Comparative studies of D-myoinositol 1,4,5-trisphosphate and synthetic 6-deoxy D-myoinositol 1,4,5-trisphosphate: binding and calcium release activity in rat parotid microsomes.

In this study, we report our data on the binding of D-myoinositol(1,4,5)P3 and of 6-deoxy D-myoinositol(1,4,5)P3 to a rat parotid microsomal fraction and their effect on Ca2+ release. The binding affinity and the potency of 6-deoxy Ins(1,4,5)P3 to induce Ca2+ release are about 100 times lower than those of Ins(1,4,5)P3. However, maximal concentrations of both inositol trisphosphates induce similar calcium efflux and present comparable displacement of radioligand binding. Experiments were performed to exclude that the microsomal preparations used display rapid metabolism of Ins(1,4,5)P3 or 6-deoxy Ins(1,4,5)P3 during binding and Ca2+ release. We also report that, in permeabilized rat parotid acini preparations, 6-deoxy Ins(1,4,5)P3 is about 100 times less potent than Ins(1,4,5)P3 in inducing Ca2+ release. These data indicate that removal of the hydroxyl group in position 6 of the Ins(1,4,5)P3 molecule severely reduces its binding affinity which seems, in a large part at least, responsible for the reported loss of potency in mobilizing Ca2+. Nevertheless, 6-deoxy Ins(1,4,5)P3 seems to be a full agonist for the release of Ca2+.     

Ca2(+)-mobilising properties of synthetic fluoro-analogues of myo-inositol 1,4,5-trisphosphate and their interaction with myo-inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase

The ability of two fluoro-analogues of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) to mobilize intracellular Ca2+ stores in SH-SY5Y neuroblastoma cells has been investigated. DL-2-deoxy-2-fluoro-scyllo-Ins(1,4,5)P3 (2F-Ins(1,4,5)P3) and DL-2,2-difluoro-2-deoxy-myo-Ins(1,4,5)P3 (2,2-F2-Ins(1,4,5)P3) were full agonists (EC50s 0.77 and 0.41 microM respectively) and slightly less potent than D-Ins(1,4,5)P3 (EC50 0.13 microM), indicating that the axial 2-hydroxyl group of Ins(1,4,5)P3 is relatively unimportant in receptor binding and stimulation of Ca2+ release. Both analogues mobilized Ca2+ with broadly similar kinetics and were substrates for Ins(1,4,5)P3 3-kinase but, qualitatively, were slightly poorer than Ins(1,4,5)P3. 2F-Ins(1,4,5)P3 was a weak substrate for Ins(1,4,5)P3 5-phosphatase but 2,2-F2-Ins(1,4,5)P3 was apparently not hydrolysed by this enzyme, although it inhibited its activity potently (Ki = 26 microM).     

Ins(1,4,5)P3 metabolism and the family of IP3-3Kinases

The release of Ca2+ from intracellular stores is triggered by the second messenger inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3). The regulation of this process is critically important for cellular homeostasis. Ins(1,4,5)P3 is rapidly metabolised, either to inositol (1,4)-bisphosphate (Ins(1,4)P2) by inositol polyphosphate 5-phosphatases or to inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) by one of a family of inositol (1,4,5)P3 3-kinases (IP3-3Ks). Three isoforms of IP3-3K have now been identified in mammals; they have a conserved C-terminal catalytic domain, but divergent N-termini. This review discusses the metabolism of Ins(1,4,5)P3, compares the IP3-3K isoforms and addresses potential mechanisms by which their activity might be regulated.     

A salt-activated inositol 1,3,4,5-tetrakisphosphate 3-phosphatase at the inner surface of the human erythrocyte membrane.

