Inositol Trisphosphate-induced Calcium Release Research Articles

Non-receptor-tyrosine Kinases Integrate Fast Glucocorticoid Signaling in Hippocampal Neurons

Despite numerous descriptions of rapid effects of corticosterone on neuronal function, the intracellular mechanisms responsible for these changes remain elusive. The present comprehensive analysis reveals that signaling from a membrane-located G protein-coupled receptor activates PKC, Akt/PKB, and PKA, which subsequently trigger the phosphorylation of the tyrosine kinases Pyk2, Src, and Abl. These changes induce rapid cytoskeletal rearrangements (increased PSD-95 co-clustering) within the post-synaptic density; these events are accompanied by increased surface NMDA receptor expression, reflecting corticosterone-induced inhibition of NMDA receptor endocytosis. Notably, none of these signaling mechanisms require de novo protein synthesis. The observed up-regulation of ERK1/2 (downstream of NMDA receptor signaling) together with the fact that c-Abl integrates cytoplasmic and nuclear functions introduces a potential mechanism through which rapid signaling initiated at the plasma membrane may eventually determine the long term integrated response to corticosterone by impacting on the transcriptional machinery that is regulated by classical, nuclear mineralocorticoid, and glucocorticoid receptors.

Transient receptor potential channels in rat renal microcirculation: Actions of angiotensin II

This study assessed the calcium-activating mechanisms mediating glomerular arteriolar constriction by angiotensin II (Ang II). Immunohistochemical and physiological studies were carried out, using antibody against transient receptor potential (TRP)-1 and an isolated perfused kidney model. Immunohistochemical experiments demonstrated that TRP-1 proteins were transcribed on both afferent and efferent arteriolar myocytes. In the first series of physiological experiments, Ang II (0.3 nmol/L) considerably constricted afferent (20.2 +/- 0.9 to 14.9 +/- 0.7 microm) and efferent arterioles (18.4 +/- 0.7 to 14.0 +/- 0.7 microm). The addition of nifedipine (1 micromol/L) restored decrements in afferent (to 20.0 +/- 0.8 microm) but not efferent arteriolar diameters. Further administration of SKF-96365 (100 micromol/L), a TRP channel blocker, reversed efferent arteriolar constriction (to 16.2 +/- 0.8 micromol/L). In the second group, although 2-aminoethoxydiphenyl borate (100 micromol/L), an inhibitor of inositol trisphosphate-induced calcium release (IP3CR), did not alter glomerular arteriolar diameters, it prevented Ang II-induced afferent arteriolar constriction and attenuated efferent arteriolar constriction (18.8 +/- 0.8 to 16.9 +/- microm). Subsequent removal of extracellular calcium abolished residual efferent arteriolar constriction (to 19.1 +/- 0.8 microm). Our data provide evidence that Ang II elicits IP3CR, possibly inducing a cellular response that activates voltage-dependent calcium channels on afferent arterioles. The present results suggest that Ang II-induced efferent arteriolar constriction involves IP3CR and calcium influx sensitive to SKF-96365.

Inhibition of inositol trisphosphate-induced calcium release by cyclicADP-ribose in A7r5 smooth-muscle cells and in 16HBE14o- bronchial mucosal cells

Ca2+ release from intracellular stores occurs via two families of intracellular channels, each with their own specific agonist: Ins(1, 4,5)P3 for the Ins(1,4,5)P3 receptor and cyclic ADP-ribose (cADPR) for the ryanodine receptor. We now report that cADPR inhibited Ins(1, 4,5)P3-induced Ca2+ release in permeabilized A7r5 cells with an IC50 of 20 microM, and in permeabilized 16HBE14o- bronchial mucosal cells with an IC50 of 35 microM. This inhibition was accompanied by an increase in specific [3H]Ins(1,4,5)P3 binding. 8-Amino-cADPR, but not 8-bromo-cADPR, antagonized this effect of cADPR. The inhibition was prevented by a whole series of inositol phosphates (10 microM) that did not affect Ins(1,4,5)P3-induced Ca2+ release, and by micromolar concentrations of PPi and various nucleotide di- or triphosphates. We propose that cADPR must interact with a novel regulatory site on the Ins(1,4,5)P3 receptor or on an associated protein. This site is neither the Ins(1,4,5)P3-binding domain, which prefers Ins(1,4,5)P3 and only binds nucleotides and PPi in the millimolar range, nor the stimulatory adenine nucleotide binding site.

