Inositol phospholipid metabolism may trigger flagellar excision in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells shed their flagella in response to environmental stress. Under favorable conditions, flagella are quickly regrown. To learn more about the signals that trigger flagellar excision and regrowth we have investigated inositol phospholipid metabolites, molecules implicated in signal transduction in several other systems. After deflagellation by low pH or mastoparan, a potent activator of G proteins, there was a rapid increase in levels of inositol 1,4,5-trisphosphate measured by use of receptor-binding assays and HPLC. This increase was concomitant with a decrease in levels of phosphatidylinositol 4,5-bisphosphate and was followed by an increase in phosphatidic acid, results consistent with activation of phospholipase C and diacylglycerol kinase. Additional experiments suggest that this activated phospholipase C is not important for flagellar regrowth but plays a role in informing the excision apparatus of the environmental stress. Addition of neomycin (an inhibitor of phospholipase C) before exposure of cells to low pH or mastoparan prevented the increase in inositol 1,4,5-trisphosphate and also prevented deflagellation. Addition of neomycin after deflagellation blocked increases in inositol 1,4,5-trisphosphate that normally followed deflagellation, but did not block flagellar assembly. Furthermore, a flagellar excision-defective mutant, fa-1, did not shed its flagella in response to low pH or mastoparan, yet both of these agents activated phospholipase C in these cells. The results suggest that activation of phospholipase C, possibly via a G protein, is a proximal step in the signal transduction pathway inducing deflagellation in Chlamydomonas.     

Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii

The molecular machinery of deflagellation can be activated in detergent permeabilized Chlamydomonas reinhardtii by the addition of Ca2+ (Sanders, M. A., and J. L. Salisbury, 1989. J. Cell Biol. 108:1751- 1760). This suggests that stimuli which induce deflagellation in living cells cause an increase in the intracellular concentration of Ca2+, but this has never been demonstrated. In this paper we report that the wasp venom peptide, mastoparan, and the permeant organic acid, benzoate, activate two different signalling pathways to trigger deflagellation. We have characterized each pathway with respect to: (a) the requirement for extracellular Ca2+; (b) sensitivity to Ca2+ channel blockers; and (c) 45Ca influx. We also report that a new mutant strain of C. reinhardtii, adf-1, is specifically defective in the acid-activated signalling pathway. Both signalling pathways appear normal in another mutant, fa-1, that is defective in the machinery of deflagellation (Lewin, R. and C. Burrascano. 1983. Experientia. 39:1397-1398; Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751-1760). We conclude that mastoparan induces the release of an intracellular pool of Ca2+ whereas acid induces an influx of extracellular Ca2+ to activate the machinery of deflagellation.     

Role of Ca2+ feedback on single cell inositol 1,4,5-trisphosphate oscillations mediated by G-protein-coupled receptors

The dynamics of inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) production during periods of G-protein-coupled receptor-mediated Ca2+ oscillations have been investigated using the pleckstrin homology (PH) domain of phospholipase C (PLC) delta1 tagged with enhanced green fluorescent protein (eGFP-PHPLCdelta1). Activation of noradrenergic alpha1B and muscarinic M3 receptors recombinantly expressed in the same Chinese hamster ovary cell indicates that Ca2+ responses to these G-protein-coupled receptors are stimulus strength-dependent. Thus, activation of alpha1B receptors produced transient base-line Ca2+ oscillations, sinusoidal Ca2+ oscillations, and then a steady-state plateau level of Ca2+ as the level of agonist stimulation increased. Activation of M3 receptors, which have a higher coupling efficiency than alpha1B receptors, produced a sustained increase in intracellular Ca2+ even at low levels of agonist stimulation. Confocal imaging of eGFP-PHPLCdelta1 visualized periodic increases in Ins(1,4,5)P3 production underlying the base-line Ca2+ oscillations. Ins(1,4,5)P3 oscillations were blocked by thapsigargin but not by protein kinase C down-regulation. The net effect of increasing intracellular Ca2+ was stimulatory to Ins(1,4,5)P3 production, and dual imaging experiments indicated that receptor-mediated Ins(1,4,5)P3 production was sensitive to changes in intracellular Ca2+ between basal and approximately 200 nM. Together, these data suggest that alpha1B receptor-mediated Ins(1,4,5)P3 oscillations result from a positive feedback effect of Ca2+ onto phospholipase C. The mechanisms underlying alpha1B receptor-mediated Ca2+ responses are therefore different from those for the metabotropic glutamate receptor 5a, where Ins(1,4,5)P3 oscillations are the primary driving force for oscillatory Ca2+ responses (Nash, M. S., Young, K. W., Challiss, R. A. J., and Nahorski, S. R. (2001) Nature 413, 381-382). For alpha1B receptors the Ca2+-dependent Ins(1,4,5)P3 production may serve to augment the existing regenerative Ca2+-induced Ca2+-release process; however, the sensitivity to Ca2+ feedback is such that only transient base-line Ca2+ spikes may be capable of causing Ins(1,4,5)P3 oscillations.     

