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At least 163 records · Page 9Linked to original sources

PubMed1993

Pharmacological differentiation of metabotropic glutamate receptors coupled to potentiation of cyclic adenosine monophosphate responses and phosphoinositide hydrolysis.

Activation of metabotropic glutamate receptors (mGluRs) results in multiple second messenger responses in rat hippocampal slices, including stimulation of phosphoinositide hydrolysis and potentiation of cyclic AMP responses induced by agonists of other receptors that are directly coupled to adenylate cyclase. Alpha 1 adrenergic receptors and H1-histaminergic receptors are similar to mGluRs in that agonists of these receptors also induce both phosphoinositide hydrolysis and potentiation of cyclic AMP responses to other agonists. In each of these cases, it is not clear whether activation of phosphoinositide hydrolysis and potentiation of cyclic AMP responses are mediated by the same or different receptor subtypes. In the present studies, the pharmacological profiles of mGluR-mediated potentiation of cyclic AMP responses and mGluR-mediated activation of phosphoinositide hydrolysis were compared to determine whether these responses are mediated by the same or distinct receptor subtypes. In addition, the authors determined the effect of mGluR activation on cyclic AMP responses in various regions of the rat brain and at different stages of postnatal development. It was found that the rank order of efficacies and potencies of mGluR agonists for potentiating cyclic AMP responses is distinct from the rank order of efficacies and potencies of the same compounds at stimulating phosphoinositide hydrolysis. Furthermore, L-serine-O-phosphate competitively blocked mGluR-mediated potentiation of cyclic AMP responses but had little or no effect on activation of phosphoinositide hydrolysis by the active isomer of trans-1-aminocyclopentane-1,3-dicarboxylic acid. These data are consistent with the hypothesis that these two responses are mediated by distinct mGluR subtypes.

Animals↗

PubMed2004

Potentiation of paclitaxel cytotoxicity in lung and esophageal cancer cells by pharmacologic inhibition of the phosphoinositide 3-kinase/protein kinase B (Akt)-mediated signaling pathway.

BACKGROUND: Constitutive activation of the phosphoinositide 3-kinase/protein kinase B survival signal transduction pathway influences the intrinsic chemoresistance of cancer cells. This study evaluates the effect of LY294002, a pharmacologic inhibitor of phosphoinositide 3-kinase, on the sensitivity of lung and esophageal cancer cells to paclitaxel (Taxol) in vitro. Materials and methods Cell viability and apoptosis of cancer cells treated with paclitaxel + LY294002 combinations were quantitated by methyl-thiazol-diphenyl-tetrazolium and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling-based ApoBrdU assays, respectively. The effect of LY294002-mediated phosphoinositide 3-kinase inhibition on protein kinase B (Akt) activation and nuclear factor-kappaB signaling was determined by Western blot analysis. Nuclear factor-kappaB transcription activity in cultured cancer cells either at baseline or after treatments with LY294002 or BAY11-0782 (a pharmacologic inhibitor of nuclear factor-kappaB) was determined by the nuclear factor-kappaB-Luciferase reporter system. RESULTS: A 4- to more than 20-fold reduction of paclitaxel IC(50) values was observed in cancer cells treated with paclitaxel + LY294002 combinations. This was paralleled with synergistic induction of apoptosis. LY294002 treatment caused a significant dose-dependent inhibition of protein kinase B (Akt) activation and suppression of nuclear factor-kappaB transcriptional activity that was accompanied by elevation of IkappaB, the intrinsic inhibitor of nuclear factor-kappaB, and concomitant reduction of nuclear factor-kappaB-regulated antiapoptotic proteins cIAP1, cIAP2, and BclXL. Direct inhibition of nuclear factor-kappaB activity by BAY11-0782 also resulted in profound enhancement of paclitaxel sensitivity and paclitaxel-mediated induction of apoptosis in lung and esophageal cancer cells. CONCLUSION: LY294002-mediated inhibition of the phosphoinositide 3-kinase/protein kinase B-dependent survival pathway with secondary suppression of nuclear factor-kappaB transcriptional activity was associated with enhancement of paclitaxel cytotoxicity in lung and esophageal cancer cells. Direct inhibition of nuclear factor-kappaB by BAY11-0782 also sensitized these cancer cells to paclitaxel, indicating that nuclear factor-kappaB may be the crucial intermediary step connecting phosphoinositide 3-kinase/protein kinase B (Akt) to the intrinsic susceptibility of cancer cells to chemotherapeutic agents.

