Inositol 1,3,4,5-tetrakisphosphate increases the duration of the inositol 1,4,5-trisphosphate-mediated Ca2+ transient

The effect of Ins 1,3,4,5-P4 on the intracellular Ca2+ mobilization produced by Ins 1,4,5-P3 has been examined in permeabilized hepatocytes. Ins 1,3,4,5-P4 did not affect the magnitude of the Ins 1,4,5-P3-mediated Ca2+ release but did inhibit re-accumulation of the released Ca2+ back into intracellular stores. This effect was not mimicked by Ins 1,3,4-P3. In hepatocytes, the re-uptake phase of the response results from Ins 1,4,5-P3 hydrolysis. Measurements using labeled substrates indicate that Ins 1,3,4,5-P4 inhibits the hydrolysis of Ins 1,4,5-P3 and vice versa. Since the removal of the 5-phosphate on Ins 1,4,5-P3 and Ins 1,3,4,5-P4 is a common step in the disposal of both compounds, it is suggested that one of the biological effects of Ins 1,3,4,5-P4 may be to slow hydrolysis of Ins 1,4,5-P3 and thereby prolong the duration of a Ca2+ transient.     

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.     

Kinetics of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate generation in dog-thyroid primary cultured cells stimulated by carbachol

The action of carbachol on the generation of inositol trisphosphate and tetrakisphosphate isomers was investigated in dog-thyroid primary cultured cells radiolabelled with [3H]inositol. The separation of the inositol phosphate isomers was performed by reverse-phase high pressure liquid chromatography. The structure of inositol phosphates co-eluting with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] standards was determined by enzymatic degradation using a purified Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. The data indicate that Ins(1,3,4,5)P4 was the only [3H]inositol phosphate which co-eluted with a [32P]Ins(1,3,4,5)P4 standard, whereas 80% of the [3H]InsP3 co-eluting with an Ins(1,4,5)P3 standard was actually this isomer. In the presence of Li+, carbachol led to rapid increases in [3H]Ins(1,4,5)P4. The level of Ins(1,4,5)P3 reached a peak at 200% of the control after 5-10 s of stimulation and fell to a plateau that remained slightly elevated for 2 min. The level of Ins(1,3,4,5)P4 reached its maximum at 20s. The level of inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] increased continuously for 2 min after the addition of carbachol. Inositol-phosphate generation was also investigated under different pharmacological conditions. Li+ largely increased the level of Ins(1,3,4)P3 but had no effect on Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin, which stimulates dog-thyroid adenylate cyclase and cyclic-AMP accumulation, had no effect on the generation of inositol phosphates. The absence of extracellular Ca2+ largely decreased the level of Ins(1,3,4,5)P4 as expected considering the Ca2(+)-calmodulin sensitivity of the Ins(1,4,5)P3 3-kinase. Staurosporine, an inhibitor of protein kinase C, increased the levels of Ins(1,4,5)P3, Ins(1,3,4,5)P4 and Ins(1,3,4)P3. This supports a negative feedback control of diacyglycerol on Ins(1,4,5)P3 generation.     

Inositol 1,3,4,5-tetrakisphosphate and inositol 1,4,5-trisphosphate act by different mechanisms when controlling Ca2+ in mouse lacrimal acinar cells

In internally perfused single lacrimal acinar cells the competitive inositol 1,4,5-trisphosphate (Ins 1,4,5-P3)-antagonist heparin inhibits the ACh-evoked K+ current response mediated by internal Ca2+ and also blocks both the Ins 1,4,5-P3-evoked transient as well as the sustained K+ current increase evoked by combined stimulation with internal Ins 1,4,5-P3 and inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4). When, during sustained stimulation with both Ins 1,4,5-P3 and Ins 1,3,4,5-P4, one of the inositol polyphosphates is removed, the K+ current declines; whereas removal of Ins 1,4,5-P3 results in an immediate termination of the response, removal of Ins 1,3,4,5-P4 only causes a very gradual and slow reduction in the current. Ins 1,3,4,5-P4 is therefore not an acute controller of Ca2+ release from stores into the cytosol, but modulates the release of Ca2+ induced by Ins 1,4,5,P3 by an unknown mechanism, perhaps by linking Ins 1,4,5 P3-sensitive and insensitive Ca2+ stores.     

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.

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.     

