Phosphatidylinositol 3-kinase facilitates bile acid-induced Ca(2+) responses in pancreatic acinar cells

Bile acids are known to induce Ca(2+) signals in pancreatic acinar cells. We have recently shown that phosphatidylinositol 3-kinase (PI3K) regulates changes in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) elicited by CCK by inhibiting sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). The present study sought to determine whether PI3K regulates bile acid-induced [Ca(2+)](i) responses. In pancreatic acinar cells, pharmacological inhibition of PI3K with LY-294002 or wortmannin inhibited [Ca(2+)](i) responses to taurolithocholic acid 3-sulfate (TLC-S) and taurochenodeoxycholate (TCDC). Furthermore, genetic deletion of the PI3K gamma-isoform also decreased [Ca(2+)](i) responses to bile acids. Depletion of CCK-sensitive intracellular Ca(2+) pools or application of caffeine inhibited bile acid-induced [Ca(2+)](i) signals, indicating that bile acids release Ca(2+) from agonist-sensitive endoplasmic reticulum (ER) stores via an inositol (1,4,5)-trisphosphate-dependent mechanism. PI3K inhibitors increased the amount of Ca(2+) in intracellular stores during the exposure of acinar cells to bile acids, suggesting that PI3K negatively regulates SERCA-dependent Ca(2+) reloading into the ER. Bile acids inhibited Ca(2+) reloading into ER in permeabilized acinar cells. This effect was augmented by phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), suggesting that both bile acids and PI3K act synergistically to inhibit SERCA. Furthermore, inhibition of PI3K by LY-294002 completely inhibited trypsinogen activation caused by the bile acid TLC-S. Our results indicate that PI3K and its product, PIP(3), facilitate bile acid-induced [Ca(2+)](i) responses in pancreatic acinar cells through inhibition of SERCA-dependent Ca(2+) reloading into the ER and that bile acid-induced trypsinogen activation is mediated by PI3K. The findings have important implications for the mechanism of acute pancreatitis since [Ca(2+)](i) increases and trypsinogen activation mediate key pathological processes in this disorder.     

Bile acids induce Ca2+ release from both the endoplasmic reticulum and acidic intracellular calcium stores through activation of inositol trisphosphate receptors and ryanodine receptors

Gallstones can cause acute pancreatitis, an often fatal disease in which the pancreas digests itself. This is probably because of biliary reflux into the pancreatic duct and subsequent bile acid action on the acinar cells. Because Ca(2+) toxicity is important for the cellular damage in pancreatitis, we have studied the mechanisms by which the bile acid taurolithocholic acid 3-sulfate (TLC-S) liberates Ca(2+). Using two-photon plasma membrane permeabilization and measurement of [Ca(2+)] inside intracellular stores at the cell base (dominated by ER) and near the apex (dominated by secretory granules), we have characterized the Ca(2+) release pathways. Inhibition of inositol trisphosphate receptors (IP(3)Rs), by caffeine and 2-APB, reduced Ca(2+) release from both the ER and an acidic pool in the granular area. Inhibition of ryanodine receptors (RyRs) by ruthenium red (RR) also reduced TLC-S induced liberation from both stores. Combined inhibition of IP(3)Rs and RyRs abolished Ca(2+) release. RyR activation depends on receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), because inactivation by a high NAADP concentration inhibited release from both stores, whereas a cyclic ADPR-ribose antagonist had no effect. Bile acid-elicited intracellular Ca(2+) liberation from both the ER and the apical acidic stores depends on both RyRs and IP(3)Rs.     

