Effect of nonhydrolyzable guanosine phosphate on IgE-mediated activation of phospholipase C and histamine release from rodent mast cells

Rat mast cells and bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE mAb, and permeabilized by ATP to introduce guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) and/or guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) into the cells. After ATP-induced lesions were resealed with Mg2+, the cells were challenged by Ag to determine the effect of the nonhydrolyzable guanosine phosphate on Ag-induced hydrolysis of phosphoinositides and histamine release. Introduction of GTP gamma S into permeabilized rat mast cells or BMMC, followed by exposure of the cells to extracellular Ca2+, resulted in histamine release, but failed to induce hydrolysis of phosphoinositides. It was also found that introduction of GTP gamma S into the cells did not synergistically enhance Ag-induced histamine release. Introduction of GDP beta S into sensitized BMMC inhibited the GTP gamma S-dependent, Ca2+-induced histamine release but failed to inhibit Ag-induced histamine release. The results suggest that GTP gamma S-dependent, Ca2+-induced histamine release and Ag-induced histamine release go through independent biochemical pathways. It was also found that introduction of GTP gamma S or GDP beta S into sensitized BMMC neither enhanced nor inhibited Ag-induced formation of inositol phosphates. These results together with previous findings that pretreatment of BMMC with either pertussis toxin or cholera toxin does not affect Ag-induced hydrolysis of phosphoinositides, indicate that a G protein is not involved in the transduction of IgE-mediated triggering signals to phospholipase C in rodent mast cells.     

Evidence for coupling of bradykinin receptors to a guanine-nucleotide binding protein to stimulate arachidonate liberation in the osteoblast-like cell line, MC3T3-E1.

The effect of bradykinin on the activation production of inositol 1,4,5-trisphosphate and prostaglandin E2 (PGE2) was examined in the murine osteoblastic cell line, MC3T3-E1. Bradykinin, at concentrations ranging from 1 to 1000 nM, stimulated the production of inositol 1,4,5-trisphosphate 2.5- to 3-fold within 10 s, and elevated cytosolic-free Ca2+, even in the absence of external Ca2+. This process is mediated through the activation of phospholipase C. Bradykinin at the same concentration also stimulated the production of PGE2 and caused a release of 3H radioactivity from the cells prelabeled with [3H]arachidonic acid, probably via the activation of phospholipase A2. Pretreatment of the cells with pertussis toxin inhibited the stimulation of PGE2 production and 3H radioactivity release, while the elevation in cytosolic Ca2+ and the production of inositol 1,4,5-trisphosphate were not altered by toxin-pretreatment. The addition of an unhydrolyzable analog of GTP, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) to the beta-escin-permeabilized cells prelabeled with [3H]arachidonic acid enhanced the release of 3H radioactivity. The simultaneous presence of bradykinin with GTP gamma S further activated the 3H radioactivity release in the beta-escin-permeabilized cells. These results provide evidence that receptors for bradykinin in the MC3T3-E1 couple stimulating arachidonate release, probably via the activation of phospholipase A2, through a guanine nucleotide binding protein sensitive to pertussis toxin.     

An islet activating protein-sensitive G protein is involved in dopamine inhibition of angiotensin and thyrotropin-releasing hormone-stimulated inositol phosphate production in anterior pituitary cells

In primary culture of anterior pituitary cells, dopamine inhibited the angiotensin (AII)-stimulated inositol phosphate production by 28 +/- 2.5% (n = 14), with an EC50 of 660 +/- 228 nM (n = 8). This effect was blocked by (+)-butaclamol, a specific dopamine receptor antagonist. RU 24926, a D2 specific agonist, but not SKF 38393, a specific D1 agonist, inhibited AII-stimulated inositol phosphate production, suggesting that this dopamine effect is mediated through a dopamine receptor of the D2 subtype. Dopamine also partially inhibited (25%) inositol phosphate production stimulated by thyrotropin-releasing hormone (TRH). Our results suggest that the dopamine-mediated inhibition of hormonally stimulated inositol phosphate production is probably not mediated through the known inhibitory effects of dopamine on cAMP and Ca2+ intracellular concentrations. Although unknown, the mechanism by which dopamine inhibited the AII and TRH-stimulated inositol phosphate production implicates a GTP binding protein sensitive to the islet activating protein (IAP) since dopamine effects were blocked by this toxin. The alpha subunit of the GTP binding protein involved could be one of the three ADP-ribosylated proteins found in anterior pituitary cells in primary cultures, the alpha o (39 kDa), the alpha i (41 kDa), and an alpha subunit of 40 kDa. Indeed, we show here that this 40-kDa IAP substrate, already described in a few tissues, is present in anterior pituitary cells. The negative coupling between dopamine receptors and the AII or TRH inositol phosphate production systems, could be implicated in the dopamine inhibition of the AII- and TRH-stimulated prolactin release since such an inhibition is blocked by IAP. Our results suggest that the negative regulation of inositol phosphate production is one of the mechanisms by which dopamine controls hormonally stimulated prolactin release.     

