Ca2+ release induced by cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is the most potent Ca²⁺-mobilizing agent known. It has been found in many different cell types, where it is synthesized from its precursor NAD⁺ by ADP-ribosyl cyclases. cADPR binds to Ca²⁺ channels in the endoplasmic reticulum membrane to activate a Ca²⁺-release mechanism. This release is itself potentiated by elevated cytoplasmic Ca²⁺ concentrations. Thus, cADPR may function as an endogenous regulator of Ca²⁺-induced Ca²⁺ release, and there is excitement that it may also function as a Ca²⁺-mobilizing second messenger.     

Bombesin-induced cytosolic Ca2+ spiking in pancreatic acinar cells depends on cyclic ADP-ribose and ryanodine receptors

Different hormones and neurotransmitters, using Ca2+ as their intracellular messenger, can generate specific cytosolic Ca2+ signals in different parts of a cell. In mouse pancreatic acinar cells, cytosolic Ca2+ oscillations are triggered by activation of acetylcholine (ACh), cholecystokinin (CCK) and bombesin receptors. Low concentrations of these three agonists all induce local Ca(2+)spikes, but in the case of bombesin and CCK these spikes can also trigger global Ca2+ signals. Here we monitor cytosolic Ca2+ oscillations induced by low (2-5 pM) concentrations of bombesin and show that, like ACh- and CCK-induced oscillations, the bombesin-elicited responses are inhibited by ryanodine(50 microM). We then demonstrate that, like CCK- but unlike ACh-induced oscillations, the responses to bombesin are abolished by intracellular infusion of the cyclic ADP ribose (cADPr) antagonist 8-NH2-cADPr (20 microM). We conclude that in mouse pancreatic acinar cells, bombesin, CCK and ACh all produce local Ca2+ spikes by recruiting common oscillator units composed of ryanodine and inositol trisphosphate receptors. However, bombesin and CCK also recruit cADPr receptors, which may account for the global Ca2+ signals that can be evoked by these two agonists. Our new results indicate that each Ca2+ -mobilizing agonist, acting on mouse pancreatic acinar cells, recruits a unique combination of intracellular Ca2+ channels.     

Ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by calmodulin

The effects of calmodulin (CaM) and CaM antagonists on microsomal Ca(2+) release through a ryanodine-sensitive mechanism were investigated in rat pancreatic acinar cells. When caffeine (10 mM) was added after a steady state of ATP-dependent (45)Ca(2+) uptake into the microsomal vesicles, the caffeine-induced (45)Ca(2+) release was significantly increased by pretreatment with ryanodine (10 microM). The presence of W-7 (60 microM), a potent inhibitor of CaM, strongly inhibited the release, while W-5 (60 microM), an inactive CaM antagonist, showed no inhibition. Inhibition of the release by W-7 was observed at all caffeine concentrations (5-30 mM) tested. The presence of exogenously added CaM (10 microg/ml) markedly increased the caffeine (5-10 mM)-induced (45)Ca(2+) release and shifted the dose-response curve of caffeine-induced (45)Ca(2+) release to the left. Cyclic ADP-ribose (cADPR, 2 microM)-induced (45)Ca(2+) release was enhanced by the presence of ryanodine (10 microM). cADPR (2 microM)- or ryanodine (500 microM)-induced (45)Ca(2+) release was also inhibited by W-7 (60 microM), but not by W-5 (60 microM), and was stimulated by CaM (10 microg/ml). These results suggest that the ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by CaM.     

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.     

Endoplasmic reticulum Ca2+ store: regulation of Ca2+ release and reuptake by intracellular and extracellular Ca2+ in pancreatic acinar cells