Effects of secretagogues and bile acids on mitochondrial membrane potential of pancreatic acinar cells: comparison of different modes of evaluating DeltaPsim

In this study, we investigated the effects of secretagogues and bile acids on the mitochondrial membrane potential of pancreatic acinar cells. We measured the mitochondrial membrane potential using the tetramethylrhodamine-based probes tetramethylrhodamine ethyl ester and tetramethylrhodamine methyl ester. At low levels of loading, these indicators appeared to have a low sensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and no response was observed to even high doses of cholecystokinin. When loaded at high concentrations, tetramethylrhodamine methyl ester and tetramethylrhodamine ethyl ester undergo quenching and can be dequenched by mitochondrial depolarization. We found the dequench mode to be 2 orders of magnitude more sensitive than the low concentration mode. Using the dequench mode, we resolved mitochondrial depolarizations produced by supramaximal and by physiological concentrations of cholecystokinin. Other calcium-releasing agonists, acetylcholine, JMV-180, and bombesin, also produced mitochondrial depolarization. Secretin, which employs the cAMP pathway, had no effect on the mitochondrial potential; dibutyryl cAMP was also ineffective. The cholecystokinin-induced mitochondrial depolarizations were abolished by buffering cytosolic calcium. A non-agonist-dependent calcium elevation induced by thapsigargin depolarized the mitochondria. These experiments suggest that a cytosolic calcium concentration rise is sufficient for mitochondrial depolarization and that the depolarizing effect of cholecystokinin is mediated by a cytosolic calcium rise. Bile acids are considered possible triggers of acute pancreatitis. The bile acids taurolithocholic acid 3-sulfate, taurodeoxycholic acid, and taurochenodeoxycholic acid, at low submillimolar concentrations, induced mitochondrial depolarization, resolved by the dequench mode. Our experiments demonstrate that physiological concentrations of secretagogues and pathologically relevant concentrations of bile acids trigger mitochondrial depolarization in pancreatic acinar cells.     

A novel role for Bcl-2 in regulation of cellular calcium extrusion

The antiapoptotic protein Bcl-2 plays important roles in Ca(2+) signaling by influencing inositol triphosphate receptors and regulating Ca(2+)-induced Ca(2+) release. Here we investigated whether Bcl-2 affects Ca(2+) extrusion in pancreatic acinar cells. We specifically blocked the Ca(2+) pumps in the endoplasmic reticulum and assessed the rate at which the cells reduced an elevated cytosolic Ca(2+) concentration after a period of enhanced Ca(2+) entry. Because external Ca(2+) was removed and endoplasmic reticulum Ca(2+) pumps were blocked, Ca(2+) extrusion was the only process responsible for recovery. Cells lacking Bcl-2 restored the basal cytosolic Ca(2+) level much faster than control cells. The enhanced Ca(2+) extrusion in cells from Bcl-2 knockout (Bcl-2 KO) mice was not due to increased Na(+)/Ca(2+) exchange activity, because removal of external Na(+) did not influence the Ca(2+) extrusion rate. Overexpression of Bcl-2 in the pancreatic acinar cell line AR42J decreased Ca(2+) extrusion, whereas silencing Bcl-2 expression (siRNA) had the opposite effect. Loss of Bcl-2, while increasing Ca(2+) extrusion, dramatically decreased necrosis and promoted apoptosis induced by oxidative stress, whereas specific inhibition of Ca(2+) pumps in the plasma membrane (PMCA) with caloxin 3A1 reduced Ca(2+) extrusion and increased necrosis. Bcl-2 regulates PMCA function in pancreatic acinar cells and thereby influences cell fate.     

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 μM and a peak-plateau signal at 500 μM, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.     

Regulation of free cytosolic Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. I. Efficiency of transport and interactions between cations.

