2,5-Di-(tert-butyl)-1,4-benzohydroquinone mobilizes inositol 1,4,5-trisphosphate-sensitive and -insensitive Ca2+ stores

In permeabilized rat hepatocytes a maximal concentration (25 microM) of 2,5-di-(tert-butyl)-1,4-benzohydroquineone (tBuBHQ) mobilized 70% of sequestere Ca2+ and a half-maximal effect was produced by 1.7 microM tBuBHQ. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) stimulated release of about 40% of the intracellular Ca2+ stores. Combined applications of a range of tBuBHQ concentrations with a maximal concentration of Ins(1,4,5)P3 demonstrated that tBuBHQ has slight selectivity for the Ca2+ transport process of the Ins(1,4,5)P3-sensitive stores. We conclude that the Ins(1,4,5)P3-sensitive stores are a subset of those sensitive to tBuBHQ and that the latter is therefore unlikely to prove useful as a tool to discriminate Ins(1,4,5)P3-sensitive and -insensitive Ca2+ stores though it may provide opportunities to design more selective agents.     

Inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation in Ca2+-mobilizing-hormone-activated cells.

The inositol trisphosphate liberated on stimulation of guinea-pig hepatocytes, pancreatic acinar cells and dimethyl sulphoxide-differentiated human myelomonocytic HL-60 leukaemia cells is composed of two isomers, the 1,4,5-trisphosphate and the 1,3,4-trisphosphate. Inositol 1,4,5-trisphosphate was released rapidly, with no measurable latency on hormone stimulation, and, consistent with its proposed role as an intracellular messenger for Ca2+ mobilization, there was good temporal correlation between its formation and Ca2+-mediated events in these tissues. There was a definite latency before an increase in the formation of inositol 1,3,4-trisphosphate could be detected. In all of these tissues, however, it formed a substantial proportion of the total inositol trisphosphate by 1 min of stimulation. In guinea-pig hepatocytes, where inositol trisphosphate increases for at least 30 min after hormone application, inositol 1,3,4-trisphosphate made up about 90% of the total inositol trisphosphate by 5-10 min. In pancreatic acinar cells, pretreatment with 20 mM-Li+ caused an increase in hormone-induced inositol trisphosphate accumulation. This increase was accounted for by a rise in inositol 1,3,4-trisphosphate; inositol 1,4,5-trisphosphate was unaffected. This finding is consistent with the observation that Li+ has no effect on Ca2+-mediated responses in these cells. The role, if any, of inositol 1,3,4-trisphosphate in cellular function is unknown.     

Presence of a putative vesicular inositol 1,4,5-trisphosphate-sensitive nucleoplasmic Ca2+ store

The inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are widely localized in both the heterochromatin and euchromatin regions. We found recently the presence of nucleoplasmic complexes that are composed of phospholipids, IP(3)R/Ca(2+) channels, and Ca(2+) storage protein chromogranin B (CGB). Close examination and 3D image reconstruction of these complexes revealed numerous vesicular structures with an average diameter of approximately 50 nm that are primarily interspersed between the heterochromatins. IP(3) rapidly released Ca(2+) from these structures, but other inositol phosphates, inositol 1,4-bisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate, failed to release Ca(2+). Addition of heparin or IP(3)R antibody blocked the IP(3)-induced Ca(2+) releases, indicating the release of Ca(2+) through the IP(3)R/Ca(2+) channels. Given the presence of the IP(3)R/Ca(2+) channels and Ca(2+) storage protein CGB in these vesicular structures, we postulate that these vesicles are the IP(3)-sensitive nucleoplasmic Ca(2+) stores. Abundance of the vesicular Ca(2+) stores between the heterochromatins appeared to imply critical roles these vesicular Ca(2+) stores play in controlling the Ca(2+) concentrations of the chromosomes.     

The redistribution of Ca2+ stores with inositol 1,4,5-trisphosphate receptor to the cleavage furrow in a microtubule-dependent manner.

