
711
712
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
D. Distribution of Cellular Organelles and
Polarized Calcium Signaling
In pancreatic acinar cells, nocodazole (a substance
that disrupts microtubular networks) inhibits IP3-induced
Physiol Rev • VOL
localized apical calcium spikes. Interestingly, treatment
with nocodazole also caused a redistribution of the ER,
resulting in a considerable decrease of ER density in the
apical region. Other substances that modify the status of
the microtubular network, colchicine and taxol, also inhibited local calcium spikes. These data argue that microtubule-dependent positioning of the ER in the apical region of pancreatic acinar cells is essential for maintaining
localized calcium signals (82).
Pancreatic acinar cells also provide another example
of polarized distribution of cellular organelles. Most of the
active mitochondria in these cells are clustered around
secretory granule region. This strategic positioning of
mitochondria allows them to play a barrier function in
limiting propagation of calcium signals. Indeed, inhibition
of mitochondrial metabolism (and therefore mitochondrial calcium uptake) with antimycin and oligomycin or
with carbonyl cyanide m-chlorophenylhydrasone (CCCP)
converts IP3-induced, local calcium oscillations into
global calcium transients (291). These results were challenged by studies of Gonzales et al. (98), who found that
mitochondrial calcium accumulation in pancreatic acinar
cells occurs with a time delay of ⬃11 s after the rise of
cytosolic calcium. In this paper, the mitochondrial clustering in the perigranular region was not found. On the
basis of their apparent slow Ca2⫹ uptake and uniform
distribution, the authors concluded that mitochondria do
not participate in limiting calcium signals to the apical
part of the cell.
However, the study by Straub et al. (268), mentioned
previously, confirmed the importance of mitochondria to
localization of apical calcium signaling in pancreatic acinar cells. Using IP3 uncaging, they also found that local
apical calcium signals are converted into global elevations by inhibition of mitochondria. The authors also
confirmed that mitochondria were concentrated mainly in
the perigranular region. These findings further demonstrated the barrier role of mitochondria in limiting the
spread of calcium signals from the apical region. In the
same paper, staining of the RyRs by BODIPY-ryanodine
showed that they were also concentrated in the perigranular region, although there was some diffuse staining
throughout the rest of the cytoplasm. The experiments of
this group indicate that mitochondria can play an important role in modulating the nearby RyRs. Inhibition of this
mitochondrial modulation makes propagation of the wave
through the area enriched with RyRs more likely (268).
Recent results from our group demonstrated the existence in pancreatic acinar cells of three distinct groups
of mitochondria: perigranular (described previously), perinuclear, and subplasmalemmal. Fast calcium uncaging
experiments showed that each group is capable of rapid
calcium uptake. The individual positioning of each group
of mitochondria allows them to influence distinct calcium
signals (216).
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
cells was reported by Nathanson’s group (150). This was
confirmed recently in what seems to be a very detailed study
from Pandol’s group (80). RT-PCR identified RyR isoforms 1,
2, and 3 in both rat pancreas and dispersed pancreatic acini.
Isoforms 1 and 2 were found using single-cell RT-PCR on
cells that were individually selected under a microscope.
The subcellular distribution of these isoforms of RyRs in
pancreatic acinar cells was studied using Western blot analysis. RyR1 and RyR2 were present in all fractions of the ER
(both rough and smooth). Immunofluorescence labeling using general antibodies against RyRs stained most of the
cytoplasm but was excluded from the nucleus. PalmitoylCoA, an activator of RyRs, released calcium from the stores
of permeabilized pancreatic acinar cells. Notably, the release was induced in both the apical and basal regions of
the cells. Upon palmitoyl-CoA activation, basal release
occurred simultaneously or immediately before the release in apical region. Alone, neither ryanodine nor caffeine had much effect. However, both substances potentiated the releasing action of palmitoyl-CoA (80).
Strong evidence in favor of RyR-dependent propagation of calcium signals into basal part of pancreatic acinar
cells was recently presented by Straub and colleagues
from Yule’s group (268). In their experiments, strong uncaging of IP3 results in global calcium signals. The application of ryanodine to inhibit RyRs limits these uncaginginduced transients to the region slightly larger than the
trigger zone. In other words, when RyRs were inhibited,
the IP3-induced apical responses were not converted into
global responses. The conclusion from this work was that
IP3 triggers responses in the apical part of pancreatic
acinar cells, but the propagation of the calcium wave
depends on RyRs (268). The role of cADP-ribose receptors in converting local apical signals into global calcium
waves was demonstrated in studies by Cancela et al. (37).
These views of calcium wave propagation in pancreatic
acinar were recently given further weight by work which
used region-specific uncaging of IP3 and cADP-ribose to
investigate the role of IP3Rs and RyRs (149). Leite et al.
(149) concluded from their studies that release from
IP3Rs in the apical region coordinates with cADP-ribosesensitized release from basal RyRs to generate global
ACh-induced calcium waves. NAADP is another candidate
for the role of “globalization” messenger. With the use of
whole cell electrophysiological recordings, it was found
that globalization of local ACh-induced calcium signals in
pancreatic acinar cells can be achieved by infusion of
NAADP (40).
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
E. Calcium Propagation Through the ER Lumen:
Signaling by “Tunneling”
The geography of cellular organelles in pancreatic
acinar cells presents an interesting puzzle in terms of
effective calcium signaling. The problem is that the apical
region of these cells is packed with secretory granules.
That leaves very little space for ER, which is the major
calcium-releasing store. Despite there being only a very
small volume of ER in the apical region, it must be able to
generate repetitive local calcium spikes, which are quite
resistant to removal of external calcium, and provide
effective initiation signals for propagating calcium waves.
How does the cell manage this when it would seem that
there is so little calcium stored in ER located in the
secretory granule region? It seems that the cell has resolved this problem by connecting the small calcium
stores in the apical part of the cell with a massive calcium
store, namely, the basally located ER. Direct conformation of ER connectivity was achieved by monitoring
FRAP. Mag-fluo 4 equilibrated quickly in the ER lumen of
pancreatic acinar cells after local photobleaching. This
dye is a particularly suitable indicator for such experiments, since it is much more fluorescent in ER than in the
cytosol. Similar fast equilibration (confirming luminal
connectivity of ER) was found for Mag-fura 2 (218). Uncaging of calcium was used to directly visualize calcium
movement in ER. Cells were loaded with the caged calcium compound NP-EGTA by incubation with the AM
form of this probe. After that, the cytosolic NP-EGTA was
Physiol Rev • VOL
removed via a patch pipette (the pipette also contained
high concentration of calcium buffer, to prevent any calcium changes in the cytosol). Localized uncaging of NPEGTA trapped in the ER lumen allowed monitoring of
calcium movements inside this organelle. In experiments
of Park et al. (218), fast intra-ER calcium movement was
found, confirming that calcium can indeed “tunnel” from
one side of the cell to the other (218).
Fast calcium movement through the ER lumen suggests that after calcium release, the apical calcium stores
can be effectively refilled by calcium moving from the
basal side through the lumen of ER (183, 218). This tunneling of calcium from the basal to the apical part of the
cell means that the apical stores effectively become undepletable. ER continuity is not peculiar to pancreatic
acinar cells. In hepatocytes, the connectivity of the stores
was assessed by using Mn2⫹ quench of fura 2 in the
cellular organelles (109). This study indicated that the
stores in intact hepatocytes were luminally connected,
and this connectivity was lost after disruption of the
cytoskeleton (using cytochalasin B). With the use of
FRAP of GFP fusion proteins targeted to the aqueous
phase of ER lumen (61, 273), ER continuity was also
demonstrated in nonepithelial cells.
Measurements of calcium binding capacity indicated
relatively weak calcium buffering in the ER compared
with the cytosol. This should make the ER route very
convenient for fast calcium translocation (182). Another
advantage of this transport route is that the cell could
move calcium through the ER tunnel without risk of
triggering calcium-induced calcium release. This should
make the “through ER” reloading route safer than via the
cytosol. For small and intermediate doses of agonists, one
can envisage bidirectional calcium fluxes in the pancreatic acinar cell: apical-to-basal flux in the cytoplasm and
simultaneous basal-to-apical movement through the ER
lumen.
In contrast, the major IP3-dependent store in SMG
duct cells seems to be disconnected from the basal ER. As
was mentioned above, Mikoshiba’s group (314) described
small vesicles containing IP3Rs located in the apical part
of these cells.
F. Polarization in Distribution of Calcium Pumps
and Other Cytoplasmic Calcium
Removal Mechanisms
Calcium pumps are essential in shaping calcium signals in exocrine secretory cells. They maintain the resting
calcium level, provide intracellular calcium stores with
releasable calcium, and play an essential role in recovery
of cytosolic calcium after calcium release from the store.
Calcium pumps are divided into two classes: plasma
membrane calcium pumps (PMCA) and SERCA (43, 160).
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
In hepatocytes, elements of the ER and mitochondria
are positioned closely to one another so that IP3-induced
calcium release has considerable effects on adjacent mitochondria. IP3-induced loss of calcium from the ER was
reflected in a rise of intramitochondrial calcium and increase of mitochondrial metabolism (108). Conversely, mitochondria can modulate IP3-induced calcium release. Inhibition of mitochondria results in an enhancement of calcium
loss from ER upon IP3 stimulation. The authors of this study
interpret this effect as an indication of mitochondrial suppression of positive calcium feedback on IP3Rs. So, under
normal conditions, by taking up some of the calcium that
comes out of the ER, the mitochondria make it less likely
that calcium-induced calcium release will occur. The ability
of mitochondria to effectively take calcium released from
ER is based on the neighborliness of the two organelles.
Such structural juxtaposition of ER and mitochondria has
been demonstrated by Rizzuto et al. (239) using organelletargeted GFP in HeLa cells. Interestingly, nonuniform distribution of mitochondria produces heterogeneity in IP3 sensitivity of different ER regions. The regions with a high density
of mitochondria are less sensitive to IP3 than the regions
with lower density (108).
713
714
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
Physiol Rev • VOL
tions. In isolated hepatocytes, inhibitors of PMCA pumps,
glucagon-(19O29) and carboxyeosin, increased the frequency of oscillations triggered by calcium-mobilizing
agonists (102). The distribution of PMCA in hepatocytes is
nonuniform, with the pumps found predominantly in the
blood sinusoidal regions of the cells (134).
With the use of pancreatic acinar cells, direct measurements of calcium extrusion fluxes at the cellular level
were made by placing a single isolated cell into a very
small droplet (⬃10⫺10 liter) that contains fluorescent calcium indicator. This “droplet technique” allows simultaneous measurements of intracellular calcium concentration and calcium extrusion (using different calcium
probes in the cell and in the extracellular droplet solution) (282, 283). The technique was used to characterize
calcium dependency of calcium extrusion (33) and for
measurements of calcium buffering of the cytoplasm
(182). The extrusion of calcium appears to be very active.
When maximally stimulated, calcium pumps of plasma
membrane can decrease total cellular calcium content by
more than 400 ␮M/min (33). It was possible to resolve
oscillations of calcium extrusion rate that occurred synchronously with spikes of intracellular calcium (284). It
was estimated that during global calcium transients triggered by secretagogues, ⬎20% of the calcium released
from the store is extruded from the cell by the PMCA
pumps (284).
The localization of calcium extrusion at the singlecell level was studied after the development of the “indicator-dextran” technique in our laboratory (16, 281). This
technique utilizes calcium indicators linked to heavy dextrans (mol wt from 70,000 to 500,000) in combination with
confocal microscopy. Under usual experimental conditions, when calcium is extruded from the cell, it would
diffuse away quickly and be diluted in the vast volume of
extracellular solution, making it impossible to measure.
The idea behind the dextran-indicator technique is to
slow down diffusion of extruded calcium so that the
fluorescence changes in the extracellular solution can be
resolved close to the site of extrusion. The labeled high
molecular weight dextrans in the extracellular solution
are both calcium indicators and immobile (in comparison
with small ions like calcium) calcium buffers. This means
that they simultaneously slow down calcium diffusion
and measure the calcium level. Free calcium concentration is kept low (0.2– 0.4 ␮M) so that the indicator dextran
is able to bind calcium. Combined imaging of intracellular
calcium (e.g., using fura-red) and of calcium extrusion
with calcium green-1 dextran is possible (15). The sensitivity of the method was high enough to allow detection of
calcium released by exocytosis of a single secretory granule (14). The technique was used to demonstrate that
extrusion occurs preferentially from the apical regions of
the pancreatic acinar cells (15, 17), suggesting that PMCA
are mainly localized in this part of the cell. This was very
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
Recently, immunofluorescent staining revealed a
highly polarized distribution of different types of SERCA
pumps in pancreatic acinar cells. Pumps of SERCA2a type
were found almost exclusively at the luminal pole of these
cells, while SERCA2b was found preferentially in the
basal region (147). There is considerable similarity in the
distribution of IP3Rs and SERCA2a calcium pumps. Close
colocalization between calcium pumps and calcium release channels leads to suggestion of short-range interactions between the two mechanisms. Thapsigargin, the
specific inhibitor of SERCA pumps, depletes the calcium
store of pancreatic acinar cell (183). It was also shown to
induce a cytoplasmic calcium rise as calcium leaks out of
the store. Interestingly, thapsigargin induces a diffuse,
spatially homogeneous calcium rise (293). Recently (and
unexpectedly), it was found that thapsigargin not only
produces uniform calcium release itself, but also abolishes the apical-to-basal polarity of calcium release triggered by carbachol. In the presence of thapsigargin (but
evidently before the calcium store is completely discharged), carbachol induces a uniform calcium rise (147).
This further suggests that there is a close functional relationship between pumps and release channels. The mechanism of this interaction is not yet clear. One possible
effect of the calcium pump activity could be formation of
high-affinity calcium buffering in close vicinity to the
calcium-releasing channels. As a transporter, the calcium
pump is relatively slow, with a maximum rate of a few
tens of ions per second. However, because of its sluggishness, it must be expressed in relatively high concentrations and its calcium binding sites form a buffer of high
affinity. Such buffering should be an effective mechanism
for both localizing calcium responses to a particular region of the cell and also for introducing a delay in the
propagation of calcium waves. Total calcium buffering in
pancreatic acinar cells is very considerable (182), but the
nature and the distribution of buffers are a complete
enigma in this and in many other cell types. It seems
feasible that the calcium-binding sites of the pumps could
form a substantial portion of this buffering.
SERCA2b has also been localized in cells of the SMG.
It was found predominantly in the apical pole of duct cells
and, in acinar cells, it is close to luminal and lateral
regions of plasma membrane (147). Interestingly, in these
cell types, the apical-to-basal polarity of SERCA2b distribution is exactly opposite to pancreatic acinar cells (see
above). SERCA3 was found in the basal pole of both types
of submandibular gland cells but was absent in pancreatic
acinar cells (147).
PMCA are responsible for setting the resting level of
free calcium in the cytosol. This, of course, has important
consequences for the total calcium content of the cell and
for the level of loading of calcium stores. Recent experiments showed that PMCA pumps also have an important
role in the regulation of the frequency of calcium oscilla-
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
soon confirmed by immunostaining, which directly demonstrated preferential localization of plasma membrane
calcium pumps in the luminal and lateral domains of the
plasma membrane of pancreatic acinar cells (147). Much
less pronounced punctate staining with antibodies against
PMCA could be found on the basal membrane of the
pancreatic acinar cells (147). PMCA was also preferentially localized to the luminal regions of submandibular
acinar cells and submandibular duct cells (147). The preferential distribution of PMCA pumps in the apical region
seems logical since it suggests that the region of the cell
that specializes in calcium release also has greater ability
to restore the resting calcium level.
trodes to measure changes in calcium on the basolateral
side and in the lumen of the epithelium. They described
that there is a net movement of calcium from the blood to
the lumen and suggest that the Ca2⫹ which enters the
lumen may act as a messenger that influences neighboring
cells through an interaction with the Ca2⫹-sensing receptor (26, 44).
Transcellular movement of calcium in secretory epithelia occurs in several stages. First, an influx mechanism
on the basal membrane must exist to move calcium from
the extracellular fluid into the cell. There must be a mechanism for the movement of the calcium across the cytosol
of the cell. Calcium is then extruded into the lumen across
the apical plasma membrane, perhaps by exocytosis of
secretory vesicles or by active transport of Ca2⫹. These
and other mechanisms underlying the process of transcellular calcium transport are shown in Figure 2.
A. Transcellular Transport Process
The nonuniform distribution of calcium channels and
calcium extrusion mechanisms can result in considerable
epithelial transcellular calcium transport. This calcium
transport is clearly an important aspect of body calcium
homeostasis, but it is not always immediately clear
whether it is essential for epithelial function or simply a
consequence of the use of calcium as an intracellular
messenger. One clear example of the functional importance of transport of calcium into the exocrine fluid is
found in mammary epithelia, where the process serves a
clear nutritional purpose. Calcium concentration in rabbit
milk can approach 100 mM (258). Amazingly, this is approximately a million times higher than the level of free
calcium inside the cell and 50 times higher than total
calcium in the blood. Human milk also contains a large
amount of calcium, with the level around 12 mM (103).
Calcium secretion occurs synchronously with the major
milk proteins casein and lactose, indicating that the transport is transcellular rather than paracellular and that it
occurs via Golgi-derived secretory vesicles (201).
Calcium released from stimulated pancreas also correlates with secreted protein, indicating that the mechanism of calcium release is mainly via exocytosis (165,
286). The total calcium concentration of pancreatic juice
is in the range of a few millimolar (165).
In saliva, an approximate calcium concentration of 1
mM was reported (173). The results of measurements of
calcium release from SMG could also be interpreted as an
indication of release by transcellular transport. ACh and
epinephrine cause 45Ca2⫹ release from prelabeled gland,
indicating that these agonists release calcium that is
“bound intracellularly” (205).
Recent, elegant experiments have directly demonstrated the transepithelial movement of calcium following
stimulation of intact gastric mucosa (44). Caroppo et al.
(44) used carefully positioned Ca2⫹-sensitive microelecPhysiol Rev • VOL
B. Calcium Entry and Its Role in Maintenance
of Calcium Stores and Transcellular
Calcium Transport
There is little information about the exact molecular
mechanism that starts the transcellular calcium movement in secretory epithelium, namely, calcium entry into
the basal part of the cells. However, the process of calcium influx has been extensively studied.
For transcellular calcium transport to be sustained,
calcium extrusion from the cell should be balanced by
appropriate influx pathways. These pathways should
compensate for the apical calcium loss and allow reloading of the calcium stores. In the presence of calciumreleasing secretagogues, calcium stores of different types
of epithelial cells quickly become completely depleted if
the extracellular solution does not contain calcium at
concentrations sufficient to sustain calcium influx (57,
228, 284). One of the mechanisms of calcium influx is
activated by the depletion of the intracellular calcium
stores. This influx pathway was termed “capacitative calcium entry” (CCE) (235). Exocrine secretory cells were
among the cell types in which this influx was characterized (5, 32, 277). In addition to this entry pathway, many
exocrine cell types have alternative calcium influx mechanisms that can be activated by hormones or neurotransmitters (261). Two pathways of calcium influx were characterized in hepatocytes. Depletion of internal calcium
stores by inhibitors of SERCA pumps induces CCE. Adenosine (292) and vasopressin (133) were able to further
increase the influx. These results suggest the presence of
two types of entry pathway.