Regulation of free cytosolic Ca2+ concentration in the rod outer segments (ROS) isolated from bovine retinas was examined with the fluorescent Ca(2+)-indicating dye fluo-3. In situ calibration of cytosolic fluo-3 was done in the presence of the Ca2+ ionophore A23187 and yielded a dissociation constant of 500 nM for the Ca(2+)-fluo-3 complex. Ca2+ influx in Ca(2+)-depleted ROS was completely abolished when internal Na+ was removed suggesting that Ca2+ influx exclusively occurred via Na-Ca-K exchange. The most striking observation was that Na-Ca-K exchange could mediate a rapid increase in cytosolic free Ca2+ over the most of the usable indicating range of fluo-3 (from 10 nM to 2 microM), even when exposed to free external Ca2+ concentrations as low as 10 nM. From a comparison between changes in free Ca2+ and changes in total Ca2+, we conclude that physiologically occurring changes in cytosolic free Ca2+ are mediated by exchange fluxes less than 1% of the maximal Na-Ca-K exchange flux. The Na-Ca-K exchanger could mediate both K(+)-dependent and K(+)-independent Ca2+ influx; Li+ caused a complete inhibition of K(+)-independent Ca2+ influx, but had no effect on K(+)-dependent Ca2+ influx. We examined the complex interactions of alkali cations with Ca2+ influx and discuss the results in terms of a three-site model for the Na-Ca-K exchanger (Schnetkamp, P. P. M. and Szerencsei, R. T. (1991) J. Biol. Chem. 266, 189-197). Ca2+ competed with one Mg2+ ion or two Na+ ions for binding to a common site. High K+ concentration greatly diminished the ability of Na+ and Mg2+ to compete with Ca2+ for this common site on the exchanger protein. As a result, high internal K+ induced a conformation of the exchange protein that kinetically favoured Ca2+ extrusion.     

Changes in the nature of calcium transport systems on the porcine sperm plasma membrane during epididymal maturation.

Comparative studies of 45Ca(2+)-transport across the plasma membrane were performed using porcine caput, corpus and cauda epididymal sperm. The Ca(2+)-uptake is dependent on the presence of the substrates for respiration and is sensitive to verapamil. The Ca(2+)-efflux is mediated by both Na(+)-dependent and -independent systems. In the immature sperm in caput epididymis, Na(+)-independent efflux is predominant, but it is gradually replaced by Na(+)-dependent efflux during the epididymal transit. The net activity of Ca2+ accumulation into sperm increases with the epididymal maturation.     

Ionic regulation of the cardiac sodium-calcium exchanger

The Na(+)-Ca(2+) exchanger (NCX) links transmembrane movements of Ca(2+) ions to the reciprocal movement of Na(+) ions. It normally functions primarily as a Ca(2+) efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca(2+) influx under certain conditions. Na(+) and Ca(2+) ions exert complex regulatory effects on NCX activity. Ca(2+) binds to two regulatory sites in the exchanger's central hydrophilic domain, and this interaction is normally essential for activation of exchange activity. High cytosolic Na(+) concentrations, however, can induce a constitutive activity that by-passes the need for allosteric Ca(2+) activation. Constitutive NCX activity can also be induced by high levels of phopshotidylinositol-4,5-bisphosphate (PIP?) and by mutations affecting the regulatory calcium binding domains. In addition to promoting constitutive activity, high cytosolic Na(+) concentrations also induce an inactivated state of the exchanger (Na(+)-dependent inactivation) that becomes dominant when cytosolic pH and PIP? levels fall. Na(+)-dependent inactivation may provide a means of protecting cells from Ca(2+) overload due to NCX-mediated Ca(2+) influx during ischemia.     

Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate--the role of pH and sodium

Although Zn(2+) homeostasis in neurons is tightly regulated and its destabilization has been linked to a number of pathologies including Alzheimer's disease and ischemic neuronal death, the primary mechanisms affecting intracellular Zn(2+) concentration ([Zn(2+) ](i)) in neurons exposed to excitotoxic stimuli remain poorly understood. The present work addressed these mechanisms in cultured hippocampal neurons exposed to glutamate and glycine (Glu/Gly). [Zn(2+)](i) and intracellular Ca(2+) concentration were monitored simultaneously using FluoZin-3 and Fura-2FF, and intracellular pH (pH(i)) was studied in parallel experiments using 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Glu/Gly applications under Na(+)-free conditions (Na(+) substituted with N-methyl-D-glucamine(+)) caused Ca(2+) influx, pH(i) drop, and Zn(2+) release from intracellular stores. Experimental maneuvers resulting in a pH(i) increase during Glu/Gly applications, such as stimulation of Na(+) -dependent pathways of H(+) efflux, forcing H(+) efflux via gramicidin-formed channels, or increasing extracellular pH counteracted [Zn(2+)](i) elevations. In the absence of Na(+), the rate of [Zn(2+)](i) decrease could be correlated with the rate of pH(i) increase. In the presence of Na(+), the rate of [Zn(2+) ](i) decrease was about twice as fast as expected from the rate of pH(i) elevation. The data suggest that Glu/Gly-induced cytosolic acidification promotes [Zn(2+) ](i) elevations and that Na(+) counteracts the latter by promoting pH(i)-dependent and pH(i)-independent mechanisms of cytosolic Zn(2+) clearance.     

