Serotonin-induced alterations in inositol phospholipid metabolism in human platelets

When human platelets were incubated for 5 min with [32P]orthophosphate and then stimulated with serotonin, the 32P content of phosphatidylinositol (PI) increased within seconds, compared with the control. The 32P content of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) only slightly increased during the first minute after addition of serotonin and became more apparent on prolonged stimulation. These changes were not caused by serotonin-induced change in the specific activity of ATP. Using inorganic phosphate determination for the chemical quantification of different inositol phospholipid pools, we found that the platelet PI content remained nearly constant; the amount of PIP increased while that of PIP2 decreased. When the platelets were first prelabeled for 80 min with [32P]orthophosphate, the changes in 32P-labeled inositol phospholipids after addition of serotonin were similar to their changes in mass. When the platelet inositol phospholipids were labeled with myo-[2-3H]inositol, serotonin induced an increase in [3H]inositol phosphates. From these data, it is concluded in addition to the earlier-reported effects on phospholipid metabolism (de Chaffoy de Courcelles, D. et al. (1985) J. Biol. Chem. 260, 7603-7608) that serotonin induces: a very rapid formation of PI; and alterations in inositol phospholipid interconversion that cannot be explained solely as a resynthesis process of PIP2.     

Metabolic regulation during early frog development: flow of glycolytic carbon into phospholipids in Xenopus oocytes and fertilized eggs

32P-labeled glucose 6-phosphate, [32P]phosphoenolpyruvate, and [gamma-32P]ATP were injected into oocytes and fertilized eggs of Xenopus laevis, and the incorporation of the 32P label was followed into phospholipids. Several classes of phospholipids incorporated 32P label from the injected glycolytic intermediates, including lysophosphatidic acid, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol phosphates, inferring de novo synthesis of these lipids from dihydroxyacetone phosphate or glycerol 3-phosphate. Injection of [gamma-32P]ATP into oocytes and fertilized eggs led to labeling of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate, indicating an active phosphatidylinositol cycle in resting oocytes and fertilized eggs. Maturation and fertilization of the oocyte led to a qualitative change in phosphatidylinositol metabolism, increased labeling of phosphatidylinositol phosphate compared to phosphatidylinositol bisphosphate (either from glycerol 3-phosphate or from ATP). This change occurs late in the maturation process, and the new pattern of phosphatidylinositol metabolism is maintained during the rapid cleavage stages of early embryogenesis.     

Modulation of inositol phospholipid metabolism by polyamines.

At low concentrations of Mg2+, incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in plasma membranes isolated from human polymorphonuclear leucocytes was enhanced 2-4-fold by the polyamines spermidine and spermine. Polyamines had no effects on inositol phospholipid phosphorylation at high concentrations of Mg2+. At 1 mM-Mg2+, [32P]PIP2 synthesis was maximally enhanced by 2 mM-spermine and 5 mM-spermidine, whereas putrescine only slightly enhanced synthesis. Spermine decreased the EC50 (concn. for half-maximal activity) for Mg2+ in [32P]PIP2 synthesis from 5 mM to 0.5 mM. Spermine did not modulate the Km for ATP for [32P]PIP or [32P]PIP2 synthesis. Spermine also decreased the EC50 for PI in [32P]PIP synthesis. In contrast, spermine elevated the apparent Vmax, without affecting the EC50 for PIP, for [32P]PIP2 synthesis. Spermine and spermidine also inhibited the hydrolysis of [32P]PIP2 by phosphomonoesterase activity. Therefore polyamines appear to activate inositol phospholipid kinases by eliminating the requirements for super-physiological concentrations of Mg2+. Polyamine-mediated inhibition of polyphosphoinositide hydrolysis would serve to potentiate further their abilities to promote the accumulation of polyphosphoinositides in biological systems.     