We reported that microinjection of Ca2+ store-enriched microsome fractions from cultured CHO cells and mouse cerebella to dividing newt eggs induced extra-cleavage furrows via inositol 1,4,5-trisphosphate-induced Ca2+ release (Mitsuyama et al., 1999). Our observation strongly suggested that Ca2+ stores with inositol 1,4,5-trisphosphate receptor (IP3R) induce and position a cleavage furrow, as Ca2+-releasing machinery, and that such is itself a putative cleavage stimulus. For confirmation, we immunocytochemically examined mitotic CHO cells using antibodies against Ca2+ store-related proteins. We found that polar dominant Ca2+ stores with IP3R during metaphase were re-distributed to the future cleavage cortex just preceding the onset of furrowing, and that this redistributing IP3R was present on microtubule bundles. When a microsome fraction from sacro/endoplasmic reticulum Ca2+-ATPase (SERCA)-GFP stably expressing CHO cells was microinjected into dividing newt eggs and observed by confocal microscopy, the microinjected Ca2+ stores with IP3R moved linearly toward the next cleavage furrow and this movement was blocked by nocodazole, a microtubule-depolarizing agent, but not by cytochalasin B, an F-actin-depolarizing agent. These observations strongly suggest that Ca2+ stores with IP3R are transferred and accumulate to the cleavage furrow by microtubule-based motility, as a cleavage stimulus.     

Stereospecific mobilization of intracellular Ca2+ by inositol 1,4,5-triphosphate. Comparison with inositol 1,4,5-trisphosphorothioate and inositol 1,3,4-trisphosphate.

The isolated activation segment of pig procarboxypeptidase A binds two Tb3+ ions in a strong and specific way. In contrast, the binding of Ca2+, Cd2+ and Mg2+ is weak. The binding of Tb3+ increases the resistance of the isolated activation segment against proteolysis and competes for the binding of the carbocyanine dye Stains-All. This dye forms complexes with the activation segment showing spectral properties similar to those observed with EF-hand structures. The presented results support a previous hypothesis on the existence of two regions in the activation segment of pancreatic procarboxypeptidases structurally related to Ca2+-binding domains of the EF-hand protein family.     

The M-phase-promoting factor modulates the sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate via the actin cytoskeleton

The resumption of the meiotic cycle (maturation) induced by 1-methyladenine in prophase-arrested starfish oocytes is indicated by the breakdown of the germinal vesicle and is characterized by the increased sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate (InsP3) to InsP3 starting at the animal hemisphere (where the germinal vesicle was originally located) and propagating along the animal/vegetal axis of the oocyte. This initiates Ca2+ signals around the germinal vesicle before nuclear envelope breakdown. Previous studies have suggested that the final activation of the maturation-promoting factor (MPF), a cyclin-dependent kinase, which is the major element controlling the entry of eukaryotic cells into the M phase, occurs in the nucleus. MPF is then exported to the cytoplasm where its activity is autocatalytically amplified following a similar animal/vegetal spatial pattern. We have investigated whether activated MPF was involved in the increased sensitivity of the Ca2+ response to InsP3. We have found that the development of increased sensitivity of the Ca2+ stores to InsP3 receptors together with the Ca2+ signals in the perinuclear region was blocked in oocytes treated with the specific MPF inhibitor roscovitine. That the nuclear MPF activation is indeed required for changes of the InsP3 receptors sensitivity was shown by enucleating or by dissecting oocytes into vegetal and animal hemispheres prior to the addition of 1-MA. MPF activity 50 min after 1-methyladenine addition was much lower in the enucleated oocytes and in the vegetal hemisphere, which did not contain the germinal vesicle, as compared with the animal hemisphere, which did contain it. The Ca2+ increase induced by InsP3 under these experimental conditions correlated with the changes in actin cytoskeleton induced by MPF.     