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.     

Cholera toxin modulation of angiotensin II-stimulated inositol phosphate production in cultured vascular smooth muscle cells.

Activation of phospholipase C by angiotensin II in vascular smooth muscle has been postulated to be mediated by an unidentified GTP-binding protein (G-protein). Using a permeabilized preparation of myo-[3H]inositol-labelled cultured vascular smooth muscle cells, we examined the ability of a non-hydrolysable analogue of GTP, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to stimulate inositol phosphate formation. GTP[S] (5 min exposure) stimulated inositol polyphosphate release by up to 3.8-fold in a dose-dependent manner, with an EC50 (concn. producing half-maximal stimulation) of approx. 50 microM. Inositol bisphosphate (IP2) and inositol trisphosphate (IP3) accumulations were also stimulated by NaF (5-20 mM). Furthermore, angiotensin II-induced inositol phosphate formation could be potentiated by a submaximal concentration of GTP[S] (10 microM), and this treatment appeared to interfere with the normal termination mechanism of the initial hormonal signal. The G-protein mediating angiotensin II-stimulated phospholipase C activation was insensitive to pertussis toxin at an exposure time and concentration which were sufficient to completely ADP-ribosylate all available substrate (100 ng/ml, 16 h). In contrast, a similar incubation with cholera toxin markedly inhibited angiotensin II-stimulated IP2 and IP3 release by 67 +/- 6% and 62 +/- 6% respectively. Cholera toxin appeared to inhibit angiotensin II stimulation of phospholipase C by a dual mechanism: it caused a 45% decrease in angiotensin II receptor number, and also inhibited G-protein transduction as assessed by GTP[S]-stimulated IP2 formation. This latter inhibition may be secondary to an increase in cyclic AMP, since it could be simulated by addition of dibutyryl cyclic AMP. Thus angiotensin II-stimulated inositol phosphate formation is cholera-toxin-sensitive, and is mediated by a pertussis-toxin-insensitive G-protein, which may be involved directly in termination of early signal generation.     

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)     

Coupling of the thyrotropin-releasing hormone receptor to phospholipase C by a GTP-binding protein distinct from the inhibitory or stimulatory GTP-binding protein

Thyrotropin-releasing hormone (TRH) stimulated a rapid rise in inositol trisphosphate (IP3) formation and prolactin release from 7315c tumor cells. The potencies (half-maximal) of TRH in stimulating IP3 formation and prolactin release were 100 +/- 30 and 140 +/- 30 mM, respectively. Pretreatment of the cells with pertussis toxin (for up to 24 h) had no effect on either process. Pretreatment of the cells with cholera toxin (30 nM for 24 h) also failed to affect basal or TRH-stimulated IP3 formation. TRH was also able to stimulate IP3 formation with a half-maximal potency of 118 +/- 10 nM in a lysed cell preparation of 7315c cells; the TRH-stimulated formation of IP3 was enhanced by GTP. 5'-Guanosine gamma-thiotriphosphate (GTP gamma S) and 5'-guanylyl imidodiphosphate (Gpp(NH)p), nonhydrolyzable analogs of GTP, stimulated IP3 formation in the absence of TRH with half-maximal potencies of 162 +/- 50 and 7500 +/- 4300 nM, respectively. In contrast to the lack of effect of pertussis toxin on the TRH receptor system, treatment of 7315c cells with pertussis toxin for 3 h or longer completely abolished the ability of morphine, an opiate agonist, to inhibit either adenylate cyclase activity or prolactin release. During this 3-h treatment, pertussis toxin was estimated to induce the endogenous ADP ribosylation of more than 70% of Ni, the inhibitory GTP-binding protein. GTP gamma S and Gpp(NH)p inhibited cholera toxin-stimulated adenylate cyclase activity (presumably by acting at Ni) with half-maximal potencies of 25 +/- 9 and 240 +/- 87 nM, respectively. Finally, Gpp(NH)p was also able to inhibit the [32P]ADP ribosylation of Ni with a half-maximal potency of 300 nM. These results suggest that a novel GTP-binding protein, distinct from Ni, couples the TRH receptor to the formation of IP3.     

