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

ABSTRACT: 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.

SUBMITTER: Irvine RF

PROVIDER: S-EPMC1144170 | biostudies-other | 1984 Aug

REPOSITORIES: biostudies-other

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Specificity of inositol phosphate-stimulated Ca2+ mobilization from Swiss-mouse 3T3 cells.

Project description:Pure samples of inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and inositol 1,2-cyclic 4,5-trisphosphate were prepared and tested for their ability to mobilize calcium from intracellular stores in a permeabilized Swiss mouse 3T3 cell preparation. In this system inositol 1,4,5-trisphosphate mobilizes Ca2+ with a half-maximal dose of 0.3 microM. Inositol 1,2-cyclic 4,5-trisphosphate mobilized Ca2+ to the same extent with a half-maximal dose of 0.3 microM, whereas inositol 1,3,4-trisphosphate required a half-maximal dose of approx. 9 microM to give the same effect. Inositol 1,3,4,5-tetrakisphosphate was ineffective up to 20 microM and at that concentration did not antagonize the mobilization induced by inositol 1,4,5-trisphosphate. The relevance of these findings to the function of the inositol tris/tetrakis-phosphate pathway is discussed.

| S-EPMC1147412 | biostudies-other

Oxidized glutathione causes sensitization of calcium release to inositol 1,4,5-trisphosphate in permeabilized hepatocytes.

Project description:The effects of GSSG on Ca2+ mobilization by Ins(1,4,5)P3 were studied in permeabilized rat hepatocytes. Incubation with GSSG (2 mM) increased the sensitivity to Ins(1,4,5)P3 for Ca2+ release, with no effect on the size of the Ca2+ pool that could be released with maximal concentrations of Ins(1,4,5)P3. GSSG decreased the EC50 for Ins(1,4,5)P3 from a control value of 578 +/- 23 nM to 137 +/- 21 nM. GSSG had no effect on the metabolism of Ins(1,4,5)P3 in permeabilized cells, and sensitization of Ca2+ release was still observed when the poorly metabolizable analogue inositol 1,4,5-trisphosphorothioate was used. GSSG did not affect the ATP-dependent Ca2+ pump or the extent of loading of intracellular Ca2+ pools. In addition, the enhancement of Ins(1,4,5)P3-sensitivity by GSSG occurred under conditions where the Ca2+ pumps were blocked with thapsigargin or by chelation of medium Ca2+ just before Ins(1,4,5)P3 addition. The effect of GSSG was time- and dose-dependent, maximal effects being observed after 5 min incubation with 2 mM-GSSG. Cystine mimicked the GSSG-induced increase in Ins(1,4,5)P3-sensitivity, and the effects could be reversed by dithiothreitol (DTT). DTT, GSH glutathione and cysteine had no effect when added alone. Other agents known to react with protein thiols, including N-ethylmaleimide, p-chloromercuribenzoic acid and Ag+, did not affect the sensitivity to Ins(1,4,5)P3, but were inhibitors of ATP-dependent Ca2+ uptake. The data suggest that the sensitivity of the intracellular Ca2+ pools to release by Ins(1,4,5)P3 can be modulated by the formation of mixed disulphides with GSSG or other oxidized thiols.

| S-EPMC1132667 | biostudies-other

Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor.

Project description:Swiss 3T3 cells incubated for 60 h with [3H]inositol incorporated radioactivity into phosphatidylinositol (PI) and the two polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). On stimulation with platelet-derived growth factor (PDGF) there were significant increases in the levels of inositol 1-phosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). The effect of PDGF and IP3 on Ca2+ mobilization was studied in both intact cells and in 'leaky' cells that had been permeabilized with saponin. In intact cells, PDGF stimulated the efflux of 45Ca2+, whereas IP3 had no effect. Conversely, IP3 stimulated 45Ca2+ efflux from 'leaky' cells, which were insensitive to PDGF. 'Leaky' cells, which accumulated 45Ca2+ to a steady state within 20 min, were found to release approx. 40% of the label within 1 min after addition of 10 microM-IP3. This stimulation of 45Ca2+ release by IP3 was reversible and was also dose-dependent, with a half-maximal effect at approx. 0.3 microM. It seems likely that an important action of PDGF on Swiss 3T3 cells is to stimulate the hydrolysis of PIP2 to form IP3 and diacylglycerol, both of which may function as second messengers. Our results indicate that IP3 mobilizes intracellular Ca2+, and we propose that diacylglycerol may act through C-kinase to activate the Na+/H+ antiport. By generating two second messengers, PDGF can simultaneously elevate the intracellular level of Ca2+ and alkalinize the cytoplasm by lowering the level of H+.

| S-EPMC1144161 | biostudies-other

Mass measurement of inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol in bombesin-stimulated Swiss 3T3 mouse fibroblasts.