Evidence for bile acid-evoked oscillations of Ca2(+)-dependent K+ permeability unrelated to a D-myo-inositol 1,4,5-trisphosphate effect in isolated guinea pig liver cells

In single liver cells, the D-myo-inositol 1,4,5-triphosphate (InsP3)-dependent agonists such as noradrenaline and angiotensin II evoke oscillations in intracellular calcium [Ca2+]i resulting mostly from the periodic release and reuptake of calcium from intracellular stores. In the present work, we have reexamined the effects of these agonists and investigated whether the natural bile acid taurolithocholic acid 3-sulfate (TLC-S), which permeabilizes the endoplasmic reticulum, could initiate oscillations of [Ca2+]i. Oscillations of [Ca2+]i were monitored with the Ca2(+)-dependent K+ permeability in whole-cell voltage-clamped guinea pig liver cells. Our results confirm the presence of two types of oscillations induced by hormones. They could be distinguished by their frequency periods. The fast (type I) had periods ranging from 5 to 12 s and the slow (type II) from 60 to 240 s. They have been respectively attributed to second messenger- and receptor-controlled oscillations, respectively. Our results also show that TLC-S, as noradrenaline and angiotensin II, induced the activation of this Ca(+)-dependent K+ current and was able to reproduce both types of oscillations. The bile acid effect was not blocked by intracellular perfusion of heparin known to inhibit both InsP3 binding and InsP3-evoked Ca2+ release in several tissues. In these conditions, TLC-S only evoked type I oscillations, suggesting that these fluctuations could originate from a mechanism that is independent of InsP3 and is an intrinsic property of internal Ca2+ stores.     

Calcium signalling and pancreatic cell death: apoptosis or necrosis?

Secretagogues, such as cholecystokinin and acetylcholine, utilise a variety of second messengers (inositol trisphosphate, cADPR and nicotinic acid adenine dinucleotide phosphate) to induce specific oscillatory patterns of calcium (Ca(2+)) signals in pancreatic acinar cells. These are tightly controlled in a spatiotemporal manner, and are coupled to mitochondrial metabolism necessary to fuel secretion. When Ca(2+) homeostasis is disrupted by known precipitants of acute pancreatitis, for example, hyperstimulation or non-oxidative ethanol metabolites, Ca(2+) stores (endoplasmic reticulum and acidic pool) become depleted and sustained cytosolic [Ca(2+)] elevations replace transient signals, leading to severe consequences. Sustained mitochondrial depolarisation, possibly via opening of the mitochondrial permeability transition pore (MPTP), elicits cellular ATP depletion that paralyses energy-dependent Ca(2+) pumps causing cytosolic Ca(2+) overload, while digestive enzymes are activated prematurely within the cell; Ca(2+)-dependent cellular necrosis ensues. However, when stress to the acinar cell is milder, for example, by application of the oxidant menadione, release of Ca(2+) from stores leads to oscillatory global waves, associated with partial mitochondrial depolarisation and transient MPTP opening; apoptotic cell death is promoted via the intrinsic pathway, when associated with generation of reactive oxygen species. Apoptosis, induced by menadione or bile acids, is potentiated by inhibition of an endogenous detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1), suggesting its importance as a defence mechanism that may influence cell fate.     

