Imaging of intracellular calcium stores
in single permeabilized lens cells
GRANT C. CHURCHILL1 AND CHARLES F. LOUIS1,2
Departments of 1Biochemistry and 2Veterinary PathoBiology,
University of Minnesota, St. Paul, Minnesota 55108
inositol 1,4,5-trisphosphate; cyclic ADP-ribose; calcium pools;
mag-fura 2; epithelium
MANY EXTRACELLULAR SIGNALS increase cytosolic Ca21 by
activating the release of Ca21 from intracellular stores
(2). In the best-characterized mechanism for Ca21 release,
the second messenger inositol 1,4,5-trisphosphate (IP3 )
activates intracellular Ca21 channels (2). Although its
mechanism is not as well characterized as that of IP3,
cyclic ADP-ribose (cADPR) also activates Ca21 release
from intracellular stores in sea urchin eggs (6, 20) and
certain mammalian cells (5, 9, 11, 13, 14, 18).
Intracellular Ca21 stores have commonly been characterized by their sensitivity to Ca21-releasing agents,
either physiological or pharmacological. Most cells contain Ca21 pools that are sensitive to IP3, thapsigargin,
and ionomycin (2), and some cells contain a Ca21 pool
that is sensitive to cADPR (5, 6, 9, 11, 13, 14, 18, 20).
These Ca21 pools overlap to varying degrees, suggesting that the intracellular Ca21 stores are functionally
organized into distinct compartments sensitive to only
certain agents (2, 6, 9, 11, 13, 14, 18, 20). The location of
intracellular Ca21 stores sensitive to IP3, inhibitors of
the intracellular Ca21 pump, activators of ryanodine
receptors, or ionomycin has been revealed by direct
imaging of permeabilized cells, the organelles of which
contain a Ca21-sensitive dye (11, 12, 15–17, 26, 27, 29).
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C426
In contrast, the location of cADPR-sensitive Ca21 stores
has not been defined, because the cells containing the
Ca21 stores that have been imaged were unresponsive
to cADPR (17, 29). Nevertheless, it has been demonstrated that certain intracellular Ca21 stores such as
the envelope of isolated nuclei (10) and isolated zymogen granules (9) are sensitive to cADPR and IP3.
The ocular lens is a transparent tissue containing
only two cell types: fiber cells, which lack organelles
and make up the bulk of its mass, and epithelial cells,
which contain organelles and form a single layer on its
anterior surface (22). In the lens the loss of Ca21
homeostasis is implicated in the loss of transparency
and cataract formation (7), so it is important to better
define the mechanisms by which Ca21 is regulated in
the lens. Duncan and co-workers (8) demonstrated that
permeabilized human lens cells in suspension exhibit
thapsigargin-sensitive, ATP-dependent Ca21 uptake and
IP3-mediated Ca21 release. However, the location of the
intracellular Ca21 stores, their sensitivity to other Ca21releasing agents, and the overlap among the various
Ca21 pools are unknown for lens cells of any species.
The objective of this study was to characterize the
intracellular Ca21 stores of mammalian lens cells in
terms of their distribution and sensitivity to Ca21releasing agents. Intracellular Ca21 stores in permeabilized cells were imaged with mag-fura 2 and fluorescence microscopy (15). A sheep lens cell culture system
(28) was used in which the cells exhibit agonistmediated Ca21 signaling (4) as well as cell-to-cell Ca21
waves (3). We conclude that lens cells contain IP3-,
cADPR-, and thapsigargin-sensitive intracellular Ca21
stores that are distributed throughout the cell, as well
as Ca21 stores that are insensitive to all these agents
that are localized in a juxtanuclear region.
MATERIALS AND METHODS
Materials. cADPR and 8-amino-cADPR were generous gifts
from Dr. Timothy Walseth (University of Minnesota). Sheep
eyes were obtained from John Morrell (Sioux Falls, SD).
Medium 199 and Hanks’ balanced salt solution (HBSS) were
obtained from GIBCO (Grand Island, NY). Fetal calf serum
was obtained from Hyclone (Logan, UT). Mag-fura 2-AM was
obtained from Molecular Probes (Eugene, OR). Ionomycin,
thapsigargin, D-IP3, and L-IP3 were obtained from LC Laboratories (Woburn, MA). Saponin, digitonin, ADP-ribose, b-NAD1,
ATP, creatine, creatine kinase (porcine), heparin (6-kDa
average fragment size), and all other chemicals were obtained
from Sigma Chemical (St. Louis, MO).