Functional changes of rat brain microsomal membrane surface after learning task depending on dietary fatty acids.

Biochemical characteristics of brain microsomal membranes were examined before and after the brightness-discrimination learning tasks in rats that were fed either safflower oil (alpha-linolenate-deficient) or perilla oil (alpha-linolenate-sufficient) diets. We detected small changes in the chain elongation system for polyunsaturated fatty acids in microsomes, whereas no significant difference was detected in the inositol trisphosphate-induced calcium release and ATP-induced calcium uptake profiles of microsomes between the two dietary groups. The calcium ion-induced aggregation rate of microsomes was determined in both groups. We found that the aggregation rate of microsomes in the safflower oil group was significantly greater than that in the perilla oil group. The difference in susceptibility of microsomal membrane phospholipids to phospholipase A2 between the groups was obvious, and the amount of released fatty acids by phospholipase A2 from the perilla oil group microsomes was nearly half of that from the safflower oil group microsomes after the learning task. Susceptibility of sialic acids on the brain microsomal membranes to exogenous sialidase was different only after the learning task in the safflower and perilla oil groups. These results suggest that the biochemical characteristics of membrane surfaces of brain microsomes are affected significantly by the learning task itself in a dietary oil-dependent manner.

Reversible Desensitization of Inositol Trisphosphate-induced Calcium Release Provides a Mechanism for Repetitive Calcium Spikes

Repetitive transient increases in cytosolic calcium concentration (calcium spikes or calcium oscillations) are a common mode of signal transduction in receptor-mediated cell activation. Repetitive calcium spikes are initiated by phospholipase C-mediated production of inositol 1,4,5-trisphosphate (InsP3) and are thought to be generated by a positive feedback mechanism in which calcium potentiates its own release, a negative feedback mechanism by which calcium release is terminated, and a slow recovery process that defines the time interval between calcium spikes. The molecular mechanisms that terminate each calcium spike and define the spike frequency are not yet known. Here we show, in intact rat basophilic leukemia cells, that calcium responses induced by InsP3 are diminished for a period of 30-60 s following an InsP3-induced calcium spike. The sensitivity of calcium release for InsP3 was probed by UV laser-mediated photorelease of InsP3, and calcium responses were monitored by fluorescence calcium imaging. A maximal loss in sensitivity (desensitization) was observed for InsP3 increases that resulted in a near maximal calcium spike and was expressed as an 80-100% reduction in the calcium response to an equal amount of InsP3, released 10 s after the first UV pulse. When the amount of released InsP3 in the second pulse was increased 2-3-fold, desensitization was overcome and a second calcium response of equal amplitude to the first was produced. A power dependence of 3.2 was measured between the amount of released InsP3 and the amplitude of the triggered calcium response, explaining how a small decrease in InsP3 sensitivity can lead to a nearly complete reduction in the calcium response. Desensitization was abolished by the addition of the calcium buffers BAPTA and EGTA and could be induced by microinjection of calcium, suggesting that it is a calcium-dependent process. Half-maximal desensitization was observed at a free calcium concentration of 290 nM and increased with a power of 3.7 with peak calcium concentration. These studies suggest that reversible desensitization of InsP3-induced calcium release serves as a "saw-tooth" parameter that controls the termination of each spike and the frequency of calcium spikes.

Inositol trisphosphate-induced calcium release in the generation of calcium oscillations in bovine eggs.

