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.     

Phorbol ester inhibits phosphoinositide hydrolysis and calcium mobilization in cultured astrocytoma cells

In cultured human 1321N1 astrocytoma cells, muscarinic receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates, and mobilization of intracellular Ca2+. Treatment of these cells with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) completely blocks the carbachol-stimulated formation of [3H]inositol mono-, bis-, and trisphosphate ( [3H]InsP, [3H]InsP2, and [3H]InsP3). The concentrations of PMA that give half-maximal and 100% inhibition of carbachol-induced [3H]InsP formation are 3 nM and 0.5 microM, respectively. Inactive phorbol esters (4 alpha-phorbol 12,13-didecanoate and 4 beta-phorbol), at 1 microM, do not inhibit carbachol-stimulated [3H]InsP formation. The KD of the muscarinic receptor for [3H]N-methyl scopolamine is unchanged by PMA treatment, while the IC50 for carbachol is modestly increased. PMA treatment also abolishes carbachol-induced 45Ca2+ efflux from 1321N1 cells. The concomitant loss of InsP3 formation and Ca2+ mobilization is strong evidence in support of a causal relationship between these two responses. In addition, our finding that PMA blocks hormone-stimulated phosphoinositide turnover suggests that there may be feedback regulation of phosphoinositide metabolism through the Ca2+- and phospholipid-dependent protein kinase.     

Mastoparan inhibits phosphoinositide hydrolysis via pertussis toxin-insensitive [corrected] G-protein in human astrocytoma cells

Mastoparan inhibited [3H]inositol phosphate accumulation induced by carbachol as well as cyclic AMP accumulation induced by isoproterenol in 1321N1 human astrocytoma cells. Mastoparan inhibited GTP gamma S-induced, but not Ca2(+)-induced, [3H]inositol phosphate accumulation in membrane preparations with an IC50 of approximately 10 microM. The inhibitory effect of mastoparan on carbachol-induced [3H]inositol phosphate accumulation was resistant to pertussis toxin (IAP) treatment in intact cells. These results suggest that mastoparan inhibits phospholipase C in human astrocytoma cells via a GTP binding protein, which is not a substrate for IAP.     

Long-term phorbol ester treatment down-regulates protein kinase C and sensitizes the phosphoinositide signaling pathway to hormone and growth factor stimulation. Evidence for a role of protein kinase C in agonist-induced desensitization

Exposure of a nontransformed, continuous line of epithelial cells derived from rat liver (WB cells) to epidermal growth factor, angiotensin II, [Arg8]vasopressin, and epinephrine resulted in rapid accumulation of the inositol phosphates (InsP) InsP1, InsP2, and InsP3. Although short-term (5-60 min) pretreatment of WB cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly attenuated InsP accumulation in response to all agonists, the inhibitory effects on the InsP response were lost after 2 h incubation with PMA; and, with extended (6-24 h) preincubation, a time-dependent potentiation of the InsP response to angiotensin II, epidermal growth factor and [Arg8]vasopressin was observed. The InsP response during a 15-min challenge with angiotensin II in cells pretreated for 18 h with 600 nM and 10 microM PMA was increased by 2-3-fold and 4-6-fold, respectively. Long-term (18 h) treatment with 600 nM and 10 microM PMA caused a similar 90-100% loss of measurable Ca2+/phospholipid-dependent enzyme (protein kinase C) activity in cytosolic and soluble particulate fractions. The effects of long-term PMA pretreatment do not represent a general enhancement of hormone responsiveness since the InsP response to epinephrine was not affected. In control cells, the InsP response to angiotensin II and epinephrine desensitized very rapidly. Long-term pretreatment with PMA greatly reduced the contribution of agonist-induced desensitization to the angiotensin II response; in contrast, the extent of desensitization occurring during incubation of WB cells with epinephrine was unaltered by long-term treatment with PMA suggesting that an additional mechanism may be involved in alpha 1-adrenergic receptor desensitization. No PMA-induced change in resting levels of [3H]phosphoinositides or the metabolism of exogenous [3H]inositol 1,4,5-trisphosphate by WB homogenates occurred. Stimulation of InsP formation in intact cells by NaF and activation of phospholipase C by GTP gamma S in membranes both were unaltered by short-term or long-term PMA pretreatment. These data are consistent with the idea that following long-term treatment of WB cells with PMA, the occurrence of agonist-induced desensitization of receptors linked to the phosphoinositide/Ca2+ signaling system is reduced, apparently at least in part due to the loss of contribution of a negative feedback regulatory role of protein kinase C.     

