Overexpression of Rab3D enhances regulated amylase secretion from pancreatic acini of transgenic mice.

Rab3D, a member of the ras-related GTP-binding protein Rab family, is localized to secretory granules of various exocrine tissues such as acinar cells of the pancreas, chief cells of the stomach, and parotid and lacrimal secretory cells. To elucidate the function of Rab3D in exocytosis, we have generated transgenic mice that over-express Rab3D specifically in pancreatic acinar cells. Hemagglutinin-tagged Rab3D was localized to zymogen granules by immunohistochemistry, and was shown to be present on zymogen granule membranes by Western blotting; both results are similar to previous studies of endogenous Rab3D. Secretion measurements in isolated acinar preparations showed that overexpression of Rab3D enhanced amylase release. Amylase secretion from intact acini of transgenic mice 5 min after 10 pM cholecystokinin octapeptide (CCK) stimulation was enhanced by 160% of control. In streptolysin-O-permeabilized acini of transgenic mice, amylase secretion induced by 100 microM GTP-gamma-S was enhanced by 150%, and 10 microM Ca2+-stimulated amylase secretion was augmented by 206% of that of the control. To further elucidate Rab3D involvement in stimulus-secretion coupling, we examined the effect of CCK on the rate of GTP binding to Rab3D. Stimulation of permeabilized acini with 10 pM CCK increased the incorporation of radiolabeled GTP into HA-tagged Rab3D. These results indicate that overexpression of Rab3D enhances secretagogue-stimulated amylase secretion through both calcium and GTP pathways. We conclude that Rab3D protein on zymogen granules plays a stimulatory role in regulated amylase secretion from pancreatic acini.

Effects of alanine on insulin-secreting cells: patch-clamp and single cell intracellular Ca2+ measurements.

The effects of alanine, glucose and tolbutamide on insulin-secreting cells (RINm5F) have been investigated using patch-clamp and single cell intracellular Ca2+ measurements. When directly challenged with the amino acid L-alanine (2-10 mM) the cells underwent a sharp depolarization, which led to the generation of Ca2+ spike potentials and an increase in [Ca2+]i. The L-alanine-induced depolarization was associated with a net inward membrane current but no measurable change in the resistance of the cell. The latter effect was found to be in contrast to the actions of glucose (5-10 mM) and tolbutamide (100 microM), both of which depolarized cells and raised [Ca2+]i by an increase in the input resistance of the cell membrane, due to the closure of ATP-sensitive potassium channels. In the complete absence of external Na+ (by replacement with 140 mM NMDG+), L-alanine had no effects on either the membrane potential or [Ca2+]i. Similarly, replacing Na+ with NMDG+ in the continued presence of the amino acid resulted in a repolarization of the membrane and an attenuation of the L-alanine-induced rise in [Ca2+]i. The Na+ channel blocker TTX (1-2 microM) had no effects on the alanine-evoked electrical activity. Exchange of the L-form of the amino acid with the D-stereoisomer had similar actions to those of removing external Na+, since D-alanine had no effects on the membrane potential or [Ca2+]i. The actions of L-alanine were also found to be mimicked by the N-methylated amino acid analogue methylamino isobutyric acid (MeAIB) (2-10 mM), suggesting that the A-type electrogenic amino acid cotransport system operates in the RINm5F insulin-secreting cell line.

Endothelin increases [Ca2+]i in rat pancreatic acinar cells by intracellular release but fails to increase amylase secretion.

In individual fura-2 loaded cells of rat pancreatic acini endothelin-1 (ET-1) (10-50 nM) induced sustained oscillations in [Ca2+]i. At higher concentrations a larger, but transient increase in [Ca2+]i was observed, which was largely unaffected by removal of extracellular Ca2+. ET-1 induced the release of Ca2+i from the same store as cholecystokinin (CCK), but with less potency. At concentrations of endothelin which transiently increased Ca2+, ET-1 increased the accumulation of inositol phosphates. Specific binding sites for 125I-endothelin were demonstrated on rat pancreatic acini. A single class of binding sites was identified with an apparent Kd 108 +/- 12 pM and Bmax of 171 +/- 17 fmol/mg for ET-1. The relative potency order for displacing [125I]ET was ET-1 greater than ET-2 greater than ET-3. In contrast to CCK and the non-phorbol ester tumour promoter Thapsigargin (TG) which induce both transient and sustained components of [Ca2+]i elevation, ET-1 failed to increase amylase release over the range 100 pM-1 microM.

Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria.