Uptake and phosphorylation of phosphatidylinositol by rat liver nuclei. Role of phosphatidylinositol transfer protein

The incorporation of phosphatidyl[2-3H]inositol ([3H]PI) from vesicles or microsomal membranes into rat liver nuclei is greatly stimulated by phosphatidylinositol transfer protein (PI-TP). The nuclei are able to phosphorylate [3H]PI, with the production of phosphatidylinositol 4-phosphate (PIP). Recovery of tritiated inositol trisphosphate, inositol phosphate, glycerophosphoinositol and inositol, suggests that in isolated nuclei a large set of enzymes of the PI cycle is present, similar to the enzymes involved in the plasma membrane PI cycle. Incubation with [gamma-32P]ATP shows that isolated nuclei are able to phosphorylate endogenous PI to PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). In the presence of exogenous PI and detergent the synthesis of PIP is increased, indicating that in nuclei the PI pool is suboptimal for the PI-kinase activity. The present study suggests that PI-TP may be involved in providing substrates for PI metabolism at the nuclear level.     

Regulation of brain phosphatidylinositol-4-phosphate kinase by GTP analogues. A potential role for guanine nucleotide regulatory proteins

Incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4,5-bisphosphate (PIP2) in membranes isolated from rat brain was enhanced in a concentration-dependent manner by the GTP analogue guanosine 5'-O-(thio)triphosphate (GTP gamma S). In contrast, neither the labeling of phosphatidylinositol 4-phosphate in the same membranes nor PIP kinase activity in the soluble fraction were stimulated by GTP gamma S. Synthesis of [32P]PIP2 was not stimulated by GTP, GDP, GMP, or ATP; however, the stimulatory effects of GTP gamma S were antagonized by GTP, GDP, and guanosine 5'-O-thiodiphosphate (GDP beta S). The nucleotide-stimulated labeling of PIP2 was not due to protection of [gamma-32P] ATP from hydrolysis, activation of PIP2 hydrolysis by phospholipase C, or inhibition of PIP2 hydrolysis by its phosphomonoesterase. Therefore, phosphatidylinositol 4-phosphate kinase activity in brain membranes may be regulated by a guanine nucleotide regulatory protein. This system may enhance the resynthesis of PIP2 following receptor-mediated activation of phospholipase C.     

Plasma membrane-associated phosphatase activities hydrolyzing [32P]phosphotyrosyl histones and [32P]phosphatidylinositol phosphate

We describe a procedure of preparing [32P]phosphotyrosyl histones with minimal contamination by 32P-labeled lipids; the latter was usually found to be mixed with the phosphoproteins when the cell membrane-enriched fraction of A-431 cells was used as a source of tyrosine kinase. The phosphatase activities previously found to be associated with the plasma membranes of a human astrocytoma were resolved using purified [32P]phosphotyrosyl histones and [32P]phosphatidylinositol phosphate. In comparison with the phosphotyrosyl protein phosphatase, the phosphatidylinositol phosphate phosphatase activity is more active over a broad range of pH values, and its activity is inhibited by fluoride, zinc chloride, and lower concentrations of vanadate.     

Turnover of the phosphomonoester groups of polyphosphoinositol lipids in unstimulated human platelets

The metabolic activity of the polyphosphoinositol lipids in unstimulated human platelets was studied by short-term labelling with [32P]Pi, by replacement of [32P]Pi from pre-labelled platelets with unlabelled phosphate, and by depriving the cells of metabolic ATP. Under short-term labelling conditions, the 4- and 5-phosphate groups of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] had the same specific 32P radioactivity as the gamma-phosphate of metabolic ATP. The specific 32P radioactivity of the 1-phosphates of phosphatidylinositol, PtdIns4P and PtdIns(4,5)P2 was similar, but only 4-13% compared to that of the ATP-gamma-phosphate. When [32P]Pi pre-labelled platelets were incubated with up to 25 mM of unlabelled phosphate, the displacement of the 32P label from PtdIns4P, PtdIns(4,5)P2 and metabolic ATP followed similar kinetics. Inhibition of ATP regeneration in platelets pre-labelled with [32P]Pi resulted in a rapid fall in metabolic ATP with a much slower fall in [32P]PtdIns(4,5)P2, whereas [32P]PtdIns4P increased initially. However, ATP turnover was not abolished, as indicated by the marked (25% of the control) incorporation of extracellular [32P]Pi into PtdIns4P and PtdIns(4,5)P2 in metabolically inhibited platelets. This low phosphate turnover may explain the relative resistance of PtdIns4P and PtdIns(4,5)P2 to metabolic inhibition. We conclude that PtdIns4P and PtdIns(4,5)P2 are present as a single metabolic pool in human platelets. Turnover of the 4- and 5-phosphates of PtdIns4P and PtdIns(4,5)P2 in unstimulated platelets is as rapid as that of the gamma-phosphate of metabolic ATP, and accounts for about 7% of basal ATP consumption.     

