Bioscience Reports, Vol. 7, No. 4, 1987
Intracellular Ca 2 § in Pancreatic Acinar Cells"
Regulation and Role in Stimulation of
Enzyme Secretion
Robert L. Dormer, Graham R. Brown, Claire Doughney and
Margaret A. McPherson
Received April 9, 1987
KEY WORDS: Ca2+; pancreas; secretion; exocytosis.
Abbreviations Used
EGTA: (ethylene dioxy) diethylene-dinitrilotetraacetic acid. BAPTA: 1,2-bis (2-aminophenoxy) ethane
NNN',N'-tetraacetic acid. InsP3: inositol trisphosphate; Ins-l,4,5P 3 and Ins-l,3,4P3, isomers of inositol
trisphosphate with the position of phosphate groups assigned. Ins-l,3,4,5P4: inositol tetrakisphosphate.
Evidence for a primary role for intracellular Ca 2 + in the stimulation of pancreatic
enzyme secretion is reviewed. Measurements of cytoplasmic free Ca 2 + concentration
have allowed direct demonstration of its importance in triggering enzyme secretion
and defined the concentration range over which membrane Ca 2 § pumps must work to
regulate intracellular Ca 2 +. Current evidence suggests a key role for the Ca z § MgATPase of rough endoplasmic reticulum in regulating intracellular Ca 2+ and
accumulating a Ca 2+ store which is released by the action of inositol-l,4,5
trisphosphate following stimulation of secretion.
INTRODUCTION
TJhe pancreatic acinar cell was the first cell type in which the kinetics and
compartments involved in the synthesis and secretion of proteins was established (1).
T])e cell is highly polarized in that secretion of digestive enzymes by exocytosis of
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff
CF4 4XN, UK.
333
0144-8463/87/0400-0333505,00/0 9 1987 Plenum Publishing Corporation
334
Dormer, Brown, Doughney and McPherson
preformed storage granules occurs across the apical membrane which is delineated
from the basolateral membrane by tight junctions (2). Receptors for the primary
stimulators of enzyme secretion, acetylcholine and cholecystokinin (CCK), have been
demonstrated by ligand binding (3) and, in the case of CCK, specifically localized to
the basolateral membrane (4). The acinar cells of some species (cat, rat and guinea-pig)
also possess receptors for vasoactive intestinal peptide and secretin (3) coupled to
adenylate cyclase, occupancy of which also results in stimulation of enzyme secretion
(5).
EARLY E V I D E N C E FOR Ca 2 + AS A R E G U L A T O R OF E N Z Y M E
SECRETION
Many earlier studies of post-receptor events in the stimulation of enzyme
secretion followed the experimental design pioneered by Douglas (6) and used his
criteria to establish a role for Ca 2 +. The main criteria tested were (a) that removal of
extracellular Ca 2 + should inhibit stimulation of secretion and (b) that stimulators of
secretion should increase Ca 2 + uptake into the cell. It was soon evident, however, that
the data did not fit the simple model proposed (6): that stimulators increased the Ca / +
permeability of the cell membrane causing uptake of Ca z + which triggered secretion
(7-9). Further studies of the effects of extracellular Ca 2 + removal on stimulation of
secretion (10-13) led to the present consensus that enzyme release can be stimulated
normally for 10-15 min in the absence of extracellular Ca 2 +, but can only be sustained
in its presence. Studies of 45Ca2 + fluxes have been difficult to interpret particularly as
results have been accumulated using various types of in vitro preparation (whole
pancreas (7), isolated fragments (8-10, 12), dissociated single cells (14-16) or isolated
acini (13, 17)). However, there was general agreement that 45Ca2+ efflux was
stimulated by cholinergic and CCK-agonists in a manner not markedly dependent on
extracellular Ca 2 + (7, 9, 14). This supported the hypothesis (7) that Ca 2 + was released
from an intracellular site, and explained the initial independence of stimulation of
secretion on extracellular Ca / +. Increased influx of 45Ca2 + was also reported (15-17)
and where net Ca 2 + flux was measured (16-18) it was clearly shown that an initial net
efflux was followed by net influx, consistent with the requirement for extracellular
Ca z + for sustained stimulation of secretion.