Amplification of Ca2+ signaling by diacylglycerol-mediated inositol 1,4,5-trisphosphate production

Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP(3) produced is particularly critical to determining the spatio-temporally coordinated Ca(2+)-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca(2+)-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity.     

Comparison of and chromogranin effect on inositol 1,4,5-trisphosphate sensitivity of cytoplasmic and nucleoplasmic inositol 1,4,5-trisphosphate receptor/Ca2+ channels

The nucleus also contains the inositol 1,4,5-trisphosphate receptor (IP3R)/Ca2+ channels in the nucleoplasm proper independent of the nuclear envelope or the cytoplasm. The nuclear IP3R/Ca2+ channels were shown to be present in small IP3-dependent nucleoplasmic Ca2+ store vesicles, yet no information is available regarding the IP3 sensitivity of nuclear IP3R/Ca2+ channels. Here, we show that nuclear IP3R/Ca2+ channels are 3-4-fold more sensitive to IP3 than cytoplasmic ones in both neuroendocrine PC12 cells and nonneuroendocrine NIH3T3 cells. Given the presence of phosphoinositides and phospholipase C and the importance of IP3-mediated Ca2+ signaling in the nucleus, the high IP3 sensitivity of nuclear IP3R/Ca2+ channels seemed to reflect the physiological needs of the nucleus to finely control the IP3-dependent Ca2+ concentrations. It was further shown that the IP3R/Ca2+ channels of secretory cells are 7-8-fold more sensitive to IP3 than those of nonsecretory cells. This difference appeared to result from the presence of secretory cell marker protein chromogranins (thus secretory granules) in secretory cells; expression of chromogranins in NIH3T3 cells increased the IP3 sensitivity of both nuclear and cytoplasmic IP3R/Ca2+ channels by approximately 4-6-fold. In contrast, suppression of chromogranin A expression in PC12 cells changed the EC50 of IP3 sensitivity for cytoplasmic IP3R/Ca2+ channels from 17 to 47 nM, whereas suppression of chromogranin B expression changed the EC50 of cytoplasmic IP3R/Ca2+ channels from 17 to 102 nM and the nuclear ones from 4.3 to 35 nM. Given that secretion is the major function of secretory cells and is under a tight control of intracellular Ca2+ concentrations, the high IP3 sensitivity appears to reflect the physiological roles of secretory cells.     

Chronic muscarinic stimulation of SH-SY5Y neuroblastoma cells suppresses inositol 1,4,5-trisphosphate action. Parallel inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ mobilization and inositol 1,4,5-trisphosphate binding.

The possibility that chronic activation of the phosphoinositide-mediated signaling pathway modifies the Ca(2+)-mobilizing action of inositol 1,4,5-trisphosphate (InsP3) was examined. SH-SY5Y human neuroblastoma cells were exposed to carbachol, permeabilized electrically, loaded with 45Ca2+, and 45Ca2+ mobilization in response to exogenous InsP3 was assessed. In control permeabilized cells, InsP3 released 65 +/- 2% of sequestered 45Ca2+ (EC50 = 0.32 +/- 0.05 microM). Pre-treatment with carbachol reduced both maximal InsP3-induced 45Ca2+ release (to 34 +/- 3%, with half-maximal and maximal inhibition at approximately 3 and 6 h, respectively) and the potency of InsP3 (EC50 = 0.92 +/- 0.13 microM). This inhibitory effect of carbachol was half-maximal at approximately 5 microM, was mediated by muscarinic receptors, and was reversible following withdrawal of agonist. Pretreatment with phorbol 12,13-dibutyrate did not alter the maximal effect of InsP3 but doubled its EC50. Evidence suggesting that the inhibitory effects of carbachol pretreatment resulted from altered Ca2+ homeostasis was not forthcoming; both 45Ca2+ uptake and release induced by ionomycin and thapsigargin were identical in control and pretreated permeabilized cells, as were the characteristics of reuptake of released Ca2+. In contrast, carbachol pretreatment, without altering the affinity of InsP3 (Kd = 64 +/- 7 nM), reduced the density of [32P]InsP3-binding sites from 2.0 +/- 0.1 to 1.0 +/- 0.1 pmol/mg protein with a time course essentially identical to that for the reduction in responsiveness to InsP3. This effect was not mimicked by pretreatment of cells with phorbol 12,13-dibutyrate. These data indicate that chronic activation of phosphoinositide hydrolysis can reduce the abundance of InsP3 receptors and that this causes a reduction in size of the InsP3-sensitive Ca2+ store. This modification, possibly in conjunction with a protein kinase C-mediated event, appears to account for the carbachol-induced suppression of InsP3 action. As intracellular InsP3 mass remained elevated above basal for at least 24 h after addition of carbachol, suppression of the Ca(2+)-mobilizing activity of InsP3 represents an important adaptive response to cell stimulation that can limit the extent to which intracellular Ca2+ is mobilized.     

