Inositol polyphosphates and intracellular calcium release

The hydrolysis of inositol lipids triggered by the occupation of cell surface receptors generates several intracellular messengers. Many different inositol phosphate isomers accumulate in stimulated cells. Of these D-myo-inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) is responsible for discharging Ca2+ from intracellular stores. Specific membrane binding sites for Ins 1,4,5-P3 have been detected. The properties of these sites and their possible relationship to the calcium release process is reviewed. Ins 1,4,5-P3 binding sites may be present in discrete subcellular structures ("calciosomes"). Kinetic and some electrophysiological evidence indicates that Ins 1,4,5-P3 acts to open a Ca2+ channel. Recent progress on the purification of the receptor from neuronal tissues is summarized. Phosphorylation of Ins 1,4,5-P3 by a specific kinase results in the production of D-myo-inositol 1,3,4,5-tetraphosphate (Ins 1,3,4,5-P4). This inositol phosphate has been reported to increase the entry of Ca2+ across the plasma membrane, activate nonspecific ion channels in the plasma membrane, alter the Ca2+ content of the Ins 1,4,5-P3-releasable store, and bind to and alter the activity of certain enzymes. These data and the possible biological significance of Ins 1,3,4,5-P4 are discussed.     

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.     

The effects of inositol trisphosphates and inositol tetrakisphosphate on Ca2+ release and Cl- current pattern in the Xenopus laevis oocyte.

We report that Ins(1,3,4,5)P4 releases calcium from intracellular stores of intact Xenopus laevis oocytes, as indicated by two different techniques, Ca2(+)-sensitive microelectrodes and a fura-2 imaging system. Ins(1,3,4,5)P4 releases only 20% as much Ca2+ as the same amount of Ins(1,4,5)P3. This effect is not due to the conversion of the injected Ins(1,3,4,5)P4 to Ins(1,4,5)P3, which is known to release Ca2+, because the amount of [3H]Ins(1,3,4,5)P4 that is converted to Ins(1,4,5)P3 is extremely small, as determined using HPLC. Examination of the different current patterns induced by Ins(1,4,5)P3 and Ins(1,3,4,5)P4, when injected into voltage-clamped oocytes, provided further evidence that the Ins(1,3,4,5)P4 was not being converted back to Ins(1,4,5)P3. We investigated the effects of four compounds, three inositol trisphosphates (Ins(1,4,5)P3, Ins(2,4,5)P3, and Ins(1,3,4)P3), and Ins(1,3,4,5)P4, on Cl- current conductance in order to examine (1) the possible role of Ins(1,3,4,5)P4 in cell activation and (2) the relationships between intracellular Ca2+ and the activation of Cl- currents. Immature stage VI Xenopus laevis oocytes were voltage-clamped and injected with Ins(1,4,5)P3, Ins(2,4,5)P3, and Ins(1,3,4)P3. Ins(1,4,5)P3 and Ins(2,4,5)P3 triggered Ca2(+)-dependent Cl- currents, but Ins(1,3,4)P3 did not trigger currents nor did it release intracellular Ca2+. Ins(2,4,5)P3 was fourfold less effective at inducing the immediate Cl- current pulse than Ins(1,4,5)P3. The Cl- current pattern was quite dependent on the amount of Ins(1,4,5)P3 injected into the oocyte. Low amounts of Ins(1,4,5)P3 triggered only an immediate single Cl- current pulse, whereas large amounts triggered the immediate single pulse, followed by a quiescent period, followed by oscillating Cl- currents. In contrast to the response of Ins(1,4,5)P3, injection of Ins(1,3,4,5)P4 triggered only oscillating Cl- currents whose magnitude, but not pattern, was dependent on the amount injected into the cell. The currents generated by Ins(1,3,4,5)P4 resemble the oscillating Cl- currents triggered by large amounts of Ins(1,4,5)P3 and Ins(2,4,5)P3. Ins(1,3,4,5)P4, unlike Ins(1,4,5)P3 and Ins(2,4,5)P3, rarely caused an immediate Cl- current pulse, but caused an immediate release of calcium. Therefore, we suggest that the oscillating currents are only indirectly dependent on calcium. These [Ca2+]i and conductance measurements suggest that both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 have roles in intracellular Ca2+ regulation.     