Using the divalent cation ionophore A23187, it was possible to add a third
criterion, that artificial introduction of C a 2 + into the cell stimulated enzyme secretion
(19, 20). Iwatsuki and Petersen (21) also showed that iontophoresis of Ca / + into the
acinar cell caused similar changes of membrane depolarization and decreased input
resistance to those evoked by acetylcholine.
THE I M P O R T A N C E OF C Y T O P L A S M I C FREE
Ca 2 +
By the late 1970s it was, therefore, possible to assert with some confidence that
Ca 2 + was an important intracellular messenger for the stimulation of enzyme secretion
by acetylcholine and CCK, and that it was initially released from an intracellular site,
the nature of which was undetermined. It had long been recognised that the
concentration of free Ca 2 § in the cytoplasm of cells was less than micromolar (see
22, 23) and that this was maintained by Ca 2 § pumps in various cellular membranes
Pancreatic Acinar Cell Ca2* Regulation
335
(:22, 24). Cytoplasmic free Ca 2 § concentration can therefore, only be measured in
intact cells using indicators that detect Ca 2 + in the range 0.1-10/~M (23). Pioneering
work using Ca 2 §
photoproteins (25, 26) and the dye, arsenazo III (27) in
giant cells of invertebrates had established the feasibility of making such
measurements, as well as adding considerably to our knowledge of intracellular Ca 2 +
regulation.
The importance of measuring the cytoplasmic free Ca 2 + concentration in the
pancreatic acinar cell therefore, was (a) that changes could be directly correlated with
secretory responses and (b) the range of free Ca 2 + over which membrane pumps would
have to operate in order to regulate intracellular Ca z § would be defined. In addition, it
could be determined whether other putative messengers act by altering intracellular
Ca 2 § and proposed mechanisms by which Ca z § stimulates exocytosis would also have
to operate within the defined range of free Ca 2 + concentrations.
MEASUREMENTS
OF CYTOPLASMIC
FREE
Ca 2 §
F o u r groups of indicators of free Ca 2 + are presently available which are suitable
for measurement of cytoplasmic Ca 2+ concentration (see 23). A Ca 2+ selective
microelectrode was first used in mouse pancreatic fragments (28) and a resting
concentration of 0.43 # M reported. 1 0 - T M acetylcholine, which caused maximal
stimulation of amylase release, increased the free Ca 2§ concentration to approx.
0.6#M, whereas a further increase of 0.96pM, caused by 10 .5 acetylcholine,
provoked no stimulation of amylase release. This lack of stimulation by high
acetylcholine concentration was not observed by others in this type of preparation (8).
Ca a § -selective microelectrodes have the disadvantage that they respond too slowly to
changes in free Ca 2 § (1-3 s) to be suitable to determine the kinetics of stimulusinduced changes. By contrast, Ca 2 +-activated photoproteins respond rapidly (rate
constant for onset of light emission 100-280s -1) but are technically difficult to
incorporate into intact small cells (23). A method of swelling in hypotonic media was
developed for isolated rat pancreatic acini (29, 30) such that the photoprotein aequorin
and other impermeant molecules could be incorporated into cells which retained
normal secretory responsiveness. It was demonstrated that an increased free Ca 2+
concentration preceded the stimulation of amylase release by carbachol, measured
simultaneously in perfused cells (29) although the calculated free Ca 2 § concentrations
(1-2 p M resting, rising to approx. 4/~M) were higher than reported by other methods
(28, Table 1). The development of the fluorescent indicator quin 2 (31), which could be
introduced into cells as the permeant ester, opened the way for measurements of
cytoplasmic free Ca 2 § by a number of groups. As can be seen in Table 1, the resting free
Ca 2+ concentration measured using quin 2, ranged from 0.09-0.18 #M. A transient
rise in free Ca 2 + in response to cholinergic or CCK-agonists was shown (29, 32-37)
with maximally-stimulated increases ranging from 3-fold, to a concentration of
0.!35 g M (33), to 11 to 12-fold, to a concentration of 1.3 # M (34). Ochs et al. (35)
showed that the lag before an increase in free Ca z + concentration could be detected
and the time taken to reach the peak of the transient, decreased with increasing
carbachol concentration up to the maximum for stimulation of amylase release. Thus,
336
Table 1.