The spatial distribution of inositol 1,4,5-trisphosphate receptor isoforms shapes Ca2+ waves

Cytosolic Ca(2+) is a versatile second messenger that can regulate multiple cellular processes simultaneously. This is accomplished in part through Ca(2+) waves and other spatial patterns of Ca(2+) signals. To investigate the mechanism responsible for the formation of Ca(2+) waves, we examined the role of inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms in Ca(2+) wave formation. Ca(2+) signals were examined in hepatocytes, which express the type I and II InsP3R in a polarized fashion, and in AR4-2J cells, a nonpolarized cell line that expresses type I and II InsP3R in a ratio similar to what is found in hepatocytes but homogeneously throughout the cell. Expression of type I or II InsP3R was selectively suppressed by isoform-specific DNA antisense in an adenoviral delivery system, which was delivered to AR4-2J cells in culture and to hepatocytes in vivo. Loss of either isoform inhibited Ca(2+) signals to a similar extent in AR4-2J cells. In contrast, loss of the basolateral type I InsP3R decreased the sensitivity of hepatocytes to vasopressin but had little effect on the initiation or spread of Ca(2+) waves across hepatocytes. Loss of the apical type II isoform caused an even greater decrease in the sensitivity of hepatocytes to vasopressin and resulted in Ca(2+) waves that were much slower and delayed in onset. These findings provide evidence that the apical concentration of type II InsP3Rs is essential for the formation of Ca(2+) waves in hepatocytes. The subcellular distribution of InsP3R isoforms may critically determine the repertoire of spatial patterns of Ca(2+) signals.     

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.     

1,6-Diaminohexane contributes to the hexamethylene bisacetamide-induced erythroid differentiation pathway by stimulating Ca2+ release from inositol 1,4,5-trisphosphate-sensitive stores and promoting Ca2+ influx

Hexamethylene bisacetamide (HMBA) stimulates Ca(2+) signals in murine erythroleukemia (MEL) cells serving as an important component of the HMBA-induced pathway that promotes differentiation to the erythroid phenotype. We observed that 1,6-diaminohexane (DAH) triggered a more rapid and robust increase in MEL cell Ca(2+) levels compared to HMBA and the monodeacetylated N-acetyl-1,6-diaminohexane (NADAH), and that polyamine deacetylase inhibition completely abolished the ability of HMBA and NADAH to induce Ca(2+) signals in MEL cells. Our work indicates that DAH mediates Ca(2+) signal propagation via its ability to activate inositol 1,4,5-trisphosphate (IP(3)) receptors, as we observed similar Ca(2+) release characteristics and heparin sensitivity of DAH and IP(3) in permeabilized MEL cells. Finally, we observed that the DAH-induced Ca(2+) release pathway robustly coupled to a Ca(2+) influx pathway that could be distinguished from thapsigargin-induced Ca(2+) influx by its unusual insensitivity to 2-aminoethoxydiphenyl borate.     