Antineoplastic Agents, Phytogenic↗

PubMed1999

Akt/PKB localisation and 3' phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction.

Protein kinase B (PKB or Akt) is a mitogen-regulated protein kinase involved in the protection of cells from apoptosis, the promotion of cell proliferation and diverse metabolic responses [1]. Its activation is initiated by the binding of 3' phosphorylated phosphoinositide lipids to its pleckstrin homology (PH) domain, resulting in the induction of activating phosphorylation at residues Thr308 and Ser473 by upstream kinases such as phosphoinositide-dependent protein kinase-1 (PDK1) [2]. Adhesion of epithelial cells to extracellular matrix leads to protection from apoptosis via the activation of phosphoinositide (PI) 3-kinase and Akt/PKB through an unknown mechanism [3] [4]. Here, we use the localisation of Akt/PKB within the cell to probe the sites of induction of PI 3-kinase activity. In fibroblasts, immunofluorescence microscopy showed that endogenous Akt/PKB localised to membrane ruffles at the outer edge of the cell following mitogen treatment as did green fluorescent protein (GFP) fusions with full-length Akt/PKB or its PH domain alone. In epithelial cells, the PH domain of Akt/PKB localised to sites of cell-cell and cell-matrix contact, distinct from focal contacts, even in the absence of serum. As this localisation was disrupted by PI 3-kinase inhibitory drugs and by mutations that inhibit interaction with phosphoinositides, it is likely to represent the sites of constitutive 3' phosphoinositide generation that provide a cellular survival signal. We propose that the attachment-induced, PI-3-kinase-mediated survival signal in epithelial cells is generated not only by cell-matrix interaction but also by cell-cell interaction.

3T3 Cells↗

PubMed2001

CD28 co-stimulates TCR/CD3-induced phosphoinositide turnover in human T lymphocytes.

Upon engagement of TCR with peptide-MHC complexes displayed on the surface of antigen-presenting cells, T lymphocytes undergo a sustained elevation of intracellular Ca(2+) concentration([Ca(2+)](i)), which is required for cytokine production. In the present work, we investigate how inositol lipid metabolism can be activated for a prolonged time to ensure a sustained link between receptor triggering and downstream signaling effectors. Four lines of evidence indicate that an extensive phosphoinositide turnover induced by TCR and CD28 engagement allows this task to be accomplished: (i) continuous phosphoinositide breakdown is required for a sustained [Ca(2+)](i )increase in antigen-stimulated T cells; (ii) TCR triggering results in a continuous release of inositol phosphates from the cell membrane paralleled by a massive and sustained phosphoinositide re-synthesis due to free inositol re-incorporation; (iii) TCR-induced phosphoinositide turnover is strongly increased by CD28 ligation; and (iv) CD28 engagement augments and sustains the TCR-induced [Ca(2+)](i )increase. Our results show that the T cell pool of phosphoinositides is continuously re-formed during T cell-APC cognate interaction, thereby explaining how sustained receptor triggering can transduce an equally sustained [Ca(2+)](i) increase. Importantly, our data identify a novel step in the signaling cascade where co-stimulation converges with TCR-generated signals to sustain and amplify the activation process.

CD28 Antigens↗

PubMed1990

Phosphoinositide metabolism in human prostate cancer cells in vitro.

To understand better the mechanism by which 5-alpha-dihydrotestosterone (5-alpha-DHT) influences prostate epithelial cell function, we examined the effects of 5-alpha-DHT on phosphoinositide metabolism in human prostate cancer cell lines. Androgen receptor-positive LN-CaP cells showed dose-responsive, steady-state elevations in phosphoinositide metabolism when treated with 5-alpha-DHT. The intracellular pool of 3H-myoinositol decreased and the incorporation of 3H-myoinositol into cellular lipids increased with increasing concentrations of 5-alpha-DHT. 5-alpha-DHT increased the release of 3H-inositol phosphates into the media. The inactive stereoisomer, 5-beta-DHT, did not increase phosphoinositide metabolism. In androgen receptor-negative cells, phosphoinositide metabolism was not altered by 5-alpha-DHT. The slow induction of phosphoinositide metabolism by 5-alpha-DHT suggests that the effects may be mediated through other factors that serve as intermediates in 5-alpha-DHT modulation of intracellular signalling. We conclude that this modulation involves increased turnover of phosphatidylinositol, incorporation of myoinositol into cellular lipids, and alterations in the aqueous intracellular myoinositol pool size, possibly as a result of altered transport mechanisms.

