Receptor-activated cytoplasmic Ca2+ oscillations in pancreatic acinar cells: generation and spreading of Ca2+ signals.

Receptor-activated cytoplasmic Ca2+ oscillations have been investigated using both single cell microfluorometry and voltage-clamp recording of Ca(2+)-dependent Cl- current in single internally perfused acinar cells. In these cells there is direct experimental evidence showing that the ACh-evoked [Ca2+]i fluctuations are due to an inositol trisphosphate-induced small steady Ca2+ release which in turn evokes repetitive Ca2+ spikes via a caffeine-sensitive Ca(2+)-induced Ca2+ release process. There is indirect evidence suggesting that receptor-activation in addition to generating the Ca2+ releasing messenger, inositol trisphosphate, also produces another regulator involved in the control of Ca2+ signal spreading. Intracellular inositol trisphosphate or Ca2+ infusion produce short duration repetitive spikes confined to the cytoplasmic area close to the plasma membrane, but these signals can be made to progress throughout the cell by addition of caffeine or by receptor activation.     

Different patterns of receptor-activated cytoplasmic Ca2+ oscillations in single pancreatic acinar cells: dependence on receptor type, agonist concentration and intracellular Ca2+ buffering.

Agonist-specific cytosolic Ca2+ oscillation patterns can be observed in individual cells and these have been explained by the co-existence of separate oscillatory mechanisms. In pancreatic acinar cells activation of muscarinic receptors typically evokes sinusoidal oscillations whereas stimulation of cholecystokinin (CCK) receptors evokes transient oscillations consisting of Ca2+ waves with long intervals between them. We have monitored changes in the cytosolic Ca2+ concentration ([Ca2+]i) by measuring Ca2(+)-activated Cl- currents in single internally perfused mouse pancreatic acinar cells. With minimal intracellular Ca2+ buffering we found that low concentrations of both ACh (50 nM) and CCK (10 pM) evoked repetitive short-lasting Ca2+ spikes of the same duration and frequency, but the probability of a spike being followed by a longer and larger Ca2+ wave was low for ACh and high for CCK. The probability that the receptor-evoked shortlasting Ca2+ spikes would initiate more substantial Ca2+ waves was dramatically increased by intracellular perfusion with solutions containing high concentrations of the mobile low affinity Ca2+ buffers citrate (10-40 mM) or ATP (10-20 mM). The different Ca2+ oscillation patterns normally induced by ACh and CCK would therefore appear not to be caused by separate mechanisms. We propose that specific receptor-controlled modulation of Ca2+ signal spreading, either by regulation of Ca2+ uptake into organelles and/or cellular Ca2+ extrusion, or by changing the sensitivity of the Ca2(+)-induced Ca2+ release mechanism, can be mimicked experimentally by different degrees of cytosolic Ca2+ buffering and can account for the various cytosolic Ca2+ spike patterns.     

Acetylcholine-evoked increase in the cytoplasmic Ca2+ concentration and Ca2+ extrusion measured simultaneously in single mouse pancreatic acinar cells.

The intracellular free Ca2+ concentration ([free Ca2+]i) was measured simultaneously with the Ca2+ extrusion from single isolated mouse pancreatic acinar cells placed in a microdroplet of extracellular solution using the fluorescent probes fura-2 and fluo-3. The extracellular solution had a low total calcium concentration (15-35 microM), and acetylcholine (ACh), applied by microionophoresis, therefore only evoked a transient elevation of [free Ca2+]i lasting about 2-5 min. The initial sharp rise in [free Ca2+]i from about 100 nM toward 0.5-1 microM was followed within seconds by an increase in the total calcium concentration in the microdroplet solution ([Ca]o). The rate of this rise of [Ca]o was dependent on the [free Ca2+]i elevation, and as [free Ca2+]i gradually decreased Ca2+ extrusion declined with the same time course. Ca2+ extrusion following ACh stimulation was not influenced by removal of all Na+ in the microdroplet solution indicating that the Ca2+ extrusion is not mediated by Na(+)-Ca2+ exchange but by the Ca2+ pump. The amount of Ca2+ extruded during the ACh-evoked transient rise in [free Ca2+]i corresponded to a decrease in the total intracellular Ca concentration of about 0.7 mM which is close to previously reported values (0.5-1 mM) for the total concentration of mobilizable calcium in these cells. Our results therefore demonstrate directly the ability of the Ca2+ pump to rapidly remove the large amount of Ca2+ released from the intracellular pools during receptor activation.     

