How far does phospholipase C activity depend on the cell calcium concentration? A study in intact cells.

The dependence of phospholipase C activity on the cytosolic Ca2+ concentration ([Ca2+]i) was studied in intact liver cells treated with the Ca2+-mobilizing hormone vasopressin, or not so treated. Phospholipase C (PLC) activity was estimated from the formation of [3H]inositol trisphosphate (InsP3) and the degradation of [3H]phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The [Ca2+]i of the cells was clamped from 29 to 1130 nM by quin2 loading. This wide concentration range was obtained by loading the hepatocytes with a high concentration of the Ca2+ indicator in low-Ca2+ medium or by using the Ca2+ ionophore ionomycin in medium containing Ca2+. In resting cells, in which [Ca2+]i was 193 nM, treatment with 0.1 microM-vasopressin which stimulates liver PLC maximally, tripled InsP3 content and raised [Ca2+]i to 2 microM within 15 s. Lowering [Ca2+]i partially decreased cell InsP3 content as well as the ability of vasopressin to stimulate InsP3 formation maximally. At 29 nM, the lowest Ca2+ concentration obtained in isolated liver cells, basal InsP3 content was 64% of that measured in control cells. Addition of vasopressin no longer affected [Ca2+]i, but significantly increased InsP3 by 200%, although less than in the controls (300%). The maintenance of the greater part of the PLC response at constant [Ca2+]i indicated that, in the liver, InsP3 formation does not result from an increase in [Ca2+]i. The effects of lowering [Ca2+]i were reversible. When low cell [Ca2+]i was restored to a normal value, resting InsP3 content and the ability of vasopressin to stimulate InsP3 formation maximally by 300% were also restored. Raising [Ca2+]i from 193 to 1130 nM had little effect on the InsP3 content or the vasopressin-mediated increase in InsP3. In agreement with the stimulation of PLC activity by vasopressin, cell [3H]PtdInsP2 and total PtdInsP2 were degraded by application of this hormone for 15 s. In contrast, when [Ca2+]i was lowered to 29 nM, basal [3H]PtdInsP2 and total PtdInsP2 were increased by about 30%, [3H]PtdInsP2 was further increased by vasopressin, but total PtdInsP2 was not changed. These results show that, in intact hepatocytes, PLC is little affected by [Ca2+]i concentrations above 193 nM, but is partially dependent on Ca2+ below that value. They suggest that, in addition to activating PLC activity, vasopressin might stimulate PtdInsP2 synthesis, presumably via phosphatidylinositol-phosphate kinase, and that this pathway might predominate in cells with low [Ca2+]i.     

Irregular spiking in free calcium concentration in single, human platelets. Regulation by modulation of the inositol trisphosphate receptors.

Fluorescence ratio imaging indicates that immobilized, aspirin-treated platelets, loaded with Fura-2, respond to inositol 1,4,5-trisphosphate- (InsP3)-generating agonists such as thrombin by high-frequency, irregular rises in cytosolic [Ca2+]i with spikes that vary in peak level and peak-to-peak interval. This differs from the regular [Ca2+]i oscillations observed in other, larger cells. We found that the thiol-reactive compounds thimerosal (10 microm) and U73122 (10 microm) evoked similar irregular Ca2+ responses in platelets, but in this case in the absence of InsP3 generation. Thrombin-induced spiking was acutely abolished by inhibiting phospholipase C or elevating intracellular cAMP levels, while spiking with sulfhydryl reagents was only partially blocked by cAMP elevation. Confocal laser scanning microscopy using fluo-3-loaded platelets indicated that, with all agonists or conditions, the irregular spikes were almost instantaneously raised in various regions within a single platelet. When using saponin-permeabilized platelets, we found that InsP3-induced Ca2+ release from stores was stimulated by modest Ca2+ concentrations, pointing to a mechanism of InsP3-dependent Ca2+-induced Ca2+ release (CICR). This process was completely inhibitable by heparin. The Ca2+ release by InsP3, but not the CICR sensor, was negatively regulated by cAMP elevation. Thimerosal treatment did not release Ca2+ from intracellular stores, but markedly potentiated the stimulatory effect of InsP3. In contrast, U73122 caused a heparin/cAMP-insensitive Ca2+ leak from stores that differed from those used by InsP3. Taken together, these results demonstrate that InsP3 receptor channels play a crucial role in the irregular, spiking Ca2+ signal of intact platelets, even when induced by agents such as thimerosal or U73122 which do not stimulate InsP3 formation. The irregular Ca2+ release events appear to be subjected to extensive regulation by: (a) InsP3 level, (b) the potentiating effect of elevated Ca2+ on InsP3 action via CICR, (c) InsP3 channel sensitization by sulfhydryl (thimerosal) modification, (d) InsP3 channel-independent Ca2+ leak with U73122, and (e) down-regulation via cAMP elevation. The observation that individual Ca2+ peaks were generated in various parts of a platelet at similar intervals and amplitudes points to effective cooperation of the various stores in the Ca2+-release process.     