The localization of the human erythrocyte membrane Ins(1,3,4,5)P4 3-phosphatase was investigated by saponin permeabilization of resealed 'isoionic' erythrocyte ghosts. This enzyme is active at the inner face of the plasma membrane, at the same site as a specific 5-phosphatase that degrades both Ins (1,4,5)P3 and Ins(1,3,4,5)P4. In the presence of EDTA, Ins(1,4,5)P3 was the only product of Ins(1,3,4,5)P4 metabolism. However, when Mg2+ was present both the 5-phosphatase and the 3-phosphatase attacked Ins (1,3,4,5)P4, directly forming Ins(1,3,4)P3 and Ins(1,4,5)P3;some Ins(1,4)P2 was also formed as a product of 5-phosphatase attack on the liberated Ins(1,4,5)P3. The Ins(1,3,4,5)P4 3-phosphatase was potently activated by KCl, thus making the route of metabolism of Ins(1,3,4,5)P4 by erythrocyte ghosts strikingly sensitive to variations in ionic strength: at 'cytosolic' K+ and Mg2+ levels, 3-phosphatase activity slightly predominated over 5-phosphatase. Ins(1,3,4,5)P4 3-phosphatase was potently inhibited by Ins-(1,3,4,5,6)P5 and InsP6 at levels lower than those often observed within cells. This leaves open the question as to whether the cellular function of inositol polyphosphate 3-phosphatase is to participate in a physiological cycle that interconverts Ins(1,3,4,5)P4 and Ins(1,4,5)P3 or to metabolize other inositol polyphosphates in the cytosol compartment of cells.     

Inositol 1,4,5 trisphosphate is inactivated by a 5-phosphatase in stamen hair cells of Tradescantia

Inositol 1,4,5 trisphosphate [Ins(1,4,5)P3] is produced from the hydrolysis of phosphatidylinositol 4,5 bisphosphate, and as part of a second-messenger signal transduction mechanism, induces release of Ca2+ from internal stores in both plant and animal systems. It is less well established how the active Ins(1,4,5)P3 is inactivated. Studies in animal cells have demonstrated two separate metabolic pathways. Ins(1,4,5)P3 can be hydrolyzed by a 5-phosphatase or phosphorylated by a 3-kinase, resulting in the formation of Ins(1,4)P2 and Ins(1,3,4,5)P4, respectively, neither of which is able to mobilize intracellular Ca2+. Plant cell extracts have been reported to have hydrolytic and kinase activities that produce Ins(1,4)P2, and Ins(4,5)P2 and Ins(1,4,5,6)P4 from Ins(1,4,5)P3. These results offer little insight into the enzyme activities in the intact plant cell since the observed activities might be confined to intracellular compartments that have little if any impact on the signaling events within the cytosol that require Ins(1,4,5)P3. To resolve the mechanism of Ins(1,4,5)P3 inactivation, we microinjected stamen hair cells of Tradescantia virginiana L. with nonhydrolysable analogs of Ins(1,4,5)P3 that have been previously shown to cause Ca2+ release from intracellular stores. Our results indicate a sustained cytosolic [Ca2+] increase when cells were injected with the 5-phosphatase-insensitive 5-monophosphorothioate derivative of Ins(1,4,5)P3, in contrast to a brief transient when injected with the 3-kinase-insensitive 3-fluoro-3-deoxy Ins(1,4,5)P3 analog. We conclude that the 5-phosphatase pathway is the preferred pathway for Ins(1,4,5)P3 inactivation in the stamen hair cells of Tradescantia.     

Use of phosphorofluoridate analogues of D-myo-inositol 1,4,5-trisphosphate to assess the involvement of ionic interactions in its recognition by the receptor and metabolising enzymes

D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] analogues fluoridated at 4- or 5-phosphate or both were analysed to assess the involvement of ionic interactions between the phosphates of Ins(1,4,5)P3 and the proteins that recognize it, such as metabolic enzymes and the InsP3 receptor. These analogues were effective in inhibiting type I Ins(1,4,5)P3 5-phosphatase activity with much the same potency as Ins(1,4,5)P3, although the enzyme showed a lower Km value as pH values increased. In contrast, the analogues were less potent ligands than Ins(1,4,5)P3 in both the assay of [3H]Ins(1,4,5)P3 binding to the receptors and the phosphorylation of [3H]Ins(1,4,5)P3 catalysed by Ins(1,4,5)P3 3-kinase. These results suggest that ionic interactions with the dianionic 4- and 5-phosphates of Ins(1,4,5)P3 are involved in recognition by the receptor and the kinase, but not by the phosphatase.     

