A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL.

Intracellular Ca2+ signalling evoked by Ca2+ mobilizing agonists, like angiotensin II in the adrenal gland, involves the activation of inositol(1,4,5)trisphosphate(InsP3)-mediated Ca2+ release from internal stores followed by activation of a Ca2+ influx termed capacitative calcium entry. Here we report the amino acid sequence of a functional capacitative Ca2+ entry (CCE) channel that supports inward Ca2+ currents in the range of the cell resting potential. The expressed CCE channel opens upon depletion of Ca2+ stores by InsP3 or thapsigargin, suggesting that the newly identified channel supports the CCE coupled to InsP3 signalling.     

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.     

Characterization of a membrane protein from brain mediating the inhibition of inositol 1,4,5-trisphosphate receptor binding by calcium.

Inositol 1,4,5-trisphosphate (InsP3) is a component of the phosphoinositide second-messenger system which mobilizes Ca2+ from intracellular stores. Recently, an InsP3 receptor binding protein from rat cerebellar membranes was solubilized and purified to homogeneity. The potent inhibition by Ca2+ of [3H]InsP3 binding to the InsP3 receptor in cellular membranes is not apparent in the purified receptor. The Ca2+-dependent inhibition of [3H]InsP3 binding in the crude homogenate (concn. giving 50% inhibition = 300 nM) can be restored by addition of solubilized cerebellar membranes to the purified receptor. In the present study, we further characterize the protein in solubilized membranes which confers Ca2+-sensitivity to the receptor, and which we term 'calmedin'. Calmedin appears to be a neutral membrane protein with an estimated Mr of 300,000 by gel filtration in the presence of Triton X-100. Calmedin confers a Ca2+-sensitivity to InsP3 receptor binding, which can be completely reversed by 10 min incubation with EDTA and therefore does not represent Ca2+-dependent proteinase action. Calmedin effects on the purified InsP3 receptor depend on Ca2+ binding to the calmedin, although Ca2+ also binds directly to the InsP3 receptor. The regional distribution of calmedin differs from that of the InsP3 receptor in the brain, suggesting that it also mediates other Ca2+-dependent functions. Calmedin activity in peripheral tissues is much lower than in brain.     

Structure of a novel InsP3 receptor.

Inositol 1,4,5-trisphosphate (InsP3) constitutes a major intracellular second messenger that transduces many growth factor and neurotransmitter signals. InsP3 causes the release of Ca2+ from intracellular stores by binding to specific receptors that are coupled to Ca2+ channels. One such receptor from cerebellum has previously been extensively characterized. We have now determined the full structure of a second, novel InsP3 receptor which we refer to as type 2 InsP3 receptor as opposed to the cerebellar type 1 InsP3 receptor. The type 2 InsP3 receptor has the same general structural design as the cerebellar type 1 InsP3 receptor with which it shares 69% sequence identity. Expression of the amino-terminal 1078 amino acids of the type 2 receptor demonstrates high affinity binding of InsP3 to the type 2 receptor with a similar specificity but higher affinity than observed for the type 1 receptor. These results demonstrate the presence of several types of InsP3 receptor in brain and raise the possibility that intracellular Ca2+ signaling may involve multiple pathways with different regulatory properties dependent on different InsP3 receptors.     

Spatial and temporal aspects of Ca2+ signaling mediated by P2Y receptors in cultured rat hippocampal astrocytes