Differential contribution of plasmalemmal Na/Ca exchange isoforms to sodium-dependent calcium influx and NMDA excitotoxicity in depolarized neurons

Inhibition of Na(+),K(+)-ATPase during NMDA applications greatly increased NMDA-induced excitotoxicity in primary cultures of forebrain neurons (FNs), but not in cerebellar granule cells (CGCs). Because Na(+),K(+)-ATPase inhibition promotes reversal of plasmalemmal Na(+)/Ca(2+) exchangers, we compared the activities of reversed K(+)-independent (NCX) and K(+)-dependent (NCKX) Na(+)/Ca(2+) exchangers in these cultures. To this end, we measured gramicidin-induced and Na(+)-dependent elevation in cytosolic [Ca(2+)] ([Ca(2+)](c)) that represents Ca(2+) influx via reversed NCX and NCKX; NCX activity was dissected out by removing external K(+). The [Ca(2+)](c) elevations mediated by NCX alone, and NCX plus NCKX combined, were 17 and 6 times more rapid in FNs than in CGCs, respectively. Northern blot analysis showed that FNs preferentially express NCX1 whereas CGCs expressed NCX3. Differences in expression of other isoforms (NCX2, NCKX2, NCKX3 and NCKX4) were less pronounced. We tested whether the NCX or NCKX family of exchangers contributes most to the toxic NMDA-induced Ca(2+) influx in depolarized neurons. We found that in FNs, inhibition of NCX alone was sufficient to significantly limit NMDA excitotoxicity, whereas in CGCs, inhibition of both NCX and NCKX was required. The data suggest that the high activity of NCX isoforms expressed in FNs, possibly NCX1, sensitizes these neurons to NMDA excitotoxicity.     

Failure of calcium microdomain generation and pathological consequences

Normal physiological regulation depends on Ca(2+) microdomains, because there is a need to spatially separate Ca(2+) regulation of different cellular processes. It is only possible to generate local Ca(2+) signals transiently; so, there is an important functional link between Ca(2+) spiking and microdomains. The pancreatic acinar cell provides a useful cell biological model, because of its clear structural and functional polarization. Although local Ca(2+) spiking in the apical (granular) microdomain regulates fluid and enzyme secretion, prolonged global elevations of the cytosolic Ca(2+) concentration are associated with the human disease acute pancreatitis, in which proteases in the granular region become inappropriately activated and digest the pancreas and its surroundings. A major cause of pancreatitis is alcohol abuse and it has now been established that fatty acid ethyl esters and fatty acids, non-oxidative alcohol metabolites, are principally responsible for causing the acinar cell damage. The fatty acid ethyl esters release Ca(2+) from the endoplasmic reticulum and the fatty acids inhibit markedly mitochondrial ATP generation, which prevents the acinar cell from disposing of the excess Ca(2+) in the cytosol. Because of the abolition of ATP-dependent Ca(2+) pump activity, all intracellular Ca(2+) concentration gradients disappear and the most important part of the normal regulatory machinery is thereby destroyed. The end stage is necrosis.     

Pancreatic islet function in omega-3 fatty acid-depleted rats: alteration of calcium fluxes and calcium-dependent insulin release