Project description:Two specific and selective assays were used to measure changes in the mass of Ins(1,4,5)P3 and sn-1,2-diacylglycerol in bombesin-stimulated Swiss 3T3 cells. The results demonstrate that the increase in Ins(1,4,5)P3 was extremely rapid, but transient, returning to basal levels by 30 s. In contrast, the increase in sn-1,2-diacylglycerol was biphasic: the first phase mirrored the transient Ins(1,4,5)P3 response, whereas the second phase was sustained and occurred in the absence of elevated Ins(1,4,5)P3. The possible source of the second phase of diacylglycerol is discussed.

| S-EPMC1136930 | biostudies-other

Characterization of the inositol 1,4,5-trisphosphate-induced calcium release from permeabilized endocrine cells and its inhibition by decavanadate and p-hydroxymercuribenzoate.

Project description:The inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ compartment of endocrine cells was studied with alpha-toxin- and digitonin-permeabilized rat insulinoma (RINA2) and rat pheochromocytoma (PC12) cells. The Ca2+ uptake was ATP-dependent, and submicromolar concentrations of IP3 specifically released the stored Ca2+. Half-maximal Ca2+ release was observed with 0.25-0.5 mumol of IP3/l, and the amount of Ca2+ released due to IP3 could be enhanced by additional loading of the Ca2+ compartment. Consecutive additions of the same concentration of IP3 for 1-2 h always released the same amount of Ca2+ without desensitization, providing an ideal basis to further characterize the IP3-induced Ca2+ release. Here we describe for the first time a reversible inhibitory effect of decavanadate on the IP3-induced Ca2+ release. Among the vanadium species tested (decavanadate, oligovanadate and monovanadate), only decavanadate was inhibitory, with a half-maximal effect at 5 mumol/l in both cell types. The effect of decavanadate could be overcome by increasing the amount of sequestered Ca2+ or added IP3. Decavanadate did not affect the ATP-driven Ca2+ uptake but oligovanadate was inhibitory on Ca2+ uptake. p-Hydroxymercuribenzoate (pHMB) at concentrations between 10 and 30 mumol/l also inhibited the Ca2+ release due to IP3. Thiol compounds such as dithiothreitol (DTT; 1 mmol/l) added before pHMB removed all its inhibitory effect on the IP3-induced Ca2+ release, whereas the inhibition caused by decavanadate was unaffected by DTT. Thus, the decavanadate-dependent inhibition functions by a distinctly different mechanism than pHMB and could serve as a specific tool to analyse various aspects of the IP3-induced Ca2+ release within endocrine cells.

| S-EPMC1133232 | biostudies-other

Effect of adenine nucleotides on myo-inositol-1,4,5-trisphosphate-induced calcium release.

Project description:The effects of a whole series of adenine nucleotides on Ins(1,4,5)P3-induced Ca2+ release were characterized in permeabilized A7r5 smooth-muscle cells. Several adenine nucleotides activated the Ins(1, 4,5)P3 receptor. It was observed that 3'-phosphoadenosine 5'-phosphoulphate, CoA, di(adenosine-5')tetraphosphate (Ap4A) and di(adenosine-5')pentaphosphate (Ap5A) were more effective than ATP. Ap4A and Ap5A also interacted with a lower EC50 than ATP. In order to find out how these adenine nucleotides affected Ins(1,4, 5)P3-induced Ca2+ release, we have measured their effect on the response of permeabilized A7r5 cells to a progressively increasing Ins(1,4,5)P3 concentration. Stimulatory ATP and Ap5A concentrations had no effect on the threshold Ins(1,4,5)P3 concentration for initiating Ca2+ release, but they stimulated Ca2+ release in the presence of supra-threshold Ins(1,4,5)P3 concentrations by increasing the co-operativity of the release process. Inhibition of the Ins(1,4,5)P3-induced Ca2+ release at higher ATP concentrations was associated with a further increase in co-operativity and also with a shift in threshold towards higher Ins(1,4,5)P3 concentrations. ATP had no effect on the non-specific Ca2+ leak in the absence of Ins(1,4,5)P3. We conclude that the adenine-nucleotide-binding site can be activated by many different adenine nucleotides. Binding of these compounds to the transducing domain of the Ins(1,4,5)P3 receptor increases the efficiency of transmitting Ins(1,4,5)P3 binding to channel opening. The inhibition by high ATP concentrations is exerted at a different site, related to Ins(1,4,5)P3 binding.

| S-EPMC1218609 | biostudies-other

Hormone-induced calcium oscillations depend on cross-coupling with inositol 1,4,5-trisphosphate oscillations.