Androgen Antagonists↗

PubMed1994

Wortmannin inhibits serum-induced activation of phosphoinositide 3-kinase and proliferation of CHRF-288 cells.

Activated phosphoinositide 3-kinase has been suggested to be involved in cytoskeletal reorganization and mitogenesis. Lysophosphatidic acid has been found to trigger several "classic" signal transduction pathways and also accounts for the ability of serum to stimulate focal adhesion and stress fiber formation in fibroblasts. We present evidence that serum or lysophosphatidic acid activates phosphoinositide 3-kinase in CHRF-288 cells (a leukemic cell line derived from megakaryoblasts), leading to transient accumulation of phosphatidylinositol(3,4,5)P3 and increased phosphatidylinositol(3,4)P2, and stimulates phospholipase C. Exposure of CHRF cells to serum promotes cell proliferation, whereas exposure to lysophosphatidic acid does not. Wortmannin, a potent inhibitor of phosphoinositide 3-kinase, inhibits 3-phosphorylated phosphoinositide accumulation and cell proliferation without inhibiting phospholipase C. We propose that activation of phosphoinositide 3-kinase is required for the full proliferative response of CHRF cells exposed to serum but, as gauged by our findings for lysophosphatidic acid, not sufficient to induce proliferation.

Androstadienes↗

PubMed1993

Homologous desensitization of the endothelin-1 receptor mediated phosphoinositide response in cultured neonatal rat cardiomyocytes.

The goal of the present study was to identify the molecular mechanism underlying desensitization of endothelin-1 receptor-mediated phosphoinositide response in cultured neonatal rat heart cells. Endothelin elicited a concentration-dependent (EC50 = 2.2 x 10(-9) M) increase of inositol-phosphate production with a much higher potency than phenylephrine (EC50 = 1.4 x 10(-6) M). Endothelin-1 (10(-8) M) evoked phosphoinositide turnover in the presence of 10 mM LiCl, which was greatly attenuated after 30-45 min of continuous stimulation with agonist, apparently resulting in a total absence of further inositol-phosphate accumulation. However, when the uncompetitive inositol monophosphatase inhibitor Li+ was only present during the last 30 min of 150 min incubation, the inositol-phosphate accumulation was decreased to a steady state of 33% of the initial rate. The loss of responsiveness of cardiomyocytes to endothelin-1 was not brought about by a limiting supply of phospholipase C substrate phosphatidylinositol 4,5-bisphosphate. A very rapid resynthesis of this substrate took place as its level remained almost constant during 45 min stimulation with 10(-8) M endothelin-1 while the accumulation of inositol-phosphates was at least 15-fold higher than the initial cellular phosphatidylinositol 4,5-bisphosphate content. After 120 min preincubation of cells with 10(-9) M endothelin-1 the activation of phospholipase C by a second higher dose (10(-8) M) was severely (67%) inhibited at the same time leaving the induction of phosphoinositide turnover by phenylephrine (10(-4) M) virtually intact. Preincubation with phenylephrine (3 x 10(-6) M) also led to inhibition of the phenylephrine (10(-4) M)-mediated inositol-phosphate response (36% inhibition) while the endothelin-1 (10(-8) M) response was not affected. Addition of a direct activator of protein kinase C, phorbol 12-myristate 13-acetate, led to inhibition of the endothelin-1 evoked phosphoinositide turnover but the rate of desensitization was not affected. Inhibition of protein kinase C with staurosporine did not alter the time course of desensitization. In conclusion, the activity of the phosphoinositide cycle in cardiomyocytes is homologously desensitized after stimulation with endothelin-1. The desensitization is not likely to be due to either depletion of phospholipase C substrate or to the activation of protein kinase C by inositol 1,4,5-trisphosphate-mobilized Ca2+ and elevated 1,2-diacylglycerol levels.