Dormer, Brown, Doughney and McPherson
Measurement of cytoplasmic free
Ca 2 +
concentration in isolated pancreatic acini using quin 2
Estimated Ca 2 + concentration
Unstimulated *Peak **Sustained
t Agonist dose-dependence
for Ca 2+ v. amylase
release
Species
Stimulus
Mouse
Mouse
Mouse
Mouse
Rat
Rat
Rat
Rat
Guineapig
Guinea pig
Carbachol
Carbachol
Carbachol
CCK
Carbachol
Carbachol
Caerulein
Caerulein
0.18
0.09
0.10
0.10
0.14
0A2
0,14
0.12
0.86
0.54
1.30
1.25
0.80
0.35
0.85
0.44
-0.25
0.31
0.31
0.25
0.12
0.25
0.12
Carbachol
0.10
0.80
0.10
--
37
CCK
0.10
0.80
0.10
--
37
-Coincident to maximum
Coincident to maximum
Shift to right (10-fold)
Coincident to maximum
Shift to right (10-fold)
Shift to right (10-fold)
Shift to right (100-fold)
Reference
32
35
34
34
36
33
36
33
concentration at peak of Ca 2+ transient using agonist concentration causing max stimulation of
amylase release.
** Steady Ca 2+ concentration obtained at least 3 min after peak of Ca 2+ transient.
? Shifts indicate the ratio of lowest agonist concentration at which increases in free Ca 2 + concentration
and amylase release were observed.
* Ca 2+
at a maximal concentration of 10 . 6 M there was a lag of approx. I s before the Ca 2 +
concentration began to rise to a peak at approx. 5 s. There is disagreement as to
whether the free Ca z + concentration returns to the resting level during sustained
stimulation (up to 15 rain) and whether the dose-dependence for stimulation of
amylase release and free Ca a+ concentration coincide (see Table 1).
It is now possible, however, to set an approx, range of free cytoplasmic Ca 2 §
concentrations from a resting value of 0.1-0.4#M to a transiently stimulated
maximum value of 0.4-1.3 #M. The observed variation in values of free Ca z+
concentration measured during stimulation may be because quin 2 has to be
accumulated in the cytoplasm at between 0.5-1 m M (31, 32, 35) to obtain reliable
fluorescent signals and this large increase in buffering capacity may attenuate the Ca 2 +
transients (see 23). In addition, quin 2 is relatively insensitive to Ca 2 + concentrations
above 0.5/~M (31). These discrepancies may be resolved by use of the more recently
developed indicator fura 2 (38) which can be used at up to 10-fold lower concentrations
in the cytoplasm (see 33).
Nevertheless, the ability to measure and manipulate free Ca 2 + in intact cells has
allowed further direct criteria to be established to demonstrate Ca z + as a messenger for
stimulation of enzyme secretion. Firstly, buffering the rise in free Ca 2 + with chelators
should block stimulation: using the hypotonic swelling method (30), E G T A or
BAPTA incorporated into isolated acini to 1-2 mM, gave an 80 ~o inhibition of the
maximum stimulation of amylase release by carbachol. Similar concentrations of quin
2+BAPTA
(incorporated as acetomethoxyesters) also inhibited carbachol
stimulation of amylase release and free Ca 2+ in isolated acini (35). Laugier and
Petersen (39) had previously shown that injection of E G T A into acinar cells reduced
acetylcholine-induced membrane depolarization and resistance reduction although
the amount of E G T A injected and its effect on secretion were not determined.
Pancreatic Acinar Cell Caz+ Regulation
337
Secondly, it was now possible to show directly that treatment of cells with divalent
cation ionophores caused graded increases in free Ca 2+ concentration which
correlated with increases in amylase release (29, 35, 36). An important finding was that
for a similar rise in free Ca 2 + concentration, the degree of stimulation of amylase
release by ionophores was less than that caused by physiological agonists. One
explanation for this is that ionophores release Ca 2 + from inappropriate stores and this
alters the subsequent secretory response. In addition, it has been shown (36) that
ionophores do not mimic the physiological Ca 2 + transient but cause a prolonged rise.
The second possibility, which will be discussed in a later section, is that other
intracellular messengers such as inositol phosphates and diacylglycerol which are
generated by the physiological stimulator may be important in stimulating secretion.
REGULATION OF INTRACELLULAR
Ca 2 +
BY M E M B R A N E
Ca 2 +
PUMPS
The Unstimulated Cell
Pancreatic mitochondria (40, 41) and rough endoplasmic reticulum membranes
(42, 43) actively sequester Ca 2 § and have been suggested to contribute to regulation of
the cytoplasmic Ca 2 § concentration. Since the total acinar cell Ca / + content remains
constant (17), energy-dependent extrusion must also occur across the cell membrane.