Phosphoinositide phosphorylation and hydrolysis in pancreatic islet cell membrane

Membranes were isolated from dispersed rat pancreatic islet cells by attachment to Sephadex beads. When these membranes were exposed to [gamma-32P]ATP, formation of 32P-labeled phosphatidate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate was observed. Carbamylcholine, added 10 s prior to lipid extraction, caused a dose-related fall in 32P-labeled phospholipids. The effect of the cholinergic agent was suppressed by atropine, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, and verapamil, and simulated, in part, by an increase in Ca2+ concentration. When the membranes were derived from islet cells prelabeled with [U-14C]arachidonate, carbamylcholine stimulation, in addition to decreasing labeled polyphosphoinositides, was accompanied by an increased production of labeled diacylglycerol, without a concomitant increase in labeled phosphatidylinositol. These results indicate that activation of a plasma membrane-associated phospholipase C directed against polyphosphoinositides represents a primary event in the functional response of the pancreatic beta cell to cholinergic agents.     

脂多糖增加小鼠巨噬细胞多磷酸肌醇脂代谢转换

多磷酸肌醇脂（这里指ＰＩＰ和ＰＩＰ２）的代谢在细胞信息传递和膜运转中起着重要的作用．脂多糖（ｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ,ＬＰＳ）于激活小鼠腹腔巨噬细胞（Ｍφ）初期（０．５～１ｈ）和后期（１６ｈ）明显增加来自［γ－３２Ｐ］ＡＴＰ的３２Ｐ参入Ｍφ的ＰＩＰ和ＰＩＰ２,３２Ｐ参入ＰＩＰ２的显著性大于ＰＩＰ．ＬＰＳ的这种作用在其激活Ｍφ的初期不受酪氨酸蛋白激酶抑制剂ｇｅｎｉｓｔｅｉｎ、蛋白激酶Ａ激动剂（ｆｏｒｓｋｏｌｉｎ）及百日咳毒素的影响;但佛波酯（ＰＭＡ）长时间预处理的Ｍφ（其ＰＫＣ活性被耗竭）再受ＬＰＳ刺激,［３２Ｐ］ＰＩＰ２水平较ＬＰＳ刺激未受ＰＭＡ预处理的Ｍφ明显降低．结果表明,ＬＰＳ于激活Ｍφ的初期和后期更显著增加ＰＩ４Ｐ－５激酶的活性,导致ＰＩＰ２合成增加,ＰＩＰ２合成的增加可能与Ｍφ激活时不同时期所表达的功能相关．     

Chemoattractant receptor-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate in human polymorphonuclear leukocyte membranes. Requirement for a guanine nucleotide regulatory protein

Incubation of plasma membranes from human polymorphonuclear leukocytes (PMNs) with [gamma-32P]ATP in the presence of MgCl2 resulted in the formation of 32P-labeled phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). Membranes from PMN specific and azurophil granules synthesized only PIP, suggesting that PIP2 metabolism is confined to the plasma membrane in PMNs. Further incubations of the labeled plasma membranes for 60 s in the presence of 1 mM CaCl2 resulted in the hydrolysis of approximately 40 and 50% of the labeled PIP and PIP2, respectively. In the presence of 2 microM added CaCl2, PIP and PIP2 levels were unchanged by incubation with either the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) at 0.1 microM or by 10 microM GTP; however, addition of fMet-Leu-Phe plus GTP together resulted in a 11 and 28% decrease in PIP and PIP2, respectively. These treatments had no effect on PA levels. No additional radiolabeled organic-soluble products were detected after treatment with fMet-Leu-Phe plus GTP. Incubation of intact PMNs, with the Bordetella pertussis toxin (islet-activating protein) eliminated the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in the isolated plasma membranes, but did not inhibit PIP2 degradation in the presence of 1 mM CaCl2. These results provide the first direct evidence that the fMet-Leu-Phe receptor in PMN membranes is coupled to polyphosphoinositide hydrolysis through an islet-activating protein-sensitive guanine nucleotide regulatory protein.     