We investigated the effect of cytosolic and extracellular Ca2+ on Ca2+ signals in pancreatic acinar cells by measuring Ca2+ concentration in the cytosol([Ca2+]c) and in the lumen of the ER([Ca2+]Lu). To control buffers and dye in the cytosol, a patch-clamp microelectrode was employed. Acetylcholine released Ca2+ mainly from the basolateral ER-rich part of the cell. The rate of Ca2+ release from the ER was highly sensitive to the buffering of [Ca2+]c whereas ER Ca2+ refilling was enhanced by supplying free Ca2+ to the cytosol with [Ca2+]c clamped at resting levels with a patch pipette containing 10 mM BAPTA and 2 mM Ca2+. Elevation of extracellular Ca2+ to 10 mM from 1 mM raised resting [Ca2+]c slightly and often generated [Ca2+]c oscillations in single or clustered cells. Although pancreatic acinar cells are reported to have extracellular Ca2+-sensing receptors linked to phospholipase C that mobilize Ca2+ from the ER, exposure of cells to 10 mM Ca2+ did not decrease [Ca2+]Lu but rather raised it. From these findings we conclude that 1) ER Ca2+ release is strictly regulated by feedback inhibition of [Ca2+]c, 2) ER Ca2+ refilling is determined by the rate of Ca2+ influx and occurs mainly in the tiny subplasmalemmal spaces, 3) extracellular Ca2+-induced [Ca2+]c oscillations appear to be triggered not by activation of extracellular Ca2+-sensing receptors but by the ER sensitised by elevated [Ca2+]c and [Ca2+]Lu.     

IP(3)and cyclic ADP-ribose induced Ca(2+) release from intracellular stores of pancreatic acinar cells from rat in primary culture

We have measured Ca(2+)concentration changes in intracellular Ca(2+)stores ([Ca(2+)](store)) of rat pancreatic acinar cells in primary culture in response to the Ca(2+)mobilizing substances inositol-1,4,5-trisphosphate (IP(3)) and cyclic ADP-ribose (cADPr) using the Ca(2+)-sensitive dye mag Fura-2. We found that in this cell model IP(3)releases Ca(2+)in a quantal manner. Higher Ca(2+)concentration in the stores allowed a response to lower IP(3)concentrations ([IP(3)]) indicating that the sensitivity of IP(3)receptors to IP(3)is regulated by the Ca(2+)concentration in the stores. Cyclic ADPr, that modifies 'Ca(2+)-induced-Ca(2+)-release' (CICR), was also able to release Ca(2+)from intracellular stores of pancreatic acinar cells in primary culture. In comparison to the Ca(2+)ionophore ionomycin, which induced a maximal decrease (100%) in [Ca(2+)](store), a hypermaximal [IP(3)] (10 microM) dropped [Ca(2+)](store)by 87% and cADPr had no further effect. Cyclic ADPr reduced [Ca(2+)](store)by only 56% and subsequent IP(3)addition caused further maximal decrease in [Ca(2+)](store). Furthermore, a maximal [IP(3)] caused the same decrease in [Ca(2+)](store)in all regions of the cell, whereas cADPr dropped the [Ca(2+)](store)between 20 and 80% in different cell regions. From these data we conclude that in primary cultured rat pancreatic acinar cells at least three types of Ca(2+)stores exist. One type possessing both cADPr receptors and IP(3)receptors, a second type possessing only IP(3)receptors, and a third type whose Ca(2+)can be released by ionomycin but neither by IP(3)nor by cADPr.     

Effect of GTP and Ca2+ on inositol 1,4,5-trisphosphate induced Ca2+ release from permeabilized rat exocrine pancreatic acinar cells

The effects of Ca2+ and GTP on the release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3) sensitive Ca2+ compartment were investigated with digitonin permeabilized rat pancreatic acinar cells. The amount of Ca2+ released due to IP3 directly correlated with the amount of stored Ca2+ and was found to be inversely proportional to the medium free Ca2+ concentration. Ca2+ release induced by 0.18 microM IP3 was half maximally inhibited at 0.5 microM free Ca2+, i.e. at concentrations observed in the cytosol of pancreatic acinar cells. GTP did not cause Ca2+ release on its own, but a single addition of GTP (20 microM) abolished the apparent desensitization of the Ca2+ release which was observed during repeated IP3 applications. This effect of GTP was reversible. GTP gamma S could not replace GTP. Desensitization still occurred when GTP gamma S was added prior to GTP. The reported data indicate that GTP, stored Ca2+ and cytosolic free Ca2+ modulate the IP3 induced Ca2+ release.     

