Identification in extracts from AR4-2J cells of inositol 1,4,5-trisphosphate by its susceptibility to inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase.

Renal brush-border membrane vesicles from rat kidney cortex were irradiated in frozen state with a gamma-radiation source. Initial rates of influx into these vesicles were estimated for substrates such as L-glutamic acid, L-alanine, L-proline and L-leucine to establish the molecular sizes of their carriers. Transport was measured in initial-rate conditions to avoid artifacts arising from a decrease in the driving force caused by a modification of membrane permeability. Initial rates of Na(+)-independent uptakes for those four substrates appeared unaffected in the dose range used (0-6 Mrad), indicating that the passive permeability of the membrane towards these substrates was unaffected. However, at higher doses of irradiation the Na+ influx and the intravesicular volume evaluated by the uptake of glucose at equilibrium were altered by radiation. Thus Na(+)-dependent influx values were corrected for volume changes, and the corrected values were used to compute radiation-inactivation sizes of the transport systems. Their respective values for L-glutamic acid, L-proline, L-leucine and L-alanine carriers were 250, 224, 293 and 274 kDa. The presence of the free-radicals scavenger benzoic acid in the frozen samples during irradiation did not affect the uptake of glucose, phosphate and alkaline phosphatase activity. These results indicate that freezing samples in a cryoprotective medium was enough to prevent secondary inactivation of transporters by free radicals. Uptakes of beta-alanine and L-lysine were much less affected by radiation. The radiation-inactivation size of the Na(+)-dependent beta-alanine carrier was 127 kDa and that of the L-lysine carrier was 90 kDa.     

Ca2+ dependence of inositol 1,4,5-trisphosphate 3-kinase activity in the cytosol fraction of pig coronary artery

1. The activity of inositol 1,4,5-trisphosphate 3-kinase in subcellular fractions of smooth muscles of the pig coronary artery was examined. 2. Incubation of [3H]inositol 1,4,5-trisphosphate (IP3) with muscle homogenates produced more polar 3H-radioactivity (probably as inositol 1,3,4,5-tetrakisphosphate, IP4) than IP3, in the Mg2+- and ATP-dependent manner, thereby indicating the presence of IP3 3-kinase activity in homogenates of the muscle. 3. Most of the kinase activity was present in the cytosol fraction. The enzyme activity was reversibly activated by Ca2+ with a half-maximal effective concentration of 2.5 x 10(-7) M. 4. The calmodulin antagonists, W-7 and chlorpromazine inhibited the Ca2+-activated enzyme activity.     

Ca2+/calmodulin independent inositol 1,4,5-trisphosphate 3-kinase activity in guinea pig peritoneal macrophages

1. The Ca2+/calmodulin (CaM) independent activity of inositol 1,4,5-trisphosphate (InsP3) 3-kinase in macrophages could be separated from the dependent activity by serial column chromatography, gel filtration, Orange A and DEAE-5PW. 2. An InsP3 analog which has an aminobenzoyl group on the 2nd carbon of the inositol ring inhibited the conversion of [3H]InsP3 to [3H]InsP4 (inositol 1,3,4,5-tetrakisphosphate) in a dose-dependent manner. The concentration required for half-maximal inhibition (IC50) with the Ca2+/CaM independent enzyme activity was also dependent on the free Ca2+ concentration, as with the dependent activity. 3. These results suggest that a conformational change in the enzyme occurs in response to a change in free Ca2+ concentration, and thus the potency to recognize the InsP3 analog would change, even when the Ca2+/CaM independent enzyme activity was used.     

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.     

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.     