High-affinity inositol 1,3,4,5-tetrakisphosphate receptor from cerebellum: solubilization, partial purification and characterization

Proteins which bind with high affinity Ins 1,3,4,5-P4 or Ins 1,4,5-P3 were solubilized from porcine cerebellar membranes. Both binding activities were separated by heparin-agarose chromatography. The Ins 1,3,4,5-P4 receptor was partially purified with an approximately 1000-fold enrichment as compared to the membrane preparation. In the receptor-enriched preparation the Ins 1,3,4,5-P413 binding protein had an affinity (Kd) for Ins 1,3,4,5-P4 of 4.6 nM. Ins 1,3,4,5,6-P5 displaced [3H]Ins 1,3,4,5-P4 binding with a comparable affinity. The Ins 1,3,4,5-P4 binding protein displayed high selectivity for Ins 1,3,4,5-P4 over other inositol-phosphates (IC50 for Ins 1,4,5,6-P4 150 nM, for Ins-P6 1 microM and for Ins 1,3,4-P3 5 microM). Most importantly, Ins 1,4,5-P3 did not displace [3H]Ins 1,3,4,5-P4 binding at concentrations up to 10 microM. Binding of Ins 1,3,4,5-P4 was maximal in the pH range between 4.5 and 6, was stable with Ca2+ concentration varied from 1 nM to 1 mM, and was suppressed by heparin (IC50 about 2 nM). The high affinity receptor for Ins 1,3,4,5-P4 reported here, which is distinct from the Ins 1,4,5-P3 receptor might allow to evaluate the possible functional role of Ins 1,3,4,5-P4 in the cellular signal transduction.     

Inositol tetrakisphosphate-induced sequestration of Ca2+ replenishes an intracellular pool sensitive to inositol trisphosphate

In a permeable neoplastic rat liver epithelial (261B) cell system, inositol 1,3,4,5-tetrakisphosphate--Ins(1,3,4,5)P4--induces sequestration of Ca2+ released by inositol 2,4,5-trisphosphate--Ins(2,4,5)P3; a non-metabolized inositol trisphosphate (InsP3) isomer--and Ca2+ added exogenously in the form of CaCl2. Studies were performed to identify the Ca2+ pool filled after Ins(1,3,4,5)P4 treatment. Both Ins(2,4,5)P3 and inositol 1,4,5-trisphosphate--Ins(1,4,5)P3--dose-dependently release Ca2+ from permeable 261B cells--Ins(1,4,5)P3 having a threefold greater potency--but differ in that Ca2+ released by Ins(1,4,5)P3 is readily sequestered, while the Ca2+ released by Ins(2,4,5)P3 is not. Maximal release of Ca2+ by 6 microM Ins(2,4,5)P3 blocked the action of Ins(1,4,5)P3, demonstrating that these two isomers influence the same intracellular Ca2+ pool through a shared membrane receptor. Addition of 2 microM Ins(2,4,5)P3 to discharge partially the Ca2+ pool reduced the amount of Ca2+ released by a maximal dose of Ins(1,4,5)P3 (2 microM). Ins(1,3,4,5)P4 combined with Ins(2,4,5)P3 produced a Ca2+ release and sequestration response, which replenished the InsP3-sensitive pool as indicated by a recovery of full Ca2+ release by 2 microM Ins(1,4,5)P3. Induction of Ca2+ sequestration by Ins(1,3,4,5)P4 occurred dose-dependently, with a half-maximal response elicited at a dose of 0.9 microM. Further studies of the effect of Ins(1,3,4,5)P4 apart from the influence of Ins(2,4,5)P3 using a model in which the Ca2+ levels are raised by an exogenous addition of CaCl2 showed that Ins(1,4,5)P3 released twice the amount of Ca2+ from the storage pool following Ins(1,3,4,5)P4-induced Ca2+ sequestration. These results demonstrate that the Ca2+ uptake induced by Ins(1,3,4,5)P4 preferentially replenishes the intracellular Ca2+ storage sites regulated by Ins(1,4,5)P3 and Ins(2,4,5)P3.     

