Differential regulation of cholecystokinin- and muscarinic-receptor-mediated phosphoinositide turnover in Flow 9000 cells.

We have explored the hypothesis that the apparent greater efficiency of cholecystokinin (CCK-8) receptor-second messenger coupling compared with that of muscarinic receptor in Flow 9000 cells is due to differential feedback inhibitory control mechanisms. Pretreatment of Flow 9000 cells with the tumour-promoting protein kinase C (PKC)-activating agent 12-O-tetradecanoylphorbol 13-acetate (TPA) produced a time- and dose-dependent inhibition of CCK-8 and acetylcholine (ACh) stimulation of inositol phosphate production. The inhibition by TPA of ACh-induced PI (phosphoinositide) response involved reduction of the maximal response, but no change in the concentration of ACh required to evoke a half-maximal response. In contrast, TPA inhibition of CCK-8 responses could be overcome by increasing the CCK-8 concentrations. Flow 9000 cells pretreated with TPA exhibited a 52-68% reduction in [3H]quinuclidinyl benzilate ([3H]QNB) binding capacity, whereas [125I]CCK-8 binding was unchanged. In saponin-permeabilized Flow 9000 cells, TPA pretreatment had no effect on guanosine 5'-[gamma-thio]triphosphate (GTP[S])-induced inositol phosphate formation, indicating that G-protein linkage to phosphoinositidase C (PIC) was not affected. However, TPA significantly inhibited the potentiating effect of GTP[S] on CCK-8 and ACh activation of PI response, suggesting that the coupling between the receptors and the G-protein was impaired. The PKC-activator 1-oleoyl-2-acetylglycerol (OAG), a diacylglycerol analogue, also significantly reduced CCK-8 and ACh stimulation of inositol phosphate accumulation in these cells. Our results are consistent with the hypothesis that muscarinic activation of PI hydrolysis is subjected to rapid feedback inhibition via the 1,2-diacylglycerol-PKC pathway. CCK-receptor activation of PI turnover is modulated to a lesser extent, and this may partially explain apparent differences in the efficiency of receptor-second messenger coupling. It is proposed that TPA acting through PKC exerts its inhibitory action on muscarinic-agonist-mediated PI response mainly at the receptor level, whereas the inhibitory effect on CCK-8 response is at a site close to the receptor-G-protein coupling step.     

Guanine nucleotides stimulate hydrolysis of phosphatidylinositol and polyphosphoinositides in permeabilized Swiss 3T3 cells

Hydrolysis-resistant analogues of GTP specifically stimulate the formation of [3H]inositol mono-, bis- and trisphosphates by saponin-permeabilized Swiss 3T3 cells prelabelled with [3H]inositol. Each inositol phosphate is formed largely by hydrolysis of its parent lipid and not by dephosphorylation of inositol 1,4,5-trisphosphate [(1,4,5)IP3]. Although hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is most sensitive to guanine nucleotides, hydrolysis of phosphatidyl-inositol (PI) and phosphatidylinositol 4-phosphate (PIP) is quantitatively more important. These results suggest that a guanine nucleotide-dependent regulatory protein(s) (G-protein) is involved in regulating the hydrolysis of PI and PIP, as well as PIP2, and so may allow formation of diacylglycerol (DG) without simultaneous production of (1,4,5)IP3 and mobilization of intracellular Ca2+.     

Polyamines inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis. Studies with permeabilized GH3 cells.

[3H]Inositol-labelled GH3 rat anterior pituitary tumour cells were permeabilized with digitonin and were incubated at 37 degrees C in the presence of ATP and Mg2+. [3H]Polyphosphoinositide breakdown and [3H]inositol phosphate production were stimulated by hydrolysis-resistant GTP analogues and by Ca2+. Of the nucleotides tested, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) was the most effective stimulus. Activation by GTP gamma S appeared to be mediated by a guanine nucleotide-binding (G) protein as GTP gamma S-stimulated [3H]inositol phosphate production was inhibited by other nucleotides with a potency order of GTP = GDP = guanosine 5'-[beta-thio]diphosphate greater than ITP greater than GMP greater than UTP = CTP = adenosine 5'-[gamma-thio]triphosphate. The stimulatory effects of 10 microM-GTP gamma S on [3H]inositol phosphate levels were reversed by spermine and spermidine with IC50 values of approx. 0.25 and 2 mM respectively. Putrescine was inhibitory only at higher concentrations. Similarly, GTP gamma S-induced decreases in [3H]polyphosphoinositide levels were reversed by 2.5 mM-spermine. The inhibitory effects of spermine were not overcome by supramaximal concentrations of GTP gamma S. In contrast, [3H]inositol phosphate production stimulated by addition of 0.3-0.6 mM-Ca2+ to incubation media was only partially inhibited by spermine (5 mM), and spermine was not inhibitory when added Ca2+ was increased to 1 mM. These data show that polyamines, particularly spermine, inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis with a marked selectivity towards the stimulatory effects of GTP gamma S.     