Two approaches have been used to assess the relative importance of the membrane
pumps: one is to purify the organelles and study their Ca z §
properties
and the other, to permeabilise the plasma membrane and study the ability of the
remaining cell components to regulate free Ca 2 § in the medium. Using the latter
approach, Streb and Schulz (44) showed that isolated cells made permeable to
molecules at least as large as lactate dehydrogenase by incubating in Ca2§
medium, could buffer free Ca 2 § to 0.42 #M. This is in agreement with the value for
cytoplasmic free Ca / § concentration measured by Ca 2 §
microelectrode (28)
though higher than those measured with quin 2 (Table 1). Addition of inhibitors of
mitochondrial Ca 2 § uptake only affected the rate of attainment of this free Ca a §
concentration suggesting that the primary intracellular regulator of the resting free
Ca 2 § is non-mitochondrial. No investigation of the Ca / §
of Ca 2 § uptake
by isolated mitochondria has been reported. However, Ca 2§ transport has been
studied in more detail in purified rough endoplasmic reticulum and plasma membranes
from rat pancreatic acinar cells. Table 2 summarizes the reported Ca 2 § sensitivities of
these membranes. There is agreement that Ca 2§ transport by rough endoplasmic
reticulum (42, 43) is more sensitive to free Ca 2 § than transport by plasma membrane
vesicles whether driven etectrogenically by a Ca 2+, Mg-ATPase (45) or by Na/Ca
counter transport (46). The Ca / §
activity of the acinar cell plasma membrane
has been difficult to demonstrate in the presence of milimolar Mg 2+ concentrations
(47, 48), unlike the erythrocyte membrane Ca 2 +, Mg-ATPase (49). It is not clear
whether this is due to the presence of other ATP phosphohydrolases, such as the ectoATPase described by Hamlyn and Senior (50), which mask the Ca2+-activated
ATPase or to a genuine difference in properties from the erythrocyte Ca 2 +, MgATPase. However, the pancreatic plasma membrane Ca2+-ATPase activity is
stimulated by calmodulin (47; A1-Mutairy, A. R. and Dormer, R. L., unpublished data)
Dormer, Brown, Doughney and McPherson
338
Table 2. Sensitivity to free Caz+ concentration of Caz +-translocating activities in purified membranes of
rat pancreatic acinar cells
Sensitivity to free Caz+
(#M)
Membrane
Rough endoplasmic
reticulum
Plasma membrane
Activity
Ca2+ uptake
Caz+ uptake
Caz+ , Mg-ATPase
Ca2+, Mg-ATPase
Ca2+ uptake
Ca/ +, Mg-ATPase
Ca2+, Mg-ATPase
Na/Ca
Countertransport
89max
Max
Reference
0.16
0.5
0.17
0.3
0.88
1.73
0.65
0.7
2
0.7
0.8
10"
88**
17
42
43
54
43
45
47
48
0.62
10
46
* Measured in the presence of 0.3 mM Mgz+.
** Measured in the presence of 1-2 #M Mg2+.
t Measured in the presence of 0.2 mM Mg2§
which increases its sensitivity to Ca 2 +, although at this stage the complex curves
obtained are difficult to interpret with regard to intracellular Ca 2 + regulation.
Thus, in the unstimulated acinar cell where the average cytoplasmic free Ca 2 +
concentration can be taken as 0 . 2 # M , the rate of Ca 2+ removal by the rough
endoplasmic reticulum would be approx. 10 nmol/min per mg protein (42) whereas
that by the plasma membrane is of the order of 0.1 for electrogenic Ca 2 + pumping (45)
and approx. 0.02 for N a / C a countertransport (46). If the various pumps are evenly
distributed in the total membrane areas (an assumption which has not been examined
directly) the contribution by the plasma membrane may be even less owing to the
approx. 12-fold greater area of the rough endoplasmic reticulum than the plasma
membrane (51).
The Stimulated Cell
As discussed above, earlier evidence had indicated that the initial stimulation of
enzyme release by acetylcholine or C C K resulted in the release of Ca 2 + from an
intracellular store. The initial rise in cytoplasmic free Ca 2+ would, therefore, be
predicted to be independent of extraeellular Ca z +. However, measurements using quin
2 have been contradictory in that the peak of the stimulated Ca 2 + transient was not
markedly affected in the absence of extracellular Ca z + in two studies (35, 37), reduced
by 50% in another (34) and completely abolished in the microelectrode study (28).