Relationship between cytosolic Ca2+ concentration and amylase release in rat parotid acinar cells following muscarinic stimulation.

Carbachol (CCh), a muscarinic-cholinergic agonist, increased both cytosolic free calcium concentration ([Ca2+]i) and amylase release in rat parotid acinar cells or acini in a dose-dependent manner. Treatment of acinar cells with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), or the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA), strongly attenuated the increases in [Ca2+]i evoked by CCh, but amylase release from acini was not significantly suppressed by the treatment with TMB-8 or BAPTA. Low concentrations (0.02-0.5 microM) of ionomycin, a Ca2+ ionophore, caused increases in [Ca2+]i comparable to those induced by CCh, but the same concentrations had only a little effect on amylase release. The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated amylase release in quantities similar to those induced by CCh, although TPA alone did not cause any change in [Ca2+]i. Combined addition of TPA and ionomycin potentiated only modestly amylase release stimulated by TPA alone. Staurosporine, a protein kinase C-inhibitor, similarly inhibited both the CCh- and TPA-induced amylase release. These results suggest that an increase in [Ca2+]i elicited by CCh does not play an essential role for inducing amylase release in rat parotid acini. Amylase release by muscarinic stimulation may be mediated mainly by activation of protein kinase C.     

Apical Ca2+-activated potassium channels in mouse parotid acinar cells

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.     

Molecular identification of Ca2+-activated K+ channels in parotid acinar cells

We used molecular biological and patch-clamp techniques to identify the Ca(2+)-activated K(+) channel genes in mouse parotid acinar cells. Two types of K(+) channels were activated by intracellular Ca(2+) with single-channel conductance values of 22 and 140 pS (in 135 mM external K(+)), consistent with the intermediate and maxi-K classes of Ca(2+)-activated K(+) channels, typified by the mIK1 (Kcnn4) and mSlo (Kcnma1) genes, respectively. The presence of mIK1 mRNA was established in acinar cells by in situ hybridization. The electrophysiological and pharmacological properties of heterologously expressed mIK1 channels matched those of the native current; thus the native, smaller conductance channel is likely derived from the mIK1 gene. We found that parotid acinar cells express a single, uncommon splice variant of the mSlo gene and that heterologously expressed channels of this Slo variant had a single-channel conductance indistinguishable from that of the native, large-conductance channel. However, the sensitivity of this expressed Slo variant to the scorpion toxin iberiotoxin was considerably different from that of the native current. RT-PCR analysis revealed the presence of two mSlo beta-subunits (Kcnmb1 and Kcnmb4) in parotid tissue. Comparison of the iberiotoxin sensitivity of the native current with that of parotid mSlo expressed with each beta-subunit in isolation and measurements of the iberiotoxin sensitivity of currents in cells from beta(1) knockout mice suggest that parotid acinar cells contain approximately equal numbers of homotetrameric channel proteins from the parotid variant of the Slo gene and heteromeric proteins composed of the parotid Slo variant in combination with the beta(4)-subunit.     

Ca2+-dependent protein kinase--a modulation of the plasma membrane Ca2+-ATPase in parotid acinar cells