ATP produces a variety of Ca2+ responses in astrocytes. To address the complex spatio-temporal Ca2+ signals, we analyzed the ATP-evoked increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured rat hippocampal astrocytes using fura-2 or fluo-3 based Ca2+ imaging techniques. ATP at less than 10 nM produced elementary Ca2+ release event "puffs" in a manner independent of extracellular Ca2+. Stimulation with higher ATP concentrations (3 or 10 micro M) resulted in global Ca2+ responses such as intercellular Ca2+ wave. These Ca2+ responses were mainly mediated by metabotropic P2Y receptors. ATP acting on both P2Y1 and P2Y2 receptors produced a transient Ca2+ release by inositol 1,4,5-trisphosphate (InsP3). When cells were stimulated with ATP much longer, the transient [Ca2+]i elevation was followed by sustained Ca2+ entry from the extracellular space. This sustained rise in [Ca2+]i was inhibited by Zn2+ (<10 micro M), an inhibitor of capacitative Ca2+ entry (CCE). CCE induced by cyclopiazonic acid or thapsigargin and Ca2+ entry evoked by ATP share the same pharmacological profile in astrocytes. Taken together, the hierarchical Ca2+ responses to ATP were observed in hippocampal astrocytes, i.e., puffs, global Ca2+ release by InsP3, and CCE in response to depletion of InsP3-sensitive Ca2+ stores. It should be noted that these Ca2+ signals and their modulation by Zn2+ could occur in the hippocampus in situ since both ATP and Zn2+ are rich in the hippocampus and could be released by excitatory stimulation.     

A Novel Effect of Cyclic AMP on Capacitative Ca2+ Entry in Cultured Rat Cerebellar Astrocytes

One of the most important intracellular Ca2+ regulatory mechanisms in nonexcitable cells, "capacitative Ca2+ entry" (CCE), has not been adequately studied in astrocytes. We therefore investigated whether CCE exists in cultured rat cerebellar astrocytes and studied the roles of cyclic AMP (cAMP) and protein kinase C (PKC) in CCE. We found that (1) at least two different intracellular Ca2+ stores, the endoplasmic reticulum and mitochondria, are present in cerebellar astrocytes; (2) CCE does exist in these cells and can be inhibited by Ni2+, miconazole, and SKF 96365; (3) CCE can be directly enhanced by an increase in intracellular cAMP, as 8-bromoadenosine 3',5'-cyclic monophosphate (8-brcAMP), forskolin, and isobutylmethylxanthine have stimulatory effects on CCE; and (4) neither of the two potent protein kinase A (PKA) inhibitors, H8 and H89, nor a specific PKA agonist, Sp-adenosine 3',5'-cyclic monophosphothioate, had a significant effect on cAMP-enhanced Ca2+ entry. The [Ca2+]i increase was not due to a release from calcium stores, hyperpolarization of the membrane potential, inhibition of calcium extrusion, or a change in pHi, suggesting that cAMP itself probably acts as a novel messenger to modulate CCE. We also conclude that activation of PKC results in an increase in CCE. cAMP and PKC seem to modulate CCE by different pathways.     

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.     

Inositol tetrakisphosphate as a second messenger: confusions, contradictions, and a potential resolution.

The second messenger function of inositol 1,4,5-trisphosphate (InsP3) is now well-defined--it mobilizes Ca2+ from intracellular stores so that cystolic Ca2+ increases. However, the function of inositol 1,3,4,5-tetrakisphosphate (InsP4) has proved much more difficult to fathom, as it has been reported to exert a wide variety of effects in a collection of experimental systems. In this review, a proposed molecular mechanism for InsP4's actions is discussed; it is suggested that InsP4 is the second messenger that controls Ca2+ entry into cells, and that it does so by binding to a receptor which itself interacts, directly or indirectly, with the receptor for InsP3. It is proposed that this is InsP4's true physiological function, but the mechanism by which it exerts this function has led to confusing data concerning its action, and also to some misconceptions about how inositol phosphates control Ca2+ entry.     