Bovine eggs exhibit repetitive rises in intracellular calcium concentration ([Ca2+]i) in response to fertilization. The signaling pathways and Ca2 release mechanisms involved in their generation are not well characterized. This study examined the presence of a GTP-binding protein (G-protein) signaling pathway as well as the role of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R)-mediated Ca2+ release and ryanodine receptor (RyR)-mediated Ca2+ release, the two Ca2+ receptors/channels most often thought to participate in the generation of [Ca2+]i oscillations, by injecting appropriate agonists and antagonists and monitoring their effects on Ca2+ release and pronucleus formation, injection of guanosine 5'-0-(3-thiotriphosphate) (GTP gamma [S]), which promotes G-protein-mediated phosphoinositide turnover, induced, at high concentrations, repetitive [Ca2+]i rises. Low concentrations of GTP gamma [S] were ineffective. Injection of inositol trisphosphorothioate (InsP3S3), a nonmetabolizable analogue of InsP3, evoked an immediate Ca2+ release followed by [Ca2R]i oscillations. The GTP gamma [S]- and InsP3S3-induced oscillations showed a rapid attenuation in amplitude and were terminated in about 30-60 min. Thimerosal, a thiol oxidizing agent, caused repetitive rises in [Ca2+]i by sensitizing Ca2+ injection-induced Ca2+ release. Injection of ryanodine, which stimulates Ca(2+)-induced Ca2+ release via the RyR, did not induce [Ca2+]i oscillations; and eggs into which it was preinjected exhibited normal [Ca2+]i oscillations in response to thimerosal. Preinjection of heparin, a competitive InsP3R antagonist, blocked in a dose-dependent manner the Ca2+ response to InsP3 and thimerosal, and its injection into fertilized oscillating eggs inhibited [Ca2+]i oscillations in all eggs.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of inositol trisphosphate-induced calcium release by caffeine is prevented by ATP

We have investigated the effect of various methylxanthines on the basal and InsP3-stimulated unidirectional 45Ca2+ efflux from permeabilized A7r5 cells under different experimental conditions. We report that caffeine and theophylline inhibit the InsP3-induced Ca2+ release, whereas the basal Ca2+ leak remained largely unaffected. The effect on InsP3-induced Ca2+ release was specific, since isocaffeine was not effective. The inhibition occurred similarly in the absence or presence of extravesicular Ca2+ and was not associated with a decrease in the [3H]InsP3 binding to the receptor. ATP and MgATP (5 mM) prevented the inhibition, suggesting that caffeine may interact with an ATP-binding site on the InsP3 receptor or some associated protein.

Myo-inositol trisphosphate-mediated calcium release from internal stores of Entamoeba histolytica

Calcium mobilisation from internal stores of the parasitic protozoan Entamoeba histolytica was studied by fluorescence measurements of the calcium indicator quin 2 and 45Ca 2+ incorporation studies in saponin-permeabilised amoebae. Prior energy-dependent calcium sequestration was found to be necessary for subsequent release of calcium by inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3). Both Ins(1,4,5)P 3 and inositol 2,4,5-trisphosphate (Ins(2,4,5)P 3) could release calcium equally well from permeabilised E. histolytica with similar EC 50 (concentration which produced half maximal release) values for calcium release. Ins(1,4,5)P 3-mediated calcium release occurred from a vesicular store, was sensitive to prior treatment by heparin and was attenuated by prior addition of a lower concentration of Ins(1,4,5)P 3. cAMP failed to influence inositol trisphosphate induced calcium release, indicating the absence of control mechanisms through cAMP-dependent phosphorylation. GTP neither induced calcium release nor could potentiate inositol trisphosphate mediated calcium mobilisation. A saturating concentration of Ins(1,4,5)P 3 could release 50% of radiolabelled calcium sequestered by energy-dependent mechanisms in E. histolytica. The energy-dependent calcium sequestration was inhibited by vanadate and the calcium antagonist Diltiazem but not by dicyclohexylcarbodiimide (DCCD), suggesting the involvement of an endoplasmic reticulum-like structure in calcium storage. Binding studies showed specific association of [ 3H]Ins(1,4,5)P 3 to crude membrane fractions of E. histolytica, which was significantly inhibited by heparin in a dose-dependent manner. IC 50 (concentration which produced half-maximal inhibition) values for displacement of radiolabelled Ins(1,4,5)P 3 binding by unlabelled Ins(1,4,5)P 3 and Ins(2,4,5)P 3 were estimated to be 0.99 μM for both isomers. Our results suggested that Ins(1,4,5)P 4-mediated calcium release from internal stores of E. histolytica most probably occurred in an inositol trisphosphate receptor-dependent manner.