Regulation of inositol 1,4,5-trisphosphate 3-kinases by calcium and localization in cells

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) 3-kinases (IP(3)Ks) are a group of calmodulin-regulated inositol polyphosphate kinases (IPKs) that convert the second messenger Ins(1,4,5)P(3) into inositol 1,3,4,5-tetrakisphosphate. However, what they contribute to the complexities of Ca(2+) signaling, and how, is still not fully understood. In this study, we have used a simple Ca(2+) imaging assay to compare the abilities of various Ins (1,4,5)P(3)-metabolizing enzymes to regulate a maximal histamine-stimulated Ca(2+) signal in HeLa cells. Using transient transfection, we overexpressed green fluorescent protein-tagged versions of all three mammalian IP(3)K isoforms, including mutants with disrupted cellular localization or calmodulin regulation, and then imaged the Ca(2+) release stimulated by 100 microm histamine. Both localization to the F-actin cytoskeleton and calmodulin regulation enhance the efficiency of mammalian IP(3)Ks to dampen the Ins (1,4,5)P(3)-mediated Ca(2+) signals. We also compared the effects of the these IP(3)Ks with other enzymes that metabolize Ins(1,4,5)P(3), including the Type I Ins(1,4,5)P(3) 5-phosphatase, in both membrane-targeted and soluble forms, the human inositol polyphosphate multikinase, and the two isoforms of IP(3)K found in Drosophila. All reduce the Ca(2+) signal but to varying degrees. We demonstrate that the activity of only one of two IP(3)K isoforms from Drosophila is positively regulated by calmodulin and that neither isoform associates with the cytoskeleton. Together the data suggest that IP(3)Ks evolved to regulate kinetic and spatial aspects of Ins (1,4,5)P(3) signals in increasingly complex ways in vertebrates, consistent with their probable roles in the regulation of higher brain and immune function.     

Depolarization and Agonist-Stimulated Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulation in Rat Cerebral Cortex

Muscarinic receptor stimulation or depolarization with elevated extracellular K+ induced rapid and sustained increases in mass accumulations of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] in cerebral cortex slices. Synergistic but transient responses of both inositol polyphosphate second messengers were observed when slices were stimulated with carbachol under depolarizing conditions; this synergy was observed as an increase in the maximal responsiveness, with no significant change in EC50 values for carbachol. Omission of buffer Ca2+ ([Ca2+]e 10-20 microM) reduced basal Ins(1,4,5)P3 and Ins(1,3,4,5)P4 concentrations; the relative stimulatory effects of muscarinic receptor stimulation were maintained, but the effects of depolarization were markedly attenuated under these conditions. A component of the response to depolarization appeared to be indirectly mediated by the release of acetylcholine, because the K(+)-evoked increase in Ins(1,3,4,5)P4 was enhanced by the cholinesterase inhibitor physostigmine, and was partially attenuated by atropine. An additive suppression by nitrendipine suggests that entry of Ca2+ through L-type Ca2+ channels may serve to accelerate phosphorylation of Ins(1,4,5)P3 by 3-kinase. Norepinephrine did not significantly increase Ins(1,4,5)P3 or Ins(1,3,4,5)P4 accumulation; however, in the presence of depolarizing K+, norepinephrine caused a dramatic increase in Ins(1,3,4,5)P4 mass accumulation. In contrast, the excitatory amino acid quisqualate caused significant increases in the mass accumulations of both inositol polyphosphates measured, with no further increase being observed under depolarizing conditions. The results are discussed with respect to the interactive effects of agonist and depolarization stimuli on inositol polyphosphate accumulation which might more accurately reflect the conditions pertaining in vivo.     

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.     

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.     

Interaction of synthetic D-6-deoxy-myo-inositol 1,4,5-trisphosphate with the Ca2(+)-releasing D-myo-inositol 1,4,5-trisphosphate receptor, and the metabolic enzymes 5-phosphatase and 3-kinase.

The ability of D-6-deoxy-myo-inositol 1,4,5-trisphosphate [6-deoxy-Ins(1,4,5)P3], a synthetic analogue of the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], to mobilise intracellular Ca2+ stores in permeabilised SH-SY5Y neuroblastoma cells was investigated. 6-Deoxy-Ins(1,4,5)P3 was a full agonist (EC50 = 6.4 microM), but was some 70-fold less potent than Ins (1,4,5)P3 (EC50 = 0.09 microM), indicating that the 6-hydroxyl group of Ins(1,4,5)P3 is important for receptor binding and stimulation of Ca2+ release, but is not an essential structural feature. 6-Deoxy-Ins(1,4,5)P3 was not a substrate for Ins (1,4,5)P3 5-phosphatase, but inhibited both the hydrolysis of 5-[32P]+ Ins (1,4,5)P3 (Ki 76 microM) and the phosphorylation of [3H]Ins(1,4,5)P3 (apparent Ki 5.7 microM). 6-Deoxy-Ins (1,4,5)P3 mobilized Ca2+ with different kinetics to Ins(1,4,5)P3, indicating that it is probably a substrate for Ins (1,4,5)P3 3-kinase.     

