Cyclic GMP mediates the agonist-stimulated increase in plasma membrane calcium entry in the pancreatic acinar cell

The present studies were performed to determine the role of cyclic GMP in regulating agonist mediated calcium entry in the pancreatic acinar cell. In guinea pig-dispersed pancreatic acini the findings demonstrated that carbachol stimulated a transient 20-40-fold rise in cellular cyclic GMP followed by a sustained 3-4-fold rise in cellular cyclic GMP. The guanylate cyclase inhibitor, 6-anilino-5,8-quinolinedione (LY83583), caused a dose-dependent inhibition of carbachol-stimulated increases in cellular cyclic GMP both during the initial transient large increase in cyclic GMP and the sustained increase in cyclic GMP. LY83583 also inhibited cellular Ca2+ influx during carbachol stimulation and reloading of the agonist-sensitive pool of Ca2+ at the termination of carbachol stimulation with atropine. The effect of the inhibition on reloading of the agonist-sensitive pool was secondary to its effects on the plasma membrane C2+ entry. The addition of dibutyryl cyclic GMP to LY83583-treated acini restored Ca2+ influx across the plasma membrane. Nitroprusside increased both cellular cyclic GMP and the rate of Ca2+ influx. During periods when plasma membrane Ca2+ entry was activated, cellular cyclic GMP levels were increased. These results suggest that agonist-induced increases in cellular cyclic GMP are necessary and sufficient to mediate the effects of the agonist on the plasma membrane Ca2+ entry mechanism.     

Early changes in pancreatic acinar cell calcium signaling after pancreatic duct obstruction

Intracellular Ca(2+)-changes not only participate in important signaling pathways but have also been implicated in a number of disease states including acute pancreatitis. To investigate the underlying mechanisms in an experimental model mimicking human gallstone-induced pancreatitis, we ligated the pancreatic duct of Sprague-Dawley rats and NMRI mice for up to 6 h and studied intrapancreatic changes including the dynamics of [Ca(2+)](i) in isolated acini. In contrast to bile duct ligation, pancreatic duct obstruction induced intra-pancreatic trypsinogen activation, leukocytosis, hyperamylasemia, and pancreatic edema and increased lung myeloperoxidase activity. Although resting [Ca(2+)](i) in isolated acini rose by 45% to 205 +/- 7 nmol, the acetylcholine- and cholecystokinin (CCK)-stimulated calcium peaks as well as the amylase secretion declined, but neither the [Ca(2+)](i)-signaling pattern nor the amylase output in response to the Ca(2+)-ATPase inhibitor thapsigargin nor the secretin-stimulated amylase release were impaired by pancreatic duct ligation. On the single cell level pancreatic duct ligation reduced the percentage of cells in which submaximal secretagogue stimulation was followed by a physiological response (i.e. Ca(2+) oscillations) and increased the percentage of cells with a pathological response (i.e. peak plateau or absent Ca(2+) signal). Moreover, it reduced the frequency and amplitude of Ca(2+) oscillation as well as the capacitative Ca(2+) influx in response to secretagogue stimulation. Serum pancreatic enzyme elevation as well as trypsinogen activation was significantly reduced by pretreatment of animals with the calcium chelator BAPTA-AM. These experiments suggest that pancreatic duct obstruction rapidly changes the physiological response of the exocrine pancreas to a Ca(2+)-signaling pattern that has been associated with premature digestive enzyme activation and the onset of pancreatitis, both of which can be prevented by administration of an intracellular calcium chelator.     

Cyclic GMP regulates free cytosolic calcium in the pancreatic acinar cell

The present studies were performed in order to measure the effects of cyclic GMP (cGMP) on the regulation of free cytosolic calcium [( Ca2+]i) in the pancreatic acinar cell. In guinea pig dispersed pancreatic acini the findings demonstrated that the Ca2+ ionophore, Br A23187, caused a sustained increase in [Ca2+]i in the presence of 3 mM CaCl2 in the media and a transient 20 fold rise in cellular cGMP followed by a sustained 3-4 fold rise in cellular cGMP. Increasing cellular cGMP with nitroprusside, hydroxylamine or dibutyryl cGMP had no effect on resting [Ca2+]i. However, these agents attenuated the increase in [Ca2+]i resulting from Br A23187-induced Ca2+ influx. Nitroprusside also attenuated the carbachol-induced sustained rise in [Ca2+]i that resulted from Ca2+ influx. The nitroprusside effect on carbachol-stimulated acini occurred without decreasing Ca2+ influx across the plasma membrane or alteration in the mobilization of Ca2+ from the intracellular agonist-sensitive pool. Inhibition of the increase in cellular cGMP caused by Br A23187 by the guanylate cyclase inhibitor, 6-anilino-5,8-quinolinedione (LY83583), resulted in augmentation of the increase in [Ca2+]i. This augmentation was reversed with dibutyryl cGMP. These results indicated that cGMP regulated [Ca2+]i in the pancreatic acinar cell. The mechanism involves the removal of Ca2+ from the cytoplasm.     