Stimulation of hepatic inositol 1,4,5-trisphosphate kinase activity by Ca2+-dependent and -independent mechanisms.

A cytosolic fraction derived from rat hepatocytes was used to investigate the regulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] kinase, the enzyme which converts Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. The activity was doubled by raising the free Ca2+ concentration of the assay medium from 0.1 microM to 1.0 microM. A 5 min preincubation of the hepatocytes with 100 microM-dibutyryl cyclic AMP (db.cAMP) plus 100 nM-tetradecanoylphorbol acetate (TPA) resulted in a 40% increase in Ins(1,4,5)P3 kinase activity when subsequently assayed at 0.1 microM-Ca2+. This effect was smaller at [Ca2+] greater than 0.5 microM, and absent at 1.0 microM-Ca2+. Similar results were obtained after preincubation with 100 microM-db.cAMP plus 300 nM-vasopressin (20% increase at 0.1 microM-Ca2+; no effect at 1.0 microM-Ca2+). Preincubation with vasopressin, db.cAMP or TPA alone did not alter Ins(1,4,5)P3 kinase activity. It is proposed that these results, together with recent evidence implicating Ins(1,3,4,5)P4 in the control of Ca2+ influx, could be relevant to earlier findings that hepatic Ca2+ uptake is synergistically stimulated by cyclic AMP analogues and vasopressin.     

Inositol 1,3,4,5-tetrakisphosphate stimulates calcium release from bovine adrenal microsomes by a mechanism independent of the inositol 1,4,5-trisphosphate receptor.

In bovine adrenal microsomes, Ins(1,4,5)P3 binds to a specific high-affinity receptor site (Kd = 11 nM) with low affinity for two other InsP3 isomers, Ins(1,3,4)P3 and Ins(2,4,5)P3. In the same subcellular fractions Ins(1,4,5)P3 was also the most potent stimulus of Ca2+ release of all the inositol phosphates tested. Of the many inositol phosphates recently identified in angiotensin-II-stimulated adrenal glomerulosa and other cells, Ins(1,3,4,5)P4 has been implicated as an additional second messenger that may act in conjunction with Ins(1,4,5)P3 to elicit Ca2+ mobilization. In the present study, an independent action of Ins(1,3,4,5)P4 was observed in bovine adrenal microsomes. Heparin, a sulphated polysaccharide which binds to Ins(1,4,5)P3 receptors in several tissues, inhibited both the binding of radiolabelled Ins(1,4,5)P3 and its Ca2(+)-releasing activity in adrenal microsomes. In contrast, heparin did not inhibit the mobilization of Ca2+ by Ins(1,3,4,5)P4, even at doses that abolished the Ins(1,4,5)P3 response. Such differential inhibition of the Ins(1,4,5)P3- and Ins(1,3,4,5)P4-induced Ca2+ responses by heparin indicates that Ins(1,3,4,5)P4 stimulates the release of Ca2+ from a discrete intracellular store, and exerts this action via a specific receptor site that is distinct from the Ins(1,4,5)P3 receptor.     

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.     