Cell culture. Primary cultures of cells isolated from the
equatorial region of fresh sheep lenses were prepared as
described previously (28). The sheep lens epithelial cells used
in this study were cultured for 4–34 days.
0363-6143/99 $5.00 Copyright r 1999 the American Physiological Society
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Churchill, Grant C., and Charles F. Louis. Imaging of
intracellular calcium stores in single permeabilized lens cells.
Am. J. Physiol. 276 (Cell Physiol. 45): C426–C434, 1999.—
Intracellular Ca21 stores in permeabilized sheep lens cells
were imaged with mag-fura 2 to characterize their distribution and sensitivity to Ca21-releasing agents. Inositol 1,4,5trisphosphate (IP3 ) or cyclic ADP-ribose (cADPR) released
Ca21 from intracellular Ca21 stores that were maintained by
an ATP-dependent Ca21 pump. The IP3 antagonist heparin
inhibited IP3- but not cADPR-mediated Ca21 release, whereas
the cADPR antagonist 8-amino-cADPR inhibited cADPR- but
not IP3-mediated Ca21 release, indicating that IP3 and cADPR
were operating through separate mechanisms. A Ca21 store
sensitive to IP3, cADPR, and thapsigargin appeared to be
distributed throughout all intracellular regions. In some cells
a Ca21 store insensitive to IP3, cADPR, thapsigargin, and
2,4-dinitrophenol, but not ionomycin, was present in a juxtanuclear region. We conclude that lens cells contain intracellular Ca21 stores that are sensitive to IP3, cADPR, and thapsigargin, as well as a Ca21 store that appears insensitive to all
these agents.
IMAGING CA21 STORES IN LENS CELLS
Data analysis and presentation. Data were analyzed only
from regions that exhibited a stable mag-fura 2 fluorescence
ratio for at least 5 min in the absence of a Ca21-releasing
agent. The results are from single experiments that are
representative of the most frequently observed response for a
given treatment. The number of independent experiments (n)
is taken as the response of a field of 5–30 cells to a given
treatment. All experiments were repeated a minimum of
three but typically many more times.
Ca21 concentrations are not presented as absolute values
but, rather, as the ratio of mag-fura 2 fluorescence at 340-nm
excitation to that at 380-nm excitation, because the amount of
Mg21 bound to mag-fura 2 is unknown, making the estimate
of Ca21 concentration potentially inaccurate (16, 27). Nevertheless, changes in Ca21 concentration are reflected by the
mag-fura 2 340 nm-to-380 nm fluorescence ratio, which is
directly proportional to the concentration of free Ca21 (dissociation constant 5 53 µM) (15, 25).
RESULTS
Monitoring Ca21 concentration in intracellular stores
with mag-fura 2. The first step in this study was to
determine whether, as reported previously in other cell
types (15), the Ca21 concentration in intracellular
stores in lens cells could be directly monitored with
mag-fura 2. After mag-fura 2 loading but before permeabilization, most cells exhibited a similar fluorescence
ratio throughout the cells (Fig. 1Ba, ratio, cell 2);
however, certain cells exhibited a higher fluorescence
ratio (Fig. 1Ba, ratio, cell 1), likely because of the
presence of mag-fura 2 in intracellular compartments.
After the addition of the intracellular buffer with 300
nM Ca21, the cells began to round up and pull apart
from one another (Fig. 1Bb), and the mag-fura 2 ratio
decreased in the cells that had an elevated fluorescence
ratio (Fig. 1, A and Bb, ratio). This decrease in the
mag-fura 2 ratio was apparently due to the release of
Ca21 from intracellular stores and was likely due to the
mechanical strain imposed on the cells during the
change in morphology, since switching to low-Ca21concentration medium can trigger a transient increase
in cytosolic Ca21 concentration (data not shown). After
incubation for ,9 min in an intracellular buffer containing digitonin (10 µg/ml) without ATP, most cells were
permeabilized on the basis of a loss of fluorescence
intensity that was most evident over nuclear regions
(Fig. 1Bc, 340 nm). The overall decrease in fluorescence
intensity with permeabilization is not evident in Fig.