Considering the insufficient supply of long-chain polyunsaturated omega-3 fatty acids often prevailing in Western populations, this report deals mainly with alterations of Ca(2+) fluxes and Ca(2+)-dependent insulin secretory events in isolated pancreatic islets from omega-3-depleted rats. In terms of (45)Ca(2+) handling, the islets from omega-3-depleted rats, compared with those from normal animals, displayed an unaltered responsiveness to an increase in extracellular K(+) concentration, a lower inflow rate and lower fractional outflow rate of the divalent cation, and higher (45)Ca(2+)-labeled cellular pool(s) at isotopic equilibrium. The latter anomaly was corrected 120 min after intravenous injection of a novel medium-chain triglyceride-fish oil (MCT:FO) emulsion, distinct from a control omega-3-poor MCT-olive oil (MCT:OO) emulsion. At 8.3 mM D-glucose, insulin release was higher in islets from omega-3-depleted rats vs. control animals, coinciding with a higher cytosolic Ca(2+) concentration. The relative magnitude of the increase in insulin output attributable to a rise in D-glucose as well as extracellular Ca(2+) or K(+) concentration, to the absence vs. presence of verapamil and to the presence vs. absence of extracellular Ca(2+), theophylline, phorbol 12-myristate 13-acetate, or Ba(2+), was always more pronounced in islets from omega-3-depleted rats injected with the MCT:OO compared with the MCT:FO emulsion. A comparable situation prevailed when comparing islets from noninjected omega-3-depleted and normal rats. In light of these and previous findings, we propose that an impairment of Na(+),K(+)-ATPase activity plays a major, although not an exclusive, role in the perturbation of Ca(2+) fluxes and Ca(2+)-dependent secretory events in the islets from omega-3-depleted rats.     

Mitochondrial calcium transport: mechanisms and functions

Ca(2+)transport across the mitochondrial inner membrane is facilitated by transporters having four distinct sets of characteristics as well as through the Ca(2+)-induced mitochondrial permeability transition pore (PTP). There are two modes of inward transport, referred to as the Ca(2+)uniporter and the rapid mode or RaM. There are also two distinct mechanisms mediating outward transport, which are not associated with the PTP, referred to as the Na(+)-dependent and the Na(+)-independent Ca(2+)efflux mechanisms. Several important functions have been proposed for these mechanisms, including control of the metabolic rate for cellular energy (ATP) production, modulation of the amplitude and shape of cytosolic Ca(2+)transients, and induction of apoptosis through release of cytochrome c from the mitochondrial inter membrane space into the cytosolic space.The goals of this review are to survey the literature describing the characteristics of the mechanisms of mitochondrial Ca(2+)transport and their proposed physiological functions, emphasizing the more recent contributions, and to consider how the observed characteristics of the mitochondrial Ca(2+)transport mechanisms affect our understanding of their functions.     

Identification of cyclic ADP-ribose-dependent mechanisms in pancreatic muscarinic Ca(2+) signaling using CD38 knockout mice

We showed that muscarinic acetylcholine (ACh)-stimulation increased the cellular content of cADPR in the pancreatic acinar cells from normal mice but not in those from CD38 knockout mice. By monitoring ACh-evoked increases in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) using fura-2 microfluorimetry, we distinguished and characterized the Ca(2+) release mechanisms responsive to cADPR. The Ca(2+) response from the cells of the knockout mice (KO cells) lacked two components of the muscarinic Ca(2+) release present in wild mice. The first component inducible by the low concentration of ACh contributed to regenerative Ca(2+) spikes. This component was abolished by ryanodine treatment in the normal cells and was severely impaired in KO cells, indicating that the low ACh-induced regenerative spike responses were caused by cADPR-dependent Ca(2+) release from a pool regulated by a class of ryanodine receptors. The second component inducible by the high concentration of ACh was involved in the phasic Ca(2+) response, and it was not abolished by ryanodine treatment. Overall, we conclude that muscarinic Ca(2+) signaling in pancreatic acinar cells involves a CD38-dependent pathway responsible for two cADPR-dependent Ca(2+) release mechanisms in which the one sensitive to ryanodine plays a crucial role for the generation of repetitive Ca(2+) spikes.     