2,5-Di-(tert-butyl)-1,4-benzohydroquinone rapidly elevates cytosolic Ca2+ concentration by mobilizing the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool

2,5-Di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a potent inhibitor of liver microsomal ATP-dependent Ca2+ sequestration (Moore, G. A., McConkey, D. J., Kass, G. E. N., O'Brien, P. J., and Orrenius, S. (1987) FEBS Lett. 224, 331-336), produced a concentration-dependent, rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) in isolated rat hepatocytes (EC50 = 1-2 microM). The amplitude of the [Ca2+]i increase was essentially identical with that produced by vasopressin, but the tBuBHQ-stimulated [Ca2+]i increase remained sustained for 15-20 min. Vasopressin added 2-3 min after tBuBHQ caused [Ca2+]i to rapidly return to basal levels; however, tBuBHQ added after vasopressin resulted in a Ca2+ transient rather than a sustained [Ca2+]i elevation. Ca2+ influx was not stimulated in tBuBHQ-treated hepatocytes, but was markedly enhanced upon addition of vasopressin. Depletion of the endoplasmic reticular Ca2+ pool by the addition of vasopressin to hepatocytes incubated in low Ca2+ medium virtually abolished the tBuBHQ-mediated [Ca2+]i rise and vice versa. In saponin-permeabilized hepatocytes, tBuBHQ released Ca2+ from the same nonmitochondrial, ATP-dependent Ca2+ pool which was released by inositol 1,4,5-trisphosphate. Furthermore, tBuBHQ-induced Ca2+ release in saponin-permeabilized cells was not inhibited by neomycin, and tBuBHQ did not produce any apparent accumulation of inositol phosphates in intact hepatocytes. The rate of passive efflux of Ca2+ from Ca2+-loaded hepatic microsomes was unaltered by tBuBHQ. Thus, tBuBHQ inhibits ATP-dependent Ca2+ sequestration via a direct effect on the endoplasmic reticulum Ca2+ pump, resulting in net Ca2+ release and elevation of [Ca2+]i. Taken together, our results show that in the absence of hormonal stimuli, excess Ca2+ is only slowly cleared from the hepatocyte cytosol, indicating that the basal rate of Ca2+ removal by the plasma membrane Ca2+ pump and mitochondria is slow. Furthermore, Ca2+-mobilizing hormones appear to stimulate an active process of Ca2+ removal from hepatocyte cytosol which does not depend on re-uptake into the endoplasmic reticulum.     

NADPH oxidase activation increases the sensitivity of intracellular Ca2+ stores to inositol 1,4,5-trisphosphate in human endothelial cells

Many stimuli that activate the vascular NADPH oxidase generate reactive oxygen species and increase intracellular Ca(2+), but whether NADPH oxidase activation directly affects Ca(2+) signaling is unknown. NADPH stimulated the production of superoxide anion and H(2)O(2) in human aortic endothelial cells that was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and was significantly attenuated in cells transiently expressing a dominant negative allele of the small GTP-binding protein Rac1, which is required for oxidase activity. In permeabilized Mag-indo 1-loaded cells, NADPH and H(2)O(2) each decreased the threshold concentration of inositol 1,4,5-trisphosphate (InsP(3)) required to release intracellularly stored Ca(2+) and shifted the InsP(3)-Ca(2+) release dose-response curve to the left. Concentrations of H(2)O(2) as low as 3 microm increased the sensitivity of intracellular Ca(2+) stores to InsP(3) and decreased the InsP(3) EC(50) from 423.2 +/- 54.9 to 276.9 +/- 14. 4 nm. The effect of NADPH on InsP(3)-stimulated Ca(2+) release was blocked by catalase and by diphenyleneiodonium and was not observed in cells lacking functional Rac1 protein. Thus, NADPH oxidase-derived H(2)O(2) increases the sensitivity of intracellular Ca(2+) stores to InsP(3) in human endothelial cells. Since Ca(2+)-dependent signaling pathways are critical to normal endothelial function, this effect may be of great importance in endothelial signal transduction.     