Elegant experiments using microinjection of IP3
(271) and photolysis of a caged analog of IP3 (106) indicated that this second messenger can activate calcium
influx in hepatocytes by two mechanisms: one by empty-
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
IV. TRANSCELLULAR CALCIUM TRANSPORT
715
716
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
ing the internal stores and inducing CCE and the other by
direct action of IP3 on calcium influx channels. The nature
of both types of influx pathways is not known.
Two types of calcium influx mechanisms with different sensitivity to pharmacological agents were recently
described in pancreatic acinar cells (32, 300). One of
the pathways is suppressed by inhibitors of tyrosine kinase (32, 143, 231, 320), and the other was inhibited by
La3⫹ (32).
In gastric parietal cells, stimulation with carbachol
did not augment the influx induced by depletion of the
calcium stores with thapsigargin. From these experiments, the authors concluded that a single calcium entry
pathway (of capacitative nature) is present in this cell
type (198). Capacitative calcium influx was characterized
Physiol Rev • VOL
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
FIG. 2. Schematic representation of a generic secretory epithelial
cell showing possible calcium transport pathways underlying transcellular calcium movement. Transcellular transport is split into three sections: 1) calcium entry at the basolateral membrane, 2) movement of
calcium across the cell, and 3) apical exit of calcium into the lumen.
Movement of calcium is depicted by arrows.
in parotid acinar cells (277). The influx has two components with differing affinity to external calcium (48, 49).
It has been widely suggested that proteins from the
Trp family could mediate capacitative calcium influx. In a
human submandibular cell line, expression of Trp1 protein resulted in increase of calcium influx, whereas a
decrease of the endogenous Trp1 level, by transfection
with antisense of trp1, suppressed the influx (155). Trp1
was also found in the plasma membranes of acinar and
duct cells of rat SMG (155). These results allowed the
authors to suggest that Trp1 could mediate the calcium
influx in these cells (155).
It is conceivable that some of the calcium import
mechanisms that have been recently identified in absorptive epithelia are also used by secretory epithelia. Substantial progress has been made in identifying molecular
mechanisms of calcium influx in calcium-absorptive epithelium. Calcium absorption via transcellular transport is
an important component of regulation of calcium content
of blood and interstitial fluid. The transport is regulated
by parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. It has been recognized for some
time that such transport should include entry through
the channels of the apical membrane and extrusion by
Ca2⫹-ATPases or Na⫹/Ca2⫹ exchangers on the basal
membrane (85).
One of the recent exciting developments that occurred on the interface of epithelial physiology and the
calcium signaling research fields was the discovery of a
new type of calcium influx channel. An apically located
epithelial calcium channel (ECaC) was identified and
characterized in calcium transporting cells of distal
nephrons, proximal small intestine, and placenta (117).
The family of ECaC expanded very soon after the discovery of ECaC when the new calcium transporting channel
CaT1 was identified in rat duodenum (223). CaT1 has 75%
sequence homology to ECaC. The kidney-specific analog
of CaT1 and ECaC, CaT2, was recently identified (amino
acid identity 73.4 and 84.2% correspondingly) (222). These
findings were followed by the discovery of the human
analog of CaT1 (hCaT1), which has broad distribution
throughout different cell types of absorptive and secretory epithelium (224).
Interestingly, the expression pattern of these new
types of calcium channels broadly mirrors that of vitamin
D-sensitive calcium-binding proteins. Tissues that express
ECaC also express calbindin-D9K and/or calbindin-D28
(187). CaT2 channel is also coexpressed with calbindinD28 (222). One of the possible functions of calcium buffers could be to decrease free calcium concentration in the
immediate vicinity of the calcium influx channels. Several
types of calcium channels are known to be inhibited by
elevated cytosolic Ca2⫹. Therefore, by binding Ca2⫹ and
removing negative calcium feedback on calcium channels
and maintaining the chemical gradient, increased buffer-
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
granule as a target, thereby releasing their contents into
the lumen through a single pore. The maximum rate of
occurrence of exocytotic events was around 1 per cell per
minute (200).
Another mechanism of calcium extrusion from the
apical membrane of secretory epithelial cells is via PMCA.
As described in previous sections, calcium extrusion mediated by calcium pumps preferentially occurs from the
apical parts of pancreatic acinar cells (15, 17). Immunostaining also indicated the highest density of PMCA in the
luminal and also lateral parts of pancreatic acinar cells
(147). A similar distribution of pumps was found in submandibular acinar and duct cells (147).
It must be noted that there is an inherent problem
with distinguishing between exocytotic and pump-mediated calcium extrusion. The problem is that intracellular
calcium elevations that trigger secretory responses in
these cell types should simultaneously stimulate calcium
extrusion by calcium pumps of apical plasma membrane.
If the same intracellular calcium rise triggers both exocytosis and calcium extrusion by the pumps, then this could
result in correlation between calcium and secreted proteins, even if the calcium does not come from the vesicles.
It is clear, therefore, that a correlation between calcium
and secreted protein cannot be considered as a definitive
proof of exocytotic calcium release. The indicator-dextran technique distinguishes between the two types of
calcium efflux. Sharp spikes of extracellular calcium are a
signature of quantal, exocytotic calcium release, whereas
a more smooth calcium appearance in the extracellular
solution indicates extrusion by calcium pumps of the
plasma membrane. Both types of transport were characterized in SMG acinar cells (14).
D. Movement of Calcium Across the Cell
C. Extrusion of Calcium at the Apical Membrane
The total calcium in secretory vesicles of different
exocrine cells is very high (245, 251). This supports the
notion that exocytosis could make an important contribution to transcellular calcium transport. Recently, the
process of calcium release via exocytosis of secretory
vesicles was directly visualized in submandibular acinar
cells using the indicator-dextran technique (14). The rate
of the exocytotic calcium release was impressively high:
several hundred micromolar of total cellular calcium per
minute. The technique also allowed estimation of the total
amount of calcium released during an exocytotic event;
this was 3.5 ⫻ 10⫺18 mol (14). Using MP microscopy to
observe stimulated pancreatic acinar cells, Nemoto et al.
(200) were able to show that the calcium released through
an exocytotic pore may come from more than one granule. After the initial exocytotic event at the plasma membrane, several granules can fuse together using the first
Physiol Rev • VOL
In pancreatic acinar cells, the movement of calcium
that enters the basal part of the cell was investigated
using local reloading of calcium stores (183). This technique involves the cell-attached patch-clamp configuration and can be subdivided into the following steps. 1)
Cell-attached patch-clamp configuration is used on isolated cells loaded with calcium indicator (usually fura 2).
After establishing a tight seal with the plasma membrane
(gigaohm resistance), intracellular calcium stores are unloaded by stimulation with supramaximal dose of calcium-releasing agonist. This is done in calcium-free extracellular solution. Calcium is released from the stores and
pumped away from the cytoplasm by Ca2⫹-ATPases of the
plasma membrane. 2) Local reloading of calcium stores is
induced by removal of the calcium-releasing agonist from
extracellular solution. The patch pipette contains high
calcium concentration (e.g., 10 mM), whereas the rest of
extracellular solution is calcium free. Thus calcium can
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
ing could stimulate calcium entry and transcellular transport. Calbindins may potentiate calcium influx, stimulate
calcium extrusion, and also prevent formation of large
intracellular calcium gradients. The recently identified
calcium channels were also colocalized, at the cellular
level, with calcium transporters. In kidney connecting
tubule, ECaC was colocalized not only with calbindin-D28
but also with Na⫹/Ca2⫹ exchanger and with plasma membrane calcium pumps (116). Colocalization of ECaC with
calbindin-D9 and PMCA (but not with Na⫹/Ca2⫹ exchanger) was found in intestine (116).
The mechanisms underlying calcium absorption are
generically similar to those used by calcium-secreting
epithelia, but they are applied in the opposite direction.
By transposing the calcium channels and moving the calcium pumps to opposite membranes, it should be possible
to convert a calcium-absorptive epithelium to a calciumsecreting epithelium. This may well have been a tempting
option for evolution. If the instruments of transcellular
calcium transport were already available, then why not
change the direction of the transport by simply changing
the location of those proteins? In this respect, it is interesting to note that the calcium uptake at the basal membrane of mammary cells is stimulated by vitamin D (179).
Also, transcripts of hCaT1, one of the recently discovered
ECaC, are present in both pancreas and salivary glands
(224). Indeed, recent work shows that CaT1 displays
some of the electrophysiological channel properties of
Icrac, a well-characterized current activated by store depletion (317). This suggests that this protein, although
originally characterized as a calcium absorption mechanism in gut epithelia, could have an important role in a
wide range of tissues as one of the major routes for
store-operated calcium entry (317).
717
718
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
Physiol Rev • VOL
important upon secretagogue stimulation (165). This is
understandable if the same mechanisms underlie both
transcellular transport and generation of intracellular calcium signals.
A substantial portion of calcium in the exocrine secretory fluids (e.g., saliva, pancreatic juice) is buffered,
leaving only part of the calcium in the ionized form (165,
173, 185). The composition and effectiveness of buffering
probably changes along the system of the ducts. In intraacinar ducts, the proteins secreted by the cells probably
continue to buffer calcium. Downstream, in larger ducts,
the role of proteins in buffering probably declines,
whereas small molecules like bicarbonate become more
important.
E. Pathophysiology and Physiology
of Calcium Secretion
For the mammary gland, the nutritional function of
transcellular calcium transport is clear. However, for
other types of secretory epithelium, we have a peculiar
situation where the pathology of the process is much
clearer than the physiology. Precipitation of calcium, resulting in formation of calcium stones, occurs in liver
(138), gallbladder (96), as well as pancreatic (185) and
salivary ducts (31). Special proteins are secreted by the
glands to prevent the formation of calcium stones. The
pancreas secretes lithostathine, a glycoprotein that inhibits grows of calcite crystals (88, 92). Interestingly, lithostathine is also present in neurofibrillary tangles of patients
with Alzheimer’s disease (45, 70). In other tissue fluids,
the prevention of the growth of calcium-containing stones
is mediated by nephrocalcin (189, 190), renal lithostathine
(304), and low-molecular-weight acidic gallstone proteins
(207, 259). It is clear, then, that there is considerable
danger of calcium crystallization in ducts of exocrine
glands and that special mechanisms have evolved to prevent this pathological action of calcium.
So, what is this secreted calcium useful for? Why
take the risk? One possible answer to this question is that
calcium in the exocrine secretory fluids is actually useless. The risk of having the calcium delivered into the
ducts and necessity to reabsorb it later may simply be
payment for its intracellular functions (e.g., for its role in
protein packaging in the secretory granules or for its
second messenger activity). Alternatively, calcium could
have important functions in the ducts. In this respect it is
useful to note that amylase secreted by salivary glands
and by pancreas requires calcium for its activation (3, 238,
264) and that the same applies for trypsin secreted by
pancreas (238). Therefore, certain minimal free calcium
levels are required for effective digestion. If the calcium
level in food is not sufficient or not released sufficiently
quickly, it could be useful to supplement it with some
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
only enter the cell via the minute area limited by the
gigaseal-forming patch pipette. 3) After a period of reloading, a second stimulation with calcium-releasing secretagogue is used to reveal the fate of the locally entered
calcium. In our experiments, the calcium rise induced by
such second stimulation started far away from the place
of calcium entry. There was no rise in cytosolic calcium
during the local reloading phase. These experiments
strongly suggest that calcium moved through the continuous lumen of ER from its place of entry at one side of the
cell (basal region) to the region of primary release (apical) at the opposite part of the cell (183, 229). In this way,
calcium can traverse the cell without entering the bulk
cytoplasm and is available for later, secretagogue-stimulated release from the apical region. The released calcium
would then be extruded across the luminal membrane by
calcium pumps. Thus the complete process of transcellular calcium transport could be formed from the following
components: basal influx, uptake into ER, tunneling in ER
lumen, release from apically located ER terminals, and
extrusion by calcium pumps located on the luminal membrane (Fig. 2).
From the ER, the transcellular transport could take
an alternative route. Calcium could be delivered to the
Golgi by vesicular transport from the ER. This can result
in loading of calcium into Golgi-derived secretory vesicles. Vesicles may also take calcium directly from the
cytoplasm of a cell as has been suggested for mammary
epithelium (258). As described in section IVA and Figure 2,
such transcellular pathways would terminate by exocytotic calcium release.
It is also possible that some of the calcium entering
the cell is initially taken up by mitochondria (Fig. 2).
Recent studies suggest fast and effective mitochondrial
accumulation of calcium which enters the cell (93, 184,
216). The relative contribution of these transcellular pathways in different types of secretory epithelium is not yet
clear.
Although in mammary epithelium the secreted calcium is moved from the interstitial fluid to the ducts
mainly via the transcellular pathway, the situation is less
clear in other secretory epithelium where there is an
important paracellular component. The efficiency of paracellular transport is regulated by secretagogues. For example, stimulation with carbachol and pancreozymin
causes an increase of paracellular permeability in pancreas (129). Paracellular permeability could also be regulated by intracellular calcium. The mobilization of intracellular calcium resulted in an increase of tight junctional
permeability in liver (157). The relative importance of the
paracellular and transcellular pathways depends on the
type and on the status (stimulated or unstimulated) of the
tissue. The pancreas presents an interesting example
where paracellular transport dominates in the unstimulated organ while the transcellular route becomes more
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
V. POLARITY OF INTERCELLULAR
CALCIUM SIGNALING
Exocrine secretory cells combine together to form
functional units in which individual cells communicate
via gap junctions. This communication allows calcium
waves activated in the most responsive cells to propagate
through the other cells in the unit and, thereby, coordinate their functions (Fig. 3A). In this section, we consider
examples of such polarized activation and intercellular
propagation of calcium signals.
A. Mechanisms Underlying the Polarity
of Intercellular Calcium Waves
Hepatocytes are connected by gap junctions (99),
which are permeable to Ca2⫹ and to IP3 (249). In isolated
hepatocyte cell couplets, hormonal stimulation (194) induces intracellular calcium transients and propagation of
a calcium-releasing signal from one cell to the other. The
ability to propagate the calcium signals between the couplet is abolished by treatment with octanol, an inhibitor of
gap junctions (194). This suggests that there is a polarized
response whereby the initial activation of calcium signals
in the more sensitive cell of the doublet is followed by
propagation of a calcium-releasing stimulus through the
gap junctions to the other cell.
At the more complex level of the perfused liver,
Nathanson et al. (196) found that vasopressin-triggered
calcium waves in hepatocytes originate in the pericentral
regions of the organ and then propagate from neighbor
cell to neighbor cell in the direction of the portal venules.
Tordjmann et al. (294) directly compared the sensitivity to
stimulation of pericentral and periportal hepatocytes
(294). Pericentral hepatocytes were more responsive than
periportal hepatocytes to norepinephrine and to vasopressin stimulation, in broad agreement with the data of
Nathanson et al. (196). However, the variations in sensitivity seem to be agonist specific since no difference was
Physiol Rev • VOL
found in sensitivity of the two groups to angiotensin II.
Furthermore, the regional sensitivity to ATP was opposite
to that for norepinephrine and vasopressin. Periportal
hepatocytes were more responsive to ATP than their pericentral counterparts (294).
IP3-induced calcium release was similar in permeabilized periportal and pericentral hepatocytes, suggesting
that the difference of sensitivity to agonists is not due to
variations in IP3 sensitivity of the calcium stores. All this
pointed toward the fact that the heterogeneity of the
responses in the two groups must be determined by variations in events upstream of IP3 production (294). Immunohistochemistry confirmed that there is a higher density
of vasopressin V1a receptors in hepatocytes of the pericentral region. This immediately suggests that it is the
nonuniformity of receptor distribution that underlies the
polarized activation and then propagation of calcium
waves (196). A gradient of vasopressin receptor mRNA
density in hepatic planes was reported (211). High levels
of V1a mRNA were found in pericentral hepatocytes and
lower levels in periportal hepatocytes (211). The largest
gradients of V1a mRNA were found in early stages of
development. The pericentral-to-periportal gradient was
maximal on postnatal day 21 when “. . .V1aR mRNA (is)
most abundant in hepatocytes surrounding central veins
and virtually absent in periportal hepatocytes” (211). This
stage of development should therefore provide an interesting model for fluorescent imaging studies of polarity of
initiation and propagation of calcium waves.
Later work from the laboratory of Claret and Combettes (295) confirmed that periportal hepatocytes have
lower levels of V1aR mRNA. This study also reports that
pericentral hepatocytes have a higher density of vasopressin binding sites and more substantial vasopressin-induced IP3 production than periportal hepatocytes (295).
These findings are in line with reported pericentral activation and periportal propagation of vasopressin-induced
calcium waves in hepatic planes (196). The intercellular
gradient of receptor density and of IP3 production was
therefore considered to be the decisive factor in determining the polarity of intercellular calcium waves (Fig.
3B). A mathematical model of polarized propagation of
calcium waves, which takes into account gradient of vasopressin sensitivity and IP3 movement through gap junctions, was developed on the basis of these findings (71).
Glucagon receptors also show polarized expression
in liver. There is a gradient of the receptor density. A high
density of glucagon receptors was found in membranes of
hepatocytes in pericentral regions. A low density of the
glucagon receptors is found in hepatocytes of periportal
regions (19).
Robb-Gaspers and Thomas (240) used fluorescent
microscopy to monitor propagation of calcium signals in
intact perfused liver. The same cell was repeatedly responsible for initiation of consecutive propagating cal-
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
additional calcium that is secreted by the exocrine glands.
Pancreatic acinar cells also require calcium for endocytosis. At low levels of extracellular calcium, endocytosis
is inhibited (168).
Recent work from Riccardi’s group (28) indicates the
presence of calcium receptors in pancreatic ducts. This
study argues that calcium released from the pancreatic
acinar cells regulates fluid secretion by the duct epithelial
cells.
New technologies used for live tissue imaging (e.g.,
two-photon microscopy) will hopefully soon provide us
with important information on the dynamics of transcellular calcium transport and maybe on the functions of the
transported calcium.
719
720
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
cium waves. The authors of this study consider that there
are particular “pacemakers for each lobule” (240) that are
the cells with highest sensitivity to the small doses of
agonists.
The polarity of vasopressin-induced calcium waves
appears to be a dose-dependent phenomenon. Stimulation
with low levels of vasopressin resulted in initiation of the
calcium signals in the periportal region of the lobule.