Novel regulation of calcium inhibition of the inositol 1,4,5-trisphosphate receptor calcium-release channel

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R), a Ca2+-release channel localized to the endoplasmic reticulum, plays a critical role in generating complex cytoplasmic Ca2+ signals in many cell types. Three InsP3R isoforms are expressed in different subcellular locations, at variable relative levels with heteromultimer formation in different cell types. A proposed reason for this diversity of InsP3R expression is that the isoforms are differentially inhibited by high cytoplasmic free Ca2+ concentrations ([Ca2+]i), possibly due to their different interactions with calmodulin. Here, we have investigated the possible roles of calmodulin and bath [Ca2+] in mediating high [Ca2+]i inhibition of InsP3R gating by studying single endogenous type 1 InsP3R channels through patch clamp electrophysiology of the outer membrane of isolated Xenopus oocyte nuclei. Neither high concentrations of a calmodulin antagonist nor overexpression of a dominant-negative Ca2+-insensitive mutant calmodulin affected inhibition of gating by high [Ca2+]i. However, a novel, calmodulin-independent regulation of [Ca2+]i inhibition of gating was revealed: whereas channels recorded from nuclei kept in the regular bathing solution with [Ca2+] approximately 400 nM were inhibited by 290 muM [Ca2+]i, exposure of the isolated nuclei to a bath solution with ultra-low [Ca2+] (<5 nM, for approximately 300 s) before the patch-clamp experiments reversibly relieved Ca2+ inhibition, with channel activities observed in [Ca2+]i up to 1.5 mM. Although InsP3 activates gating by relieving high [Ca2+]i inhibition, it was nevertheless still required to activate channels that lacked high [Ca2+]i inhibition. Our observations suggest that high [Ca2+]i inhibition of InsP3R channel gating is not regulated by calmodulin, whereas it can be disrupted by environmental conditions experienced by the channel, raising the possibility that presence or absence of high [Ca2+]i inhibition may not be an immutable property of different InsP3R isoforms. Furthermore, these observations support an allosteric model in which Ca2+ inhibition of the InsP3R is mediated by two Ca2+ binding sites, only one of which is sensitive to InsP3.     

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.     

Inositol trisphosphate is required for the propagation of calcium waves in Xenopus oocytes.

Stimuli which act through the second messenger inositol 1,4,5-trisphosphate (InsP3) often increase free intracellular Ca2+ concentration ([Ca2+]i) in a localized subcellular area. Actively propagated Ca2+ waves then extend this focal Ca2+ signal to other parts of the cell. To understand how cells may control the spatial distribution of Ca2+, we investigated the mechanism by which Ca2+ waves propagate through the cytoplasm of Xenopus oocytes. Heparin, which inhibits the binding of InsP3 to its receptor, prevented the migration of Ca2+ waves induced by a poorly metabolized InsP3 (InsP3S3). This result suggested that Ca2+ waves move through the cell via the serial release of Ca2+ from InsP3-sensitive stores. Interventions which caused a localized increase in [Ca2+]i without elevations of InsP3 did not trigger Ca2+ waves. In the presence of a Ins-P3S3, however, endogenously released or locally injected Ca2+ elicited Ca2+ waves. A cooperative interaction between Ca2+ and InsP3 may therefore be responsible for the propagation of Ca2+ waves.     