The nature of the Ca z + store released upon stimulation of secretion has been
investigated directly by determining changes in 45Ca 2 + and total content of subcellular
fractions isolated from control and stimulated cells (42, 52-54). This led to early
proposals that mitochondria (52) or a component of a microsomal fraction (53) would
be the site of Ca 2 + release. In addition, evidence was also presented for an initial
release of Ca z+ from the plasma membrane in that: (a) a rapid effect of E G T A
( < 1 min) mimicked acetylcholine-induced membrane depolarization and decreased
Pancreatic Acinar Cell Ca2+ Regulation
339
resistance (10). (b) La 3+ blocked 45Ca2+ efflux from isolated cells stimulated by
carbachol and when fixed cells were analysed by electron microscopy, La 3 § was only
observed on the surface membrane (55). (c) Carbachol caused Ca 2+ release from
saponin-permeabilized cells in which a chemical messenger would be expected to
diffuse away too rapidly except to act at the plasma membrane (56).
More recently, loss of total Ca 2 + from rough endoplasmic reticulum membranes
purified from carbachol stimulated acini has been demonstrated (54). This Ca 2 + was
not regained during sustained stimulation unless receptor occupancy was blocked by
atropine. This is consistent with analysis of 45Ca2 § fluxes in which it had been
suggested that the "trigger pool" was not refilled during sustained stimulation by
carbachol (18).
It has also been shown that when rough endoplasmic reticulum membranes were
isolated from stimulated acini, the rate of active 4SCaZ § uptake (42) and Ca 2 +, MgATPase activity (54) was increased relative to controls. This activity returned to
control levels when carbachol stimulation was blocked with atropine (54). The effect
could be mimicked in isolated membranes by treatment with A23187 which released
actively accumulated Ca 2+ (42) and stimulated ATPase activity (54). The results
suggested that Ca 2+ accumulated in the endoplasmic reticulum lumen has an
inhibitory effect on ATPase activity.
The hypothesis that the rough endoplasmic reticulum is the site of intracellular
Ca 2+ release has been strengthened by the discovery of Ins-l,4,5P 3 as a putative
messenger linking receptor occupancy to Ca 2 § release (see 57, 58). Streb e t al. (59) first
showed in pancreatic acinar cells, that Ins-l,4,5P 3 released Ca 2+ from a nonmitochondrial store and this compound was later shown to release Ca 2 + directly from
isolated rough endoplasmic reticulum membranes (60, 61). In addition, the amount of
Ca 2 + released from isolated membranes was shown to be similar to that released from
membranes isolated from carbachol-stimulated acini (54, 61).
INOSITOL PHOSPHOLIPID BREAKDOWN
The original demonstration of increased phospholipid turnover in response to
acetylcholine was made by Hokin and Hokin in 1953 (62). The hypothesis that
stimulation of phosphoinositide metabolism was linked to changes in intracellutar
C a z + (63) led to the recognition that stimulation of a variety of cells by effectors which
mobilize cellular Ca 2 § leads to the breakdown of polyphosphoinositides to release
water-soluble inositol phosphates and the lipid moiety diacylglycerol (57, 58, 64). In
order to establish inositol phosphates as intracellular messengers, it will be necessary
to fulfil the same criteria as have been discussed above for Ca 2+. At present, no
methods have been reported which allow direct measurements of inositol phosphate
concentrations in intact cells; current evidence depends upon measuring changes in
radioactivity in phosphoinositides or inositoI phosphates in cells prelabeled with
radiolabeled myo-inositol. Thus, it has been shown in acinar cells that breakdown of
phosphatidylinositol-4,5-bisphosphate occurred within 30s of stimulation by
carbachol or caerulein (65-67) and was independent of stimulation of Ca 2+
mobilization (65) or a rise in cytoplasmic free Ca 2 § (67). Earlier evidence also showed
340
Dormer, Brown,Doughneyand McPherson
that InsP3 was increased in response to these secretagogues (68, 69). However, reports
of inositol polyphosphate formation in cholinergically-stimulated parotid (70) and
cerebral cortical (71) slices demonstrated formation of another InsP3 isomer Clns1,3,4Pa) and of Ins-1,3,4,5P 4 which were not separated from Ins-l,4,5Pa in earlier
studies on pancreatic acinar cells (68, 69). These inositol polyphosphates have recently
been identified in isolated acini stimulated with carbachol or caerulein (61, 72) by
separation on high-performance anion-exchange columns. Ins-l,4,5Pa and Ins1,3,4,5P4 were shown to be increased within 5 s of stimulation by carbachol and
maintained at this level for up to 15 rain. By contrast, as was shown in salivary acinar
cells (70, 73), Ins-l,3,4P 3 was not significantly increased until 10-15 s following
stimulation, but accumulated markedly over 10-15 min at which time it was the
predominantly labellad inositol polyphosphate. Using isolated acinar cells, Merritt et
al. (74) found only a transient increase in Ins-l,4,5P3 in response to caerulein with no
effect of carbachol. The reason for this discrepancy is not clear, except that isolated
acinar cells have markedly lower secretory responses than isolated acini (75, 76).