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells

We have examined the effect of the Ca²⁺ (Mg²⁺)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent ⁴⁵Ca²⁺ uptake into IP₃-sensitive Ca²⁺ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca²⁺ uptake was inhibited by TG up to 10 nm (apparent K_i4.2 nm, Ca²⁺ pool I). An additional increase of inhibition up to 85–90% of total Ca²⁺ uptake could be achieved at 15 to 20 nm of TG (apparent K_i12.1 nm, Ca²⁺ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent K_i10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca²⁺ uptake. About 30–40% of total Ca²⁺ uptake was inhibited by 100 m of vanadate (apparent K_i18 m, Ca²⁺ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent K_i300 m) or by TG up to 10 nm (Ca²⁺ pool I). The amount of IP₃-induced Ca²⁺ release was constant at 25% over a wide range of Ca²⁺ filling. About 10–20% remained unreleasable by IP₃. Reduction of IP₃ releasable Ca²⁺ in the presence of inhibitors showed similar dose-response curves as Ca²⁺ uptake (apparent K_i 3.0 nm for IP₃-induced Ca²⁺ release as compared to 4.2 nm for Ca²⁺ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca²⁺ pump fills the IP₃-sensitive Ca²⁺ pool I. At TG concentrations >10 nm which blocked Ca²⁺ pool II the apparent K_i values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent K_i values were 18 m for Ca²⁺ uptake and 7 m for Ca²⁺ release (Ca²⁺ pool II). At vanadate concentrations up to 1 mm the apparent K_i values were 300 and 200 m, respectively (Ca²⁺ pool I). Both Ca²⁺ pools I and II also showed different sensitivities to IP₃. Dose-response curves for IP₃ in the absence of inhibitors (control) showed an apparent K_m value for IP₃ at 0.6 m. In the presence of TG (inhibition of Ca²⁺ pool I) the curve was shifted to the left with an apparent K_m for IP₃ at 0.08 m. In the presence of vanadate (inhibition of Ca²⁺ pool II), the apparent K_m for IP₃ was 2.1 m. These data allow the conclusion that there are at least three different Ca²⁺ uptake mechanisms present in pancreatic acinar cells: TG- and IP₃ insensitive but highly vanadate-sensitive Ca²⁺ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca²⁺ pools with different TG-, vanadate- and IP₃-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.     

Monoacetyldiglycerides as new Ca2+ mobilizing agents in rat pancreatic acinar cells

Several monoacetyldiglycerides were synthesized from glycerol in search for new Ca²⁺ mobilizing agent in vitro. All monoacetyldiglycerides except linolenoyl and phenlycyclopropylcarbonyl derivatives showed activity toward Ca²⁺ release in pancreatic acinar cells. Linoleoyl and docosahexaenoyl derivatives were chosen for further test and exhibited unique activity.     

Ca2(+)-sensitivity of inositol 1,4,5-trisphosphate-mediated Ca2+ release in permeabilized pancreatic acinar cells.

Hormonal and phorbol ester pretreatment of pancreatic acinar cells markedly decreases the Ins(1,4,5)P3-induced release of actively stored Ca2+ [Willems, Van Den Broek, Van Os & De Pont (1989) J. Biol. Chem. 264, 9762-9767]. Inhibition occurred at an ambient free Ca2+ concentration of 0.1 microM, suggesting a receptor-mediated increase in Ca2(+)-sensitivity of the Ins(1,4,5)P3-operated Ca2+ channel. To test this hypothesis, the Ca2(+)-dependence of Ins(1,4,5)P3-induced Ca2+ release was investigated. In the presence of 0.2 microM free Ca2+, permeabilized cells accumulated 0.9 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Uptake into this pool increased 2.2- and 3.3-fold with 1.0 and 2.0 microM free Ca2+ respectively. At 0.2, 1.0 and 2.0 microM free Ca2+, Ins(1,4,5)P3 maximally released 0.53 (56%), 0.90 (44%) and 0.62 (20%) nmol of Ca2+/mg of acinar protein respectively. Corresponding half-maximal stimulatory Ins(1,4,5)P3 concentrations were calculated to be 0.5, 0.6 and 1.4 microM, suggesting that the affinity of Ins(1,4,5)P3 for its receptor decreases beyond 1.0 microM free Ca2+. The possibility that an inhibitory effect of sub-micromolar Ca2+ is being masked by the concomitant increase in size of the releasable store is excluded, since Ca2+ release from cells loaded in the presence of 0.1 or 0.2 microM free Ca2+ and stimulated at higher ambient free Ca2+ was not inhibited below 1.0 microM free Ca2+. At 2.0 and 10.0 microM free Ca2+, Ca2+, Ca2+ release was inhibited by approx. 30% and 75% respectively. The results presented show that hormonal pretreatment does not lead to an increase in Ca2(+)-sensitivity of the release mechanism. Such an increase in Ca2(+)-sensitivity to sub-micromolar Ca2+ is required to explain sub-micromolar oscillatory changes in cytosolic free Ca2+ by a Ca2(+)-dependent negative-feedback mechanism.     