Stoichiometry of contraction and Ca2+ mobilization by inositol 1,4,5-trisphosphate in isolated gastric smooth muscle cells

Smooth muscle cells were isolated from the circular muscle layer of guinea pig stomach and permeabilized by brief exposure to saponin. Both permeabilized and intact muscle cells contracted in response to cholecystokinin octapeptide (CCK-8) and acetylcholine, but only permeabilized muscle cells contracted in response to inositol 1,4,5-trisphosphate (InsP3). The contractile response to InsP3 was prompt (peak less than 5 s), concentration-dependent (EC50-0.3 microM), and insensitive to antimycin or oligomycin. Contraction induced by either InsP3 or CCK-8 was accompanied by a concentration-dependent increase in free Ca2+ that was directly correlated with the magnitude of contraction. Both InsP3 and CCK-8 caused rapid net efflux of Ca2+ from cells preloaded with 45Ca2+. Contraction, increase in free Ca2+ concentration, and net 45Ca2+ efflux elicited by a combination of maximal concentrations of InsP3 and CCK-8 were not significantly different from those elicited by maximal concentrations of either agent alone. Repeated stimulation of single muscle cells with either InsP3 or CCK-8 in Ca2+-free medium caused eventual loss of the contractile response to all agents. The response to all agents was restored upon re-exposure of the cell to a cytosol-like concentration of Ca2+, implying equal access of InsP3 and receptor-linked agonists to the same intracellular Ca2+ store. The results demonstrate that InsP3 mimics the effects of receptor-linked agonists on contraction and mobilization of intracellular Ca2+ in permeabilized smooth muscle cells that retain the functional properties of intact smooth muscle cells and support a role for InsP3 as membrane-derived messenger responsible for mobilization of intracellular Ca2+ in smooth muscle cells.     

Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase in rat and bovine brain tissues

Inositol 1,4,5-trisphosphate (Ins P3) 3-kinase catalyzes the ATP-dependent phosphorylation of Ins P3 to Inositol 1,3,4,5-tetrakisphosphate (Ins P4). Ca2+/calmodulin (CaM)-sensitivity of Ins P3 3-kinase was measured in the crude soluble fraction from rat brain and different anatomic regions of bovine brain. Kinase activity was inhibited in the presence of EGTA (free Ca2+ below 1 nM) as compared to Ca2+ (10 microM free Ca2+) or Ca2+ (10 microM free Ca2+) and CaM (1 microM). Ca2+-sensitivity was also seen for the cAMP phosphodiesterase measured under the same assay conditions, but was not for the Ins P3 5-phosphatase. DEAE-cellulose chromatography of the soluble fraction of rat brain or bovine cerebellum resolved a Ca2+/CaM-sensitive Ins P3 3-kinase (maximal stimulation at 1 microM Ins P3 substrate level was 2.0-3.0 fold).     

Calmodulin activates inositol 1,4,5-trisphosphate 3-kinase activity in pig aortic smooth muscle.

The effects of calmodulin (CaM) on inositol 1,4,5-trisphosphate (InsP3) 3-kinase activity in pig aortic smooth muscle were examined. The cytosol fraction of muscle cells, containing 1.2-2.0 micrograms of CaM/mg of cytosol protein (thus 0.12-0.2%, w/w), showed a Ca2+-dependent InsP3 3-kinase activity, and there was no further activation by exogenous addition of CaM purified from dog brain. (NH4)2SO4 fractionation of the cytosol fraction revealed that a 20-60%-satd.-(NH4)2SO4 fraction was rich in the enzyme activity, and the activity without exogenous CaM was still dependent on Ca2+, although the CaM content in this fraction was minute (0.013-0.016%, w/w). The kinase activity observed in the absence of exogenous CaM became insensitive to Ca2+ when a 20-60%-satd.-(NH4)2SO4 fraction was applied to a DEAE-cellulose column, but exogenous addition of CaM increased the enzyme activity from 80-120 to 450 pmol/min per mg of protein, with addition of 10 microM free Ca2+. A fraction separated by DEAE-cellulose chromatography was applied to a CaM affinity column. The kinase activity was retained on the column in the presence of Ca2+, and was eluted by lowering the free Ca2+ concentration by adding EGTA. These results directly show that CaM activates InsP3 3-kinase activity and the enzyme becomes sensitive to Ca2+.     

