Inhibition Of Phosphoinositide Hydrolysis Research Articles

Inhibition of Gαq-dependent PLC-β1 activity by PKG and PKA is mediated by phosphorylation of RGS4 and GRK2

In smooth muscle of the gut, G(q)-coupled receptor agonists activate preferentially PLC-beta1 to stimulate phosphoinositide (PI) hydrolysis and inositol 1,4,5-trisphosphate (IP(3)) generation and induce IP(3)-dependent Ca(2+) release. Inhibition of Ca(2+) mobilization by cAMP- (PKA) and cGMP-dependent (PKG) protein kinases reflects inhibition of PI hydrolysis by both kinases and PKG-specific inhibitory phosphorylation of IP(3) receptor type I. The mechanism of inhibition of PLC-beta1-dependent PI hydrolysis has not been established. Neither G(q) nor PLC-beta1 was directly phosphorylated by PKA or PKG in gastric smooth muscle cells. However, both kinases 1) phosphorylated regulator of G protein signaling 4 (RGS4) and induced its translocation from cytosol to plasma membrane, 2) enhanced ACh-stimulated association of RGS4 and Galpha(q).GTP and intrinsic Galpha(q).GTPase activity, and 3) inhibited ACh-stimulated PI hydrolysis. RGS4 phosphorylation and inhibition of PI hydrolysis were blocked by selective PKA and PKG inhibitors. Expression of RGS4(S52A), which lacks a PKA/PKG phosphorylation site, blocked the increase in GTPase activity and the decrease in PI hydrolysis induced by PKA and PKG. Blockade of PKA-dependent effects was only partial. Selective phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which contains a RGS domain, by PKA augmented ACh-stimulated GRK2:Galpha(q).GTP association; both effects were blocked in cells expressing GRK2(S685A), which lacks a PKA phosphorylation site. Inhibition of PI hydrolysis induced by PKA was partly blocked in cells expressing GRK2(S685A) and completely blocked in cells coexpressing GRK2(S685A) and RGS4(S52A) or Galpha(q)(G188S), a Galpha(q) mutant that binds GRK2 but not RGS4. The results demonstrate that inhibition of PLC-beta1-dependent PI hydrolysis by PKA is mediated via stimulatory phosphorylation of RGS4 and GRK2, leading to rapid inactivation of Galpha(q).GTP. PKG acts only via phosphorylation of RGS4.

Stimulatory effect of endothelin-1 on Na-dependent phosphate transport and its signaling mechanism in osteoblast-like cells.

Endothelin-1 (ET-1) has been reported to modulate bone metabolism both in vivo and in vitro. In the present study, we investigated the effect of ET-1 on inorganic phosphate (Pi) transport in osteoblast-like cells, which is now considered to be important for the initiation of bone matrix calcification. ET-1 time- and dose-dependently stimulated Na-dependent Pi transport in mouse calvaria-derived osteoblast-like MC3T3-E1 cells, and this effect was dependent on transcriptional and translational process. Kinetic analysis indicated that the change in Pi transport activity induced by ET-1 was due to alteration in the number of the Pi transporter. BQ123, a selective antagonist for ET(A) receptor, suppressed the ET-1-induced Pi transport, but BQ788, a selective antagonist for ET(B) receptor, had no effect. The inhibition of phosphoinositide hydrolysis by phospholipase C (PLC) partially attenuated the Pi transport by ET-1. Propranolol, which inhibits phosphatidic acid phosphohydrolase, also suppressed ET-1-induced Pi transport. On the contrary, indomethacin did not affect the stimulatory effect of Pi transport by ET-1. Calphostin C, a protein kinase C (PKC) inhibitor, significantly blunted the stimulatory effect of ET-1 on Pi transport. Combined effect of PMA and ET-1 on Pi transport was not additive. Pi transport induced by ET-1 was also suppressed in PKC down-regulated cells. In conclusion, the results of the present study indicate that in MC3T3-E1 osteoblast-like cells, ET-1 acting through ET receptor links to a stimulation of Pi transport via activation of PKC through both phosphoinositide and phosphatidylcholine hydrolyses.