Guanosine 5'-O-(3-thiotrisphosphate) potentiates both thrombin- and platelet-derived growth factor-induced inositol phosphate release in permeabilized vascular smooth muscle cells. Signaling mechanisms distinguished by sensitivity to pertussis toxin and phorbol esters

We compared the mechanisms by which thrombin and platelet-derived growth factor (PDGF) activate phospholipase C in cultured vascular smooth muscle cells. Thrombin caused a transient (less than 5 min) increase in inositol trisphosphate (IP3) while PDGF caused a sustained (greater than 10 min) increase. Both pertussis toxin and phorbol 12-myristate 13-acetate (PMA) inhibited the thrombin-induced increase in IP3 but neither agent affected the PDGF-induced increase in IP3. To examine the role of GTP binding (G) proteins in the activation of phospholipase C by these two hormones, GTP analogues were introduced into saponin-permeabilized cells. In the absence of hormones, guanosine 5'-O-(3-thiotrisphosphate) (GTP gamma S) caused a progressive increase in IP3 release which was inhibited 55% by PMA (200 ng/ml). In the presence of thrombin, GTP gamma S caused synergistic increase in IP3 release. The synergism between GTP gamma S and thrombin was virtually eliminated by 10 min prior exposure to PMA (200 ng/ml). When PDGF was the hormonal agonist, GTP gamma S also caused synergistic increase in IP3 release and guanosine 5'-O-(2-thiodiphosphate) blunted PDGF-induced IP3 release. However, in contrast to thrombin, the synergism between GTP gamma S and PDGF was unaffected by PMA. Thus, thrombin and PDGF activate phospholipase C by signal transduction systems which differ in kinetic properties and in sensitivity to PMA and pertussis toxin. Despite these differences, both systems appear to involve GTP binding proteins at some step.     

Adenosine Inhibits Histamine-Induced Phosphoinositide Hydrolysis Mediated via Pertussis Toxin-Sensitive G Protein in Human Astrocytoma Cells

The effect of adenosine on phosphoinositide hydrolysis was examined in 1321N1 human astrocytoma cells. Adenosine, L-N6-phenylisopropyladenosine (L-PIA), and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was L-PIA greater than adenosine greater than NECA, a finding indicating that A1-class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by L-PIA was blocked by an adenosine receptor antagonist, 8-phenyltheophylline. Stimulation of A1-class adenosine receptors inhibited isoproterenol-stimulated cyclic AMP accumulation as well as histamine-induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet-activating protein (IAP)]. L-PIA also inhibited guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-stimulated accumulation of inositol phosphates in membrane preparations, and 8-phenyl-theophylline antagonized the inhibition. L-PIA could not inhibit GTP gamma S-induced accumulation of inositol phosphates in IAP-treated membranes. Gi/Go, purified from rabbit brain, inhibited GTP gamma S-stimulated accumulation of inositol phosphates in a concentration-dependent manner in membrane preparations. These results suggest that stimulation of A1-class adenosine receptors interacts with the IAP-sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells.     

Coupling of bradykinin receptors to phospholipase C in cultured fibroblasts is mediated by a G-protein

In cultured foreskin fibroblasts, bradykinin stimulates inositol phosphate generation, arachidonic acid release, and Na+/H+ exchange, with doses of 1-3 nM yielding half-maximal stimulation. Binding of 3H-bradykinin to these cells demonstrates a single receptor site with a Kd of 2.0 nM and a Bmax of 91 fmoles/mg protein. Bradykinin analogs of the B2 type inhibit this binding. GTP synergizes with bradykinin to stimulate phosphatidylinositol turnover in permeabilized fibroblasts and GTP-gamma-S decreases the Bmax of bradykinin binding to fibroblast membranes, indicating that a G-protein couples the receptor to phospholipase C. Pretreatment of fibroblasts with either cholera or pertussis toxin enhances bradykinin stimulation of inositol phosphate accumulation.     