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.     

Regulation of bombesin-stimulated inositol 1,4,5-trisphosphate generation in Swiss 3T3 fibroblasts by a guanine-nucleotide-binding protein.

The stimulation of inositol phosphate generation by bombesin and GTP analogues was studied in Swiss 3T3 cells permeabilized by electroporation. Bombesin-stimulated inositol phosphate generation is potentiated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and inhibited by guanosine 5'-[beta-thio]diphosphate at all peptide concentrations tested, with no change in the EC50 value (concn. giving half-maximal response) for the agonist. Kinetic analysis showed that, although bombesin-stimulated [3H]InsP3 generation in [3H]inositol-labelled cells was rapid (maximal by 5-10 s), the response to GTP[S] alone displayed a distinct lag time of 20-30 s. This lag time was significantly decreased by the addition of bombesin, suggesting that in this system agonist-stimulated GTP/GDP exchange occurs. In addition, bombesin-stimulated generation of Ins(1,4,5)P3 mass at 10 s was enhanced by GTP[S] in the absence of a nucleotide response alone, a result consistent with this proposal. Pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent inhibition of bombesin-, but not GTP[S]-, stimulated inositol phosphate generation. Furthermore, although PMA pretreatment did not affect the lag time for InsP3 formation in response to GTP[S] alone, the degree of synergy between bombesin and the nucleotide was severely decreased at early time points. The results therefore demonstrate that the high-affinity bombesin receptor is coupled via a G-protein to phospholipase C in a manner consistent with a general model for receptor-G-protein interactions and that this coupling is sensitive to phosphorylation by protein kinase C.     

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)     

Regulation of membrane-associated and cytosolic phospholipase C activities in human platelets by guanosine triphosphate

The effects of guanine nucleotides, thrombin, and platelet cytosol (100,000 X g supernatant) on the hydrolysis of polyphosphoinositides by phospholipase C was examined in isolated platelet membranes labeled with [3H]inositol. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) (10 microM) caused a 2-fold stimulation of polyphosphoinositide hydrolysis, compared to background. GTP gamma S (10 microM) plus thrombin (1 unit/ml) stimulated the release of inositol triphosphate, inositol diphosphate, and inositol phosphate 500, 300, and 250%, respectively, compared to GTP gamma S alone. Cytosol prepared from unlabeled platelets slightly increased the release of inositol phosphates from [3H]inositol-labeled membranes. Addition of cytosol plus GTP gamma S (10 microM), however, resulted in a 300% enhancement of the release of inositol phosphates compared to membranes incubated with thrombin and GTP gamma S. The stimulatory effects of cytosol and GTP gamma S on polyphosphoinositide hydrolysis were also observed when membranes were replaced by sonicated lipid vesicles prepared from a total platelet lipid extract. These data suggest that PIP2 hydrolysis in platelets is catalyzed by a soluble phospholipase C which is regulated by a GTP-binding regulatory protein.     

Guanosine 5''-O-Thiotriphosphate and Sodium Fluoride Activate Polyphosphoinositide Hydrolysis in Rat Cortical Membranes by Distinct Mechanisms

NaF and guanosine 5'-O-thiotriphosphate [GTP(S)] stimulated the accumulation of [3H]inositol monophosphate ([3H]InsP) in rat brain cortical membranes, with half-maximal stimulation at 2 mM and 1 microM, respectively. Calcium also increased basal [3H]InsP formation over a range of concentrations from 10(-7) to 10(-4) M. The stimulatory effect of GTP(S) (30 microM) on [3H]InsP production was insensitive to Ca2+, whereas NaF-evoked [3H]InsP formation was dependent on Ca2+ concentrations. Guanosine 5'-O-thiodiphosphate significantly attenuated GTP(S)- but not NaF-stimulated [3H]InsP production. Coincubation of GTP(S) (30 microM) and submaximal concentrations of NaF (1 or 3 mM) stimulated [3H]InsP formation to a degree that was nearly additive with that produced by either drug alone. However, the resultant accumulation of [3H]InsP in the presence of maximally effective concentrations of GTP(S) and NaF was not different from that produced by NaF alone. Incubation of cortical membranes with GTP(S) and NaF for 1 min stimulated the accumulation of [3H]inositol bisphosphate (InsP2) but not [3H]InsP. [3H]InsP2 production elicited by GTP(S) was markedly enhanced by the muscarinic cholinergic agonist carbachol. In contrast, NaF-stimulated [3H]InsP2 formation was not potentiated by carbachol. Our findings of different characteristics of GTP(S) and fluoride activation of polyphosphoinositide (PPI) hydrolysis suggest that separate regulatory mechanisms are involved in these two modes of stimulation in brain membranes. Activation of PPI hydrolysis by fluoride may be mediated by a direct stimulation of PPI phosphodiesterase or by activating a putative guanine nucleotide regulatory protein at a location distinct from the GTP-binding site.     