Inhibition of Phosphoinositide Metabolism or Chelation of Intracellular Calcium Blocks FSH-Induced but Not Spontaneous Meiotic Resumption in Mouse Oocytes

Mammalian oocytes are arrested at the diplotene phase of the first meiotic division until ovulation. In the mouse, germinal vesicle breakdown (GVBD) and progression to metaphase II is thought to be triggered by a positive signal originating in the follicular cells following stimulation by the luteinizing hormone (LH) surge. Isolated, fully grown oocytes can also undergo spontaneous reinitiation of meiosisin vitroin the absence of gonadotrophin stimulation. To investigate the mechanism of meiotic resumption, inhibitors of phosphoinositide metabolism and an intracellular calcium chelator were used during maturationin vitrounder different conditions. In a series of experiments, isolated cumulus cell–oocyte complexes (COCs) maintained in meiotic arrest by hypoxanthine were induced to resume meiosis by treatment with follicle-stimulating hormone (FSH). Under these conditions, both LiCl and neomycin, which inhibit phosphoinositide hydrolysis, produced a dose-dependent inhibitory effect on meiotic resumption. Similar results were obtained when FSH-induced meiotic resumption was observed in the presence of the acetoxymethyl ester form of 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA/AM), an intracellular calcium chelator. In hypoxanthine-arrested oocytes, GVBD induced by epidermal growth factor (EGF), which mimics FSH action inin vitromaturation, was also repressed by LiCl and neomycin. Conversely, meiotic resumption triggered by a pulse of 8-bromo-cyclic adenosine monophosphate (8-Br cAMP) was not affected by these two inhibitors. In experiments in which oocytes were cultured under conditions which permit spontaneous meiotic maturation, resumption of meiosis was not affected by either inhibition of phosphoinositide hydrolysis or chelation of intracellular calcium. Therefore, it appears that meiotic resumption induced by hormone stimulation requires activation of the phosphoinositide pathway and mobilization of intracellular calcium. In contrast, spontaneous maturation probably occurs through a different mechanism because it is not affected by inhibition of this signaling pathway.

Inhibition of phosphoinositide hydrolysis and cell growth of Swiss 3T3 cells by myristoylated phospholipase C inhibitor peptides.

It has been demonstrated that the phospholipase C-gamma (PLC-gamma) molecule contains within it a phospholipase C inhibitor (PCI) region and that synthetic peptides based on the sequence of this region (PCI peptides) suppress the enzymatic activity of PLC isoforms [Y. Homma and T. Takenawa (1992) J. Biol. Chem. 267, 21884-21889]. In order to improve the permeability of the plasma membrane to PCI peptides, we synthesized myristoylated PCI peptides, myr-GLYRKAMRLRYPV [myr-PCI(Y)] and myr-GLFRKAMRLRFPV [myr-PCI(F)], which are identical except for the replacement of the two tyrosine residues in myr-PCI(Y) by phenylalanines in myr-PCI(F), and examined their inhibitory activity on PLC enzymes in vitro and in vivo. This fatty acid modification potentiated the inhibitory activity of the original PCI peptides and both myr-PCI(Y) and myr-PCI(F) suppressed the PIP2-hydrolyzing activity of purified PLC isoforms in vitro. The Ki values of myr-PCI(Y) and myr-PCI(F) for purified PLC-gamma1 were 3.5 and 55 microM, respectively. Myr-PCI(Y) at concentrations in the sub-micromolar range significantly suppressed IP3 formation induced by EGF, PDGF, bombesin, or serum in Swiss 3T3 cells. Furthermore, myr-PCI(Y) also strongly inhibited cell proliferation induced by these stimuli. The inhibitory effect on IP3 formation and proliferation of myr-PCI(F) was much less potent than that of myr-PCI(Y). These results suggest that myristoylated PCI peptides could be applied to living cells as specific inhibitors of PLC signaling pathways and that PLC pathways are at least in part required for growth in Swiss 3T3 cells.

Thromboxane A 2 receptor-mediated signal transduction in rabbit aortic smooth muscle cells

1. 1. 9,11-Epithio-11,12-methenothromboxane A 2 (STA 2), a stable analogue of thromboxane A 2 (TXA 2), contracted rabbit aortic smooth muscles (RASM) and accumulated [ 3H]inositol phosphates in cultured RASM cells. The contraction and phosphoinositide hydrolysis were competitively inhibited by TXA 2 receptor antagonists, including ONO NT-126, S-145, SQ29548, KW3635, GR32191B and ONO3708. 2. 2. STA 2 inhibited [ 3H]ONO NT-126 binding in a concentration-dependent manner in membranes derived from cultured aortic smooth muscle cells, but GTPγS, a stable GTP analogue, did not affect STA 2-induced inhibition of [ 3H]ONO NT-126 binding. 3. 3. The time course analysis revealed that STA 2 rapidly decreased inositol phosphate level and thereafter increased. Pertussis toxin did not attenuate but rather increased STA 2-induced phosphoinositide hydrolysis. 4. 4. TXA 2 receptor stimulation results in at least two signaling pathways in RASM cells: stimulation and inhibition of phosphoinositide hydrolysis.