1Bc, because the camera’s amplification (gain and kV
settings) was increased to compensate for the loss of
dye from the cytosol. The remaining fluorescence was
concentrated in certain regions that are likely organelles with trapped dye, which appeared as evenly
fluorescent regions or punctate vesicular compartments (Fig. 1Bc, 340 nm). After the addition of 3 mM
ATP, the mag-fura 2 fluorescence ratio increased rapidly for ,5 min and then more slowly over the next 20
min (Fig. 1A). These data demonstrate that mag-fura 2
can be used to monitor the Ca21 concentration in the
intracellular stores of sheep lens cells.
IP3 releases Ca21 from intracellular stores. That the
Ca21 concentration in intracellular stores could be
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Monitoring intracellular store Ca21 concentration. Cells
were maintained in HBSS supplemented with 10 mM HEPES
(HBSS-H, pH 7.2) before they were loaded with mag-fura 2 by
incubation in 6 µM mag-fura 2-AM for 1 h at 37°C, which
promotes compartmentalization of the dye into intracellular
organelles (15). After they were loaded, cells were rinsed
three times with HBSS-H and incubated for 20 min at 37°C to
promote complete hydrolysis of the AM ester. The glass
coverslip with attached cells formed the bottom of a microincubation culture chamber (model MS 200D, Medical Systems,
Greenvale, NY). The chamber was mounted on the stage of an
inverted epifluorescence microscope (model IM 35, Zeiss)
supported on a vibration-isolated table (Technical Manufacturing, Peabody, MA). Cells were viewed through a 340, 1.3-NA,
oil-immersion objective lens (Fluor 40, Nikon, Melville, NY).
Mag-fura 2 was excited with light from a 50-W mercury
lamp alternately filtered to 340 or 380 nm. Fluorescence
emission was filtered to .510 nm, focused with a 320 lens,
and monitored with a silicon intensified target camera (model
VE-1000, Dage-MTI, Michigan City, IN). The camera’s gain
and kilovolts were set to manual and initially adjusted for the
fluorescence intensity of intact cells but were increased to
monitor permeabilized cells to compensate for the smaller
fluorescence due to the loss of cytosolic dye. Images were captured
and processed with the software package Image-1/Fluorescence
(Universal Imaging, West Chester, PA). Unprocessed images
were recorded in digital form to an optical memory disk recorder
(model TQ-3031F, Panasonic, Secaucus, NJ). Images were captured at 512 3 480 pixels at 256 intensity values. The software
performed a background subtraction and a shading correction
and then calculated the 340 nm-to-380 nm ratios for each
matched pixel pair from the 340- and 380-nm intensity images
and displayed the resulting image in pseudocolor. Thresholding
was used to eliminate pixels that were too dim at either wavelength to calculate an accurate ratio.
Cells were permeabilized by exchanging the HBSS-H with
an intracellular buffer lacking ATP and containing saponin
(50 µg/ml) or digitonin (10–15 µg/ml). The intracellular buffer
was composed of (in mM) 120 KCl, 20 NaCl, 10 HEPES, 3
MgSO4, 1 EGTA, 0.75 CaCl2, 3 (or 5) dithiothreitol, 3 Na2ATP,
and 30 creatine phosphate and 10 U/ml creatine kinase. Free
Ca21 concentration was calculated to be 300 nM. The extent of
permeabilization was monitored on-line. When ,75% of the
cells showed a decrease in fluorescence intensity due to the
loss of cytosolic mag-fura 2, the detergent was washed out by
several exchanges of intracellular buffer supplemented with
ATP (equivalent to 10–30 times the chamber volume). Unless
otherwise noted, images were captured every 30 s to minimize photobleaching of the mag-fura 2.
Additions of chemicals and solution changes. The major
problem encountered with solution additions or exchanges
was that the fluid flow caused the permeabilized cells to
detach from the coverslip. Therefore, to minimize fluid flows
during solution additions, a 1 3 1-cm piece of Kimwipe was
placed on the edge of the chamber in contact with the bathing
solution. New solutions were transferred onto the edge of the
Kimwipe with a pipette, which allowed gravity flow of the
solution into the chamber at a relatively slow rate. Solutions were
removed with an aspirator, the height of which could be adjusted
to control the volume in the chamber (0.3–5 ml). Ca21-releasing
agents were added to the cells by dilution of the stock solution
(never .1% of the final volume bathing the cells) into ,100 µl
of the intracellular buffer and addition of this to the chamber.