These were followed by formation of calcium waves propagating through cell-cell contacts to the pericentral region
of the lobule. This is actually the opposite direction of
propagation to that found in the works discussed above.
Physiol Rev • VOL
However, when higher concentrations of vasopressin
were applied, the wave started in the pericentral region, in
agreement with the other studies (240). The results obtained by this group demonstrate the complexity of the
pattern of activation and propagation of intercellular calcium signals in liver. Interestingly, whereas the rate of
intracellular calcium spreading was not dependent on
hormone concentration, the propagation from cell to cell
was accelerated by increased hormone doses (240).
It is important to keep in mind that intercellular
waves propagate across intact tissue in which many different types of cell may closely coexist. An interesting and
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
FIG. 3. Modes of intercellular calcium wave propagation. A: example of polarized intercellular calcium wave.
Sequential confocal images of intact liver depicting coordinated calcium waves traveling across the tissue as a result of
vasopressin (50 pM) stimulation (with 1.3 nM bradykinin prestimulation). Calcium elevation, measured with fluo 3, is
shown in red and overlays transmitted images of the tissue. The direction of waves is shown by the white arrows, which
overlay the first image. Images were captured every 8 s, and the total area of each image is 226 ⫻ 171 ␮m. B and C:
representative diagrams of putative mechanisms underlying the initiation and propagation of calcium waves from cell to
cell across epithelial tissue. The wave is propagated through gap junctions by diffusible messengers [x, probably Ca2⫹
or inositol 1,4,5-trisphosphate (InsP3)]. B: in this model (created on the basis of hepatocyte data), the wave is initiated
in the cell that has the greatest density of agonist (vasopressin) receptors. Cells with a greater number of agonist
receptors will display a larger production of IP3 and will, thus, be more sensitive. The wave propagates to neighboring
cells through gap junctions and spreads across the tissue in the direction of lower receptor density. C: model depicting
an alternative mechanism from the salivary gland. Agonist receptor density is homogeneous across the tissue, but some
cells have significantly more intracellular calcium release channels (InsP3 receptors in this case). The calcium wave is
initiated in these cells because they have greater sensitivity to release of calcium from stores for a given amount of InsP3
production. The wave then travels to neighboring cells via gap junctions. [A from Patel et al. (220), copyright Nature
Publishing Group. B from Tordjmann et al. (295), copyright Oxford University Press.]
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
B. Propagation of Intercellular Calcium Waves
The signals initiated in pioneer cells propagate to
neighboring cells through gap junctions. Gap junctionbased intercellular calcium spreading was also found in
the salivary glands of blowfly. The propagation of calcium
signals occurs through regions of the basal plasma membranes where gap junctions are concentrated (326).
Pancreatic acinar cells are linked by gap junctions (127,
128, 177). More than 200 individual pancreatic acinar cells
can be connected, as visualized by fluorescent dye (Lucifer
yellow) transfer from a single, injected cell (76). Electrical
coupling could be resolved between cells in the acinus that
were located as far as 100 ␮m apart. High doses of the
calcium-releasing secretagogues ACh, caerulin, or bombesin
produced reversible uncoupling of this link (126).
Stimulation of pancreatic acini with small and intermediate doses of the secretagogues CCK and carbachol
Physiol Rev • VOL
(CCh) produced calcium elevations that propagated as an
intercellular calcium wave through the cells of the acinus
(322). These waves started repeatedly in the same cells of
the acinus, once again reinforcing the notion of the existence of pacemaker cells. The injection of IP3 into one of
the cells of the cluster of pancreatic acinar cells resulted
in calcium signals in the injected cell and in a few of its
neighbors (up to 5 cells distance from the injected cell).
When the cluster was stimulated with small doses of CCK,
injection of calcium into a single cell resulted in calcium
elevations in the neighboring cells as well. However, in
unstimulated clusters of pancreatic acinar cells, calcium
had a much shorter intercellular range. Indeed, injection
of calcium into one of the cells of the unstimulated cluster
resulted in transient calcium rise in the injected cell but
failed to elevate calcium in surrounding cells. This led to
the conclusion that both IP3 and calcium are involved in
propagation of intercellular calcium waves (322).
The propagation of IP3 and calcium between cells
involves movement through gap junctions. Small doses of
CCK and CCh do not affect junctional connectivity of the
pancreatic acinar cells, whereas large concentrations of
these secretagogues blocked gap junctions. Gap junction
connectivity was also blocked by a very high calcium rise
induced by large calcium injection (322). Interestingly, a
recent study showed that ACh-induced uncoupling is prevented by inhibition of NO production by NG-nitro-L-arginine, an inhibitor of NO production, while cGMP was able
to restore gap junction connectivity (47). This finding pinpoints the messenger cascade involved in secretagogue-induced uncoupling in pancreatic acinar cells (47) and also
relates to the facilitation, by NO released from the endothelial cells, of wave propagation in hepatocytes (220).
Increase of gap junction permeability by NO⫺
2 synchronized oscillations in neighboring cells, which were triggered
by CCK-J (267). Importantly, increase of gap junction permeability resulted in enhancement of amylase secretion.
This increase in sensitivity manifests itself as a shift of the
dose-response curves into the range of smaller concentrations. This was found for a number of secretagogues. Particularly impressive shifts, resulting in an increase in sensitivity of more than one order of magnitude, were found for
CCK analogs CCK-8 and CCK-J. These experiments indicate
the functional importance of intercellular communication
and the influence that the most sensitive “pacemaker” cells
could exert on the rest of the acinus via gap junctions. The
gap junction communication increases the responsiveness
of the tissue by making it as sensitive to the agonist as its
most sensitive individual cells. Inhibition of gap junctions by
octanol decreased stimulated amylase secretion by shifting
the dose-response curves for secretagogues toward higher
concentrations (267).
Not all agonist-induced calcium oscillations propagate through networks of pancreatic acinar cells. Although extended CCK-induced transients can propagate
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
potentially important description of interaction between
two different cell types was recently reported for hepatocytes and endothelial cells (220). Nitric oxide (NO), released from endothelial cells after stimulation by bradykinin, is able to facilitate the spreading of vasopressininduced calcium waves in hepatocytes. In intact perfused
liver, low doses of vasopressin induce calcium waves that
engage a few cells but fail to propagate through the entire
hepatic plate. These incomplete calcium waves are considerably extended by bradykinin stimulation of endothelial cells (see Fig. 3A) (220). Elegant experiments on
cultured hepatocytes and on cocultures of hepatocytes
with endothelial cells confirmed that NO released from
endothelial cells is responsible for this facilitation of the
propagation of calcium waves (220).
Different tissues seem to exhibit different properties
with regard to initiation of intercellular waves. In the
SMG, the pacemaker cells that probably initiate calcium
waves in the ductal epithelium were identified in work
from Mikoshiba’s laboratory (314). Unlike hepatocytes in
liver lobules, the submandibular cells in interlobular
ducts were highly heterogeneous with respect to abundance of IP3Rs. Some, but not all, cells of the interlobular
ducts display very strong immunostaining for IP3Rs.
These pioneer cells display fast calcium responses to
stimulation with secretagogues and probably propagate
calcium signals into neighboring cells of the duct which
respond more slowly (this was suggested but not shown
directly in the paper) (314). This type of intercellular
polarity is shown schematically in Figure 3C.
We have therefore two types of heterogeneity that
can define the pacemaker cells: 1) a difference in receptor
density (as was found in hepatocytes) or 2) a difference in
abundance of intracellular calcium release channels (as
described in SMG ducts) (see Fig. 3).
721
722
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
VI. POLARIZED CALMODULIN SIGNALING
A. General Properties of CaM
CaM is an abundant and ubiquitous calcium binding
protein (53). In humans, the protein is encoded by three
genes CALM1, CALM2, and CALM3 that reside on different chromosomes (79, 299). Amazingly, these different
genes with substantially different nucleotide sequences
encode the same protein (79, 299). The acidic protein is
small (mol wt 17,000 and composed of 148 amino acid
residues; for review of properties, structure, and mechanisms of interactions, see Refs. 51, 53, 206). CaM belongs
to the EF-hand family of proteins. All members of this
Physiol Rev • VOL
family contain one or more calcium-binding EF-hand motifs. CaM has four such motifs, which bind calcium with
intermediate affinity (Kd from 0.5 to 5 ␮M; Ref. 53). Other
EF-hand proteins include parvalbumin, troponin C , calbindin, recoverin, and a number of other small calcium
binding proteins (30, 122). Binding of calcium changes the
conformation of CaM, exposing hydrophobic groups which
allow interaction with CaM’s target proteins (53, 206).
B. General Functions of CaM
Formation of the Ca-CaM complex is an essential
step of numerous calcium signaling cascades. Calciumbound CaM (Ca-CaM) binds to different types of Ca2⫹ATPases of the plasma membrane to activate these pumps
(100, 130; for review, see Refs. 43, 269). The ability of
Ca-CaM to stimulate smooth muscle myosin light-chain
kinase (MLCK) means that it can activate cellular motor
functions (4). In many receptor-effector pathways, calcium binding to CaM is followed by downstream activation of Ca2⫹/CaM-dependent protein kinases (CaMK)
(176, 188). Also, the prominent phosphatase calcineurin is
activated by CaM in a calcium-dependent manner (53,
248, 315). The list of CaM binding proteins is long and
continues to grow. For some recent additions to this list
see Reference 53.
1. Interactions with calcium channels
CaM has an antagonistic effect on IP3Rs. It binds to
type 1 receptors (263, 312) and inhibits their function
(115, 180, 219, 263). The CaM inhibition of IP3Rs can be
calcium independent (219, 263) or calcium dependent
(180, 312). Indeed, the declining slope of the bell-shaped
curve of calcium sensitivity of the IP3R1 (20), which
signifies inhibition of calcium release by high calcium,
may be explained by inhibition of the receptors by calciumbound CaM (180). Other types of IP3Rs are also sensitive to
CaM. IP3R2 and IP3R3 are inhibited by CaM (2).
Contrary to its inhibitory action on IP3Rs, CaM seems
to have a mainly stimulatory action on RyRs. CaM potentiates calcium release mediated by RyR1 (but not RyR2)
by increasing sensitivity to calcium-induced calcium release (86). The stimulatory action of cADP-ribose was
also potentiated by CaM (280).
2. Nuclear functions of CaM
CaM is involved in regulation of some major nuclear
functions such as nuclear transport (275), mitotic progression (298), and gene expression (23, 62, 78, 174, 310).
A rapid expansion of knowledge was initiated upon discovery of the involvement of the CaM pathway in regulation of the transcription factor CREB (cAMP response
binding protein) (23, 60, 62, 78, 174, 257, 310). In some cell
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
from cell to cell, the short-lasting calcium spikes induced
by ACh are not transmitted to neighboring cells (225).
This diversity in the effects of different agonists may
enlarge the spectrum of available tissue responses. It is
possible to envisage that everything from small, subcellular spikes to long-lasting, transepithelial waves could be
generated by specific agonist stimulation.
The gap junction pathway is not the only route for
propagation of intercellular calcium waves. For example,
ATP released from the cells undergoing calcium elevation
can stimulate the nearby cells and serve as the propagating factor for calcium waves (74, 209, 214). However, such
“paracrine” ATP-based forms of intercellular propagation
do not seem to be prominent in secretory epithelium,
whereas direct cell-to-cell communication via gap junctions is essential for a number of exocrine tissues (194,
267). Recently, the propagation of calcium waves through
gap junctions was directly visualized (214). These experiments were performed on HeLa cells expressing GFPlabeled connexins (214), but the process of gap junction
propagation of calcium waves is probably similar for
many cell types.
An interesting form of intercellular calcium signaling
that involves calcium extrusion and calcium detection by
neighboring cells via Ca2⫹-sensing receptor (CaR) was
described by Hofer et al. (119). In secretory exocrine units
(such as liver, pancreas, and salivary and lacrimal glands),
where the cells are very tightly packaged, there is the
possibility of large changes in local extracellular calcium
levels due to calcium extrusion from cells undergoing
calcium responses. Nearby cells, which express the CaR,
may detect these changes leading to generation of signals
of their own. The tissue morphology, together with widespread tissue expression of the CaR (including exocrine
secretory epithelium, Ref. 28), suggests that there may be
an important role for this newly discovered pathway in
calcium wave propagation. This can also be important for
interaction of the same type (e.g., acinar-acinar cells) or
different cells within a tissue (e.g., acinar-duct cells) (28).
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
types, this pathway involves nuclear CaMKIV (23, 62, 310).
CaM can translocate to the nucleus to regulate gene expression. It was found that nuclear translocation of CaM
links calcium influx into a cell and CREB-dependent gene
expression (62). The activation of CREB was shown to
reflect a balance of CaM-dependent phosphorylation (by
CaMKIV) and CaM-dependent dephosphorylation by calcineurin (62).
C. CaM Signaling in Exocrine Secretory Cells
1. Effects on calcium signaling
Physiol Rev • VOL
in rat hepatocytes. Pretreatment of cells with CaM antagonists (calmidazolium or CGs 9343B) abolished thapsigargin-induced SOC in hepatocytes. However, the application of CaM antagonists was ineffective when SOC was
already established. “Secretion-like coupling” between
the membrane of the calcium store and the plasma membrane was shown to be essential for activation of SOC in
some cell types (221, 246). In chromaffin cells, CaM amplifies secretion by seemingly affecting the late stages of
exocytosis of secretory vesicles (46, 136). CaM could
activate secretion-like coupling in the same way as it
activates secretion. Alternatively, CaM could exert its
effect via interaction with IP3Rs that were recently shown
to activate SOC (139).
It is interesting to note that CaM interacts with connexins, the gap junction-forming proteins (296). In particular, the interaction with connexin-32, which is the major
type of connexin in gap junctions of exocrine cells, suggests that it may be involved in the cell-to-cell communication underlying intercellular propagation of calcium signals (see sect. III).
2. CaM in the nuclei of exocrine cells
CaM was found in nuclei of hepatocytes, where it
may play a role in condensation of chromatin (232). Orrenius and co-workers (203, 204) demonstrated that isolated nuclei of hepatocytes can accumulate calcium. The
process of calcium accumulation was prevented by the
potent CaM inhibitor calmidazolium. IP3 was able to release calcium from isolated nuclei that had been loaded in
a CaM-dependent manner (203, 204). Later studies confirmed the effect of calmidazolium on nuclear calcium
uptake and specified the region of calcium accumulation
as the nuclear envelope (91). The same paper also confirmed the existence of IP3- and cADP-ribose-induced release from the nuclear envelope of hepatocytes (91). Interestingly, both CaM and MLCK are present in the nuclei
of hepatocytes (10). It was suggested that CaM could
activate some kind of “nuclear contractile system,” the
functions of which are not currently known.
In extracts from mammary epithelial cells, CaM was
shown to form complexes with estrogen receptors (22,
288). CaM is also required for activation of the promotor,
which is responsive to the estrogen receptor in these
cells. Here, CaM could be playing an important role in
regulation of estrogen-induced proliferation of mammary
epithelial cells (22).
3. Effects on exocrine secretion
CaM has effects on secretion in a number of exocrine
cell types. In liver, CaM antagonists chlorpromazine
(CPZ), trifluoperazine (TFP), and W-7 were shown to
inhibit both basal bile flow and bile flow stimulated by
taurocholic acid (111). The mechanism of this inhibition
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
CaM influences various components of calcium signaling pathways in exocrine secretory cells. Calcium release triggered by cADP-ribose or ryanodine in microsomes from pancreatic acini is potentiated by CaM (212).
CaM was also able to potentiate cADP-ribose-induced
calcium release in digitonin-permeablized salivary gland
acinar cells (313). In the same preparation, CaM inhibited
IP3-induced calcium release (313). Experiments with hepatic microsomes showed somewhat different results.
cADP-ribose did not release calcium from this preparation either in the presence or absence of CaM (153).
CaM may modulate calcium release indirectly. PKA
has been shown to phosphorylate and inhibit IP3Rs, as
shown recently in pancreatic acinar cells (94). Phosphodiesterase is one of the classical targets for CaM activation. CaM-dependent phosphodiesterases are expressed
in the whole pancreas (12). By activating the breakdown
of cAMP by phosphodiesterases, CaM could activate
IP3Rs via decreased PKA-dependent inhibition. This effect
should be particularly prominent during cytosolic calcium
elevations.
Colocalization of IP3R3 and CaM-dependent protein
kinase II (CaMKII) was described in the apical region of
pancreatic acinar cells, gastric parietal cells, intestinal
enterocytes, and some other cell types (171). Spatial colocalization indicates the possibility of functional interactions of the two proteins in apical regions of these cells.
In pancreatic acinar cells, CaM increases the sensitivity to calcium of plasma membrane calcium pumps (7).
Other work describes that CaM antagonists, trifluoperazine and compound 48/50, inhibit calcium efflux across
plasma membranes of pancreatic acinar cells (161). In
liver, a high density of PMCA pumps is present is bile
canaliculi. Interestingly, the Ca2⫹-ATPase in the plasma
membrane of hepatocytes seems to be CaM insensitive
(11). Studies of Hurley and Martinez (121) suggested the
presence of CaM-dependent calcium pumps in submandibular acinar cells. CaM-dependent calcium pumps were
also identified in the basal and lateral membranes of
parietal cells (186).
A recent study by Cao and Chatton (41) implicated
CaM in activation of store-operated calcium entry (SOC)
723
724
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
Physiol Rev • VOL
Secretion of H⫹ in parietal cells is mediated by the
H -K⫹-ATPase. CCh stimulates H⫹ secretion, which is
calcium-dependent and inhibited by W-7, suggesting that
CaM is involved in activation of H⫹ secretion (252). W-7
also inhibited H⫹ secretion stimulated by histamine (253).
A number of proteins that are phosphorylated by CaMdependent kinases were found in the cytosol of parietal
cells (175). CaMKII was found throughout the canalicular,
F-actin domain of parietal cells (171). An important step
in activation of secretion of H⫹ from parietal cells is the
fusion of tubulovesicles containing H⫹-K⫹-ATPase with
the apical plasma membrane of the cells. CaMKII was
identified in tubulovesicles of parietal cells and was suggested to play a role in motor activity of tubulovesicles
and consequently in H⫹ secretion (73).
⫹
D. CaM Translocation and Oscillations
of CaM Distribution
Pancreatic acinar cells are well-suited for studies of
translocation of CaM due to their easily distinguishable
polarity. The movement of CaM in live pancreatic acinar
cells was studied using confocal imaging of CaM labeled
with a fluorescent probe (DTAF-CaM). Stimulation of pancreatic acinar cells with a supramaximal dose of CCK
triggers transient movement and redistribution of CaM.