Inhibition of the type 1 inositol 1,4,5-trisphosphate-sensitive Ca2+ channel by calmodulin antagonists

This study describes the effects of a number of calmodulin antagonists on the cerebellar type 1 inositol 1,4,5-trisphosphate (InsP3) receptor. All the antagonists tested (trifluoperazine, fluphenazine, chlorpromazine and calmidazolium) inhibited the extent of InsP3-induced Ca2+ release (IICR) with similar IC(50) values (between 60 and 85 microM). They did not affect the efficacy of InsP3 to release Ca2+, since the concentrations of InsP3 required to cause half-maximal release was little affected in the presence of these agents. In addition, these agents did not affect InsP3 binding to its receptor. Stopped-flow studies to determine the rate constants of IICR showed this process to be biphasic with a fast and slow component. All the calmodulin antagonists appeared to reduce the rate constants for Ca2+ release in a phase-specific manner, preferentially reducing the fast phase component. Chlorpromazine (75 microM) appeared to have the most potent effect on the fast phase rate constant, reducing it from 1.0 to 0.08 s(-1), while only reducing the rate constant for the slow phase about twofold (0.2-0.08 s(-1)). The fact that calmodulin itself inhibits both IICR and InsP3 binding, while these calmodulin antagonists also reduce Ca2+ release and do not affect InsP3 binding, suggests that the mechanism of action of these agents is unlikely to be due to the reversal of the modulatory action of calmodulin on this receptor.     

Mechanism of Ca2+ inhibition of inositol 1,4,5-trisphosphate (InsP3) binding to the cerebellar InsP3 receptor.

Ca2+ efficiently inhibits binding of inositol 1,4,5-trisphosphate (InsP3) to the InsP3 receptor in cerebellar membranes but not to the purified receptor. We have now investigated the mechanism of action by which Ca2+ inhibits InsP3 binding. Our results suggest that Ca2+ does not cause the stable association of a Ca(2+)-binding protein with the receptor. Instead, Ca2+ leads to the production of a soluble, heat-stable, low molecular weight substance from cerebellar membranes that competes with InsP3 for binding. This inhibitory substance probably represents endogenously generated InsP3 as judged by the fact that it co-purifies with InsP3 on anion-exchange chromatography, competes with [3H]InsP3 binding in a pattern similar to unlabeled InsP3, and is in itself capable of releasing 45Ca2+ from permeabilized cells. A potent Ca(2+)-activated phospholipase C activity producing InsP3 was found in cerebellar microsomes that exhibited a Ca2+ dependence identical to the Ca(2+)-dependent inhibition of InsP3 binding. Together these results suggest that the Ca(2+)-dependent inhibition of InsP3 binding to the cerebellar receptor is due to activation of a Ca(2+)-sensitive phospholipase C enriched in cerebellum. Nevertheless, Ca2+ probably also modulates the InsP3 receptor function by a direct interaction with the receptor that does not affect InsP3 binding but regulates InsP3-dependent channel gating.     

The effect of external calcium and pH on inositol trisphosphate-mediated calcium release from cerebellum microsomal fractions.

Binding of D-myo-inositol 1,4,5-trisphosphate (InsP3) to rat cerebellum membranes has previously been shown to be stimulated by alkaline pH and inhibited by low concentrations of Ca2+ [Worley, Baraban, Suppatopone, Wilson & Snyder (1987) J. Biol. Chem. 262, 12132-12136]. In the present study, Scatchard analysis of InsP3 binding to cerebellum microsomes indicates that the effects of Ca2+ and pH are exerted through changes in the apparent affinity of the receptor without effects on maximal binding. The influence of extravesicular Ca2+ and pH on InsP3-mediated 45Ca2+ release was investigated. Extravesicular Ca2+ inhibited InsP3-mediated Ca2+ release. The inhibitory effect of Ca2+ was most marked when a sub-optimal concentration of InsP3 was used. An increase in extravesicular pH produced a decrease in the concentration of InsP3 that yielded half-maximal Ca2+ release. Regulation of the affinity of the InsP3 receptor by Ca2+ and pH can qualitatively account for the observed effects of these factors on InsP3-mediated Ca2+ release. Feedback inhibition of InsP3 binding by Ca2+ could provide a mechanism to generate Ca2+ oscillations, particularly under hormonal conditions that produce sub-optimal elevations of InsP3 concentration.     