Inositol trisphosphate isomers, but not inositol 1,3,4,5-tetrakisphosphate, induce calcium influx in Xenopus laevis oocytes

To investigate the mechanisms by which inositol phosphates regulate cytosolic free Ca2+ concentration ([Ca2+]c), we injected Xenopus oocytes with inositol phosphates and measured Ca2+-activated Cl- currents as an assay of [Ca2+]c. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) injection (0.1-10.0 pmol) induced an initial transient Cl- current (I1) followed by a second more prolonged Cl- current (I2). Both currents were Ca2+-dependent, but the source of Ca2+ was different. Release of intracellular Ca2+ stores produced I1, whereas influx of extracellular Ca2+ produced I2; Ca2+-free bathing media and inorganic calcium channel blockers (Mn2+, Co2+) did not alter I1 but completely and reversibly inhibited I2. Injection of the Ins(1,4,5)P3 metabolite, inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (0.2-10.0 pmol) generated a Ca2+-dependent Cl- current with superimposed current oscillations that resulted from release of intracellular Ca2+, not Ca2+ influx. Injection of the Ins(1,3,4,5)P4 metabolite, inositol 1,3,4-trisphosphate (10.0 pmol), or the synthetic inositol trisphosphate isomer, inositol 2,4,5-trisphosphate (1.0-10.0 pmol), mimicked the effect of Ins(1,4,5)P3, stimulating an I1 resulting from release of intracellular Ca2+ and an I2 resulting from influx of extracellular Ca2+. The results indicate that several inositol trisphosphate isomers stimulate both release of intracellular Ca2+ and influx of extracellular Ca2+. Ins(1,3,4,5)P4 also stimulated release of intracellular Ca2+, but it was neither sufficient nor required for Ca2+ influx.     

Inositol 1,3,4,5-tetrakisphosphate-induced release of intracellular Ca2+ in SH-SY5Y neuroblastoma cells.

Inositol-polyphosphate-induced Ca2+ mobilization was investigated in saponin-permeabilized SH-SY5Y human neuroblastoma cells. Ins(1,4,5)P3 induced a dose-related release from intracellular Ca2+ stores with an EC50 (concn. giving half-maximal effect) of 0.1 microM and a maximal release of 70%. Ins(1,3,4)P3, DL-Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5 did not evoke Ca2+ mobilization in these cells when used at concentrations up to 10 microM. However, Ins(1,3,4,5)P4 was found to release Ca2+ in a dose-related manner, but the response was dependent on the source of Ins(1,3,4,5)P4 used. When commercially available D-Ins(1,3,4,5)P4 was used, the EC50 and maximal response values were 1 microM and 50% respectively, compared with values for chemically synthesized DL-Ins(1,3,4,5)P4 of 2 microM and 25%. The enhanced maximal response of commercial D-Ins(1,3,4,5)P4 was decreased by pretreatment with rat brain crude Ins(1,4,5)P3 3-kinase and was therefore concluded to be indicative of initial Ins(1,4,5)P3 contamination of the Ins(1,3,4,5)P4 preparation. When metabolism of DL-Ins(1,3,4,5)P4 (10 microM) in these cells at 25 degrees C was investigated by h.p.l.c., substantial amounts of Ins(1,4,5)P3 (0.2 microM) and Ins(1,3,4)P3 (0.8 microM) were found to be produced within 3 min. Analysis of DL-Ins(1,3,4,5)P4 incubation with cells at 4 degrees C, however, indicated that metabolism had been arrested ([3H]Ins(1,4,5)P3 detection limits were estimated to be approx. 0.01 microM). When chemically synthesized DL-Ins(1,3,4,5)P4 and incubation conditions of low temperature were used, the Ca2(+)-releasing properties of this compound were established to be 1 microM and 19% for the EC50 and maximal response values respectively. The results obtained strongly suggest that Ins(1,3,4,5)P4 alone has the ability to release intracellular Ca2+. However, in the presence of sub-maximal concentrations of Ins(1,4,5)P3, Ca2+ release appears to be synergistic with Ins(1,3,4,5)P4, but at supramaximal concentrations not even additive effects are observed.     