Pertussis Toxin-Insensitive G Protein Mediates Carbachol Activation of Phospholipase D in Rat Pheochromocytoma PC12 Cells

In the present study, an activation mechanism for phospholipase D (PLD) in [3H]palmitic acid-labeled pheochromocytoma PC12 cells in response to carbachol (CCh) was investigated. PLD activity was assessed by measuring the formation of [3H]phosphatidylethanol ([3H]PEt), the specific marker of PLD activity, in the presence of 0.5% (vol/vol) ethanol. CCh caused a rapid accumulation of [3H]-PEt, which reached a plateau within 1 min, in a concentration-dependent manner. The [3H]PEt formation by CCh was completely antagonized by atropine, demonstrating that the CCh effect was mediated by the muscarinic acetylcholine receptor (mAChR). A tumor promoter, phorbol 12-myristate 13-acetate (PMA), also caused an increase in [3H]-PEt content, which reached a plateau at 30-60 min after exposure, but an inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not. Although a protein kinase C (PKC) inhibitor, staurosporine (5 microM), blocked PMA-induced [3H]PEt formation by 77%, it had no effect on the CCh-induced formation. These results suggest that mAChR-induced PLD activation is independent of PKC, whereas PLD activation by PMA is mediated by PKC. NaF, a common GTP-binding protein (G protein) activator, and a stable analogue of GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), also stimulated [3H]PEt formation in intact and digitonin-permeabilized cells, respectively. GTP, UTP, and CTP were without effect. Furthermore, guanosine 5'-O-(2-thiodiphosphate) significantly inhibited CCh- and GTP gamma S-induced [3H]PEt formation in permeabilized cells but did not inhibit the formation by PMA, and staurosporine (5 microM) had no effect on [3H]PEt formation by GTP gamma S.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters

Polymorphonuclear leukocytes (PMNs) activate phospholipase C via a guanine nucleotide regulatory (G) protein. Pretreatment of the PMNs with pertussis toxin (PT) or 4-beta-phorbol 12-myristate 13-acetate (PMA) inhibited chemoattractant-induced inositol trisphosphate generation. To determine the loci of inhibition by PT and PMA, G protein-mediated reactions in PMN plasma membranes were examined. Plasma membranes prepared from untreated and PMA-treated PMNs demonstrated equivalent ability of a GTP analogue to suppress high affinity binding of the chemoattractant-N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) to its receptor. The rate, but not the extent, of high affinity binding of GTP gamma[35S] to untreated PMN membranes was stimulated up to 2-fold by preincubation with 1 microM fMet-Leu-Phe. The ability of fMet-Leu-Phe to stimulate the rate of GTP gamma S binding was absent in membranes prepared from PT-treated PMNs, but remained intact in membranes from PMA-treated cells. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) via phospholipase C could be activated in untreated PMN membranes by either fMet-Leu-Phe plus GTP or GTP gamma S alone at low concentrations of Ca2+ (0.1-1 microM). Membranes prepared from PT-treated PMNs degraded PIP2 upon exposure to GTP gamma S, but not fMet-Leu-Phe plus GTP. In contrast, membranes prepared from phorbol ester-treated PMNs did not hydrolyze PIP2 when incubated with GTP gamma S. Treatment with PT or PMA did not affect the ability of 1 mM Ca2+ to activate PIP2 hydrolysis in PMN membranes, indicating that neither treatment directly inactivated phospholipase C. Therefore, PT appears to block coupling of the chemoattractant receptors to G protein activation, while phorbol esters disrupt coupling of the activated G protein to phospholipase C. The phorbol ester-mediated effect may mimic a negative feedback signal induced by protein kinase C activation by diacylglycerol generated upon activation of phospholipase C.     

