Cardiotoxin from cobra venom increases the level of phosphatidylinositol 4-monophosphate and phosphatidylinositol kinase activity in two cell lines

In rat basophilic leukemia-2H3 (RBL-2H3) and Madin-Darby canine kidney (MDCK) cells, cardiotoxin from cobra venom induced a marked decrease in the level of [3H] phosphatidylinositol and a corresponding increase in the level of [3H]phosphatidylinositol 4-monophosphate over the course of 20 min as demonstrated in cells that had been labeled to equilibrium with [3H]inositol. The effect was dependent on the concentration (5-30 micrograms/ml) of the toxin. In plasma membrane-enriched fractions isolated from the two cell lines, the cardiotoxin enhanced the endogenous activity of phosphatidylinositol kinase especially at temperatures above 14 degrees C. In RBL-2H3 cells, cardiotoxin also induced release of substantial amounts of histamine and lactate dehydrogenase. The release of histamine, but not of lactate dehydrogenase, was totally dependent on external calcium and this release probably represented an exocytotic response of the cells to cardiotoxin. Although, initially, treatment with the toxin did not impair antigen-induced hydrolysis of inositol phospholipids or prevent the antigen-induced rise in the concentration of cytosol Ca2+, prolonged exposure to the toxin did result in a progressive loss of responsiveness of RBL-2H3 cells to antigen.     

A wasp venom mastoparan-induced polyphosphoinositide breakdown in rat peritoneal mast cells

The phospholipid metabolism of rat peritoneal mast cells stimulated with mastoparan, a secretagogue purified from wasp venom, was investigated. Mastoparan at 20 micrograms/ml caused a rapid secretion of histamine. Mastoparan induced a transient decrease of phosphatidylinositol 4,5-biphosphate on 32P labeling and generation of a water-soluble degradation product, inositol trisphosphate on [3H]inositol labeling, suggesting the activation of phospholipase C upon stimulation.     

Lithium-induced accumulation of inositol 1-phosphate during cholecystokinin octapeptide- and acetylcholine-stimulated phosphatidylinositol breakdown in dispersed mouse pancreas acinar cells

Dispersed mouse pancreas acinar cells were prepared in which phosphatidylinositol had been labeled with myo[2-3H]inositol. During incubation with 0.3 microM cholecystokinin octapeptide (CCK-8) for 15 min, there was a loss of [3H]phosphatidylinositol radioactivity (23%) and a 3-fold gain in trichloroacetic acid-soluble radioactivity. Replacement of NaCl by up to 58 mM LiCl did not significantly affect the amount of CCK-8-stimulated [3H]phosphatidylinositol breakdown or the gain in acid-soluble radioactivity. However, in normal medium, the product of phosphatidylinositol breakdown was almost all inositol, whereas in Li+-containing medium, the product was almost all inositol 1-phosphate. Similar results were obtained with acetylcholine which, in the presence of Li+, gave a dose-responsive increase in inositol 1-phosphate over the concentration range of 0.1 to 10 microM. No increased accumulation of [3H]inositol diphosphate or [3H]inositol triphosphate was detected in stimulated cells. Time courses in the presence of Li+ indicated that the formation of inositol 1-phosphate preceded the formation of inositol. Addition of up to 50 mM myoinositol to the incubation medium showed no diluting effect on the amount of [3H]inositol 1-phosphate found. The accumulation of inositol 1-phosphate is presumably due to the known ability of Li+ to inhibit myoinositol 1-phosphatase. The results provide clear evidence that stimulated phosphatidylinositol breakdown involves a phospholipase C type of phosphodiesterase activity. 1.25 mM Li+ gave half-maximal inositol 1-phosphate accumulation. This is close to the range of plasma Li+ levels which is used therapeutically in psychiatric disorders. In unstimulated cells, [3H]inositol 1-phosphate accumulation in the presence of Li+ corresponded to a breakdown rate for [3H]phosphatidylinositol of 2 to 3%/h.     