Cyclic ADP-ribose regulation of ryanodine receptors involved in agonist evoked cytosolic Ca2+ oscillations in pancreatic acinar cells.

We have investigated the role of the ryanodine-sensitive intracellular Ca2+ release channel (ryanodine receptor) in the cytosolic Ca2+ oscillations evoked in pancreatic acinar cells by acetylcholine (ACh) or cholecystokinin (CCK). Ryanodine abolished or markedly inhibited the agonist evoked Ca2+ spiking, but enhanced the frequency of spikes evoked by direct internal inositol trisphosphate (InsP3) application. We have also investigated the possibility that cyclic ADP-ribose (cADP-ribose), the putative second messenger controlling the ryanodine receptor, plays a role in Ca2+ oscillations. We found that cADP-ribose could itself induce repetitive Ca2+ spikes localized in the secretory pole and that these spikes were blocked by ryanodine, but also by the InsP3 receptor antagonist heparin. Our results indicate that both the ryanodine and the InsP3 receptors are involved in Ca2+ spike generation.     

Heparin inhibits the inositol 1,4,5-trisphosphate-dependent, but not the independent, calcium release induced by guanine nucleotide in vascular smooth muscle

The effects of heparin on the release of intracellular Ca2+, assessed by tension development in saponin-permeabilized rabbit main pulmonary artery, were determined. Heparin inhibited (IC50 = 5 micrograms/ml) inositol 1,4,5-trisphosphate (InsP3)-induced, but not caffeine-induced, Ca2+ release. The initial (InsP3-dependent) component of GTP gamma S-induced Ca2+-release was also inhibited by heparin, but the InsP3-independent component was resistant to both heparin and procaine. These results support the existence of a G protein activated mechanism of Ca2+ release that is not mediated by InsP3 or by Ca2+-induced Ca2+ release.     

G-proteins in the signal-transduction pathways of Dictyostelium discoideum

The functional interaction of surface cAMP receptors with effector enzymes via G-proteins was investigated in Dictyostelium discoideum. Several experimental conditions were used to investigate signal transduction, such as reduced temperatures, use of down-regulated cells and of mutants. The results are presented as a model describing the complex interaction between multiple forms of the surface cAMP receptor and different G-proteins that are responsible for the generation of the second messengers, cAMP, cGMP, InsP3 and Ca2+.     

Pulmonary artery smooth muscle cells from normal subjects and IPAH patients show divergent cAMP-mediated effects on TRPC expression and capacitative Ca2+ entry

Pulmonary vascular remodeling due to overgrowth of pulmonary artery smooth muscle cells (PASMC) is a major cause for the elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased cytosolic Ca(2+) concentration, resulting from enhanced capacitative Ca(2+) entry (CCE) and upregulated transient receptor potential (TRP) channel expression, is involved in stimulating PASMC proliferation. The current study was designed to determine the impact of cAMP, a second messenger that we hypothesized would blunt aspects of PASMC activity, as a possible contributor to IPAH pathophysiology. Short-term (30 min) pretreatment with forskolin (FSK; 10 muM), a direct activator of adenylyl cyclase, in combination with the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 200 muM), attenuated CCE in PASMC from normal subjects, patients without pulmonary hypertension (NPH), and patients with IPAH. The FSK-mediated CCE inhibition was independent of protein kinase A (PKA), because the PKA inhibitor H89 negligibly affected the decrease in CCE produced by cAMP. By contrast, longer (4 h) treatment with FSK (with IBMX) attenuated CCE in normal and NPH PASMC but enhanced CCE in IPAH PASMC. This enhancement of CCE was abolished by PKA inhibition and associated with an upregulation of TRPC3. In addition, cAMP increased TRPC1 mRNA expression in IPAH (but not in normal or NPH) PASMC, an effect blunted by H89. Furthermore, iloprost, a prostacyclin analog that increases cAMP, downregulated TRPC3 expression in IPAH PASMC and FSK-mediated cAMP increase inhibited IPAH PASMC proliferation. Although a rapid rise in cellular cAMP decreases CCE by a PKA-independent mechanism, sustained cAMP increase inhibits CCE in normal and NPH PASMC but increases CCE via a PKA-dependent pathway in IPAH PASMC. The divergent effect of cAMP on CCE parallels effects on TRPC expression. The results suggest that the combined use of a PKA inhibitor and cAMP-elevating drugs may provide a novel approach for treatment of IPAH.     