Thyrotropin-releasing hormone and GTP activate inositol trisphosphate formation in membranes isolated from rat pituitary cells

Stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by a phospholipase C to produce inositol trisphosphate (InsP3) and 1,2-diacylglycerol appears to be the initial step in signal transduction for a number of cell-surface interacting stimuli, including thyrotropin-releasing hormone (TRH). In suspensions of membranes isolated from rat pituitary (GH3) cells that were prelabeled to isotopic steady state with [3H]inositol and incubated with ATP, [3H] PtdIns(4,5)P2, and [3H]phosphatidylinositol 4-phosphate, the polyphosphoinositides, and [3H]InsP3 and [3H]inositol bisphosphate, the inositol polyphosphates, accumulated. TRH and GTP stimulated the accumulation of [3H]inositol polyphosphates in time- and concentration-dependent manners; half-maximal effects occurred with 10-30 nM TRH and with 3 microM GTP. A nonhydrolyzable analog of GTP also stimulated [3H] inositol polyphosphate accumulation. Moreover, when TRH and GTP were added together their effects were more than additive. Fixing the free Ca2+ concentration in the incubation buffer at 20 nM, a value below that present in the cytoplasm in vivo did not inhibit stimulation by TRH and GTP of [3H]inositol polyphosphate accumulation. ATP was necessary for basal and stimulated accumulation of [3H]inositol polyphosphates, and a nonhydrolyzable analog of ATP could not substitute for ATP. These data demonstrate that TRH and GTP act synergistically to stimulate the accumulation of InsP3 in suspensions of pituitary membranes and that ATP, most likely acting as substrate for polyphosphoinositide synthesis, was necessary for this effect. These findings suggest that a guanine nucleotide-binding regulatory protein is involved in coupling the TRH receptor to a phospholipase C that hydrolyzes PtdIns(4,5)P2.     

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)     

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.     

Concerted CMP-Dependent [3H]Inositol Labeling of Phosphoinositides and Agonist Activation of Phospholipase C in Rat Brain Cortical Membranes

[3H]Inositol ([3H]Ins) labeling of phosphoinositides was studied in rat brain cortical membranes. [3H]Ins was incorporated into a common lipid pool through both CMP-dependent and independent mechanisms. These are as follows: (1) a reverse reaction catalyzed by phosphatidyl-inositol (PtdIns) synthase, and (2) the reaction performed by the PtdIns headgroup exchange enzyme, respectively. Membrane phosphoinositides prelabeled in either CMP-dependent or independent fashions were hydrolyzed by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)- and carbachol-stimulated phospholipase C. Unlike CMP-dependent labeling, however, CMP-independent incorporation of [3H]Ins into lipids was inhibited by 1 mM (0.04%) sodium deoxycholate. Thus, when PtdIns labeling and phospholipase C stimulation were studied in a concerted fashion, [3H]Ins was incorporated into lipids primarily through the PtdIns synthase-catalyzed reaction because of the presence of deoxycholate required to observe carbachol-stimulation of phospholipase C. Little direct breakdown of [3H]PtdIns was detected because production of myo-[3H]inositol 1-monophosphate was minimal and myo-[3H]inositol 1,4-bisphosphate was the predominant product. Although PtdIns labeling and 3H-polyphosphoinositide formation were unaffected by GTP gamma S and carbachol and had no or little lag period, GTP gamma S- and carbachol-stimulated appearance of 3H-Ins phosphates exhibited an appreciable lag (10 min). Also, flux of label from [3H]Ins to 3H-Ins phosphates was restricted to a narrow range of free calcium concentrations (10-300 nM). These results show the concerted activities of PtdIns synthase, PtdIns 4-kinase, and phospholipase C, and constitute a simple assay for guanine nucleotide-dependent agonist stimulation of phospholipase C in a brain membrane system using [3H]Ins as labeled precursor.     

