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.     

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.     

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.     

Growth of Pollen Tubes of Papaver rhoeas Is Regulated by a Slow-Moving Calcium Wave Propagated by Inositol 1,4,5-Trisphosphate

A signaling role for cytosolic free Ca2+ ([Ca2+]i) in regulating Papaver rhoeas pollen tube growth during the self-incompatibility response has been demonstrated previously. In this article, we investigate the involvement of the phosphoinositide signal transduction pathway in Ca2+-mediated pollen tube inhibition. We demonstrate that P. rhoeas pollen tubes have a Ca2+-dependent polyphosphoinositide-specific phospholipase C activity that is inhibited by neomycin. [Ca2+]i imaging after photolysis of caged inositol (1,4,5)-trisphosphate (Ins[1,4,5]P3) in pollen tubes demonstrated that Ins(1,4,5)P3 could induce Ca2+ release, which was inhibited by heparin and neomycin. Mastoparan, which stimulated Ins(1,4,5)P3 production, also induced a rapid increase in Ca2+, which was inhibited by neomycin. These data provide direct evidence for the involvement of a functional phosphoinositide signal-transducing system in the regulation of pollen tube growth. We suggest that the observed Ca2+ increases are mediated, at least in part, by Ins(1,4,5)P3-induced Ca2+ release. Furthermore, we provide data suggesting that Ca2+ waves, which have not previously been reported in plant cells, can be induced in pollen tubes.     

Auranofin dissociates chemotactic peptide-induced generation of inositol 1,4,5-trisphosphate from the subsequent mobilization of intracellular calcium in intact human neutrophils

Auranofin, an antiarthritic gold compound, modulates a number of chemotactic factor-induced inflammatory responses in human neutrophils. In order to unravel the mechanism involved, the present study investigated the effects of auranofin on early signal transduction events in these cells. Auranofin did not affect the chemotactic peptide (fMetLeuPhe)-induced formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), neither in the presence nor in the absence of extracellular calcium ions. In contrast, there was a progressive inhibition by auranofin on the fMet-Leu-Phe-induced mobilization of intracellular calcium. This demonstrates that auranofin can dissociate the generation of Ins(1,4,5)P3 from the subsequent release of intracellular calcium, perhaps by interfering with the intracellular binding of Ins(1,4,5)P3 to its receptor. In experiments performed in electro-permeabilized cells, however, a relatively high concentration of the drug failed to abolish the specific binding of Ins(1,4,5)P3. In addition, in the same system, auranofin also failed to abolish the Ins(1,4,5)P3-induced release of Ca2+. Consequently, auranofin-mediated dissociation of fMLP-induced Ins(1,4,5)P3 formation and intracellular calcium release can not be explained merely by an antagonistic effect of auranofin on the Ins(1,4,5)P3 receptor. Instead the interaction between auranofin and the plasma membrane seems to be an initial and important part of the mechanism by which this drug interferes with the transduction signalling system.     

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.     

Developmental regulation of the Inositol 1,4,5-trisphosphate phosphatases in Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum is a microorganism in which growth and development are strictly separated. Starvation initiates a developmental program in which extracellular cAMP plays a major role as a signal molecule. In response to cAMP several second messengers are produced, including cAMP, cGMP and inositol 1,4,5-trisphosphate, (Ins(1,4,5)P3). Ins(1,4,5)P3 levels are controlled by the activation of phosphoinositidase C and the activity of the Ins(1,4,5)P3-degrading phosphatases. In Dictyostelium discoideum two major routes for the dephosphorylation of Ins(1,4,5)P3 are present: a 5-phosphatase, which hydrolyses Ins(1,4,5)P3 at the 5-position producing Ins(1,4)P2 as in vertebrate cells, and a 1-phosphatase which removes the 1-phosphate, giving Ins(4,5)P2, as in plants. In this paper we show that at the onset of development both the 1-phosphatase and the 5-phosphatase are present in equal amounts. During development the 5-phosphatase disappears leaving the 1-phosphatase as the single enzyme to remove Ins(1,4,5)P3. We conclude that during development Dictyostelium discoideum switches from a mixed type of Ins(1,4,5)P3 degradation to a more plant-like degradation pathway.     