Bradykinin-evoked acetylcholine release via inositol trisphosphate-dependent elevation in free calcium in neuroblastoma x glioma hybrid NG108-15 cells

The mechanism underlying the bradykinin (BK)-induced increase of acetylcholine (ACh) release was studied in neuroblastoma x glioma hybrid NG108-15 cells and their synapses formed onto mouse muscle cells. External application of BK or iontophoretic injection of extrinsic inositol 1,4,5-trisphosphate (InsP3) into the cytoplasm of NG108-15 cells produced membrane hyperpolarization in the hybrid cells and an increase in the frequency of miniature end-plate potentials (MEPPs) in paired myotubes. Ba2+ blocked the hyperpolarization in response to BK, but facilitation of MEPPs was still observed. InsP3-dependent facilitation of MEPPs was also observed in cells where the InsP3 injections produced no detectable hyperpolarization or even depolarization. Real-time quantitative monitoring of intracellular free Ca2+ concentration [( Ca2+]i) with fura-2 in single NG108-15 cells showed that BK application or InsP3 injection induced an elevation of [Ca2+]i which coincided in time with membrane hyperpolarization recorded from the same cell. The [Ca2+]i rise produced by InsP3 injection started from the single site of injection and that produced by BK began from a deep compartment of the cytoplasm of the NG108-15 cells. The BK- and InsP3-evoked facilitation of MEPPs and the [Ca2+]i rise were relatively independent of extracellular Ca2+. These findings suggest that the BK-induced ACh release results not from membrane potential changes but from a transient InsP3-dependent elevation of [Ca2+]i.     

Inositol trisphosphate and ryanodine receptors share a common functional Ca2+ pool in cerebellar Purkinje neurons

Changes in the intracellular free calcium concentration ([Ca2+]i) control many important processes in excitable and nonexcitable cells. In cerebellar Purkinje neurons, increases in [Ca2+]i modulate excitability by turning on calcium-activated potassium and chloride conductances, and modifying the synaptic efficacy of inhibitory and excitatory inputs to the cell. Calcium release from the intracellular stores plays an important role in the regulation of [Ca2+]i. Purkinje neurons contain both inositol trisphosphate (InsP3) and ryanodine (Ry) receptors. With the exception of the dendritic spines, where only InsP3 receptors are found, InsP3 and Ry receptors are present in the entire cell. The distribution of the two calcium release channels, however, is not uniform, and it has been suggested that InsP3 and Ry receptors use separate Ca2+ pools. The functional properties of InsP3 and Ry Ca2+ pools were investigated by flash photolysis and single-cell microspectrofluorimetry. It was found that depletion of ryanodine-sensitive Ca2+ stores renders InsP3 incapable of releasing more Ca2+ from the stores. Abolishing calcium-induced calcium release by blocking ryanodine receptors with ruthenium red did not have a significant effect on InsP3-evoked Ca2+ release. It is concluded that InsP3 receptors use the same functional Ca2+ pool as that utilized by Ry receptors in Purkinje neurons.     

The thiol reagent, thimerosal, evokes Ca2+ spikes in HeLa cells by sensitizing the inositol 1,4,5-trisphosphate receptor.

The thiol reagent, thimerosal, has been shown to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in several cell types, and to cause Ca2+ spikes in unfertilized hamster eggs. Using single cell video-imaging we have shown that thimerosal evokes repetitive Ca2+ spikes in intact Fura-2-loaded HeLa cells that were similar in shape to those stimulated by histamine. Both thimerosal- and histamine-stimulated Ca2+ spikes occurred in the absence of extracellular (Ca2+ o), suggesting that they result from mobilization of Ca2+ from intracellular stores. Whereas histamine stimulated formation of inositol phosphates, thimerosal, at concentrations that caused sustained Ca2+ spiking, inhibited basal and histamine-stimulated formation of inositol phosphates. Thimerosal-evoked Ca2+ spikes are therefore not due to the stimulated production of inositol 1,4,5-trisphosphate (InsP3). The effects of thimerosal on Ca2+ spiking were probably due to alkylation of thiol groups on intracellular proteins because the spiking was reversed by the thiol-reducing compound dithiothreitol, and the latency between addition of thimerosal and a rise in [Ca2+]i was greatly shortened in cells where the intracellular reduced glutathione concentration had been decreased by preincubation with DL-buthionine (S,R)-sulfoximine. In permeabilized cells, thimerosal caused a concentration-dependent inhibition of Ca2+ accumulation, which was entirely due to inhibition of Ca2+ uptake into stores because thimerosal did not affect unidirectional 45Ca2+ efflux from stores preloaded with 45Ca2+. Thimerosal also caused a concentration-dependent sensitization of InsP3-induced Ca2+ mobilization: half-maximal mobilization of Ca2+ stores occurred with 161 +/- 20 nM InsP3 in control cells and with 62 +/- 5 nM InsP3 after treatment with 10 microM thimerosal. We conclude that thimerosal can mimic the effects of histamine on intracellular Ca2+ spiking without stimulating the formation of InsP3 and, in light of our results with permeabilized cells, suggest that thimerosal stimulates spiking by sensitizing cells to basal InsP3 levels.     