Activation of phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase by cAMP in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, cAMP-dependent phosphorylation plays an essential role at the start of the cell cycle. It has also recently been demonstrated that the breakdown of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol is a requisite process for cell proliferation (Uno, I., Fukami, K., Kato, H., Takenawa, T., and Ishikawa, T. (1988) Nature 333, 188-190). To clarify the relationship between the cAMP- and inositol phospholipid-mediated signal transduction systems, alterations in the inositol phospholipid metabolism of cAMP mutants were examined. The incorporation of [32P]Pi into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was markedly reduced in ras2, which produces low levels of cAMP, and increased in bcy1, which produces cAMP-independent protein kinase. The incorporation of [32P]Pi into ATP and phosphatidylinositol (PI) was almost the same in wild type, ras1, ras2, and bcy1 yeast strains. The addition of exogenous cAMP to cyr1-2 caused a tremendous increase in [32P]Pi incorporation into PIP and PIP2 without any effect on incorporation into ATP and PI, suggesting that cAMP plays an important role in polyphosphoinositide synthesis. We therefore examined the activities of PI and PIP kinases, the enzymes that catalyze the sequential steps from PI to PIP2 via PIP. The activities of both kinases were found to be very low in the membranes of cry1-2 and ras2 but very high in the membranes of bcy1 and ras1 ras2 bcy1 strain cells. The addition of cAMP to cyr1-2 cells caused the activation of PI and PIP kinases. Furthermore, the treatment of membranes with cAMP or dibutyryl cAMP caused the activation of PI kinase in wild type, ras1, cry1-2, and ras2 strains, but not in bcy1 strain cells. The effect was most prominent in membranes from cyr1-2 and ras2 cells. These results show that cAMP-dependent phosphorylation enhances polyphosphoinositide synthesis through activation of PI and PIP kinase, an effect which may lead to the enhanced production of inositol 1,4,5-trisphosphate and diacylglycerol.     

Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands.

The molecular mechanisms underlying the ability of muscarinic agonists to enhance the metabolism of inositol phospholipids were studied using rat parotid gland slices prelabelled with tracer quantities of [3H]inositol and then washed with 10 mM unlabelled inositol. Carbachol treatment caused rapid and marked increases in the levels of radioactive inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and an accumulation of label in the free inositol pool. There were much less marked changes in the levels of [3H]phosphatidylinositol, [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate. At 5 s after stimulation with carbachol there were large increases in [3H]inositol 1,4-bisphosphate and [3H]inositol 1,4,5-trisphosphate, but not in [3H]inositol 1-phosphate. After stimulation with carbachol for 10 min the levels of radioactive inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate greatly exceeded the starting level of radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively. When carbachol treatment was followed by addition of sufficient atropine to block all the muscarinic receptors the radioactive inositol phosphates rapidly returned towards control levels. The carbachol-evoked changes in radioactive inositol phosphate and phospholipid levels were blocked in the presence of 2,4-dinitrophenol (an uncoupler of oxidative phosphorylation). The results suggest that muscarinic agonists stimulate a polyphosphoinositide-specific phospholipase C and that these lipids are continuously replenished from the labelled phosphatidylinositol pool. [3H]Inositol 1-phosphate in the stimulated glands probably arises via hydrolysis of inositol 1,4-bisphosphate and not directly from phosphatidylinositol.     

Inhibition of CDP-DG: inositol transferase by inostamycin.

Inostamycin, a novel microbial secondary metabolite, inhibited [3H]inositol and 32P1 incorporation into phosphatidylinositol (PtdIns) induced by epidermal growth factor (EGF) in cultured A431 cells, the IC50 being 0.5 micrograms/ml, without inhibiting macromolecular synthesis. The drug inhibited cellular inositol phosphate formation only when it was added at the same time as labeled inositol. It was found to inhibit in vitro CDP-DG:inositol transferase activity of the A431 cell membrane, the IC50 being about 0.02 micrograms/ml. It did not inhibit tyrosine kinase, PtdIns phospholipase C, or PtdIns kinase. Therefore, inhibition of PtdIns turnover by inostamycin must be due to the inhibition of CDP-DG:inositol transferase. Thus, inostamycin is a novel inhibitor of CDP-DG:inositol transferase.     