Barley (Hordeum vulgare L.) inositol monophosphatase: gene structure and enzyme characteristics

The cellular myo-inositol (Ins) pool is important to many metabolic and signaling pathways in plants. Ins monophosphatase (IMPase; EC 3.1.3.25) activity is essential for the de novo synthesis of myo-Inositol (Ins), and for recycling of Ins in Ins(1,4,5)P3. However, proteins encoded by at least one family of IMP genes also have L-galactose-1-P phosphatase activity important to ascorbic acid synthesis, indicating a bifunctionality that links these two branches of carbon metabolism. As part of research into the regulation of Ins synthesis and supply during seed development, the barley IMP-1 gene and gene products were studied. The 1.4 kb barley IMP-1 promoter contains one low temperature response element (RE), two heat shock REs, one gibberellin and two auxin REs, and five sugar REs. Barley IMP-1 is expressed in all tissues assayed, and expression levels were not greatly altered by abiotic stress treatments. Reduced use of Ins for Ins P6 synthesis in developing seed of barley low phytic acid (lpa) mutants results in Ins accumulation, and IMP-1 expression is reduced in proportion to the increase in Ins level. The barley recombinant enzyme had a lower Km, indicating higher affinity, for D/L-Ins(3)P1 (Km = 9.7 microM) as compared with reported Km (Ins P1) values for other eukaryotic IMPases (43-330 microM) or with a reported Km (L-Gal-1P) of 150 microM for a kiwifruit (Actinidia deliciosa) enzyme. These and other data indicate that the barley IMP-1 gene is regulated at least in part in response to Ins metabolic needs, and that the enzyme it encodes displays catalytic properties well suited for a role in Ins synthesis, in addition to other roles as an L-gal-1-P phosphatase important to ascorbate synthesis, or as an IMPase important to Ins(1,4,5)P3 signal recycling.     

Aluminum Inhibition of the Inositol 1,4,5-Trisphosphate Signal Transduction Pathway in Wheat Roots: A Role in Aluminum Toxicity?

In crop plants, aluminum (Al) rhizotoxicity is a major problem worldwide; however, the cause of Al toxicity remains elusive. The effects of Al on the inositol 1,4,5-trisphosphate (Ins[1,4,5]P3)-mediated signal transduction pathway were investigated in wheat roots. Exogenously applied Al (50 [mu]M) rapidly inhibited root growth (<2 hr) but did not affect general root metabolism. An Ins(1,4,5)P3 transient was generated in root tips, either before or after exposure to Al for 1 hr, by treating the roots with H2O2 (10 mM). Background (unstimulated) levels of Ins(1,4,5)P3 were similar in both Al-treated and Al-untreated root apices. However, H2O2-stimulated levels of Ins(1,4,5)P3 in root apices showed a significant (>50%) reduction after Al exposure in comparison with untreated controls, indicating that Al may be interfering with the phosphoinositide signaling pathway. When phospholipase C (PLC) was assayed directly in the presence of Al or other metal cations in microsomal membranes, AlCl3 and Al-citrate specifically inhibited PLC action in a dose-dependent manner and at physiologically relevant Al levels. Al exposure had no effect on inositol trisphosphate dephosphorylation or on a range of enzymes isolated from wheat roots, suggesting that Al exposure may specifically target PLC. Possible mechanisms of PLC inhibition by Al and the role of Ins(1,4,5)P3 in Al toxicity and growth are discussed. This study provides compelling evidence that the phytotoxic metal cation Al has an intracellular target site that may be integrally involved in root growth.     

Different patterns of agonist-stimulated increases of 3H-inositol phosphate isomers and cytosolic Ca2+ in bovine adrenal chromaffin cells: comparison of the effects of histamine and angiotensin II

Bovine adrenal chromaffin cells (BCC) were used to compare histamine- and angiotensin II-induced changes of inositol mono-, bis-, and trisphosphate (InsP1, InsP2, and InsP3, respectively) isomers, intracellular free Ca2+ ([Ca2+]i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca2+]i by 200 nM 3-4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four- to fivefold increases of Ins(1,4,5)P3 that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P3 formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II- and histamine-stimulated signal transduction. The metabolism of Ins(1,4,5)P3 by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P2 and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5-tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P3, Ins(1,3)P2, and Ins(3,4)P2, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P2 was the only other InsP2 besides Ins(1,4)P2 to accumulate within 1 min of agonist treatment [Ins(3,4)P2 did not increase]. These results support a correlation between the time course of Ins(1,4,5)P3 formation and the time course of [Ca2+]i transients and illustrate that Ca2(+)-mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca2+]i changes in BCC. Different patterns of second-messenger formation are likely to be important in signal recognition and may encode agonist-specific information.     