Two components of hormone-evoked calcium release from intracellular stores of pancreatic acinar cells.

Dispersed pancreatic acini loaded with Fura 2 were used to study the effect of hormonal stimulation on [Ca2+]i (free cytosolic Ca2+ concentration). Stimulation of acini with cholecystokinin octapeptide or carbachol resulted in two components of increase in [Ca2+]i. The maximal increase in [Ca2+]i and the time to maximum for both components was dependent on hormone concentration. The first component reached a maximum after 2-10 s of stimulation, whereas the second component required 30-60 s of stimulation for maximal effect. Both components of the [Ca2+]i increase can be observed in the presence or absence of Ca2+ in the incubation medium. The two components of Ca2+ release from intracellular stores showed similar dependency on agonist concentration. Termination of cell stimulation with specific antagonist revealed two, kinetically separated, rates of decrease in [Ca2+]i. The initial decrease in [Ca2+]i, was completed within 2.5-7 s, whereas the secondary decrease in [Ca2+]i, back to resting values, required approx. 40 s. The magnitude of the antagonist-induced initial (rapid) and secondary (slow) decrease in [Ca2+]i was dependent on the duration of cell stimulation. Hence it appears that stimulation of pancreatic acinar cells with Ca2+-mobilizing hormones results in two, kinetically separated, components of Ca2+ release from intracellular stores.     

Agonist-stimulated pathways of calcium signaling in pancreatic acinar cells.

In pancreatic acinar cells stimulation of different intracellular pathways leads to different patterns of Ca2+ signaling. Bombesin induces activation of both phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and phospholipase D (PLD). The latter leads to generation of diacylglycerol (DAG) in addition to that produced by activation of PIP2-PLC. Strong activation of protein kinase C (PKC) results in inhibition of Ca(2+)-induced Ca2+ release from Ca2+ pools arranged in sequence to the luminally located IP3-sensitive Ca2+ pools. Consequently the Ca2+ wave which starts in the luminal cell pole is slower in the presence of bombesin (5 microm/s) as compared to that in the presence of acetylcholine (17 microm/s) which activates PIP2-PLC but not PLD. Activation of high-affinity CCK-receptors triggers a Ca2+ wave with slow propagation (5 microm/s) due to stimulation of phospholipase A2 (PLA2) and generation of arachidonic acid, which in turn leads to inhibition of Ca(2+)-induced Ca2+ release. Low-affinity CCK-receptors are coupled to both PIP2-PLC and PLD.     

Low extracellular pH induces damage in the pancreatic acinar cell by enhancing calcium signaling

Low extracellular pH (pHe) occurs in a number of clinical conditions and sensitizes to the development of pancreatitis. The mechanisms responsible for this sensitization are unknown. Because abnormal Ca(2+) signaling underlies many of the early steps in the pathogenesis of pancreatitis, we evaluated the effect of decreasing pHe from 7.4 to 7.0 on Ca(2+) signals in the acinar cell. Low pHe significantly increased the amplitude of cerulein-induced Ca(2+) signals. The enhancement in amplitude was localized to the basolateral region of the acinar cell and was reduced by pretreatment with ryanodine receptor (RYR) inhibitors. Because basolateral RYRs also have been implicated in the pathogenesis of pancreatitis, we evaluated the effects of RYR inhibitors on pancreatitis responses in acidic conditions. RYR inhibitors significantly reduced the sensitizing effects of low pHe on zymogen activation and cellular injury. These findings suggest that enhanced RYR-mediated Ca(2+) signaling in the basolateral region of the acinar cell is responsible for the injurious effects of low pHe on the exocrine pancreas.     

The regulation of exocytosis in the pancreatic acinar cell.