Ca2+ homeostasis in permeabilized human neutrophils. Characterization of Ca2+-sequestering pools and the action of inositol 1,4,5-triphosphate

The regulation of Ca2+ transport by intracellular compartments was studied in digitonin-permeabilized human neutrophils, using a Ca2+-selective electrode. When incubated in a medium containing ATP and respiratory substrates, the cells lowered within 6 min the ambient [Ca2+] to a steady state of around 0.2 microM. A vesicular ATP-dependent and vanadate-sensitive non-mitochondrial pool maintained this low [Ca2+] level. In the absence of ATP, a higher Ca2+ steady state of 0.6 microM was seen, exhibiting the characteristics of a mitochondrial Ca2+ "set point." Both pools were shown to act in concert to restore the previous ambient [Ca2+] following its elevation. Thus, the mitochondria participate with the other pool(s) in decreasing [Ca2+] to the submicromolar range whereas only the nonmitochondrial pool(s) lowers [Ca2+] to the basal level. The action of inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a few cell types was studied. IP3 released (detectable within 2 s) Ca2+ accumulated in the ATP-dependent pool(s) but had no effect on the mitochondria. The response was transient and resulted in desensitization toward subsequent IP3 additions. Under experimental conditions in which the ATP-dependent Ca2+ influx was blocked, the addition of IP3 resulted in a very large Ca2+ release from nonmitochondrial pool. The results strongly suggest that IP3 is a second messenger mediating intracellular Ca2+ mobilization in human neutrophils. Furthermore, the nonmitochondrial pool appears to have independent influx and efflux pathways for Ca2+ transport, a Ca2+ ATPase (the influx component) and an IP3-sensitive efflux component activated during Ca2+ mobilization.     

Synergistic control of Ca2+ mobilization in permeabilized mouse L1210 lymphoma cells by inositol 2,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate.

L1210 lymphoma cells were permeabilized with digitonin, and the ability of Ins(2,4,5)P3 and Ins(1,3,4,5)P4 to mobilize intracellular Ca2+ was studied. At high doses of Ins(2,4,5)P3 Ca2+ was rapidly released from intracellular stores, and prior or subsequent addition of Ins(1,3,4,5)P4 had no discernible effect. However, the Ca2(+)-mobilizing action of low (threshold or just above) concentrations of Ins(2,4,5)P3 was markedly enhanced by Ins(1,3,4,5)P4, which alone caused no mobilization of Ca2+; this phenomenon was shown not to be due to protection of Ins(2,4,5)P3 by the Ins(1,3,4,5)P4 against hydrolysis. The ability of the pre-addition of Ins(1,3,4,5)P4 to enhance subsequent Ins(2,4,5)P3-induced Ca2+ mobilization was always seen whether or not the free Ca2+ concentration was low (pCa = 7) or high (pCa = 6). However, at low Ca2+, Ins(1,3,4,5)P4 could cause a further mobilization if added after the Ins(2,4,5)P3, whereas at higher Ca2+ values Ins(1,3,4,5)P4 was only able to affect Ca2+ if added before Ins(2,4,5)P3. These effects of Ins(1,3,4,5)P4 were not, at the same concentration, mimicked by a random mixture of InsP4 isomers obtained by partial acid hydrolysis of phytic acid, by Ins(1,3,4)P3 or by Ins(1,3,4,5,6)P5, and they were shown not to be due to enzymic generation of Ins(1,4,5)P3 from Ins(1,3,4,5)P4 by (a) the absence of any detectable production of Ins(1,4,5)P3 if radiolabelled Ins(1,3,4,5)P4 was used, or (b) the observation that Ins(1,3,4,5,6)P5 could mimic Ins(1,3,4,5)P4 provided that higher doses were used; this inositol phosphate, when added radiolabelled, yielded only trace quantities of D/L-Ins(1,4,5,6)P4, which itself does not mobilize Ca2+. We interpret these results overall to mean that in these cells there is a small proportion of the Ins(2,4,5)P3-mobilizable Ca2+ pools which can only be mobilized in the presence of Ins(1,3,4,5)P4 [or at the least, Ins(1,3,4,5)P4 can help Ins(2,4,5)P3 to gain access to them]. The significance of this conclusion is discussed in the light of current concepts of the second messenger function of Ins(1,3,4,5)P4.     