An increase of CaM concentration starts in the secretory
granule region and is followed by much slower accumulation of CaM in the nucleus. CaM concentration in secretory granule region of the cell peaked at ⬃40 s after the
start of CCK stimulation, while the maximum of the nuclear CaM content was reached ⬃130 s later. The beginning of the nuclear rise of CaM was delayed by a few tens
of seconds compared with the calcium signal. The concentration of CaM in basal part of the cell declined during
CCK stimulation, indicating that basal CaM was temporarily donated to the secretory granule region and nucleus
(59). A similar pattern of redistribution was triggered by
supramaximal doses of ACh. The redistribution of CaM
depended on the duration of ACh application. Brief applications favored translocation to the apical region,
whereas more prolonged stimulation produces more substantial nuclear movement. Agonist-induced redistribution of CaM was mimicked by the calcium ionophore
ionomycin, suggesting that a cytosolic calcium rise is
sufficient to produce the observed redistribution. Experiments with TA-CaM (a form of labeled CaM that increases its fluorescence upon calcium binding; Refs. 297,
298) indicated that stimulation with secretagogues resulted in preferential formation of calcium-CaM complexes in the secretory region and in the nucleus of pancreatic acinar cells (279). Nuclear translocation of CaM is
not unique for pancreatic acinar cells and was reported in
different cell types and under different conditions (152,
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
is not clear, and the authors had considered the possibility that inhibition could be due to general cytotoxic effect
of the CaM antagonists.
Several papers report the involvement of CaM in
stimulated exocytosis in pancreatic acinar cells. The concentration of CaM in pancreas is high, at ⬃5 ␮M (303).
Pancreatic acinar cells also express CaMKII and CaMKIV
(316). The CaM inhibitor W-7 and the CaMKII inhibitor
KN-62 reduced stimulated amylase secretion (123). Cyclosporin A, an inhibitor of CaM-dependent protein phosphatase PP2B (or calcineurin), also inhibits amylase release
triggered by CCK and by CCh (104). The results indicate
the involvement of CaM and its downstream phosphatase
in regulated calcium-dependent exocytosis from pancreatic acinar cells. The authors were also able to identify
and characterize a 24-kDa protein that is dephosphorylated by cyclosporin A-dependent phosphatase (104, 105)
and probably serves as a mediator of Ca-CaM-dependent
protein secretion in pancreas.
Another study describes that W-7 partially inhibited
CCK-8-induced calcium release and completely inhibited
amylase secretion. Cyclosporin A partially inhibited secretion induced by CCK-8. These data confirm the involvement of CaM and PP2B in regulation of calcium-dependent secretion in pancreatic acinar cells (178).
CCK stimulation of pancreatic acinar cells leads to
activation of CaMKIV. W-7 and the CaMK inhibitor K252-a
(but not KN-62, the specific inhibitor of CaMKII) suppressed calcium oscillations and inhibited CCK-induced
amylase secretion (145). These results suggest that CaM
and probably CaMKIV are providing some “positive feedback mechanism” necessary for activation of oscillatory
calcium signaling and secretion in pancreatic acinar cells
(145). Another study from the same group describes that
calcium-releasing secretagogues CCK-8, CCh, and CCKOPE (an agonist of high-affinity CCK-A receptors) activate both CaMKII and CaMKIV in pancreatic acinar cells
(316). Wide-spectrum CaMK inhibitors KT-5926 and
K-252a inhibited enzyme secretion induced by calciumreleasing secretagogues. In this study, KN-62 was again
unable to inhibit secretion (316), confirming that CaMKII
is not involved. Secretion was also unaffected by ML-9, an
inhibitor of MLCK. The data confirm previous findings by
the same group that CaMKIV, but not CaMKII, is involved
in activation of calcium-dependent enzyme secretion
(316). This particular result actually contradicts a study
by a different group that implicated CaMKII in regulated
enzyme secretion in pancreas (123).
It is interesting to note that in pancreatic acinar cells,
a CaM-dependent kinase is present on the zymogen granule membrane. This kinase was shown to regulate the
Na⫹/H⫹ exchanger of zymogen granules (6). CaM can also
regulate other types of H⫹ transporters. It was suggested
that, in hepatocytes, CaM and calcium activate the plasma
membrane Na⫹/H⫹ exchanger (8).
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
Physiol Rev • VOL
Stimulation with smaller (close to physiological)
doses of CCK produces oscillations of calcium concentration in pancreatic acinar cells, which are accompanied by
oscillations of CaM distribution (59) (Fig. 4). The pattern
of oscillations of CaM concentration was different in different parts of the cell. In the apical region, the pattern of
oscillations of CaM concentration was similar to oscillations of calcium. In both cases, oscillations start with a
prolonged initial spike followed by smaller individual
transients with complete or almost complete recovery to
the prestimulation level. In the basal region, the pattern of
oscillations was a mirror image to that in the apical part
of the cells, confirming that CaM was translocating into
the apical from the basal region. CaM concentration in the
nucleus established an elevated plateau with superimposed CaM transients that were delayed (in comparison
with calcium signals) and incomplete. The nuclei were
capable of integrating the CaM transients occurring due to
calcium oscillations (59). It is interesting to note that
small “oscillatory” concentrations of CCK are particularly
effective in maintaining elevated concentration of CaM in
the nucleus. Although supramaximal doses of CCK produced only transient translocation of CaM, small oscillatory doses are able to maintain CaM presence in the
nucleus as long as the oscillations continue. Oscillations
of intracellular calcium were recently shown to be more
efficient than sustained elevations in activating gene expression (66, 151). The ability of calcium oscillations to
maintain an elevated level of nuclear CaM could explain
this higher efficiency of oscillatory responses.
VII. SUMMARY
Stimulated exocrine epithelial cells produce calcium
signals with specific spatiotemporal signatures that regulate essential cellular tasks such as secretion. The basis of
the polarity of the intracellular calcium signals is the
heterogeneous distribution of calcium channels, pumps,
and calcium-handling organelles. Some secretory epithelial cells are able to produce calcium spikes, which are
localized to the apical region of the cell. This is based on
the positioning of the most sensitive calcium stores in that
region. The nature of the mechanism that limits the
spread of the spikes is largely unknown. However, in
some cells, strategically positioned mitochondria can play
this buffering role. To coordinate functions in different
regions of the cell, globally propagating calcium signals
are needed. The propagation of intracellular calcium
waves is mediated by calcium channels, which are also
nonuniformly distributed.
The movement of calcium from interstitial fluid to the
lumen of secretory epithelia is dependent on nonuniform
location of calcium handling mechanisms. The process
involves polarized calcium entry at the basal membrane,
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
158, 233). Particularly interesting long-distance translocation of CaM into the nucleus of hippocampal neurons was
described by Tsien and co-workers (62).
The mechanism of the nuclear translocation of CaM
is not clear. Experiments by Pruschy et al. (233) indicate
that the translocation is mediated by facilitated diffusion
(on the basis of wheat germ agglutinin sensitivity, ATP
independence, and inhibition by chilling). A later study
from the Luby-Phelps laboratory (152) suggests that CaM
diffuses freely through the nuclear pores and that the
transport is driven by a concentration gradient established by Ca-CaM binding proteins in the nucleus. The
considerable delay between the rise in calcium concentration and the beginning of nuclear CaM accumulation in
pancreatic acinar cells suggests that there may be intermediate calcium-dependent reactions between the two
processes (59). Interestingly, CaM itself activates a specific ATP-dependent mode of nuclear transport (275).
This transport is not inhibited by depletion of internal
calcium stores (275) and conceivably could mediate CaM
translocation into the nucleus, especially when it is triggered by calcium release. In this situation, calcium triggers a positive feedback mechanism whereby CaM stimulates its own transport into the nucleus.
After partial hepatectomy, CaM concentration in the
cytosol of hepatocytes transiently increased and was followed by translocation of CaM into the nuclei (234). The
increase of CaM content in nuclei of liver cells after
partial hepatectomy was very substantial (⬃3-fold), and it
occurred in the same time frame as the rise of DNA
synthesis (255). Some of the CaM entering the nuclei
associated with the nuclear matrix in a calcium-dependent manner (234, 255). CaM was shown to regulate phosphorylation and dephosphorylation of a number of nuclear proteins in hepatocytes (25; for review, see Ref. 10).
The only cell type in which translocation of CaM into
the secretory granule region was shown directly is pancreatic acinar cells. There are, however, indications of
similar nonuniformity of CaM signaling in other exocrine
secretory cells. For example, a preferential concentration
of CaMKII was found in apical regions of parietal gastric
cells (171). The mechanism of translocation could involve
calcium-dependent binding of CaM to its target proteins in
the apical region. This provides the concentration gradient for diffusional movement of the “free” CaM from the
basal part of the cell. Potential targets for calcium-bound
CaM in the apical part of pancreatic acinar cells are
secretory granules that contain a CaM-dependent kinase
(6) and calcium pumps of plasma membrane that also
concentrate in the apical region of the pancreatic acinar
cells (17, 147). Other CaM-binding proteins that are preferentially located in the apical region of pancreatic acinar
cells include CaMKII (171) and IP3Rs (148, 197). Binding
to these proteins could also attract some CaM into the
apical region.
725
726
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
transport across the cell, and preferential apical efflux.
The entry mechanism could involve some of the recently
identified calcium channels of Trp/ECaC/CaT families.
Apical efflux is mediated by exocytotic release with the
contents of secretory granules or extrusion by calcium
pumps. The transport of calcium across the cell could
involve movement through the cytosol or propagation
through the lumen of the ER. In pancreatic acinar cells,
the process of calcium tunneling through the lumen of the
ER probably plays an essential role in both transcellular
calcium transport and maintaining the calcium available
for release from apical stores during apically localized
Physiol Rev • VOL
calcium spiking. The relative roles of the different calcium transporting mechanisms in transcellular transport
are not completely defined for any epithelial cell types
and certainly deserve further investigation.
At the more complex tissue level, there is also heterogeneous density of calcium-regulating proteins which
underlies polarized intercellular calcium signals. Intercellular calcium waves are initiated in pioneer cells (314)
which respond first to secretagogue stimulation because
they have either more agonist receptors or more sensitive
intracellular calcium stores. This coordination of tissue
signaling is important because it makes the tissue as
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
FIG. 4. Relationship between agonist-induced calcium oscillations and calmodulin (CaM) translocation in pancreatic
acinar cells. A: graph showing cholecystokinin (CCK)-induced oscillations in intracellular calcium that are uniform in
different regions of the cell: nucleus (blue), secretory granule region (pink), and basal cytoplasm (green). Calcium levels
were measured using fura 2 imaging. B: record depicting oscillations in CaM concentration (measured by fluorescence
of DTAF-CaM using confocal microscopy) triggered by the same dose of CCK as above. Traces are from similarly
selected and similarly color-coded regions as above. CaM oscillates with different patterns in different regions of the cell.
C: schematic representation of a pancreatic acinar cell showing the CCK-induced Ca2⫹ oscillations and translocation
patterns of CaM. The calcium and CaM concentrations in different regions of the cell are shown. CCK triggers an initial
large rise in calcium followed by repetitive calcium spikes. The first movement of CaM is from the basal to the apical
region. CaM leaves this region rapidly as soon as calcium falls. There is a delayed but substantial movement of CaM into
the nucleus. Removal of CaM from the nucleus is relatively slow, allowing integration of nuclear CaM influx, leading to
a sustained rise. [From Craske et al. (59), copyright National Academy of Sciences USA.]
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
We thank Prof. O. H. Petersen and Dr. C. J. Dixon for useful
discussion and helpful advice regarding the manuscript.
We gratefully acknowledge funding from the Medical Research Council (MRC Programme Grant G8801575) and The
Wellcome Trust.
Address for reprint requests and other correspondence:
A. V. Tepikin, MRC Secretory Control Research Group, The
Physiological Laboratory, University of Liverpool, Crown Street,
Liverpool L69 3BX, UK.
REFERENCES
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
1. ADAMS SR AND TSIEN RY. Controlling cell chemistry with caged
compounds. Annu Rev Physiol 55: 755–784, 1993.
2. ADKINS CE, MORRIS SA, DE SMEDT H, SIENAERT I, TOROK K, AND TAYLOR
CW. Ca2⫹-calmodulin inhibits Ca2⫹ release mediated by type-1, -2
and -3 inositol trisphosphate receptors. Biochem J 345: 357–363,
2000.
3. AGARWAL RP AND HENKIN RI. Metal binding characteristics of human
salivary and porcine pancreatic amylase. J Biol Chem 262: 2568 –
2575, 1987.
4. ALLEN BG AND WALSH MP. The biochemical basis of the regulation of
smooth-muscle contraction. Trends Biochem Sci 19: 362–368, 1994.
5. AMBUDKAR IS. Regulation of calcium in salivary gland secretion.
Crit Rev Oral Biol Med 11: 4 –25, 2000.
6. ANDERIE I AND THEVENOD F. Evidence for involvement of a zymogen
granule Na⫹/H⫹ exchanger in enzyme secretion from rat pancreatic
acinar cells. J Membr Biol 152: 195–205, 1996.
7. ANSAH TA, MOLLA A, AND KATZ S. Ca2⫹-ATPase activity in pancreatic
Physiol Rev • VOL
26.
27.
28.
29.
30.
31.
acinar plasma membranes. Regulation by calmodulin and acidic
phospholipids. J Biol Chem 259: 13442–13450, 1984.
ANWER MS AND ATKINSON JM. Intracellular calcium-mediated activation of hepatic Na⫹/H⫹ exchange by arginine vasopressin and
phenylephrine. Hepatology 15: 134 –143, 1992.
ATHMANN C, ZENG N, SCOTT DR, AND SACHS G. Regulation of parietal
cell calcium signaling in gastric glands. Am J Physiol Gastrointest
Liver Physiol 279: G1048 –G1058, 2000.
BACHS O, AGELL N, AND CARAFOLI E. Calmodulin and calmodulinbinding proteins in the nucleus. Cell Calcium 16: 289 –296, 1994.
BACHS O, FAMULSKI KS, MIRABELLI F, AND CARAFOLI E. ATP-dependent
Ca2⫹ transport in vesicles isolated from the bile canalicular region
of the hepatocyte plasma membrane. Eur J Biochem 147: 1–7, 1985.
BARTELT DC, WOLFF DJ, AND SCHEELE GA. Calmodulin-binding proteins and calmodulin-regulated enzymes in dog pancreas. Biochem
J 240: 753–763, 1986.
BEGENISICH T AND MELVIN JE. Regulation of chloride channels in
secretory epithelia. J Membr Biol 163: 77– 85, 1998.
BELAN P, GARDNER J, GERASIMENKO O, GERASIMENKO J, MILLS CL,
PETERSEN OH, AND TEPIKIN AV. Isoproterenol evokes extracellular
Ca2⫹ spikes due to secretory events in salivary gland cells. J Biol
Chem 273: 4106 – 4111, 1998.
BELAN P, GERASIMENKO O, PETERSEN OH, AND TEPIKIN AV. Distribution
of Ca2⫹ extrusion sites on the mouse pancreatic acinar cell surface.
Cell Calcium 22: 5–10, 1997.
BELAN PV, GERASIMENKO OV, BERRY D, SAFTENKU E, PETERSEN OH,
AND TEPIKIN AV. A new technique for assessing the microscopic
distribution of cellular calcium exit sites. Pflügers Arch 433: 200 –
208, 1996.
BELAN PV, GERASIMENKO OV, TEPIKIN AV, AND PETERSEN OH. Localization of Ca2⫹ extrusion sites in pancreatic acinar cells. J Biol
Chem 271: 7615–7619, 1996.
BERRIDGE MJ. Inositol trisphosphate and calcium signalling. Nature
361: 315–325, 1993.
BERTHOUD VM, IWANIJ V, GARCIA AM, AND SAEZ JC. Connexins and
glucagon receptors during development of rat hepatic acinus. Am J
Physiol Gastrointest Liver Physiol 263: G650 –G658, 1992.
BEZPROZVANNY I, WATRAS J, AND EHRLICH BE. Bell-shaped calciumresponse curves of Ins(1,4,5)P3- and calcium-gated channels from
endoplasmic reticulum of cerebellum. Nature 351: 751–754, 1991.
BIRD GS, ROSSIER MF, OBIE JF, AND PUTNEY JW. Sinusoidal oscillations in intracellular calcium requiring negative feedback by protein kinase C. J Biol Chem 268: 8425– 8428, 1993.
BISWAS DK, REDDY PV, PICKARD M, MAKKAD B, PETTIT N, AND PARDEE
AB. Calmodulin is essential for estrogen receptor interaction with
its motif and activation of responsive promoter. J Biol Chem 273:
33817–33824, 1998.
BITO H, DEISSEROTH K, AND TSIEN RW. CREB phosphorylation and
dephosphorylation: a Ca2⫹- and stimulus duration-dependent
switch for hippocampal gene expression. Cell 87: 1203–1214, 1996.
BOOTMAN MD, COLLINS TJ, PEPPIATT CM, PROTHERO LS, MACKENZIE L,
DE SMET P, TRAVERS M, TOVEY SC, SEO JT, BERRIDGE MJ, CICCOLINI F,
AND LIPP P. Calcium signalling—an overview. Semin Cell Dev Biol
12: 3–10, 2001.
BOSSER R, ALIGUE R, GUERINI D, AGELL N, CARAFOLI E, AND BACHS O.
Calmodulin can modulate protein phosphorylation in rat liver cells
nuclei. J Biol Chem 268: 15477–15483, 1993.
BROWN EM AND MACLEOD RJ. Extracellular calcium sensing and
extracellular calcium signaling. Physiol Rev 81: 239 –297, 2001.
BROWNLEE C. Cellular calcium imaging: so, what’s new? Trends Cell
Biol 10: 451– 457, 2000.
BRUCE JI, YANG X, FERGUSON CJ, ELLIOTT AC, STEWARD MC, CASE RM,
2⫹
AND RICCARDI D. Molecular and functional identification of a Ca
(polyvalent cation)-sensing receptor in rat pancreas. J Biol Chem
274: 20561–20568, 1999.
BURDAKOV D, CANCELA JM, AND PETERSEN OH. Bombesin-induced
cytosolic Ca2⫹ spiking in pancreatic acinar cells depends on cyclic
ADP-ribose and ryanodine receptors. Cell Calcium 29: 211–216,
2001.
BURGOYNE RD AND WEISS JL. The neuronal calcium sensor family of
Ca2⫹-binding proteins. Biochem J 353: 1–12, 2001.
BURNSTEIN LS, BOSKEY AL, TANNENBAUM PJ, POSNER AS, AND MANDEL
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
sensitive to agonist as its most sensitive cells. In this way,
the tissue uses polarized calcium signals to make it much
more sensitive and economic than it would be as an
uncoordinated group of cells.
A detailed knowledge of calcium signaling pathways
requires understanding of calcium’s downstream effectors. Calcium binding to CaM is one of the most common
and important steps in regulation of calcium-dependent
cellular functions. CaM’s effects have been studied extensively by biochemical methods, but the ability to image
the Ca-CaM reaction in living cells is in its very promising
infancy. The early application of this technology has
shown that CaM distribution is dynamic and Ca-CaM signaling can be polarized. In secretory epithelial cells, there
are indications that the polarity of CaM signals could have
important consequences for secretion and gene expression.