Selective amplification of endothelin-stimulated inositol 1,4,5-trisphosphate and calcium signaling by v-src transformation of rat-1 fibroblasts.

The effects of the expression of the protein tyrosine kinase pp60v-src on endothelin- and thrombin-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) production and calcium responses were investigated in Rat-1 fibroblasts. The ability of endothelin-1 to induce the accumulation of these second messengers was dramatically amplified by v-src transformation, with 6- and 3-fold enhancements of the peak Ins(1,4,5)P3 and peak calcium responses, respectively. In contrast, thrombin-dependent responses were slightly reduced following v-src transformation, demonstrating that the augmentation of endothelin-stimulated signal transduction is a selective effect. The magnitude of the stimulated accumulation of Ins(1,4,5)P3 presumably depends upon both the functional activation of phospholipase C to produce Ins(1,4,5)P3, and the activity of the enzymes that metabolize Ins(1,4,5)P3. Although the metabolism of Ins(1,4,5)P3 was strikingly altered by expression of pp60v-src, with a bias towards the production of higher inositol polyphosphates that is consistent with an activated Ins(1,4,5)P3 3-kinase, this change could not account for the marked increase in endothelin-stimulated signaling induced by v-src transformation. This suggests that an effect of pp60v-src is expressed at the level of the plasma membrane, through an interaction with one or more components in the receptor/guanine nucleotide binding protein (G protein)/phospholipase C system that transduces the endothelin signal into Ins(1,4,5)P3 production. Preparation of membranes from normal and v-src-transformed cells showed that, while there was no change in the number of high-affinity endothelin binding sites, the release of Ins(1,4,5)P3 in response to guanine nucleotides and endothelin-1 was significantly increased following v-src transformation. In contrast, the Ins(1,4,5)P3 responses to thrombin and high Ca2+ concentrations were unaffected by transformation. Thus the selective interactions within the G protein system that couples the endothelin receptor to phospholipase C are potential sites at which the v-src transformation process may act to amplify endothelin-dependent Ins(1,4,5)P3 production.     

Bradykinin-induced generation of inositol 1,4,5-trisphosphate in fibroblasts and neuroblastoma cells: effect of pertussis toxin, extracellular calcium, and down-regulation of protein kinase C

The net content of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was measured in bradykinin (BK)-stimulated NIH3T3 fibroblasts and neuroblastoma-glioma hybrid cells (NG108-15). BK-mediated production of Ins(1,4,5)P3 was not affected by replacing the medium with Ca2+-free medium, but addition of EGTA (1mM) to Ca2+-free medium markedly prevented production of Ins(1,4,5)P3. Although pertussis toxin (PT) treatment caused ADP-ribosylation in both NIH3T3 cells and NG108-15 cells, the BK-induced Ins(1,4,5)P3 formation was considerably reduced in the former cells but not in the latter cells, suggesting that PT-sensitive and PT-insensitive GTP-binding proteins are involved in phosphoinositide phospholipase C (PI-PLC) activation in fibroblasts and neuroblastoma cells, respectively. In NG108-15 cells down-regulated in protein kinase C (PKC) by long-term exposure to phorbol 12-myristate 13-acetate (PMA), BK-stimulated Ins(1,4,5)P3 accumulation was significantly enhanced compared to control cells.     

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.     

Induction of microtubule protein synthesis in Chlamydomonas reinhardi during flagellar regeneration