Then 100 µl of the intracellular buffer were removed and
readded five times from different regions of the microincubator to ensure complete mixing of the added compound.
C427
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IMAGING CA21 STORES IN LENS CELLS
monitored in permeabilized lens cells enabled the activity of Ca21-releasing agents to be tested by directly
adding them to the medium bathing the permeabilized
cells. IP3 mobilizes Ca21 in many cell types (2), and
0.1–5 µM has been shown to mobilize Ca21 from
permeabilized human lens cells in suspension (8).
Figure 2 shows the effect of various concentrations of
IP3 on the intracellular store Ca21 concentration in a
cytoplasmic region of a permeabilized sheep lens cell.
The addition of submaximal concentrations (0.16–8
µM) of IP3 resulted in a rapid release of only a fraction
of the Ca21 that could be released with maximal
concentrations ($16 µM) of IP3 (Fig. 2). This phenomenon is termed quantal Ca21 release and is commonly
reported during IP3-mediated Ca21 release in other cell
types (2, 21).
cADPR releases Ca21 from intracellular stores. cADPR
is known to mobilize Ca21 from intracellular stores in
sea urchin eggs (6, 20), as well as in certain mammalian
cells (5, 9, 11, 13, 14, 18). To determine whether cADPR
could release Ca21 from permeabilized lens cells, cADPR
was added to the buffer bathing permeabilized sheep
lens cells. The effect of cADPR on the intracellular store
Ca21 concentration was variable; therefore, the results
from three experiments are presented, which depict the
different types of responses of permeabilized lens cells
to added cADPR. In the experiment shown in Fig. 3A,
10 µM cADPR elicited a monophasic, nonquantal decrease in the intracellular store Ca21 concentration
that did not fully deplete these Ca21 stores, even after
20 min. In the experiment shown in Fig. 3B, 0.5 µM
cADPR elicited a monophasic, nonquantal decrease in
the intracellular store Ca21 concentration; the rate of
Ca21 release was not accelerated by the addition of
higher concentrations of cADPR. In contrast, in the
experiment shown in Fig. 3C, 0.5 µM cADPR elicited a
rapid, quantal decrease in the Ca21 concentration in
intracellular stores. The subsequent addition of 5 µM
cADPR led to no further Ca21 release, whereas the
addition of 50 µM cADPR resulted in further quantal
Ca21 release (Fig. 3C). A similar quantal Ca21 release is
shown in Fig. 3D; however, in this experiment the cells
were monitored for 10 min before the first addition of
the cADPR, demonstrating that the Ca21 concentration
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Fig. 1. Permeabilization and Ca21 uptake into
intracellular stores in mag-fura 2-loaded sheep
lens epithelial cells. A: change in mag-fura 2
fluorescence ratio after lowering of extracellular Ca21, addition of digitonin, and addition of
ATP. B: field of cells presented as fluorescence
at 340-nm excitation (340 nm) and pseudocolor images (ratio). Color corresponds to ratio
as indicated by calibration scale in A. Camera’s gain and kV were increased after permeabilization (b and c) to compensate for decrease in fluorescence resulting from loss of
cytosolic mag-fura 2. Thus loss in mag-fura 2
fluorescence intensity on permeabilization is
not evident in these images, except over nuclei. Two cells outlined in pseudocolor images
in B were used to obtain graphs of Ca21
concentration vs. time in A. Cells were loaded
with mag-fura 2-AM at 37°C for 1 h, rinsed
with Hanks’ balanced salt solution-HEPES,
and imaged (a). Hanks’ balanced salt solutionHEPES was then exchanged with intracellular buffer containing 300 nM free Ca21, no
ATP, and 15 µg/ml digitonin (b). Cells were
washed with digitonin-free intracellular buffer
(c) and incubated in a buffer containing 3 mM
ATP, 30 mM creatine phosphate, and 10 U/ml
creatine kinase (d). Data are from a single
experiment representative of response observed in 599 of 911 cells in 49 experiments in
which 49 cultures were used.