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.     

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°C or at 25°C, 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°C. In contrast, with increase of [Ca²⁺]_o from 0 to 100 nmol/L at 25°C, the InsP₃ binding activity increased gradually. Then the InsP₃ binding activity was decreased drastically at higher [Ca²⁺]_o and inhibited entirely at 50 μmol/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°C. While at 25°C, the effects of 10 μmol/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.     

Modeling Ca2+ feedback on a single inositol 1,4,5-trisphosphate receptor and its modulation by Ca2+ buffers

The inositol 1,4,5-trisphosphate receptor/channel (IP₃R) is a major regulator of intracellular Ca²⁺ signaling, and liberates Ca²⁺ ions from the endoplasmic reticulum in response to binding at cytosolic sites for both IP₃ and Ca²⁺. Although the steady-state gating properties of the IP₃R have been extensively studied and modeled under conditions of fixed [IP₃] and [Ca²⁺], little is known about how Ca²⁺ flux through a channel may modulate the gating of that same channel by feedback onto activating and inhibitory Ca²⁺ binding sites. We thus simulated the dynamics of Ca²⁺ self-feedback on monomeric and tetrameric IP₃R models. A major conclusion is that self-activation depends crucially on stationary cytosolic Ca²⁺ buffers that slow the collapse of the local [Ca²⁺] microdomain after closure. This promotes burst-like reopenings by the rebinding of Ca²⁺ to the activating site; whereas inhibitory actions are substantially independent of stationary buffers but are strongly dependent on the location of the inhibitory Ca²⁺ binding site on the IP₃R in relation to the channel pore.     

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic beta-cells

The effect of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition on the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was studied in primary insulin-releasing pancreatic beta-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca(2+)-deficient medium the individual primary beta-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca(2+)](i) followed by an explosive transient elevation. The [Ca(2+)](i) transients were preferentially observed at low intracellular concentrations of the Ca(2+) indicator fura-2 and were unaffected by pre-treatment with 100 microM ryanodine. Whereas 20mM caffeine had no effect on basal [Ca(2+)](i) or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca(2+)](i) transients as well as inositol 1,4,5-trisphosphate-mediated [Ca(2+)](i) transients in response to carbachol. In striking contrast to the primary beta-cells, caffeine readily mobilized intracellular Ca(2+) in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca(2+) from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca(2+)-induced Ca(2+) release (CICR). In primary pancreatic beta-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal beta-cells.     

The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria

There are three isoforms of the inositol 1,4,5- trisphosphate receptor (InsP(3)R), each of which has a distinct effect on Ca(2+) signaling. However, it is not known whether each isoform similarly plays a distinct role in the activation of Ca(2+)-mediated events. To investigate this question, we examined the effects of each InsP(3)R isoform on transmission of Ca(2+) signals to mitochondria and induction of apoptosis. Each isoform was selectively silenced using isoform-specific small interfering RNA in Chinese hamster ovary cells, which express all three InsP(3)R isoforms. ATP-induced cytosolic Ca(2+) signaling patterns were altered, regardless of which isoform was silenced, but in a different fashion depending on the isoform. ATP also induced Ca(2+) signals in mitochondria, which were inhibited more effectively by silencing the type III InsP(3)R than by silencing either the type I or type II isoform. The type III isoform also co-localized most strongly with mitochondria. When apoptosis was induced by activation of either the extrinsic or intrinsic apoptotic pathway, induction was reduced most effectively by silencing the type III InsP(3)R. These findings provide evidence that the type III isoform of the InsP(3)R plays a special role in induction of apoptosis by preferentially transmitting Ca(2+) signals into mitochondria.