Flagellar regeneration in gametes of Chlamydomonas reinhardi is initiated within 15–20 min after flagellar amputation and proceeds at a rapid but decelerating rate until by 90 min flagellar outgrowth is 80–85% complete. Sufficient flagellar protein reserves exist in the cytoplasm to allow regeneration of flagella $\text{1}\text{3}\text{–}\text{1}\text{2}$ normal length. Nevertheless, in vivo labeling with ¹⁴C-amino acids shows that microtubule protein and other flagellar proteins are synthesized de novo during flagellar regeneration. To determine whether tubulin is synthesized continuously by gametic cells or whether its synthesis is induced as a consequence of deflagellation, we have isolated polyribosomes from deflagellated and control cells, and analyzed the proteins produced by these polyribosomes during in vitro translation. Two proteins of 53,000 and 56,000 molecular weight which co-migrate with flagellar and chick brain tubulin on SDS-polyacrylamide gels and which selectively co-assemble with chick brain tubulin during in vitro microtubule assembly are synthesized by polyribosomes (or polyadenylated mRNA) from deflagellated cells. No microtubule proteins can be detected in the translation products synthesized by polyribosomes (or mRNA) from control cells, clearly indicating that deflagellation results in the induction of tubulin synthesis.Kinetics of tubulin synthesis demonstrate that induction takes place immediately after deflagellation; polyribosomes bearing tubulin mRNA can be detected in the cytoplasm in as little as 15 min after removal of flagella. Maximal rates of tubulin synthesis occur between 45 and 90 min after deflagellation when approximately 14% of the protein being synthesized by the cell is tubulin. This estimate of tubulin synthesis based on in vitro translation data agrees well with in vivo measurements of flagellar tubulin synthesis. While high levels of tubulin production extend well beyond the period of rapid flagellar assembly, synthesis begins to decline after 90 min, and by 180 min after deflagellation only low levels of tubulin mRNA are detectable in polyribosomes.     

Chlamydomonas contains calcium stores that are mobilized when phospholipase C is activated

Mastoparan induces Ca²⁺-dependent deflagellation of the unicellular green alga Chlamydomonas moewusii Gerloff, as well as the activation of phospholipase C and the production of inositol 1,4,5-trisphosphate (InsP₃; T. Munnik et al., 1998, Planta 207: 133–145). Even in the absence of extracellular Ca²⁺, mastoparan still induces deflagellation (L.M. Quarmby and H.C. Hartzell, 1994, J Cell Biol 124: 807–815; J.A.J. van Himbergen et al., 1999, J Exp Bot, in press) suggesting that InsP₃ mediates Ca²⁺ release from intracellular stores. To test this hypothesis, cells were pre-loaded with ⁴⁵Ca²⁺ and their plasma membranes permeabilized by digitonin. Subsequent treatment of the cells with mastoparan (3.5 μM) induced release of intracellular ⁴⁵Ca²⁺. Mastoparan also activated phospholipase C in permeabilized cells, as demonstrated by the breakdown of ³²P-phosphatidylinositol 4,5-bisphosphate and the production of diacylglycerol. The mastoparan analogues mas7 and mas17 were also effective and their efficacy was correlated with their biological activity. X-ray microanalysis showed that electron-dense bodies (EDBs) are a major Ca²⁺ store in  C. moewusii. Analysis of digitonin-permeabilized cells showed that EDBs lost calcium at digitonin concentrations that released radioactivity from ⁴⁵Ca²⁺-labelled cells, suggesting that ⁴⁵Ca²⁺ monitored the content of EDBs. X-ray microanaysis of living cells treated with mastoparan also revealed that calcium was released from EDBs. Received: 30 December 1998 / Accepted: 25 June 1999     

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.     

The role of haem in the regulation of rat liver tryptophan metabolism.

Anion-exchange h.p.l.c. analysis of [3H]inositol phosphates derived from glucose-stimulated isolated pancreatic islets that had been prelabelled with myo-[3H]inositol revealed that the predominant inositol trisphosphate was the 1,3,4-isomer [Ins(1,3,4)P3]. The 1,4,5-isomer [Ins(1,4,5)P3] was also detectable, as was a more polar inositol phosphate with the chromatographic properties of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. Glucose-induced accumulation of Ins(1,3,4)P3 was augmented by Li+ and occurred after maximal accumulation of Ins(1,4,5)P3. These findings suggest a possible role for Ins(1,3,4)P3 or its probable precursor Ins(1,3,4,5)P4 in stimulus-secretion coupling in pancreatic islets.     

Phosphoinositide hydrolysis in mitogen-stimulated human peripheral-blood T lymphocytes.

Both phytohaemagglutinin and antibodies to the CD3 molecule induced proliferation and phosphoinositide hydrolysis in human peripheral-blood T lymphocytes, but the magnitude of the inositol phosphate response was small and the rate of accumulation slow [significant increases in Ins(1,4,5)P3 were observed only after 10 min]. Hence this response differs from the well-characterized Ins(1,4,5)P3 responses of many other systems. This slow response, its abrogation in Ca2+-depleted medium, the slow and maintained increase in Ca2+ as measured by Quin-2, and the ability of the Ca2+ ionophore A23187 to stimulate Ins(1,4,5)P3 accumulation all suggest that the increase in Ins(1,4,5)P3 occurs, at least in part, as a result of receptor-mediated Ca2+ influx in mitogen-stimulated T lymphocytes.     