Regulation of intracellular calcium in the mouse egg: evidence for inositol trisphosphate-induced calcium release, but not calcium-induced calcium release.

Fertilization of the mammalian egg initiates transient and repetitive release of Ca2+ from intracellular stores. The mechanism by which these Ca2+ transients are produced is not completely known. We examined the role of two principal Ca2+ release mechanisms, inositol trisphosphate-induced Ca2+ release and Ca(2+)-induced Ca2+ release, in altering intracellular Ca2+ in the mouse egg. Microinjection of inositol 1,4,5-trisphosphate (IP3) transiently elevated intracellular Ca2+ and, at higher concentrations, produced repetitive Ca2+ transients. Addition of 100 microM thimerosal, a sulfhydryl reagent, caused repetitive Ca2+ transients. IP3 and thimerosal responses were inhibited by prior injection of heparin, a competitive antagonist of IP3-induced Ca2+ release. Addition of caffeine or injection of caffeine, ryanodine, or cyclic ADP-ribose, which are known to initiate or modulate Ca(2+)-induced Ca2+ release in sea urchin eggs and other cells, produced no change in intracellular Ca2+. The response to injection of Ca2+ was not altered by prior injection of ryanodine. The magnitude of the Ca2+ transients produced by injection of IP3 was not changed by prior injection of cyclic ADP-ribose or external caffeine. We found no evidence of Ca(2+)-induced Ca2+ release from ryanodine- or caffeine-sensitive stores. It is most likely that release of Ca2+ from intracellular stores in the mouse egg is dependent on IP3-induced Ca2+ release.

Characterization of the inositol trisphosphate-sensitive and insensitive calcium stores by selective inhibition of the endoplasmic reticulum-type calcium pump isoforms in isolated platelet membrane vesicles

In mixed platelet membrane vesicles the presence of two distinct endoplasmic reticulum-type calcium pump enzymes of 100 and 97 kD molecular mass has been demonstrated. We have previously shown that both calcium pumps were recognized by polyclonal anti-sarcoplasmic reticulum calcium pump antisera [11]. In the present work we studied the effects of several calcium pump inhibitors on active calcium transport and inositol trisphosphate-induced calcium release in these vesicles in an attempt to assign the two calcium pump isoenzymes to specific calcium pools. The effect of the PL IM 430 inhibitory anti-calcium pump antibody was compared to that of other calcium pump inhibitors acting predominantly on the 100 and the 97 kD calcium pump isoforms, respectively. The PL IM 430 antibody, which recognized the 97 kD pump on Western blots and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, which inhibited phosphoenzyme formation of the same pump isoform, inhibited calcium accumulation predominantly into an inositol trisphosphate-releasable calcium pool. On the other hand, low concentration of thapsigargin, which inhibited phosphoenzyme formation mainly of the 100 kD pump isozyme, had a more pronounced effect on calcium uptake into an inositol trisphosphate-resistant pool. These data suggest that in platelets the 97 kD calcium pump isoform is likely to be associated with the inositol trisphosphate-sensitive calcium storage organelle.

Development of Inositol Trisphosphate-Induced Calcium Release Mechanism during Maturation of Hamster Oocytes

Mature hamster eggs exhibit repetitive increases in the intracellular Ca 2+ concentration ([Ca 2+] i) at fertilization, caused by inositol 1,4,5-trisphosphate (InsP 3)-induced Ca 2+ release (IICR) from stores. Oscillating Ca 2+ rises also occurred in inseminated immature oocytes at the germinal vesicle (GV) stage but the peak [Ca 2+] i of each response was about half of that in mature eggs. Like the responses to sperm in the mature egg, those in the immature oocyte were also blocked by injection of a monoclonal antibody against the InsP 3 receptor (18A10). Compared with mature eggs, immature oocytes were more sensitive to externally applied serotonin which caused repetitive IICR, but each response was smaller. The development of the IICR mechanism during oocyte maturation was investigated using iontophoretic injection of InsP 3 and Ca 2+ imaging with fura 2. In immature oocytes the rise in [Ca 2+] i with an InsP 2 pulse of 1.2 nA for 1 sec was 35% of that in mature eggs. The response increased with increasing doses of InsP 3. The nearly maximum response, obtained with a 20-nA 2-sec pulse, was 80% of that in mature eggs, suggesting that immature oocytes are less sensitive to InsP 3 but that most of Ca 2+-releasable stores are already present in the immature oocyte. IICR developed in two phases during in vivo maturation. The sensitivity to InsP 3 increased gradually between the GV stage and prometaphase of the first meiosis. Then a nonlinear dose-response relation developed during the second meiosis to metaphase II, resulting in regenerative, propagating Ca 2+ release induced by a threshold pulse of 1 nA for 1 sec in mature eggs. Similar changes occurred during in vitro maturation of oocytes isolated from follicles at GV stage and cultured for up to 16 hr. The development of IICR is thought to be a prerequisite factor for the acquisition of the ability of an egg to undergo normal fertilization.