Role of PRIP-1, a novel Ins(1,4,5)P3 binding protein, in Ins(1,4,5)P3-mediated Ca2+ signaling

PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein with a domain organization similar to phospholipase C-delta1 (PLC-delta1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P3. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P3-mediated Ca2+ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1-/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca2+ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1-/- mice than in those from wild type mice. The relative amounts of [3H]Ins(1,4,5)P3 measured in neurons labeled with [3H]inositol was also lower in cells from PRIP-1-/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,5)P3 is enhanced in cells from PRIP-1-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P3 to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P3-mediated Ca2+ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P3.     

Inositol polyphosphate 5-phosphatase-controlled Ins(1,4,5)P3/Ca2+ is crucial for maintaining pollen dormancy and regulating early germination of pollen

Appropriate pollen germination is crucial for plant reproduction. Previous studies have revealed the importance of dehydration in maintaining pollen dormancy; here, we show that phosphatidylinositol pathway-controlled Ins(1,4,5)P(3)/Ca(2+) levels are crucial for maintaining pollen dormancy in Arabidopsis thaliana. An interesting phenotype, precocious pollen germination within anthers, results from a disruption of inositol polyphosphate 5-phosphatase 12 (5PT12). The knockout mutant 5pt12 has normal early pollen development and pollen dehydration, and exhibits hypersensitive ABA responses, indicating that precocious pollen germination is not caused either by abnormal dehydration or by suppressed ABA signaling. Deficiency of 5PT13 (a close paralog of 5PT12) synergistically enhances precocious pollen germination. Both basal Ins(1,4,5)P(3) levels and endogenous Ca(2+) levels are elevated in pollen from 5pt12 mutants, and 5pt12 5pt13 double mutants show an even higher precocious germination rate along with much higher levels of Ins(1,4,5)P(3)/Ca(2+). Strikingly, exogenous Ca(2+) stimulates the germination of wild-type pollen at floral stage 12, even in very low humidity, both in vitro and in vivo, and treatment with BAPTA, a [Ca(2+)](cyt) inhibitor, reduces the precocious pollen germination rates of 5pt12, 5pt13 and 5pt12 5pt13 mutants. These results indicate that the increase in the levels of Ins(1,4,5)P(3)/Ca(2+) caused by deficiency of inositol polyphosphate 5-phosphatases is sufficient to break pollen dormancy and to trigger early germination. The study reveals that independent of dehydration, the control of Ins(1,4,5)P(3)/Ca(2+) levels by Inositol polyphosphate 5-phosphatases is crucial for maintaining pollen dormancy.     

Inositol trisphosphate analogues induce different oscillatory patterns in Xenopus oocytes.

Agonists that utilize the calcium-mobilizing second messenger inositol(1,4,5)trisphosphate Ins(1,4,5)P3 usually generate oscillations in intracellular calcium. Such oscillations, based on the periodic release of calcium from the endoplasmic reticulum, can also be induced by injecting cells with Ins(1,4,5)P3. The mechanism responsible for oscillatory activity was studied in Xenopus oocytes by injecting them with different inositol trisphosphates. The plasma membrane of Xenopus oocytes has calcium-dependent chloride channels that open in response to calcium, leading to membrane depolarization. Oscillations in calcium were thus monitored by recording membrane potential. The naturally occurring Ins(1,4,5)P3 produced a large initial transient followed by a single transient or a burst of oscillations. By contrast, two analogues (Ins(2,4,5)P3 and Ins(1,4,5)P(S)3) produced a different oscillatory pattern made up of a short burst of sharp transients. Ins(1,3,4,5)P4 had no effect when injected by itself, and it also failed to modify the oscillatory responses to either Ins(2,4,5)P3 or Ins(1,4,5)P(S)3. Both analogues failed to induce a response when injected immediately after the initial Ins(1,4,5)P3-induced response, indicating that they act on the same intracellular pool of calcium. The existence of different oscillatory patterns suggests that there may be different mechanisms for setting up calcium oscillations. The Ins(2,4,5)P3 and Ins(1,4,5)P(S)3 analogues may initiate oscillations through a negative feedback mechanism whereby calcium inhibits its own release. The two-pool model is the most likely mechanism to describe the Ins(1,4,5)P3-induced oscillations.     