Partial purification and reconstitution of inositol 1,4,5-trisphosphate receptor/Ca2+ channel of bovine liver microsomes.

The binding of inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] to bovine liver microsomes was characterized. The Ins(1,4,5)P3 receptor of the microsomes was solubilized by 1% Triton X-100 and purified by sucrose density gradient, Heparin-Sepharose, DEAE-Toyopearl, ATP-Agarose, and Ins(1,4,5)P3-Sepharose column chromatographies. More than 1,000-fold enrichment of the Ins(1,4,5)P3-binding activity was achieved. Kd values of the binding activity were 2.8 nM in microsomes and 3.0 nM in the partially purified receptor, respectively, and the binding activity was optimal in the medium containing 100 mM KCl and at pH between 7.5 and 8.5. The presence of Ca2+ failed to inhibit the binding. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PtdIns), and phosphatidylinositol-4-monophosphate [PtdIns(4)P] showed no effect on the Ins(1,4,5)P3 binding. However, soybean phospholipids asolectin and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] strongly inhibited the binding activity. PtdIns(4,5)P2 inhibited the activity competitively with a half-maximal inhibitory concentration of 30 micrograms/ml. The partially purified Ins(1,4,5)P3 receptor was reconstituted into proteoliposomes. Fluorescence measurements using Quin 2 indicated that Ins(1,4,5)P3 stimulated Ca2+ influx into the proteoliposomes. The EC50 of Ins(1,4,5)P3 on Ca2+ influx was 50 nM. This result strongly suggest that Ins(1,4,5)P3 binding protein of liver microsomes acts as a physiological Ins(1,4,5)P3 receptor/Ca2+ channel.     

Characterisation of Distinct Inositol 1,4,5-Trisphosphate-Sensitive and Caffeine-Sensitive Calcium Stores in Digitonin-Permeabilised Adrenal Chromaffin Cells

The effect of inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3] and caffeine on Ca2+ release from digitonin-permeabilised bovine adrenal chromaffin cells was examined by using the Ca2+ indicator fura-2 to monitor [Ca2+]. Permeabilised cells accumulated Ca2+ in the presence of ATP and addition of either Ins(1,4,5)P3 or caffeine released 17% or 40-50%, respectively, of the accumulated Ca2+, indicated by sustained rises in [Ca2+] in the cell suspension. Prior addition of Ins(1,4,5)P3 had no effect on the magnitude of the response to a subsequent addition of caffeine. The response to Ins(1,4,5)P3 was prevented by prior addition of caffeine or CaCl2, indicating that the Ins(1,4,5)P3 response was blocked by elevated [Ca2+]. The responses were essentially identical in the presence of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, indicating that the Ca2+ release was not from mitochondria or secretory granules and that a proton gradient was not required for Ca2+ accumulation into the Ins(1,4,5)P3- or caffeine-sensitive stores. Ca2+ release from the caffeine-sensitive store was selectively blocked by ryanodine. The Ins(1,4,5)P3-sensitive store was emptied by thapsigargin, which had no effect on caffeine responses. These data suggest that permeabilised chromaffin cells possess two distinct nonoverlapping Ca2+ stores sensitive to either Ins(1,4,5)P3 or caffeine and support previous conclusions that these stores possess different Ca2(+)-ATPases.     

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.     

Hyperosmotic stress induces a rapid and transient increase in inositol 1,4,5-trisphosphate independent of abscisic acid in Arabidopsis cell culture

Phospholipid metabolism is involved in hyperosmotic-stress responses in plants. To investigate the role of phosphoinositide-specific phospholipase C (PI-PLC)-a key enzyme in phosphoinositide turnover-in hyperosmotic-stress signaling, we analyzed changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content in response to hyperosmotic shock or salinity in Arabidopsis thaliana T87 cultured cells. Within a few s, a hyperosmotic shock, caused by mannitol, NaCl, or dehydration, induced a rapid and transient increase in Ins(1,4,5)P3. However, no transient increase was detected in cells treated with ABA. Neomycin and U73122, inhibitors of PI-PLC, inhibited the increase in Ins(1,4,5)P3 caused by the hyperosmotic shock. A rapid increase in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in response to the hyperosmotic shock also occurred, but the rate of increase was much slower than that of Ins(1,4,5)P3. These findings indicate that the transient Ins(1,4,5)P3 production was due to the activation of PI-PLC in response to hyperosmotic stress. PI-PLC inhibitors also inhibited hyperosmotic stress-responsive expression of some dehydration-inducible genes, such as rd29A (lti78/cor78) and rd17 (cor47), that are controlled by the DRE/CRT cis-acting element but did not inhibit hyperosmotic stress-responsive expression of ABA-inducible genes, such as rd20. Taken together, these results suggest the involvement of PI-PLC and Ins(1,4,5)P3 in an ABA-independent hyperosmotic-stress signal transduction pathway in higher plants.     