Potassium-channel blockers inhibit inositol trisphosphate-induced calcium release in the microsomal fractions isolated from the rat brain

The ionic mechanism of inositol trisphosphate (InsP3)-induced Ca2+ release was investigated in microsomes (microsomal fractions) isolated from rat brain. InsP3 stimulated Ca2+ release from microsomes incubated in media containing 100 mM-KCl. The InsP3-induced Ca2+ release was insensitive to a variety of Ca2+-channel blockers; however, the K+-channel blockers tetraethylammonium chloride (TEA; 1 mM) and 9-tetraethylammonium chloride (9-TEA; 1 mM) blocked InsP3-induced Ca2+ release. Moreover, addition of InsP3 increased 86Rb+ influx into the microsomes. The influx of 86Rb+ also was sensitive to TEA and 9-TEA. The above results suggest that InsP3-induced Ca2+ release requires an opposite flow of K+ ions, and modulation of K+ channels by TEA and 9-TEA may underlie the inhibition of InsP3-induced Ca2+ release from brain microsomes by these agents.

Heparin inhibits inositol trisphosphate-induced calcium release from permeabilized rat liver cells

Neoplastic rat liver epithelial (261B) cells made permeable by electroporation released 0.2-0.3 μM Ca 2+ from intracellular stores in response to 0.5 μM Ins(1,4,5)P 3 stimulation. This Ca 2+ release response was found to be inhibited by heparin in a dose-dependent manner (K i of 15 μg/ml). Two other glycosaminoglycans, chondroitin sulfate and hyaluronic acid, showed no inhibitory effect at doses as high as 0.2 mg/ml. Passive Ca 2+ release, and sequestration of Ca 2+ into intracellular storage sites by the action of Ca 2+-ATPase were unaffected by heparin treatment. We conclude that the inhibitory action of heparin treatment on Ca 2+ mobilization in permeabilized 261B cells is mediated through its interaction at the Ins(1,4,5)P 3 receptor binding site.

Inositol trisphosphate-induced calcium release in brain microsomes

The effects of different ionic media on Ca 2+ uptake and release in isolated brain microsomes were investigated. KCl (100 mM) provided the best medium for Ca 2+ uptake in the presence of ATP. The effect of myo-inositol 1,4,5-trisphosphate (IP 3) on Ca 2+ release was examined and was maximum at 0.2 μM. IP 3-induced Ca 2+ release was dependenton extramicrosomal free Ca 2+ concentration with maximal release at 5.0 μM free Ca 2+. Replacement of KCl by sucrose or NaCl did not show any response to IP 3. Electron microscopy showed that the microsomal fraction consisted of characteristic endoplasmic reticulum-derived vesicular profiles and were free of mitochondria or plasma membrane contamination. Our results support the concept that the endoplasmic reticulum is the target for IP 3-induced mobilization of Ca 2+ in the cell.