Project description:Receptor-mediated oscillations in cytosolic Ca(2+) concentration ([Ca(2+)]i) could originate either directly from an autonomous Ca(2+) feedback oscillator at the inositol 1,4,5-trisphosphate (IP3) receptor or as a secondary consequence of IP3 oscillations driven by Ca(2+) feedback on IP3 metabolism. It is challenging to discriminate these alternatives, because IP3 fluctuations could drive Ca(2+) oscillations or could just be a secondary response to the [Ca(2+)]i spikes. To investigate this problem, we constructed a recombinant IP3 buffer using type-I IP3 receptor ligand-binding domain fused to GFP (GFP-LBD), which buffers IP3 in the physiological range. This IP3 buffer slows hormone-induced [IP3] dynamics without changing steady-state [IP3]. GFP-LBD perturbed [Ca(2+)]i oscillations in a dose-dependent manner: it decreased both the rate of [Ca(2+)]i rise and the speed of Ca(2+) wave propagation and, at high levels, abolished [Ca(2+)]i oscillations completely. These data, together with computational modeling, demonstrate that IP3 dynamics play a fundamental role in generating [Ca(2+)]i oscillations and waves.

| S-EPMC6469397 | biostudies-literature

Caspase 3 cleavage of the inositol 1,4,5-trisphosphate receptor does not contribute to apoptotic calcium release.

Project description:An important role in the regulation of apoptotic calcium release is played by the ubiquitously expressed family of inositol 1,4,5-trisphosphate receptor (IP(3)R) channels. One model for IP(3)R activation during apoptosis is cleavage by the apoptotic protease caspase 3. Here we show that early elevations in cytosolic calcium during apoptosis do not require caspase 3 activity. We detected a robust increase in calcium levels in response to staurosporine treatment in primary human fibroblasts and HeLa cells in the presence of the caspase inhibitor Z-VAD, indicating that calcium release during the initiation of apoptosis occurs independently of caspase 3. Similar results were obtained with MCF-7 cells which lack caspase 3 expression. Stable expression of caspase 3 in MCF-7 cells and TAT-based transduction of the active recombinant caspase 3 directly into living MCF-7 cells had marginal effects on the early events leading to cytosolic calcium elevations and irreversible commitment to apoptotic cell death. Significantly, blocking IP(3) binding to the IP(3)R with an IP(3) sponge resulted in suppression of staurosporine-induced calcium release and cell death. Collectively, our results suggest that generation of IP(3) is sufficient for the initiation of apoptotic calcium signaling, and caspase 3-mediated truncation of IP(3)R channel is a consequence, not causative, of apoptotic calcium release.

| S-EPMC3572306 | biostudies-literature

Polycystin-2 activation by inositol 1,4,5-trisphosphate-induced Ca2+ release requires its direct association with the inositol 1,4,5-trisphosphate receptor in a signaling microdomain.

Project description:Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca(2+) signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP(3)R), an intracellular Ca(2+) channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP(3)R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2(-/-) mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca(2+) release in intact cells and IP(3)-induced Ca(2+) release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca(2+) signal by a local Ca(2+)-induced Ca(2+)-release mechanism, which only occurred in the presence of the TRPP2-IP(3)R interaction, and not via altered IP(3)R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca(2+) microdomains necessary for initiating Ca(2+)-induced Ca(2+) release. The data strongly suggest that defects in this mechanism may account for the altered Ca(2+) signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.

| S-EPMC2881802 | biostudies-literature

Inositol 1,3,4,5-tetrakisphosphate causes release of Ca2+ from permeabilized mouse lymphoma L1210 cells by its conversion into inositol 1,4,5-trisphosphate.

Project description:Addition of Ins(1,3,4,5)P4 at micromolar concentrations causes release of Ca2+ from electroporated L1210 cells, but not from digitonin-permeabilized cells. This was shown to be due to its conversion into Ins(1,4,5)P3, because only the electroporated cells convert Ins(1,3,4,5)P4 into Ins(1,4,5)P3. Thus electroporation appears to activate or expose an Ins(1,3,4,5)P4 3-phosphatase.

| S-EPMC1138610 | biostudies-other

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