Cross-talk between cAMP and [Ca(2+)](i) signaling pathways represents a general feature that defines the specificity of stimulus-response coupling in a variety of cell types including parotid acinar cells. We have reported recently that cAMP potentiates Ca(2+) release from intracellular stores, primarily because of a protein kinase A-mediated phosphorylation of type II inositol 1,4,5-trisphosphate receptors (Bruce, J. I. E., Shuttleworth, T. J. S., Giovannucci, D. R., and Yule, D. I. (2002) J. Biol. Chem. 277, 1340-1348). The aim of the present study was to evaluate the functional and molecular mechanism whereby cAMP regulates Ca(2+) clearance pathways in parotid acinar cells. Following an agonist-induced increase in [Ca(2+)](i) the rate of Ca(2+) clearance, after the removal of the stimulus, was potentiated substantially ( approximately 2-fold) by treatment with forskolin. This effect was prevented completely by inhibition of the plasma membrane Ca(2+)-ATPase (PMCA) with La(3+). PMCA activity, when isolated pharmacologically, was also potentiated ( approximately 2-fold) by forskolin. Ca(2+) uptake into the endoplasmic reticulum of streptolysin-O-permeabilized cells by sarco/endoplasmic reticulum Ca(2+)-ATPase was largely unaffected by treatment with dibutyryl cAMP. Finally, in situ phosphorylation assays demonstrated that PMCA was phosphorylated by treatment with forskolin but only in the presence of carbamylcholine (carbachol). This effect of forskolin was Ca(2+)-dependent, and protein kinase C-independent, as potentiation of PMCA activity and phosphorylation of PMCA by forskolin also occurred when [Ca(2+)](i) was elevated by the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid and was attenuated by pre-incubation with the Ca(2+) chelator, 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The present study demonstrates that elevated cAMP enhances the rate of Ca(2+) clearance because of a complex modulation of PMCA activity that involves a Ca(2+)-dependent step. Tight regulation of both Ca(2+) release and Ca(2+) efflux may represent a general feature of the mechanism whereby cAMP improves the fidelity and specificity of Ca(2+) signaling.     

Spontaneous [Ca2+]i fluctuations in rat chromaffin cells do not require inositol 1,4,5-trisphosphate elevations but are generated by a caffeine- and ryanodine-sensitive intracellular Ca2+ store

A considerable fraction (65%) of single rat chromaffin cells loaded with the fluorescent [Ca2+]i indicator fura-2 exhibited spontaneous rhythmic fluctuations with an average period of approximately 100 s. Parallel patch clamp experiments as well as fura-2 experiments carried out in Ca2(+)-free and other modified media in the presence of Ca2+ and Na+ channel blockers indicated an origin from intracellular stores. Appropriate concentrations of agonists (bradykinin and histamine) for receptors (B2 and H1) that trigger generation of inositol 1,4,5-trisphosphate induced increased fluctuation frequency, recruitment of silent cells, and large [Ca2+]i changes at high doses. These effects were blocked by cell pretreatment with neomycin, a drug that inhibits inositol 1,4,5-trisphosphate generation. In contrast, spontaneous fluctuations and the effects of another drug, caffeine, which also induced increased frequency and recruitment, were unaffected by neomycin. Ryanodine caused first a prolongation and then (approximately 10 min) a block of both spontaneous fluctuations and caffeine effects, where the single transients after bradykinin and histamine were maintained. Caffeine and ryanodine are known to affect selectively the process of calcium-induced Ca2+ release; this is the first demonstration of [Ca2+]i fluctuation activity arising from Ca2(+)-induced Ca2+ release in nonmuscle cells with no strict requirement for inositol 1,4,5-trisphosphate involvement.     

Glucose-induced oscillations in cytoplasmic free Ca2+ concentration precede oscillations in mitochondrial membrane potential in the pancreatic beta-cell.

Using dual excitation and fixed emission fluorescence microscopy, we were able to measure changes in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and mitochondrial membrane potential simultaneously in the pancreatic beta-cell. The beta-cells were exposed to a combination of the Ca(2+) indicator fura-2/AM and the indicator of mitochondrial membrane potential, rhodamine 123 (Rh123). Using simultaneous measurements of mitochondrial membrane potential and [Ca(2+)](i) during glucose stimulation, it was possible to measure the time lag between the onset of mitochondrial hyperpolarization and changes in [Ca(2+)](i). Glucose-induced oscillations in [Ca(2+)](i) were followed by transient depolarizations of mitochondrial membrane potential. These results are compatible with a model in which nadirs in [Ca(2+)](i) oscillations are generated by a transient, Ca(2+)-induced inhibition of mitochondrial metabolism resulting in a temporary fall in the cytoplasmic ATP/ADP ratio, opening of plasma membrane K(ATP) channels, repolarization of the plasma membrane, and thus transient closure of voltage-gated L-type Ca(2+) channels.     