Contribution of G Protein Activation to Fluoride Stimulation of Phosphoinositide Hydrolysis in Human Neuroblastoma Cells

Abstract: To examine the possibility that NaF enhances phosphoinositide-specific phospholipase C (PIC) activity in neural tissues by a mechanism independent of a guanine nucleotide binding protein (G_p), we have evaluated the contribution of G_p activation to NaF-stimulated phosphoinositide hydrolysis in human SK-N-SH neuroblastoma cells. Addition of NaF to intact cells resulted in an increase in the release of inositol phosphates (450% of control values; EC₅₀ of ～ 8 mM). Inclusion of U-73122, an aminosteroid inhibitor of guanine nucleotide-regulated PIC activity in these cells, resulted in a dose-dependent inhibition of NaF-stimulated inositol lipid hydrolysis (IC₅₀ of ～ 3.5 μM). When added to digitonin-permeabilized cells, NaF or guanosine-5′-O-thiotriphosphate (GTPγS) resulted in a three- and sevenfold enhancement, respectively, of inositol phosphate release. In the combined presence of optimal concentrations of NaF and GTPγS, inositol phosphate release was less than additive, indicative of a common site of action. Inclusion of 2–5 mM concentrations of guanosine-5′-O-(2-thiodiphosphate) (GDPβS) fully blocked phosphoinositide hydrolysis elicited by GTPγS, whereas that induced by NaF was partially inhibited (65%). However, preincubation of the cells with GDPβS resulted in a greater reduction in the ability of NaF to stimulate inositol phosphate release (87% inhibition). Both GTPγS and NaF-stimulated inositol phosphate release were inhibited by inclusion of 10 μM U-73122 (54–71%). The presence of either NaF or GTPγS also resulted in a marked lowering of the Ca²⁺ requirement for activation of PIC in permeabilized cells. These results indicate that in SK-N-SH cells, little evidence exists for direct stimulation of PIC by NaF and that the majority of inositol phosphate release that occurs in the presence of NaF can be attributed to activation of G_p.     

Involvement of a specific guanine nucleotide binding protein in receptor immunoglobulin stimulated inositol phospholipid hydrolysis

The role of a specific guanine nucleotide binding (G protein) protein in coupling murine B lymphocyte receptor immunoglobulin to inositol phospholipid hydrolysis was investigated. Using an in vitro system with isolated membranes, we have observed specific enhancement of GTP binding subsequent to ligand-induced receptor crosslinking. Induced increases were inhibited by pretreatment with pertussis toxin which catalyzed ADP-ribosylation of a 43 kDa substrate. Involvement of this G protein with receptor immunoglobulin-induced inositol phospholipid hydrolysis was evidenced by the ability of pertussis toxin to block this response. This report, then, indicates that the B lymphocyte antigen receptor belongs to a family of receptors which are linked to inositol phospholipid hydrolysis through a G protein.     

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.     

Guanine nucleotides induce Ca2+-independent insulin secretion from permeabilized RINm5F cells

The role of guanine nucleotides in insulin secretion was investigated in electrically permeabilized RINm5F cells. Ca2+ stimulated insulin release (EC50 approximately 2 microM Ca2+). The GTP stable analog, GTP gamma S, elicited insulin secretion at vanishingly low Ca2+ concentrations (less than 10(-11) M), slightly potentiated the response to intermediate Ca2+ levels, but exerted less than additive effects at maximal Ca2+ concentrations. The GDP analog, GDP beta S, inhibited both GTP gamma S- and Ca2+-stimulated secretion. The action of GTP gamma S was not mediated by cAMP, as the latter only enhanced Ca2+-induced secretion. In contrast, 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, promoted insulin release at nonstimulatory Ca2+ levels as well as potentiating the Ca2+ response. GTP analogs stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2), as assessed by inositol phosphate generation. However, this could not fully explain guanine nucleotide-induced secretion because: GTP gamma S-stimulated PtdInsP2 breakdown was totally dependent on Ca2+ and abolished at Ca2+ below 10(-11) M; at these Ca2+ levels, activators of protein kinase C were weak or ineffective secretagogues; the GTP analog Gpp(NH)p was much less effective than GTP gamma S in activating PtdInsP2 hydrolysis, while fully mimicking the effect on Ca2+-independent secretion. Both GTP gamma S-induced PtdInsP2 hydrolysis and insulin release were insensitive to pertussis toxin and cholera toxin. The findings point to a guanine nucleotide-regulated site in the activation of insulin secretion different from the known transmembrane signalling systems.     