Gonadotropin-releasing hormone activates a rapid Ca2+-independent phosphodiester hydrolysis of polyphosphoinositides in pituitary gonadotrophs

Addition of gonadotropin releasing hormone (GnRH) to pituitary cells prelabeled with [32P]Pi or with myo-[2-3H]inositol, resulted in a rapid decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate (approximately 10 s), and in [32P]phosphatidylinositol 4-phosphate (approximately 1 min), followed by increased labeling of [32P]phosphatidylinositol and [32P]phosphatidic acid (1 min). GnRH stimulated the appearance of [3H]myo-inositol 1,4,5-trisphosphate (10 s), [3H]myo-inositol 1,4-bisphosphate (15 s), and [3H]myo-inositol 1-phosphate (1 min) in the presence of Li+ (10 mM). Li+ alone stimulated the accumulation of [3H]myo-inositol 1-phosphate and [3H]myo-inositol 1,4-bisphosphate but not [3H]myo-inositol 1,4,5-trisphosphate, but had no effect on luteinizing hormone release. The effect of GnRH on inositol phosphates (Ins-P) production was dose-related (ED50 = 1-5 nM), and was blocked by a potent antagonist [D-pGlu,pClPhe,D-Trp]GnRH. Elevation of cytosolic free Ca2+ levels ([Ca2+]i), by ionomycin and A23187 from intracellular or extracellular Ca2+ pools, respectively, had no significant effect on [3H]Ins-P production. GnRH-induced [3H]Ins-P production was not dependent on extracellular Ca2+ and was noticed also after extracellular or intracellular Ca2+ mobilization by A23187 or ionomycin, respectively. The effect of GnRH on [3H]Ins-P accumulation was not affected by prior treatment of the cells with the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate or with islet-activating protein pertussis toxin. These results indicate that GnRH stimulates a rapid phosphodiester hydrolysis of polyphosphoinositides. The stimulatory effect is not mediated via an islet-activating protein-substrate, is not dependent on elevation of [Ca2+]i, neither is it negatively regulated by 12-O-tetradecanoylphorbol-13-acetate which activates Ca2+/phospholipid-dependent protein C kinase. The results are consistent with the hypothesis that GnRH-induced phosphoinositide turnover is responsible for Ca2+ mobilization followed by gonadotropin release.     

Antigen receptor-mediated regulation of sustained polyphosphoinositide turnover in a human T cell line. Evidence for a receptor-regulated pathway for production of phosphatidylinositol 4,5-bisphosphate

Stimulation of the human T cell line, Jurkat, by the addition of monoclonal antibodies reactive with the T cell antigen receptor complex (CD3/Ti) leads to sustained increases in levels of inositol 1,4,5-trisphosphate. To investigate the possibility that the production of polyphosphoinositides is regulated during CD3/Ti stimulation, we studied Jurkat cells whose inositol phospholipids had been labeled to steady state with [3H]inositol, as well as Jurkat cells during nonequilibrium labeling with [32P]orthophosphate. The addition of CD3 monoclonal antibodies led to a 4-5-fold increase in [3H]inositol trisphosphate that was sustained for greater than 20 min. Within 60 s of CD3/Ti stimulation, [3H] phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) decreased by 65 and 35%, respectively. This change in [3H]PtdIns(4,5)P2 persisted for greater than 20 min. The decrease in [3H]PtdIns4P, however, was transient, and, after 5 min, the levels of [3H]PtdIns4P were comparable in stimulated and unstimulated cells. To examine the rate of flux through inositol phospholipids, we measured the CD3/Ti-stimulated changes in the ratio, 32P cpm/3H cpm, in each inositol phospholipid. CD3/Ti stimulation led to accelerated fluxes through PtdIns(4,5)P2 and phosphatidylinositol (PtdIns) that were maintained for greater than 20 min. After the initial 30 s, however, there was no detectable effect of anti-CD3 on flux through Ptsins4p. This observation suggested that, during CD3/Ti stimulation, production of PtdIns(4,5)P2 from PtdIns might occur via a small pool of PtdIns4P with a very high turnover. The existence of such a pool was established by determining that, in stimulated cells, the 32P-specific activity of the 1-position phosphate of PtdIns(4,5)P2 was 8-10-fold that of PtdIns4P. We conclude that, during the initial 60 s of CD3/Ti stimulation, there is a substantial depletion of cellular PtdIns(4,5)P2 and PtdIns4P. Thereafter, a CD3/Ti-regulated pathway generates PtdIns(4,5)P2 from PtdIns through a small, but highly labile, pool of PtdIns4P.     