Arabidopsis PLC1 is required for secondary responses to abscisic acid signals

The role of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) in transducing the abscisic acid (ABA) signal during seed germination and in the stress responses of mature plants is poorly understood. We have considered the contributions of the phospholipase C1 (encoded by AtPLC1) and an Ins(1,4,5)P3 5-phosphatase (encoded by AtIP5PII) to ABA signaling by using a modified version of the glucocorticoid-inducible system to regulate transgene expression. In the presence of the dexamethasone (Dex) inducer, transgenic lines expressing the AtPLC1 antisense and AtIP5PII sense transgenes showed no inhibition of germination and growth by ABA, whereas in the absence of the inducer they were sensitive. In the presence of Dex, these lines accumulated lower Ins(1,4,5)P3 levels upon ABA treatment compared with that of the control transgenic lines. RNA gel blot analysis revealed a decrease in the induction of the ABA-responsive genes RD29a, KIN2, and RD22 but not COR47 in the Dex-induced transgenic plants. In transgenic lines expressing the inducible AtPLC1 sense transgene, an increase in AtPLC1 expression was not sufficient to activate the expression of ABA-responsive genes in vegetative tissues. In vitro experiments demonstrated the induced PLC1 expression when extracts were assayed in the presence of calcium, but no increase in Ins(1,4,5)P3 levels in vivo was detected, suggesting that the PLC1 enzyme was latent. Our results indicate that although an increase in PLC1 activity and increased Ins(1,4,5)P3 levels are necessary for maximal gene induction by ABA, overexpression of AtPLC1 itself is not sufficient to trigger the expression of ABA-responsive genes. We propose that AtPLC1 plays a role in secondary ABA responses.     

Role of 1,4,5-inositol triphosphate-induced Ca2+ release in pollen tube orientation

 Pollen tube reorientation is a dynamic cellular event crucial for successful fertilization. Previously, it was shown that reorientation is preceded by an asymmetric increase of cytosolic free calcium ([Ca²⁺]_c) in the side of the apex to which the cell will bend. In order to find the targets for this signal transduction pathway, the effects of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] in the reorientation process were analyzed. Ins(1,4,5)P₃ was artificially increased in different cell domains by localized photoactivation of caged Ins(1,4,5)P₃ and its effects on [Ca²⁺]_c monitored by ion confocal microscopy. It was found that photolysis of caged Ins(1,4,5)P₃ in the nuclear or subapical region resulted in a transient increase in [Ca²⁺]_c and reorientation of the growth axis, while photolysis in the apex frequently resulted in disturbed growth or tip bursting. Perfusion of the cells with the Ins(1,4,5)P₃ receptor blocker heparin prior to photoactivation inhibited the increase in [Ca²⁺]_c and no reorientation was observed. Ca²⁺ release from Ins(1,4,5)P₃-dependent stores localized in the shank of the tube thus seems to be part of the signal transduction pathway that controls tube guidance, although not the primary stimulus leading to reorientation. Received: 5 May 1998 / Accepted: 11 June 1998     

Rat inositol 1,4,5-trisphosphate 3-kinase C is enzymatically specialized for basal cellular inositol trisphosphate phosphorylation and shuttles actively between nucleus and cytoplasm