Potassium channels regulated by inositol 1,3,4,5-tetrakisphosphate and internal calcium in DDT1 MF-2 smooth muscle cells

This study was carried out to determine the intracellular components responsible for the transmembrane current evoked by stimulation of H1-histaminergic receptors in DDT1 MF-2 smooth muscle cells. Histamine elicited an outward current that was reversed below the K+ equilibrium potential and passed voltage-independent K+ channels. A histamine concentration-dependent rise in outward current and in cytoplasmic-free Ca2+ with similar time courses was observed. The histamine-induced current was not found after depletion of internal Ca2+ stores, suggesting a coupling between internal Ca2+ and K+ current. The time course of the initial increase in inositol (1,4,5)-trisphosphate (Ins (1,4,5)P3) caused by histamine differs from that of the internal Ca2+ response. However, a significant concentration-dependent increase in inositol (1,3,4,5)-tetrakisphosphate (Ins (1,3,4,5)P4) was seen during the whole stimulating period. The role of internal Ca2+, Ins (1,4,5)P3, and Ins (1,3,4,5)P4 on the outward current was also examined by the addition of these substances directly to the cytoplasm. Internal application of Ca2+ increased the amplitude and duration of the histamine-induced current whereas internal EGTA suppressed the outward current. Internal Ins (1,4,5)P3 did not affect the histamine-induced K+ current, Ins (1,3,4,5)P4 inhibited the outward current, and the combination of Ins (1,3,4,5)P4 and Ca2+ abolished this response. The noradrenaline response evoked under normal conditions is not reflected by a change in transmembrane current or a change in Ins (1,3,4,5)P4 but is associated with an increase in Ins (1,4,5)P3 and internal Ca2+. Stimulation of alpha 1-adrenoceptors, however, also evoked an outward current after the addition of Ins (1,3,4,5)P4 intracellularly. It is concluded that K+ channels, carrying the histamine outward current, are activated from the combined action of internal Ca2+ and Ins (1,3,4,5)P4.     

Recovery of hepatocytes from attack by the pore former amphotericin B.

Factors underlying the transience of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] accumulation following muscarinic stimulation of RINm5F cells were examined. Transience was not due to a protein kinase C-mediated stimulation of Ins(1,4,5)P3 dephosphorylation, since pretreatment of cells with tetradecanoyl-phorbol acetate (TPA) did not alter the rate of this conversion. However, preincubation with TPA did inhibit carbamoylcholine-stimulated Ins(1,4,5)P3 formation. In permeabilized cells, the conversion of Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] was slightly enhanced in the presence of TPA or cyclic AMP, but much more markedly by raising the Ca2+ concentration from 10(-7) M to 10(-6) or 10(-5) M. In intact cells the most rapid rate of accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 occurred in the first 2 s following stimulation, whereas the levels of inositol 1,4-bisphosphate were not increased until after 5 s. This suggests that Ins(1,4,5)P3 kinase is chiefly responsible for the early disposal of Ins(1,4,5)P3 following cellular stimulation. The results are consistent with the proposal that the transient accumulation of Ins(1,4,5)P3 is due both to its enhanced metabolism via the Ca2+-calmodulin-sensitive Ins(1,4,5)P3 kinase, as well as a down-regulation of phosphatidylinositol 4,5-bisphosphate hydrolysis.     

Production of inositol trisphosphates and inositol tetrakisphosphate in stimulated pancreatic islets

Glucose and carbamylcholine caused concentration-dependent increases in the production of total [3H]inositol phosphates in [3H]inositol-labelled rat pancreatic islets. When extracts from islets stimulated with glucose, carbamylcholine or depolarising concentrations of K+ were analysed using anion-exchange high performance liquid chromatography, increased production of [3H]Ins1,4,5-P3 was detected, and in addition, elevated levels of two other labelled compounds which co-chromatographed with Ins1,3,4-P3 and Ins1,3,4,5-P4. In the case of carbamylcholine and high K+, such an effect was apparent within 20 s, whereas glucose appeared to cause a delayed response. In the presence of 5 mM LiCl, the accumulation of Ins1,3,4-P3 was more marked. The presence of LiCl had no major influence on the levels of Ins1,4,5-P3 or Ins1,3,4,5-P4. It is suggested that the stimulation of pancreatic islets with glucose, carbamylcholine or high K+ results in the hydrolysis of inositol lipids with the production of Ins1,4,5-P3 and in addition, Ins1,3,4-P3 and Ins1,3,4,5-P4. The physiological functions of these novel inositol phosphates in islets remain to be established.     