Inositol 1,4,5-trisphosphate-induced Ca2+ release from the sarcoplasmic reticulum and contraction in crustacean muscle

Intracellular applications of a fixed amount (0.2 to 8 nmol) of inositol 1,4,5-trisphosphate (InsP3) over a brief period (2 s) into barnacle muscle fibers induced vigorous contractures. Peak tension attained during the first application depended on [InsP3]: the maximum tension evoked by the injection of 8 nmol was 1.6 kg/cm2. Peak tension during a second application of a high dose of InsP3 (greater than 10 microM) was always smaller than that during the first application. Extracellular Ca2+ could be omitted with no measurable effects on either the amplitude or time course of the contractures evoked by InsP3. Aequorin was used to measure InsP3-evoked Ca2+ release from intracellular stores in minced muscle fibers from lobster and in skinned muscle fibers from barnacle. Provided the sarcoplasmic reticulum was preloaded with Ca2+, application of InsP3 induced a transient Ca2+ release that was [InsP3] dependent. During each transient, [Ca2+] rose rapidly to a peak value (t1/2 less than 5 s) and then slowly returned (t1/2 less than 100 s) to a basal level. Maximum Ca2+ release was obtained at [InsP3] less than 100 microM and amounted to 4 nmol Ca2+/g of muscle, enough to increase [Ca2+]i from 0.1 to 8 microM had the Ca2+ release occurred in the intact fiber. Successive applications of a fixed amount of InsP3 elicited successive transient increases in Ca2+. The effects of [Ca2+] on the incorporation of [3H]inositol into the pools of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate pools were measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caffeine inhibits the agonist-evoked cytosolic Ca2+ signal in mouse pancreatic acinar cells by blocking inositol trisphosphate production.

The inhibitory effects of caffeine on receptor-activated cytosolic Ca2+ signal generation in isolated mouse pancreatic acinar cells were investigated. Using the ability of caffeine to quench Indo-1 fluorescence we measured simultaneously the free intracellular Ca2+ concentration ([Ca2+]i) and the intracellular caffeine concentration ([caffeine]i). We also measured inositol 1,4,5-trisphosphate (InsP3) production with a radioreceptor assay. When caffeine was added to the extracellular solution during a sustained receptor-activated increase in [Ca2+]i, [caffeine]i rose to its steady level within a few seconds. This was accompanied by a decrease of [Ca2+]i, which started only after [caffeine]i had reached an apparent threshold concentration (about 2 mM in the case of 0.5 microM acetylcholine (ACh) stimulation). Above this [caffeine]i level there was a linear relationship between [caffeine]i and [Ca2+]i. Throughout the caffeine exposure [Ca2+]i remained at a steady low level. Following removal of caffeine from the bath, [caffeine]i decreased to zero within seconds. There was no significant increase in [Ca2+]i until [caffeine]i had been reduced to the threshold level (about 2 mM at 0.5 microM ACh). Caffeine inhibited Ca2+ signals evoked by ACh, cholecystokinin, and ATP and also inhibited signals generated in the absence of external Ca2+. Caffeine application had the same effect as removal of agonist allowing recovery from apparent desensitization. Caffeine inhibited the agonist-evoked production of InsP3 in a dose-dependent manner. Our results demonstrate the acute and reversible dose-dependent inhibition of agonist-evoked cytosolic Ca2+ signal generation due to rapid intracellular caffeine accumulation and washout. The inhibition can be explained by the reduction of agonist-evoked InsP3 production.     