5-Methyltryptamine stimulates phospholipase C-mediated breakdown of exogenous phosphoinositides by blowfly salivary gland membranes

5-Methyltryptamine, through a GTP-dependent mechanism, stimulated breakdown of endogenous [3H]inositol-labeled phosphoinositides in membranes prepared from blowfly salivary gland homogenates through a phospholipase C exhibiting a pH optimum of approximately 7.0. Unlabeled membranes, prepared from salivary gland homogenates, hydrolyzed exogenous [3H]phosphatidylinositol 4,5-bisphosphate substrate with generation of labeled inositol phosphates. Inositol trisphosphate formation was increased approximately 200% by 10 microM guanosine 5'-(O-thio)-trisphosphate (GTP gamma S) within 30 s. 5-Methyltryptamine, in the presence of 10 microM GTP gamma S, increased the rate of inositol trisphosphate formation by approximately 500% within 30 s. Half-maximal activation of hormone-stimulated breakdown of exogenous substrate required approximately 0.05 microM GTP gamma S. [3H]Phosphatidylinositol was also hydrolyzed during incubation with membranes, resulting in the generation of inositol, glycerol phosphoinositol, and inositol monophosphate. Formation of inositol monophosphate was stimulated approximately 30% by 10 microM GTP gamma S and 10 microM 5-methyltryptamine. Neither inositol nor glycerol phosphoinositol formation was affected by hormone. These results indicate that in a cell-free system from blowfly salivary glands, 5-methyltryptamine, through a GTP-dependent mechanism, directly activates a phospholipase C which mediates phosphoinositide hydrolysis.     

Agonist-induced desensitization of cholinergically stimulated phosphoinositide breakdown is independent of endogenously activated protein kinase C in HT-29 human colon carcinoma cells.

Activation of M3 muscarinic receptors in HT-29 cells by carbachol rapidly increases polyphosphoinositide breakdown. Pretreatment of these cells with carbachol (0.1 mM) for 5 h completely inhibits the subsequent ability of carbachol to increase [3H]inositol monophosphate ([3H]InsP) accumulation, paralleled by a total loss of muscarinic binding sites. In contrast, protein kinase C (PK-C)-mediated desensitization by incubation with phorbol esters [PMA (phorbol 12-myristate 13-acetate)], leading to a time- and dose-dependent inhibition of cholinergically stimulated InsP release (95% inhibition after 4 h with 0.1 microM-PMA), is accompanied by only a 40% decrease in muscarinic receptor binding, which suggests an additional mechanism of negative-feedback control. Neither carbachol nor PMA pretreatment had any effect on receptor affinity. Incubation with carbachol for 15 min caused a small increase of membrane-associated PK-C activity (15% increase, P less than 0.05) as compared with the potency of phorbol esters (PMA) (3-4-fold increase, P less than 0.01). Long-term incubation (4-24 h) with PMA resulted in a complete down-regulation of cytosolic and particulate PK-C activity. Stimulation of InsP release by NaF (20 mM) was not affected after a pretreatment with phorbol esters or carbachol, demonstrating an intact function of G-protein and phospholipase-C (PL-C) at the effector side. Determination of PL-C activity in a liposomal system with [3H]PtdInsP2 as substrate, showed no change in PL-C activity after carbachol (13 h) and short-term PMA (2.5 h) pretreatment, whereas long-term preincubation with phorbol esters (13 h) caused a small but significant decrease in PL-C activity (19%, P less than 0.05). Our results indicate that agonist-induced desensitization of phosphoinositide turnover occurs predominantly at the receptor level, with a rapid loss of muscarinic receptors. Exogenous activation of PK-C by phorbol esters seems to dissociate the interaction between receptor and G-protein/PL-C, without major effects on total cellular PL-C activity.     

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.     

Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.     

Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of [3H]choline and [3H]phosphorylcholine ([3H]Pchol) from cells containing [3H]choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of [3H]phosphatidic acid ([3H]PA) in cells containing [3H]myristate-labeled PC. [3H]Diacylglycerol ([3H]DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with [3H]myristate and [14C]arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol. By analyzing the increase in 3H versus 14C in DAG, we estimate that the DAG that is formed in response to PMA arises largely from PC. Muscarinic receptor activation also causes formation of DAG from PC, but approximately 20% of carbachol-stimulated DAG appears to arise from hydrolysis of the phosphoinositides.