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.     

Inhibition of protein kinase C by staurosporine promotes elevated accumulations of inositol trisphosphates and tetrakisphosphate in human platelets exposed to thrombin

We have examined regulation by protein kinase C (Ca2+/phospholipid-dependent enzyme) of thrombin-induced inositol polyphosphate accumulation in human platelets. When platelets are exposed to thrombin for 10 s, the protein kinase C inhibitor staurosporine causes inositol phosphate elevations over control values of 2.7-fold (inositol 1,4,5-trisphosphate (Ins(1,4,5)P3], 1.9-fold (inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4], and 1.2-fold (inositol 1,3,4-trisphosphate). In the same period, phosphatidic acid and diacylglycerol are unaffected. The myosin light chain kinase inhibitor ML-7 has no effect on inositol phosphate accumulations. Staurosporine does not inhibit Ins(1,4,5)P3 3-kinase and 5-phosphomonoesterase activities in saponin-permeabilized platelets incubated with exogenous Ins(1,4,5)P3 unless the platelets have been exposed to thrombin and protein kinase C is consequently activated. The protein kinase C agonist beta-phorbol 12,13-dibutyrate increases the Vmax of the 3-kinase 1.8-fold, with little effect on Km. Our results provide strong evidence for a role for protein kinase C in regulating inositol phosphate levels in thrombin-activated platelets. We propose that endogenously activated protein kinase C removes Ins(1,4,5)P3 by stimulating both 5-phosphomonoesterase and Ins(1,4,5)P3 3-kinase. Initial activation of phospholipase C does not appear to be affected by such protein kinase C. Inhibition of protein kinase C by staurosporine decreases 5-phosphomonoesterase activity. The resulting elevated Ins(1,4,5)P3, as substrate for Ins(1,4,5)P3 3-kinase, promotes production of Ins(1,3,4,5)P4, which also may accumulate through decreased 5-phosphomonoesterase activity and elevated Ca2+ levels. These factors apparently counteract the inhibitory effect on 3-kinase, yielding a net increase in Ins(1,3,4,5)P4.     

Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulations in Cultured Adrenal Chromaffin Cells in Response to Bradykinin and Histamine

In previous studies it has been shown that both bradykinin and histamine increase the formation of 3H-labeled inositol phosphates in adrenal chromaffin cells prelabelled with [3H]inositol and that both these agonists stimulate release of catecholamines by a mechanism dependent on extracellular calcium. Here, we have used mass assays of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] to investigate changes in levels of these two candidates as second messengers in response to stimulation with bradykinin and histamine. Bradykinin increased the mass of Ins(1,3,4,5)P4 despite the failure in earlier studies with [3H]inositol-labelled cells to observe a bradykinin-mediated increase in content of [3H]InsP4. Bradykinin elicited a very rapid increase in level of Ins(1,4,5)P3, which was maximal at 5-10 s and then rapidly decreased to a small but sustained elevation at 2 min. The bradykinin-elicited Ins(1,3,4,5)P4 response increased to a maximum at 30-60 s and at 2 min was still elevated severalfold above basal levels. Histamine, which produced a larger overall total inositol phosphate response in [3H]inositol-loaded cells, produced significantly smaller Ins(1,4,5)P3 and Ins(1,3,4,5)P4 responses compared with bradykinin. The bradykinin stimulation of Ins(1,4,5)P3 accumulation was partially dependent on a high (1.8 mM) extracellular Ca2+ concentration, whereas the Ins(1,3,4,5)P4 response was almost completely lost when the extracellular Ca2+ concentration was reduced to 100 nM. Changes in the inositol polyphosphate second messengers are compared with the time course of bradykinin-stimulated increases in free intracellular Ca2+ concentrations and noradrenaline release.     