Inositol 1,4,5-trisphosphate concentrations increase after adherence in the macrophage-like cell line J774.1.

Several properties of macrophages change when suspended cells become adherent. To determine the intracellular signals involved in these changes, concentrations of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] were monitored during adherence of J774.1 cells, a macrophage-like cell line. When cells grown in suspension were allowed to adhere to a glass surface, there was a transient increase in InsP3 that reached a peak between 100 and 120 s after plating. Inositol mono- and bis-phosphate concentrations were also elevated 100 and 120 s after plating. Analysis of isomer distribution showed significant 3-fold increases in Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] at 100 s after plating. These values were maintained at 120 s, with the additional appearance of a 4-fold increase in inositol 1,3,4-trisphosphate. The adherence-induced generation of Ins(1,4,5)P3 was decreased, and Ins(1,3,4,5)P4 formation was blocked, in Ca2+-free medium. However, doubling intracellular [Ca2+] by addition of the Ca2+ ionophore ionomycin (1 microM) did not increase Ins(1,4,5)P3 in suspended cells. Adherence of J774.1 cells to fibronectin-coated glass also induced an increase in InsP3.     

Does inositol tetrakisphosphate play a role in the receptor-mediated control of calcium mobilization?

The evidence for and against an important role for inositol 1,3,4,5 tetrakisphosphate (Ins 1,3,4,5 P4) in receptor-mediated Ca2+ mobilization is reviewed. Data obtained from patch-clamp whole-cell current recording studies on internally perfused exocrine acinar cells show that the acetylcholine (ACh)-evoked sustained increase in Ca2+-dependent K+ current caused by an increase in [Ca2+]i cannot be mimicked by internal application of inositol 1,4,5-trisphosphate (Ins 1,4,5 P3), but only by a combination of Ins 1,4,5 P3 and Ins 1,3,4,5 P4. The sustained response evoked by Ins 1,4,5 P3 + Ins 1,3,4,5 P4 is dependent on the presence of external Ca2+ as is the effect of ACh. Only those inositol trisphosphates able to evoke Ca2+ release from internal stores can support the action of Ins 1,3,4,5 P4 in evoking responses that are acutely dependent on extracellular Ca2+ (Ca2+ influx). The various arguments presented against an involvement of Ins 1,3,4,5 P4 are discussed. The main point emerging is that most studies are inadequately controlled and it is concluded that there is a strong need for whole-cell current recording studies combined with pipette fluid exchange to be carried out in many more systems. The major problem in this field is that the precise site and mechanism of action of Ins 1,3,4,5 P4 are unknown and that the pathway for Ca2+ uptake during receptor activation is inadequately defined.     

Enhancement of the inositol 1,4,5-trisphosphate-releasable Ca2+ pool by GTP in permeabilized hepatocytes