Formation of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate from inositol 1,3,4,5-tetrakisphosphate and their pathways of degradation in RBL-2H3 cells

Previous studies with antigen-stimulated rat basophilic leukemia (RBL-2H3) cells indicated the formation of multiple isomers of each of the various categories of inositol phosphates. The identities of the different isomers have been elucidated by selective labeling of [3H]inositol 1,3,4,5-tetrakisphosphate with [32P]phosphate in the 3'-or 4',5'-positions and by following the metabolism of different radiolabeled inositol phosphates in extracts of RBL-2H3 cells. We report here that inositol 1,3,4,5-tetrakisphosphate, when incubated with the membrane fraction of extracts of RBL-2H3 cells, was converted to inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate. Further dephosphorylation of the inositol polyphosphates proceeded rapidly in whole extracts of cells, although the process was significantly retarded when ATP (2 mM) levels were maintained by an ATP-regenerating system. The degradation of inositol 1,4,5-trisphosphate proceeded with the sequential formation of inositol 1,4-bisphosphate, the inositol 4-monophosphate (with smaller amounts of the 1-monophosphate), and finally inositol. Inositol 1,3,4-trisphosphate, on the other hand, was converted to inositol 1,3-bisphosphate and inositol 3,4-bisphosphate and subsequently to inositol 4-monophosphate and inositol 1-monophosphate (stereoisomeric forms were undetermined). The possible implications of the apparent interconversion between inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in regulating histamine secretion in the RBL-2H3 cells are discussed.     

cAMP-dependent protein kinase enhances inositol 1,4,5-trisphosphate-induced Ca2+ release in AR4-2J cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, plays an important role in the control of intracellular Ca(2+). There are three subtypes of IP(3)R that are differentially distributed among cell types. AR4-2J cells express almost exclusively the IP(3)R-2 subtype. The purpose of this study was to investigate the effect of cAMP-dependent protein kinase (PKA) on the activity of IP(3)R-2 in AR4-2J cells. We showed that immunoprecipitated IP(3)R-2 is a good substrate for PKA. Using a back-phosphorylation approach, we showed that endogenous PKA phosphorylates IP(3)R-2 in intact AR4-2J cells. Pretreatment with PKA enhanced IP(3)-induced Ca(2+) release in permeabilized AR4-2J cells. Pretreatment with the cAMP generating agent's forskolin and vasoactive intestinal peptide (VIP) enhanced carbachol (Cch)-induced and epidermal growth factor (EGF)-induced Ca(2+) responses in intact AR4-2J cells. Our results are consistent with an enhancing effect of PKA on IP(3)R-2 activity. This conclusion supports the emerging concept of crosstalk between Ca(2+) signaling and cAMP pathways and thus provides another way by which Ca(2+) signals are finely encoded within non-excitable cells.     

Characterization of inositol 1,4,5-trisphosphate-stimulated calcium release from rat cerebellar microsomal fractions. Comparison with [3H]inositol 1,4,5-trisphosphate binding.

The abilities of D-myo-inositol phosphates (InsPs) to promote Ca2+ release and to compete for D-myo-[3H]-inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) binding were examined with microsomal preparations from rat cerebellum. Of the seven InsPs examined, only Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 stimulated the release of Ca2+. Ca2+ release was maximal in 4-6 s and was followed by a rapid re-accumulation of Ca2+ into the Ins(1,4,5)P3-sensitive compartment after Ins(1,4,5)P3, but not after Ins(2,4,5)P3 or Ins(4,5)P2. Ca2+ re-accumulation after Ins(1,4,5)P3 was also faster than after pulse additions of Ca2+, and coincided with the metabolism of [3H]Ins(1,4,5)P3. These data suggest that Ins(1,4,5)P3-induced Ca2+ release and the accompanying decrease in intraluminal Ca2+ stimulate the Ca2+ pump associated with the Ins(1,4,5)P3-sensitive compartment. That this effect was observed only after Ins(1,4,5)P3 may reflect differences in either the metabolic rates of the various InsPs or an effect of the Ins(1,4,5)P3 metabolite Ins(1,3,4,5)P4 to stimulate refilling of the Ins(1,4,5)P3-sensitive store. InsP-induced Ca2+ release was concentration-dependent, with EC50 values (concn. giving half-maximal release) of 60, 800 and 6500 nM for Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 respectively. Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 also competed for [3H]Ins(1,4,5)P3 binding, with respective IC50 values (concn. giving 50% inhibition) of 100, 850 and 13,000 nM. Comparison of the EC50 and IC50 values yielded a significant correlation (r = 0.991). These data provide evidence of an association between the [3H]Ins(1,4,5)P3-binding site and the receptor mediating Ins(1,4,5)P3-induced Ca2+ release.     