IMAGING CA21 STORES IN LENS CELLS
C429
certain agents. Also, to determine whether functionally
distinct Ca21 stores were also spatially distinct, as
demonstrated recently in astrocytes and myocytes (12),
three regions of interest were defined: one over the
nucleus, a second over a juxtanuclear region, and a
third over a cytoplasmic region. An experiment in
which the IP3-sensitive Ca21 stores were depleted before the addition of cADPR is shown in Fig. 5, A and C.
The addition of 16 µM IP3 released a portion of the
stored Ca21 from all three regions monitored (Fig. 5, A
and C), and the subsequent addition of 32 µM IP3 led to
the release of additional Ca21 (Fig. 5, A and C). The
subsequent addition of 30 µM cADPR failed to release
in the intracellular stores was stable until the addition
of cADPR. These results indicate that although cADPR
can mobilize Ca21 from intracellular stores in sheep
lens cells, the response to cADPR addition was more
variable than that obtained with IP3 in terms of the
rate of Ca21 release and whether the Ca21 release was
quantal.
Specificity of IP3- and cADPR-mediated Ca21 release.
To determine whether IP3 and cADPR released Ca21
from intracellular stores through specific and separate
mechanisms, the effect of specific antagonists and
analogs of these Ca21-releasing messengers was evaluated. In the first approach the responses to IP3 and
cADPR were assessed in the presence of the IP3 antagonist heparin (2) or the cADPR antagonist 8-aminocADPR (31). Heparin (100 µg/ml) blocked the Ca21
release mediated by 16 µM IP3, but not that mediated
by 10 µM cADPR (Fig. 4A). Conversely, 8-aminocADPR (40 µM) blocked the Ca21 release mediated by
10 µM cADPR, but not that mediated by 16 µM IP3 (Fig.
4B). These results indicate that IP3 and cADPR are
acting through separate mechanisms.
In the second approach the Ca21-releasing activity of
IP3 or cADPR analogs was evaluated. IP3 is an enantiomer, and the naturally occurring D-form has a 100- to
1,000-fold greater affinity than the L-form for the IP3
receptor (24). In sea urchin eggs, cADPR is formed from
b-NAD1 and is metabolized to ADPR (20). Ca21 was not
released from intracellular Ca21 stores in permeabilized lens cells after the addition of 32 µM L-IP3, 50 µM
ADPR, or 200 µM b-NAD1, but Ca21 was released after
the addition of 50 µM cADPR (Fig. 4C). Collectively,
these results indicate that IP3 and cADPR release Ca21
through specific mechanisms.
Spatial distribution and functional overlap of the
intracellular Ca21 stores sensitive to IP3, cADPR, and
thapsigargin. The next set of experiments was designed
to reveal whether lens cells contained a single intracellular Ca21 store sensitive to all Ca21-releasing agents
or multiple intracellular Ca21 stores sensitive to only
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Fig. 2. Effect of inositol 1,4,5-trisphosphate (IP3 ) concentration
([D-IP3]) on intracellular store Ca21 concentration in permeabilized
lens cells. Increasing concentrations of IP3 were added at arrows.
Mag-fura 2 ratio was obtained from a cytoplasmic region (,10 µm2 ).
Data are from a single experiment representative of response observed in 73 of 134 cells in 11 similar experiments in which 11
cultures were used.
Fig. 3. Effect of cyclic ADP-ribose (cADPR) concentration ([cADPR])
on intracellular store Ca21 concentration in permeabilized lens cells.
Indicated concentrations of cADPR were added at arrows. Different
types of responses were observed after addition of cADPR: a relatively slow monophasic decrease in Ca21 concentration (A), a slow
decrease in Ca21 concentration that did not accelerate with increasing concentrations of cADPR (B), and a relatively rapid quantal
decrease in the Ca21 concentration (C and D). Data are from single
experiments representative of rates of release observed in 77 of 336
cells (A), 14 of 336 cells (B), and 112 of 336 cells (C and D) in 36
similar experiments in which 17 cultures were used.
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IMAGING CA21 STORES IN LENS CELLS
any additional Ca21, indicating that IP3 had completely
depleted the Ca21 stores sensitive to cADPR. The
addition of thapsigargin released a small amount of
additional Ca21 from all three intracellular regions.