Turkey erythrocyte membranes as a model for regulation of phospholipase C by guanine nucleotides

Phosphoinositides of human, rabbit, rat, and turkey erythrocytes were radiolabeled by incubation of intact cells with [32P]Pi. Guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and NaF, which are known activators of guanine nucleotide regulatory proteins, caused a large increase in [32P]inositol phosphate release from plasma membranes derived from turkey erythrocytes, but had no effect on inositol phosphate formation by plasma membranes prepared from the mammalian erythrocytes. High performance liquid chromatography analysis indicated that inositol bisphosphate, inositol 1,3,4-trisphosphate, inositol 1,4,5-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate all increased by 20-30-fold during a 10-min incubation of turkey erythrocyte membranes with GTP gamma S. The increase in inositol phosphate formation was accompanied by a similar decrease in radioactivity in phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). GTP gamma S increased inositol phosphate formation with a K0.5 of 600 nM; guanosine 5'-(beta, gamma-imido)trisphosphate was 50-75% as efficacious as GTP gamma S and expressed a K0.5 of 36 microM. Although GTP alone had little effect on inositol phosphate formation, it blocked GTP gamma S-stimulated inositol phosphate formation, as did guanosine 5'-O-(2-thiodiphosphate). Turkey erythrocytes were also shown to express phosphatidylinositol synthetase activity in that incubation of cells with [3H] inositol resulted in incorporation of radiolabel into phosphatidylinositol, PIP, and PIP2. Incubation of membranes derived from [3H]inositol-labeled erythrocytes with GTP gamma S resulted in large increases in [3H] inositol phosphate formation and corresponding decreases in radiolabel in PIP and PIP2. The data suggest that, in contrast to mammalian erythrocytes, the turkey erythrocyte expresses a guanine nucleotide-binding protein that regulates phospholipase C, and as such, should provide a useful model system for furthering our understanding of hormonal regulation of this enzyme.     

P2-purinergic receptors activate a guanine nucleotide-dependent phospholipase C in membranes from HL-60 cells

We have previously determined that human neutrophils and monocytes, as well as neutrophil/monocyte progenitor cells, express a subtype of P2-purinergic receptors (for ATP) which activate the inositol phospholipid signalling system. In the present study, membranes prepared from HL-60 promyelocytic leukemia cells were used to examine the mechanism by which these ATP receptors activate phosphatidylinositol-specific phospholipase C (PI-PLC) under defined in vitro conditions. Micromolar concentrations of the receptor agonists ATP, UTP, and ATP gamma S stimulated the GTP-dependent formation of inositol bisphosphate (IP2) and inositol trisphosphate (IP3) in washed membranes prepared from undifferentiated HL-60 cells prelabeled with [3H]inositol. The stimulatory effects of these nucleotides on PI-PLC appeared to be mediated through a GTP binding protein since minimal inositol polyphosphate accumulation was observed in the absence of guanine nucleotides. The increased inositol polyphosphate formation triggered by these nucleotide receptor agonists did not result from inhibition of GTP breakdown. Neither was it a consequence of increased [3H]polyphosphatidylinositol levels resulting from enhanced activity of membrane-associated PI- or PIP-kinases. Instead, the stimulated phospholipase activity was apparently receptor-mediated. The rank order of potency observed in these in vitro membrane assays (ATP = UTP greater than ATP gamma S much greater than TTP greater than CTP much greater than beta, gamma-CH-ATP) was similar to that observed with intact HL-60 cells. This order of potency appears to distinguish the P2-purinergic receptors expressed by human phagocytic leukocytes from the P2 gamma-purinergic receptors which activate PI-PLC in turkey erythrocyte membranes.     