Evidence for an alteration in the microsomal Ca2+ release mechanism in senescent rat parotid acinar cells

Previously, we have shown that Ca2+ mobilization following an alpha 1-adrenergic receptor stimulus is reduced in parotid acinar cells from senescent rats as a result of an altered ability of inositol 1,4,5-trisphosphate (IP3) to induce Ca2+ release from a non-mitochondrial, intracellular Ca2+ store (Ishikawa, Y., et al. Biochim. Biophys. Acta 968, 203-210). We have used this model to examine the IP3-induced Ca2+ release mechanism in these cells. 45Ca2+ efflux, after exposure to (-) epinephrine, from cells of young adult (3-6 months) rats was approx. 2-fold that observed from cells from older animals (approx. 24 months) either in the presence or absence of extracellular Ca2+. Similarly, cytosolic Ca2+ levels were greater in cells of young adult rats under these same incubation conditions. However, microsomal membrane preparations, from both age groups displayed similar IP3 binding sites (Kd approximately 90 nM, Bmax approximately 850 fmol/mg protein) and ATP-dependent Ca2+ transport ability (approx. 8 nmol/mg protein.min -1). These data suggest that there is an alteration in the IP3-induced Ca2+ release mechanism in microsomal membranes of parotid glands from senescent rats which may account for the decreased Ca2+ release seen after agonist stimulation of this tissue.     

Na+/Ca2+ countertransport in plasma membrane of rat pancreatic acinar cells

Summary The presence of a coupled Na⁺/Ca²⁺ exchange system has been demonstrated in plasma membrane vesicles from rat pancreatic acinar cells. Na⁺/Ca²⁺ exchange was investigated by measuring⁴⁵Ca²⁺ uptake and⁴⁵Ca²⁺ efflux in the presence of sodium gradients and at different electrical potential differences across the membrane (=) in the presence of sodium. Plasma membranes were prepared by a MgCl₂ precipitation method and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the plasma membrane, (Na⁺+K⁺)-ATPase was enriched by 23-fold. Markers for the endoplasmic reticulum, such as RNA and NADPH cytochromec reductase, as well as for mitochondria, the cytochromec oxidase, were reduced by twofold, threefold and 10-fold, respectively. For the Na⁺/Ca²⁺ countertransport system, the Ca²⁺ uptake after 1 min of incubation was half-maximal at 0.62 mol/liter Ca²⁺ and at 20 mmol/liter Na⁺ concentration and maximal at 10 mol/liter Ca²⁺ and 150 mmol/liter Na⁺ concentration, respecitively. When Na⁺ was replaced by Li⁺, maximal Ca²⁺ uptake was 75% as compared to that in the presence of Na⁺. Amiloride (10^–3 mol/liter) at 200 mmol/liter Na⁺ did not inhibit Na⁺/Ca²⁺ countertransport, whereas at low Na⁺ concentration (25 mmol/liter) amiloride exhibited dose-dependent inhibition to be 62% at 10^–2 mol/liter. CFCCP (10^–5 mol/liter) did not influence Na⁺/Ca²⁺ countertransport. Monensin inhibited dose dependently; at a concentration of 5×10^–6 mol/liter inhibition was 80%. A SCN^– or K⁺ diffusion potential (=), being positive at the vesicle inside, stimulated calcium uptake in the presence of sodium suggesting that Na⁺/Ca²⁺ countertransport operates electrogenically, i.e. with a stoichiometry higher than 2 Na⁺ for 1 Ca²⁺. In the absence of Na⁺, did not promote Ca²⁺ uptake. We conclude that in addition to ATP-dependent Ca²⁺ outward transport as characterized previously (E. Bayerdörffer, L. Eckhardt, W. Haase & 1. Schulz, 1985,J. Membrane Biol. 84:45–60) the Na⁺/Ca²⁺ countertransport system, as characterized in this study, represents a second transport system for the extrusion of calcium from the cell. Furthermore, the high affinity for calcium suggests that this system might participate in the regulation of the cytosolic free Ca²⁺ level.     