Animals↗

PubMed1991

BW755C or staurosporine inhibits collagen-stimulated phosphoinositide phosphorylation in platelets.

Stimulation of platelets by collagen results in increased formation of the polyphosphoinositides, phosphatidylinositol phosphate (PtdInsP) and phosphatidylinositol bisphosphate (PtdInsP2) through stimulation of phosphoinositide kinase activities. We investigated a possible regulatory role of endogenous thromboxane formation and protein kinase C (PKC) activation in the induction of phosphoinositide phosphorylation following collagen stimulation, as well as following stimulation by the thromboxane mimetic, U-46619. Human platelets were prelabeled with [3H]inositol and stimulated with collagen (2 micrograms/mL) or U-46619 (1 microM), in the absence or presence of either the cyclo-oxygenase/lipoxygenase inhibitor, BW755C, or staurosporine, a putative inhibitor or PKC. Collagen stimulation resulted in a time-dependent increase in [3H]inositol-labeled PtdInsP and PtdInsP2 which was completely inhibited in the presence of BW755C. Addition of U-46619 to BW755C-treated, collagen-stimulated platelets restored the increased polyphosphoinositide formation. Stimulation of platelets with U-46619 alone also resulted in increased formation of [3H]PtdInsP and [3H]PtdInsP2, but this was not affected by the presence of BW755C. These results suggest that the collagen-induced activation of phosphoinositide kinases was dependent upon thromboxane formation, but that U-46619-induced phosphoinositide formation was rather independent of further thromboxane production. Pretreatment of platelets with staurosporine, prior to agonist addition, completely blocked the collagen-stimulated rise in radiolabeled PtdInsP and the U-46619-induced PtdInsP and PtdInsP2 generations, suggesting that protein kinase, possibly PKC, may play a role in the activation of phosphoinositide kinases by these agonists.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1991

Role of calcium in regulation of phosphoinositide signaling pathway.

Using primary neuronal cultures we have examined the role of extracellular Ca2+ in a receptor-regulated phosphoinositide turnover. We report that receptor (glutamic acid and acetylcholine)-activated phosphoinositide turnover requires the presence of extracellular Ca2+ (EC50 = 21.1 microM). The requirement for Ca2+ appears to be at an intracellular level and is highly selective for Ca2+. We also found that several inorganic and organic Ca2+ channel blockers, including La3+ and verapamil, inhibit phosphoinositide turnover. However, the pharmacological profile of these agents in this regard was distinct from their actions at the voltage-sensitive Ca2+ channels. To explain the above requirement for extracellular Ca2+ in agonist-stimulated phosphoinositide turnover and its sensitivity to Ca(2+)-channel blockers, we propose a hypothetical model suggesting that Ca2+, following IP-3-mediated mobilization, exerts a facilitatory action on the activity of receptor-phospholipase C complex. We further propose that in the absence of extracellular Ca2+ or in the presence of certain Ca(2+)-channel blockers, refilling of calciosomes is ineffectual or inhibited, causing its depletion and subsequent inactivation of agonist-stimulated phosphoinositide turnover.

Animals↗

PubMed1991

Fatty acid pattern of the different phosphoinositide fractions in human meningiomas.

Previous studies showed no differences in the phospholipid content of human meningiomas compared to normal leptomeninges, but only a higher unsaturation degree in the individual phospholipid fractions of tumors. Inasmuch as phosphoinositides play a role in the membrane responsiveness to numerous effectors, we studied the fatty acid pattern of the different phosphoinositide fractions of 14 human meningiomas of different histological origin. The fatty acid analysis revealed remarkable differences among the histological types, and, above all, among the different phosphoinositide fractions of a single tumor class. The phosphoinositides derived from transitional meningiomas appeared to be the most saturated ones, because of their low arachidonic acid content. Furthermore, in all the meningiomas, long chain polyunsaturated fatty acids were present only in the phosphatidylinositol fractions and the polyphosphorylated compounds appeared to be significantly different from the corresponding monophosphorylated ones. The possible significance of the different fatty acid distribution in the three phosphoinositide classes is discussed.