Permeabilized rat hepatocytes were used to study the effects of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and GTP on Ca2+ uptake and release by ATP-dependent intracellular Ca2+ storage pools. Under conditions where these Ca2+ pools were completely filled, maximal doses of Ins(1,4,5)P3 released only 25-30% of the sequestered Ca2+. The residual Ca2+ was freely releasable with the Ca2+ ionophore ionomycin. Addition of GTP in the absence of Ins(1,4,5)P3 did not cause Ca2+ release and had no effect on the steady-state level of Ca2+ accumulation by intracellular storage pools. However, after a 3-4-min treatment with GTP the size of the Ins(1,4,5)P3-releasable Ca2+ pool was increased by about 2-fold, with a proportional decrease in the residual Ca2+ available for release by ionomycin. In contrast to the situation with freshly permeabilized cells, permeabilized hepatocytes from which cytosolic components had been washed out exhibited direct Ca2+ release in response to GTP addition. The potentiation of Ins(1,4,5)P3-induced Ca2+ release by GTP in permeabilized hepatocytes was concentration-dependent with half-maximal effects at about 5 microM GTP. The dose response to Ins(1,4,5)P3 was not shifted by GTP; instead GTP increased the amount of Ca2+ released at all Ins(1,4,5)P3 concentrations. The effects of GTP were not mimicked by other nucleotides or nonhydrolyzable GTP analogues. In fact, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) inhibited the actions of GTP. However, this inhibition only occurred when GTP gamma S was added prior to GTP, suggesting that the GTP effect is not readily reversible once the cells have been permeabilized. Experiments using vanadate to inhibit the ATP-dependent Ca2+ uptake pump showed that Ins(1,4,5)P3 releases all of the Ca2+ within the Ins(1,4,5)P3-sensitive Ca2+ pool even in the absence of GTP. The increase of Ins(1,4,5)P3-induced Ca2+ release brought about by GTP was also unaffected by vanadate. It is concluded that GTP increases the proportion of the sequestered Ca2+ which is available for release by Ins(1,4,5)P3, either by unmasking latent Ins(1,4,5)P3-sensitive Ca2+ release sites or by allowing direct Ca2+ movement between Ins(1,4,5)P3-sensitive and Ins(1,4,5)P3-insensitive Ca2+ storage pools.     

Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin

Inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) is an important second-messenger molecule that mobilizes Ca2+ from intracellular stores in response to the occupancy of receptor by various Ca2+-mobilizing agonists. The fate of Ins-1,4,5-P3 is determined by two enzymes, a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins-1,4,5-P3 to Ins-1,3,4,5-P4, whereas the latter forms Ins-1,4-P2. Recent studies suggest that Ins-1,3,4,5-P4 might modulate the entry of Ca2+ from an extracellular source. In the current report, we describe the partial purification of the 3-kinase [approximately 400-fold purified, specific activity = 0.12 mumol/(min.mg)] from the cytosolic fraction of bovine brain and studies of its catalytic properties. We found that the 3-kinase activity is significantly activated by the Ca2+/calmodulin complex. Therefore, we propose that Ca2+ mobilized from endoplasmic reticulum by the action of Ins-1,4,5-P3 forms a complex with calmodulin, and that the Ca2+/calmodulin complex stimulates the conversion of Ins-1,4,5-P3, an intracellular Ca2+ mobilizer, to Ins-1,3,4,5-P4, an extracellular Ca2+ mobilizer. A rapid assay method for the 3-kinase was developed that is based on the separation of [3-32P]Ins-1,3,4,5-P4 and [gamma-32P]ATP by thin-layer chromatography. Using this new assay method, we evaluated kinetic parameters (Km for ATP = 40 microM, Km for Ins-1,4,5-P3 = 0.7 microM, Ki for ADP = 12 microM) and divalent cation specificity (Mg2+ much greater than Mn2+ greater than Ca2+) for the 3-kinase. Studies with various inositol polyphosphates indicate that the substrate-binding site is quite specific to Ins-1,4,5-P3. Nevertheless, Ins-2,4,5-P3 could be phosphorylated at a velocity approximately 1/20-1/30 that of Ins-1,4,5-P3.     

Ca2+-mediated generation of inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakisphosphate in pancreatic islets. Studies with K+, glucose, and carbamylcholine