In pancreatic acinar cells, inositol 1,4,5-trisphosphate (InsP(3))-dependent cytosolic calcium ([Ca(2+)](i)) increases resulting from agonist stimulation are initiated in an apical "trigger zone," where the vast majority of InsP(3) receptors (InsP(3)R) are localized. At threshold stimulation, [Ca(2+)](i) signals are confined to this region, whereas at concentrations of agonists that optimally evoke secretion, a global Ca(2+) wave results. Simple diffusion of Ca(2+) from the trigger zone is unlikely to account for a global [Ca(2+)](i) elevation. Furthermore, mitochondrial import has been reported to limit Ca(2+) diffusion from the trigger zone. As such, there is no consensus as to how local [Ca(2+)](i) signals become global responses. This study therefore investigated the mechanism responsible for these events. Agonist-evoked [Ca(2+)](i) oscillations were converted to sustained [Ca(2+)](i) increases after inhibition of mitochondrial Ca(2+) import. These [Ca(2+)](i) increases were dependent on Ca(2+) release from the endoplasmic reticulum and were blocked by 100 microM ryanodine. Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import. This effect was also abolished by ryanodine receptor (RyR) blockade. Photolysis of d-myo InsP(3) P(4(5))-1-(2-nitrophenyl)-ethyl ester (caged InsP(3)) produced either apically localized or global [Ca(2+)](i) increases in a dose-dependent manner, as visualized by digital imaging. Mitochondrial inhibition permitted apically localized increases to propagate throughout the cell as a wave, but this propagation was inhibited by ryanodine and was not seen for minimal control responses resembling [Ca(2+)](i) puffs. Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone. These data suggest that, while Ca(2+) release is initially triggered through InsP(3)R, release by RyRs is the dominant mechanism for propagating global waves. In addition, mitochondrial Ca(2+) import controls the spread of Ca(2+) throughout acinar cells by modulating RyR activation.

Agonist-dependent phosphorylation of the inositol 1,4,5-trisphosphate receptor: A possible mechanism for agonist-specific calcium oscillations in pancreatic acinar cells.

The properties of inositol 1,4,5-trisphosphate (IP3)-dependent intracellular calcium oscillations in pancreatic acinar cells depend crucially on the agonist used to stimulate them. Acetylcholine or carbachol (CCh) cause high-frequency (10-12-s period) calcium oscillations that are superimposed on a raised baseline, while cholecystokinin (CCK) causes long-period (>100-s period) baseline spiking. We show that physiological concentrations of CCK induce rapid phosphorylation of the IP3 receptor, which is not true of physiological concentrations of CCh. Based on this and other experimental data, we construct a mathematical model of agonist-specific intracellular calcium oscillations in pancreatic acinar cells. Model simulations agree with previous experimental work on the rates of activation and inactivation of the IP3 receptor by calcium (DuFour, J.-F., I.M. Arias, and T.J. Turner. 1997. J. Biol. Chem. 272:2675-2681), and reproduce both short-period, raised baseline oscillations, and long-period baseline spiking. The steady state open probability curve of the model IP3 receptor is an increasing function of calcium concentration, as found for type-III IP3 receptors by Hagar et al. (Hagar, R.E., A.D. Burgstahler, M.H. Nathanson, and B.E. Ehrlich. 1998. Nature. 396:81-84). We use the model to predict the effect of the removal of external calcium, and this prediction is confirmed experimentally. We also predict that, for type-III IP3 receptors, the steady state open probability curve will shift to lower calcium concentrations as the background IP3 concentration increases. We conclude that the differences between CCh- and CCK-induced calcium oscillations in pancreatic acinar cells can be explained by two principal mechanisms: (a) CCK causes more phosphorylation of the IP3 receptor than does CCh, and the phosphorylated receptor cannot pass calcium current; and (b) the rate of calcium ATPase pumping and the rate of calcium influx from the outside the cell are greater in the presence of CCh than in the presence of CCK.

Acetylcholine and cholecystokinin induce different patterns of oscillating calcium signals in pancreatic acinar cells.