The addition of 10 µM ionomycin released the remaining Ca21 from all the intracellular Ca21 stores in all
regions of the cell. The nuclear region appears to have
ionomycin-insensitive Ca21 stores, but this is an artifact arising from the low fluorescence intensity of
mag-fura 2 in this region after permeabilization of the
cells (Fig. 1B). The weaker the mag-fura 2 fluorescence,
the more the ratio will be influenced by noise. The pixel
intensities due to noise above the background will be
approximately equal at 340- and 380-nm excitation,
yielding a ratio of ,1. This ‘‘noise’’ ratio is averaged
with the ratio reporting Ca21, presumably 0.6, on the
basis of the other regions of the cell that have sufficient
dye to accurately report on Ca21, resulting in an
apparent ratio of 0.8.
An experiment in which the cADPR-sensitive Ca21
stores were depleted before the addition of IP3 is shown
in Fig. 5, D and F. The addition of 1 µM cADPR resulted
in a slow release of Ca21 from all three intracellular
regions (Fig. 5, D and F). The subsequent addition of 30
µM cADPR did not release appreciably more Ca21, and
all three regions reached a new steady state soon
thereafter. The addition of IP3 released additional Ca21
from all three regions, and this release was accelerated by
the addition of thapsigargin. The addition of ionomycin
rapidly released all the remaining Ca21 from all three
regions.
Thapsigargin-, IP3-, and cADPR-insensitive intracellular Ca21 stores. In some cell types such as sea urchin
eggs, the thapsigargin-sensitive Ca21 pools completely
overlap the IP3- and/or cADPR-sensitive Ca21 pools
(20); however, in other cell types, these Ca21 pools are
separable. For example, cADPR releases Ca21 from a
thapsigargin-insensitive Ca21 pool in T lymphocytes
(14), and IP3 and cADPR, but not thapsigargin, release
Ca21 from secretory granules isolated from pancreatic
acinar cells (9). Therefore, it was of interest to determine whether cADPR or IP3 could release Ca21 from a
thapsigargin-insensitive store in lens cells. To address
this issue, mag-fura 2-loaded permeabilized lens cells
were treated sequentially with thapsigargin, IP3, and
cADPR (Fig. 6). Thapsigargin almost fully depleted all
the intracellular Ca21 stores in the cytoplasmic regions
but only partially depleted the intracellular Ca21 stores
in a juxtanuclear region (Fig. 6, A and Bb). Neither the
thapsigargin-sensitive nor the thapsigargin-insensitive Ca21 stores released any additional Ca21 in response to the subsequent additions of 16 µM IP3 or 30
µM cADPR (Fig. 6A). These data demonstrate that the
thapsigargin-sensitive Ca21 stores appear to completely overlap the IP3- and cADPR-sensitive Ca21
stores and that the thapsigargin-insensitive Ca21 stores
are also IP3 and cADPR insensitive. In some cell types,
certain Ca21 stores have been reported to contain a
thapsigargin-resistant Ca21 pump with an IC50 for
thapsigargin of 5 µM compared with the more typical
IC50 of ,0.2 µM (30); therefore, a higher concentration
of thapsigargin was applied (10 µM) but was also
without effect (Fig. 6A). In contrast, ionomycin rapidly
depleted these Ca21 stores (Fig. 6, A and Bc).
Mag-fura 2 fluorescence has been shown to respond
to mitochondrial Ca21 concentration (16); therefore, it
was considered that the thapsigargin-, IP3-, and cADPRinsensitive Ca21 stores identified in sheep lens cells
might be mitochondria. Mitochondrial Ca21 can be
released by agents that dissipate their electrochemical
gradient (17). After the addition of thapsigargin to
deplete all but the thapsigargin-insensitive Ca21 stores,
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Fig. 4. Specificity of IP3- and cADPR-dependent Ca21 release from intracellular stores in permeabilized lens cells.
A: effect of IP3 antagonist heparin (100 µg/ml) on Ca21 release mediated by IP3 (16 µM) or cADPR (10 µM). B: effect
of cADPR antagonist 8-amino-cADPR (8NH2-cADPR, 40 µM) on Ca21 release mediated by cADPR (10 µM) or IP3 (16
µM). C: effect of compounds structurally similar to IP3 or cADPR on Ca21 release: nonphysiological enantiomer L-IP3
(32 µM); hydrolytic product of cADPR, ADPR (50 µM); precursor to cADPR, b-NAD1 (200 µM); and cADPR (50 µM).