Activation of phospholipase C associated with isolated rabbit platelet membranes by guanosine 5''-[gamma-thio]triphosphate and by thrombin in the presence of GTP.

Rabbit platelets were labelled with [3H]inositol and a membrane fraction was isolated in the presence of ATP, MgCl2 and EGTA. Incubation of samples for 10 min with 0.1 microM-Ca2+free released [3H]inositol phosphates equivalent to about 2.0% of the membrane [3H]phosphoinositides. Addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) caused an additional formation of [3H]inositol phosphates equivalent to 6.6% of the [3H]phosphoinositides. A half-maximal effect was observed with 0.4 microM-GTP[S]. The [3H]inositol phosphates that accumulated consisted of 10% [3H]inositol monophosphate, 88% [3H]inositol bisphosphate ([3H]IP2) and 2% [3H]inositol trisphosphate ([3H]IP3). Omission of ATP and MgCl2 led to depletion of membrane [3H]polyphosphoinositides and marked decreases in the formation of [3H]inositol phosphates. Thrombin (2 units/ml) or GTP (4-100 microM) alone weakly stimulated [3H]IP2 formation, but together they acted synergistically to exert an effect comparable with that of 10 microM-GTP[S]. The action of thrombin was also potentiated by 0.1 microM-GTP[S]. Guanosine 5'-[beta-thio]diphosphate not only inhibited the effects of GTP[S], GTP and GTP with thrombin, but also blocked the action of thrombin alone, suggesting that this depended on residual GTP. Incubation with either GTP[S] or thrombin and GTP decreased membrane [3H]phosphatidylinositol 4-phosphate ([H]PIP) and prevented an increase in [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) observed in controls. Addition of unlabelled IP3 to trap [3H]IP3 before it was degraded to [3H]IP2 showed that only about 20% of the additional [3H]inositol phosphates that accumulated with GTP[S] or thrombin and GTP were derived from the action of phospholipase C on [3H]PIP2. The results provide further evidence that guanine-nucleotide-binding protein mediates signal transduction between the thrombin receptor and phospholipase C, and suggest that PIP may be a major substrate of this enzyme in the platelet.     

Short chain alcohols activate guanine nucleotide-dependent phosphoinositidase C in turkey erythrocyte membranes

The ability of alcohols to regulate inositol lipid-specific phospholipase C (phosphoinositidase C) was examined in turkey erythrocyte ghosts prepared by cell lysis of erythrocytes which were prelabeled with [3H] inositol. Guanosine 5'-[gamma-thiotriphosphate] GTP[S] stimulated the production of both [3H]inositol bisphosphate (18-fold) and [3H]inositol trisphosphate (6-fold) in this system. The accumulation of [3H]inositol bisphosphate and [3H]inositol trisphosphate was linear up to 8 min following an initial lag period of 1-2 min. Ethanol (300 mM) reduced the lag period for [3H]inositol phosphate accumulation at submaximal GTP[S] concentrations and caused a shift to the left (3-fold) in the dose-response curve. Other short chain alcohols, methanol (300 mM), 1-propanol (200 mM), and 1-butanol (50 mM) also enhanced the accumulation of [3H] inositol phosphates in the presence of submaximal GTP[S] concentrations. Receptor activation by the purinergic agonist adenosine 5'-[beta-thio]disphosphate (ADP[S]) (10 microM) also reduced the lag period for [3H] inositol phosphate formation and shifted the GTP[S] dose response to the left (10-fold). In addition, ADP[S] increased the response to maximal GTP[S] concentrations. The formation of [3H]inositol phosphates induced by GTP[S] was associated with a concomitant decrease in labeling of both [3H]phosphatidylinositol monophosphate and [3H]phosphatidylinositol bisphosphate, but no decrease in [3H]phosphatidylinositol was observed. All of the alcohols tested enhanced the breakdown of [3H]polyphosphoinositides in the presence of GTP[S]. The dose response to guanosine 5'-[beta gamma-imino]triphosphate for [3H]inositol phosphate formation was displaced to the left by ethanol (300 mM) and ADP[S] (10 microM) (2- and 7-fold), respectively. ADP[S] also enhanced the maximal response to guanosine 5'-[beta gamma-imino]triphosphate. The [3H]inositol phosphate formation produced in response to NaF was unaffected by either ethanol or receptor activation. These results indicate that alcohols initiate an activation of phosphoinositidase C, mediated at the level of the regulatory guanine nucleotide-binding protein.     