Ca2+ entry in T cells is activated by emptying the inositol 1,4,5-triphosphate sensitive Ca2+ pool

Using alpha-linolenic acid (ALA), one of several polyunsaturated fatty acids (PUFAs) that have previously been shown to both mobilize intracellular Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool independently of IP3 production and inhibit Ca2+ influx, the relationship between Ca2+ mobilization from intracellular stores and Ca2+ influx in T cells (JURKAT) was studied. JURKAT cells were treated with 30 microM ALA to deplete the IP3-sensitive Ca2+ pool. When the intracellular free Ca2+ concentration [( Ca2+]i) returned to basal level, fatty acid free bovine serum albumin (BSA) was added to remove extracellular and membrane bound ALA. This resulted in a sustained increase in [Ca2+]i in the absence of inositol phosphates' formation. This sustained increase in [Ca2+]i was insensitive to protein kinase C activation but was inhibited by Ni2+ ions. The extent of Ca2+ influx was found to be correlated to the amount of Ca2+ initially discharged from the IP3-sensitive Ca2+ pool by sub-optimal concentrations of ALA. Ligation of the CD3 complex of the T cell antigen receptor with an anti-CD3 antibody (OKT3) during the sustained [Ca2+]i increased (induced by a sub-optimal concentration of ALA), produced a greater response. No increase in the sustained response was observed when the CD3 complex was activated in cells pretreated with an optimal concentration of ALA. In summary, Ca2+ entry in T cells is activated by emptying of the IP3-sensitive Ca2+ pool which can be dissociated from inositol phosphate production. The rate of Ca2+ influx appears to be closely correlated to the initial discharge of Ca2+ from the IP3-sensitive Ca2+ pool, suggesting that Ca2+ may first enter the depleted pool and then is released into the cytosol.     

Characterization of the inositol 1,4,5-trisphosphate-induced Ca2+ release in pancreatic beta-cells.

Pancreatic beta-cells isolated from obese-hyperglycaemic mice released intracellular Ca2+ in response to carbamoylcholine, an effect dependent on the presence of glucose. The effective Ca2+ concentration reached was sufficient to evoke a transient release of insulin. When the cells were deficient in Ca2+, the Ca2+ pool sensitive to carbamoylcholine stimulation was equivalent to that released by ionomycin. Unlike intact cells, cells permeabilized by high-voltage discharges failed to generate either inositol 1,4,5-triphosphate (InsP3) or to release Ca2+ after exposure to carbamoylcholine. However, the permeabilized cells released insulin sigmoidally in response to increasing concentrations of Ca2+. Also in the absence of functional mitochondria these cells exhibited a large ATP-dependent buffering of Ca2+, enabling the maintenance of an ambient Ca2+ concentration corresponding to about 150 nM even after several additional pulses of Ca2+. InsP3, maximally effective at 6 microM, promoted a rapid and pronounced release of Ca2+. The InsP3-sensitive Ca2+ pool was rapidly filled and lost its Ca2+ late after ATP depletion. The transient nature of the Ca2+ signal was not overcome by repetitive additions of InsP3. It was possible to restore the response to InsP3 after a delay of approx. 20 min, an effect which had less latency after the addition of Ca2+. These latter findings argue against degradation and/or desensitization as factors responsible for the transiency in InsP3 response. It is suggested that Ca2+ released by InsP3 is taken up by a part of the endoplasmic reticulum (ER) not sensitive to InsP3. On metabolism of InsP3, Ca2+ recycles to the InsP3-sensitive pool, implying that this pool indeed has a very high affinity for the ion. The presence of functional mitochondria did not interfere with the recycling process. The ER in pancreatic beta-cells is of major importance in buffering Ca2+, but InsP3 only modulates Ca2+ transport for a restricted period of time following immediately upon its formation. Thereafter the non-sensitive part of the ER takes over the continuous regulation of Ca2+ cycling.     

Ca2(+)-sensitivity of inositol 1,4,5-trisphosphate-mediated Ca2+ release in permeabilized pancreatic acinar cells.