The pancreatic acinar cell synthesises a variety of digestive enzymes. In transit through the secretory pathway, these enzymes are separated from constitutively secreted proteins and packaged into zymogen granules, which are localised in the apical pole of the cell. Stimulation of the cell by secretagogues such as acetylcholine and cholecystokinin, acting at receptors on the basolateral plasma membrane, causes the generation of an intracellular Ca(2+) signal. This signal, in turn, triggers the fusion of the zymogen granules with the apical plasma membrane, leading to the polarised secretion of the enzymes. This review describes recent advances in our understanding of the control of secretion in the acinar cell. In particular, we discuss the mechanisms underlying the sorting of digestive enzymes into the zymogen granules, the molecular components of the exocytotic "membrane fusion machine," the generation and propagation of the Ca(2+ signal and the development of new techniques for the visualisation of single granule fusion events.     

Vanadate inhibits the calcium extrusion in rat pancreatic acinar cells

Our objective was to evaluate the role of vanadate on calcium extrusion in Fura-2-loaded rat pancreatic acinar cells by digital microscopic fluorimetry and spectrofluorimetry. In the absence of extracellular calcium, perfusion of pancreatic acinar cells with 1 nM CCK-8 and 1 mM vanadate did not significantly affect the typical transient calcium spike induced by CCK-8, but the plateau phase of calcium in response to CCK-8 remained elevated. In addition, vanadate was able to inhibit calcium efflux evoked by CCK-8 when we determined directly calcium transport across plasma membrane using Calcium Green-5N hexapotassium salt (cell impermeant form) in cell populations. The effect of vanadate on calcium extrusion was strongly blocked by the sulfhydryl-reducing agent dithiothreitol (DTT). The present results demonstrate that vanadate is able to irreversibly inhibit the calcium extrusion. This effect of vanadate can be blocked using DTT, indicating that its action is probably mediated by oxidation of sulfhydryl groups of Ca2+-ATPases.     

Cyclic AMP accelerates calcium waves in pancreatic acinar cells

Cytosolic Ca(2+) (Ca(i)(2+)) flux within the pancreatic acinar cell is important both physiologically and pathologically. We examined the role of cAMP in shaping the apical-to-basal Ca(2+) wave generated by the Ca(2+)-activating agonist carbachol. We hypothesized that cAMP modulates intra-acinar Ca(2+) channel opening by affecting either cAMP-dependent protein kinase (PKA) or exchange protein directly activated by cAMP (Epac). Isolated pancreatic acinar cells from rats were stimulated with carbachol (1 muM) with or without vasoactive intestinal polypeptide (VIP) or 8-bromo-cAMP (8-Br-cAMP), and then Ca(i)(2+) was monitored by confocal laser-scanning microscopy. The apical-to-basal carbachol (1 muM)-stimulated Ca(2+) wave was 8.63 +/- 0.68 microm/s; it increased to 19.66 +/- 2.22 microm/s (*P < 0.0005) with VIP (100 nM), and similar increases were observed with 8-Br-cAMP (100 microM). The Ca(2+) rise time after carbachol stimulation was reduced in both regions but to a greater degree in the basal. Lag time and maximal Ca(2+) elevation were not significantly affected by cAMP. The effect of cAMP on Ca(2+) waves also did not appear to depend on extracellular Ca(2+). However, the ryanodine receptor (RyR) inhibitor dantrolene (100 microM) reduced the cAMP-enhancement of wave speed. It was also reduced by the PKA inhibitor PKI (1 microM). 8-(4-chloro-phenylthio)-2'-O-Me-cAMP, a specific agonist of Epac, caused a similar increase as 8-Br-cAMP or VIP. These data suggest that cAMP accelerates the speed of the Ca(2+) wave in pancreatic acinar cells. A likely target of this modulation is the RyR, and these effects are mediated independently by PKA and Epac pathways.     

Quantal size is dependent on stimulation frequency and calcium entry in calf chromaffin cells

To what extent the quantal hypothesis of transmitter release applies to dense-core vesicle (DCV) secretion is unknown. We determined the characteristics of individual secretory events in calf chromaffin cells using catecholamine amperometry combined with different patterns of stimulation. Raising the frequency of action potential trains from 0.25-10 Hz in 2 mM [Ca(2+)]o or [Ca(2+)]o from 0.25-7 mM at 7 Hz elevated the amount released per event (quantal size). With increased stimulation, quantal size rose continuously, not abruptly, suggesting that release efficiency from a single population of DCVs rather than recruitment of different-sized vesicles contributed to the effect. These results suggest that catecholamine secretion does not conform to the quantal model. Inhibition of rapid endocytosis damped secretion in successive episodes, implying an essential role for this process in the recycling of vesicles needed for continuous secretion.     