A novel cholera toxin-sensitive G-protein (Gc) regulating receptor-mediated phosphoinositide signalling in human pituitary clonal cells

Recent studies have implicated that a GTP-binding protein (G-protein) is involved in the coupling of both CCK-8 and muscarinic cholinergic receptors to phosphoinositidase C (PIC) in the human embryonic pituitary cell line, Flow 9000. Pretreatment of these cells with cholera toxin, but not pertussis toxin, inhibited the stimulation of [³H]inositol phosphate production by CCK-8 and acetylcholine. These inhibitory effects of cholera toxin could not be reproduced by treating the cells with the B-subunit of cholera toxin or cAMP-generating agents such as forskolin. These data suggest the presence of a novel G_c protein which is responsible for receptor-PIC coupling in Flow 9000 cells.     

Guanosine 5''-[gamma-thio]triphosphate-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate in HL-60 granulocytes. Evidence that the guanine nucleotide acts by relieving phospholipase C from an inhibitory constraint.

Myeloid differentiated human leukaemia (HL-60) cells contain a soluble phospholipase C that hydrolysed phosphatidylinositol 4.5-bisphosphate and was markedly stimulated by the metabolically stable GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]). Half-maximal and maximal (up to 5-fold) stimulation of inositol phosphate formation by GTP[S] occurred at 1.5 microM and 30 microM respectively. Other nucleotides (GTP, GDP, GMP, guanosine 5'-[beta-thio]diphosphate. ATP, adenosine 5'-[gamma-thio]triphosphate, UTP) did not affect phospholipase C activity, GTP[S] stimulation of inositol phosphate accumulation was inhibited by excess GDP, but not by ADP. The effect of GTP[S] on inositol phosphate formation was absolutely dependent on and markedly stimulated by free Ca2+ (median effective concn. approximately 100 nM). Analysis of inositol phosphates by anion-exchange chromatography revealed InsP3 as the major product of GTP[S]-stimulated phospholipase C activity. In the absence of GTP[S], specific phospholipase C activity was markedly decreased when tested at high protein concentrations, whereas GTP[S] stimulation of the enzyme was markedly enhanced under these conditions. As both basal and GTP[S]-stimulated inositol phosphate formation were linear with time whether studied at low or high protein concentration, these results suggest that (a) phospholipase C is under an inhibitory constraint and (b) GTP[S] relieves this inhibition, most likely by activating a soluble GTP-binding protein.     

Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: possible involvement of an inhibitory GTP-binding protein

Cultured pituitary cells prelabeled with myo-[2-3H] inositol were permeabilized by ATP4-, exposed to guanine nucleotides and resealed by Mg2+. Addition of guanosine 5'-0-(3-thio triphosphate) (GTP gamma S) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5'-0-(2-thiodiphosphate) (GDP beta S) or by neomycin. Surprisingly, addition of GDP beta S (100-600 microM) on its own resulted in a dose-related increase in [3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP greater than GTP gamma S greater than ATP greater than CTP. The stimulatory effect of UTP, ATP and CTP, but not GTP gamma S or GDP beta S, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTP gamma S decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.     

Effects of guanosine 5'-[gamma-thio]triphosphate and thrombin on the phosphoinositide metabolism of electropermeabilized human platelets