Agonist-evoked mitochondrial Ca2+ signals in mouse pancreatic acinar cells

In the present study we have investigated cytosolic and mitochondrial Ca(2+) signals in isolated mouse pancreatic acinar cells double-loaded with the fluorescent probes fluo-3 and rhod-2. Stimulation of pancreatic acinar cells with 500 nm acetylcholine caused release of Ca(2+) from intracellular stores and produced cytosolic Ca(2+) signals in form of Ca(2+) waves propagating from the luminal to the basal cell pole. The increase in the cytosolic Ca(2+) concentration was followed by Ca(2+) uptake into mitochondria. Between onset of cytosolic and mitochondrial Ca(2+) signals there was a delay of 10.7 +/- 0.4 s. Ca(2+) uptake into mitochondria could be inhibited with Ruthenium Red and carbonyl cyanide m-chlorophenylhydrazone, whereas 2,5-di-tert-butylhydroquinone, which inhibits sarco(endo)plasmic reticulum Ca(2+) ATPases, did not prevent Ca(2+) accumulation in mitochondria. Carbonyl cyanide m-chlorophenylhydrazone-induced Ca(2+) release from mitochondria could only be observed after a preceding stimulation of the cell with a physiological agonist or by treatment with 2, 5-di-tert-butylhydroquinone, indicating that under resting conditions mitochondria do not contain releasable Ca(2+) ions. Analysis of the propagation rate of acetylcholine-induced Ca(2+) waves revealed that inhibition of mitochondrial Ca(2+) uptake did not accelerate spreading of cytosolic Ca(2+) signals. Our experiments indicate that in the early phase of secretagogue-induced Ca(2+) signals, mitochondria behave as passive Ca(2+)-buffering elements and do not actively suppress spreading of Ca(2+) signals in pancreatic acinar cells.     

Inhibition of sodium-calcium exchange by ceramide and sphingosine

Na(+)/Ca(2+) exchange activity in Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger was inhibited by the short chain ceramide analogs N-acetylsphingosine and N-hexanoylsphingosine (5-15 micrometer). The sphingolipids reduced exchange-mediated Ba(2+) influx by 50-70% and also inhibited the Ca(2+) efflux mode of exchange activity. The biologically inactive ceramide analog N-acetylsphinganine had only modest effects on exchange activity. Cells expressing the Delta(241-680) and Delta(680-685) deletion mutants of the Na(+)/Ca(2+) exchanger were not inhibited by ceramide; these mutants show defects in both Na(+)-dependent and Ca(2+)-dependent regulatory behavior. Another mutant, which was defective only in Na(+)-dependent regulation, was as sensitive to ceramide inhibition as the wild-type exchanger. Inhibition of exchange activity by ceramide was time-dependent and was accelerated by depletion of internal Ca(2+) stores. Sphingosine (2.5 micrometer) also inhibited the Ca(2+) influx and efflux modes of exchange activity in cells expressing the wild-type exchanger; sphingosine did not affect Ba(2+) influx in the Delta(241-680) mutant. The effects of the exogenous sphingolipids were reproduced by blocking cellular ceramide utilization pathways, suggesting that exchange activity is inhibited by increased levels of endogenous ceramide and/or sphingosine. We propose that sphingolipids impair Ca(2+)-dependent activation of the exchanger and that in cardiac myocytes, this process serves as a feedback mechanism that links exchange activity to the diastolic concentration of cytosolic Ca(2+).     

Low affinity cholecystokinin receptor inhibits cholecystokinin- and bombesin-induced oscillations of cytosolic Ca2+ concentration

We have investigated whether low affinity cholecystokinin (CCK) receptors suppress agonist-induced rises of cytosolic free Ca(2+) concentration ([Ca(2+)]c) in pancreatic acinar cells by using properties of caffeine. A high concentration of caffeine (20 mM) completely blocked inositol 1,4,5-trisphosphate (InsP(3))-induced [Ca(2+)]c rises but spared the InsP(3)-independent long-lasting [Ca(2+)]c oscillations. In the presence of 20 mM caffeine, only high concentrations of CCK, but not bombesin or JMV-180, suppressed the caffeine-resistant CCK or bombesin-induced [Ca(2+)]c oscillations, indicating that low affinity CCK receptors inhibit agonist-induced [Ca(2+)]c oscillations. It could be one of the underlying mechanisms by which low affinity CCK receptors suppress secretion in pancreatic acinar cells.