This review highlights the wealth of information that
has been already accumulated about the generation and
properties of polarized calcium signals. The future of the
field would seem to lie at the other end of the signaling
pathway, in particular, in gaining knowledge of events
that link polarized calcium and CaM signals to cell functions. In this respect, new imaging techniques and new
probes should have a significant role to play, particularly
in studies of intact and secreting epithelia. Eventually we
should gain an understanding of complete signaling cascades: from agonist-receptor binding to specific polarized
calcium-CaM signals to activation of the cellular functions.
727
728
32.
33.
34.
35.
36.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
ID. The crystal chemistry of submandibular and parotid salivary
gland stones. J Oral Pathol 8: 284 –291, 1979.
CAMELLO C, PARIENTE JA, SALIDO GM, AND CAMELLO PJ. Sequential
activation of different Ca2⫹ entry pathways upon cholinergic stimulation in mouse pancreatic acinar cells. J Physiol 516: 399 – 408,
1999.
CAMELLO P, GARDNER J, PETERSEN OH, AND TEPIKIN AV. Calcium
dependence of calcium extrusion and calcium uptake in mouse
pancreatic acinar cells. J Physiol 490: 585–593, 1996.
CAMPOS-TOIMIL M, EDWARDSON JM, AND THOMAS P. Real-time studies
of zymogen granule exocytosis in intact rat pancreatic acinar cells.
J Physiol 528: 317–326, 2000.
CANAFF L, PETIT JL, KISIEL M, WATSON PH, GASCON-BARRE M, AND
HENDY GN. Extracellular calcium-sensing receptor is expressed in
rat hepatocytes: coupling to intracellular calcium mobilization and
stimulation of bile flow. J Biol Chem 276: 4070 – 4079, 2001.
CANCELA JM, CHURCHILL GC, AND GALIONE A. Coordination of agonistinduced Ca2⫹-signalling patterns by NAADP in pancreatic acinar
cells. Nature 398: 74 –76, 1999.
CANCELA JM, GERASIMENKO OV, GERASIMENKO JV, TEPIKIN AV, AND
PETERSEN OH. Two different but converging messenger pathways to
intracellular Ca2⫹ release: the roles of nicotinic acid adenine dinucleotide phosphate, cyclic ADP-ribose and inositol trisphosphate.
EMBO J 19: 2549 –2557, 2000.
CANCELA JM, MOGAMI H, TEPIKIN AV, AND PETERSEN OH. Intracellular
glucose switches between cyclic ADP-ribose and inositol trisphosphate triggering of cytosolic Ca2⫹ spiking. Curr Biol 8: 865– 868,
1998.
CANCELA JM AND PETERSEN OH. The cyclic ADP ribose antagonist
8-NH2-cADP-ribose blocks cholecystokinin-evoked cytosolic Ca2⫹
spiking in pancreatic acinar cells. Pflügers Arch 435: 746 –748, 1998.
CANCELA JM, VAN COPPONOLLE F, GALIONE A, TEPIKIN AV, AND PE2⫹
TERSEN OH. Transformation of local Ca
spikes to global Ca2⫹
transients: the combinatorial roles of multiple Ca2⫹ releasing messengers. EMBO J 21: 909 –919, 2002.
CAO Y AND CHATTON JY. Involvement of calmodulin in the activation
of store-operated Ca2⫹ entry in rat hepatocytes. FEBS Lett 424:
33–36, 1998.
CAPIOD T, COMBETTES L, NOEL J, AND CLARET M. Evidence for bile
acid-evoked oscillations of Ca2⫹-dependent K⫹ permeability unrelated to a D-myo-inositol 1,4,5-trisphosphate effect in isolated
guinea pig liver cells. J Biol Chem 266: 268 –273, 1991.
CARAFOLI E. Biogenesis: plasma membrane calcium ATPase: 15
years of work on the purified enzyme. FASEB J 8: 993–1002, 1994.
CAROPPO R, GERBINO A, DEBELLIS L, KIFOR O, SOYBEL DI, BROWN EM,
HOFER AM, AND CURCI S. Asymmetrical, agonist-induced fluctuations in local extracellular [Ca2⫹] in intact polarized epithelia.
EMBO J 20: 6316 – 6326, 2001.
CERINI C, PEYROT V, GARNIER C, DUPLAN L, VEESLER S, LE CAER JP,
BERNARD JP, BOUTEILLE H, MICHEL R, VAZI A, DUPUY P, MICHEL B,
BERLAND Y, AND VERDIER JM. Biophysical characterization of lithostathine. Evidences for a polymeric structure at physiological pH
and a proteolysis mechanism leading to the formation of fibrils.
J Biol Chem 274: 22266 –22274, 1999.
CHAMBERLAIN LH, ROTH D, MORGAN A, AND BURGOYNE RD. Distinct
effects of ␣-SNAP, 14 –3-3 proteins, and calmodulin on priming and
triggering of regulated exocytosis. J Cell Biol 130: 1063–1070, 1995.
CHANSON M, MOLLARD P, MEDA P, SUTER S, AND JONGSMA HJ. Modulation of pancreatic acinar cell to cell coupling during ACh-evoked
changes in cytosolic Ca2⫹. J Biol Chem 274: 282–287, 1999.
CHAUTHAIWALE JV, LOCKWICH TP, AND AMBUDKAR IS. Characteristics
of a low affinity passive Ca2⫹ influx component in rat parotid gland
basolateral plasma membrane vesicles. J Membr Biol 162: 139 –145,
1998.
CHAUTHAIWALE JV, SAKAI T, TAYLOR SE, AND AMBUDKAR IS. Presence of
two Ca2⫹ influx components in internal Ca2⫹-pool-depleted rat
parotid acinar cells. Pflügers Arch 432: 105–111, 1996.
CHENG I, QURESHI I, CHATTOPADHYAY N, QURESHI A, BUTTERS RR, HALL
AE, CIMA RR, ROGERS KV, HEBERT SC, GEIBEL JP, BROWN EM, AND
SOYBEL DI. Expression of an extracellular calcium-sensing receptor
in rat stomach. Gastroenterology 116: 118 –126, 1999.
CHEUNG WY. Calmodulin plays a pivotal role in cellular regulation.
Science 207: 19 –27, 1980.
Physiol Rev • VOL
52. CHIESA A, RAPIZZI E, TOSELLO V, PINTON P, DE VIRGILIO M, FOGARTY E,
AND RIZZUTO R. Recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signalling. Biochem J 355:
1–12, 2001.
53. CHIN D AND MEANS AR. Calmodulin: a prototypical calcium sensor.
Trends Cell Biol 10: 322–328, 2000.
54. CHU S, TANAKA S, KAUNITZ JD, AND MONTROSE MH. Dynamic regulation of gastric surface pH by luminal pH. J Clin Invest 103: 605–
612, 1999.
55. CHURCHILL GC AND GALIONE A. NAADP induces Ca2⫹ oscillations via
a two-pool mechanism by priming IP3- and cADPR-sensitive Ca2⫹
stores. EMBO J 20: 2666 –2671, 2001.
56. CLAPHAM DE. Calcium signaling. Cell 80: 259 –268, 1995.
57. COBBOLD PH, SANCHEZ-BUENO A, AND DIXON CJ. The hepatocyte
calcium oscillator. Cell Calcium 12: 87–95, 1991.
58. COOK DI, DAY ML, CHAMPION MP, AND YOUNG JA. Ca2⫹ not cyclic
AMP mediates the fluid secretory response to isoproterenol in the
rat mandibular salivary gland: whole-cell patch-clamp studies.
Pflügers Arch 413: 67–76, 1988.
59. CRASKE M, TAKEO T, GERASIMENKO O, VAILLANT C, TOROK K, PETERSEN
OH, AND TEPIKIN AV. Hormone-induced secretory and nuclear translocation of calmodulin: oscillations of calmodulin concentration
with the nucleus as an integrator. Proc Natl Acad Sci USA 96:
4426 – 4431, 1999.
60. DASH PK, KARL KA, COLICOS MA, PRYWES R, AND KANDEL ER. cAMP
response element-binding protein is activated by Ca2⫹/calmodulinas well as cAMP-dependent protein kinase. Proc Natl Acad Sci USA
88: 5061–5065, 1991.
61. DAYEL MJ, HOM EF, AND VERKMAN AS. Diffusion of green fluorescent
protein in the aqueous-phase lumen of endoplasmic reticulum.
Biophys J 76: 2843–2851, 1999.
62. DEISSEROTH K, HEIST EK, AND TSIEN RW. Translocation of calmodulin
to the nucleus supports CREB phosphorylation in hippocampal
neurons. Nature 392: 198 –202, 1998.
63. DELVALLE J, TSUNODA Y, WILLIAMS JA, AND YAMADA T. Regulation of
[Ca2⫹]i by secretagogue stimulation of canine gastric parietal cells.
Am J Physiol Gastrointest Liver Physiol 262: G420 –G426, 1992.
64. DENK W, STRICKLER JH, AND WEBB WW. Two-photon laser scanning
fluorescence microscopy. Science 248: 73–76, 1990.
65. DIXON CJ, COBBOLD PH, AND GREEN AK. Oscillations in cytosolic free
Ca2⫹ induced by ADP and ATP in single rat hepatocytes display
differential sensitivity to application of phorbol ester. Biochem J
309: 145–149, 1995.
66. DOLMETSCH RE, XU K, AND LEWIS RS. Calcium oscillations increase
the efficiency and specificity of gene expression. Nature 392: 933–
936, 1998.
67. DORMER RL. Direct demonstration of increases in cytosolic free
Ca2⫹ during stimulation of pancreatic enzyme secretion. Biosci
Rep 3: 233–240, 1983.
68. DORMER RL, POULSEN JH, LICKO V, AND WILLIAMS JA. Calcium fluxes
in isolated pancreatic acini: effects of secretagogues. Am J Physiol
Gastrointest Liver Physiol 240: G38 –G49, 1981.
69. DUCHEN MR. Contributions of mitochondria to animal physiology:
from homeostatic sensor to calcium signalling and cell death.
J Physiol 516: 1–17, 1999.
70. DUPLAN L, MICHEL B, BOUCRAUT J, BARTHELLEMY S, DESPLAT-JEGO S,
MARIN V, GAMBARELLI D, BERNARD D, BERTHEZENE P, ALESCIO-LAUTIER
B, AND VERDIER JM. Lithostathine and pancreatitis-associated protein are involved in the very early stages of Alzheimer’s disease.
Neurobiol Aging 22: 79 – 88, 2001.
71. DUPONT G, TORDJMANN T, CLAIR C, SWILLENS S, CLARET M, AND COMBETTES L. Mechanism of receptor-oriented intercellular calcium
wave propagation in hepatocytes. FASEB J 14: 279 –289, 2000.
72. ELLIS-DAVIES GC AND KAPLAN JH. Nitrophenyl-EGTA, a photolabile
chelator that selectively binds Ca2⫹ with high affinity and releases
it rapidly upon photolysis. Proc Natl Acad Sci USA 91: 187–191,
1994.
73. FAHRMANN M, JACOB P, SEIDLER U, OSTERHOFF M, MOHLIG M, AND
PFEIFFER A. Ca2⫹/calmodulin-dependent protein kinase II isoenzymes gamma and delta are both present in H⫹/K⫹-ATPase-containing rabbit gastric tubulovesicles. Eur J Biochem 266: 1036 –
1042, 1999.
74. FAM SR, GALLAGHER CJ, AND SALTER MW. P2Y(1) purinoceptor-me-
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
37.
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
75.
76.
77.
78.
79.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
Physiol Rev • VOL
95. GIOVANNUCCI DR, YULE DI, AND STUENKEL EL. Optical measurement
of stimulus-evoked membrane dynamics in single pancreatic acinar
cells. Am J Physiol Cell Physiol 275: C732–C739, 1998.
96. GLEESON D, HOOD KA, MURPHY GM, AND DOWLING RH. Calcium and
carbonate ion concentrations in gallbladder and hepatic bile. Gastroenterology 102: 1707–1716, 1992.
97. GOBEL A, KRAUSE E, FEICK P, AND SCHULZ I. IP3 and cyclic ADP-ribose
induced Ca2⫹ release from intracellular stores of pancreatic acinar
cells from rat in primary culture. Cell Calcium 29: 29 –37, 2001.
98. GONZALEZ A, SCHULZ I, AND SCHMID A. Agonist-evoked mitochondrial
Ca2⫹ signals in mouse pancreatic acinar cells. J Biol Chem 275:
38680 –38686, 2000.
99. GOODENOUGH DA. The structure of cell membranes involved in
intercellular communication. Am J Clin Pathol 63: 636 – 645, 1975.
100. GOPINATH RM AND VINCENZI FF. Phosphodiesterase protein activator
mimics red blood cell cytoplasmic activator of (Ca2⫹Mg2⫹)ATPase. Biochem Biophys Res Commun 77: 1203–1209,
1977.
101. GRAY PT. Oscillations of free cytosolic calcium evoked by cholinergic and catecholaminergic agonists in rat parotid acinar cells.
J Physiol 406: 35–53, 1988.
102. GREEN AK, COBBOLD PH, AND DIXON CJ. Effects on the hepatocyte
[Ca2⫹]i oscillator of inhibition of the plasma membrane Ca2⫹ pump
by carboxyeosin or glucagon-(19O29). Cell Calcium 22: 99 –109,
1997.
103. GREGER R AND WINDHORST U. Comprehensive Human Physiology:
From Cellular Mechanisms to Integration. Berlin: Springer-Verlag,
1996, p. 2427–2449.
104. GROBLEWSKI GE, WAGNER AC, AND WILLIAMS JA. Cyclosporin A inhibits Ca2⫹/calmodulin-dependent protein phosphatase and secretion
in pancreatic acinar cells. J Biol Chem 269: 15111–15117, 1994.
105. GROBLEWSKI GE, YOSHIDA M, BRAGADO MJ, ERNST SA, LEYKAM J, AND
WILLIAMS JA. Purification and characterization of a novel physiological substrate for calcineurin in mammalian cells. J Biol Chem 273:
22738 –22744, 1998.
106. GUIHARD G, NOEL J, AND CAPIOD T. Ca2⫹ depletion and inositol
1,4,5-trisphosphate-evoked activation of Ca2⫹ entry in single guinea
pig hepatocytes. J Biol Chem 275: 13411–13414, 2000.
107. HAGAR RE, BURGSTAHLER AD, NATHANSON MH, AND EHRLICH BE. Type
III InsP3 receptor channel stays open in the presence of increased
calcium. Nature 396: 81– 84, 1998.
108. HAJNOCZKY G, HAGER R, AND THOMAS AP. Mitochondria suppress
local feedback activation of inositol 1,4,5-trisphosphate receptors
by Ca2⫹. J Biol Chem 274: 14157–14162, 1999.
109. HAJNOCZKY G, LIN C, AND THOMAS AP. Luminal communication between intracellular calcium stores modulated by GTP and the
cytoskeleton. J Biol Chem 269: 10280 –10287, 1994.
110. HAMILL OP, MARTY A, NEHER E, SAKMANN B, AND SIGWORTH FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:
85–100, 1981.
111. HASHIMOTO N, MARUYAMA T, TODA G, IKEDA Y, SUGIYAMA Y, AND OKA H.
Effects of calmodulin antagonists on secretion of bile and bile acid.
Gastroenterol Jpn 22: 194 –202, 1987.
112. HAUGLAND RP. Molecular Probes: Handbook of Fluorescent Probes
and Reseach Chemicals. Eugene, OR: Molecular Probes, 2001.
113. HIRATA K, NATHANSON MH, BURGSTAHLER AD, OKAZAKI K, MATTEI E,
AND SEARS ML. Relationship between inositol 1,4,5-trisphosphate
receptor isoforms and subcellular Ca2⫹ signaling patterns in nonpigmented ciliary epithelia. Invest Ophthalmol Vis Sci 40: 2046 –
2053, 1999.
114. HIROSE K, KADOWAKI S, TANABE M, TAKESHIMA H, AND IINO M. Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies
complex Ca2⫹ mobilization patterns. Science 284: 1527–1530, 1999.
115. HIROTA J, MICHIKAWA T, NATSUME T, FURUICHI T, AND MIKOSHIBA K.
Calmodulin inhibits inositol 1,4,5-trisphosphate-induced calcium
release through the purified and reconstituted inositol 1,4,5trisphosphate receptor type 1. FEBS Lett 456: 322–326, 1999.
116. HOENDEROP JG, HARTOG A, STUIVER M, DOUCET A, WILLEMS PH, AND
BINDELS RJ. Localization of the epithelial Ca2⫹ channel in rabbit
kidney and intestine. J Am Soc Nephrol 11: 1171–1178, 2000.
117. HOENDEROP JG, VAN DER KEMP AW, HARTOG A, VAN DE GRAAF SF, VAN
OS CH, WILLEMS PH, AND BINDELS RJ. Molecular identification of the
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
80.
diated Ca2⫹ signaling and Ca2⫹ wave propagation in dorsal spinal
cord astrocytes. J Neurosci 20: 2800 –2808, 2000.
FAN GY, FUJISAKI H, MIYAWAKI A, TSAY RK, TSIEN RY, AND ELLISMAN
MH. Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons. Biophys J 76: 2412–
2420, 1999.
FINDLAY I AND PETERSEN OH. The extent of dye-coupling between
exocrine acinar cells of the mouse pancreas. The dye-coupled
acinar unit. Cell Tissue Res 232: 121–127, 1983.
FINDLAY I AND PETERSEN OH. Acetylcholine stimulates a Ca2⫹-dependent Cl⫺ conductance in mouse lacrimal acinar cells. Pflügers Arch
403: 328 –330, 1985.
FINKBEINER S, TAVAZOIE SF, MALORATSKY A, JACOBS KM, HARRIS KM,
AND GREENBERG ME. CREB: a major mediator of neuronal neurotrophin responses. Neuron 19: 1031–1047, 1997.
FISCHER R, KOLLER M, FLURA M, MATHEWS S, STREHLER-PAGE MA,
KREBS J, PENNISTON JT, CARAFOLI E, AND STREHLER EE. Multiple
divergent mRNAs code for a single human calmodulin. J Biol Chem
263: 17055–17062, 1988.
FITZSIMMONS TJ, GUKOVSKY I, MCROBERTS JA, RODRIGUEZ E, LAI FA,
AND PANDOL SJ. Multiple isoforms of the ryanodine receptor are
expressed in rat pancreatic acinar cells. Biochem J 351: 265–271,
2000.
FOGARTY KE, KIDD JF, TUFT DA, AND THORN P. Mechanisms underlying InsP3-evoked global Ca2⫹ signals in mouse pancreatic acinar
cells. J Physiol 526: 515–526, 2000.