Cytosolic heparin inhibits muscarinic and alpha-adrenergic Ca2+ release in smooth muscle. Physiological role of inositol 1,4,5-trisphosphate in pharmacomechanical coupling

In order to test the physiological significance of inositol 1,4,5-trisphosphate (InsP3) in pharmacomechanical coupling, we have utilized two near-physiological systems, in which relatively high molecular weight solutes can be applied intracellularly and receptor coupling is retained: beta-escin permeabilization and reversible permeabilization. We showed that in smooth muscle permeabilized with beta-escin, one of the saponin esters, alpha 1-adrenergic (phenylephrine) and muscarinic (carbachol) agonists, as well as caffeine and InsP3, cause contractions mediated by Ca2+ release. These contractions were calmodulin-dependent and blocked by depletion of Ca2+ stored in the sarcoplasmic reticulum. Intracellular heparin (Mr = about 5000), a blocker of InsP3 binding to its receptor and a specific inhibitor of InsP3-induced Ca2+ release in smooth muscles, inhibited the responses to the agonists and to InsP3, but not those to caffeine, nor did it block the enhanced contractile response to cytoplasmic Ca2+ induced by agonists and by GTP gamma S. Neomycin blocked Ca2+ release induced by carbachol, but not by caffeine. In reversibly permeabilized ileum smooth muscle cells, loaded with Fura-2 acid and heparin, the intracellular heparin inhibited Ca2+ release and contractions induced by carbachol in Ca2+-free, high K+ solution. Heparin did not inhibit the high K+ contractions (with 1.2 mM Ca2+) and had no significant inhibitory effects on carbachol-induced responses in the presence of extracellular Ca2+. These results, obtained under near-physiological conditions, support the conclusion that InsP3 is the major physiological messenger of the Ca2+ release component of pharmacomechanical coupling, but not of the components mediated by Ca2+ influx or by potentiation of the contractile response to Ca2+.     

Prolonged exposure to inositol 1,4,5-trisphosphate does not cause intrinsic desensitization of the intracellular Ca(2+)-mobilizing receptor.

The rapid release of Ca2+ from intracellular stores stimulated with inositol 1,4,5-trisphosphate (InsP3) has required superfusion or stopped-flow techniques to resolve the kinetics of Ca2+ mobilization and made it difficult to determine whether the InsP3 receptor desensitizes during prolonged stimulation. Here we have overloaded the intracellular Ca2+ stores of permeabilized rat hepatocytes by incubating them with ATP and 45Ca2+ in the presence of pyrophosphate, which precipitates Ca2+ within the lumen of the stores. Subsequent ATP removal initiated slow 45Ca2+ efflux that followed zero-order kinetics, allowing us to examine the effects of InsP3 over a prolonged time course. InsP3 produced a concentration-dependent increase in the 45Ca2+ efflux rate that was sustained for several min. The rate rapidly returned to the unstimulated level after the addition of decavanadate, a competitive antagonist of InsP3 at its receptor. Prior incubation with a submaximal concentration of InsP3 (1 microM) did not affect the subsequent enhanced rate of 45Ca2+ efflux stimulated by a higher, but still submaximal, concentration of InsP3 (3 microM). We conclude that prolonged exposure to InsP3 does not desensitize the InsP3 receptor and that intrinsic receptor desensitization cannot provide an explanation for the quantal responses to InsP3 observed in several cell types.     