O-169 - Calcium- and inositol trisphosphate-induced calcium release in skinned fibor bundles of the guinea-pig taenia caeci

O-169 - Calcium- and inositol trisphosphate-induced calcium release in skinned fibor bundles of the guinea-pig taenia caeci

Calcium dependent inositol trisphosphate-induced calcium release in the guinea-pig taenia caeci

The properties of the Ca stores in the skinned fiber bundles of the guinea-pig taenia caeci were studied by a novel method to measure Ca released from the stores using a fluorescent Ca indicator, fura-2. It is found that the capacity of the Ca stores amounts some 170μ moles/liter cell water. There are at least two Ca release mechanisms, Ca-induced Ca release (CICR) and Ca release induced by inositol-1,4,5-trisphosphate (IP 3). Only about a half of the Ca is released by the Ca-induced Ca release, while IP 3 releases most of the Ca. The rate of the IP 3-induced Ca release is found to be strongly enhanced by submicromolar concentrations of Ca 2+.

Calcium modulation of inositol 1,4,5-trisphosphate-induced calcium release from neuroblastoma x glioma hybrid (NG108-15) microsomes.

Subcellular fractions of neuroblastoma x glioma (NG108-15) hybrid cells were used to study the mechanism of inositol 1,4,5-trisphosphate-induced calcium release. A microsomal fraction, enriched in endoplasmic reticulum and plasma membranes and almost devoid of mitochondria, was the most active in inositol trisphosphate- or GTP-dependent release of calcium. Neither GTP nor inositol 1,4,5-trisphosphate affected the calcium efflux mediated by the other reagent, suggesting that inositol trisphosphate and GTP act on different calcium-sequestrating vesicles. The stimulation of calcium release by GTP was relatively slow (t1/2 = 90 s), dependent on polyethyleneglycol, and greater at 2 X 10(-5) M calcium (5 nmol X min-1 X mg-1) than at 10(-6) M calcium (0.8 nmol X min-1 X mg-1). The inositol trisphosphate-induced calcium efflux was not mimicked by inositol monophosphate; it was fast (t1/2 less than 10 s) and unaffected by 3% polyethyleneglycol. The amount of calcium released by inositol trisphosphate was greatest at 10(-6) M external calcium (1 nmol X min-1 X mg-1) and it was undetectable at 2 X 10(-5) M calcium. A feedback inhibition of the inositol trisphosphate-induced calcium release by cytoplasmic calcium provides a safety mechanism preventing deleterious effects of abnormally high calcium levels.

Inositol trisphosphate-induced calcium release and contraction in vascular smooth muscle.

Inositol 1,4,5-trisphosphate (InsP3) caused Ca release and tension development in rabbit main pulmonary artery smooth muscle permeabilized with saponin or digitonin. Both of these responses to single additions of InsP3 (0.5-30 microM) were repeatable and occurred in the presence of 0.0-1.9 mM free Mg2+. Sustained contractions were induced by InsP3. The amount of Ca released by InsP3, measured with a Ca2+-selective electrode, was also estimated to be sufficient to stimulate contraction in intact smooth muscle. Ca release was not influenced by inhibitors of mitochondrial oxidative phosphorylation. The uptake of Ca2+ from the medium into the InsP3-sensitive pool was ATP-dependent. The present results support the hypothesis that, in smooth muscle, InsP3 is the messenger, or one of the messengers, involved in transmitter-induced (pharmacomechanical) Ca release from the sarcoplasmic reticulum, which is the intracellular Ca store identified previously as the source of Ca released by norepinephrine in main pulmonary artery.

Specificity of inositol trisphosphate-induced calcium release from permeabilized Swiss-mouse 3T3 cells.

Swiss-mouse 3T3 cells permeabilized with saponin were used to study the specificity of the inositol trisphosphate-induced release of 45Ca2+ from their intracellular stores. Inositol 1,4,5-trisphosphate was the most potent compound studied (dose giving half-maximal effect 0.3 microM). 45Ca2+ was also released by inositol 2,4,5-trisphosphate, glycerophosphoinositol 4,5-bisphosphate and inositol 4,5-bisphosphate, with doses giving half-maximal effect of respectively 1.6 microM, 1.6 microM and 20 microM, but not by inositol 1,4-bisphosphate (50 microM). These data suggest that the trans-vicinal phosphates on the 4- and 5-positions are essential for the Ca2+-mobilizing effect of inositol trisphosphate, and that in addition there is a requirement for a phosphate group on the opposite side of the molecule, with a preference for the 1-position.