In all reports of stimulated inositol phospholipid turnover, measured as 32p
incorporation into phospholipids (77), breakdown of radiolabelled phosphoinositides
(66) or inositol polyphosphate formation (61, 69) the dose-response was shifted to the
right compared to that for stimulation of enzyme release. This has been observed in
other tissues and explained on the basis of a reserve of receptors for the physiological
response but not for stimulation of phosphoinositide breakdown (78). Thus, it would
have to be argued that at the lower carbachol concentrations, the amount of InsPa
produced is too small to be detected by the present method and that the primary signal
is amplified through one or more subsequent activation steps.
Three pieces of evidence support this argument: firstly, stimulation of amylase
release by a concentration of carbachol which caused no detectable increase in InsPa
formation was not affected by the removal of extracellular Ca 2 § (61). This suggests
that intracellular Ca 2 + release is used to generate the increased cytoplasmic free Ca 2 §
concentration at these low levels of stimulation.
Secondly, on the basis of the amount of actively accumulated Ca 2 § released from
isolated rough endoplasmic reticulum membranes, it was calculated (61) that 1 amol.
of Ins-l,4,5Pa has the capacity to release 420 amol Ca 2§ per cell. This would be an
overestimate if the site of Ins-l,4,5Pa-induced Ca 2§ mobilization is restricted to a
small area of rough endoplasmic reticulum but, nevertheless, provides direct evidence
for amplification of calcium release in response to Ins-l,4,5P 3.
Thirdly, previous data on 45Ca2§ fluxes in whole acini (17) showed that
stimulation of 45Ca2 § efflux, which probably represents Ca 2 § mobilization from the
internal store, was also less sensitive to the concentration of cholinergic agonist than
was stimulation of amylase release. This suggests a link between Ins-l,4,5P a formation
and intraceUular Ca 2§ mobilisation and a further amplification step from Ca 2§
mobilisation to the increased cytoplasmic free Ca 2§ concentration. It was also shown
(17) that 45Ca2 § uptake by isolated acini, which can be differentiated kinetically from
45Ca2+ efflux, is stimulated by cholinergic agonists with the same concentration
dependence as amylase release. This indicates that Ca 2 § influx into the cell contributes
to the rise in cytoplasmic free Ca 2§ concentration and is secondary to inositol
phosphate formation and calcium mobilization. This hypothesis would be
Pancreatic AcinarCell Ca2+ Regulation
341
compatible with either of two recent suggestions that stimulation of Ca z + entry is
secondary to emptying of the intracellular store (79) or is stimulated by Ins-l,3,4,5P4
(80).
The other product of phospholipase C action on phospholipids is diacylglycerol,
which is known to activate the Ca 2 +- and phospholipid-dependent protein kinase
(protein kinase C) (64). This kinase has been demonstrated in pancreatic acinar cells
and suggested to be important in phosphorylating proteins involved in regulating
exocytosis (81, 82). Increased diacylglycerol formation has been demonstrated in
response to acetylcholine (83, 84), but it is difficult to interpret these results with
re.spect to the stimulation of secretion since it was concluded (83) that not all of the
diacylglycerolformed could be derived from phosphoinositides and that stimulation of
its formation only occurred at high concentrations of secretagogue (83, 84).