Data are from single experiments representative of response observed in 29 of 50 cells in 5 similar experiments (A)
and 19 of 21 cells in 4 similar experiments (B) in which 8 cultures were used and 65 of 77 cells in 5 similar
experiments in which 5 cultures were used (C).
IMAGING CA21 STORES IN LENS CELLS
C431
the addition of the proton ionophore 2,4-dinitrophenol
did not release Ca21 from the thapsigargin-insensitive
Ca21 stores, whereas ionomycin rapidly released this
Ca21 (Fig. 7). These results indicate that the thapsigargin-, IP3-, and cADPR-insensitive Ca21 stores were not
mitochondria.
DISCUSSION
Intracellular Ca21 stores in permeabilized sheep lens
cells were imaged with mag-fura 2 to characterize their
distribution and sensitivity to Ca21-releasing agents.
The intracellular Ca21 stores exhibited ATP-dependent
Ca21 uptake and IP3- and cADPR-mediated Ca21 release. These results are consistent with the previous
report of IP3-mediated Ca21 release in permeabilized
human lens cells (8) and with the ability of an inhibitor
of phospholipase C to prevent agonist-mediated cytosolic Ca21 increases in sheep lens cells (4).
In permeabilized lens cells the response to cADPR
was less consistent than the response to IP3 in regard to
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Fig. 5. Spatial distribution and functional overlap of Ca21 stores sensitive to IP3, cADPR, thapsigargin, and
ionomycin in permeabilized lens cells. A and D: changes in mag-fura 2 fluorescence ratio of 3 subcellular regions
after addition of indicated compounds. B and E: fluorescence images of permeabilized cells showing intracellular
distribution of Ca21 stores based on mag-fura 2. C and F: images of monitored cells in which mag-fura 2 fluorescence
ratio is presented in pseudocolor according to calibration scale. Three intracellular regions used to obtain traces in A
and D are indicated and correspond to nuclear (circles), juxtanuclear (triangles), and cytoplasmic (squares) regions.
Compounds were added as indicated by arrows at concentrations and sequence as follows: 16 and 32 µM IP3, 30 µM
cADPR, 1 µM thapsigargin (Tg), and 10 µM ionomycin (iono) in A and 1 and 30 µM cADPR, 16 µM IP3, 1 µM
thapsigargin, and 10 µM ionomycin in B. Data are from single experiments representative of responses observed in
55 of 96 cells in 8 similar experiments in which 8 cultures were used and 71 of 145 cells in 11 similar experiments in
which 11 cultures were used. Ca21 was not released by initial addition of IP3 in 75 of 88 cells or cADPR in 110 of 120
cells. Thapsigargin released a portion of stored Ca21 in 204 of 208 cells, and ionomycin released all stored Ca21 in
208 of 208 cells.
C432
IMAGING CA21 STORES IN LENS CELLS
the cADPR concentration dependence and the rate of
Ca21 release. This may relate to the loss of a soluble
factor(s) from the permeabilized cells such as calmodulin, which has been shown to be required for cADPRmediated Ca21 release in sea urchin egg homogenates
(19, 20). The variable rate of Ca21 release in response to
even maximal concentrations of cADPR may relate to
the number of cADPR-activated Ca21 channels per
intracellular compartment in different lens cells in
these primary cultures. If only a few such channels are
present, then the number of channels would be the
rate-limiting step in Ca21 store depletion, and the rate
of release would likely be independent of cADPR concentration. In contrast, cells with a greater number of
cADPR-dependent channels would likely exhibit a more
pronounced concentration dependence. Although the
conditions required to improve the consistency of the
response to cADPR remain to be defined, as does the
physiological significance of cADPR in lens cells, it is
clear that cADPR can stimulate Ca21 release from
intracellular Ca21 stores in sheep lens cells.