Incubation of human platelets with myo-[3H]inositol in a low-glucose Tyrode's solution containing MnCl2 enhanced the labelling of phosphoinositides about sevenfold and greatly facilitated the measurement of [3H]inositol phosphates formed by the activation of phospholipase C. Labelled platelets were permeabilized by high-voltage electric discharges and equilibrated at 0 degree C with ATP, Ca2+ buffers and guanine nucleotides, before incubation in the absence or presence of thrombin. Incubation of these platelets with ATP in the presence or absence of Ca2+ ions led to the conversion of [3H]phosphatidylinositol to [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PtdInsP2). At a pCa of 6, addition of 100 microM GTP[gamma S] both prevented this accumulation of [3H]PtdInsP2 and stimulated its breakdown; the formation of [3H]inositol phosphates was increased ninefold. After 5 min these comprised 70% [3H]inositol monophosphate ([3H]InsP), 28% [3H]inositol bisphosphate ([3H]InsP2) and 2% [3H]inositol trisphosphate ([3H]InsP3). In shorter incubations higher percentages of [3H]InsP2 and [3H]InsP3 were found. In the absence of added Ca2+, the formation of [3H]inositol phosphates was decreased by over 90%. Incubation of permeabilized platelets with GTP[gamma S] in the presence of 10 mM Li+ decreased the accumulation of [3H]InsP and increased that of [3H]InsP2, without affecting [3H]InsP3 levels. Addition of unlabelled InsP3 decreased the intracellular hydrolysis of exogenous [32P]InsP3 but did not trap additional [3H]InsP3. These results and the time course of [3H]inositol phosphate formation suggest that GTP[gamma S] stimulated the action of phospholipase C on a pool of [3H]phosphatidylinositol 4-phosphate that was otherwise converted to [3H]PtdInsP2 and that much less hydrolysis of [3H]phosphatidylinositol to [3H]InsP or of [3H]PtdInsP2 to [3H]InsP3 occurred. At a pCa of 6, addition of thrombin (2 units/ml) to permeabilized platelets caused small increases in the formation of [3H]InsP and [3H]InsP2. This action of thrombin was enhanced twofold by 10-100 microM GTP and much more potently by 4-40 microM GTP[gamma S]. In the presence of the latter, thrombin also increased [3H]InsP3. The total formation of [3H]inositol phosphates by permeabilized platelets incubated with thrombin and GTP[gamma S] was comparable with that observed on addition of thrombin alone to intact platelets. However, HPLC of the [3H]inositol phosphates formed indicated that about 75% of the [3H]InsP accumulating in permeabilized platelets was the 4-phosphate, whereas in intact platelets stimulated by thrombin, up to 80% was the 1-phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphoinositide hydrolysis by guanosine 5''-[gamma-thio]triphosphate-activated phospholipase C of turkey erythrocyte membranes.

Phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] of turkey erythrocytes were labelled by using either [32P]Pi or [3H]inositol. Although there was little basal release of inositol phosphates from membranes purified from labelled cells, in the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) the rate of accumulation of inositol bis-, tris- and tetrakis-phosphate (InsP2, InsP3 and InsP4) was increased 20-50-fold. The enhanced rate of accumulation of 3H-labelled inositol phosphates was linear for up to 20 min; owing to decreases in 32P specific radioactivity of phosphoinositides during incubation of membranes with unlabelled ATP, the accumulation of 32P-labelled inositol phosphates was linear for only 5 min. In the absence of ATP and a nucleotide-regenerating system, no InsP4 was formed, and the overall inositol phosphate response to GTP[S] was decreased. Analyses of phosphoinositides during incubation with ATP indicated that interconversions of PtdIns to PtdIns4P and PtdIns4P to PtdIns(4,5)P2 occurred to maintain PtdIns(4,5)P2 concentrations; GTP[S]-induced inositol phosphate formation was accompanied by a corresponding decrease in 32P- and 3H-labelled PtdIns, PtdIns4P and PtdIns(4,5)P2. In the absence of ATP, only GTP[S]-induced decreases in PtdIns(4,5)P2 occurred. Since inositol monophosphate was not formed under any condition, PtdIns is not a substrate for the phospholipase C. The production of InsP2 was decreased markedly, but not blocked, under conditions where Ins(1,4,5)P3 5-phosphomonoesterase activity in the preparation was inhibited. Thus the predominant substrate of the GTP[S]-activated phospholipase C of turkey erythrocyte membranes is PtdIns(4,5)P2. Ins(1,4,5)P3 was the major product of this reaction; only a small amount of Ins(1:2-cyclic, 4,5)P3 was released. The effects of ATP on inositol phosphate formation apparently involve the contributions of two phenomena. First, the P2-receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) greatly increased inositol phosphate formation and decreased [3H]PtdIns4P and [3H]PtdIns(4,5)P2 in the presence of a low (0.1 microM) concentration of GTP[S]. ATP over the concentration range 0-100 microM produced effects in the presence of 0.1 microM-GTP[S] essentially identical with those observed with 2MeSATP, suggesting that the effects of low concentrations of ATP are also explained by a stimulation of P2-receptors. Higher concentrations of ATP also increase inositol phosphate formation, apparently by supporting the synthesis of substrate phospholipids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Guanine nucleotides stimulate production of inositol trisphosphate in rat cortical membranes.