FOGARTY KE, KIDD JF, TURNER A, SKEPPER JN, CARMICHAEL J, AND
THORN P. Microtubules regulate local Ca2⫹ spiking in secretory
epithelial cells. J Biol Chem 275: 22487–22494, 2000.
FOSKETT JK, ROIFMAN CM, AND WONG D. Activation of calcium oscillations by thapsigargin in parotid acinar cells. J Biol Chem 266:
2778 –2782, 1991.
FOSKETT JK AND WONG DC. [Ca2⫹]i inhibition of Ca2⫹ release-activated Ca2⫹ influx underlies agonist- and thapsigargin-induced
[Ca2⫹]i oscillations in salivary acinar cells. J Biol Chem 269: 31525–
31532, 1994.
FRIEDMAN PA AND GESEK FA. Cellular calcium transport in renal
epithelia: measurement, mechanisms, and regulation. Physiol Rev
75: 429 – 471, 1995.
FRUEN BR, BARDY JM, BYREM TM, STRASBURG GM, AND LOUIS CF.
Differential Ca2⫹ sensitivity of skeletal and cardiac muscle ryanodine receptors in the presence of calmodulin. Am J Physiol Cell
Physiol 279: C724 –C733, 2000.
FURUYA K, ENOMOTO K, AND YAMAGISHI S. Spontaneous calcium oscillations and mechanically and chemically induced calcium responses in mammary epithelial cells. Pflügers Arch 422: 295–304,
1993.
GEIDER S, BARONNET A, CERINI C, NITSCHE S, ASTIER JP, MICHEL R,
BOISTELLE R, BERLAND Y, DAGORN JC, AND VERDIER JM. Pancreatic
lithostathine as a calcite habit modifier. J Biol Chem 271: 26302–
26306, 1996.
GENAZZANI AA AND GALIONE A. Nicotinic acid-adenine dinucleotide
phosphate mobilizes Ca2⫹ from a thapsigargin-insensitive pool.
Biochem J 315: 721–725, 1996.
GERASIMENKO OV, GERASIMENKO JV, PETERSEN OH, AND TEPIKIN AV.
Short pulses of acetylcholine stimulation induce cytosolic Ca2⫹
signals that are excluded from the nuclear region in pancreatic
acinar cells. Pflügers Arch 432: 1055–1061, 1996.
GERASIMENKO OV, GERASIMENKO JV, TEPIKIN AV, AND PETERSEN OH.
ATP-dependent accumulation and inositol trisphosphate- or cyclic
ADP-ribose-mediated release of Ca2⫹ from the nuclear envelope.
Cell 80: 439 – 444, 1995.
GERBAUD V, PIGNOL D, LORET E, BERTRAND JA, BERLAND Y, FONTECILLA-CAMPS JC, CANSELIER JP, GABAS N, AND VERDIER JM. Mechanism
of calcite crystal growth inhibition by the N-terminal undecapeptide of lithostathine. J Biol Chem 275: 1057–1064, 2000.
GILABERT JA AND PAREKH AB. Respiring mitochondria determine the
pattern of activation and inactivation of the store-operated Ca2⫹
current ICRAC. EMBO J 19: 6401– 6407, 2000.
GIOVANNUCCI DR, GROBLEWSKI GE, SNEYD J, AND YULE DI. Targeted
phosphorylation of inositol 1,4,5-trisphosphate receptors selectively inhibits localized Ca2⫹ release and shapes oscillatory Ca2⫹
signals. J Biol Chem 275: 33704 –33711, 2000.
729
730
118.
119.
120.
121.
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
apical Ca2⫹ channel in 1,25-dihydroxyvitamin D3-responsive epithelia. J Biol Chem 274: 8375– 8378, 1999.
HOFER AM. Using low-affinity fluorescent calcium indicators and
chelators for monitoring and manipulating free [Ca2⫹] in the endoplasmic reticulum. In: Calcium Signalling: A Practical Approach,
edited by Tepikin AV. Oxford, UK: Oxford Univ. Press, 2001, p. 111.
HOFER AM, CURCI S, DOBLE MA, BROWN EM, AND SOYBEL DI. Intercellular communication mediated by the extracellular calciumsensing receptor. Nat Cell Biol 2: 392–398, 2000.
HOFER AM AND MACHEN TE. Technique for in situ measurement of
calcium in intracellular inositol 1,4,5-trisphosphate-sensitive stores
using the fluorescent indicator mag-fura-2. Proc Natl Acad Sci USA
90: 2598 –2602, 1993.
HURLEY TW AND MARTINEZ JR. Characterization of the kinetic and
regulatory properties of high-affinity Ca2⫹-ATPase activity in acinar
preparations of rat submandibular salivary glands. Arch Oral Biol
30: 587–594, 1985.
IKURA M. Calcium binding and conformational response in EF-hand
proteins. Trends Biochem Sci 21: 14 –17, 1996.
ISHIGURO H, HAYAKAWA T, KONDO T, SHIBATA T, KITAGAWA M, SAKAI Y,
SOBAJIMA H, NAKAE Y, TANIKAWA M, AND HIDAKA H. Effects of calmodulin inhibitors on amylase secretion from rat pancreatic acini.
Digestion 53: 162–170, 1992.
ITO K, MIYASHITA Y, AND KASAI H. Micromolar and submicromolar
Ca2⫹ spikes regulating distinct cellular functions in pancreatic
acinar cells. EMBO J 16: 242–251, 1997.
ITO K, MIYASHITA Y, AND KASAI H. Kinetic control of multiple forms of
Ca2⫹ spikes by inositol trisphosphate in pancreatic acinar cells.
J Cell Biol 146: 405– 413, 1999.
IWATSUKI N AND PETERSEN OH. Electrical coupling and uncoupling of
exocrine acinar cells. J Cell Biol 79: 533–545, 1978.
IWATSUKI N AND PETERSEN OH. Direct visualization of cell to cell
coupling: transfer of fluorescent probes in living mammalian pancreatic acini. Pflügers Arch 380: 277–281, 1979.
IWATSUKI N AND PETERSEN OH. Pancreatic acinar cells: the effect of
carbon dioxide, ammonium chloride and acetylcholine on intercellular communication. J Physiol 291: 317–326, 1979.
JANSEN JW, SCHREURS VV, SWARTS HG, FLEUREN-JAKOBS AM, DE PONT
JJ, AND BONTING SL. Role of calcium in exocrine pancreatic secreation. VI. Characteristics of the paracellular pathway for divalent
cations. Biochim Biophys Acta 599: 315–323, 1980.
JARRETT HW AND PENNISTON JT. Partial purification of the Ca2⫹-Mg2⫹
ATPase activator from human erythrocytes: its similarity to the
activator of 3⬘:5⬘-cyclic nucleotide phosphodiesterase. Biochem
Biophys Res Commun 77: 1210 –1216, 1977.
KASAI H AND AUGUSTINE GJ. Cytosolic Ca2⫹ gradients triggering
unidirectional fluid secretion from exocrine pancreas. Nature 348:
735–738, 1990.
KASAI H, LI YX, AND MIYASHITA Y. Subcellular distribution of Ca2⫹
release channels underlying Ca2⫹ waves and oscillations in exocrine pancreas. Cell 74: 669 – 677, 1993.
KASS GE, CHOW SC, GAHM A, WEBB DL, BERGGREN PO, LLOPIS J, AND
ORRENIUS S. Two separate plasma membrane Ca2⫹ carriers participate in receptor-mediated Ca2⫹ influx in rat hepatocytes. Biochim
Biophys Acta 1223: 226 –233, 1994.
KESSLER F, BENNARDINI F, BACHS O, SERRATOSA J, JAMES P, CARIDE AJ,
GAZZOTTI P, PENNISTON JT, AND CARAFOLI E. Partial purification and
characterization of the Ca2⫹-pumping ATPase of the liver plasma
membrane. J Biol Chem 265: 16012–16019, 1990.
KHOO KM, HAN MK, PARK JB, CHAE SW, KIM UH, LEE HC, BAY BH,
AND CHANG CF. Localization of the cyclic ADP-ribose-dependent
calcium signaling pathway in hepatocyte nucleus. J Biol Chem 275:
24807–24817, 2000.
KIBBLE AV AND BURGOYNE RD. Calmodulin increases the initial rate
of exocytosis in adrenal chromaffin cells. Pflügers Arch 431: 464 –
466, 1996.
KIDD JF AND THORN P. Intracellular Ca2⫹ and Cl⫺ channel activation
in secretory cells. Annu Rev Physiol 62: 493–513, 2000.
KIM MH, SEKIJIMA J, AND LEE SP. Primary intrahepatic stones. Am J
Gastroenterol 90: 540 –548, 1995.
KISELYOV K, XU X, MOZHAYEVA G, KUO T, PESSAH I, MIGNERY G, ZHU X,
BIRNBAUMER L, AND MUALLEM S. Functional interaction between
Physiol Rev • VOL
140.
141.
142.
143.
144.
145.
146.
147.
148.
149.
150.
151.
152.
153.
154.
155.
156.
157.
158.
159.
160.
InsP3 receptors and store-operated Htrp3 channels. Nature 396:
478 – 482, 1998.
KITAGAWA M, WILLIAMS JA, AND DE LISLE RC. Amylase release from
streptolysin O-permeabilized pancreatic acini. Am J Physiol Gastrointest Liver Physiol 259: G157–G164, 1990.
KOESTER HJ, BAUR D, UHL R, AND HELL SW. Ca2⫹ fluorescence
imaging with pico- and femtosecond two-photon excitation: signal
and photodamage. Biophys J 77: 2226 –2236, 1999.
KONIG K. Multiphoton microscopy in life sciences. J Microsc 200:
83–104, 2000.
KRAUSE E, PFEIFFER F, SCHMID A, AND SCHULZ I. Depletion of intracellular calcium stores activates a calcium conducting nonselective
cation current in mouse pancreatic acinar cells. J Biol Chem 271:
32523–32528, 1996.
KUBA K AND NAKAYAMA S. Two-photon laser-scanning microscopy:
tests of objective lenses and Ca2⫹ probes. Neurosci Res 32: 281–
294, 1998.
LANKISCH TO, NOZU F, OWYANG C, AND TSUNODA Y. High-affinity
cholecystokinin type A receptor/cytosolic phospholipase A2 pathways mediate Ca2⫹ oscillations via a positive feedback regulation
by calmodulin kinase in pancreatic acini. Eur J Cell Biol 78: 632–
641, 1999.
LEE HC AND AARHUS R. A derivative of NADP mobilizes calcium
stores insensitive to inositol trisphosphate and cyclic ADP-ribose.
J Biol Chem 270: 2152–2157, 1995.
LEE MG, XU X, ZENG W, DIAZ J, KUO TH, WUYTACK F, RACYMAEKERS L,
2⫹
AND MUALLEM S. Polarized expression of Ca
pumps in pancreatic
and salivary gland cells. Role in initiation and propagation of
[Ca2⫹]i waves. J Biol Chem 272: 15771–15776, 1997.
LEE MG, XU X, ZENG W, DIAZ J, WOJCIKIEWICZ RJ, KUO TH, WUYTACK
F, RACYMAEKERS L, AND MUALLEM S. Polarized expression of Ca2⫹
channels in pancreatic and salivary gland cells. Correlation with
initiation and propagation of [Ca2⫹]i waves. J Biol Chem 272:
15765–15770, 1997.
LEITE MF, BURGSTAHLER AD, AND NATHANSON MH. Ca2⫹ waves require sequential activation of inositol trisphosphate receptors and
ryanodine receptors in pancreatic acini. Gastroenterology 122:
415– 427, 2002.
LEITE MF, DRANOFF JA, GAO L, AND NATHANSON MH. Expression and
subcellular localization of the ryanodine receptor in rat pancreatic
acinar cells. Biochem J 337: 305–309, 1999.
LI W, LLOPIS J, WHITNEY M, ZLOKARNIK G, AND TSIEN RY. Cell-permeant caged InsP3 ester shows that Ca2⫹ spike frequency can
optimize gene expression. Nature 392: 936 –941, 1998.
LIAO B, PASCHAL BM, AND LUBY-PHELPS K. Mechanism of Ca2⫹-dependent nuclear accumulation of calmodulin. Proc Natl Acad Sci
USA 96: 6217– 6222, 1999.
LILLY LB AND GOLLAN JL. Ryanodine-induced calcium release from
hepatic microsomes and permeabilized hepatocytes. Am J Physiol
Gastrointest Liver Physiol 268: G1017–G1024, 1995.
LIPP P, BOOTMAN MD, AND COLLINS T. Photometry, video imaging,
confocal and multi-photon microscopy in calcium signalling studies. In: Calcium Signalling: A Practical Approach, edited by
Tepikin AV. Oxford, UK: Oxford Univ. Press, 2001, p. 17.
LIU X, WANG W, SINGH BB, LOCKWICH T, JADLOWIEC J, O’CONNELL B,
WELLNER R, ZHU MX, AND AMBUDKAR IS. Trp1, a candidate protein for
the store-operated Ca2⫹ influx mechanism in salivary gland cells.
J Biol Chem 275: 3403–3411, 2000.
LJUNGSTROM M AND CHEW CS. Calcium oscillations and morphological transformations in single cultured gastric parietal cells. Am J
Physiol Cell Physiol 260: C67–C78, 1991.
LLOPIS J, KASS GE, DUDDY SK, FARELL GC, GAHM A, AND ORRENIUS S.
Mobilization of the hormone-sensitive calcium pool increases hepatocyte tight junctional permeability in the perfused rat liver.
FEBS Lett 280: 84 – 86, 1991.
LUBY-PHELPS K, HORI M, PHELPS JM, AND WON D. Ca2⫹-regulated
dynamic compartmentalization of calmodulin in living smooth
muscle cells. J Biol Chem 270: 21532–21538, 1995.
LUO X, POPOV S, BERA AK, WILKIE TM, AND MUALLEM S. RGS proteins
provide biochemical control of agonist-evoked [Ca2⫹]i oscillations.
Mol Cell 7: 651– 660, 2001.
LYTTON J, WESTLIN M, BURK SE, SHULL GE, AND MACLENNAN DH.
Functional comparisons between isoforms of the sarcoplasmic or
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
122.
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
161.
162.
163.
164.
166.
167.
168.
169.
170.
171.
172.
173.
174.
175.
176.
177.
178.
179.
180.
Physiol Rev • VOL
181. MIZUNO N, NARUSE S, KITAGAWA M, ISHIGURO H, AND HAYAKAWA T.
Effects of an inhibitor of myosin light chain kinase on amylase
secretion from rat pancreatic acini. Biochem Biophys Res Commun 269: 792–797, 2000.
182. MOGAMI H, GARDNER J, GERASIMENKO OV, CAMELLO P, PETERSEN OH,
AND TEPIKIN AV. Calcium binding capacity of the cytosol and endoplasmic reticulum of mouse pancreatic acinar cells. J Physiol 518:
463– 467, 1999.
183. MOGAMI H, NAKANO K, TEPIKIN AV, AND PETERSEN OH. Ca2⫹ flow via
tunnels in polarized cells: recharging of apical Ca2⫹ stores by focal
Ca2⫹ entry through basal membrane patch. Cell 88: 49 –55, 1997.
184. MONTERO M, ALONSO MT, CARNICERO E, CUCHILLO-IBANEZ I, ALBILLOS
A, GARCIA AG, GARCIA-SANCHO J, AND ALVAREZ J. Chromaffin-cell
stimulation triggers fast millimolar mitochondrial Ca2⫹ transients
that modulate secretion. Nat Cell Biol 2: 57– 61, 2000.
185. MOORE EW AND VERINE HJ. Pancreatic calcification and stone formation: a thermodynamic model of calcium in pancreatic juice.
Am J Physiol Gastrointest Liver Physiol 252: G707–G718, 1987.
186. MUALLEM S AND SACHS G. Ca2⫹ metabolism during cholinergic stimulation of acid secretion. Am J Physiol Gastrointest Liver Physiol
248: G216 –G228, 1985.
187. MULLER D, HOENDEROP JG, MEIJ IC, VAN DEN HEUVEL LP, KNOERS NV,
DEN HOLLANDER AI, EGGERT P, GARCIA-NIETO V, CLAVERIE-MARTIN F,
AND BINDELS RJ. Molecular cloning, tissue distribution, and chromosomal mapping of the human epithelial Ca2⫹ channel (ECaC1).
Genomics 67: 48 –53, 2000.
188. NAIRN AC AND PICCIOTTO MR. Calcium/calmodulin-dependent protein kinases. Semin Cancer Biol 5: 295–303, 1994.
189. NAKAGAWA Y, ABRAM V, KEZDY FJ, KAISER ET, AND COE FL. Purification and characterization of the principal inhibitor of calcium oxalate monohydrate crystal growth in human urine. J Biol Chem 258:
12594 –12600, 1983.
190. NAKAGAWA Y, AHMED M, HALL SL, DEGANELLO S, AND COE FL. Isolation from human calcium oxalate renal stones of nephrocalcin, a
glycoprotein inhibitor of calcium oxalate crystal growth. Evidence
that nephrocalcin from patients with calcium oxalate nephrolithiasis is deficient in gamma-carboxyglutamic acid. J Clin Invest 79:
1782–1787, 1987.
191. NAKANISHI S, FUJII A, NAKADE S, AND MIKOSHIBA K. Immunohistochemical localization of inositol 1,4,5-trisphosphate receptors in
non-neural tissues, with special reference to epithelia, the reproductive system, and muscular tissues. Cell Tissue Res 285: 235–251,
1996.
192. NASH MS, YOUNG KW, CHALLISS RA, AND NAHORSKI SR. Intracellular
signalling. Receptor-specific messenger oscillations. Nature 413:
381–382, 2001.
193. NASH MS, YOUNG KW, WILLARS GB, CHALLISS RA, AND NAHORSKI SR.
Single-cell imaging of graded Ins(1,4,5)P3 production following Gprotein-coupled-receptor activation. Biochem J 356: 137–142, 2001.
194. NATHANSON MH AND BURGSTAHLER AD. Coordination of hormoneinduced calcium signals in isolated rat hepatocyte couplets: demonstration with confocal microscopy. Mol Biol Cell 3: 113–121,
1992.
195. NATHANSON MH, BURGSTAHLER AD, AND FALLON MB. Multistep mechanism of polarized Ca2⫹ wave patterns in hepatocytes. Am J
Physiol Gastrointest Liver Physiol 267: G338 –G349, 1994.
196. NATHANSON MH, BURGSTAHLER AD, MENNONE A, FALLON MB, GONZA2⫹
LEZ CB, AND SAEZ JC. Ca
waves are organized among hepatocytes
in the intact organ. Am J Physiol Gastrointest Liver Physiol 269:
G167–G171, 1995.
197. NATHANSON MH, FALLON MB, PADFIELD PJ, AND MARANTO AR. Localization of the type 3 inositol 1,4,5-trisphosphate receptor in the
Ca2⫹ wave trigger zone of pancreatic acinar cells. J Biol Chem 269:
4693– 4696, 1994.
