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.     

Phenylarsine oxide evokes intracellular calcium increases and amylase secretion in isolated rat pancreatic acinar cells

The effects of the thiol reagent, phenylarsine oxide (PAO, 10(-5)-10(-3) M ), a membrane-permeable trivalent arsenical compound that specifically complexes vicinal sulfhydryl groups of proteins to form stable ring structures, were studied by monitoring intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. PAO increased [Ca2+]i by mobilizing calcium from intracellular stores, since this increase was observed in the absence of extracellular calcium. PAO also prevented the CCK-8-induced signal of [Ca2+]i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF-4). In addition to the effects of PAO on calcium mobilization, it caused a significant increase in amylase secretion and reduced the secretory response to either CCK-8 or AlF-4. The effects of PAO on both [Ca2+]i and amylase release were reversed by the sulfhydryl reducing agent, dithiothreitol (2 mM). Pretreatment of acinar cells with high concentration of ryanodine (50 microM) reduced the PAO-evoked calcium release. However, PAO was still able to release a small fraction of Ca2+ from acinar cells in which agonist-releasable Ca2+ pools had been previously depleted by thapsigargin (0.5 microM) and ryanodine receptors were blocked by 50 microM ryanodine. We conclude that, in pancreatic acinar cells, PAO mainly releases Ca2+ from the ryanodine-sensitive calcium pool and consequently induces amylase secretion. These effects are likely to be due to the oxidizing effects of this compound.     

Involvement of cellular calcium in exocrine pancreatic insufficiency during streptozotocin-induced diabetes mellitus

This study investigates the effects of the islet hormones insulin (Ins), glucagon (Glu), and somatostatin (Som) with nerve stimulation (EFS) acetylcholine (ACh) and cholecytokinin-octapeptide (CCK-8) on amylase secretion and intracellular free calcium concentration [Ca(2+)](i) in the pancreas of age-matched control and diabetic rats. Either Ins, Glu or Som elicited small increases in amylase secretion from the pancreas of age-matched control animals compared to a much larger increase in amylase secretion with either EFS, ACh or CCK-8. Combining the islet hormones with either EFS, ACh or CCK-8 resulted in marked potentiation of amylase output. In the diabetic pancreas, the islet hormones had no effect on amylase secretion compared to diabetic control. Moreover, either EFS, ACh or CCK-8 evoked a much smaller increase in amylase output compared to age-matched control. In addition, the islet hormones failed to potentiate the secretory effects of either EFS, ACh or CCK-8. In fura-2 loaded acinar cells from age-matched control pancreas either Ins or Glu elicited a small increase in [Ca(2+)](i) whereas Som had no effect. Both ACh and CCK-8 evoked large increases in [Ca(2+)](i) compared to control. Combining either Ins, Glu or Som with either ACh or CCK-8 resulted in a marked elevation in [Ca(2+)](i) compared to the responses obtained with either the islet hormones, ACh or CCK-8 alone. In diabetic fura-2 loaded pancreatic acinar cells, the islet hormones had no effect on [Ca(2+)](i) compared to control and moreover, the responses were much smaller than those obtained in acinar cells from age-matched control. Both ACh and CCK-8 induced large increases in [Ca(2+)]( i) in diabetic acinar cells. However, combining the islet hormones with either ACh or CCK-8 failed to enhance [Ca(2+)](i) compared to the reponses obtained in acinar cells from age-matched control. The results suggests that [Ca(2+)](i) homeostasis is deranged during diabetes mellitus and this in turn is probably associated with reduced pancreatic amylase secretion.     

Dose-dependent effect of hydrogen peroxide on calcium mobilization in mouse pancreatic acinar cells.