'Quantal' Ca2+ release and the control of Ca2+ entry by inositol phosphates--a possible mechanism

The release of Ca2+ from intracellular stores by sub-optimal doses of inositol trisphosphate has been shown to be dose-related ('quantal'), and a simple model is proposed here to account for this phenomenon. It is suggested that there is a regulatory Ca2(+)-binding site on, or associated with, the luminal domain of the InsP3 receptor, which allosterically controls Ca2+ efflux, and the affinity for Ca2+ of that site is modulated by InsP3 binding to the cytoplasmic domain of the receptor; a similar mechanism applied to the ryanodine receptor might also explain some aspects of Ca2(+)-induced Ca2+ release. The stimulated entry of Ca2+ into a cell which occurs upon activation of inositide-linked receptors has been variously and confusingly proposed to be regulated by InsP3, InsP4, and/or a 'capacitative' Ca2+ pool; the mechanism of InsP3 receptor action suggested here is shown to lead to a potential reconciliation of all these conflicting proposals.     

Temperature-dependence and conformational basis of inositol 1,4,5-trisphosphate receptor regulated by Ca2+

The inositol 1,4,5-trisphosphate (InsP3) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (1:1) successfully.No effect of Ca2+ concentration on [3H]-InsP3 binding to unreconstituted InsP3 receptor could be observed either at 4℃ or at 25℃,whereas the effect of [Ca2+] on reconstituted InsP3 receptor depended on the temperature.The Ca2+ concentration outside the proteolipsome ([Ca2+]o) had no detectable effect on InsP3 binding to InsP3 receptor at 4℃.In contrast,with increase of [Ca2+]o from 0 to 100 nmol/L at 25℃,the InsP3 binding activity increased gradually.Then the InsP3 binding activity was decreased drastically at higher [Ca2+]o and inhibited entirely at 50 mol/L [Ca2+]o.Conformational studies on intrinsic fluorescence of the reconstituted InsP3 receptor and its quenching by KI and HB indicated that the global conformation of reconstituted InsP3 receptor could not be affected by [Ca2+]o at 4℃.While at 25℃,the effects of 10 m mol/L [Ca2+]o on global,membrane and cytoplasmic conformation of the reconstituted InsP3 receptor were different significantly from that of 100 nmol/L [Ca2+]o.     

Kinetics of cytosolic Ca2+ concentration after photolytic release of 1-D-myo-inositol 1,4-bisphosphate 5-phosphorothioate from a caged derivative in guinea pig hepatocytes.

The influence of 1-D-myo-inositol 1,4,5-trisphosphate (InsP3) breakdown by InsP3 5-phosphatase in determining the time course of Ca2+ release from intracellular stores was investigated with flash photolytic release of a stable InsP3 derivative, 5-thio-InsP3, from a photolabile caged precursor. The potency and Ca(2+)-releasing properties of the biologically active D isomers of 5-thio-InsP3 and InsP3 itself were compared by photolytic release in guinea pig hepatocytes. After a light flash, cytosolic free calcium concentration ([Ca2+]i) showed an initial delay before rising quickly to a peak and declining more slowly to resting levels, with time course and amplitude generally similar to those seen with photolytic release of InsP3. Differences were a three- to eightfold lower potency of 5-thio-InsP3 in producing Ca2+ release, much longer delays between photolytic release and Ca2+ efflux with low concentrations of 5-thio-InsP3 than with InsP3, and persistent reactivation of Ca2+ release, producing periodic fluctuations of cytosolic [Ca2+]i with high concentrations of 5-thio-InsP3 but not InsP3 itself. The lower potency of 5-thio-InsP3 may be a result of a lower affinity for closed receptor/channels or a lower open probability of liganded receptor/channels. The longer delays with 5-thio-InsP3 at low concentration suggest that metabolism of InsP3 by 5-phosphatase may reduce the concentration sufficiently to prevent receptor activation and may have a similar effect on InsP3 concentration during hormonal activation.(ABSTRACT TRUNCATED AT 250 WORDS)