Modelling mechanism of calcium oscillations in pancreatic acinar cells

We present a simple model for calcium oscillations in the pancreatic acinar cells. This model is based on the calcium release from two receptors, inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) through the process of calcium induced calcium release (CICR). In pancreatic acinar cells, when the Ca²⁺ concentration increases, the mitochondria uptake it very fast to restrict Ca²⁺ response in the cell. Afterwards, a much slower release of Ca²⁺ from the mitochondria serves as a calcium supply in the cytosol which causes calcium oscillations. In this paper we discuss a possible mechanism for calcium oscillations based on the interplay among the three calcium stores in the cell: the endoplasmic reticulum (ER), mitochondria and cytosol. Our model predicts that calcium shuttling between ER and mitochondria is a pacemaker role in the generation of Ca²⁺oscillations. We also consider the calcium dependent production and degradation of (1,4,5) inositol-trisphosphate (IP₃), which is a key source of intracellular calcium oscillations in pancreatic acinar cells. In this study we are able to predict the different patterns of calcium oscillations in the cell from sinusoidal to raised-baseline, high frequency and low-frequency baseline spiking.     

Folate deficiency and pancreatic acinar cell function

The present study was designed to determine the effect of folate deficiency on pancreatic acinar cell function. In the first series of experiments, three groups of rats were fed ad libitum regular rat feed, folate-deficient diet, or an equivalent amount of folate-sufficient diet. In the second series of experiments, rats were either fed ad libitum or rendered folate deficient by a purified folate-deficient diet; half of the folate-deficient group was replenished with oral folate. Body weight, pancreatic weight, DNA [methyl-14C]thymidine incorporation into DNA, RNA, [8-14C]adenine incorporation into RNA, protein content, synthesis of proteins, amylase content, and basal and bethanechol-stimulated amylase secretion were determined. The parameters were the same in the rats fed a folate-sufficient diet as in those fed a regular rat feed. Feeding a folate-deficient diet resulted in impaired DNA synthesis as evidenced by diminished incorporation of [methyl-14C]thymidine into DNA. There was no change in secretion of amylase. Similar results were obtained in the second series of experiments. These studies indicate that folate deficiency (rather than antibiotic content of the diet) impaired pancreatic function. Folate deficiency may therefore contribute to pancreatic injury in malnutrition and alcoholism.     

Secretagogue induced calcium mobilization in single pancreatic acinar cells

Microspectrofluorometry of fura-2 was utilized to monitor [Ca2+]i in single acinar cells stimulated with a cholinergic agonist and cholecystokinin. A similar amplitude of agonist induced Ca mobilization between single cell and populational approaches was observed. New findings in single cells not observable in populations of cells include: 1) the maintenance of a sustained elevation in [Ca2+]i above basal levels throughout agonist application, 2) the reloading of the agonist-sensitive Ca pool only following removal of the agonist and 3) the presence of oscillations of [Ca2+]i in response to agonist application which is enhanced at lower agonist concentrations.     

Extracellular calcium and the organization of tight junctions in pancreatic acinar cells

In pancreatic lobules incubated in Ca²⁺-free Krebs-Ringer bicarbonate solution +0.5 mM EGTA tight junctions are first disarrayed and then break up into fasciae occludentes and small fibrillar fragments, which move laterally in the plane of the plasmalemma and often wind up around the gap junctions. The interruption of the continuity of tight junctions results in the disappearance of the difference in intramembranous particle density between the lateral and luminal regions of the plasmalemma. These results are consistent with the interpretation of tight junctions as dynamic structures, probably resulting from a specific polymerization of intramembranous particles and confirm that tight junctions might have a role in establishing and maintaining the regional differences of the plasmalemma.     

Recent developments in non-excitable cell calcium entry.