Histamine-stimulated and GTP-binding proteins-mediated phospholipase A2 activation in rabbit platelets

Histamine is known to be a mediator of inflammation. In order to understand the role of histamine in platelets, we have examined the effects of histamine on arachidonic acid (AA) release, cAMP accumulation, inositol trisphosphate production, and serotonin secretion. Incubation of rabbit (and human) platelets with histamine resulted in rapid increase of [3H]AA release from the platelets prelabeled with [3H]AA. The effect of histamine was blocked by the addition of H1 receptor antagonist mepyramine. Histamine did not substantially affect the cAMP content and inositol trisphosphate production. Histamine-stimulated AA release was not observed in digitonin-permeabilized platelets, whereas histamine acted synergistically with GTP or GTP analog, guanosine 5'-(3-O-thio)triphosphate. Histamine-stimulated, and GTP analog-dependent AA release was inhibited by guanosine 5'-(2-O-thio) diphosphate. The effects of three receptor stimulants, thrombin, norepinephrine, and histamine were both diminished by 1 microgram/ml of pertussis toxin treatment and by the antiserum against GTP-binding proteins (G proteins) treatment. However, the antiserum against beta gamma subunits of G proteins inhibited the histamine effect, not thrombin effect. 4 beta-Phorbol 12-myristate 13-acetate (PMA) treatment enhanced histamine-stimulated AA release and serotonin secretion but inhibited thrombin-stimulated reactions. The effect of PMA was dose dependent and was due to enhance the coupling of histamine receptors and G proteins. The results show the existence of H1 histamine receptors which couple phospholipase A2 activation via pertussis toxin-sensitive G proteins. Histamine actions differ in sensitivities to anti-beta gamma antiserum treatment and PMA treatment from thrombin actions.     

Intracellular Calcium Pools in Neuroblastoma × Glioma Hybrid NG108–15 Cells

The intracellular nonmitochondrial calcium pools of saponin-permeabilized NG108-15 cells were characterized using inositol 1,4,5-trisphosphate (IP3) and GTP. IP3 or GTP alone induced release of 47 and 68%, respectively, of the calcium that was releasable by A23187. GTP induced release of a further 24% of the calcium after IP3 treatment, whereas IP3 induced release of a further 11% of the calcium after GTP treatment. Guanosine 5'-O-(3-thio)triphosphate had little effect on IP3-induced calcium release but completely inhibited GTP-induced calcium release. In contrast, heparin inhibited the action of IP3 but not that of GTP. The results imply the existence of at least three nonmitochondrial pools: (a) 31% is releasable by IP3 and GTP, (b) 11% is releasable by IP3 alone, and (c) 24% is releasable by GTP alone. GTP enhanced calcium uptake in the presence of oxalate with an EC50 of 0.6 microM and stimulated calcium release in the absence of oxalate with an EC50 of 0.32 microM. The similar EC50 values for these dual effects of GTP on calcium movement suggest that GTP exerts its dual action by the same mechanism.     

Calcium mobilization in permeabilized fibroblasts: effects of inositol trisphosphate, orthovanadate, mitogens, phorbol ester, and guanosine triphosphate

Utilizing a digitonin-permeabilized cell system, we have studied the release of calcium from a non-mitochondrial intracellular compartment in cultured human fibroblasts (HSWP cells). Addition of 1 mM MgATP to a monolayer of permeabilized cells in a cytosolic media buffered to 150 nM Ca with EGTA rapidly stimulates 45Ca uptake, and the subsequent addition of the putative intracellular messenger inositol trisphosphate (InsP3) induces rapid release of 85% (+/- 6% n = 6) of the 45Ca taken up in response to ATP. Mitogenic peptides (bradykinin, vasopressin, epidermal growth factor [EGF], and insulin) and orthovanadate, which are effective in mobilizing intracellular Ca in intact cells, have little or no effect when added alone to permeabilized cells. However, in the presence of GTP these agents stimulate accumulation of inositol phosphates and release Ca from the InsP3-sensitive pool. These data suggest that a GTP binding protein is involved in receptor mediated activation of phospholipase C, which leads to release of inositol phosphates. The GTP-dependent release of InsP3 and the mobilization of 45Ca from the intracellular compartment are inhibited by pretreatment of cells, prior to permeabilization, with the protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). TPA pretreatment does not affect the InsP3 stimulated Ca release. These results suggest that protein kinase C is involved in down-regulation or inhibition of phospholipase C, or the GTP binding protein responsible for relaying the mitogenic signal from the cell surface receptor to the phospholipase C activity.     