The calcium-liberating second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is converted to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) by Ins(1,4,5)P3 3-kinases (IP3Ks) that add a fourth phosphate group to the 3-position of the inositol ring. Two isoforms of IP3Ks (named A and B) from different vertebrate species have been well studied. Recently the cloning and examination of a human full-length cDNA encoding a novel isoform, termed human IP3K-C (HsIP3K-C), has been reported. In the present study we report the cloning of a full-length cDNA encoding a rat homologue of HsIP3K-C with a unique mRNA expression pattern, which differs remarkably from the tissue distribution of HsIP3K-C. Of the rat tissues examined, rat IP3K-C (RnIP3K-C) is mainly present in heart, brain, and testis and shows the strongest expression in an epidermal tissue, namely tongue epithelium. RnIP3K-C has a calculated molecular mass of approximately 74.5 kDa and shows an overall identity of approximately 75% with HsIP3K-C. A bacterially expressed, enzymatically active and Ca2+-calmodulin-regulated fragment of this isoform displays remarkable enzymatic properties like a very low Km for Ins(1,4,5)P3 ( approximately 0.2 microm), substrate inhibition by high concentrations of Ins(1,4,5)P3, allosteric product activation by Ins(1,3,4,5)P4 in absence of Ca2+-calmodulin (Ka(app) 0.52 microm), and the ability to efficiently phosphorylate a second InsP3 substrate, inositol 2,4,5-trisphosphate, to inositol 2,4,5,6-tetrakisphosphate in the presence of Ins(1,3,4,5)P4. Furthermore, the RnIP3K-C fused with a fluorescent protein tag is actively transported into and out of the nucleus when transiently expressed in mammalian cells. A leucine-rich nuclear export signal and an uncharacterized nuclear import activity are localized in the N-terminal domain of the protein and determine its nucleocytoplasmic shuttling. These findings point to a particular role of RnIP3K-C in nuclear inositol trisphosphate phosphorylation and cellular growth.     

Calcium/calmodulin-mediated gravitropic response in plants

Calcium and calmodulin (CaM) play an important role in gravity signal transduction. However, the molecular and biochemical mechanisms involved in gravity signal transduction are not clearly understood. It is becoming evident that hydrogen peroxide is involved in gravity-induced response. Recent results indicate that Ca 2+/CaM is involved in hydrogen peroxide homeostasis by regulating catalase activity in plants (Yang and Poovaiah, 2002). It is well established that auxin controls differential growth during gravitropic bending. Results indicated that an auxin-responsive gene family (SAURs) encodes for Ca 2+ /CaM-binding proteins (Yang and Poovaiah, 2000a). To investigate the effects of gravity on the expression of genes involved in Ca 2+/CaM-mediated signaling, Arabidopsis and corn seedlings were subjected to simulated microgravity using the Random Positioning Machine (RPM), and hypergravity using the MidiCAR centrifuge. The changes in mRNA levels were studied. Selective and significant differences in gene expression were observed in simulated microgravity- and hypergravity- treated plants. The relevance of these genes in gravity signal perception and transduction is discussed.     

Degradation of inositol 1,3,4,5-tetrakisphosphates by porcine brain cytosol yields inositol 1,3,4-trisphosphate and inositol 1,4,5-trisphosphate

Inositol 1,3,4,5-tetrakisphosphates (Ins(1,3,4,5)P4), 32P-labelled in positions 4 and 5 were prepared enzymatically, using [4-32P]-phosphatidylinositol 4-phosphate (PtdInsP) and [5-32P]phosphatidylinositol 4,5-bisphosphate (PtdInsP2) as substrates, respectively. Degradation studies of Ins(1,3,4,5)P4, using an enriched phosphatase preparation from porcine brain cytosol, led to the formation of two inositol trisphosphate isomers which were identified as inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). This novel degradation pathway of Ins(1,3,4,5)P4 to Ins(1,4,5)P3 provides an additional source for the generation of Ins(1,4,5)P3, involving a 3-phosphatase.     

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.     

Intercellular communication between follicular angiotensin receptors and Xenopus laevis oocytes: medication by an inositol 1,4,5- trisphosphate-dependent mechanism

In Xenopus laevis oocytes, activation of angiotensin II (AII) receptors on the surrounding follicular cells sends a signal through gap junctions to elevate cytoplasmic calcium concentration ([Ca2+]i) within the oocyte. The two major candidates for signal transfer through gap junctions into the oocyte during AII receptor stimulation are Ins(1,4,5)P3 and Ca2+. In [3H]inositol-injected follicular oocytes, AII stimulated two- to fourfold increases in phosphoinositide hydrolysis and production of inositol phosphates. Injection of the glycosaminoglycan, heparin, which selectively blocks Ins(1,4,5)P3 receptors, prevented both AII-stimulated and Ins(1,4,5)P3-induced Ca2+ mobilization in Xenopus follicular oocytes but did not affect mobilization of Ca2+ by ionomycin or GTP. These results indicate that the AII-regulated process of gap junction communication between follicular cells and the oocyte operates through an Ins(1,4,5)P3-dependent mechanism rather than through transfer of Ca2+ into the ooplasm and subsequent Ca(2+)-induced Ca2+ release.     