Receptor-activated cytoplasmic calcium (Ca2+) oscillations have been investigated in single pancreatic acinar cells by microfluorimetry (Fura-2 as indicator). At submaximal concentrations of the agonists acetylcholine (ACh) and cholecystokinin octapeptide (CCK-8), both give rise to oscillatory changes in the cytosolic free calcium concentration ([Ca2+]i). The patterns of oscillations are markedly and consistently different for each of these two agonists. The ACh induced oscillations are superimposed upon a median elevation in background [Ca2+]i. The CCK-8 induced oscillations are of longer duration with [Ca2+]i returning to prestimulus levels between the discrete spikes. The ACh induced oscillations are rapidly abolished upon removal of extracellular Ca2+ while the CCK-8 induced oscillations persist for many minutes in the absence of external Ca2+. The CCK-8, but not the ACh, induced oscillations are increased in duration by the protein kinase C (PKC) inhibitor staurosporine and abolished by the PKC activating phorbol ester PMA. It is clear that CCK-8 and ACh do not activate receptor transduction mechanisms in an identical manner to generate oscillating [Ca2+]i signals.

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.

Oscillations of cytosolic calcium in single pancreatic acinar cells stimulated by acetylcholine.

The changes in cytosolic free calcium concentration [( Ca2+]i) were monitored (fura-2) in single, isolated, mouse pancreatic acinar cells stimulated by acetylcholine (ACh). Responses to ACh at concentrations between 10(-7) and 5 x 10(-7) M are marked by the appearance of regular, sinusoidal, oscillations in [Ca2+]i. At 37 degrees C the oscillations are transient, being seen only in the initial rising phase of the calcium signal. At 30 degrees C regular oscillations can be maintained throughout the period of ACh application. This study reports that release of intracellular calcium and influx of extracellular calcium are both involved in the generation of these oscillatory calcium signals.

CCK antagonists reveal that CCK-8 and JMV-180 interact with different sites on the rat pancreatic acinar cell CCKA receptor.

The ability of CCKA antagonists to inhibit full and partial CCK agonists of the rat pancreatic acinar cell CCKA receptor has been studied. When isolated rat pancreatic acini were superfused with CCK-8 (10 pM-1 nM) or CCK-4 (1 microM), an increase in [Ca2+]i signal was initiated. Concurrent superfusion of either L-364,718 (0.1 microM) or lorglumide (10 microM), chemically distinct, specific, potent antagonists of the CCKA receptor, resulted in a rapid inhibition of the [Ca2+]i signal initiated by all concentrations of CCK-8. In contrast, Ca2+ oscillations, initiated by JMV-180 (25 nM-1 microM), a partial agonist analogue of CCK-8, were essentially unaffected by concurrent superfusion of either L-364,718 or lorglumide. When JMV-179, an analogue of JMV-180 that exhibits characteristics of a pure antagonist, was superfused concurrently with either CCK-8 or JMV-180, Ca2+ oscillations were inhibited, even in the presence of 0.1 microM L-364,718. In a similar fashion, amylase secretion stimulated by CCK-8 was markedly attenuated by L-364,718, lorglumide, and JMV-179, whereas secretion stimulated by JMV-180 was only inhibited by JMV-179. A model is proposed to reconcile this data, based on the assumption that JMV-180 and CCK-8 interact with discrete sites on the CCKA receptor, which are differentially affected by the binding of antagonists. This model may also explain how a single receptor may transduce multiple signals in response to different agonists.

Ca2+ signaling through secretagogue and growth factor receptors on pancreatic AR42J cells.

Intracellular signaling by an increase in [Ca2+]i was observed in pancreatic AR42J cells in response to agonists whose receptors are G-protein coupled including cholecystokinin (CCK), bombesin, carbachol, substance P, pituitary adenylate cyclase activating peptide (PACAP), bradykinin, ATP, calcitonin gene related peptide (CGRP), and in response to growth factors EGF and FGF whose receptors are tyrosine kinases. The response to growth factors was smaller both in magnitude and in the percentage of cells responding but was independent of extracellular Ca2+. CCK and carbachol induced sizeable increases in inositol phosphates while growth factors did not. The responses to both carbachol and EGF, however, were blocked by the phospholipase C inhibitor U73122. The tyrosine kinase inhibitor, genestein, blocked the response to EGF but not that to CCK. These data are consistent with two types of signaling mechanisms in AR42J cells. Secretagogues act on receptors which couple through G proteins to induce a large amount of inositol phosphate production and subsequent intracellular Ca2+ mobilization. Growth factors act on receptors which signal through tyrosine kinase activity and in this cell type produced limited amounts of inositol phosphate and a smaller increase in intracellular Ca2+.

A method for determining the dependence of calcium oscillations on inositol trisphosphate oscillations.