Guanine nucleotide effects on catecholamine secretion from digitonin-permeabilized adrenal chromaffin cells

The nonhydrolyzable GTP analogue guanosine 5'-(beta, gamma-imido)triphosphate (GMP-PNP) produced an ATP-dependent but Ca2+-independent stimulation of [3H]norepinephrine release from permeabilized chromaffin cells. This stimulation of secretion was 25-35% of the secretion induced by 10 microM Ca2+. A similar Ca2+-independent stimulation was produced by other non-hydrolyzable GTP analogues. No effect was seen with a variety of other nucleotides, including GTP. The GMP-PNP effect was specifically inhibited by low concentrations of guanine nucleotides. Addition of cAMP did not mimic the Ca2+-independent GMP-PNP effect, but did slightly enhance Ca2+-dependent secretion. Pretreatment with pertussis toxin had no effect on Ca2+-dependent secretion or on the GMP-PNP effect. There was no detectable diglyceride or inositol phosphate produced during GMP-PNP treatment, and addition of diglyceride and inositol trisphosphate did not induce secretion. Guanosine 5'-(beta-thio)diphosphate (GDP-beta-S), in addition to its ability to inhibit the GMP-PNP effect, partially inhibited Ca2+-dependent secretion. At 10 microM free Ca2+, the effects of GMP-PNP and Ca2+ were nonadditive. In fact, secretion in the presence of both GMP-PNP and 10 microM Ca2+ was slightly less than secretion due to Ca2+ alone. These data suggest that a guanine nucleotide-dependent process interacts in some way with one or more components of the normal Ca2+-dependent secretory pathway. However, it may not be an intrinsic part of the mechanism underlying Ca2+-dependent secretion.     

GTP and cytosol stimulate phosphoinositide hydrolysis in isolated platelet membranes

Hydrolysis of polyphosphoinositides by phospholipase C was examined in isolated membranes prepared from [32P]labelled platelets. In the presence of GTP gamma S, thrombin increased the release of inositol triphosphate and inositol biphosphate approximately 500%. GTP gamma S alone stimulated release 2 fold. Maximal activation of thrombin-induced phosphoinositide hydrolysis was observed at 10 uM GTP. Although addition of calcium had no effect, 2 mM EGTA completely inhibited inositolphosphate release. Addition of high speed supernatant to [32P]labelled membranes stimulated the release of inositolphosphates. This hydrolysis was further enhanced by the addition of GTP. These data demonstrate that the breakdown of polyphosphoinositides in isolated platelet membranes is dependent on GTP and stimulated by platelet cytosol.     

Pertussis toxin-sensitive GTP-binding proteins may regulate phospholipase A2 in response to thrombin in rabbit platelets

Incubation of rabbit platelets with thrombin resulted in rapid accumulations of inositol trisphosphate (IP3) in [3H]inositol-labeled platelets, increases of [3H]arachidonic acid [( 3H]AA) release, and [3H]serotonin secretion from the platelets prelabeled with these labeled compounds. The experiments using phospholipase A2 or C inhibitor suggested that not only phospholipase C but also phospholipase A2 activity plays an important role in serotonin secretion. We then studied the regulatory mechanisms of phospholipase A2 activity. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), guanyl-5'-(beta,gamma-iminio)triphosphate), or AlF4- caused a significant liberation of AA in digitonin-permeabilized platelets but not in intact platelets. Thrombin-stimulated AA release was not observed in permeabilized platelets, whereas thrombin acted synergistically with GTP or GTP analogs to stimulate AA release. GTP analog-stimulated AA release was inhibited by guanosine 5'-(2-O-thio)diphosphate) and was also inhibited by decreased Mg2+ concentrations. Thrombin-induced, GTP-dependent AA release, but not IP3 formation, was diminished by 100 ng/ml of pertussis toxin, associated with ADP-ribosylation of membrane 41-kDa protein(s). Thrombin-stimulated AA release from intact platelets and GTP gamma S-stimulated release from permeabilized platelets were both markedly dependent on Ca2+. However, Ca2+ addition could not enhance AA release without GTP gamma S even when Ca2+ was increased up to 10(-4) M in permeabilized platelets. The results show that thrombin-stimulated AA release from rabbit platelets is mainly mediated by phospholipase A2 activity, not by phospholipase C activity, and that Ca2+ is an important factor to the activation of phospholipase A2 but is not the sole factor to the regulation. GTP-binding protein(s) is involved in receptor-mediated activation of phospholipase A2.