The guanine nucleotides guanosine 5'[beta, gamma-imido]triphosphate (Gpp[NH]p), guanosine 5'-[gamma-thio]-triphosphate (GTP gamma S), GMP, GDP and GTP stimulated the hydrolysis of inositol phospholipids by a phosphodiesterase in rat cerebral cortical membranes. Addition of 100 microM-Gpp[NH]p to prelabelled membranes caused a rapid accumulation of [3H )inositol phosphates (less than 30 s) for up to 2 min. GTP gamma S and Gpp [NH]p caused a concentration-dependent stimulation of phosphoinositide phosphodiesterase with a maximal stimulation of 2.5-3-fold over control at concentrations of 100 microM. GMP was as effective as the nonhydrolysable analogues, but much less potent (EC50 380 microM). GTP and GDP caused a 50% stimulation of the phospholipase C at 100 microM and at higher concentrations were inhibitory. The adenine nucleotides App[NH]p and ATP also caused small stimulatory effects (64% and 29%). The guanine nucleotide stimulation of inositide hydrolysis in cortical membranes was selective for inositol phospholipids over choline-containing phospholipids. Gpp[NH]p stimulated the production of inositol trisphosphate and inositol bisphosphate as well as inositol monophosphate, indicating that phosphoinositides are substrates for the phosphodiesterase. EGTA (33 microM) did not prevent the guanine nucleotide stimulation of inositide hydrolysis. Calcium addition by itself caused inositide phosphodiesterase activation from 3 to 100 microM which was additive with the Gpp[NH]p stimulation. These data suggest that guanine nucleotides may play a regulatory role in the modulation of the activity of phosphoinositide phosphodiesterase in rat cortical membranes.     

Guanine nucleotide-dependent pertussis-toxin-insensitive stimulation of inositol phosphate formation by carbachol in a membrane preparation from human astrocytoma cells.

The efficacy of muscarinic-receptor agonists for stimulation of inositol phosphate formation and Ca2+ mobilization in intact 1321N1 human astrocytoma cells is correlated with their capacity for formation of a GTP-sensitive high-affinity binding complex in membranes from these cells [Evans, Hepler, Masters, Brown & Harden (1985) Biochem. J. 232, 751-757]. These observations prompted the proposal that a guanine nucleotide regulatory protein serves to couple muscarinic receptors to the phospholipase C involved in phosphoinositide hydrolysis in 1321N1 cells. Inositol phosphate (InsP) formation was measured in a cell-free preparation from 1321N1 cells to provide direct support for this idea. The formation of InsP3, InsP2 and InsP1 was increased in a concentration-dependent manner (K0.5 approximately 5 microM) by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in washed membranes prepared from myo-[3H]inositol-prelabelled 1321N1 cells. Both GTP[S] and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated InsP formation by 2-3-fold over control; GTP, GDP and GMP were much less efficacious. Millimolar concentrations of NaF also stimulated the formation of inositol phosphates in membrane preparations from 1321N1 cells. In the presence of 10 microM-GTP[S], the muscarinic cholinergic-receptor agonist carbachol stimulated (K0.5 approximately 10 microM) the formation of InsP above that achieved with GTP[S] alone. The effect of carbachol was completely blocked by atropine. The order of potency of nucleotides for stimulation of InsP formation in the presence of 500 microM-carbachol was GTP[S] greater than p[NH]ppG greater than GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate Gi (the inhibitory guanine nucleotide regulatory protein), had no effect on InsP formation in the presence of GTP[S] or GTP[S] plus carbachol. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not Gi is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells.     