Hormonal and phorbol ester pretreatment of pancreatic acinar cells markedly decreases the Ins(1,4,5)P3-induced release of actively stored Ca2+ [Willems, Van Den Broek, Van Os & De Pont (1989) J. Biol. Chem. 264, 9762-9767]. Inhibition occurred at an ambient free Ca2+ concentration of 0.1 microM, suggesting a receptor-mediated increase in Ca2(+)-sensitivity of the Ins(1,4,5)P3-operated Ca2+ channel. To test this hypothesis, the Ca2(+)-dependence of Ins(1,4,5)P3-induced Ca2+ release was investigated. In the presence of 0.2 microM free Ca2+, permeabilized cells accumulated 0.9 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Uptake into this pool increased 2.2- and 3.3-fold with 1.0 and 2.0 microM free Ca2+ respectively. At 0.2, 1.0 and 2.0 microM free Ca2+, Ins(1,4,5)P3 maximally released 0.53 (56%), 0.90 (44%) and 0.62 (20%) nmol of Ca2+/mg of acinar protein respectively. Corresponding half-maximal stimulatory Ins(1,4,5)P3 concentrations were calculated to be 0.5, 0.6 and 1.4 microM, suggesting that the affinity of Ins(1,4,5)P3 for its receptor decreases beyond 1.0 microM free Ca2+. The possibility that an inhibitory effect of sub-micromolar Ca2+ is being masked by the concomitant increase in size of the releasable store is excluded, since Ca2+ release from cells loaded in the presence of 0.1 or 0.2 microM free Ca2+ and stimulated at higher ambient free Ca2+ was not inhibited below 1.0 microM free Ca2+. At 2.0 and 10.0 microM free Ca2+, Ca2+, Ca2+ release was inhibited by approx. 30% and 75% respectively. The results presented show that hormonal pretreatment does not lead to an increase in Ca2(+)-sensitivity of the release mechanism. Such an increase in Ca2(+)-sensitivity to sub-micromolar Ca2+ is required to explain sub-micromolar oscillatory changes in cytosolic free Ca2+ by a Ca2(+)-dependent negative-feedback mechanism.     

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.     

Formation and metabolism of inositol 1,3,4,5-tetrakisphosphate in liver

The inositol lipid pools of isolated rat hepatocytes were labeled with [3H]myo-inositol, stimulated maximally with vasopressin and the relative contents of [3H]inositol phosphates were measured by high performance liquid chromatography. Inositol 1,4,5-trisphosphate accumulated rapidly (peak 20 s), while inositol 1,3,4-trisphosphate and a novel inositol phosphate (ascribed to inositol 1,3,4,5-tetrakisphosphate) accumulated at a slower rate over 2 min. Incubation of hepatocytes with 10 mM Li+ prior to vasopressin addition selectively augmented the levels of inositol monophosphate, inositol 1,4-bisphosphate, and inositol 1,3,4-trisphosphate. A kinase was partially purified from liver and brain cortex which catalyzed an ATP-dependent phosphorylation of [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. Incubation of purified [3H]inositol 1,3,4,5-tetrakisphosphate with diluted liver homogenate produced initially inositol 1,3,4-trisphosphate and subsequently inositol 1,3-bisphosphate, the formation of which could be inhibited by Li+. The data demonstrate that the most probable pathway for the formation of inositol 1,3,4,5-tetrakisphosphate is by 3-phosphorylation of inositol 1,4,5-trisphosphate by a soluble mammalian kinase. Degradation of both compounds occurs first by a Li+-insensitive 5-phosphatase and subsequently by a Li+-sensitive 4-phosphatase. The prolonged accumulation of both inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in vasopressin-stimulated hepatocytes suggest that they have separate second messenger roles, perhaps both relating to Ca2+-signalling events.     

Metabolism of inositol 1,3,4,5-tetrakisphosphate by human erythrocyte membranes. A new mechanism for the formation of inositol 1,4,5-trisphosphate.

Human erythrocyte membranes metabolize inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] to inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] in the presence of Mg2+. In the absence of Mg2+ a less rapid conversion of Ins(1,3,4,5)P4 into Ins(1,4,5)P3 was revealed. Such an enzyme activity, if present in hormonally sensitive cells, could provide a mechanism for maintaining constant concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, important for stimulation of Ca2+ entry after Ca2+ mobilization.     

Mass measurements of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in a neuronal cell line stimulated with bradykinin: inositolphosphate response shows desensitization.

In a neuronal cell line (108CC15, NG108-15) the levels of inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4), as measured by receptor binding assays, rise transiently after stimulation with bradykinin (EC50 approx. 150 nM). Maximal InsP3 level of 354 pmol/mg protein (15-fold basal level) is obtained at 10-15 s after addition of bradykinin, the InsP4 level rises maximally to 78 pmol/mg protein (14-fold basal level) at 20-30 s. In a rat glioma cell line, bradykinin (2 microM) causes a fast 6-fold increase in InsP3 and InsP4 levels. In the neuronal cells the bradykinin-dependent rise of the inositolphosphate levels is diminished with reduced extracellular Ca2+ concentration. However, depletion of internal Ca2+ stores does not affect the bradykinin-induced rise in InsP3 and InsP4 levels. Homologous desensitization to bradykinin occurs in the signal transduction pathway already at the production of inositolphosphates, since after a 2 min stimulation with bradykinin the rise in cellular masses of InsP3 and InsP4, inducible by a following second bradykinin stimulus, is substantially reduced.     