Internal Ca2+ mobilization and secretion in bovine adrenal chromaffin cells

Since secretion from intact bovine adrenal chromaffin cells in response to depolarization by nicotine is triggered by a rise in the concentration of intracellular Ca2+ ([Ca2+]i) to about 200-300 nM above basal, it has been assumed that the failure of the inositol 1,4,5-trisphosphate (InsP3)-mobilizing muscarinic agonists to induce secretion reflects the fact that the 50 nM rise in [Ca2+]i they elicit is insufficient to trigger the exocytotic machinery. A recent report, however, has demonstrated that some of the nicotine-induced rise in [Ca2+]i could originate from the InsP3-releasable Ca2+ store. The role of this Ca2+ store in secretion from bovine adrenal chromaffin cells is therefore unclear. In order to investigate in more detail the role of the InsP3-sensitive Ca2+ store in secretion from these cells, we have used a combination of an InsP3-mobilizing muscarinic agonist and the sesquiterpene lactone thapsigargin (TG), which releases internal Ca2+ without concomitant breakdown of inositol lipids or protein kinase C activation, to examine the events which follow depletion of the releasable Ca2+ store in these cells. Monitoring [Ca2+]i using Fura-2 demonstrated that TG released Ca2+ from the InsP3-sensitive store and, additionally, that the Ca2+ response to TG was composed of two distinct, temporally separated, components: a) a slow (1 min) increase in [Ca2+]i to approximately 50 nM above basal that was independent of extracellular Ca2+ and b) the maintenance of this level at a new steady-state that was dependent on the continual entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism mediating regenerative intercellular Ca2+ waves in the blowfly salivary gland.

Intercellular Ca2+ signaling in intact salivary glands of the blowfly Calliphora erythrocephala was studied by fluorimetric digital imaging combined with microinjection of putative messenger molecules. Iontophoretic injection of D-myo-inositol 1,4, 5-trisphosphate (InsP3) into salivary gland cells evoked regenerative intercellular Ca2+ waves that spread through the impaled cell and several rows of surrounding cells. Ca2+ increases induced by microinjection of Ca2+ ions were confined to the injected cells and their nearest neighbors. Depletion of intracellular Ca2+ stores by thapsigargin pre-treatment did not alter the time course of the Ca2+ increase caused by Ca2+ injection. However, activation of Ca2+ release became clearly evident when Ca2+ was injected in the presence of serotonin (5-HT). Under these conditions, injection of Ca2+ triggered intercellular Ca2+ waves that consecutively passed through >10 cells. The phospholipase C inhibitor U73122 blocked 5-HT-induced Ca2+ increases but did not affect InsP3-dependent Ca2+ spiking and intercellular Ca2+ wave propagation. The results demonstrate that propagation of agonist-evoked Ca2+ waves in the blowfly salivary gland requires supra-basal [InsP3] but does not depend on feedback activation of phospholipase C. We conclude that the intra- and intercellular transmission of these Ca2+ waves is mediated by diffusion of Ca2+ and Ca2+-induced Ca2+ release via the InsP3 receptor channel.     

Novel model of calcium and inositol 1,4,5-trisphosphate regulation of InsP3 receptor channel gating in native endoplasmic reticulum

The InsP3R Ca(2+)-release channel has biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing high [Ca2+]i inhibition. To determine whether relieving Ca2+ inhibition is sufficient for activation, we examined single-channels in low [Ca2+]i in the absence of InsP3 by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent activities with low open probability Po (approximately 0.03) were observed in [Ca2+]i < 5 nM, whereas none were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i in the absence of InsP3 and demonstrate that the channel can be active when all of its ligand-binding sites are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies, the tetrameric channel can adopt six conformations, the equilibria among which are controlled by two inhibitory, one activating Ca(2+)-binding, and one InsP3-binding sites in a manner similar to the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the relative affinity for Ca2+ of one of the inhibitory sites in different channel conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent second inhibitory site.     

Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 insp3 receptors

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3-induced Ca2+ release and low gain Ca2+ -induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.     