The inhibition of platelet-activating factor-induced platelet activation by oleic acid is associated with a decrease in polyphosphoinositide metabolism

In an earlier study (Miwa, M., Hill, C., Kumar, R., Sugatani, J., Olson, M. S., and Hanahan, D. J. (1987) J. Biol. Chem. 262, 527-530) it was shown that an inhibitor of platelet-activating factor (PAF), a powerful endogenous mediator of platelet aggregation, was present in freeze-clamped perfused livers. Subsequently, we determined that this substance was a mixture of unsaturated free fatty acids (FFA). Among these FFA, oleic acid between 10 and 100 microM was found to be a potent inhibitor of PAF-induced platelet aggregation and serotonin secretion. Consequently, in order to understand the molecular mechanism of oleic acid action, we investigated the effects of this FFA on several biochemical events associated with platelet aggregation induced by PAF. The effect of oleic acid and/or PAF on the level of [32P]phosphatidylinositol 4-phosphate (PIP) and [32P]phosphatidylinositol 4,5-bisphosphate (PIP2) was examined by using platelets labeled with [32P]phosphate. Oleic acid induced a dose-dependent decrease in the levels of [32P]PIP and [32P]PIP2; a maximal decrease in [32P]PIP and [32P]PIP2 of approximately 50 and 25%, respectively, was observed within seconds after the addition of 20 microM oleic acid and persisted for at least 15 min. Oleic acid did not induce the formation of [3H]inositol phosphates in platelets prelabeled with [3H]inositol, suggesting that the decrease in [32P]PIP and [32P]PIP2 was not due to a stimulation of phospholipase C. In contrast to oleic acid, PAF induced a dose-dependent increase in the [32P]PIP level, reaching a maximum of approximately 200% 3 min after the addition of 1 nM PAF to the platelets. This increase in [32P]PIP was accompanied by platelet aggregation and secretion, and a close correlation was established between the [32P]PIP level and the degree of aggregation. Oleic acid and PAF, when added together to the platelets, interacted by affecting the level of [32P]PIP and [32P]PIP2 in an opposite way since the decrease in the level of [32P]PIP and [32P] PIP2 induced by oleic acid was partially reversed by an excess of PAF. The decrease in the levels of [32P] PIP and [32P]PIP2 caused by oleic acid was associated with an inhibition of platelet aggregation induced by PAF. Interestingly, oleic acid did not block [3H]PAF binding to platelets but inhibited the PAF-induced phosphorylation of platelet proteins of 20 kDa and 40 kDa. These results suggest that inhibition of the PAF response by oleic acid may be at one of the steps in the signal transduction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylation of phosphatidylinositol associated with the nicotinic acetylcholine receptor of Torpedo californica

When isolated, detergent solubilized and affinity chromatographically purified nicotinic acetylcholine receptor of Torpedo californica electric organ is incubated with [gamma-32P]ATP/Mg2+, phosphatidylinositol 4-phosphate (PIP) is formed from receptor associated phosphatidylinositol (PI). This receptor associated endogenous kinase activity is enhanced by orthovanadate and, remarkably, also by acetylcholine. Exogenously added PI-kinase only increases the phosphorylation rate if vanadate is present. PIP as the main phosphorylation product (up to 95%) remains bound to the beta-, gamma- and delta-subunits of the receptor and to the receptor associated v-protein. The alpha-subunits do not carry 32p phosphate; no phosphatidylinositol 4,5-bisphosphate formation has been observed. Concomitant to lipid phosphorylation tyrosine and serine residues are phosphorylated (5% of total incorporated 32P phosphate).     