A guanine nucleotide-dependent regulatory protein couples substance P receptors to phospholipase C in rat parotid gland

Electrically permeabilized cells of rat parotid gland, prelabelled with [3H]-inositol, synthesized [3H]-inositol phosphates (IP3 and IP2) when stimulated with alpha 1-adrenergic, muscarinic-cholinergic, and substance P receptor-agonists. Non-hydrolyzable analogues of GTP (GTP gamma S and GppNHp) also stimulated [3H]-IP3 formation by permeabilized cells and they potentiated the stimulation by receptor-agonists. These effects of guanine nucleotides occurred only with GTP analogues and only in permeabilized cells indicating an intracellular site of action. NaF stimulated [3H]-IP3 accumulation, an effect that was not entirely attributable to the ability of F- to inhibit (1,4,5)IP3 degradation. These results suggest that a guanine nucleotide-dependent regulatory protein couples Ca2+-mobilizing receptors to phospholipase C in parotid gland.     

Beta-adrenergic receptor-mediated phospholipase C activation independent of cAMP formation in turkey erythrocyte membranes.

The effect of the beta-adrenergic receptor agonist isoproterenol on guanine nucleotide-dependent phospholipase C (PLC) activity was examined in turkey erythrocyte membranes prepared from [3H]inositol-labeled turkey erythrocytes. In the presence of guanosine 5'-(gamma-thiotriphosphate) (GTP[S]) isoproterenol caused a dose-dependent stimulation of [3H]inositol phosphate ([3H]InsP) formation. The activation of PLC by GTP[S] occurred after an initial lag period of 1-2 min and was followed by a sustained rate of [3H]InsP formation which remained linear for 4-5 min. Isoproterenol decreased the lag period for GTP[S]-induced [3H]InsP formation and increased PLC activity at all time points following this lag. Consequently, isoproterenol shifted the dose-response curve for GTP[S] to the left (10-fold) and increased the maximal response. The EC50 value for isoproterenol-induced activation of PLC was 104 +/- 17 nM. Isoproterenol also potentiated GTP-dependent PLC activity but was ineffective in stimulating the enzyme in the presence of AIF4-. The PLC activation by isoproterenol was completely inhibited by propanolol and atenolol but was unaffected by prazosin or yohimbine. Although GTP[S] and isoproterenol could increase cAMP formation in this membrane preparation, the isoproterenol-induced stimulation of PLC occurred in the absence of ATP and was independent of cAMP formation. Furthermore, addition of cAMP, 8-bromo-cAMP, forskolin, or either the regulatory or catalytic subunits of cAMP-dependent protein kinase failed to stimulate [3H]InsP formation and had no effect on the responses elicited by GTP[S] and isoproterenol. Isoproterenol also stimulated [3H]InsP2 and [3H]InsP3 production in intact erythrocytes. Cholera toxin had no effect on [3H]InsP formation in the intact cells under conditions where it stimulated cAMP accumulation. In addition, the activation of PLC by GTP[S] and isoproterenol was unaffected in membranes prepared from cholera toxin-treated erythrocytes. These data demonstrate that stimulation of turkey erythrocyte beta-adrenergic receptors by isoproterenol results in a direct activation of guanine nucleotide-dependent PLC.     

Carbachol and histamine stimulation of guanine-nucleotide-dependent phosphoinositide hydrolysis in rat brain cortical membranes.

Guanine nucleotides have been shown to stimulate phosphoinositide breakdown in brain membranes, but no potentiation of such an effect by agonist was demonstrated. We have studied the effect of carbachol and histamine on guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulation of inositol phosphates formation in [3H]inositol-labelled rat brain cortical membranes. In this preparation, GTP[S] enhancement of phosphoinositide hydrolysis required the presence of MgATP and low Ca2+ concentration (100 nM). Carbachol potentiation of the GTP[S] effect was only observed when 1 mM-deoxycholate was also added. Under these conditions, stimulated production of [3H]inositol phosphates was linear for at least 15 min, and [3H]inositol bisphosphate [( 3H]IP2) accounted for approx. 80%, whereas the amount of [3H]inositol trisphosphate [( 3H]IP3) was very low. Stimulation by GTP[S] was concentration-dependent (half-maximal effect at 0.86 microM), and its maximal effect (815% over basal) was increased by 1 mM-carbachol (1.9-fold) and -histamine (1.7-fold). Both agonists decreased the slope index of the GTP[S] concentration/effect curve to values lower than unity, suggesting the appearance of some heterogeneity in the population of guanine-nucleotide-binding proteins (G-proteins) involved. The carbachol and histamine effects were also concentration-dependent, and were inhibited by atropine and mepyramine respectively. Fluoroaluminate stimulated phosphoinositide hydrolysis to a higher extent than GTP[S] plus carbachol, and these stimulations were not additive, indicating that the same polyphosphoinositide phospholipase C-coupled G-protein mediates both effects.     