Spontaneous channel activity of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Application of allosteric modeling to calcium and InsP3 regulation of InsP3R single-channel gating

The InsP3R Ca2+ release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca2+]i. To determine if relieving Ca2+ inhibition is sufficient for channel activation, we examined single-channel activities in low [Ca2+]i in the absence of InsP3, by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent channel activities with low open probability Po ( approximately 0.03) were observed in [Ca2+]i < 5 nM with the same frequency as in the presence of InsP3, whereas no activities were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i of the channel to be 1.2-4.0 nM in the absence of InsP3, and demonstrate that the channel can be active when all of its ligand-binding sites (including InsP3) are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca2+]i and InsP3 regulation of steady-state channel gating behavior of types 1 and 3 InsP3R isoforms, including spontaneous InsP3-independent channel activities, the tetrameric channel can adopt six different conformations, the equilibria among which are controlled by two inhibitory and one activating Ca2+-binding and one InsP3-binding sites in a manner outlined in the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the Ca2+ affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent low-affinity inhibitory site. The model also suggests that besides the ligand-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel Po being less than unity. The validity of this model was established by its successful quantitative prediction of channel behavior after it had been exposed to ultra-low bath [Ca2+].     

Inositol 1,4,5-trisphosphate increases myoplasmic [Ca2+] in isolated muscle fibers. Depolarization enhances its effects

Inositol 1,4,5-trisphosphate (InsP3) has been proposed as an intracellular messenger which mobilizes calcium from the sarcoplasmic reticulum, during excitation-contraction coupling in skeletal muscle. We have measured the myoplasmic free calcium concentration ([Ca2+]i) by means of calcium selective microelectrodes in intact fibers isolated from Leptodactylus insularis microinjected with InsP3. In muscle fibers bathed in normal Ringer, the mean resting [Ca2+]i was 0.11 +/- 0.01 microM (M +/- SEM, n = 30). The microinjection of 0.3, 0.5 and 1 microM InsP3 induced transient increments in the [Ca2+]i to 0.35 +/- 0.02 microM (n = 9), to 0.53 +/- 0.03 microM (n = 11) and 0.94 +/- 0.06 microM (n = 10) respectively. Microinjection of 0.3, 0.5 and 1 microM InsP3 in muscle fibers incubated in low Ca2+ solution induced increments in [Ca2+]i similar to those observed in fibers bathed with normal Ringer. The microinjection of 0.3, 0.5 and 1 microM InsP3 in muscle fibers partially depolarized with 10 mM [K+]o induced transient enhancements of the resting [Ca2+]i that were greater than the transients observed in the normally polarized muscle. In partially depolarized fibers microinjected with 0.3, 0.5 and 1 microM InsP3, the [Ca2+]i was changed to 1.45 +/- 0.14 microM (n = 20), to 3.37 +/- 0.34 microM (n = 7) and to 7.43 +/- 0.70 microM (n = 6) respectively. In all partially depolarized fibers these increments in [Ca2+]i were associated with local contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2*- that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2*--triggered Ca2+ mobilization preceded a loss in mitochondrial membrane potential that was independent of other oxidants and mitochondrially derived ROS. Activation of apoptosis occurred selectively in response to O2*- and could be prevented by [Ca2+]i buffering. This study provides evidence that O2*- facilitates an InsP3R-linked apoptotic cascade and may serve a critical function in I/R injury and inflammation.     

Desynchronising effect of the endothelium on intracellular Ca2+ concentration dynamics in vascular smooth muscle cells of rat mesenteric arteries