198. NEGULESCU PA AND MACHEN TE. La3⫹ and pH sensitivity of Ca2⫹
entry and intracellular store filling in gastric parietal cells. Am J
Physiol Gastrointest Liver Physiol 269: G770 –G778, 1995.
199. NEHER E AND AUGUSTINE GJ. Calcium gradients and buffers in bovine
chromaffin cells. J Physiol 450: 273–301, 1992.
200. NEMOTO T, KIMURA R, ITO K, TACHIKAWA A, MIYASHITA Y, IINO M, AND
KASAI H. Sequential-replenishment mechanism of exocytosis in pancreatic acini. Nat Cell Biol 3: 253–258, 2001.
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
165.
endoplasmic reticulum family of calcium pumps. J Biol Chem 267:
14483–14489, 1992.
MAHEY R, ALLEN BG, BRIDGES MA, AND KATZ S. Regulation of calcium
transport in pancreatic acinar plasma membranes from guinea pig.
Mol Cell Biochem 112: 155–162, 1992.
MAK DO, MCBRIDE S, AND FOSKETT JK. Inositol 1,4,5-trisphosphate
activation of inositol trisphosphate receptor Ca2⫹ channel by ligand
tuning of Ca2⫹ inhibition. Proc Natl Acad Sci USA 95: 15821–15825,
1998.
MAK DO, MCBRIDE S, AND FOSKETT JK. Regulation by Ca2⫹ and
inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3
InsP3 receptor channels. Ca2⫹ activation uniquely distinguishes
types 1 and 3 InsP3 receptors. J Gen Physiol 117: 435– 446, 2001.
MAK DO, MCBRIDE S, RAGHURAM V, YUE Y, JOSEPH SK, AND FOSKETT
JK. Single-channel properties in endoplasmic reticulum membrane
of recombinant type 3 inositol trisphosphate receptor. J Gen
Physiol 115: 241–256, 2000.
MARTEAU C AND GEROLAMI A. Influence of hypercalcemia on ionized
calcium concentration in pancreatic juice of the dog. J Lab Clin
Med 123: 565–573, 1994.
MARTIN SC AND SHUTTLEWORTH TJ. Ca2⫹ influx drives agonist-activated [Ca2⫹]i oscillations in an exocrine cell. FEBS Lett 352: 32–36,
1994.
MARTY A, EVANS MG, TAN YP, AND TRAUTMANN A. Muscarinic response in rat lacrimal glands. J Exp Biol 124: 15–32, 1986.
MARUYAMA Y. Effects of external calcium on acetylcholine-evoked
increases in membrane capacitance in rat pancreatic acinar cells.
Pflügers Arch 413: 438 – 440, 1989.
MARUYAMA Y. Selective activation of exocytosis by low concentrations of ACh in rat pancreatic acinar cells. J Physiol 492: 807– 814,
1996.
MARUYAMA Y, GALLACHER DV, AND PETERSEN OH. Voltage and Ca2⫹activated K⫹ channel in baso-lateral acinar cell membranes of
mammalian salivary glands. Nature 302: 827– 829, 1983.
MATOVCIK LM, MARANTO AR, SOROKA CJ, GORELICK FS, SMITH J, AND
GOLDENRING JR. Co-distribution of calmodulin-dependent protein
kinase II and inositol trisphosphate receptors in an apical domain
of gastrointestinal mucosal cells. J Histochem Cytochem 44: 1243–
1250, 1996.
MATOZAKI T, ZHU WY, TSUNODA Y, GOKE B, AND WILLIAMS JA. Intracellular mediators of bombesin action on rat pancreatic acinar
cells. Am J Physiol Gastrointest Liver Physiol 260: G858 –G864,
1991.
MATSUO S AND LAGERLOF F. Relationship between total and ionized
calcium concentrations in human whole saliva and dental plaque
fluid. Arch Oral Biol 36: 525–527, 1991.
MATTHEWS RP, GUTHRIE CR, WAILES LM, ZHAO X, MEANS AR, AND
MCKNIGHT GS. Calcium/calmodulin-dependent protein kinase types
II and IV differentially regulate CREB-dependent gene expression.
Mol Cell Biol 14: 6107– 6116, 1994.
MAYER P, MOHLIG M, SEIDLER U, ROCHLITZ H, FAHRMANN M, SCHATZ H,
HIDAKA H, AND PFEIFFER A. Characterization of gamma- and deltasubunits of Ca2⫹/calmodulin-dependent protein kinase II in rat
gastric mucosal cell populations. Biochem J 297: 157–162, 1994.
MEANS AR. Regulatory cascades involving calmodulin-dependent
protein kinases. Mol Endocrinol 14: 4 –13, 2000.
MEDA P, FINDLAY I, KOLOD E, ORCI L, AND PETERSEN OH. Short and
reversible uncoupling evokes little change in the gap junctions of
pancreatic acinar cells. J Ultrastruct Res 83: 69 – 84, 1983.
MEYER-ALBER A, HOCKER M, FETZ I, FORNEFELD H, WASCHULEWSKI IH,
FOLSCH UR, AND SCHMIDT WE. Differential inhibitory effects of
serine/threonine phosphatase inhibitors and a calmodulin antagonist on phosphoinositol/calcium- and cyclic adenosine monophosphate-mediated pancreatic amylase secretion. Scand J Gastroenterol 30: 384 –391, 1995.
MEZZETTI G, MONTI MG, CASOLO LP, PICCININI G, AND MORUZZI MS.
1,25-Dihydroxycholecalciferol-dependent calcium uptake by
mouse mammary gland in culture. Endocrinology 122: 389 –394,
1988.
MICHIKAWA T, HIROTA J, KAWANO S, HIRAOKA M, YAMADA M, FURUICHI
T, AND MIKOSHIBA K. Calmodulin mediates calcium-dependent inactivation of the cerebellar type 1 inositol 1,4,5-trisphosphate receptor. Neuron 23: 799 – 808, 1999.
731
732
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
Physiol Rev • VOL
MA. A rat kidney-specific calcium transporter in the distal
nephron. J Biol Chem 275: 28186 –28194, 2000.
PENG JB, CHEN XZ, BERGER UV, VASSILEV PM, TSUKAGUCHI H, BROWN
EM, AND HEDIGER MA. Molecular cloning and characterization of a
channel-like transporter mediating intestinal calcium absorption.
J Biol Chem 274: 22739 –22746, 1999.
PENG JB, CHEN XZ, BERGER UV, WEREMOWICZ S, MORTON CC, VASSILEV
PM, BROWN EM, AND HEDIGER MA. Human calcium transport protein
CaT1. Biochem Biophys Res Commun 278: 326 –332, 2000.
PETERSEN CC AND PETERSEN OH. Receptor-activated cytoplasmic
Ca2⫹ spikes in communicating clusters of pancreatic acinar cells.
FEBS Lett 284: 113–116, 1991.
PETERSEN CC, TOESCU EC, AND PETERSEN OH. Different patterns of
receptor-activated cytoplasmic Ca2⫹ oscillations in single pancreatic acinar cells: dependence on receptor type, agonist concentration and intracellular Ca2⫹ buffering. EMBO J 10: 527–533, 1991.
PETERSEN CC, TOESCU EC, POTTER BV, AND PETERSEN OH. Inositol
triphosphate produces different patterns of cytoplasmic Ca2⫹ spiking depending on its concentration. FEBS Lett 293: 179 –182, 1991.
PETERSEN OH. Stimulus-secretion coupling: cytoplasmic calcium
signals and the control of ion channels in exocrine acinar cells.
J Physiol 448: 1–51, 1992.
PETERSEN OH, BURDAKOV D, AND TEPIKIN AV. Polarity in intracellular
calcium signaling. Bioessays 21: 851– 860, 1999.
PETERSEN OH, IWATSUKI N, PHILPOTT HG, LAUGIER R, PEARSON GT,
DAVISON JS, AND GALLACHER DV. Membrane potential and conductance changes evoked by hormones and neurotransmitters in mammalian exocrine gland cells. Methods Cell Biol 23: 513–530, 1981.
PFEIFFER F, SCHMID A, AND SCHULZ I. Capacitative Ca2⫹ influx and a
Ca2⫹-dependent nonselective cation pathway are discriminated by
genistein in mouse pancreatic acinar cells. Pflügers Arch 430: 916 –
922, 1995.
PORTOLES M, FAURA M, RENAU-PIQUERAS J, IBORRA FJ, SAEZ R, GUERRI
C, SERRATOSA J, RIUS E, AND BACHS O. Nuclear calmodulin/62 kDa
calmodulin-binding protein complexes in interphasic and mitotic
cells. J Cell Sci 107: 3601–3614, 1994.
PRUSCHY M, JU Y, SPITZ L, CARAFOLI E, AND GOLDFARB DS. Facilitated
nuclear transport of calmodulin in tissue culture cells. J Cell Biol
127: 1527–1536, 1994.
PUJOL MJ, SORIANO M, ALIGUE R, CARAFOLI E, AND BACHS O. Effect of
alpha-adrenergic blockers on calmodulin association with the nuclear matrix of rat liver cells during proliferative activation. J Biol
Chem 264: 18863–18865, 1989.
PUTNEY JW JR. A model for receptor-regulated calcium entry. Cell
Calcium 7: 1–12, 1986.
RECH F, ROHLICEK V, AND SCHMID A. A method of resolution improvement by the measurement of cell membrane capacitance. Physiol
Res 45: 421– 425, 1996.
RENARD-ROONEY DC, HAJNOCZKY G, SEITZ MB, SCHNEIDER TG, AND
THOMAS AP. Imaging of inositol 1,4,5-trisphosphate-induced Ca2⫹
fluxes in single permeabilized hepatocytes. Demonstration of both
quantal and nonquantal patterns of Ca2⫹ release. J Biol Chem 268:
23601–23610, 1993.
RINDERKNECHT H. Pancreatic secretory enzymes. In: The Pancreas:
Biology, Pathobiology and Disease, edited by Go VLW, Dimagno
EP, Gardner JD, Lebenthal E, Reber HA, and Scheele GA. New
York: Raven, 1993, p. 219.
RIZZUTO R, PINTON P, CARRINGTON W, FAY FS, FOGARTY KE, LIFSHITZ
LM, TUFT RA, AND POZZAN T. Close contacts with the endoplasmic
reticulum as determinants of mitochondrial Ca2⫹ responses. Science 280: 1763–1766, 1998.
ROBB-GASPERS LD AND THOMAS AP. Coordination of Ca2⫹ signaling
by intercellular propagation of Ca2⫹ waves in the intact liver. J Biol
Chem 270: 8102– 8107, 1995.
ROBINSON JP. Principles of confocal microscopy. Methods Cell Biol
63: 89 –106, 2001.
ROMOSER VA, HINKLE PM, AND PERSECHINI A. Detection in living cells
of Ca2⫹-dependent changes in the fluorescence emission of an
indicator composed of two green fluorescent protein variants
linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J Biol Chem 272: 13270 –13274, 1997.
ROONEY TA, SASS EJ, AND THOMAS AP. Characterization of cytosolic
GER
223.
224.
225.
226.
227.
228.
229.
230.
231.
232.
233.
234.
235.
236.
237.
238.
239.
240.
241.
242.
243.
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
201. NEVILLE MC AND PEAKER M. The secretion of calcium and phosphorus into milk. J Physiol 290: 59 – 67, 1979.
202. NGUYEN QT, CALLAMARAS N, HSIEH C, AND PARKER I. Construction of
a two-photon microscope for video-rate Ca2⫹ imaging. Cell Calcium 30: 383–393, 2001.
203. NICOTERA P, MCCONKEY DJ, JONES DP, AND ORRENIUS S. ATP stimulates Ca2⫹ uptake and increases the free Ca2⫹ concentration in
isolated rat liver nuclei. Proc Natl Acad Sci USA 86: 453– 457, 1989.
204. NICOTERA P, ORRENIUS S, NILSSON T, AND BERGGREN PO. An inositol
1,4,5-trisphosphate-sensitive Ca2⫹ pool in liver nuclei. Proc Natl
Acad Sci USA 87: 6858 – 6862, 1990.
205. NIELSEN SP AND PETERSEN OH. Transport of calcium in the perfused
submandibular gland of the cat. J Physiol 223: 685– 697, 1972.
206. O’NEIL KT AND DEGRADO WF. How calmodulin binds its targets:
sequence independent recognition of amphiphilic alpha-helices.
Trends Biochem Sci 15: 59 – 64, 1990.
207. OKIDO M, SHIMIZU S, OSTROW JD, AND NAKAYAMA F. Isolation of a
calcium-regulatory protein from black pigment gallstones: similarity with a protein from cholesterol gallstones. Hepatology 15: 1079 –
1085, 1992.
208. OKUBO Y, KAKIZAWA S, HIROSE K, AND IINO M. Visualization of IP3
dynamics reveals a novel AMPA receptor-triggered IP3 production
pathway mediated by voltage-dependent Ca2⫹ influx in Purkinje
cells. Neuron 32: 113–122, 2001.
209. OSIPCHUK Y AND CAHALAN M. Cell-to-cell spread of calcium signals
mediated by ATP receptors in mast cells. Nature 359: 241–244,
1992.
210. OSIPCHUK YV, WAKUI M, YULE DI, GALLACHER DV, AND PETERSEN OH.
Cytoplasmic Ca2⫹ oscillations evoked by receptor stimulation, Gprotein activation, internal application of inositol trisphosphate or
Ca2⫹: simultaneous microfluorimetry and Ca2⫹ dependent Cl⫺ current
recording in single pancreatic acinar cells. EMBO J 9: 697–704, 1990.
211. OSTROWSKI NL, YOUNG WS III, KNEPPER MA, AND LOLAIT SJ. Expression of vasopressin V1a and V2 receptor messenger ribonucleic
acid in the liver and kidney of embryonic, developing, and adult
rats. Endocrinology 133: 1849 –1859, 1993.
212. OZAWA T. Ryanodine-sensitive Ca2⫹ release mechanism of rat pancreatic acinar cells is modulated by calmodulin. Biochim Biophys
Acta 1452: 254 –262, 1999.
213. PADFIELD PJ, DING TG, AND JAMIESON JD. Ca2⫹-dependent amylase
secretion from pancreatic acinar cells occurs without activation of
phospholipase C linked G-proteins. Biochem Biophys Res Commun 174: 536 –541, 1991.
214. PAEMELEIRE K, MARTIN PE, COLEMAN SL, FOGARTY KE, CARRINGTON
WA, LEYBAERT L, TUFT RA, EVANS WH, AND SANDERSON MJ. Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin
43, 32, or 26. Mol Biol Cell 11: 1815–1827, 2000.
215. PAREKH AB AND PENNER R. Store depletion and calcium influx.
Physiol Rev 77: 901–930, 1997.
216. PARK MK, ASHBY MC, ERDEMLI G, PETERSEN OH, AND TEPIKIN AV.
Perinuclear, perigranular and sub-plasmalemmal mitochondria
have distinct functions in the regulation of cellular calcium transport. EMBO J 20: 1863–1874, 2001.
217. PARK MK, LOMAX RB, TEPIKIN AV, AND PETERSEN OH. Local uncaging of
caged Ca2⫹ reveals distribution of Ca2⫹-activated Cl⫺ channels in
pancreatic acinar cells. Proc Natl Acad Sci USA 98: 10948–10953,
2001.
218. PARK MK, PETERSEN OH, AND TEPIKIN AV. The endoplasmic reticulum
as one continuous Ca2⫹ pool: visualization of rapid Ca2⫹ movements and equilibration. EMBO J 19: 5729 –5739, 2000.
219. PATEL S, MORRIS SA, ADKINS CE, O’BEIRNE G, AND TAYLOR CW. Ca2⫹independent inhibition of inositol trisphosphate receptors by calmodulin: redistribution of calmodulin as a possible means of regulating Ca2⫹ mobilization. Proc Natl Acad Sci USA 94: 11627–
11632, 1997.
220. PATEL S, ROBB-GASPERS LD, STELLATO KA, SHON M, AND THOMAS AP.
Coordination of calcium signalling by endothelial-derived nitric
oxide in the intact liver. Nat Cell Biol 1: 467– 471, 1999.
221. PATTERSON RL, VAN ROSSUM DB, AND GILL DL. Store-operated Ca2⫹
entry: evidence for a secretion-like coupling model. Cell 98: 487–
499, 1999.
222. PENG JB, CHEN XZ, BERGER UV, VASSILEV PM, BROWN EM, AND HEDI-
CALCIUM AND CALMODULIN IN SECRETORY EPITHELIA
244.
245.
246.
247.
248.
250.
251.
252.
253.
254.
255.
256.
257.
258.
259.
260.
261.
262.
263.
264.
Physiol Rev • VOL
265.
266.
267.
268.
269.
270.
271.
272.
273.
274.
275.
276.
277.
278.
279.
280.
281.
282.
283.
284.
285.
286.
lase. II. Effects of pH, substrate and ions on the activity of the
enzyme. Ann Clin Lab Sci 7: 310 –317, 1977.
SLAWIK M, ZDEBIK A, HUG MJ, KERSTAN D, LEIPZIGER J, AND GREGER R.
Whole-cell conductive properties of rat pancreatic acini. Pflügers
Arch 432: 112–120, 1996.
SMITH JP, YELAMARTY RV, KRAMER ST, AND CHEUNG JY. Effects of
cholecystokinin on cytosolic calcium in pancreatic duct segments
and ductal cells. Am J Physiol Gastrointest Liver Physiol 264:
G1177–G1183, 1993.
STAUFFER PL, ZHAO H, LUBY-PHELPS K, MOSS RL, STAR RA, AND MUAL2⫹
LEM S. Gap junction communication modulates [Ca ]i oscillations
and enzyme secretion in pancreatic acini. J Biol Chem 268: 19769 –
19775, 1993.
STRAUB SV, GIOVANNUCCI DR, AND YULE DI. Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol
1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria. J Gen Physiol 116: 547–560, 2000.
STREHLER EE AND ZACHARIAS DA. Role of alternative splicing in
generating isoform diversity among plasma membrane calcium
pumps. Physiol Rev 81: 21–50, 2001.
STRICKER SA AND WHITAKER M. Confocal laser scanning microscopy
of calcium dynamics in living cells. Microsc Res Tech 46: 356 –369,
1999.
STRIGGOW F AND BOHNENSACK R. Inositol 1,4,5-trisphosphate activates receptor-mediated calcium entry by two different pathways
in hepatocytes. Eur J Biochem 222: 229 –234, 1994.
STUENKEL EL, TSUNODA Y, AND WILLIAMS JA. Secretagogue induced
calcium mobilization in single pancreatic acinar cells. Biochem
Biophys Res Commun 158: 863– 869, 1989.
SUBRAMANIAN K AND MEYER T. Calcium-induced restructuring of
nuclear envelope and endoplasmic reticulum calcium stores. Cell
89: 963–971, 1997.