Pleckstrin Inhibits Phosphoinositide Hydrolysis Initiated by G-protein-coupled and Growth Factor Receptors. A ROLE FOR PLECKSTRIN'S PH DOMAINS

Pleckstrin is a 40-kDa protein present in platelets and leukocytes that contains two PH domains separated by a 150-residue intervening sequence. Pleckstrin is a major substrate for protein kinase C, but its function is unknown. The present studies examine the effects of pleckstrin on second messenger generation. When expressed in cos-1 or HEK-293 cells, pleckstrin inhibited 1) the G alpha-mediated activation of phospholipase C beta initiated by thrombin, M1-muscarinic acetylcholine, and angiotensin II receptors, 2) the stimulation of phospholipase C beta by constitutively active Gq alpha, 3) the G beta gamma-mediated activation of phospholipase C beta caused by alpha 2A-adrenergic receptors, and 4) the tyrosine phosphorylation-mediated activation of phospholipase C gamma caused by Trk A. However, pleckstrin had no effect on either the stimulation or inhibition of adenylyl cyclase. The inhibition of phosphoinositide hydrolysis caused by pleckstrin was similar in magnitude to that caused by activating protein kinase C with phorbol 12-myristate 13-acetate (PMA). When combined, pleckstrin and PMA had an additive effect, inhibiting phosphoinositide hydrolysis by as much as 90%. Structure-function analysis highlighted the role of pleckstrin's N-terminal PH domain in these events. Although deleting the C-terminal PH domain had no effect, deleting the N-terminal PH domain abolished activity (but not expression) and mutating a highly conserved tryptophan residue within the N-terminal PH domain decreased activity by one-third. Notably, however, a pleckstrin variant in which the N-terminal PH domain was replaced with a second copy of the C-terminal PH domain was nearly as active as native pleckstrin. These results show that: 1) pleckstrin can inhibit pathways leading to both phospholipase C beta- and phospholipase C gamma-mediated phosphoinositide hydrolysis, 2) this inhibition affects activation of phospholipase C beta mediated by either G alpha or G beta gamma, but does not affect the regulation of adenylyl cyclase activity by G alpha or G beta gamma, 3) although pleckstrin is a substrate for protein kinase C, the effects of pleckstrin and PMA are at least partially independent, 4) the inhibition caused by pleckstrin appears to be mediated by the PH domain at the N terminus, rather than the C terminus of the molecule, and 5) location of the two PH domains within the molecule clearly contributes to their individual activity.2+1

Effects of the phospholipase-C inhibitor, U73122, on signaling and secretion in pituitary gonadotrophs.

The effects of inhibition of phosphoinositide hydrolysis by U73122 [1-(6-[17 beta-3-methoxyestra-1,3,5- (10) triene-17-yl] amino/hexyl) 1H-pyrroledione] and neomycin on agonist-stimulated intracellular signaling and secretory responses were analyzed in cultured pituitary cells and alpha T3-1 gonadotrophs. GnRH (100 nM)- and endothelin-1 (ET-1; 100 nM)-induced inositol (1,4,5)-trisphosphate and diacylglycerol formation in normal cells and immortalized gonadotrophs were reduced by U73122 in a concentration-dependent manner, with IC50 values of about 2 microM and complete inhibition at 10 microM U73122. Neomycin also reduced GnRH- and ET-induced inositol phosphate production in both cell types. Agonist-induced intracellular Ca2+ responses were also inhibited in both cell types by U73122 and neomycin at the same concentrations that inhibited their inositol phosphate responses. In cultured pituitary cells, agonist-induced LH release was inhibited by U73122 and neomycin in a dose-dependent manner. In perifused pituitary cells, U73122 completely inhibited GnRH- and ET-1-induced LH release, but after 10 min caused a progressive and substantial increase in basal LH release. In static cultures, U73122 inhibited agonist-induced LH response at low concentrations (up to 3 microM), but stimulated LH release at higher concentrations due to direct activation of exocytosis by the compound. When added alone, U73122 caused a concentration-dependent increase in LH release with an EC50 of about 7 microM and a maximum response similar that that elicited by GnRH. The stimulatory action of U73122 on LH release was not reduced in the absence of extracellular Ca2+. In contrast to cultured pituitary cells, alpha T3-1 gonadotrophs showed only constitutive exocytosis that was not affected by either neomycin or U73122. These results demonstrate that GnRH and ET(A) receptors are coupled to the phosphoinositide/Ca2+ transduction system in pituitary gonadotrophs, and provide evidence for the dependence of agonist-regulated exocytosis on this signaling pathway. The ability of U73122 to stimulate LH release could reflect an additional action of the compound at late steps in the exocytic pathway.