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.     

Elevated carbon dioxide blunts mammalian cAMP signaling dependent on inositol 1,4,5-triphosphate receptor-mediated Ca2+ release

Elevated CO(2) is generally detrimental to animal cells, suggesting an interaction with core processes in cell biology. We demonstrate that elevated CO(2) blunts G protein-activated cAMP signaling. The effect of CO(2) is independent of changes in intracellular and extracellular pH, independent of the mechanism used to activate the cAMP signaling pathway, and is independent of cell context. A combination of pharmacological and genetic tools demonstrated that the effect of elevated CO(2) on cAMP levels required the activity of the IP(3) receptor. Consistent with these findings, CO(2) caused an increase in steady state cytoplasmic Ca(2+) concentrations not observed in the absence of the IP(3) receptor or under nonspecific acidotic conditions. We examined the well characterized cAMP-dependent inhibition of the isoform 3 Na(+)/H(+) antiporter (NHE3) to demonstrate a functional relevance for CO(2)-mediated reductions in cellular cAMP. Consistent with the cellular biochemistry, elevated CO(2) abrogated the inhibitory effect of cAMP on NHE3 function via an IP(3) receptor-dependent mechanism.     

The size of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores depends on inositol 1,4,5-trisphosphate concentration.

An explanation of the complex effects of hormones on intracellular Ca2+ requires that the intracellular actions of Ins(1,4,5)P3 and the relationships between intracellular Ca2+ stores are fully understood. We have examined the kinetics of 45Ca2+ efflux from pre-loaded intracellular stores after stimulation with Ins(1,4,5)P3 or the stable phosphorothioate analogue, Ins(1,4,5)P3[S]3, by simultaneous addition of one of them with glucose/hexokinase to rapidly deplete the medium of ATP. Under these conditions, a maximal concentration of either Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 evoked rapid efflux of about half of the accumulated 45Ca2+, and thereafter the efflux was the same as occurred under control conditions. Submaximal concentrations of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 caused a smaller rapid initial efflux of 45Ca2+, after which the efflux was similar whatever the concentration of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 present. The failure of submaximal concentrations of Ins(1,4,5)P3 and Ins(1,4,5)P3[S]3 to mobilize fully the Ins(1,4,5)P3-sensitive Ca2+ stores despite prolonged incubation was not due either to inactivation of Ins(1,4,5)P3 or to desensitization of the Ins(1,4,5)P3 receptor. The results suggest that the size of the Ins(1,4,5)P3 sensitive Ca2+ stores depends upon the concentration of Ins(1,4,5)P3.     

Intracellular Ca2+ mobilization by arachidonic acid. Comparison with myo-inositol 1,4,5-trisphosphate in isolated pancreatic islets

Previous studies have demonstrated that myo-inositol 1,4,5-trisphosphate (IP3) mobilizes Ca2+ from the endoplasmic reticulum (ER) of digitonin-permeabilized islets and that an increase in intracellular free Ca2+ stimulates insulin release. Furthermore, glucose stimulates arachidonic acid metabolism in islets. In digitonin-permeabilized islets, exogenous arachidonic acid at concentrations between 1.25 to 10 microM elicited significant Ca2+ release from the ER at a free Ca2+ concentration of 0.1 microM. Arachidonic acid-induced Ca2+ release was not due to the metabolites of arachidonic acid. Arachidonic acid induced a rapid release of Ca2+ within 2 min. Comparison of arachidonic acid-induced Ca2+ release with IP3-induced Ca2+ release revealed a similar molar potency of arachidonic acid and IP3. The combination of both arachidonic acid and IP3 resulted in a greater effect on Ca2+ mobilization from the ER than either compound alone. The mass of endogenous arachidonic acid released by islets incubated with 28 mM glucose was measured by mass spectrometric methods and was found to be sufficient to achieve arachidonic acid concentrations equal to or exceeding those required to induce release of Ca2+ sequestered in the ER. These observations indicate that glucose-induced arachidonic acid release could participate in glucose-induced Ca2+ mobilization and insulin secretion by pancreatic islets, possibly in cooperation with IP3.