By directly imaging intracellular Ca21 stores in
permeabilized lens cells, we have demonstrated that
IP3 and cADPR can release Ca21 from stores that
appear to be distributed throughout the entire cell. The
Fig. 7. Effect of 2,4-dinitrophenol (DNP) on thapsigargin-insensitive
intracellular Ca21 stores. Changes in mag-fura 2 fluorescence ratio of
2 intracellular regions after addition of 1 µM thapsigargin, 100 µM
2,4-dinitrophenol, and 10 µM ionomycin are indicated by arrows.
Traces correspond to a cytoplasmic region (k) and a juxtanuclear
region (l). Data are from a single experiment representative of
responses observed in 7 similar experiments in which 7 cultures were
used, and 35 of 88 cells showed a juxtanuclear thapsigargininsensitive Ca21 store that was insensitive to 2,4-dinitrophenol.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on June 18, 2017
Fig. 6. Characterization of a thapsigargin-insensitive intracellular
Ca21 store in permeabilized lens cells. A: changes in mag-fura 2
fluorescence ratio of 2 intracellular regions after addition of indicated
compounds. B: fluorescence images of permeabilized cell showing
intracellular distribution of Ca21 stores retaining mag-fura 2 (340
nm); mag-fura 2 fluorescence ratio is presented in pseudocolor
according to calibration scale. Two intracellular regions used to
obtain traces in A are indicated in B and correspond to a juxtanuclear
region (k) and a cytoplasmic region (l). Compounds were added as
indicated by arrows at concentrations and sequence as follows: 1 µM
thapsigargin, 16 µM IP3, 30 µM cADPR, 10 µM thapsigargin, and 10
µM ionomycin. Data are from a single experiment representative of
response observed in 6 similar experiments in which 6 cultures were
used, and 31 of 68 cells showed a juxtanuclear thapsigargininsensitive Ca21 store that was sensitive to only ionomycin.
IP3- and cADPR-sensitive Ca21 stores in lens cells
appeared to be functionally distinct in terms of the
relative overlap of the these two Ca21 pools. In some
experiments the IP3- and cADPR-sensitive Ca21 pools
completely overlapped, whereas in other experiments
cADPR released only a portion of the IP3-releasable
Ca21 pool. Similarly, the portion of the thapsigarginsensitive Ca21 pool released by IP3 was variable. Taken
together, these results indicate heterogeneity in the
Ca21 stores in lens cells. This heterogeneity was not
manifested as spatially distinct Ca21 stores sensitive to
IP3 or cADPR, however, indicating that the Ca21 stores
might be organized into distinct compartments on a
scale that is below the spatial resolution of conventional microscopy or organized along the axis through
which the cells are viewed. Inasmuch as conventional
microscopy was used in this study, only the average
Ca21 in all stores through the thickness of the cell was
detected.
Regardless of the intracellular organization of the
Ca21 stores, our data indicate the presence of at least
three types of functionally distinct Ca21 stores in lens
cells. These Ca21 pools may represent physically distinct Ca21 stores with different complements of Ca21
channels and pumps. In one type of Ca21 store there
would be channels activated by IP3 and cADPR as well
as a thapsigargin-sensitive Ca21 pump. In the second
type of Ca21 store there would be Ca21 channels activated by IP3 but not cADPR as well as a thapsigarginsensitive Ca21 pump. In the third type of Ca21 store
there would be no Ca21 channels activated by either IP3
or cADPR and no thapsigargin-sensitive Ca21 pump.
The relative abundance of each of these Ca21 stores in
any intracellular region would determine the degree of
functional overlap among the various Ca21 pools in that
region.
This third type of Ca21 store that was insensitive to
IP3, cADPR, and thapsigargin was functionally and
spatially distinct from the Ca21 stores sensitive to these
IMAGING CA21 STORES IN LENS CELLS
We thank Tim Walseth (University of Minnesota) for generously
providing cyclic ADP-ribose and 8-amino-cyclic ADP-ribose and for
helpful discussions.
G. C. Churchill was partially supported by a doctoral dissertation
fellowship awarded by the Graduate School, University of Minnesota.
This research was supported by National Eye Institute Grant
EY-05684.
Address for reprint requests: C. F. Louis, Dept. of Veterinary
PathoBiology, University of Minnesota, 1988 Fitch Ave., Rm. 295, St.
Paul, MN 55108.
Received 15 April 1998; accepted in final form 12 October 1998.
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