Adult↗

PubMed1985

[3H]Inositol incorporation into phosphoinositides of pig reticulocytes.

Phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) of pig reticulocytes were extensively labelled when these cells were incubated with [3H]inositol. In marked contrast, a total lack of [3H]inositol labelling of phosphoinositides was observed in mature erythrocytes. Phosphoinositides of both reticulocytes and mature erythrocytes were labelled with 32P but the labelling in reticulocytes was several-fold higher than in mature erythrocytes. Inclusion of Ca2+ (2 mM)+ ionophore A23187 (2 micrograms/ml) during the labelling experiments substantially reduced the radioactivity incorporation into phosphoinositides of reticulocytes. When [3H]inositol-prelabelled reticulocytes were treated with Ca2+ + A23187 the levels of radioactive PI and PIP2 did not change significantly. However, the PIP pool exhibited a remarkable sensitivity to Ca2+ as shown by a 75% increase in its radioactivity over the control. The ability to incorporate [3H]inositol into phosphoinositides remains transitorily intact in the reticulocyte stage. Thus, pig reticulocytes offer a suitable model in which to explore the physiological role of phosphoinositides in relation to cellular maturation process.

Animals↗

PubMed1986

Evidence that phosphoinositide response is mediated by alpha 1-adrenoceptor stimulation, but not linked with excitation-contraction coupling in cardiac muscle.

Phosphoinositide metabolism is known to be associated with neuronal or humoral stimulation of excitable cells. The present study examined whether the phosphoinositide response is involved in such events using isolated rat papillary muscles labeled with [3H]inositol. It was found that neither increase in the stimulation frequencies (0-2 Hz) nor prolongation of the pulse duration (10-70 msec) altered the labeling of phosphoinositides and the accumulation of [3H]inositol phosphates in this preparation. However, phenylephrine, a known alpha 1-agonist, was capable of provoking the breakdown of phosphoinositides associated with a positive inotropic effect in this preparation. We report the evidence that phosphoinositide response is mediated by alpha 1-adrenoceptor stimulation, but not linked with excitation-contraction coupling in cardiac muscle.

Animals↗

PubMed1986

Complete dissociation between the activation of phosphoinositide turnover and of NADPH oxidase by formyl-methionyl-leucyl-phenylalanine in human neutrophils depleted of Ca2+ and primed by subthreshold doses of phorbol 12,myristate 13,acetate.

Evidences have been provided by many laboratories that the activation of the NADPH oxidase in neutrophils by formyl-methionyl-leucyl-phenylalanine (FMLP) is strictly linked to a transduction pathway that involves the stimulation, via GTP binding protein, of the phosphoinositide turnover and the increase in [Ca2+]i. The results presented in this paper demonstrate that FMLP can activate the NADPH oxidase by triggering a transduction pathway completely independent of phosphoinositide turnover and Ca2+ changes. In fact: i) Ca2+-depleted neutrophils do not respond to FMLP with the activation of phosphoinositide hydrolysis and NADPH oxidase. Both the responses are restored by the addition of exogenous Ca2+. ii) In Ca2+-depleted neutrophils phorbol-myristate-acetate (PMA) activates the NADPH oxidase. iii) The pretreatment of Ca2+-depleted neutrophils with non stimulatory doses of PMA restores the activation of the NADPH oxidase but not of the turnover of phosphoinositides by FMLP. This priming effect of PMA and the role of this phosphoinositide and Ca2+-independent pathway for the stimulation of the NADPH oxidase by receptors mediated stimuli are discussed.

Calcium↗

PubMed1989

Inhibition of receptor-coupled phosphoinositide hydrolysis by sulfur-containing amino acids in rat brain slices.