Lipid phosphorylation in chloroplast envelopes. Evidence for galactolipid CTP-dependent kinase activities

Lipid phosphorylation takes place within the chloroplast envelope. In addition to phosphatidic acid, phosphatidylinositol phosphate, and their corresponding lyso-derivatives, we found that two novel lipids underwent phosphorylation in envelopes, particularly in the presence of carrier-free [gamma-(32)P]ATP. These two lipids incorporated radioactive phosphate in chloroplasts in the presence of [gamma-(32)P]ATP or [(32)P]P(i) and light. Interestingly, these two lipids were preferentially phosphorylated in envelope membranes in the presence [gamma-(32)P]CTP, as the phosphoryl donor, or [gamma-(32)P]ATP, when supplemented with CDP and nucleoside diphosphate kinase II. The lipid kinase activity involved in this reaction was specifically inhibited in the presence of cytosine 5'-O-(thiotriphosphate) (CTPgammaS) and sensitive to CTP chase, thereby showing that both lipids are phosphorylated by an envelope CTP-dependent lipid kinase. The lipids were identified as phosphorylated galactolipids by using an acid hydrolysis procedure that generated galactose 6-phosphate. CTPgammaS did not affect the import of the small ribulose-bisphosphate carboxylase/oxygenase subunit into chloroplasts, the possible physiological role of this novel CTP-dependent galactolipid kinase activity in the chloroplast envelope is discussed.     

Cationic amphiphilic drugs perturb the metabolism of inosititides and phosphatidic acid in photoreceptor membranes

Incubation of purified bovine photoreceptor rod outer segments with [gamma-32P]ATP resulted in the labeling of phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid (PA) with little labeling of phosphatidylinositol 4,5-bisphosphate (PIP2). Propranolol inhibited in a dose-dependent manner the labeling of PA and enhanced that of PIP. Various cationic amphiphilic drugs also were tested for these effects. Propranolol had the same effects on high-speed rat brain particulate material. While this particular preparation displayed more labeling of PIP2, propranolol was ineffective, as it was on retinal PIP-kinase. Ca2+-activated polyphosphoinositide phosphodiesterase activity in nerve-ending membranes also was inhibited by propranolol. It is concluded that cationic amphiphilic drugs can inhibit diacylglycerol kinase and the polyphosphoinositide phosphodiesterase and stimulate the phosphatidylinositol-kinase (but not PIP-kinase).     

In vitro synthesis of 32P-labelled phosphatidylinositol 4,5-bisphosphate and its hydrolysis by smooth muscle membrane-bound phospholipase C

For studies of phospholipase C (PLC) activity in cell-free systems, 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2) was prepared enzymatically by phosphorylating phosphatidylinositol 4-phosphate (PIP) in the presence of [gamma-32P]ATP using a PIP kinase partially purified from bovine retinae. PLC activity was determined by incubating membranes of DDT1 MF-2 cells with 32P-PIP2 and measuring remaining non-hydrolyzed substrate as well as accumulation of the hydrolysis product, inositol trisphosphate (IP3). Guanine nucleotides stimulated PIP2 hydrolysis and IP3 release. Additional increase in IP3 accumulation was observed with adrenaline plus guanine nucleotides.     

Stimulation of phosphoinositides breakdown by the heat stable E. coli enterotoxin in rat intestinal epithelial cells

Rat intestinal epithelial cells were labelled with [32P]Pi and extracted, and the phospholipids were analysed by thin-layer chromatography. 32P-incorporation in phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-phosphate (PIP2) were measured in control and heat stable enterotoxin (ST)-treated cells. ST was found to induce rapid degradation of PIP and PIP2. The degradation of inositol lipids was accompanied by an increase of water soluble inositol phosphate (IP1, IP2, IP3) compounds. There was a two-fold increase of radioactivity in IP2 and IP3 but no significant change was observed in IP1. Phospholipase C activity was increased tenfold with substrate PIP2 in ST-pretreated cells. The present study indicates that ST triggers another second messenger system by increasing the PIP2 hydrolysis with the enzyme phospholipase C.     

The inositide cycle in bovine photoreceptor membranes

Various enzymic steps in the inositide cycle were investigated in purified bovine retinal rod outer segments (ROS). Incubation of ROS with [gamma-32P]ATP resulted in a rapid labeling of phosphatidic acid and phosphatidylinositol 4-phosphate (PIP), while little radio-tracer was recovered from phosphatidylinositol 4,5-bisphosphate (PIP2). This can be explained by the relatively low activity of PIP-kinase activity in ROS as compared to the remainder of the retina. Similarly, relatively little phosphodiesteratic activity degraded PIP2 and PIP in ROS when 32P labeled phosphoinositides in synaptic membranes (heat-treated to inactivate endogenous enzymes) were used. Although light exposure of ROS did cause rapid rhodopsin phosphorylation, no enzymic steps of the cycle were changed, even when ROS were obtained from retinas excised from cows dark-adapted by unilateral eye patching the day prior to kill. These studies do not support the view that light is an agonist of the inositide cycle in mammalian photoreceptors.     