Role of extracellular Ca2+ in acetylcholine-induced repetitive Ca2+ release in submandibular gland acinar cells of the rat

Acetylcholine (ACh) caused repetitive transient Cl⁻ currents activated by intracellular Ca²⁺ in single rat submandibular grand acinar cells. As the concentration of ACh increased the amplitude and the frequency of the transient Cl⁻ currents increased. These responses occurred also in the absence of extracellular Ca²⁺ but disappeared after several minutes. Repetitive transient Cl⁻ currents were restored by readmission of Ca²⁺ to the extracellular solution. The higher the concentration of extracellular Ca²⁺ readmitted, the larger the amplitude of the transient Cl⁻ currents. Ca²⁺ entry through a store-coupled pathway was detected by application of Ca²⁺ to the extracellular solution during a brief cessation of stimulation with ACh. In these experiments too, the higher the concentration of Ca²⁺, the larger the transient Cl⁻ currents activated by Ca²⁺ released from the stores. The time course of decrease in total charge movements of repetitive transient responses to ACh with removal of extracellular Ca²⁺ depended on a decrease in charge movements of each transient event rather than a decrease in frequency of the repetitive events. The decrease of charge movements of each transient event was due to a decrease in its amplitude rather than its duration. The results suggest that in this cell type an amplitude-modulated mechanism is involved in repetitive Ca²⁺ release and that Ca²⁺ entry is essential to maintain the repetitive release of Ca²⁺. The results further suggest that the magnitude of Ca²⁺ entry determines the number of unitary stores filled with Ca²⁺ which can synchronously respond to ACh. © 1996 Wiley-Liss, Inc.     

Ca2+ release dynamics in parotid and pancreatic exocrine acinar cells evoked by spatially limited flash photolysis

Intracellular calcium concentration ([Ca(2+)](i)) signals are central to the mechanisms underlying fluid and protein secretion in pancreatic and parotid acinar cells. Calcium release was studied in natively buffered cells following focal laser photolysis of caged molecules. Focal photolysis of caged-inositol 1,4,5 trisphosphate (InsP(3)) in the apical region resulted in Ca(2+) release from the apical trigger zone and, after a latent period, the initiation of an apical-to-basal Ca(2+) wave. The latency was longer and the wave speed significantly slower in pancreatic compared with parotid cells. Focal photolysis in basal regions evoked only limited Ca(2+) release at the photolysis site and never resulted in a propagating wave. Instead, an apical-to-basal wave was initiated following a latent period. Again, the latent period was significantly longer under all conditions in pancreas than parotid. Although slower in pancreas than parotid, once initiated, the apical-to-basal wave speed was constant in a particular cell type. Photo release of caged-Ca(2+) failed to evoke a propagating Ca(2+) wave in either cell type. However, the kinetics of the Ca(2+) signal evoked following photolysis of caged-InsP(3) were significantly dampened by ryanodine in parotid but not pancreas, indicating a more prominent functional role for ryanodine receptor (RyR) following InsP(3) receptor (InsP(3)R) activation. These data suggest that differing expression levels of InsP(3)R, RyR, and possibly cellular buffering capacity may contribute to the fast kinetics of Ca(2+) signals in parotid compared with pancreas. These properties may represent a specialization of the cell type to effectively stimulate Ca(2+)-dependent effectors important for the differing primary physiological role of each gland.     