In some cell types, oscillations in the concentration of free intracellular calcium ([Ca2+]) are accompanied by oscillations in the concentration of inositol 1,4,5-trisphosphate ([IP3]). However, in most cell types it is still an open question as to whether oscillations in [IP3] are necessary for Ca2+ oscillations in vivo, or whether they merely follow passively. Using a wide range of models, we show that the response to an artificially applied pulse of IP3 can be used to distinguish between these two cases. Hence, we show that muscarinic receptor-mediated, long-period Ca2+ oscillations in pancreatic acinar cells depend on [IP3] oscillations, whereas short-period Ca2+ oscillations in airway smooth muscle do not.

Calcium oscillations in a triplet of pancreatic acinar cells.

We use a mathematical model of calcium dynamics in pancreatic acinar cells to investigate calcium oscillations in a ring of three coupled cells. A connected group of cells is modeled in two different ways: 1), as coupled point oscillators, each oscillator being described by a spatially homogeneous model; and 2), as spatially distributed cells coupled along their common boundaries by gap-junctional diffusion of inositol trisphosphate and/or calcium. We show that, although the point-oscillator model gives a reasonably accurate general picture, the behavior of the spatially distributed cells cannot always be predicted from the simpler analysis; spatially distributed diffusion and cell geometry both play important roles in determining behavior. In particular, oscillations in which two cells are in synchrony, with the third phase-locked but not synchronous, appears to be more dominant in the spatially distributed model than in the point-oscillator model. In both types of model, intercellular coupling leads to a variety of synchronous, phase-locked, or asynchronous behaviors. For some parameter values there are multiple, simultaneous stable types of oscillation. We predict 1), that intercellular calcium diffusion is necessary and sufficient to coordinate the responses in neighboring cells; 2), that the function of intercellular inositol trisphosphate diffusion is to smooth out any concentration differences between the cells, thus making it easier for the diffusion of calcium to synchronize the oscillations; 3), that groups of coupled cells will tend to respond in a clumped manner, with groups of synchronized cells, rather than with regular phase-locked periodic intercellular waves; and 4), that enzyme secretion is maximized by the presence of a pacemaker cell in each cluster which drives the other cells at a frequency greater than their intrinsic frequency.

U73122 inhibits Ca2+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells.

Stimulation of rat pancreatic acinar cells with low concentrations of phosphatidylinositol (PI)-linked secretagogues induces [Ca2+]i oscillations, without measurable changes in the formation of inositol 1,4,5-trisphosphate. Therefore, we tested U73122 a new phospholipase C inhibitor to determine if PI turnover is necessary for the generation of [Ca2+]i oscillations. In acini prelabeled with [3H]inositol, PI hydrolysis on stimulation with either cholecystokinin or carbachol was inhibited dose-dependently by U73122, with a maximal effect seen at 10 microM; the formation of inositol 1,4,5-trisphosphate, measured using a radioreceptor assay, was also similarly inhibited. By contrast secretin- or vasoactive intestinal peptide-stimulated production of cAMP was unaffected by 10 microM U73122. These studies indicate that U73122 is a relatively specific inhibitor of G-protein-mediated phospholipase C activation in pancreatic acini. In fura-2-loaded acini, U73122 inhibited the increases in [Ca2+]i stimulated by these high concentrations of secretagogues which can be demonstrated to elicit PI turnover. The [Ca2+]i signal generated by directly stimulating G-proteins with sodium fluoride was also inhibited by U73122; however, the [Ca2+]i rise induced by thapsigargin was unaffected. These data indicate that the mechanism of inhibition was distal to the occupation of cell surface receptors but did not involve an interference of Ca2+ metabolism in general. When [Ca2+]i oscillations were elicited by low concentrations of cholecystokinin or carbachol, U73122 rapidly inhibited the oscillating [Ca2+]i signal. In contrast, oscillations induced by an analogue of cholecystokinin, JMV-180, which does not stimulate changes in PI metabolism at any concentration, were unaffected. This indicates that cholecystokinin- and carbachol-induced oscillations are probably initiated by small, localized changes in PI metabolism, which are not readily detectable. However, the inability of U73122 to inhibit JMV-180-induced oscillations indicates that PI metabolism may not necessarily be a prerequisite for the generation of [Ca2+]i oscillations.

Mastoparan induces oscillations of cytosolic Ca2+ in rat pancreatic acinar cells.