Adrenergic-agonist-induced Ca2+ fluxes in rat parotid cells are not Na+-dependent.

We investigated the hypothesis that extracellular Na+ is required for the rapid mobilization of Ca2+ by rat parotid cells after adrenergic stimulation. When Na+ salts in the media were osmotically replaced with either choline chloride (+atropine) or sucrose, efflux of 45Ca2+ from preloaded cells, caused by 10 microM-(-)-adrenaline, was unchanged. Similarly adrenaline stimulated 45Ca2+ uptake into cells under nonsteady-state conditions in the presence or absence of Na+. Monensin, a Na+ ionophore, was able to elicit a modest increase in 45Ca2+ efflux, compared with controls. Studies of net 45Ca2+ flux, performed under near-steady-state conditions, showed that adrenaline caused net 45Ca2+ accumulation, whereas monensin caused net 45Ca2+ release. The effect of monensin required the presence of Na+ in the incubation medium. Both 1 mM-LaCl3 and 0.1 mM-D-600 prevented adrenaline-stimulated 45Ca2+ uptake into cells, but had no effect on monensin-induced changes. We conclude that (1) the rapid mobilization of Ca2+ by adrenergic agonists seen in rat parotid cells does not require a Na+out greater than Na+in gradient and (2) the nature of the monensin effect is quite different from the adrenergic-agonist-induced response.     

Feedback inhibition of Ca2+ release by Ca2+ is the underlying mechanism of agonist-evoked intracellular Ca2+ oscillations in pancreatic acinar cells.

Oscillations of free intracellular Ca2+ concentration ([Ca2+]i) are known to occur in many cell types during physiological cell signaling. To identify the basis for the oscillations, we measured both [Ca2+]i and extracellular Ca2+ concentration ([Ca2+]o) to follow the fate of Ca2+ during stimulation of [Ca2+]i oscillations in pancreatic acinar cells. [Ca2+]i oscillations were initiated by either t-butyloxycarbonyl-Tyr(SO3)-Nle-Gly-Tyr-Nle-Asp-2-phenylethyl ester (CCK-J), which mobilized Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-insensitive pool, or low concentration of cholecystokinin octapeptide (CCK-OP), which mobilized Ca2+ from the IP3-sensitive internal pool. Little Ca2+ efflux occurred during the oscillations triggered by CCK-J or CCK-OP in spite of a large average increase in [Ca2+]i. When internal store Ca2+ pumps were inhibited with thapsigargin (Tg) during [Ca2+]i oscillations, a rapid Ca2+ efflux occurred similar to that measured in intensely stimulated, nonoscillatory cells. Tg also stimulated 45Ca efflux from internal pools of cells stimulated with CCK-J or a low concentration of CCK-OP. Hence, a large fraction of the Ca2+ released during each spike is reincorporated by the internal store Ca2+ pumps. Surprisingly, when the increase in [Ca2+]i during stimulation of oscillations was prevented by loading the cells with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, a persistent activation of Ca2+ release and Ca2+ efflux occurred. This was reflected as a persistent increase in [Ca2+]o in cells suspended at low [Ca2+]o or persistent efflux of 45Ca from internal stores of cells maintained at high [Ca2+]o. Since agonist-stimulated Ca2+ release evidently remains activated when [Ca2+]i is highly buffered, the primary mechanism determining Ca2+ oscillations must include an inhibition of Ca2+ release by [Ca2+]i. Loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid had no apparent effect on the levels or kinetics of IP3 formation in agonist-stimulated cells. This suggests that [Ca2+]i regulated the oscillation by inhibition of Ca2+ release independent of its possible effects on cellular levels of IP3.     