A novel receptor-mediated regulation mechanism of type I inositol polyphosphate 5-phosphatase by calcium/calmodulin-dependent protein kinase II phosphorylation

D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)) are both substrates of the 43-kDa type I inositol polyphosphate 5-phosphatase. Transient and okadaic acid-sensitive inhibition by 70-85% of Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) 5-phosphatase activities was observed in homogenates from rat cortical astrocytes, human astrocytoma 1321N1 cells, and rat basophilic leukemia RBL-2H3 cells after incubation with carbachol. The effect was reproduced in response to UTP in rat astrocytic cells and Chinese hamster ovary cells overexpressing human type I 5-phosphatase. Immunodetection as well as mass spectrometric peptide mass fingerprinting and post-source decay (PSD) sequence data analysis after immunoprecipitation permitted unambiguous identification of the major native 5-phosphatase isoform hydrolyzing Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) as type I inositol polyphosphate 5-phosphatase. In ortho-(32)P-preincubated cells, the phosphorylated 43 kDa-enzyme could be identified after receptor activation by immunoprecipitation followed by electrophoretic separation. Phosphorylation of type I 5-phosphatase was blocked after cell preincubation in the presence of Ca(2+)/calmodulin kinase II inhibitors (i.e. KN-93 and KN-62). In vitro phosphorylation of recombinant type I enzyme by Ca(2+)/calmodulin kinase II resulted in an inhibition (i.e. 60-80%) of 5-phosphatase activity. In this study, we demonstrated for the first time a novel regulation mechanism of type I 5-phosphatase by phosphorylation in intact cells.     

Rapid down-regulation of the type I inositol 1,4,5-trisphosphate receptor and desensitization of gonadotropin-releasing hormone-mediated Ca2+ responses in alpha T3-1 gonadotropes

Despite no evidence for desensitization of phospholipase C-coupled gonadotropin-releasing hormone (GnRH) receptors, we previously reported marked suppression of GnRH-mediated Ca(2+) responses in alphaT3-1 cells by pre-exposure to GnRH. This suppression could not be accounted for solely by reduced inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) responses, thereby implicating uncoupling of Ins(1,4,5)P(3) production and Ca(2+) mobilization (McArdle, C. A., Willars, G. B., Fowkes, R. C., Nahorski, S. R., Davidson, J. S., and Forrest-Owen, W. (1996) J. Biol. Chem. 271, 23711-23717). In the current study we demonstrate that GnRH causes a homologous and heterologous desensitization of Ca(2+) signaling in alphaT3-1 cells that is coincident with a rapid (t((12)) < 20 min), marked, and functionally relevant loss of type I Ins(1,4,5)P(3) receptor immunoreactivity and binding. Furthermore, using an alphaT3-1 cell line expressing recombinant muscarinic M(3) receptors we show that the unique resistance of the GnRH receptor to rapid desensitization contributes to a fast, profound, and sustained loss of Ins(1,4,5)P(3) receptor immunoreactivity. These data highlight a potential role for rapid Ins(1,4,5)P(3) receptor down-regulation in homologous and heterologous desensitization and in particular suggest that this mechanism may contribute to the suppression of the reproductive system that is exploited in the major clinical applications of GnRH analogues.     

Effect of inositol trisphosphate and calcium on oscillating elevations of intracellular calcium in Xenopus oocytes

Stimulation of many nonexcitable cells by Ca2(+)-mobilizing receptor agonists causes oscillating elevations of the intracellular free Ca2+ concentration ((Ca2+]i), rather than a continuous increase. It has been proposed that the frequency at which [Ca2+]i oscillates determines the biological response. Because the occurrence of [Ca2+] oscillations is observed together with endogenous inositol polyphosphate (InsPs) production or following InsPs application, we injected Xenopus laevis oocytes with InsPs and monitored Ca2(+)-activated Cl- currents as an assay of [Ca2+]i. Microinjection of the poorly metabolizable inositol trisphosphate (InsP3) derivatives inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) and inositol 1,4,5-trisphosphorothioate (Ins(1,4,5) P3S3) induced [Ca2+]i oscillations. The frequency at which [Ca2+]i oscillated increased with the injected dose, indicating that the frequency-generating mechanism lies distal to InsP3 production and that generation of oscillations does not require either oscillation of InsP3 levels or InsP3 metabolism. Injections of high doses of Ins(1,4,5)P3 or Ins(2,4,5)P3 inhibited ongoing oscillations, whereas Ca2+ injections decreased the amplitude of Ins(2,4,5)P3-induced oscillations without altering their frequency. Injections of the Ins(1,4,5)P3 metabolite inositol 1,3,4,5-tetrakisphosphate also caused oscillations whose frequency was related to the injected dose, although inositol tetrakisphosphate injection induced an increase in the cellular level of Ins(1,4,5)P3. The results suggest a multicomponent oscillatory system that includes the InsP3 target as well as a Ca2(+)-sensitive step that modulates amplitude.