Microfluorimetry of fura-2 was used to monitor [Ca2+]i in single cells stimulated with the G-protein activating agent mastoparan. Mastoparan induced the generation of [Ca2+]i oscillations, which in contrast to oscillations induced by low concentrations of CCK were acutely dependent on the presence of extracellular Ca2+. Oscillations were inhibited by phorbol ester. Sodium fluoride, a known activator of G-proteins, gave similar results. Both mastoparan and CCK induced turnover of inositol phosphates, at concentrations higher than necessary to induce oscillations.

Pilocarpine and carbachol exhibit markedly different patterns of Ca2+ signaling in rat pancreatic acinar cells.

The effects of the partial muscarinic agonist pilocarpine on physiological responses were investigated in rat pancreatic acinar cells and compared with carbachol, a full muscarinic agonist, together with previous results using JMV-180, a partial agonist of CCK-A receptors. Pilocarpine was found to stimulate amylase release from isolated pancreatic acini in a concentration-dependent manner. At a maximal concentration (10 microM), pilocarpine was only capable of stimulating 63% of the secretion stimulated by a maximal concentration of carbachol. Moreover pilocarpine did not induce a decrease in secretion at supramaximal concentrations as does carbachol. In acini loaded with fura-2, superfusion of pilocarpine resulted exclusively in generation of intracellular Ca2+ concentration ([Ca2+]i) oscillations at all concentrations tested (0.3 microM-1 mM), in marked contrast to high concentrations of full agonists, which result in a biphasic sustained increase in [Ca2+]i. In common with low concentrations of other secretagogues that stimulate [Ca2+]i oscillations, pilocarpine at all concentrations was only able to stimulate a very small increase in phosphoinositide (PI) hydrolysis. In acini previously incubated with [3H]inositol, pilocarpine was shown to stimulate PI hydrolysis 27% above basal, compared with 872% for carbachol. To ascertain if this small degree of PI hydrolysis seen with pilocarpine is responsible for the generation of [Ca2+]i oscillations, an inhibitor of phospholipase C-linked processes, U-73122, which has been shown to inhibit Ca2+ oscillations induced by carbachol and CCK but not JMV-180 was tested. This agent rapidly inhibited pilocarpine-stimulated oscillations, indicating that in contrast to JMV-180, oscillations induced by pilocarpine are the result of PI hydrolysis.

Tyrosine kinase inhibitors attenuate "capacitative" Ca2+ influx in rat pancreatic acinar cells.

The effect of several tyrosine kinase inhibitors was tested on Ca2+ influx mediated by thapsigargin-and CCh-induced intracellular store depletion. Genestein inhibited Ca2+ influx in a concentration dependent manner without affecting Ca2+ release or Ca2+ pumping activity. A measureable effect was observed at 3 microM with total inhibition of influx seen at 100 microM. Tyrphostin A25 (300 microM; 78% inhibition) and methyl 2,5 dihydroxycinnamate (10 microM; 51% inhibition) also inhibited Ca2+ influx. The degree of attenuation was not markedly altered by preincubation of the inhibitors. Genestein also inhibited Ca2+ influx induced by CCh. These data indicate that inhibition of Ca2+ influx could in part underlie the previously reported inhibition of enzyme secretion by these agents.

Halothane and octanol block Ca2+ oscillations in pancreatic acini by multiple mechanisms.

This study has investigated halothane and octanol, reported inhibitors of gap junction permeability, for their effects on acinar cell intracellular Ca2+ concentration ([Ca2+]i) signaling. Halothane and octanol alone at maximal concentrations induced a sustained rise in [Ca2+]i of 23 +/- 4 and 29 +/- 5 nM, respectively. Cholecystokinin (CCK, 20 pM) induced [Ca2+]i oscillations in single acinar cells within the acinus to a peak of 275 +/- 17 nM, rising from a basal level of 55 +/- 3 nM. These oscillations were completely abolished by superfusion with both halothane (4 mM) and octanol (1 mM), concentrations that blocked the spread of Lucifer yellow from cell to cell within an acinus. Lower concentrations of octanol markedly reduced the oscillation frequency (0.2 and 0.5 mM octanol: reduction in oscillation frequency of 69 +/- 6 and 43 +/- 6%, respectively). These agents however, over the same concentration range, also exhibited similar inhibitory effects on [Ca2+]i oscillations in single cells dispersed from the acinus (reduction in oscillation frequency of 75 +/- 10 and 32 +/- 12% for 0.2 and 0.5 mM octanol, respectively), suggesting additional effects other than on gap junctions. Halothane inhibited inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production in response to both 1 and 10 nM CCK (31 and 40% inhibition, respectively), possibly explaining its effects on [Ca2+]i oscillations, whereas octanol showed no significant inhibition. Octanol, unlike halothane, blocked Ins(1,4,5)P3-induced Ca2+ release from permeabilized acini, an effect that was most pronounced at a more physiological Ins(1,4,5)P3 concentration. Octanol did not affect Ins(1,4,5)P3 binding to Ins(1,4,5)P3 receptor preparation. In conclusion, although halothane and octanol block gap junction permeability in pancreatic acinar cells, these agents also affect Ins(1,4,5)P3 production and Ca2+ mobilization in response to agonist stimulation.