Characterization of phospholipase C-mediated phosphatidylinositol degradation in rat heart ventricle

Phosphoinositide-specific phospholipase C (PI-PLC) activity was investigated in the rat heart ventricle. Incubation of ventricle homogenate or 100,000g supernatant fraction with [3H]myoinositol or [3H]arachidonate-labeled phosphatidylinositol in the presence of Ca2+ resulted in a decrease in phosphatidylinositol with a concomitant increase in water-soluble [3H]inositol phosphate or [3H]diglyceride, respectively. Total overt homogenate PI-PLC activity could be accounted for in the supernatant fraction. Neutral, zwitterionic, cationic, or anionic detergents did not unmask membrane-associated activity. While cytosolic phospholipase C was active against a pure phosphatidylinositol substrate in the presence of Ca2+, no hydrolytic activity was detected when phosphatidylinositol was presented as a component (4-5%) of a mixture of phospholipids. However, addition of deoxycholate to the incubation mixture (pH 6.5, Ca2+ 10(-3) M) containing mixed phospholipids resulted in the exclusive hydrolysis of inositol phospholipids. Ventricular supernatant phospholipase C-mediated phosphatidylinositol degradation has a sharp pH optimum at 5.5 and a specific requirement for Ca2+. Activity is maximal at 1 to 2 X 10(-3) M Ca2+, with inhibition occurring at higher levels. Under optimized conditions phosphatidylinositol is hydrolyzed at a rate of 20-25 nmol/min/mg protein. Multivalent cations inhibit Ca2+-dependent PI-PLC activity while monovalent cations and anions have no effect. There is no apparent selectivity for specific fatty acid moieties on phosphatidylinositol. Soluble PI-PLC is inhibited by sulfhydryl reagents, neomycin, mepacrine, trifluoperazine, and propranolol. Chlorpromazine, dibucaine, and tetracaine exert a biphasic influence, stimulating at lower and inhibiting at higher concentrations.     

Prolactin induces the formation of inositol bisphosphate and inositol trisphosphate in cultured mouse mammary gland explants

The effect of prolactin on [3H]inositol metabolism in cultured mouse mammary gland explants derived from 12-14-day pregnant mice was determined. In mammary gland explants that were prelabeled by culturing the tissues with 3 microCi/ml myo-[3H]inositol for 48 h, the levels of 3H in inositol derivatives were determined. The temporal effect of prolactin on the quantity of 3H present in phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP), phosphatidylinositol bisphosphate (PIP2) and various inositol phosphate containing fractions were examined. Prolactin significantly stimulated the accumulation of 3H label in inositol monophosphate (IP1), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) 1-3 h after addition of prolactin. An effect of prolactin on the accumulation of inositol derivatives was not apparent at prolactin-exposure periods of less than 60 min; nor was an effect of prolactin apparent when exposure periods of 4 h or longer were employed. Prolactin did not significantly decrease the 3H label in PI, PIP or PIP2 except at 1 and 2 h. These data when considered with other apropos studies are compatible with the conclusion that the turnover of inositol lipid derivatives may be involved in the mechanism by which prolactin regulates metabolic processes in the mammary gland. The primary action of prolactin on mammary cells, however, would not appear to involve its action on the metabolism of the inositol derivatives in view of the extended time required (1 h) before effects of prolactin on perturbations of inositide metabolism are manifested.     

Differential regulation by phosphatidylinositol 4,5-bisphosphate of pituitary plasma-membrane and cytosolic phosphoinositide kinases.

Regulation of phosphatidylinositol kinase (EC 2.7.1.67) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase (EC 2.7.1.68) was investigated in highly enriched plasma-membrane and cytosolic fractions derived from cloned rat pituitary (GH3) cells. In plasma membranes, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] added exogenously enhanced incorporation of [32P]phosphate from [gamma-32P]MgATP2- into PtdIns(4,5)P2 and PtdIns4P to 150% of control; half-maximal effect occurred with 0.03 mM exogenous PtdIns(4,5)P2. Exogenous PtdIns4P and phosphatidylinositol (PtdIns) had no effect. When plasma membranes prepared from cells prelabelled to isotopic steady state with [3H]inositol were used, there was a MgATP2- dependent increase in the content of [3H]PtdIns(4,5)P2 and [3H]PtdIns4P that was enhanced specifically by exogenous PtdIns(4,5)P2 also. Degradation of 32P- and 3H-labelled PtdIns(4,5)P2 and PtdIns4P within the plasma-membrane fraction was not affected by exogenous PtdIns(4,5)P2. Phosphoinositide kinase activities in the cytosolic fraction were assayed by using exogenous substrates. Phosphoinositide kinase activities in cytosol were inhibited by exogenously added PtdIns(4,5)P2. These findings demonstrate that exogenously added PtdIns(4,5)P2 enhances phosphoinositide kinase activities (and formation of polyphosphoinositides) in plasma membranes, but decreases these kinase activities in cytosol derived from GH3 cells. These data suggest that flux of PtdIns to PtdIns4P to PtdIns(4,5)P2 in the plasma membrane cannot be increased simply by release of membrane-associated phosphoinositide kinases from product inhibition as PtdIns(4,5)P2 is hydrolysed.     