Effect of pertussis toxin and neomycin on G-protein-regulated polyphosphoinositide phosphodiesterase. A comparison between HL60 membranes and permeabilized HL60 cells.

Dictyostelium discoideum homogenates contain phosphatase activity which rapidly dephosphorylates Ins(1,4,5)P3 (D-myo-inositol 1,4,5-trisphosphate) to Ins (myo-inositol). When assayed in Mg2+, Ins(1,4,5)P3 is dephosphorylated by the soluble Dictyostelium cell fraction to 20% Ins(1,4)P2 (D-myo-inositol 1,4-bisphosphate) and 80% Ins(4,5)P2 (D-myo-inositol 4,5-bisphosphate). In the particulate fraction Ins(1,4,5)P3 5-phosphatase is relatively more active than the Ins(1,4,5)P3 1-phosphatase. CaCl2 can replace MgCl2 only for the Ins(1,4,5)P3 5-phosphatase activity. Ins(1,4)P2 and Ins(4,5)P2 are both further dephosphorylated to Ins4P (D-myo-inositol 4-monophosphate), and ultimately to Ins. Li+ ions inhibit Ins(1,4,5)P3 1-phosphatase, Ins(1,4)P2 1-phosphatase, Ins4P phosphatase and L-Ins1P (L-myo-inositol 1-monophosphate) phosphatase activities; Ins(1,4,5)P3 1-phosphatase is 10-fold more sensitive to Li+ (half-maximal inhibition at about 0.25 mM) than are the other phosphatases (half-maximal inhibition at about 2.5 mM). Ins(1,4,5)P3 5-phosphatase activity is potently inhibited by 2,3-bisphosphoglycerate (half-maximal inhibition at 3 microM). Furthermore, 2,3-bisphosphoglycerate also inhibits dephosphorylation of Ins(4,5)P2. These characteristics point to a number of similarities between Dictyostelium phospho-inositol phosphatases and those from higher organisms. The presence of an hitherto undescribed Ins(1,4,5)P3 1-phosphatase, however, causes the formation of a different inositol bisphosphatase isomer [Ins(4,5)P2] from that found in higher organisms [Ins(1,4)P2]. The high sensitivity of some of these phosphatases for Li+ suggests that they may be the targets for Li+ during the alteration of cell pattern by Li+ in Dictyostelium.     

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.     

The Croonian lecture, 1988. Inositol lipids and calcium signalling

The response of cells to many external stimuli requires a decoding process at the membrane to transduce information into intracellular messengers. A major decoding mechanism employed by a variety of hormones, neurotransmitters and growth factors depends on the hydrolysis of a unique inositol lipid to generate two key second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Here I examine the second messenger function of Ins(1,4,5)P3 in controlling the mobilization of calcium. We know most about how this messenger releases calcium from internal reservoirs but less is known concerning the entry of external calcium. One interesting possibility is that Ins(1,4,5)P3 might function in conjunction with its metabolic product Ins(1,3,4,5)P4 to control calcium entry through a mechanism employing a region of the endoplasmic reticulum as a halfway house during the transfer of calcium from outside the cell into the cytoplasm. The endoplasmic reticulum interposed between the plasma membrane and the cytosol may function as a capacitor to insure against the cell being flooded with external calcium. When stimulated, cells often display remarkably uniform oscillations in intracellular calcium. At least two oscillatory patterns have been recognized suggesting the existence of separate mechanisms both of which may depend upon Ins(1,4,5)P3. In one mechanism, oscillations may be driven by periodic pulses of Ins(1,4,5)P3 produced by receptors under negative feedback control of protein kinase C. The other oscillatory mechanism may depend upon Ins(1,4,5)P3 unmasking a process of calcium-induced calcium release from the endoplasmic reticulum. The function of these calcium oscillations is still unknown. This Ins(1,4,5)P3/calcium signalling system is put to many uses during the life history of a cell.(ABSTRACT TRUNCATED AT 250 WORDS)