Effect of glucocorticoid on prostaglandin F2α-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2

It is well known that osteoporosis is a common complication of patients with glucocorticoid excess. We showed previously that prostaglandin (PG) F2 alpha stimulates the synthesis of PGE2, a potent bone resorbing agent, and that the activation of protein kinase C amplifies the PGF2 alpha-induced PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of dexamethasone on PGE2 synthesis induced by PGF2 alpha in MC3T3-E1 cells. The pretreatment with dexamethasone significantly inhibited the PGE2 synthesis in a dose-dependent manner in the range between 0.1 and 10 nmol/l in these cells. This effect of dexamethasone was dependent on the time of pretreatment up to 8 h. Dexamethasone also inhibited PGE2 synthesis induced by melittin, known as a phospholipase A2 activator. Furthermore, dexamethasone significantly inhibited the enhancement of PGF2 alpha- or melittin-induced PGE2 synthesis by 12-O-tetradecanoylphorbol-13-acetate, known as a protein kinase C activator. In addition, dexamethasone significantly inhibited PGF2 alpha-induced formation of inositol phosphates in a dose-dependent manner between 0.1 and 10 nmol/l in MC3T3-E1 cells. These results strongly suggest that glucocorticoid inhibits PGF2 alpha-induced PGE2 synthesis through the inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2 in osteoblast-like cells.

Modulatory effect of 4 β-phorbol 12-myristate 13-acetate (PMA) on carbachol-induced Ca 2+ mobilization in rat parotid acinar cells

Treatment of rat parotid acinar cells with 4 β-phorbol 12-myristate 13-acetate (PMA) significantly inhibited an increase in cytosolic free Ca 2+ concentration ([Ca 2+] i) induced by carbachol (CCh), a muscarinic agonist. The CCh-induced increase in [Ca 2+] i was also inhibited by another active phorbol ester, 4 β-phorbol 12,13-dibutyrate, but not by 4 α-phorbol 12,13-didecanoate, which does not activate protein kinase C. The treatment with PMA had no effect on increases in [Ca 2+] i evoked by ionomycin and thapsigargin, which do not stimulate phosphoinositide hydrolysis. In contrast, an increase in [Ca 2+] i induced by NaF, a direct activator of GTP-binding proteins, was delayed in the presence of PMA. The formation of inositol phosphates in response to CCh was suppressed significantly by PMA treatment. In radioligand binding assays, PMA did not directly interfere with the specific binding of [ 3H] quinuclidinyl benzilate ([ 3H]QNB), a muscarinic antagonist, to plasma membranes. Furthermore, the [ 3H]QNB binding to plasma membranes prepared from the PMA-pretreated cells was not different from that to the control membranes. These results indicate that PMA attenuated the CCh-induced increase in [Ca 2+] i through inhibition of phosphoinositide hydrolysis. Activation of protein kinase C may play a role in negative-feedback control of the muscarinic pathway in rat parotid acinar cells.

Reduction of phosphoinositide hydrolysis by l-amino-3-phosphonopropionate may be caused by the inhibition of synthesis of phosphatidylinositols

l-Amino-3-phosphonopropionate ( l-AP3), a putative antagonist of metabotropic glutamate receptors, inhibited the formation of [ 3H]inositol phosphates induced by (1S,3R)1-aminocyclopentane-1,3-dicarboxylic acid in rat hippocampal slices. The inhibition was accompanied by a decrease in the levels of [ 3H]phosphatidylinositols ([ 3H]PIs). Preincubation of slices with l-AP3 inhibited the incorporation of [ 3H] myo-inositol into PI fractions. The effects of l-AP3 was in contrast with those of a typical receptor antagonist, atropine; atropine inhibited carbachol-induced phosphoinositide hydrolysis, but the levels of [ 3H]PIs were not affected. These findings suggest that the inhibition of phosphoinositide hydrolysis by l-AP3 is not due to the receptor antagonism but may be caused by the inhibition of synthesis of PIs.