Release and sequestration of Ca2+ by a caffeine- and ryanodine-sensitive store in a sub-population of human SH-SY5Y neuroblastoma cells

We have used single cell fluorescence imaging techniques to examine the role that ryanodine receptors play in the stimulus-induced Ca(2+) responses of SH-SY5Y cells. The muscarinic agonist methacholine (1mM) resulted in a Ca(2+) signal in 95% of all cells. Caffeine (30 mM) however stimulated a Ca(2+) signal in only 1-7% of N-type (neuroblastic) cells within any given field. The caffeine response was independent of extracellular Ca(2+), regenerative in nature, and abolished in a use-dependent fashion by ryanodine. In caffeine-responsive cells, the magnitude of the methacholine-induced Ca(2+) signal was inhibited by 75.07 +/- 5.51% by pretreatment with caffeine and ryanodine, suggesting that the caffeine-sensitive store may act as a Ca(2+) source after muscarinic stimulation. When these data were combined with equivalent data from non-caffeine-responsive cells, the degree of apparent inhibition was significantly reduced. In contrast, after store depletion by caffeine, the Ca(2+) signal induced by 55 mM K(+) was potentiated 2.5-fold in the presence of ryanodine, suggesting that the store may act a Ca(2+) sink after depolarisation. We conclude that a caffeine- and ryanodine-sensitive store can act as a Ca(2+) source and sink in SH-SY5Y cells, and that effects of the store can become obscured if data from caffeine-insensitive cells are not excluded.     

Interaction of caffeine-, IP3- and vanadate-sensitive Ca2+ pools in acinar cells of the exocrine pancreas

Summary Previous studies have shown the existence of functionally distinguishable inositol 1,4,5-trisphosphate- (IP₃) sensitive and IP₃-insensitive nonmitochondrial intracellular Ca²⁺ pools in acinar cells of the exocrine pancreas. For further characterization of Ca²⁺ pools, endoplasmic reticulum (ER) membrane vesicles were separated by Percoll gradient centrifugation which allowed us to distinguish five discrete fractions designatedP ₁ toP ₅ from the top to the bottom of the gradient. Measuring Ca²⁺ uptake and Ca²⁺ release with a Ca²⁺ electrode, we could differentiate three nonmitochondrial intracellular Ca²⁺ pools; (i) an IP₃-sensitive Ca²⁺ pool (IsCaP), vanadate- and caffeine-insensitive, (ii) a caffeine-sensitive Ca²⁺ pool (CasCaP), vanadate- and IP₃-insensitive, and (iii) a vanadate-sensitive Ca²⁺ pool (VasCaP), neither IP₃- nor caffeine-sensitive, into which Ca²⁺ uptake is mediated via a Ca²⁺ ATPase sensitive to vanadate at 10^–4 mol/liter. A fourth Ca²⁺ pool is neither IP₃- nor caffeine- or vanadate-sensitive. Percoll fractionP ₁ contained essentially the IsCaP, CasCaP and VasCaP and was mainly used for studies on Ca²⁺ uptake and Ca²⁺ release.When membrane vesicles were incubated in the presence of caffeine (2×10^–2 mol/liter), Ca²⁺ uptake up to the steady state [Ca²⁺] did not appear to be altered as compared to the control Ca²⁺ uptake. However, in control vesicles spontaneous Ca²⁺ release occurred after the steady state had been reached, whereas cfffeine-pretreated vesicles did not spontaneously release Ca²⁺. Addition of IP₃ at steady state [Ca²⁺] induced similar Ca²⁺ release followed by Ca²⁺ reuptake in both caffeine-pretreated and control vesicles. However, when caffeine was acutely added at steady state, Ca²⁺ was released from all Ca²⁺ pools including the IsCaP. Following Ca²⁺ reuptake after IP₃ had been added, a second addition of IP₃ to control vesicles induced further but smaller Ca²⁺ release, and a third addition resulted in a steady Ca²⁺ efflux by which all Ca²⁺ that had been taken up was released. This steady Ca²⁺ release started at a Ca²⁺ concentration between 5.5–8 ×10^–7 mol/liter and could also be induced by the IP₃ analogue inositol 1,4,5-trisphosphorothioate (IPS₃) or by addition of Ca²⁺ itself. Ruthenium red (10^–5 mol/liter) inhibited both caffeine-induced as well as Ca²⁺-induced but not IP₃-induced Ca²⁺ release. Heparin (100 g/m) inhibited IP₃-but not caffeine-induced Ca²⁺ release. The data indicate the presence of at least three separate Ca²⁺ pools in pancreatic acinar cells: the IsCaP, CasCaP and VasCaP. During Ca²⁺ uptake these Ca²⁺ pools appear to be separate. However, when steady state is reached, we assume that these Ca²⁺ pools come into contact and total Ca²⁺ release from all three pools can occur. The mechanism of this contact of Ca²⁺ pools is not clear but seems to be different from that induced by GTP in the presence of polyethylene glycol, which probably involves fusion of membranes.     