We have employed confocal laser scanning microscopy to investigate how intracellular free calcium concentration ([Ca2+]i) is influenced by hydrogen peroxide (H2O2) in collagenase-dispersed mouse pancreatic acinar cells. In the absence of extracellular calcium, treatment of cells with increasing concentrations of H2O2 resulted in an increase in [Ca2+]i, indicating the release of calcium from intracellular stores. Micromolar concentrations of H2O2 induced an oscillatory pattern, whereas 1 mmol H2O2/L caused a slow and sustained increase in [Ca2+]i. H2O2 abolished the typical calcium release stimulated by thapsigargin or by the physiological agonist cholecystokinin octapeptide (CCK-8). Depletion of either agonist-sensitive or mitochondrial calcium pools was unable to prevent calcium release induced by 1 mmol H2O2/L, but depletion of both stores abolished it. Additionally, lower H2O2 concentrations were able to release calcium only after depletion of mitochondrial calcium stores. Treatment with either the phospholipase C inhibitor U-73122 or the inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor xestospongin C did not modify calcium release from the agonist-sensitive pool induced by 100 micromol H2O2/L, suggesting the involvement of a mechanism independent of IP3 generation. In addition, H2O2 reduced amylase release stimulated by CCK-8. Finally, either the H2O2-induced calcium mobilization or the inhibitory effect of H2O2 on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulphydryl reducing agent. We conclude that H2O2 at micromolar concentrations induces calcium release from agonist-sensitive stores, and at millimolar concentrations H2O2 can also evoke calcium release from the mitochondria. The action of H2O2 is mediated by oxidation of sulphydryl groups of calcium ATPases independently of IP3 generation.     

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.     

Cytoplasmic Ca2+ oscillations evoked by receptor stimulation, G-protein activation, internal application of inositol trisphosphate or Ca2+: simultaneous microfluorimetry and Ca2+ dependent Cl- current recording in single pancreatic acinar cells.

The effects of acetylcholine (ACh), cholecystokinin (CCK), internally applied GTP-gamma-S, inositol trisphosphate [Ins (1,4,5) P3] or Ca2+ on the cytoplasmic free Ca2+ concentration [( Ca2+]i) were assessed by simultaneous microfluorimetry (fura-2) and measurement of the Ca2(+)-dependent Cl- current (patch-clamp whole-cell recording) in single internally perfused mouse pancreatic acinar cells. ACh (0.1-0.2 microM) evoked an oscillating increase in [Ca2+]i measured in the cell as a whole (microfluorimetry) which was synchronous with oscillations in the Ca2(+)-dependent Cl- current reporting [Ca2+]i close to the cell membrane. In the same cells a lower ACh concentration (0.05 microM) evoked shorter repetitive Cl- current pulses that were not accompanied by similar spikes in the microfluorimetric recording. When cells did not respond to 0.1 microM ACh, caffeine (1 mM) added on top of the sustained ACh stimulus resulted in [Ca2+]i oscillations seen synchronously in both types of recording. CCK (10 nM) also evoked [Ca2+]i oscillations, but with much longer intervals between slightly broader Ca2+ pulses. Internal perfusion with 100 microM GTP-gamma-S evoked [Ca2+]i oscillations with a similar pattern. Ins (1,4,5) P3 (10 microM) evoked repetitive shortlasting spikes in [Ca2+]i that were only seen in the Cl- current traces, except in one small cell where these spikes were also observed synchronously in the microfluorimetric recording. Caffeine (1 mM) broadened these Ca2+ pulses. [Ca2+]i was also directly changed, bypassing the normal signalling process, by infusion of a low or high Ca2+ solution into the pipette.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synchronized oscillations in cytoplasmic free calcium concentration in confluent bradykinin-stimulated bovine pulmonary artery endothelial cell monolayers

Bradykinin-evoked rises in [Ca2+]i were measured in fura-2-loaded bovine pulmonary artery endothelial cell monolayers by dual wavelength excitation fluorimetry. In monolayers seeded thinly and grown to confluence, bradykinin, in the presence of external Ca2+, evoked a rise in [Ca2+]i composed of an initial peak and subsequent oscillating plateau. In the absence of external Ca2+, bradykinin evoked a rise in [Ca2+]i which then returned to the basal value without oscillating. In monolayers seeded near confluent density, the bradykinin-evoked peak in [Ca2+]i was followed by a steady plateau which showed no oscillation. The addition of the phorbol ester, phorbol 12,13-dibutyrate, to a monolayer during bradykinin-evoked oscillations abolished the oscillations and lowered [Ca2+]i partway back toward the basal level. The addition of the protein kinase C inhibitor, H7, did not abolish oscillatory activity, although the frequency of oscillation was reduced. These results indicate that synchronized oscillatory activity can occur in endothelial cell monolayers. It is suggested that these oscillations are dependent on intercellular coupling developed when the cells are grown to confluence and that the mechanism responsible for generating oscillations in [Ca2+]i requires extracellular Ca2+ and involves protein kinase C.     

Modulation of [Ca2+]i signaling dynamics and metabolism by perinuclear mitochondria in mouse parotid acinar cells

Parotid acinar cells exhibit rapid cytosolic calcium signals ([Ca2+]i) that initiate in the apical region but rapidly become global in nature. These characteristic [Ca2+]i signals are important for effective fluid secretion, which critically depends on a synchronized activation of spatially separated ion fluxes. Apically restricted [Ca2+]i signals were never observed in parotid acinar cells. This is in marked contrast to the related pancreatic acinar cells, where the distribution of mitochondria has been suggested to contribute to restricting [Ca2+]i signals to the apical region. Therefore, the aim of this study was to determine the mitochondrial distribution and the role of mitochondrial Ca2+ uptake in shaping the spatial and temporal properties of [Ca2+]i signaling in parotid acinar cells. Confocal imaging of cells stained with MitoTracker dyes (MitoTracker Green FM or MitoTracker CMXRos) and SYTO dyes (SYTO-16 and SYTO-61) revealed that a majority of mitochondria is localized around the nucleus. Carbachol (CCh) and caged inositol 1,4,5-trisphosphate-evoked [Ca2+]i signals were delayed as they propagated through the nucleus. This delay in the CCh-evoked nuclear [Ca2+]i signal was abolished by inhibition of mitochondrial Ca2+ uptake with ruthenium red and Ru360. Likewise, simultaneous measurement of [Ca2+]i with mitochondrial [Ca2+] ([Ca2+]m), using fura-2 and rhod-FF, respectively, revealed that mitochondrial Ca2+ uptake was also inhibited by ruthenium red and Ru360. Finally, at concentrations of agonist that evoke[Ca2+]i oscillations, mitochondrial Ca2+ uptake, and a nuclear [Ca2+] delay, CCh also evoked a substantial increase in NADH autofluorescence. This autofluorescence exhibited a predominant perinuclear localization that was also sensitive to mitochondrial inhibitors. These data provide evidence that perinuclear mitochondria and mitochondrial Ca2+ uptake may differentially shape nuclear [Ca2+] signals but more importantly drive mitochondrial metabolism to generate ATP close to the nucleus. These effects may profoundly affect a variety of nuclear processes in parotid acinar cells while facilitating efficient fluid secretion.     

Release of intracellular Ca2+ and elevation of inositol trisphosphate by secretagogues in parietal and chief cells isolated from rabbit gastric mucosa

The fluorescent intracellular Ca2+ indicator, fura2/AM, was used to determine the effects of carbachol, cholecystokinin octapeptide (CCK-8), gastrin and histamine on intracellular Ca2+ ([Ca2+]i) in parietal cells from rabbit gastric mucosa enriched to more than 95% purity by a new Nycodenz gradient/centrifugal elutriation technique. Changes in [Ca2+]i in response to the same agonists were also measured in enriched chief cells. Carbachol, histamine, gastrin and CCK-8 increased parietal cell [Ca2+]i with the response to carbachol greater than CCK -8 = histamine = gastrin. Prestimulation with msximal doses of carbachol blocked histamine-induced increases in [Ca2+]i. In chief cells, carbachol increased [Ca2+]i but to a lesser degree than CCK-8, while histamine had no significant effect on [Ca2+]i. Neither removal of extracellular Ca2+ coupled with acute addition of 1 mM EGTA nor addition of the Ca2+-channel blocker nicardipine prevented agonist-induced changes in [Ca2+]i in either cell type. In the presence and absence of 10 mM LiCl2, carbachol and CCK-8 were found to increase inositol trisphosphate (IP3) content in both parietal and chief cells while histamine had no significant effect on this phosphoinositide hydrolysis product. From these results and previous observations with gastric glands (Chew, C.S. (1986) Am. J. Physiol. 13, G814-G823) we conclude that: carbachol, CCK-8, gastrin and histamine increase parietal cell [Ca2+]i initially by release of Ca2+ from the same intracellular store(s); the release of [Ca2+]i in response to carbachol and CCK-8 in both chief and parietal cells appear to be mediated by IP3; however, other mechanisms may be involved in histamine-induced release of parietal cell Ca2+.     

Spontaneous and agonist-induced calcium oscillations in pituitary gonadotrophs

Basal and receptor-regulated changes in cytoplasmic calcium concentration ([Ca2+]i) were monitored by fluorescence analysis in individual rat pituitary gonadotrophs loaded with the calcium-sensitive dye indo-1. Most gonadotrophs exhibited low amplitude spontaneous oscillations in basal [Ca2+]i that were interspersed by quiescent periods and abolished by removal of extracellular Ca2+ or addition of calcium channel blockers. Such random fluctuations in [Ca2+]i, which reflect the operation of a plasma membrane oscillator, were not coupled to basal gonadotropin secretion. The physiological agonist GnRH induced high amplitude [Ca2+]i oscillations; when a threshold [Ca2+]i level was reached, a cytoplasmic oscillator began to generate extremely regular Ca2+ transients. The time required to reach the threshold [Ca2+]i level was inversely correlated with agonist dose; the frequency, but not the amplitude, of agonist-induced Ca2+ spiking increased with agonist concentration. The duration of the latent period decreased and the frequency of Ca2+ spiking increased with the increase in ambient temperature. At high GnRH concentrations, the calcium transients merged into biphasic responses similar to those observed in cell suspensions at all GnRH concentrations. The presence of spontaneous fluctuations in basal [Ca2+]i did not significantly change the patterns of agonist-induced [Ca2+]i responses. Also, removal of extracellular Ca2+ did not interfere with the frequency or amplitude of Ca2+ spikes, but caused the loss of the plateau phase. Blockade of intracellular Ca(2+)-ATPase pumps by thapsigargin was usually accompanied by a subthreshold increase in [Ca2+]i. In such cells the agonist-induced oscillatory pattern was transformed into the biphasic response. In about 10% of the cells, however, high thapsigargin concentrations induced coarse [Ca2+]i oscillations; subsequent stimulation of such cells with GnRH was ineffective. The cytoplasmic oscillatory and biphasic responses may represent a mechanism for differential activation of Ca(2+)-dependent enzymes and their dependent cellular processes, including hormone secretion. The membrane oscillator is probably responsible for refilling of agonist-sensitive pools during and after agonist stimulation.     

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

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

Calcium oscillations induced by lindane in peritoneal macrophages of mice: control by the maturation stage of the macrophage

Mouse resident peritoneal macrophages loaded with Fluo-3 were examined for changes in cytosolic calcium concentration ([Ca2+]i) after stimulation with gamma-hexachlorocyclohexane (Lindane or gamma-HCCH). These studies, realized on macrophage populations, or single cells, by digital imaging microscopy, sought to determine the role of calcium influx on cyclical changes according to maturation stages of macrophages. Single cell analysis of [Ca2+]i changes in macrophages, after gamma-HCCH exposure in 600 microM extracellular calcium, demonstrated that: 1) these [Ca2+]i variations were asynchronous oscillations with the same frequency (1.7 min-1), and 2) these [Ca2+]i variations in macrophages were not at the same [Ca2+]i level. This heterogeneity could be correlated to a cell size partition of the macrophage population (10.1 +/- 0.44 and 11.45 +/- 0.43 microns). In the presence of 100 microM calcium, gamma-HCCH induced a calcium influx into the two subpopulations, but the calcium oscillations appeared only in small macrophages. In the largest ones, [Ca2+]i slowly decreased back down to the basal level. The cell size variation could be correlated to a phenotypic heterogeneity, linked to the differenciation stage of the cell. Peroxydase activity showed that small macrophages were in fact exudate macrophages and the largest ones were resident macrophages. Inhibition of the oscillatory patterns by a decrease in the extracellular calcium concentration ([Ca2+]ext) or by lanthanum chloride (LaCl3) addition is indicative of the important role of calcium influx in the triggering of oscillations. The calcium influx was transient and induced inositol phosphate (InsP3) production in macrophages. The maintainance of these calcium oscillations depended on calcium mobilization from intracellular calcium stores by InsP3, since neomycin and 8-(diethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8) abolished the oscillations. gamma-HCCH induced a transient calcium entry which triggered phospholipase C activation and the associated [Ca2+]i oscillations. However, we showed that differences in cell responses were observed in relationship with the differentiation stage of the mouse peritoneal macrophages, and with the extracellular calcium concentration.     

Norepinephrine acts on the KATP channel and produces different effects on [Ca2+]i in oscillating and non-oscillating HIT-T15 cells

Norepinephrine (NE) is an inhibitor of insulin secretion that acts, in part, by decreasing intracellular free calcium ([Ca2+]i). We examined the effects of NE on [Ca2+]i in individual HIT-T15 cells loaded with indo 1. Cells were categorized as oscillators or non-oscillators on the basis of the pattern of the calcium response to glucose and the effect of NE on [Ca2+]i was subsequently measured in each cell. NE caused a simple decrease in [Ca2+]i in nonoscillators. In oscillators, NE decreased the amplitude and frequency of the oscillations. Furthermore, the duration of the NE effect in oscillators was longer than in non-oscillators. NE did not affect the rise in [Ca2+]i elicited by depolarizing concentrations of 20 mM or 35 mM KCl alone, or in the presence of 20 mM KCl, 100 microM diazoxide, and 10 mM glucose. In other experiments, NE had no effect on [Ca2+]i when the KATP channels were fully clamped with diazoxide or tolbutamide. We conclude that the action of NE to decrease [Ca2+]i in both oscillators and non-oscillators is mediated via activation of the KATP channel. Despite this common mechanism, NE exerts different effects on oscillating and non-oscillating cells.     

Spatial and temporal aspects of ACh-induced [Ca2+]i oscillations in porcine tracheal smooth muscle

This study evaluated the relationship between regional elevation in intracellular calcium concentration ([Ca2+]i) induced by acetylcholine (ACh) and the global cellular responses in porcine tracheal smooth muscle (TSM) cells. Regional (approximately 1.5 microm3) and global (whole cell) changes in [Ca2+]i were measured in fluo-3 loaded TSM cells using real-time confocal microscopy. Regional responses appeared as propagating [Ca2+]i oscillations whereas global responses reflected the spatiotemporal integration of these regional responses. Within a region, [Ca2+]i oscillations were 'biphasic' with initial higher frequencies, followed by slower steady-state oscillations. With increasing ACh concentration, the peak (maximum value relative to 0 nM) of regional [Ca2+]i oscillations remained relatively constant, whereas both frequency and propagation velocity increased. In contrast, the global spatiotemporal integration of the regional oscillatory responses appeared as a concentration-dependent increase in peak as well as mean cellular [Ca2+]i. We conclude that the significance of ACh-induced [Ca2+]i oscillations lies in the establishment of mean [Ca2+]i level for slower Ca2+-dependent physiological processes via modulation of oscillation frequency and propagation velocity.     

Pulsatile Ca2+ extrusion from single pancreatic acinar cells during receptor-activated cytosolic Ca2+ spiking.

Ca2+ extrusion was measured simultaneously with the free intracellular Ca2+ concentration ([Ca2+]i) from single pancreatic acinar cells placed in microdroplets of extracellular solution (Tepikin, A. V., Voronina, S. G., Gallacher, D. V., and Petersen, O. H. (1992) J. Biol. Chem. 267, 3569-3572). Submaximal stimulation with cholecystokinin usually evoked discrete cytosolic Ca2+ spikes and each of these spikes was associated with a discrete and virtually synchronous pulse of Ca2+ extrusion into the extracellular microdroplet solution. When ACh evoked repetitive discrete [Ca2+]i spikes, each spike was also accompanied by a discrete pulse of Ca2+ extrusion. The velocity of Ca2+ extrusion oscillated with a time course similar to that of [Ca2+]i. The extracellular solution in our experiments had a low total calcium concentration (15-35 microM) and only a limited number of [Ca2+]i spikes (2-8) could be evoked. The magnitudes of the [Ca2+]i spikes and the amounts of Ca2+ extruded during each spike gradually decreased in each experiment. During the first cholecystokinin-evoked cytosolic Ca2+ spike the Ca2+ extrusion corresponded to a loss of 15-70% (mean value 39% +/- 12) of the mobilizable cellular calcium pool. The substantial pulsatile Ca2+ extrusion occurring synchronously with the receptor-activated cytosolic Ca2+ spikes is therefore an important element in repetitively bringing back [Ca2+]i to the resting level.     

Phosphatidic acid increases inositol-1,4,5,-trisphosphate and [Ca2+]i levels in neonatal rat cardiomyocytes.

Phosphatidic acid (PA), which can be synthesized de novo, or as a product of phosphatidylcholine hydrolysis and/or phosphorylation of 1,2-diacylglycerol (DAG), mediates diverse cellular functions in various cell types, including cardiomyocytes. We set out to characterize the effect of PA on intracellular free calcium ([Ca2+]i) and inositol-1,4,5-trisphosphate (IP(3)) levels in primary cultures of neonatal rat cardiomyocytes. Addition of PA led to rapid, concentration and time dependent increases in both IP(3) and [Ca2+]i levels in adherent cells. There was strong correlation in the concentration-response relationships between IP(3) and [Ca2+]i increases evoked by PA. Incubation with the sarcoplasmic reticulum (SR) Ca2+ pump inhibitor, cyclopiazonic acid (CPA), significantly attenuated the PA evoked [Ca2+]i increase but had no significant effect on IP(3) accumulation. The phospholipase C (PLC) inhibitor, D-609, attenuated both IP(3) and [Ca2+]i elevations evoked by PA whereas staurosporine (STS), a potent and non-selective PKC inhibitor, had no significant effect on either. Another PLC inhibitor, U73122, but not its inactive analog, U73343, also inhibited PA evoked increases in [Ca2+]i. Depletion of extracellular calcium attenuated both basal and PA evoked increases in [Ca2+]i. The PLA(2) inhibitors, bromophenylacyl-bromide (BPB) and CDP-choline, had no effect on PA evoked [Ca2+]i responses. Neither the DAG analog, dioctanoylglycerol, nor the DAG kinase inhibitor, R59949, affected PA evoked changes in [Ca2+]i. Taken together, these data indicate that PA, in a manner independent of PKC, DAG, or PLA(2), may enhance Ca2+ release from IP(3) sensitive SR Ca(2+) stores via activation of PLC in neonatal rat cardiomyocytes.     

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.     

Stimulus-Induced Oscillations in Guard Cell Cytosolic Free Calcium

Ca2+ is implicated as a second messenger in the response of stomata to a range of stimuli. However, the mechanism by which stimulus-induced increases in guard cell cytosolic free Ca2+ ([Ca2+]i) are transduced into different physiological responses remains to be explained. Oscillations in [Ca2+]i may provide one way in which this can occur. We used photometric and imaging techniques to examine this hypothesis in guard cells of Commelina communis. External Ca2+ ([Ca2+]e), which causes an increase in [Ca2+]i, was used as a closing stimulus. The total increase in [Ca2+]i was directly related to the concentration of [Ca2+]e, both of which correlated closely with the degree of stomatal closure. Increases were oscillatory in nature, with the pattern of the oscillations dependent on the concentration of [Ca2+]e. At 0.1 mM, [Ca2+]e induced symmetrical oscillations. In contrast, 1.0 mM [Ca2+]e induced asymmetric oscillations. Oscillations were stimulus dependent and modulated by changing [Ca2+]e. Experiments using Ca2+ channel blockers and Mn2+-quenching studies suggested a role for Ca2+ influx during the oscillatory behavior without excluding the possible involvement of Ca2+ release from intracellular stores. These data suggest a mechanism for encoding the information required to distinguish between a number of different Ca2+-mobilizing stimuli in guard cells, using stimulus-specific patterns of oscillations in [Ca2+]i.     

Effects of CCK-8 on the cytoplasmic free calcium concentration in isolated rat islet cells.

The C-terminal octapeptide of cholecystokinin (CCK-8) is known to stimulate insulin secretion. We examined its effects on the cytoplasmic free calcium concentration ([Ca2+]IC) in isolated rat pancreatic islet cells. At 8.3 mM glucose and 1.28 mM Ca2+, CCK-8 (100 nM) rapidly increased [Ca2+]IC to a short-lived peak, whereafter the [Ca2+]IC, within 1.5 minutes, fell to values below baseline. CCK-8 also rapidly increased the [Ca2+]IC at 3.3 mM glucose and in a calcium deficient medium. However, either at low glucose or in the absence of extracellular Ca2+, the post-peak [Ca2+]IC did not fall below baseline levels. The CCKA receptor antagonist, L-364,718 (20 nM), inhibited the effects of CCK-8 on [Ca2+]IC. The results suggest that CCK-8 in islet cells liberates calcium from intracellular stores by activating CCKA receptors.     

Cytosolic Ca2+ oscillations by gastrin releasing peptide in single HIT-T15 cells.

In single, superfused, FURA-2AM loaded insulin producing HIT-T15 cells, gastrin releasing peptide (GRP) induced a peak in cytoplasmnic Cu2+ ([Ca2+]i) followed by a sustained (high GRP concentrations) or oscillatory (low GRP concentrations) [Ca2+]i pattern. The GRP (25-50 microM)-induced [Ca2+]i oscillations ceased upon removal of glucose or addition of thapsigargin (1 microM), EGTA (2 mM), or diazoxide (200 microM), whereas nifedipine (10 microM) reduced their amplitude (by 35%). Both protein kinase C (PKC)-activation or PKC-inhibition disrupted GRP induced [Ca2+]i oscillations. GRP induced [Ca2+]i oscillations in insulin producing cells therefore rely on intracellular Ca2+ mobilization, voltage-dependent and voltage-independent Ca2+ entry mechanisms and the integrity of protein kinase C.