However, exogenous diacylglycerol as well as phorbol esters, which are activators
of' protein kinase C (64), stimulate enzyme release (36, 37, 85, 86) without increasing
cytoplasmic free Ca 2+ (36, 37). In addition, these agents potentiate stimulation of
enzyme release elicited by Ca 2 + ionophores (36, 86). This might explain why the latter
cause less amylase release for a given rise in cytoplasmic free Ca 2 + than physiological
stimulators as discussed above. However, a recent paper showed that an inhibitor of
protein kinase C (H-7) augmented carbachol-stimulated amylase release (84) and
failed to inhibit stimulation by phorbol esters. These results suggest that extrapolation
from effects of phorbol esters to the involvement of protein kinase C in the stimulation
of secretion should be treated with caution and that protein kinase C may have
inhibitory effects on exocytosis.
THE Ca 2 + SENSITIVITY OF EXOCYTOSIS
Baker and Knight (87), introduced an important new technique for studying
regulation of exocytosis. By permabilizing cells with an intense electric field, direct
access of extracellular compounds to the site of exocytosis was obtained. This was
applied to pancreatic acinar cells by Knight and Koh (88) who showed that increasing
free Ca 2 + in the range 1-10 #M stimulated exocytosis of amylase but not leakage of
lactate dehydrogenase. However, the sensitivity to Ca 2+ was lower than predicted
from the range of cytoplasmic free Ca / + established by measurements in intact cells.
Interestingly, this was the range measured using aequorin, incorporated into isolated
acini by hypotonic swelling (29), raising the possibility that permeabilization methods
cause loss of factors necessary for physiological regulation of intracellular Ca 2 + and
the stimulation of secretion. A second possibility, suggested by Knight and Koh, was
that protein kinase C action increases the sensitivity of exocytosis to Ca 2 + since a
phorbol ester caused activation at lower concentrations of Ca z + (88).
SUMMARY
Ca 2 + is the sole regulator whose importance in the stimulation of enzyme release
has been established by direct measurement and manipulation of its concentration in
342
Dormer, Brown, Doughney and McPherson
intact cells. In the unstimulated ceil, evidence suggests that the rough endoplasmic
reticulum possesses a Ca 2 +, Mg-ATPase of sufficient sensitivity to accumulate Ca 2 §
which can be released upon stimulation of secretion. Negative feedback by
intraluminal Ca a + may prevent over-accumulation of Ca 2+ in the endoplasmic
reticulum by inhibiting the ATPase when the store is filled. Under these conditions
Ca z + extrusion across the basolateral membrane by a Ca a §
or N a + / C a 1+
counter-transport may become important.
U p o n stimulation by acetylcholine or C C K , phosphatidylinositol-4,5
bisphosphate is rapidly cleaved to form Ins-l,4,5P 3 which releases Ca 2+ from the
rough endoplasmic reticulum thus contributing to the rise in cytoplasmic free Ca z +.
Ca 2 + influx is also stimulated, possibly by the action of Ins-1,3,4,5P, and this might
also contribute to the initial rise in cytoplasmic free Ca 2 +. Increases in free Ca z § would
be expected to increase the concentration of Ca4-calmodulin in the cell; this would
activate the plasma membrane Ca 2 § -ATPase causing increased Ca 2-- efflux, as well as
Ca 2 +/calmodulin-kinase which m a y be involved in the stimulation of exocytosis (89).
Sustained stimulation of secretion (10min) is dependent on increased Ca 2+
influx, yet evidence suggests that the endoplasmic reticulum Ca / + store is not refilled.
This m a y be due to sustained action of Ins-l,4,5P 3 effectively short-circuiting the
endoplasmic reticulum membrane. However, this would be energetically wasteful in
terms of ATPase activity and the need to resynthesise phosphatidylinositol-4,5
bisphosphate by ATP-dependent phosphorylation. It has been suggested in other cell
types (24) that Ca 2 + entering the cell is accumulated by mitochondria. However, an
earlier subfractionation study (53) showed that during carbachol stimulation of
45Ca 2 + uptake by isolated acini, there was a marked increase in the 45Ca2 + content of
secretory granules, without a change in total Ca 2 + content. This suggests a flux of
Ca 2 § through this pool and provides an alternative explanation that Ca z + taken up by
the endoplasmic reticulum is removed via the secretory pathway.
ACKNOWLED GEMENTS
We are grateful to the Medical Research Council, the Welsh Scheme for the
Development of Health and Social Research and the Cystic Fibrosis Trust, U K for
financial support.
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