Relationship between secretagogue-induced Ca2+ release and inositol polyphosphate production in permeabilized pancreatic acinar cells

We have previously shown that inositol trisphosphate (IP3) releases Ca2+ from a nonmitochondrial pool of permeabilized rat pancreatic acinar cells (Streb, H., Irvine, R. F., Berridge, M. J., and Schulz, I. (1984) Nature 306, 67-69). This pool was later identified as endoplasmic reticulum (Streb, H., Bayerdorffer, E., Haase, W., Irvine, R. F., and Schulz, I. (1984) J. Membr. Biol. 81, 241-253). As IP3 is produced by hydrolysis of phosphatidylinositol bisphosphate on activation of many "Ca2+-mobilizing receptors," our observation supported the proposal that IP3 functions as a second messenger to release Ca2+ from the endoplasmic reticulum. We have here used the same preparation of permeabilized acinar cells to study the relationship of secretagogue-induced Ca2+ release and IP3 production. We show that: 1) secretagogue-induced Ca2+ release in permeabilized cells is accompanied by a parallel production of inositol trisphosphate. 2) When the secretagogue-induced increase in intracellular free Ca2+ concentration was abolished by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering, secretagogue-induced IP3 production was unimpaired. 3) When secretagogue-induced IP3 production was reduced by inhibiting phospholipase C with neomycin, secretagogue-induced Ca2+ release was also abolished. 4) When the IP3 breakdown was reduced either by lowering the free Mg2+ concentration of the incubation medium or by adding 2.3-diphosphoglyceric acid, the rise in IP3 and the release of Ca2+ induced by secretagogues were both increased. These results further support the role of IP3 as a second messenger to induce Ca2+ mobilization.     

FK506 blocks intracellular Ca2+ oscillations in bovine adrenal glomerulosa cells

The inositol 1,4,5-trisphosphate (InsP(3)) receptor is a ligand-gated Ca(2+) channel playing an important role in the control of intracellular Ca(2+). In the study presented here, we demonstrate that angiotensin (AngII), phorbol ester (PMA), and FK506 significantly increase the level of InsP(3) receptor phosphorylation in intact bovine adrenal glomerulosa cells. With a back-phosphorylation approach, we showed that the InsP(3) receptor is a good substrate for protein kinase C (PKC) and that FK506 increases the level of PKC-mediated InsP(3) receptor phosphorylation. With a microsomal preparation from bovine adrenal cortex, we showed that PKC enhances the release of Ca(2+) induced by a submaximal dose of InsP(3). We also showed that FK506 blocks intracellular Ca(2+) oscillations in isolated adrenal glomerulosa cells by progressively increasing the intracellular Ca(2+) concentration to a high plateau level. This effect is consistent with an inhibitory role of FK506 on calcineurin dephosphorylation of the InsP(3) receptor, thus keeping the receptor in a phosphorylated, high-conductance state. Our results provide further evidence for the crucial role of the InsP(3) receptor in the regulation of intracellular Ca(2+) oscillations and show that FK506, by maintaining the phosphorylated state of the InsP(3) receptor, causes important changes in the Ca(2+) oscillatory process.     

Free cytoplasmic Ca2+ concentration oscillations in thapsigargin-treated parotid acinar cells are caffeine- and ryanodine-sensitive.

The microsomal Ca-ATPase inhibitor thapsigargin induces in rat salivary acinar cells [Ca2+]i oscillations which, though similar to those activated by agonists, are independent of inositol phosphates or inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ stores (Foskett, J. K., Roifman, C., and Wong, D. (1991) J. Biol. Chem. 266, 2778-2782). To examine whether the oscillation mechanism resides in another, thapsigargin- and IP3-insensitive intracellular store, we examined the effects of caffeine and ryanodine, known modulators of Ca2+ release from sarcoplasmic reticulum in excitable cells. Oscillations were induced by caffeine (1-20 mM) in nonoscillating thapsigargin-treated acinar cells, which required the continued presence of caffeine, whereas caffeine was without effect or reduced oscillation amplitude in oscillating cells. Ryanodine (10-50 microM) inhibited oscillations in most of the cells. These results suggest that Ca2+ oscillations in parotid acinar cells are driven by periodic Ca2+ release from an IP3-insensitive Ca2+ store with properties similar to sarcoplasmic reticulum of excitable cells.     

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.