Regulation of polyphosphoinositide synthesis in cardiac membranes

The relative distribution of phosphatidylinositol (PI) and phosphatidylinositol-4-phosphate (PIP) kinase activities in enriched cardiac sarcolemma (SL), sarcoplasmic reticulum (SR), and mitochondrial fractions was investigated. PI and PIP kinase activities were assayed by measuring 32P incorporation into PIP and phosphatidylinositol 4,5-bisphosphate (PIP2) from endogenous and exogenous PI in the presence of [gamma-32P]ATP. PI and PIP kinase activities were present in SL, SR, and mitochondrial fractions prepared from atria and ventricles although the highest activities were found in SL. A similar membrane distribution was found for PI kinase activity measured in the presence of detergent and exogenous PI. PI and PIP kinase activities were detectable in the cytosol providing exogenous PI and PIP and Triton X-100 were present. Further studies focused on characterizing the properties and regulation of PI and PIP kinase activities in ventricular SL. Alamethacin, a membrane permeabilizing antibiotic, increased 32P incorporation into PIP and PIP2 4-fold. PI and PIP kinase activities were Mg2+ dependent and plateaued within 15-20 min at 25 degrees C. Exogenous PIP and PIP2 (0.1 mM) had no effect on PIP and PIP2 labeling in SL in the absence of Triton X-100 but inhibited PI kinase activity in the presence of exogenous PI and Triton X-100. Apparent Km's of ATP for PI and PIP kinase were 133 and 57 microM, respectively. Neomycin increased PIP kinase activity 2- to 3-fold with minor effects on PI kinase activity. Calmidazolium and trifluoperazine activated PI kinase activity 5- to 20-fold and completely inhibited PIP kinase activity. Quercetin inhibited PIP kinase 66% without affecting PI kinase activity. NaF and guanosine 5'-O-(3-thiotriphosphate) had no effect on PI and PIP kinase activities, indicating that these enzymes were not modulated by G proteins. The probability that PIP and PIP2 synthesis in cardiac sarcolemma is regulated by product inhibition and phospholipase C was discussed.     

Stimulation of polyphosphoinositide hydrolysis by thrombin in membranes from human fibroblasts.

One of the earliest actions of thrombin in fibroblasts is stimulation of a phospholipase C (PLC) that hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. In membranes prepared from WI-38 human lung fibroblasts, thrombin activated an inositol-lipid-specific PLC that hydrolysed [32P]PIP2 and [32P]phosphatidylinositol 4-monophosphate (PIP) to [32P]IP3 and [32P]inositol 1,4-bisphosphate (IP2) respectively. Degradation of [32P]phosphatidylinositol was not detected. PLC activation by thrombin was dependent on GTP, and was completely inhibited by a 15-fold excess of the non-hydrolysable GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]). Neither ATP nor cytosol was required. Guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) also stimulated polyphosphoinositide hydrolysis, and this activation was inhibited by GDP[S]. Stimulation of PLC by either thrombin or p[NH]ppG was dependent on Ca2+. Activation by thrombin required Ca2+ concentrations between 1 and 100 nM, whereas stimulation of PLC activity by GTP required concentrations of Ca2+ above 100 nM. Thus the mitogen thrombin increased the sensitivity of PLC to concentrations of free Ca2+ similar to those found in quiescent fibroblasts. Under identical conditions, another mitogen, platelet-derived growth factor, did not stimulate polyphosphoinositide hydrolysis. It is concluded that an early post-receptor effect of thrombin is the activation of a Ca2+- and GTP-dependent membrane-associated PLC that specifically cleaves PIP2 and PIP. This result suggests that the cell-surface receptor for thrombin is coupled to a polyphosphoinositide-specific PLC by a GTP-binding protein that regulates PLC activity by increasing its sensitivity to Ca2+.     

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.