Molecular definition of a novel inositol polyphosphate metabolic pathway initiated by inositol 1,4,5-trisphosphate 3-kinase activity in Saccharomyces cerevisiae

The production of inositol polyphosphate (IPs) and pyrophosphates (PP-IPs) from inositol 1,4,5-trisphosphate (I(1,4,5)P3) requires the 6-/3-/5-kinase activity of Ipk2 (also known as Arg82 and inositol polyphosphate multikinase). Here, we probed the distinct roles for I(1,4,5)P3 6- versus 3-kinase activities in IP metabolism and cellular functions reported for Ipk2. Expression of either I(1,4,5)P3 6- or 3-kinase activity rescued growth of ipk2-deficient yeast at high temperatures, whereas only 6-kinase activity enabled growth on ornithine as the sole nitrogen source. Analysis of IP metabolism revealed that the 3-kinase initiated the synthesis of novel pathway consisting of over eleven IPs and PP-IPs. This pathway was present in wild-type and ipk2 null cells, albeit at low levels as compared with inositol hexakisphosphate synthesis. The primary route of synthesis was: I(1,4,5)P3 --> I(1,3,4,5)P4 --> I(1,2,3,4,5)P5 --> PP-IP4 --> PP2-IP3 and required Kcs1 (or possibly Ipk2), Ipk1, a novel inositol pyrophosphate synthase, and then Kcs1 again, respectively. Mutation of kcs1 ablated this pathway in ipk2 null cells and overexpression of Kcs1 in ipk2 mutant cells phenocopied IP3K expression, confirming it harbors a novel 3-kinase activity. Our work provides a revised genetic map of IP metabolism in yeast and evidence for dosage compensation between IPs and PP-IPs downstream of I(1,4,5)P3 in the regulation of nucleocytoplasmic processes.     

Distinct roles of inositol 1,4,5-trisphosphate receptor types 1 and 3 in Ca2+ signaling

Three subtypes of inositol 1,4,5-trisphosphate receptor (IP(3)R1, IP(3)R2, and IP(3)R3) Ca(2+) release channel share basic properties but differ in terms of regulation. To what extent they contribute to complex Ca(2+) signaling, such as Ca(2+) oscillations, remains largely unknown. Here we show that HeLa cells express comparable amounts of IP(3)R1 and IP(3)R3, but knockdown by RNA interference of each subtype results in dramatically distinct Ca(2+) signaling patterns. Knockdown of IP(3)R1 significantly decreases total Ca(2+) signals and terminates Ca(2+) oscillations. Conversely, knockdown of IP(3)R3 leads to more robust and long lasting Ca(2+) oscillations than in controls. Effects of IP(3)R3 knockdown are surprisingly similar in COS-7 cells that predominantly (>90% of total IP(3)R) express IP(3)R3, suggesting that IP(3)R3 functions as an anti-Ca(2+)-oscillatory unit without contributing to peak amplitude of Ca(2+) signals, irrespective of its relative expression level. Therefore, differential expression of the IP(3)R subtype is critical for various forms of Ca(2+) signaling, and, particularly, IP(3)R1 and IP(3)R3 have opposite roles in generating Ca(2+) oscillations.     

Turkey erythrocytes possess a membrane-associated inositol 1,4,5-trisphosphate 3-kinase that is activated by Ca2+ in the presence of calmodulin.