Influx of calcium into cells following stimulation of cell surface receptors is a key process controlling cellular activity. However, despite intensive research, there is still no consensus on precisely how calcium entry is controlled in electrically no n-excitable cells. In particular, the regulation of depletion-activated or 'capacitative' calcium entry continues to be a focus of debate. Work published in the last 2 years has lent new impetus to the so-called 'conformational coupling' theory, although evidence for the existence of soluble messengers between the ER and the plasma membrane also continues to appear. In addition, there remains disagreement on whether intra-store [Ca(2+)] has to fall below a threshold before Ca(2+)entry is activated. A further major question is the identity of the putative depletion-operated Ca(2+)channel or channels. Here discussion has largely focussed on whether homologue(s) of the Drosophila TRP ('Transient Receptor Potential') protein is/are the elusive channel, or at least a part of it. Finally, it remains possible that Ca(2+)entry mechanisms other than depletion-activated channels may be important in agonist-evoked Ca(2+)influx. This commentary summarizes recent developments in the field, and highlights both current debates and critical unsolved questions.     

A model of calcium waves in pancreatic and parotid acinar cells

We construct a mathematical model of Ca(2+) wave propagation in pancreatic and parotid acinar cells. Ca(2+) release is via inositol trisphosphate receptors and ryanodine receptors that are distributed heterogeneously through the cell. The apical and basal regions are separated by a region containing the mitochondria. In response to a whole-cell, homogeneous application of inositol trisphosphate (IP(3)), the model predicts that 1), at lower concentrations of IP(3), the intracellular waves in pancreatic cells begin in the apical region and are actively propagated across the basal region by Ca(2+) release through ryanodine receptors; 2), at higher [IP(3)], the waves in pancreatic and parotid cells are not true waves but rather apparent waves, formed as the result of sequential activation of inositol trisphosphate receptors in the apical and basal regions; 3), the differences in wave propagation in pancreatic and parotid cells can be explained in part by differences in inositol trisphosphate receptor density; 4), in pancreatic cells, increased Ca(2+) uptake by the mitochondria is capable of restricting Ca(2+) responses to the apical region, but that this happens only for a relatively narrow range of [IP(3)]; and 5), at higher [IP(3)], the apical and basal regions of the cell act as coupled Ca(2+) oscillators, with the basal region partially entrained to the apical region.     

Compartmentation of calcium in digitonin-disrupted guinea pig pancreatic acinar cells

The treatment of guinea pig pancreatic acinar cells with digitonin leads to disruption of the plasma membrane, as judged by the liberation of cytosolic enzymes, without significant alteration of the mitochondrial membrane. The transport of calcium by the particulate residue was studied, and two different pools could be distinguished. One was supported by ATP or ADP, succinate providing the respiratory substrate, and was sensitive to the inhibitors, Ruthenium red and azide. The other pool needed the presence of ATP, ADP being ineffective, and also was unaffected by Ruthenium red or by azide, but was stimulated several-fold by oxalate. The Ruthenium red-sensitive calcium pool has characteristics resembling those of the transport of calcium by a mitochondrial fraction prepared from digitonin-treated acinar cells. In contrast, the Ruthenium red-insensitive calcium transport has characteristics resembling those of a microsomal fraction obtained from guinea pig pancreas. When the transport of calcium in digitonized cells was assayed at a calcium concentration range of 10(-8)-10(-4) M, preferential Ruthenium red-insensitive calcium transport could be observed at submicromolar calcium concentrations.     

Identification of SNAREs involved in regulated exocytosis in the pancreatic acinar cell.

The molecular basis of exocytotic membrane fusion in the pancreatic acinar cell was investigated using an in vitro assay that measures both zymogen granule-plasma membrane fusion and granule-granule fusion. These two fusion events were differentially sensitive to Ca(2+), suggesting that they are controlled by different Ca(2+)-sensing mechanisms. Botulinum neurotoxin C (BoNT/C) treatment of the plasma membranes caused cleavage of syntaxin 2, the apical isoform of this Q-SNARE, but did not affect syntaxin 4, the basolateral isoform. BoNT/C also cleaved syntaxin 3, the zymogen granule isoform. BoNT/C treatment of plasma membranes abolished granule-plasma membrane fusion, whereas toxin treatment of the granules reduced granule-plasma membrane fusion and abolished granule-granule fusion. Tetanus toxin cleaved granule-associated synaptobrevin 2 but caused only a small reduction in both granule-plasma membrane fusion and granule-granule fusion. Our results indicate that syntaxin 2 is the isoform that mediates fusion between zymogen granules and the apical plasma membrane of the acinar cell. Syntaxin 3 mediates granule-granule fusion, which might be involved in compound exocytosis. In contrast, the major R-SNARE on the zymogen granule remains to be identified.