Activation of calcium oscillations by thapsigargin in parotid acinar cells.

The tumor promoter thapsigargin releases Ca2+ from intracellular stores by specific inhibition of microsomal Ca-ATPase activity without inositol phosphate formation. Recent studies of the actions of thapsigargin support the concept that the level of Ca2+ within the inositol (1,4,5)-trisphosphate (IP3)-sensitive intracellular pool regulates the Ca2+ permeability of the plasma membrane. We examined the effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) in single rat parotid cells using digital fluorescence microscopy. In the absence of extracellular Ca2+ (Ca2+o), thapsigargin transiently increased [Ca2+]i. Following the thapsigargin-induced [Ca2+]i transient, carbachol in the continued absence of Ca2+o was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes Ca2+ from the IP3-sensitive store. In the converse experiment, carbachol prevented a rise of [Ca2+]i by thapsigargin, suggesting that the IP3- and thapsigargin-sensitive Ca2+ pools are the same. Depletion of Ca2+ from the IP3-sensitive pool by thapsigargin enhanced plasma membrane Ca2+ permeability. Thapsigargin triggered sustained Ca2+ oscillations in Ca2(+)-containing medium which are highly reminiscent of agonist-induced oscillations in these cells. Carbachol addition rapidly raised IP3 levels during oscillations triggered by thapsigargin but did not elevate [Ca2+]i, indicating that the IP3-sensitive pool remains continuously depleted during [Ca2+]i fluctuations. The results from this study rule out the involvement of the IP3-sensitive pool in the mechanisms involved in thapsigargin-induced (and by analogy, agonist-induced) oscillations in parotid cells.     

Effect of thapsigargin on cytosolic Ca2+ level and amylase release in rat parotid acinar cells.

At concentrations greater than 0.01 microM, thapsigargin (ThG) dose-dependently caused an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in rat parotid acinar cells, as measured by the fluorescent Ca(2+)-indicator fura-2. In the absence of extracellular Ca2+, a transient increase in [Ca2+]i by ThG was observed, and subsequent addition of carbachol (CCh) did not produce a further [Ca2+]i response, suggesting that ThG released Ca2+ from the CCh-sensitive intracellular Ca2+ pool. Since ThG did not stimulate formation of inositol phosphates, the ThG-induced Ca2+ mobilization is independent of phosphoinositide breakdown. High concentrations (greater than 0.1 microM) of ThG induced amylase release from rat parotide acini, but the effect was very poor as compared with that of CCh or the protein kinase C activator, PMA (phorbol 12-myristate 13-acetate). Combined addition of ThG and PMA modestly potentiated amylase release induced by PMA alone. These results support the view that amylase release by muscarinic stimulation is mediated mainly by activation of protein kinase C rather than a rise in [Ca2+]i, although Ca2+ may modulate the secretory response.     

Modelling the transition from simple to complex Ca2+oscillations in pancreatic acinar cells

A mathematical model is proposed which systematically investigates complex calcium oscillations in pancreatic acinar cells. This model is based on calcium-induced calcium release via inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) and includes calcium modulation of inositol (1,4,5) trisphosphate (IP₃) levels through feedback regulation of degradation and production. In our model, the apical and the basal regions are separated by a region containing mitochondria, which is capable of restricting Ca²⁺ responses to the apical region. We were able to reproduce the observed oscillatory patterns, from baseline spikes to sinusoidal oscillations. The model predicts that calcium-dependent production and degradation of IP₃ is a key mechanism for complex calcium oscillations in pancreatic acinar cells. A partial bifurcation analysis is performed which explores the dynamic behaviour of the model in both apical and basal regions.     