Sulfur-containing amino acids were found to inhibit norepinephrine-stimulated [3H]phosphoinositide hydrolysis in rat cortical slices. Of the amino acids tested, L-cysteine was the most potent, inhibiting the response by 42 and 85% at concentrations of 50 and 500 microM respectively. L-Cystine and L-serine-O-sulfate also inhibited the response to norepinephrine, but to a lesser degree than did L-cysteine. L-Homocysteic acid slightly potentiated phosphoinositide hydrolysis at a concentration of 100 microM, but caused inhibition at 500 microM. L-Cysteine sulfinate produced effects intermediate to those of L-cysteine and L-homocysteic acid, having no effect on the response to norepinephrine at 50 microM, but causing 84% inhibition at 500 microM. The D-isomers of cysteine and homocysteic acid were much less potent than were the L-isomers. Examination of the time course of the inhibition of norepinephrine-stimulated [3H]phosphoinositide hydrolysis by L-cysteine showed that it was inhibited almost completely after 15, 30, 45 and 60 min of incubation. L-Cysteine and L-homocysteic acid caused similarly strong inhibitions of the production of [3H]inositol monophosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. The hydrolysis of [3H]phosphoinositides stimulated by norepinephrine in slices from rat hippocampus and striatum were inhibited by L-cysteine to an extent similar to that occurring in cortical slices. These results demonstrate that several sulfur-containing amino acids, some of which have been proposed to be endogenous excitatory amino acid neurotransmitters, effectively modulate the response to norepinephrine of the phosphoinositide second messenger system in rat brain.

Amino Acids, Sulfur↗

PubMed1991

Differential effects of propranolol on the IgE-dependent, or calcium ionophore-stimulated, phosphoinositide hydrolysis and calcium mobilization in a mast (RBL 2H3) cell line.

Our previous studies demonstrated that propranolol, an inhibitor of phosphatidic acid phosphohydrolase (PAPase) (EC 3.1.3.4) blocks the IgE-dependent mediator release from a rat mast (RBL 2H3) cell line. To continue these studies, we examined the ability of propranolol to inhibit the IgE-dependent or ionomycin-mediated phosphoinositide hydrolysis and calcium mobilization in RBL 2H3 cells. RBL 2H3 cells, sensitized with mouse monoclonal anti-trinitrophenol IgE (anti-TNP IgE), were stimulated to release both histamine and peptidoleukotrienes (LT) in response to a suboptimal concentration of trinitrophenol-ovalbumin conjugate (TNP-OVA) or ionomycin. Preincubation of the cells with d,l-propranolol (300 microM) significantly (P less than 0.05) inhibited the effects of both TNP-OVA and ionomycin on histamine and LT release. There was no difference in potency for the different isomers of propranolol, indicating that these effects were not a consequence of an effect on beta 2-adrenergic receptors. TNP-OVA produced a rapid hydrolysis of phosphoinositides resulting in a time-dependent increase in mono- (IP1), di- (IP2), tri- (IP3), and total inositol phosphate production. Ionomycin also produced a rapid increase in total inositol phosphate production; however, this largely reflected an accumulation of IP1. Both secretagogues produced a rapid elevation in cytosolic free calcium ([Ca2+]i); however, the effect of ionomycin maximized within a much shorter time frame than the effect of TNP-OVA. The effects of TNP-OVA on phosphoinositide hydrolysis and increase in [Ca2+]i were inhibited by propranolol over exactly the same concentration range as the effects of this compound on TNP-OVA-stimulated mediator release. In contrast, propranolol had no effect on the increase in [Ca2+]i and phosphoinositide hydrolysis in response to ionomycin. Taken together, these results suggest that PAPase/phospholipase D (PLD) (EC 3.1.4.4) activation may be a prerequisite for both IgE-dependent and ionomycin-stimulated mediator release from RBL 2H3 cells. Although other explanations are possible, the data further suggest that receptor-mediated, but not ionophore-stimulated, phosphoinositide hydrolysis and [Ca2+]i in RBL 2H3 cells may be regulated by a propranolol-sensitive pathway involving possible activation of PAPase.

Animals↗

PubMed1992

Chlorpromazine increases the turnover of metabolically active phosphoinositides and elevates the steady-state level of phosphatidylinositol-4-phosphate in human platelets.