Chemotactic peptide, calcium and guanine nucleotide regulation of phospholipase C activity in membranes from DMSO-differentiated HL60 cells

Membranes prepared from DMSO-differentiated HL60 cells labeled with [3H]inositol hydrolyze polyphosphoinositides in a Ca2+-dependent manner, generating inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). Incubation of membranes with GTP or GTP gamma S reduces the concentration of Ca2+ required for activation. This nucleotide effect is potentiated by formyl-Met-Leu-Phe (FMLP). Pertussis toxin inhibits FMLP-induced augmentation, but not the induction of IP2/IP3 formation by GTP or GTP gamma S. These results suggest that differentiated HL60 cells contain a membrane-associated phospholipase C that degrades polyphosphoinositides and that activation of this enzyme is mediated by at least two guanine nucleotide binding proteins, one of which is linked to FMLP receptors and is pertussis toxin sensitive.     

Kinetic analysis of guanosine 5'-O-(3-thiotriphosphate) effects on phosphatidylinositol turnover in NRK cell homogenates

Addition of the guanine nucleotide analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to [3H]inositol-labeled NRK cell homogenates resulted in rapid breakdown of cellular polyphosphoinositides. GTP gamma S stimulated phospholipase C, resulting in a more than 4-fold increase in the hydrolysis rates of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bis(phosphate) (PIP2). No significant effect of GTP gamma S on direct phosphatidylinositol (PI) hydrolysis was detected. There was an increase in water-soluble inositols, with inositol tris(phosphate) (IP3) levels increasing at least 10 times over the decrease seen in PIP2, indicating that PIP kinase activity was also accelerated following GTP gamma S addition. Inositol 1,4,5-tris(phosphate) peaked rapidly after GTP gamma S addition (less than 2 min) while inositol 1,3,4-tris-(phosphate) was produced more slowly and leveled off after approximately 10 min. The differential equations describing conversion between intermediates in the PI turnover pathway were solved and fitted to data obtained from both [3H]inositol and [32P]phosphate fluxes by nonlinear least-squares analysis. GTP gamma S effects on the pseudo-first-order rate constants for the lipase, kinase, and phosphatase steps were determined from the analysis. From these measurements it can be estimated that, in the presence of GTP gamma S and calcium buffered to 130 nM, hydrolysis of PIP2 accounts for at least 10 times as much diacylglycerol as direct PI breakdown despite the 100-fold excess of PI over PIP2. From the kinetic model it is predicted that small changes in the activities of PI and PIP kinases can have large but different effects on the level of IP3 and diacylglycerol following GTP gamma S addition. These results argue that regulation of PI and PIP kinases may be important for determining both cellular IP3 and diacylglycerol levels.     

Plasma membrane associated phospholipase C from human platelets: synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5'-O-(3-thiotriphosphate)

The effects of thrombin and GTP gamma S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous [3H]inositol-labeled membranes or with lipid vesicles containing either [3H]phosphatidylinositol or [3H]phosphatidylinositol 4,5-bisphosphate. GTP gamma S (1 microM) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP3), inositol bisphosphate (IP2), or inositol phosphate (IP) from [3H]inositol-labeled membranes. IP2 and IP3, but not IP, from [3H]inositol-labeled membranes were, however, stimulated 3-fold by GTP gamma S (1 microM) plus thrombin (1 unit/mL). A higher concentration of GTP gamma S (100 microM) alone also stimulated IP2 and IP3, but not IP, release. In the presence of 1 mM calcium, release of IP2 and IP3 was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) by platelet membrane associated PLC was also markedly enhanced by GTP gamma S (100 microM) or GTP gamma S (1 microM) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP2 was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP gamma S (100 microM) or calcium (1 mM) dependent PIP2 breakdown, while TPA inhibited GTP gamma S-dependent but not calcium-dependent phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)