SWATTON JE, MORRIS SA, CARDY TJ, AND TAYLOR CW. Type 3 inositol
trisphosphate receptors in RINm5F cells are biphasically regulated
by cytosolic Ca2⫹ and mediate quantal Ca2⫹ mobilization. Biochem
J 344: 55– 60, 1999.
SWEITZER TD AND HANOVER JA. Calmodulin activates nuclear protein
import: a link between signal transduction and nuclear transport.
Proc Natl Acad Sci USA 93: 14574 –14579, 1996.
TAKAHASHI A, CAMACHO P, LECHLEITER JD, AND HERMAN B. Measurement of intracellular calcium. Physiol Rev 79: 1089 –1125, 1999.
TAKEMURA H AND PUTNEY JW JR. Capacitative calcium entry in
parotid acinar cells. Biochem J 258: 409 – 412, 1989.
TAKEMURA H, YAMASHINA S, AND SEGAWA A. Millisecond analyses of Ca2⫹
initiation sites evoked by muscarinic receptor stimulation in exocrine
acinar cells. Biochem Biophys Res Commun 259: 656– 660, 1999.
TAKEO T, CRASKE M, TOROK K, PETERSEN OH, AND TEPIKIN AV. Intracellular distribution of calmodulin and calcium-calmodulin binding in
nucleus and cytoplasm of pancreatic acinar cells. J Physiol 506: 74,
1998.
TANAKA Y AND TASHJIAN AH. Calmodulin is a selective mediator of
Ca2⫹-induced Ca2⫹ release via the ryanodine receptor-like Ca2⫹
channel triggered by cyclic ADP-ribose. Proc Natl Acad Sci USA 92:
3244 –3248, 1995.
TEPIKIN AV. Measuring calcium extrusion. In: Calcium Signalling:
A Practical Approach, edited by Tepikin AV. Oxford, UK: Oxford
Univ. Press, 2001, p. 197.
TEPIKIN AV, KOSTYUK PG, SNITSAREV VA, AND BELAN PV. Extrusion of
calcium from a single isolated neuron of the snail Helix pomatia.
J Membr Biol 123: 43– 47, 1991.
TEPIKIN AV, LLOPIS J, SNITSAREV VA, GALLACHER DV, AND PETERSEN OH.
The droplet technique: measurement of calcium extrusion from single
isolated mammalian cells. Pflügers Arch 428: 664– 670, 1994.
TEPIKIN AV, VORONINA SG, GALLACHER DV, AND PETERSEN OH. Pulsatile Ca2⫹ extrusion from single pancreatic acinar cells during receptor-activated cytosolic Ca2⫹ spiking. J Biol Chem 267: 14073–
14076, 1992.
TERUEL MN, CHEN W, PERSECHINI A, AND MEYER T. Differential codes
for free Ca2⫹-calmodulin signals in nucleus and cytosol. Curr Biol
10: 86 –94, 2000.
TEUFEL H, STOCK P, ROHRMOSER H, AND FORELL MM. Calcium secretion in the isolated perfused canine pancreas. Res Exp Med 176:
51– 68, 1979.
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
249.
calcium oscillations induced by phenylephrine and vasopressin in
single fura-2-loaded hepatocytes. J Biol Chem 264: 17131–17141, 1989.
ROONEY TA, SASS EJ, AND THOMAS AP. Agonist-induced cytosolic
calcium oscillations originate from a specific locus in single hepatocytes. J Biol Chem 265: 10792–10796, 1990.
ROOS N. A possible site of calcium regulation in rat exocrine
pancreas cells: an X-ray microanalytical study. Scanning Microsc
2: 323–329, 1988.
ROSADO JA, GRAVES D, AND SAGE SO. Tyrosine kinases activate
store-mediated Ca2⫹ entry in human platelets through the reorganization of the actin cytoskeleton. Biochem J 351: 429 – 437, 2000.
ROSENZWEIG SA, MILLER LJ, AND JAMIESON JD. Identification and
localization of cholecystokinin-binding sites on rat pancreatic
plasma membranes and acinar cells: a biochemical and autoradiographic study. J Cell Biol 96: 1288 –1297, 1983.
RUSNAK F AND MERTZ P. Calcineurin: form and function. Physiol Rev
80: 1483–1521, 2000.
SAEZ JC, CONNOR JA, SPRAY DC, AND BENNETT MV. Hepatocyte gap
junctions are permeable to the second messenger, inositol 1,4,5trisphosphate, and to calcium ions. Proc Natl Acad Sci USA 86:
2708 –2712, 1989.
SANCHEZ-BUENO A, DIXON CJ, WOODS NM, CUTHBERTSON KS, AND
COBBOLD PH. Inhibitors of protein kinase C prolong the falling
phase of each free-calcium transient in a hormone-stimulated hepatocyte. Biochem J 268: 627– 632, 1990.
SASAKI S, NAKAGAKI I, MORI H, AND IMAI Y. Intracellular calcium store
and transport of elements in acinar cells of the salivary gland
determined by electron probe X-ray microanalysis. Jpn J Physiol
33: 69 – 83, 1983.
SCHEPP W, BROSCH E, TATGE C, SCHUSDZIARRA V, AND CLASSEN M.
Cholinergic stimulation of isolated rat parietal cells: role of calcium, calmodulin and protein kinase C. Clin Physiol Biochem 7:
137–148, 1989.
SCHEPP W, SCHNEIDER J, HEIM HK, RUOFF HJ, SCHUSDZIARRA V, AND
CLASSEN M. A calmodulin antagonist inhibits histamine-stimulated
acid production by isolated rat parietal cells. Regul Pept 17: 209 –
220, 1987.
SEGAWA A, TERAKAWA S, YAMASHINA S, AND HOPKINS CR. Exocytosis in
living salivary glands: direct visualization by video-enhanced microscopy and confocal laser microscopy. Eur J Cell Biol 54: 322–
330, 1991.
SERRATOSA J, PUJOL MJ, BACHS O, AND CARAFOLI E. Rearrangement of
nuclear calmodulin during proliferative liver cell activation. Biochem Biophys Res Commun 150: 1162–1169, 1988.
SHAW PJ. Computer reconstruction in three-dimensional fluorescence microscopy. In: Electronic Light Microscopy. New York:
Wiley-Liss, 1993, p. 211.
SHENG M, THOMPSON MA, AND GREENBERG ME. CREB: a Ca2⫹-regulated transcription factor phosphorylated by calmodulin-dependent
kinases. Science 252: 1427–1430, 1991.
SHENNAN DB AND PEAKER M. Transport of milk constituents by the
mammary gland. Physiol Rev 80: 925–951, 2000.
SHIMIZU S, SABSAY B, VEIS A, OSTROW JD, REGE RV, AND DAWES LG.
Isolation of an acidic protein from cholesterol gallstones, which
inhibits the precipitation of calcium carbonate in vitro. J Clin
Invest 84: 1990 –1996, 1989.
SHIN DM, LUO X, WILKIE TM, MILLER LJ, PECK AB, HUMPHREYS-BEHER
MG, AND MUALLEM S. Polarized expression of G protein-coupled
receptors and an all-or-none discharge of Ca2⫹ pools at initiation
sites of [Ca2⫹]i waves in polarized exocrine cells. J Biol Chem 276:
44146 – 44156, 2001.
SHUTTLEWORTH TJ. What drives calcium entry during [Ca2⫹]i oscillations?— challenging the capacitative model. Cell Calcium 25:
237–246, 1999.
SHUTTLEWORTH TJ AND THOMPSON JL. Evidence for a non-capacitative
Ca2⫹ entry during [Ca2⫹] oscillations. Biochem J 316: 819 – 824, 1996.
SIPMA H, DE SMET P, SIENAERT I, VANLINGEN S, MISSIAEN L, PARYS JB,
AND DE SMEDT H. Modulation of inositol 1,4,5-trisphosphate binding
to the recombinant ligand-binding site of the type-1 inositol 1,4,5trisphosphate receptor by Ca2⫹ and calmodulin. J Biol Chem 274:
12157–12162, 1999.
SKY-PECK HH AND THUVASETHAKUL P. Human pancreatic alpha-amy-
733
734
MICHAEL C. ASHBY AND ALEXEI V. TEPIKIN
Physiol Rev • VOL
307. WILLIAMS JA, KORC M, AND DORMER RL. Action of secretagogues on
a new preparation of functionally intact, isolated pancreatic acini.
Am J Physiol Cell Physiol 235: C517–C524, 1978.
308. WOJCIKIEWICZ RJ, ERNST SA, AND YULE DI. Secretagogues cause
ubiquitination and down-regulation of inositol 1,4,5-trisphosphate
receptors in rat pancreatic acinar cells. Gastroenterology 116:
1194 –1201, 1999.
309. WOODS NM, CUTHBERTSON KS, AND COBBOLD PH. Repetitive transient
rises in cytoplasmic free calcium in hormone-stimulated hepatocytes. Nature 319: 600 – 602, 1986.
310. WU GY, DEISSEROTH K, AND TSIEN RW. Activity-dependent CREB
phosphorylation: convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein
kinase pathway. Proc Natl Acad Sci USA 98: 2808 –2813, 2001.
311. XU X, ZENG W, POPOV S, BERMAN DM, DAVIGNON I, YU K, YOWE D,
OFFERMANNS S, MUALLEM S, AND WILKIE TM. RGS proteins determine
signaling specificity of Gq-coupled receptors. J Biol Chem 274:
3549 –3556, 1999.
312. YAMADA M, MIYAWAKI A, SAITO K, NAKAJIMA T, YAMAMOTO-HINO M, RYO
Y, FURUICHI T, AND MIKOSHIBA K. The calmodulin-binding domain in
the mouse type 1 inositol 1,4,5-trisphosphate receptor. Biochem J
308: 83– 88, 1995.
313. YAMAKI H, MORITA K, KITAYAMA S, IMAI Y, ITADANI K, AKAGAWA Y, AND
DOHI T. Cyclic ADP-ribose induces Ca2⫹ release from caffeineinsensitive Ca2⫹ pools in canine salivary gland cells. J Dent Res 77:
1807–1816, 1998.
314. YAMAMOTO-HINO M, MIYAWAKI A, SEGAWA A, ADACHI E, YAMASHINA S,
FUJIMOTO T, SUGIYAMA T, FURUICHI T, HASEGAWA M, AND MIKOSHIBA K.
Apical vesicles bearing inositol 1,4,5-trisphosphate receptors in the
Ca2⫹ initiation site of ductal epithelium of submandibular gland.
J Cell Biol 141: 135–142, 1998.
315. YANG SD, TALLANT EA, AND CHEUNG WY. Calcineurin is a calmodulindependent protein phosphatase. Biochem Biophys Res Commun
106: 1419 –1425, 1982.
316. YOSHIDA H, NOZU F, LANKISCH TO, MITAMURA K, OWYANG C, AND
TSUNODA Y. A possible role for Ca2⫹/calmodulin-dependent protein
kinase IV during pancreatic acinar stimulus-secretion coupling.
Biochim Biophys Acta 1497: 155–167, 2000.
317. YUE L, PENG JB, HEDIGER MA, AND CLAPHAM DE. CaT1 manifests the
pore properties of the calcium-release-activated calcium channel.
Nature 410: 705–709, 2001.
318. YULE DI, ERNST SA, OHNISHI H, AND WOJCIKIEWICZ RJ. Evidence that
zymogen granules are not a physiologically relevant calcium pool.
Defining the distribution of inositol 1,4,5-trisphosphate receptors in
pancreatic acinar cells. J Biol Chem 272: 9093–9098, 1997.
319. YULE DI AND GALLACHER DV. Oscillations of cytosolic calcium in
single pancreatic acinar cells stimulated by acetylcholine. FEBS
Lett 239: 358 –362, 1988.
320. YULE DI, KIM ET, AND WILLIAMS JA. Tyrosine kinase inhibitors attenuate “capacitative” Ca2⫹ influx in rat pancreatic acinar cells. Biochem Biophys Res Commun 202: 1697–1704, 1994.
321. YULE DI, LAWRIE AM, AND GALLACHER DV. Acetylcholine and cholecystokinin induce different patterns of oscillating calcium signals
in pancreatic acinar cells. Cell Calcium 12: 145–151, 1991.
322. YULE DI, STUENKEL E, AND WILLIAMS JA. Intercellular calcium waves
in rat pancreatic acini: mechanism of transmission. Am J Physiol
Cell Physiol 271: C1285–C1294, 1996.
323. ZACHARIAS DA, BAIRD GS, AND TSIEN RY. Recent advances in technology for measuring and manipulating cell signals. Curr Opin
Neurobiol 10: 416 – 421, 2000.
324. ZENG W, XU X, POPOV S, MUKHOPADHYAY S, CHIDIAC P, SWISTOK J, DANHO
W, YAGALOFF KA, FISHER SL, ROSS EM, MUALLEM S, AND WILKIE TM. The
N-terminal domain of RGS4 confers receptor-selective inhibition of G
protein signaling. J Biol Chem 273: 34687–34690, 1998.
325. ZIMMERMANN B. Control of InsP3-induced Ca2⫹ oscillations in permeabilized blowfly salivary gland cells: contribution of mitochondria. J Physiol 525: 707–719, 2000.
326. ZIMMERMANN B. Subcellular organization of agonist-evoked Ca2⫹
waves in the blowfly salivary gland. Cell Calcium 27: 297–307, 2000.
327. ZIMPRICH F, TOROK K, AND BOLSOVER SR. Nuclear calmodulin responds rapidly to calcium influx at the plasmalemma. Cell Calcium
17: 233–238, 1995.
82 • JULY 2002 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.32.247 on July 4, 2017
287. THOMAS AP, RENARD DC, AND ROONEY TA. Spatial and temporal
organization of calcium signalling in hepatocytes. Cell Calcium 12:
111–126, 1991.
288. THOMPSON MP, PIAZZA GJ, BROWER DP, BINGHAM EW, AND FARRELL
HM. Isolation and characterization of bovine mammary calmodulin. J Dairy Sci 70: 1551–1556, 1987.
289. THORN P, GERASIMENKO O, AND PETERSEN OH. Cyclic ADP-ribose
regulation of ryanodine receptors involved in agonist evoked cytosolic Ca2⫹ oscillations in pancreatic acinar cells. EMBO J 13:
2038 –2043, 1994.
290. THORN P, LAWRIE AM, SMITH PM, GALLACHER DV, AND PETERSEN OH.
Local and global cytosolic Ca2⫹ oscillations in exocrine cells evoked
by agonists and inositol trisphosphate. Cell 74: 661– 668, 1993.
291. TINEL H, CANCELA JM, MOGAMI H, GERASIMENKO JV, GERASIMENKO OV,
TEPIKIN AV, AND PETERSEN OH. Active mitochondria surrounding the
pancreatic acinar granule region prevent spreading of inositol
trisphosphate-evoked local cytosolic Ca2⫹ signals. EMBO J 18:
4999 –5008, 1999.
292. TINTON SA, CHOW SC, BUC-CALDERON PM, AND KASS GE. Adenosine
stimulates calcium influx in isolated rat hepatocytes. Eur J Biochem 238: 576 –581, 1996.
293. TOESCU EC, LAWRIE AM, PETERSEN OH, AND GALLACHER DV. Spatial
and temporal distribution of agonist-evoked cytoplasmic Ca2⫹ signals in exocrine acinar cells analysed by digital image microscopy.
EMBO J 11: 1623–1629, 1992.
294. TORDJMANN T, BERTHON B, COMBETTES L, AND CLARET M. The location of
hepatocytes in the rat liver acinus determines their sensitivity to
calcium-mobilizing hormones. Gastroenterology 111: 1343–1352, 1996.
295. TORDJMANN T, BERTHON B, JACQUEMIN E, CLAIR C, STELLY N, GUILLON
G, CLARET M, AND COMBETTES L. Receptor-oriented intercellular
calcium waves evoked by vasopressin in rat hepatocytes. EMBO J
17: 4695– 4703, 1998.
296. TOROK K, STAUFFER K, AND EVANS WH. Connexin 32 of gap junctions
contains two cytoplasmic calmodulin-binding domains. Biochem J
326: 479 – 483, 1997.
297. TOROK K AND TRENTHAM DR. Mechanism of 2-chloro-(⑀-aminoLys75)-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin interactions with smooth muscle myosin light chain kinase and
derived peptides. Biochemistry 33: 12807–12820, 1994.
298. TOROK K, WILDING M, GROIGNO L, PATEL R, AND WHITAKER M. Imaging
the spatial dynamics of calmodulin activation during mitosis. Curr
Biol 8: 692– 699, 1998.
299. TOUTENHOOFD SL, FOLETTI D, WICKI R, RHYNER JA, GARCIA F, TOLON R,
AND STREHLER EE. Characterization of the human CALM2 calmodulin gene and comparison of the transcriptional activity of CALM1,
CALM2 and CALM3. Cell Calcium 23: 323–338, 1998.
300. TSUNODA Y AND TASHIRO Y. Distinct characteristics of receptoroperated Ca2⫹ influx and refilling in pancreatic acinar cells. Biochem Biophys Res Commun 256: 579 –583, 1999.
301. VAN DE PUT FH AND ELLIOTT AC. Imaging of intracellular calcium
stores in individual permeabilized pancreatic acinar cells. Apparent
homogeneous cellular distribution of inositol 1,4,5-trisphosphatesensitive stores in permeabilized pancreatic acinar cells. J Biol
Chem 271: 4999 –5006, 1996.
302. VAN DE PUT FH AND ELLIOTT AC. The endoplasmic reticulum can act
as a functional Ca2⫹ store in all subcellular regions of the pancreatic acinar cell. J Biol Chem 272: 27764 –27770, 1997.
303. VANDERMEERS A, VANDERMEERS-PIRET MC, RATHE J, KUTZNER R, DELFORGE A, AND CHRISTOPHE J. A calcium-dependent protein activator
of guanosine 3⬘:5⬘-monophosphate phosphodiesterase in bovine
and rat pancreas. Isolation, properties and levels in vivo. Eur
J Biochem 81: 379 –386, 1977.
304. VERDIER JM, DUSSOL B, CASANOVA P, DAUDON M, DUPUY P, BERTHEZENE P,
BOISTELLE R, BERLAND Y, AND DAGORN JC. Evidence that human kidney
produces a protein similar to lithostathine, the pancreatic inhibitor of
CaCO3 crystal growth. Eur J Clin Invest 22: 469 – 474, 1992.
305. WAKASUGI H, KIMURA T, HAASE W, KRIBBEN A, KAUFMANN R, AND
SCHULZ I. Calcium uptake into acini from rat pancreas: evidence for
intracellular ATP-dependent calcium sequestration. J Membr Biol
65: 205–220, 1982.
306. WAKUI M, POTTER BV, AND PETERSEN OH. Pulsatile intracellular
calcium release does not depend on fluctuations in inositol
trisphosphate concentration. Nature 339: 317–320, 1989.