Non-permeabilizing concentrations (< 40 microM) of chlorpromazine (CPZ) increase the radioactivity of phosphatidylinositol-4-phosphate (PIP) in platelets pre-labelled with [32P]Pi, but the biochemical mechanisms underlying this increase are poorly understood. Incubation of [32P]Pi-labelled, gel-filtered platelets with 25 microM CPZ for 10 min increased: (1) the mass of PIP from 315 to 476 nmol/10(11) platelets but not the total inositol phospholipid mass, (2) the specific phosphodiester radioactivities in phosphatidylinositol (PI), PIP and phosphatidylinositol-4,5-bisphosphate (PIP2) by 34, 63 and 37%, respectively, and (3) the specific phosphomonoester radioactivities in PIP and PIP2 by 53 and 10%, respectively. In control platelets (no CPZ) the specific radioactivity of the phosphodiester was the same in PI, PIP and PIP2, and the specific radioactivity in the phosphomonoester in PIP and PIP2 was 55% of that of the gamma-phosphoryl in ATP, measured as metabolically active, actin-bound ADP. These results suggest that 55% of each of PI, PIP and PIP2 constitutes a metabolic pool which is labelled by 32P in the platelets, while the remainder is in a metabolically inactive pool and not labelled. CPZ has two major effects: (1) CPZ interferes with the kinase and phosphohydrolase reactions that maintain the steady-state level of PIP in the metabolic phosphoinositide pool, resulting in a 92% increase in the PIP level of this pool, and (2) CPZ causes synthesis (45% in 10 min) of new phosphodiester in the metabolically active phosphoinositides by tentative stimulation of the turnover of the phosphoinositide cycle, de novo phosphoinositide synthesis and/or diacylglycerol formation through phospholipases C and D. The marked alteration by CPZ of phosphoinositide metabolism may be part of the mechanism by which this drug effects its psychotropic action.

Adenosine Diphosphate↗

PubMed1992

Adrenalectomy increases phosphoinositide hydrolysis induced by norepinephrine or excitatory amino acids in rat hippocampal slices.

Phosphoinositide hydrolysis induced by norepinephrine, quisqualate, or trans-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD), but not by carbachol, was approximately 50% greater in hippocampal slices from adrenalectomized (14 days) rats compared with controls. These changes appeared to be selective for the hippocampus because no effects of adrenalectomy on phosphoinositide hydrolysis were detected in cortical or striatal slices. The enhanced response to norepinephrine in hippocampal slices after adrenalectomy was observed throughout the effective concentration range of norepinephrine, was not influenced by in vitro addition of corticosterone, was not mimicked or altered by incubation with dibutyryl cyclic adenosine 3',5'-monophosphate (AMP), and did not appear to be due to impaired inhibition of the response to norepinephrine which was elicited by activation of protein kinase C or by inclusion of an inhibitory concentration of quisqualate. These findings indicate that adrenalectomy either removes an inhibitory influence of glucocorticoids on the phosphoinositide system in the hippocampus or that the neurodegeneration of granule cells in the dentate gyrus following adrenalectomy is associated with neurotransmitter-selective increases in phosphoinositide hydrolysis. These data provide further evidence that glucocorticoids modify signal transduction in the brain and extends their known influence to the phosphoinositide second messenger system.

Adrenalectomy↗

PubMed1988

Differential effects of maitotoxin on ATP secretion and on phosphoinositide breakdown in rat pheochromocytoma cells.

Maitotoxin (MTX) induced exocytotic secretion of ATP from PC12 rat pheochromocytoma cells. The threshold for stimulation of secretion was at concentrations of about 2 ng/ml of MTX. Maximal release occurred at 40 ng/ml. MTX-induced ATP release required the presence of calcium in the extracellular medium and could be inhibited by nifedipine, a specific blocker of voltage-dependent calcium channels. In addition to the effects on ATP secretion from PC12 cells, MTX stimulated the breakdown of phosphoinositides, as measured by the accumulation of [3H]inositol phosphates. Maximal stimulation of phosphoinositide breakdown was reached at only 0.5-1.0 ng/ml MTX. MTX at concentrations required to evoke ATP release (greater than 2 ng/ml) had lesser or no effect on phosphoinositide breakdown. Although stimulation of phosphoinositide breakdown by MTX was dependent on extracellular calcium, it was insensitive to the calcium channel blockers nifedipine, D-600 and cobalt ions. The different concentration range required to elicit these responses and the varying sensitivity to calcium channel blockers indicate that MTX-evoked secretion and MTX-stimulated phosphoinositide breakdown are independent phenomena in PC12 cells.

Adenosine Triphosphate↗