Role of protein kinase C in the phosphatidylserine-induced inhibition of DNA synthesis in blood mononuclear cells

The mechanism of immunosuppressant activity of phosphatidylserine has been studied in peripheral blood mononuclear cells depleted or not of monocytes. After the addition of phosphatidylserine, mass determinations and uptake of labeled compound demonstrate its transfer into the cells. Phosphatidylserine incorporation causes a 2.5-fold increase of membrane-bound protein kinase C activity. The activation of translocated enzyme is indicated by the inhibition of phosphoinositide hydrolysis, an early feedback effect induced by activated protein kinase C. This action of phosphatidylserine is reproduced by tetradecanoylphorbolacetate and is prevented by the protein kinase C inhibitor, staurosporine. Consistently, phosphatidylserine (8 nmol/106 cells) decreases by 46% the production of inositol phosphates in cells responding to phytohemagglutinin. The decrease of phosphoinositide signal pathway as well as the inhibition of mitogen-induced DNA synthesis are produced at the same phosphatidylserine concentration and are equally manifest in total mononuclear cells or in preparations depleted of monocytes. However, only in the presence of monocytes does tetradecanoylphorbolacetate enhance the action of phospholipid, decreasing its IC50 from 13–15 μM to 7 μM. Thus, the data suggest that a reaction driven by protein kinase-C and a factor released by activated monocytes are involved in the phosphatidylserine-induced inhibition of lymphocyte DNA synthesis.

Neuropeptide modulation of muscarinic receptors and function in cerebral cortex of young and senescent rats

The possible influence of several neuropeptides on muscarinic receptor binding and function in fronto-parietal cortex of young and senescent Fischer 344 rats was examined. Low concentrations (100 nM) of cholecystokinin, neurotensin and vasoactive intestinal polypeptide (VIP), added in vitro, enhanced carbachol-stimulated phosphoinositide metabolism in cortical miniprisms from both young and senescent rats, while somatostatin was ineffective. Interestingly, the VIP receptor antagonist [d-parachloro-Phe 6, Leu 17]VIP shifted the dose-response curve for carbachol significantly to the right, indicating inhibition of phosphoinositide hydrolysis. No direct actions of neuropeptides on the number or affinity of [ 3H]-quinuclidinyl benzilate binding sites nor on agonist conformation states of the muscarinic receptor were noted in cortex from young animals. The neuropeptide modulation of phosphoinositide metabolism was selective for muscarinic systems, as norepinephrine-stimulated phosphoinositide hydrolysis was not altered. Pretreatment with hemicholinium-3, an inhibitor of high-affinity choline uptake, did not prevent the neuropeptide effects, indicating the interaction was probably postsynaptic. It is possible that pharmacologic manipulation of peptidergic processes could improve cholinergic neurotransmission in brain.

CCK A receptor antagonism inhibits mechanisms underlying CCK-8-stimulated insulin release in isolated rat islets

The influence of the cholecystokinin (CCK) A receptor antagonist, L-364,718, on β-cell activation was examined in isolated perifused prelabelled rat islets. Insulin secretion and 3H efflux from myo-[2- 3H]inositol-prelabelled islets (reflecting phosphoinositide hydrolysis) stimulated by CCK-8 (100 nM) were both inhibited by L-364,718, partially at 1 nM and totally at 10 nM. 45Ca 2+ efflux from prelabelled islets was markedly stimulated by CCK-8. This stimulation was inhibited equally by 1 and 10 nM L-364,718. CCK-8 stimulated the 86Rb + efflux (reflecting K + movements) from prelabelled islets, which probably reflects an indirect effect of CCK-8 due to opening of Ca 2+-activated K + channels. This 86Rb + efflux was inhibited by L-364,718 at 10 nM but not affected by L-364,718 at 1 nM. It is concluded that insulin secretion, phosphoinositide hydrolysis, Ca 2+ and K + movements stimulated by CCK-8 in isolated islets are all events mediated by CCK A receptors. The L-364,718-induced inhibition of phosphoinositide hydrolysis was most closely correlated to the inhibition of insulin secretion. This suggests that induction of cellular events activated through stimulation of phosphoinositide hydrolysis is a major mechanism underlying CCK-8-stimulated insulin secretion.