Intercellular calcium waves in rat pancreatic acini: mechanism of transmission.

Digital-imaging microfluorimetry, together with microinjection of marker/messenger molecules, was utilized to investigate intercellular Ca2+ signaling in rat pancreatic acinar cells. Stimulation of acini with low concentrations of secretagogues [< 100 pM cholecystokinin (CCK), < 1 microM carbachol (CCh)] resulted in asynchronous but coordinated increases in Ca2+ that appeared to pass in a "wavelike" fashion between cells. In contrast, at higher supermaximal concentrations of agonists (> 300 pM CCK, > 1 microM CCh), which induce a large "peak-and-plateau" intracellular Ca2+ signal, all cells in the acinus appeared to increase Ca2+ concentration ([Ca2+]) in synchrony. Microinjection of lissarhodamine, a marker of gap-junctional permeability, into cells previously loaded with fura 2 allowed the simultaneous measurement of gap-junctional coupling and [Ca2+]. Stimulation with supermaximal concentrations of agonists resulted in the attenuation of junctional permeability, whereas, during stimulation with physiological concentrations of agonist, junctional communication remained operable. Injection of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] into one cell of an acinar cluster resulted in the generation of a Ca2+ signal in the injected cell and adjacent cells. In contrast, injection of CaCl2 itself did not result in propagation of the signal. When CaCl2 was injected into cells that had been previously stimulated with a threshold concentration of CCK, propagation of a signal was observed between cells. On the basis of these data, a model is proposed in which Ca2+ acts as coagonist with Ins(1,4,5)P3 to potentiate the Ca(2+)-releasing action of Ins(1,4,5)P3 and, by diffusion of the two molecules through gap junctions, underlies intercellular signaling in acinar cells. Gap-junctional communication may be an important factor in amplifying a threshold signal produced in one cell throughout the acinus, resulting in enhanced stimulated secretion in acinar preparations compared with preparations of isolated cells.

Extracellular ATP mediates Ca2+ signaling in cultured myenteric neurons via a PLC-dependent mechanism.

In the myenteric plexus, ATP is released as a neurotransmitter by "purinergic" nerves, relaxing visceral smooth muscle. We report a signal transduction mechanism for ATP in cultured myenteric neurons involving receptor-mediated release of intracellular Ca2+ stores. Primary cultures of myenteric neurons from guinea pigs taenia coli were loaded with the Ca2+ indicator fura 2-acetoxymethyl ester (AM) and examined using digital imaging microscopy. Superfusion of single neurons with ATP (0.01-1,000 microM) resulted in concentration-dependent increases in intracellular Ca2+ concentration ([Ca2+]i) that were independent of extracellular Ca2+. Decrements in peak [Ca2+]i were seen with repetitive ATP exposure. Responsiveness of myenteric neurons to purinergic agonists (100 microM) was consistent with action at a neuronal P 2y purinoceptor: 2-chloro-ATP = ATP = 2-methyl-thio-ATP (MeSATP) > ADP > alpha, beta-MeATP = beta,gamma-MeATP > AMP > adenosine. ATP-evoked Ca2+ transients were inhibited dose dependently by suramin, a nonspecific P2 antagonist, and reactive blue 2, a specific P 2y antagonist. ATP and cyclopiazonic acid (30 microM) appear to release an identical intracellular Ca2+ store. Preincubation with the aminosteroid U-73122 (10 microM) inhibited ATP-evoked Ca2+ transients by 71 +/- 7%, whereas phorbol ester pretreatment (phorbol 12-myristate 13-acetate, 100 nM, 5 min) caused a 76 +/- 4% inhibition. Peak [Ca2+]i evoked by ATP was not affected by preincubation with pertussis toxin (100 ng/ml, 24 h) or nifedipine (10 microM). These data suggest a signal transduction mechanism for ATP in cultured myenteric neurons involving purinoceptor-mediated activation of phospholipase C (PLC), with release of D-myo-inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores.