Inhibitors of Bcl-2 protein family deplete ER Ca2+ stores in pancreatic acinar cells

Physiological stimulation of pancreatic acinar cells by cholecystokinin and acetylcholine activate a spatial-temporal pattern of cytosolic [Ca⁺²] changes that are regulated by a coordinated response of inositol 1,4,5-trisphosphate receptors (IP₃Rs), ryanodine receptors (RyRs) and calcium-induced calcium release (CICR). For the present study, we designed experiments to determine the potential role of Bcl-2 proteins in these patterns of cytosolic [Ca⁺²] responses. We used small molecule inhibitors that disrupt the interactions between prosurvival Bcl-2 proteins (i.e. Bcl-2 and Bcl-xl) and proapoptotic Bcl-2 proteins (i.e. Bax) and fluorescence microfluorimetry techniques to measure both cytosolic [Ca⁺²] and endoplasmic reticulum [Ca⁺²]. We found that the inhibitors of Bcl-2 protein interactions caused a slow and complete release of intracellular agonist-sensitive stores of calcium. The release was attenuated by inhibitors of IP₃Rs and RyRs and substantially reduced by strong [Ca²⁺] buffering. Inhibition of IP₃Rs and RyRs also dramatically reduced activation of apoptosis by BH3I-2′. CICR induced by different doses of BH3I-2′ in Bcl-2 overexpressing cells was markedly decreased compared with control. The results suggest that Bcl-2 proteins regulate calcium release from the intracellular stores and suggest that the spatial-temporal patterns of agonist-stimulated cytosolic [Ca⁺²] changes are regulated by differential cellular distribution of interacting pairs of prosurvival and proapoptotic Bcl-2 proteins.     

Miniature Ca2+ channels in excised plasma-membrane patches: activation by IP3

 In the present work, we characterized the receptor properties and the conductive features of the inositol (1,4,5)-trisphosphate (IP₃)-activated Ca²⁺ channels present in excised plasma-membrane patches obtained from mouse macrophages and A431 cells. We found that the receptor properties of the channels tested were similar to those of the IP₃ receptor (IP₃R) expressed in the endoplasmic reticulum (ER) membrane. These properties include activation by IP₃, inhibition by heparin, time-dependent inactivation by high IP₃ concentrations, activation by guanosine 5′o-thiotriphosphate and regulation by arachidonic acid. On the other hand, in terms of conductive properties, the channel closely resembles Ca²⁺-release-activated Ca²⁺ channels (I _crac). These conductive properties include extremely low conductance (≈1 pS), very high selectivity for Ca²⁺ over K⁺ (P _Ca/P _K>1000), inactivation by high intracellular Ca²⁺ concentration and, importantly, strong inward rectification. Notably, the same channel was activated by: (1) agonists in the cell-attached mode of channel recording, and (2) cytosolic IP₃ after patch excision. Although the possibility cannot be completely excluded that a novel type of IP₃R is expressed exclusively in the plasma membrane, in their entirety our findings suggest that the plasma membrane of mouse macrophages and A431 cells contains I _crac-like Ca²⁺ channels coupled to an IP₃-responsive protein which displays properties similar to those of the IP₃R expressed in the ER membrane. Received: 16 June 1998 / Received after revision: / 24 August 1998 / Accepted: 1 September 1998     

Novel Ca2+ signalling mechanisms in vascular myocytes: symposium overview*

This commentary presents the proceedings of the symposium sponsored by Cardiovascular Section of American Physiological Society in San Diego, CA on 12 April 2003. The major focus of this symposium was on the actions and physiological relevance of several novel Ca²⁺ signalling mechanisms in vascular smooth muscle (VSM) cells. Five important topics were presented in this symposium including the discovery and roles of cyclic ADP‐ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) in mediating Ca²⁺ release, Ca²⁺ sparks and activation of plasma membrane K_Ca channels in VSM cells, the role of cADPR‐mediated activation of ryanodine receptors in the control of vascular tone, the role of [Ca²⁺]_i in mechanotransduction in the arterioles, and interactions of mitochondrial Ca²⁺ release and SR Ca²⁺ mobilization. The purpose of this symposium was to promote discussions and exchange of ideas between scientists with interests in Ca²⁺ signalling mechanisms and those with interests in vascular physiology and pharmacology. The cross‐fertilization of ideas is expected to greatly advance our understanding of the physiological and pharmacological relevance of these new Ca²⁺ signalling mechanisms.     

Reduced cholecystokinin receptor phosphorylation and restored signalling in protein kinase C down-regulated rat pancreatic acinar cells

 Receptor phosphorylation in response to agonist stimulation is a key regulatory principle in signal transduction. Previous work has suggested the concerted action of protein kinase C (PKC) and a staurosporine-insensitive receptor kinase in homologous phosphorylation of the cholecystokinin (CCK) receptor in freshly isolated rat pancreatic acinar cells [Gates, Ulrich, Miller (1993) Am J Physiol 264:G840–G847]. The present study shows that down-regulation of PKC by prolonged (2 h) treatment with 0.1 μM 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly reduced basal CCK receptor phosphorylation as well as that induced by TPA (0.1 μM) and cholecystokinin-(26-33)-peptide amide (CCK₈, 0.1 μM). The phosphorylation level reached was the same with both stimulants and equalled basal phosphorylation in untreated control cells. The absence of any CCK₈-stimulated phosphorylation reflecting the activity of a putative staurosporine-insensitive receptor kinase raises the intriguing possibility that a basal level of PKC-mediated receptor phosphorylation is required for the action of such a receptor kinase. Immunoblot analysis revealed that the decrease in receptor phosphorylation coincided with a marked reduction of PKC-α and, to a lesser extent, PKC-ɛ. In addition, TPA-induced inhibition of the increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) evoked by the high-affinity CCK receptor agonist JMV-180 was completely reversed. The time-course of recovery closely matched that of the reduction of PKC-α. Finally, digital imaging microscopy of individual PKC down-regulated cells revealed a marked increase in the duration of JMV-180-evoked oscillatory changes in [Ca²⁺]_i. Taken together, the present findings are in agreement with the idea that PKC-α-mediated receptor phosphorylation leads to a shortening of the duration of the [Ca²⁺]_i oscillations and eventually to inhibition of high-affinity Ca²⁺ signalling through the native CCK receptor in pancreatic acinar cells. Received: 15 September 1997 / Accepted: 2 October 1997     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

IP3-induced Ca2+ release in A7r5 vascular smooth-muscle cells represents a partial emptying of the stores and not an all-or-none Ca2+ release of separate quanta

 There is still no agreement on the mechanism of the intracellular action of low concentrations of inositol 1,4,5-trisphosphate (IP₃). Intracellular Ca²⁺ stores may transiently release some Ca²⁺ before they become insensitive to IP₃. Alternatively, stores with a low IP₃ threshold may lose all their Ca²⁺ and the others none. We now report that the IP₃ threshold was not correlated with the extent of Ca²⁺ release in permeabilized A7r5 smooth-muscle cells. In contrast, the maximum rate of release, which was changed either by varying the level of IP₃ receptor (IP₃R) activation, or by changing the concentration of IP₃R at a constant level of IP₃R activation, was directly related to the extent of Ca²⁺ release. We conclude that IP₃-induced Ca²⁺ release reflects partial emptying of the stores and not all-or-none Ca²⁺ release of separate quanta. Received: 22 October 1998 / Received after revision: 15 December 1998 / Accepted: 11 January 1999     

Plasticity of cholinoreceptors of neurons of the common snail after effects on inositol-1,4,5-triphosphate-and Ca2+-dependant mobilization of stored Ca2+ and the level of phosphatidic acid

The influences on the depth of extinction of the inward current induced by acetylcholine (the ACh-current) of a number of compounds affecting the mobilization of stored Ca²⁺ and the intracellular level of Ca²⁺-mobilizing second messengers, namely, inositol-1,4,5-trisphosphate (IP₃), inositol hexakisphosphate, TMB-8 (an inhibitor of (IP₃)-dependant Ca²⁺ mobilization), tetracaine (an inhibitor of Ca²⁺-dependant mobilization of Ca²⁺), as well as phospholipase D, which leads to the formation of phosphatidic acid through the hydrolysis of phosphatidylcholine, were investigated in identified RPa3 and LPa3 neurons of the common snail using the two-electrode voltage clamp technique for the recording of the potential on the membrane. The participation of IP₃, of IP₃-dependant, and Ca²⁺-dependant mobilized intracellular Ca²⁺, as well as phosphatidic acid in the regulation of the plasticity of the cholinoreceptors of the neurons was demonstrated. M. V. Lomonosov Moscow State University. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I. P. Pavlova, Vol. 44, No. 4-5, pp. 796–805, July–October, 1994.     

Frequency-dependent mitochondrial Ca2+ accumulation regulates ATP synthesis in pancreatic β cells

Pancreatic β cells respond to increases in glucose concentration with enhanced metabolism, the closure of ATP-sensitive K⁺ channels and electrical spiking. The latter results in oscillatory Ca²⁺ influx through voltage-gated Ca²⁺ channels and the activation of insulin release. The relationship between changes in cytosolic and mitochondrial free calcium concentration ([Ca²⁺]_cyt and [Ca²⁺]_mit, respectively) during these cycles is poorly understood. Importantly, the activation of Ca²⁺-sensitive intramitochondrial dehydrogenases, occurring alongside the stimulation of ATP consumption required for Ca²⁺ pumping and other processes, may exert complex effects on cytosolic ATP/ADP ratios and hence insulin secretion. To explore the relationship between these parameters in single primary β cells, we have deployed cytosolic (Fura red, Indo1) or green fluorescent protein-based recombinant-targeted (Pericam, 2mt8RP for mitochondria; D4ER for the ER) probes for Ca²⁺ and cytosolic ATP/ADP (Perceval) alongside patch-clamp electrophysiology. We demonstrate that: (1) blockade of mitochondrial Ca²⁺ uptake by shRNA-mediated silencing of the uniporter MCU attenuates glucose- and essentially blocks tolbutamide-stimulated, insulin secretion; (2) during electrical stimulation, mitochondria decode cytosolic Ca²⁺ oscillation frequency as stable increases in [Ca²⁺]_mit and cytosolic ATP/ADP; (3) mitochondrial Ca²⁺ uptake rates remained constant between individual spikes, arguing against activity-dependent regulation (“plasticity”) and (4) the relationship between [Ca²⁺]_cyt and [Ca²⁺]_mit is essentially unaffected by changes in endoplasmic reticulum Ca²⁺ ([Ca²⁺]_ER). Our findings thus highlight new aspects of Ca²⁺ signalling in β cells of relevance to the actions of both glucose and sulphonylureas.     

Suppression of Ca2+ oscillations induced by cholecystokinin (CCK) and its analog OPE in rat pancreatic acinar cells by low-level protein kinase C activation without transition of the CCK receptor from a high- to low-affinity state

Cholecystokinin (CCK) analogs, JMV-180 and OPE, release Ca²⁺ from intracellular stores and induce oscillations in the concentration of cytosolic Ca²⁺ ([Ca²⁺]_i), but do not generate a detectable rise in inositol 1,4,5-trisphosphate (InsP ₃) levels. In contrast, high concentrations of CCK elevate InsP ₃, as well [Ca²⁺]_i, to a peak which decreases to near basal levels without oscillations. The mechanisms which underlie inhibition of [Ca²⁺]_i oscillations observed with high CCK concentrations are unclear, but are believed to involve a low-affinity CCK receptor state. Alternately, CCK analogs may be weak partial agonists of the phospholipase C pathway, whereas native CCK, as a full agonist of this pathway, stimulates low levels of protein kinase C (PKC) activity. Preincubation of acini with 1 nM 12 O-tetradecanoyl-phorbol 13-acetate (TPA) for 15 min at 37°C did not affect OPE binding to acini, but abolished OPE-induced (at 1 M) [Ca²⁺]_i oscillations without affecting the initial [Ca²⁺]_i spike. These transformed OPE-induced [Ca²⁺]_i responses mimicked those induced by supramaximal CCK octapeptide (CCK-8) concentrations. Inhibition of [Ca²⁺]_i oscillations by 1 nM TPA was reversed by the PKC inhibitor staurosporine (0.2 M). After [Ca²⁺]_i oscillations were induced with OPE or low concentrations of CCK-8 (20 pM), 1 nM TPA caused a gradual slowing of oscillation frequency over 15–20 min without affecting [Ca²⁺]_i spike amplitude. In contrast, 1 M TPA inhibited OPE binding and caused a more generalized inhibition of OPE- and CCK-evoked Ca²⁺ signals. These data suggest that inhibitory effects of low-level PKC activation on agonist-evoked Ca²⁺ signalling are distinct from the effects of high-level PKC activation by 1 M TPA, and do not require the transition of the CCK receptor from a high-affinity to a low-affinity state.     

A model of electrical activity and cytosolic calcium dynamics in vascular endothelial cells in response to fluid shear stress

A mathematical model is proposed to describe the intracellularCa ²⁺ (Ca _i) transient and electrical activity of vascular endothelial cells (VEC) elicited by fluid shear stress (τ). The intracellularCa ²⁺ store of the model VEC is comprised of aCa _i-sensitive (sc) and an inositol (1,4,5)-trisphosphate (IP ₃)-sensitive compartment (dc). The dc [Ca ²⁺] is refilled by the sc whose [Ca ²⁺] is the same as extracellular [Ca ²⁺].IP ₃ produced by the τ-deformed mechanoreceptors discharges the dcCa ²⁺ into the cytosol. The increase of cytosolic[Ca ²⁺] inducesCa ²⁺ release (CICR) from the sc. The raisedCa _i activates aCa _i-activatedK ⁺ current (I _{K, Ca}) and inhibitsIP ₃ production. The cell membrane potential is determined byI _{K, Ca}, voltage-dependentNa ⁺ andK ⁺ currents. Steady τ>0.1 dyne/cm² elicits aCa _i varies sigmoidally withLog ₁₀(τ) with a maximal peakCa _i of 150 nM at τ=4 dynes/cm². Step increases of τ fail to elicit aCa ²⁺ response in cells previously stimulated by a lower shear. TheCa ²⁺ response gradually decreases with repetitive τ stimuli. Pulsatile shear elicits two to three times higherCa _i and hyperpolarizes the cell more than steady shear of the same magnitude. The simulatedCa ²⁺ responses to τ are quantitatively and qualitatively similar to those observed in cultured VEC. The model provides a possible explanation of why the vasodilating stimulus is greater for pulsatile flow than for nonpulsatile flow.     

Inositol 1,4,5-triphosphate receptors and NAD(P)H mediate Ca signaling required for hypoxic preconditioning of hippocampal neurons

Exposure of neurons to a non-lethal hypoxic stress greatly reduces cell death during subsequent severe ischemia (hypoxic preconditioning, HPC). In organotypic cultures of rat hippocampus, we demonstrate that HPC requires inositol triphosphate (IP₃) receptor-dependent Ca²⁺ release from the endoplasmic reticulum (ER) triggered by increased cytosolic NAD(P)H. Ca²⁺ chelation with intracellular BAPTA, ER Ca²⁺ store depletion with thapsigargin, IP₃ receptor block with xestospongin, and RNA interference against subtype 1 of the IP₃ receptor all blunted the moderate increases in [Ca²⁺]_i (50–100 nM) required for tolerance induction. Increases in [Ca²⁺]_i during HPC and neuroprotection following HPC were not prevented with NMDA receptor block or by removing Ca²⁺ from the bathing medium. Increased NAD(P)H fluorescence in CA1 neurons during hypoxia and demonstration that NADH manipulation increases [Ca²⁺]_i in an IP₃R-dependent manner revealed a primary role of cellular redox state in liberation of Ca²⁺ from the ER. Blockade of IP₃Rs and intracellular Ca²⁺ chelation prevented phosphorylation of known HPC signaling targets, including MAPK p42/44 (ERK), protein kinase B (Akt) and CREB. We conclude that the endoplasmic reticulum, acting via redox/NADH-dependent intracellular Ca²⁺ store release, is an important mediator of the neuroprotective response to hypoxic stress.     

Two distinct signaling pathways for regulation of spontaneous local Ca2+ release by phospholipase C in airway smooth muscle cells

Spontaneous local Ca²⁺ release events have been observed in airway smooth muscle cells (SMCs), but the underlying mechanisms are largely unknown. Considering that each type of SMCs may use its own mechanisms to regulate local Ca²⁺ release events, we sought to investigate the signaling pathway for spontaneous local Ca²⁺ release events in freshly isolated mouse airway SMCs using a laser scanning confocal microscope. Application of ryanodine to block ryanodine receptors (RyRs) abolished spontaneous local Ca²⁺ release events, indicating that these events are RyR-mediated Ca²⁺ sparks. Inhibition of inositol 1,4,5-triphosphate receptors (IP₃Rs) by 2-aminoethoxydiphenyl-borate (2-APB) or xestospongin-C significantly blocked the activity of Ca²⁺ sparks. Under patch clamp conditions, dialysis of IP₃ to activate IP₃Rs increased the activity of local Ca²⁺ events in control cells but had no effect in ryanodine-pretreated cells. The RyR agonist caffeine augmented the frequency of Ca²⁺ sparks in cells pretreated with and without 2-APB or xestospongin-C. The specific phospholipase C (PLC) blocker U73122 decreased the activity of Ca²⁺ sparks and prevented xestospongin-C from producing the inhibitory effect. The protein kinase C (PKC) activator 1-oleoyl-2-acetyl-glycerol or phorbol-12-myristate-13-acetate inhibited Ca²⁺ sparks, whereas the PKC inhibitor chelerythrine, PKCɛ inhibitory peptide, or PKCɛ gene knockout produced an opposite effect. Collectively, our data suggest that the basal activation of PLC regulates the activity of RyR-mediated, spontaneous Ca²⁺ sparks in airway SMCs through two distinct signaling pathways: a positive IP₃-IP₃R pathway and a negative diacylglycerol–PKCɛ pathway.     

Inositol Triphosphate and Ryanodine Receptors in the Control of the Cholinosensitivity of Common Snail Neurons by the Na,K Pump During Habituation

The effects of the Na,K pump inhibitor ouabain on habituation of the common snail to tactile stimulation were identical to the ouabain-induced modification of the decrease in the cholinosensitivity of defensive behavior command neurons in the common snail in a cellular model of habituation. Studies addressed the effects of intracellularly delivered ligands of two types of Ca²⁺ depot receptors — inositol triphosphate (IP₃) receptors and ryanodine receptors — on the action of ouabain in the cellular analog of habituation. The IP₃ receptor antagonist heparin (0.1 mM), the IP₃ receptor agonist inositol triphosphate (0.1 mM), and the ryanodine-dependent Ca²⁺ mobilization inhibitor dantrolene (0.1 mM) prevented ouabain from modifying the depression of the evoked acetylcholine current. The ryanodine agonist/antagonist ryanodine was used at two concentrations (0.1 and 1 mM) and neither had any effect on the action of ouabain. It is concluded that Ca²⁺ mobilized from intracellular Ca²⁺ depots via IP₃ receptors is involved in the neuronal mechanism of regulation of the habitation of the common snail to tactile stimulation by the Na,K pump.__________Translated from Zhurnal Vysshei Nervnoi Deyatel’nosti, Vol. 54, No. 4, pp. 554–564, July–August, 2004.     

Trisk 32 regulates IP(3) receptors in rat skeletal myoblasts

To date, four isoforms of triadins have been identified in rat skeletal muscle. While the function of the 95-kDa isoform in excitation–contraction coupling has been studied in detail, the role of the 32-kDa isoform (Trisk 32) remains elusive. Here, Trisk 32 overexpression was carried out by stable transfection in L6.G8 myoblasts. Co-localization of Trisk 32 and IP₃ receptors (IP₃R) was demonstrated by immunocytochemistry, and their association was shown by co-immunoprecipitation. Functional effects of Trisk 32 on IP₃-mediated Ca²⁺ release were assessed by measuring changes in [Ca²⁺]_i following the stimulation by bradykinin or vasopressin. The amplitude of the Ca²⁺ transients evoked by 20 μM bradykinin was significantly higher in Trisk 32-overexpressing (p < 0.01; 426 ± 84 nM, n = 27) as compared to control cells (76 ± 12 nM, n = 23). The difference remained significant (p < 0.02; 217 ± 41 nM, n = 21, and 97 ± 29 nM, n = 31, respectively) in the absence of extracellular Ca²⁺. Similar observations were made when 0.1 μM vasopressin was used to initiate Ca²⁺ release. Possible involvement of the ryanodine receptors (RyR) in these processes was excluded, after functional and biochemical experiments. Furthermore, Trisk 32 overexpression had no effect on store-operated Ca²⁺ entry, despite a decrease in the expression of STIM1. These results suggest that neither the increased activity of RyR, nor the amplification of SOCE, is responsible for the differences observed in bradykinin- or vasopressin-evoked Ca²⁺ transients; rather, they were due to the enhanced activity of IP₃R. Thus, Trisk 32 not only co-localizes with, but directly contributes to, the regulation of Ca²⁺ release via IP₃R.     

ATP depletion inhibits Ca2+ release, influx and extrusion in pancreatic acinar cells but not pathological Ca2+ responses induced by bile

Here, we describe novel mechanisms limiting a toxic cytosolic Ca²⁺ rise during adenosine 5′-triphosphate (ATP) depletion. We studied the effect of ATP depletion on Ca²⁺ signalling in mouse pancreatic acinar cells. Measurements of ATP in isolated cells after adenovirus-mediated expression of firefly luciferase revealed that the cytosolic ATP concentration fell from approximately 1 mM to near zero after treatment with oligomycin plus iodoacetate. ATP depletion resulted in the inhibition of Ca²⁺ extrusion, which was accompanied by a remarkably synchronous inhibition of store-operated Ca²⁺ influx. Alternative inhibition of Ca²⁺ extrusion by carboxyeosin had a much smaller effect on Ca²⁺ influx. The coordinated metabolic inhibition of Ca²⁺ influx and extrusion suggests the existence of a common ATP-dependent master regulator of both processes. ATP-depletion also suppressed acetylcholine (ACh)-induced Ca²⁺ oscillations, which was due to the inhibition of Ca²⁺ release from internal stores. This could be particularly important for limiting Ca²⁺ toxicity during periods of hypoxia. In contrast, metabolic control of Ca²⁺ influx and Ca²⁺ release from internal stores spectacularly failed to prevent large toxic Ca²⁺ responses induced by bile acids—activators of acute pancreatitis (a frequent and often fatal disease of the exocrine pancreas). The bile acids taurolithocholic acid 3-sulphate (TLC-S), taurochenodeoxycholic acid (TCDC) and taurocholic acid (TC) were used in our experiments. Neither Ca²⁺ release from internal stores nor Ca²⁺ influx triggered by bile acids were inhibited by ATP depletion, emphasising the danger of these pathological mechanisms. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Low-conductivity calcium channels in the macrophage plasma membrane: Activation by inositol-1,4,5-triphosphate

Local voltage clamping was applied to mouse macrophage plasma membrane to study calcium channels activated by inositol-1,4,5-triphosphate (IP₃) and blocked by heparin. These channels were clearly distinguished from IP₃-activated channels of the endoplasmic reticulum by their low conductivity (about 1 pSm for 100 mM Ca²⁺), high selectivity for Ca²⁺ relative to K⁺ (P_Ca:P_K>1000), calcium inactivation, and activation on hyperpolarization; these properties allowed them to be assigned to the I_CRAC family. On the other hand, the properties of the IP₃ receptors of these channels (IP₃R), i.e., the dose-dependent effect of IP₃, the IP₃ desensitization of the receptor, and the sensitivity to micromolar concentrations of heparin and arachidonic acid were close to those of the endoplasmic reticulum IP₃ receptor. The most likely intepretation of these data is that IP₃R are not located in the endoplsmic reticulum, but, acting via some kind of conformational change occurring on binding of IP₃, transmit a signal from the endoplasmic reticulum to the highly selective Ca²⁺ channels. This point of view is in agreement with the published “coupling model” [1]. Translated from Rosiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 84, No. 5-6, pp. 417–425, May–June, 1998.     

Decrease of acidity inside zymogen granules inhibits acetylcholine-or inositol trisphosphate-evoked cytosolic Ca2+ spiking in pancreatic acinar cells

 In isolated pancreatic acinar cells application of the proton-potassium ionophore nigericin or the proton-sodium ionophore monensin led to a reduction of acidity inside the zymogen granules which could be visualized in an imaging system by a rapid reduction in the intragranular quinacrine fluorescence. Cytosolic Ca²⁺ spikes in response to acetylcholine stimulation or intracellular inositol trisphosphate application were assessed by recording Ca²⁺ -sensitive ionic currents in the patch clamp whole-cell recording configuration. Both nigericin and monensin evoked marked reductions in frequency and amplitude of spikes and in many experiments abolished spiking altogether. The Ca²⁺ -sensitive membrane currents could still be activated after nigericin or monensin treatment since subsequent application of the Ca²⁺ ionophore ionomycin evoked a large current response. The decrease in intragranular acidity would appear to inhibit intracellular Ca²⁺ release perhaps due to a reduction in the free intragranular Ca²⁺ concentration.