The kinetics of the intracellular Ca2+ concentration ([Ca2+]i) of vascular smooth muscle cells (VSMCs) in rat small mesenteric arteries was investigated by confocal laser scanning microscopy using the fluorescent Ca2+ indicator fluo-3 AM. One micromole noradrenaline (NA) induced randomly distributed transient elevations of [Ca2+]i in several single VSMCs which were weakly temporally coupled. Higher NA concentrations of 3 or 10 microM, however, induced strongly synchronised [Ca2+]i oscillations in VSMCs. In preparations with intact endothelium, the synchronisation of [Ca2+]i signals was attenuated by acetylcholine (ACh) but augmented by the NO synthase antagonist L-NAME, pointing to a desynchronising effect of the endothelium even under basal conditions. In preparations with or without intact endothelium sodium nitroprusside (SNP) as well as the gap-junction uncoupler heptanol reversibly desynchronised the [Ca2+]i transients. The effect of ACh but not that of SNP was influenced by L-NAME. Propagated intracellular [Ca2+]i waves had a velocity of 25 microm/s. The phase shift of [Ca2+]i oscillations between single VSMCs were maximally 2s and independent of the distance of up to 90 microm between individual cells. Therefore, we consider intercellular [Ca2+]i waves to be too slow to account for the synchronisation of [Ca2+]i oscillations.We conclude that the coupling of [Ca2+]i signals in vascular smooth muscle cells is not constant but highly regulated by NA and by endothelium derived NO. Oscillations of vessel contraction at high sympathetic tone may be induced by synchronisation of [Ca2+]i transients of distinct VSMCs whereas endothelium derived NO inhibits vasomotion by desynchronising [Ca2+]i transients of single VSMCs.     

Critical intracellular Ca2+ concentration for all-or-none Ca2+ spiking in single smooth muscle cells.

Neurotransmitters induce contractions of smooth muscle cells initially by mobilizing Ca2+ from intracellular Ca2+ stores through inositol 1,4,5-trisphosphate (InsP3) receptors. Here we studied roles of the molecules involved in Ca2+ mobilization in single smooth muscle cells. A slow rise in cytoplasmic Ca2+ ([Ca2+]i) in agonist-stimulated smooth muscle cells was followed by a wave of rapid regenerative Ca2+ release as the local [Ca2+]i reached a critical concentration of approximately 160 nM. Neither feedback regulation of phospholipase C nor caffeine-sensitive Ca(2+)-induced Ca2+ release was found to be required in the regenerative Ca2+ release. These results indicate that Ca(2+)-dependent feedback control of InsP3-induced Ca2+ release plays a dominant role in the generation of the regenerative Ca2+ release. The resulting Ca2+ release in a whole cell was an all-or-none event, i.e. constant peak [Ca2+]i was attained with agonist concentrations above the threshold value. This finding suggests a possible digital mode involved in the neural control of smooth muscle contraction.     

ATP regulation of recombinant type 3 inositol 1,4,5-trisphosphate receptor gating

A family of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channels plays a central role in Ca2+ signaling in most cells, but functional correlates of isoform diversity are unclear. Patch-clamp electrophysiology of endogenous type 1 (X-InsP3R-1) and recombinant rat type 3 InsP3R (r-InsP3R-3) channels in the outer membrane of isolated Xenopus oocyte nuclei indicated that enhanced affinity and reduced cooperativity of Ca2+ activation sites of the InsP3-liganded type 3 channel distinguished the two isoforms. Because Ca2+ activation of type 1 channel was the target of regulation by cytoplasmic ATP free acid concentration ([ATP](i)), here we studied the effects of [ATP]i on the dependence of r-InsP(3)R-3 gating on cytoplasmic free Ca2+ concentration ([Ca2+]i. As [ATP]i was increased from 0 to 0.5 mM, maximum r-InsP3R-3 channel open probability (Po) remained unchanged, whereas the half-maximal activating [Ca2+]i and activation Hill coefficient both decreased continuously, from 800 to 77 nM and from 1.6 to 1, respectively, and the half-maximal inhibitory [Ca2+]i was reduced from 115 to 39 microM. These effects were largely due to effects of ATP on the mean closed channel duration. Whereas the r-InsP3R-3 had a substantially higher Po than X-InsP3R-1 in activating [Ca2+]i (< 1 microM) and 0.5 mM ATP, the Ca2+ dependencies of channel gating of the two isoforms became remarkably similar in the absence of ATP. Our results suggest that ATP binding is responsible for conferring distinct gating properties on the two InsP3R channel isoforms. Possible molecular models to account for the distinct regulation by ATP of the Ca2+ activation properties of the two channel isoforms and the physiological implications of these results are discussed. Complex regulation by ATP of the types 1 and 3 InsP3R channel activities may enable cells to generate sophisticated patterns of Ca2+ signals with cytoplasmic ATP as one of the second messengers.