The role of Ca2+ in the generation of inositol phosphates was investigated using rat pancreatic islets after steady state labeling with myo-[2-3H]inositol. Depolarizing K+ concentrations (24 mM) evoked early (2 s) increases in inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) as measured by high performance anion-exchange chromatography. The increase in Ins-1,4,5-P3 was transient and was followed by a more pronounced rise in Ins-1,3,4-P3. These effects were dependent on the presence of extracellular Ca2+ but were not secondary to release of either neurotransmitters or metabolites of arachidonic acid. K+ also promoted the breakdown of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) and of the other phosphoinositides. Glucose (16.7 mM) was less marked in its effects but still promoted rapid increases in Ins-1,3,4,5-P4 (2 s) and Ins-1,4,5-P3 (10 s) and a slower rise in Ins-1,3,4-P3 (30 s). The levels of all three metabolites rose steadily over 10 min stimulation. These responses to glucose could be largely, although not entirely, inhibited by depletion of extracellular Ca2+ or by Ca2+ channel blockade with verapamil (20 microM). Carbamylcholine (0.5 mM) was the most potent stimulus used evoking early rises in Ins-1,4,5-P3 and Ins-1,3,4,5-P4 (2 s) followed by Ins-1,3,4-P3 (10 s), effects which were only partially dependent on extracellular Ca2+. The results suggest that a Ca2+-mediated PtdIns-4,5-P2 hydrolysis accounts for most of the Ins-1,4,5-P3 generated in response to glucose but not carbamylcholine. In addition, glucose may exert effects on inositol phosphate metabolism which are Ca2+ independent.     

Agonist-induced calcium signaling is impaired in fibroblasts overproducing inositol 1,3,4,5-tetrakisphosphate.

The proposed Ca(2+)-signaling actions of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), formed by phosphorylation of the primary Ca(2+)-mobilizing messenger, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), were analyzed in NIH 3T3 and CCL39 fibroblasts transfected with rat brain Ins(1,4,5)P3 3-kinase. In such kinase-transfected cells, the conversion of Ins(1,4,5)P3 to Ins(1,3,4,5)P4 during agonist stimulation was greatly increased, with a concomitant reduction in Ins(1,4,5)P3 levels and attenuation of both the cytoplasmic Ca2+ increase and the Ca2+ influx response. This reduction in Ca2+ signaling was observed during activation of receptors coupled to guanine nucleotide-binding proteins (thrombin and bradykinin), as well as with those possessing tyrosine kinase activity. Single-cell Ca2+ measurements in CCL39 cells revealed that the smaller averaged Ca2+ response of enzyme-transfected cells was due to a marked increase in the number of cells expressing small and slow Ca2+ increases, in contrast to the predominantly large and rapid Ca2+ responses of vector-transfected controls. There was no evidence that high Ins(1,3,4,5)P4 levels promote Ca2+ mobilization, Ca2+ entry, or Ca2+ sequestration. These data indicate that Ins(1,4,5)P3 is the major determinant of the agonist-induced Ca2+ signal in fibroblasts and that Ins(1,3,4,5)P4 does not appear to contribute significantly to this process. Instead, Ins(1,4,5)P3 3-kinase may serve as a negative regulator of the Ca(2+)-phosphoinositide signal transduction mechanism.     

Thrombin and phorbol ester stimulate inositol 1,3,4,5-tetrakisphosphate 3-phosphomonoesterase in human platelets

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which mobilizes intracellular Ca2+, is metabolized either by dephosphorylation to inositol 1,4-bisphosphate(Ins-(1,4)P2) or by phosphorylation to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). It has been shown in vitro that Ins(1,3,4,5)P4 is also dephosphorylated by a 5-phosphomonoesterase to inositol 1,3,4-trisphosphate. However, we have found that exogenous Ins(1,3,4,5)P4 is dephosphorylated to predominantly Ins(1,4,5)P3 in saponin-permeabilized platelets in the presence of KCl (40-160 mM). This inositol polyphosphate 3-phosphomonoesterase activity is independent of Ca2+ (0.1-100 microM), and it was also observed when the ionic strength of the incubation medium was increased with Na+. The action of KCl appears to be due to activation of a 3-phosphomonoesterase as well as an inhibition of the 5-phosphomonoesterase, because the dephosphorylation of Ins(1,4,5)P3 to Ins(1,4)P2 was completely inhibited by KCl. The 3-phosphomonoesterase may be regulated by a protein kinase C, since both thrombin and phorbol dibutyrate increase 3-phosphomonoesterase activity and this is inhibited by staurosporine. The formation of Ins(1,4,5)P3 from Ins(1,3,4,5)P4 reported here provides an additional pathway for the formation of the Ca2+-mobilizing second messenger in stimulated cells.