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

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

Simplification of adenophostin A defines a minimal structure for potent glucopyranoside-based mimics of D-myo-inositol 1,4,5-trisphosphate

The synthesis of 1-O-[(3S,4R)-3-hydroxytetrahydrofuran-4-yl]-alpha-D-glucopyranosid e 3,4,3'-trisphosphate (7), a novel Ca2+ mobilising agonist at the Ins(1,4,5)P3 receptor, designed by excision of two motifs of adenophostin A is reported, defining a potential minimal structure for potent glucopyranoside-based agonists of Ins(1,4,5)P3 receptors.     

Characteristics of inositol trisphosphate-mediated Ca2+ release from permeabilized hepatocytes

Ca2+ release triggered by inositol trisphosphate (Ins(1,4,5)P3) has been measured in saponin-permeabilized hepatocytes with 45Ca2+ or Quin 2. The initial rate of Ca2+ release was not greatly affected by the incubation temperature (175 +/- 40 pmol X s-1 X mg dry weight-1, at 30 degrees C versus 133 +/- 24 pmol X s-1 X mg dry weight-1 at 4 degrees C). The amount of Ca2+ released by Ins(1,4,5)P3 was not affected by pH (6.5-8.0). La3+ (100 microM) markedly inhibited the effect of 1 microM Ins(1,4,5)P3. The possibility that La3+ chelates Ins(1,4,5)P3 cannot be excluded since the effect of La3+ could be overcome by increasing the Ins(1,4,5)P3 concentration. Ins(1,4,5)P3-mediated Ca2+ release showed a requirement for permeant cations in the incubation medium. Optimal release was observed with potassium gluconate. Other monovalent cations, with the exception of Li+, can substitute for K+. Permeant anions, at concentrations above 40 mM, inhibited Ca2+ release produced by Ins(1,4,5)P3. Cl-, Br-, I-, and SO2-4 were equally effective as inhibitors. Ins(1,4,5)P3 also caused the release of 54Mn2+ and 85Sr2+ accumulated by the permeabilized hepatocytes. Our results are consistent with Ins(1,4,5)P3 promoting the membrane translocation of divalent cations through an ion channel rather than an ion carrier. The translocation of positive charge through this channel is balanced by ancillary movements of monovalent cations and anions across the reticular membranes. The transport systems responsible for these compensatory ion movements may represent a potential site for the regulation of the hormone-mediated Ca2+ signal.     

Inositol 1,4,5-trisphosphate and inositol 1,2-cyclic 4,5-trisphosphate are minor components of total mass of inositol trisphosphate in thrombin-stimulated platelets. Rapid formation of inositol 1,3,4-trisphosphate

Stimulation of human platelets by thrombin leads to rises of both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) within 10 s. The mass of Ins(1,4,5)P3 was measured in platelet extracts after conversion to [3-32P]Ins(1,3,4,5)P4 with Ins(1,4,5)P3 3-kinase and [gamma-32P]ATP. Basal levels were equivalent to 0.2 microM and rose to 1 microM within 10 s of stimulation by thrombin. The mass of Ins(1,3,4)P3 was more than 10-fold greater than that of Ins(1,4,5)P3 between 10 and 60 s of thrombin stimulation. These results indicate that the majority of InsP3 liberated by phospholipase C in stimulated platelets must be the non-cyclic Ins(1,4,5)P3 in order to allow rapid phosphorylation by Ins(1,4,5)P3 3-kinase to Ins(1,3,4,5)P4 and then dephosphorylation to Ins(1,3,4)P3 by 5-phosphomonoesterase. A significant proportion of the InsP3 extracted from thrombin-stimulated platelets under neutral conditions is resistant to Ins(1,4,5)P3 3-kinase but susceptible after acid treatment, implying the presence of inositol 1,2-cyclic 4,5-trisphosphate (Ins(1,2cyc4,5)P3. The relative proportion of Ins(1,2cyc4,5)P3 increases with time. We suggest that such gradual accumulation is attributable to the relative insensitivity of this compound to hydrolytic and phosphorylating enzymes. Therefore, early Ca2+ mobilization in platelets is more likely to be effected by Ins(1,4,5)P3 than by Ins(1,2cyc4,5)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.