Turkey erythrocytes contain soluble and particulate kinase activities which catalyse the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. The particle-bound activity accounts for approximately one-quarter of the total cellular Ins(1,4,5)P3 kinase, when assayed at a [Ca2+] of 10 nM. The particle-bound Ins(1,4,5)P3 kinase is not washed from the membrane by 0.6 M-KCl, yet may be solubilized by a variety of detergents. This suggests that it is an intrinsic membrane protein. The product of the membrane-bound Ins(1,4,5)P3 kinase is inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], identifying the enzyme as an Ins(1,4,5)P3 3-kinase. In the presence of calmodulin, the membrane-associated Ins(1,4,5)P3 3-kinase is activated as [Ca2+] is increased over the range 0.2-1.0 microM. Under these conditions, the rates of dephosphorylation of Ins(1,3,4,5)P4 and Ins(1,4,5)P3 by phosphatases in the membrane fraction are unchanged.     

Chemoattractant and guanosine 5''-[gamma-thio]triphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3H]inositol by electroporation.

The analysis of the inositol cycle in Dictyostelium discoideum cells is complicated by the limited uptake of [3H]inositol (0.2% of the applied radioactivity in 6 h), and by the conversion of [3H]inositol into water-soluble inositol metabolites that are eluted near the position of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] on anion-exchange h.p.l.c. columns. The uptake was improved to 2.5% by electroporation of cells in the presence of [3H]inositol; electroporation was optimal at two 210 microseconds pulses of 7 kV. Cells remained viable and responsive to chemotactic signals after electroporation. The intracellular [3H]inositol was rapidly metabolized to phosphatidylinositol and more slowly to phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. More than 85% of the radioactivity in the water-soluble extract that was eluted on Dowex columns as Ins(1,4,5)P3 did not co-elute with authentic [32P]Ins(1,4,5)P3 on h.p.l.c. columns. Chromatography of the extract by ion-pair reversed-phase h.p.l.c. provided a good separation of the polar inositol polyphosphates. Cellular [3H]Ins(1,4,5)P3 was identified by (a) co-elution with authentic [32P]Ins(1,4,5)P3 and (b) degradation by a partially purified Ins(1,4,5)P3 5-phosphatase from rat brain. The chemoattractant cyclic AMP and the non-hydrolysable analogue guanosine 5'-[gamma-thio]triphosphate induced a transient accumulation of radioactivity in Ins(1,4,5)P3; we did not detect radioactivity in inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In vitro, Ins(1,4,5)P3 was metabolized to inositol 1,4- and 4,5-bisphosphate, but not to Ins(1,3,4,5)P4 or another tetrakisphosphate isomer. We conclude that Dictyostelium has a receptor- and G-protein-stimulated inositol cycle which is basically identical with that in mammalian cells, but the metabolism of Ins(1,4,5)P3 is probably different.     

Interaction with the inositol 1,4,5-trisphosphate receptor promotes Ca2+ sequestration in permeabilised insulin-secreting cells.

Electropermeabilised insulin-secreting RINm5F cells sequestered Ca2+, resulting in a steady-state level of the ambient free Ca2+ concentration corresponding to 723 +/- 127 nM (mean +/- SEM, n = 10), as monitored by a Ca(2+)-selective minielectrode. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) promoted a rapid and pronounced release of Ca2+. This Ca2+ was resequestered and a new steady-state Ca2+ level was attained, which was always lower (460 +/- 102 nM, n = 10, P less than 0.001) than the steady-state Ca2+ level maintained before the addition of Ins(1,4,5)P3. Whereas the initial reuptake of Ca2+ subsequent to Ins(1,4,5)P3 stimulation was relatively slow, the later part of reuptake was fast as compared to the reuptake phases of a pulse addition of extraneous Ca2+. In the latter case the uptake of Ca2+ resulted in a steady-state level similar to that found in the absence of Ins(1,4,5)P3. Addition of Ins(1,4,5)P3 under this condition resulted in a further Ca2+ uptake and thus a lower steady-state Ca2+ level. Heparin, which binds to the Ins(1,4,5)P3 receptor, also lowered the steady-state free Ca2+ concentration. In contrast to Ins(1,4,5)P3, inositol 1,3,4,5-tetrakisphosphate was without effect on Ca2+ sequestration. These findings are consistent with the presence of a high-affinity Ins(1,4,5)P3 receptor promoting continuous release of Ca2+ under basal conditions and/or the Ins(1,4,5)P3 receptor being actively involved in Ca2+ sequestration.