Effects of a glycine antagonist on the inhibition of phosphoinositide hydrolysis in guinea pig brain slices by N-methyl-D-aspartate

Effects of a glycine antagonist on the inhibition of phosphoinositide hydrolysis in guinea pig brain slices by N-methyl-D-aspartate

Modulation of phosphoinositide metabolism in rat brain slices by excitatory amino acids, arachidonic acid, and GABA

In rat brain slices the synthesis of [3H]phosphoinositides and the production of [3H]inositol monophosphate (IP1) induced by norepinephrine (NE) were inhibited by glutamate. Calcium concentrations were varied to test if these inhibitory effects of glutamate were mediated by a calcium-dependent process. Although reducing calcium or addition of the calcium antagonist verpamil reduced the inhibitory effects of glutamate, these results were equivocal because reduced calcium directly decreased agonist-induced [3H]phosphoinositide synthesis. The inhibitory effects of glutamate were mimicked by quisqualate in a dose-dependent manner, but none of a variety of excitatory amino acid receptor antagonists modified the inhibition caused by quisqualate. It is suggested that glutamate activates a quisqualate-sensitive receptor (for which an antagonist is not available) and causes inhibition of phosphoinositide hydrolysis mediated in part by a direct or indirect inhibitory effect of calcium on phosphoinositide synthesis. Modulatory effects of arachidonic acid were examined because glutamate and calcium can activate phospholipase A2. Arachidonic acid caused a rapid and dose-dependent inhibition of [3H]phosphoinositide synthesis and of NE-stimulated [3H]IP1 production. A similar inhibition of the response to carbachol also occurred. The inhibition caused by arachidonic acid was unchanged by addition of inhibitors of cyclooxygenase or lipoxygenase. Activation of phospholipase A2 with melittin caused inhibitory effects similar to those of arachidonic acid. Inhibitors of phospholipase A2 were found to impair phosphoinositide metabolism, likely due to their lack of specificity for phospholipase A2. Further studies were carried out in slices that were prelabelled with [3H]inositol in an attempt to separate modulatory effects on [3H]phosphoinositide synthesis and agonist-stimulated [3H]IP1 production. Several excitatory amino acid agonists inhibited NE-stimulated [3H]IP1 production. This inhibitory interaction could be due to impaired synthesis of [3H]phosphoinositides because, even though the slices were prelabeled, addition of unlabelled inositol reduced NE-stimulated [3H]IP1 production, indicating that continuous regeneration of [3H]phosphoinositides is required. In contrast to the inhibitory effects of the excitatory amino acids, gamma-aminobutyric acid (GABA) enhanced the response to NE in cortical and hippocampal slices. GABA also enhanced the response to carbachol in hippocampal and striatal slices and to ibotenic acid in hippocampal slices. Baclofen potentiated the response to NE similarly to the effect of GABA and baclofen partially blocked the inhibitory effect of arachidonic acid but did not alter that of quisqualate.

Inhibition of phosphoinositide hydrolysis by the novel neurotoxinβ-N-oxalyl- l-α, β-diaminopropionic acid ( l-BOAA)

Inhibition by a recently isolated neurotoxic amino acid, β-N-oxalyl- l-α, β-diaminopropionic acid, ( l-BOAA), of stimulated phosphoinositide hydrolysis was studied in rat brain cerebral cortical slices. l-BOAA inhibited the norepinephrine-stimulated response but did not affect hydrolysis induced by 55 mM K +, carbachol in the presence of 20 mM K +. The inhibition was concentration-dependent with anIC 50 of 300 μM. This inhibition was insensitive to the excitatory amino acid antagonists, γ-glutamylglycine, glutamic acid diethyl ether, CNQX, AP-4, AP-7, or kynurenate. Thus, we propose that the l-BOAA-mediated inhibition of the norepinephrine-stimulated response was due to an interaction at a novel site, which may also be sensitive to quisqualate (see discussion). The mechanism of the inhibition is still unknown but was not prevented by inhibition of phospholipase A 2 or polyamine synthesis and it was not affected by blockade of chloride channels. However, the presence of 20 mM K + completely blocked the inhibitory effect of l-BOAA on norepinephrine-stimulated phosphoinositide hydrolysis.

O-130 - Mastoparan inhibits [35S]GTPγS binding with an inhibition of phosphoinositide hydrolysis in membrane preparations derived from human astrocytoma cells

O-130 - Mastoparan inhibits [35S]GTPγS binding with an inhibition of phosphoinositide hydrolysis in membrane preparations derived from human astrocytoma cells