Ryanodine receptor expression is associated with intracellular Ca2+ release in rat parotid acinar cells

The ryanodinereceptor mediates intracellularCa²⁺ mobilization in muscle andnerve, but its physiological role in nonexcitable cells is less welldefined. Like adenosine 3',5'-cyclic monophosphate andinositol 1,4,5-trisphosphate, cyclic ADP-ribose (0.3-5 µM) andADP (1-25 µM) produced a concentration-dependent rise incytosolic Ca²⁺ in permeabilizedrat parotid acinar cells. Adenosine and AMP were less effective.Ryanodine markedly depressed theCa²⁺-mobilizing action of theadenine nucleotides and forskolin in permeabilized cells and waslikewise effective in depressing the action of forskolin in intactcells. Cyclic ADP-ribose-evoked Ca²⁺ release was enhanced bycalmodulin and depressed by W-7, a calmodulin inhibitor. Afluorescently labeled ligand,4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indacene-3-propionic acid-glycyl ryanodine, was synthesized to detect the expression anddistribution of ryanodine receptors. In addition, ryanodine receptorexpression was detected in rat parotid cells with a sequence highlyhomologous to a rat skeletal muscle type 1 and a novel brain type 1 ryanodine receptor. These findings demonstrate the presence of aryanodine-sensitive intracellularCa²⁺ store in rat parotid cellsthat shares many of the characteristics of stores in muscle and nerveand may mediate Ca²⁺-inducedCa²⁺ release or a modified form ofthis process.

     

Octanoate increases cytosolic Ca2+ concentration and membrane conductance in ovine pancreatic acinar cells

 In order to investigate the cellular mechanisms involved in amylase release in response to stimulation with short-chain fatty acids, changes in intracellular calcium concentration ([Ca²⁺]_i), membrane current and amylase release were measured in pancreatic acinar cells of sheep. Both octanoate and acetylcholine raised [Ca²⁺]_i in acinar cells in a concentration-dependent manner. The rise in [Ca²⁺]_i in response to the stimulation with octanoate (10 mmol ⋅ l^-1) was reduced in a medium without CaCl₂, but was markedly enhanced by reintroduction of CaCl₂ into the medium up to 2.56 mmol ⋅ l^-1. Perfusion of the cells with a medium containing octanoate (5 mmol ⋅ l^-1) or acetylcholine (0.5 μmol ⋅ l^-1) immediately raised inward current across the cell membrane at a holding-membrane potential of −30 mV. The inward current became greater as the holding potential became more negative. The equilibrium potential was 1.8 mV and 3.9 mV for octanoate and acetylcholine, respectively, being consistent with that for Cl^-. Although intracellular application of octanoate through a patch-clamp pipette also raised inward current after several minutes in some cells (4 out of 12), this possibility was significantly smaller than that for extracellular application. In other cells, even though the intracellular application of octanoate did not cause an increase in current, it always caused responses immediately after introduction of the fatty acid into the medium. Stimulation with fatty acid as well as acetylcholine raised amylase release in a concentration-dependent manner in cells dispersed from tissue segments with crude collagenase and trypsin inhibitor. Without trypsin inhibitor, crude collagenase significantly and selectively reduced the octanoate (10 mmol ⋅ l^-1)-induced amylase release. Dispersion with crude collagenase and trypsin significantly reduced both responses induced by octanoate and acetylcholine (5.5 μmol ⋅ l^-1). We conclude that fatty acids and acetylcholine increase [Ca²⁺]_i, which consequently evokes a rise in transmembrane ion (Cl^-) conductance and amylase release, and that trypsin-sensitive protein(s) in the cell membrane are involved in secretory processes activated by stimulation with fatty acids in ovine pancreatic acinar cells. Accepted: 14 May 1996