Effects of in vitro hypoxia on depolarization-stimulated accumulation of inositol phosphates in synaptosomes

The effects of potassium and in vitro histotoxic hypoxia (i.e. KCN) on phosphatidylinositol turnover in rat cortical synaptosomes were determined. [2-3H] Inositol prelabelled rat synaptosomes were prepared from cerebral cortex slices that had been incubated with [2-3H] inositol. Depolarization with 60 mM KCl increased [2-3H] inositol phosphates in a time dependent manner. Depolarization with 60 mM KCl increased [2-3H] inositol trisphosphate transiently at 5 s. K+ induced rapid formation of [2-3H]-inositol bisphosphate and maintained an elevated level for at least 5 min. K+ stimulated gradual formation of [2-3H] inositol monophosphate with time. One minute of hypoxia enhanced potassium-stimulated [2-3H] inositol bisphosphate formation. However, 30 min of hypoxia impaired potassium-stimulated accumulation of [2-3H] inositol phosphates. The effects of histotoxic hypoxia were all dependent upon calcium in the medium and on K+-depolarization. Thus, hypoxia altered the K+-induced accumulation of inositol phosphates in prelabelled synaptosomes in a time dependent, biphasic manner that was calcium dependent.     

Purification to homogeneity of protein kinase C from bovine brain--identity with the phorbol ester receptor

The calcium- and phospholipid-dependent kinase activity (protein kinase C) was isolated from bovine brains by a combination of DEAE-cellulose chromatography, gel filtration and hydrophobic chromatography on octyl-Sepharose and phenyl-Sepharose. The phorbol ester receptor co-purifies with the protein kinase C throughout the procedure yielding a homogeneous protein of 79 500 daltons on SDS-polyacrylamide gels. The purified kinase incorporated approximately 5000 nmol phosphate into substrate/min/mg protein at saturating concentrations of Ca2+ and phosphatidyl serine. Reciprocal plots of protein kinase activity at varying phosphatidyl serine concentrations were biphasic and yielded two apparent Ka values for phosphatidyl serine of 0.6-2 and 35-80 micrograms/ml). These apparent Ka values were reduced 2- to 3-fold by either diolein (20 micrograms/ml) or phorbol-12,13-dibutyrate (10 micrograms/ml). The protein binds [3H]phorbol-12,13-dibutyrate ( [3H]PDB) with high affinity (Ka = 15 nM) in a phosphatidyl serine-dependent manner. At saturating phosphatidyl serine concentrations 0.89 mol [3H]PDB are bound per mol protein. The identification of protein kinase C as the phorbol ester receptor is discussed with respect to the function and regulation of this protein.     

Phosphatidylinositol Metabolism During In Vitro Hypoxia

The effects of in vitro histotoxic hypoxia (0.5 mM KCN) on potassium-stimulated phosphatidylinositol turnover were determined. In rat cortical slices that were prelabeled with [2-3H]inositol, depolarization with 60 mM KCl increased [2-3H]inositol monophosphate and [2-3H]inositol bisphosphate accumulation in a Ca2+-dependent manner. At early times (10 s and 1 min), histotoxic hypoxia enhanced potassium-stimulated [2-3H]inositol monophosphate and inositol bisphosphate accumulation. Under basal conditions, hypoxia did not alter the accumulation of [2-3H]inositol phosphates. These results are consistent with the following hypothesis. The hypoxic-induced increase in cytosolic free calcium that we reported previously may lead to the early stimulation of inositol phosphates formation during hypoxia through activation of phospholipase C. The impairment of inositol phosphates formation during more prolonged hypoxia may be due to negative feedback regulation of the phosphatidylinositol cascade by protein kinase C or to a reduction in ATP levels.     

Scrape-loaded p21ras down-regulates agonist-stimulated inositol phosphate production by a mechanism involving protein kinase C.

The effect of scrape-loaded [Val-12]p21ras on agonist-stimulated phosphatidylinositol 4,5-bisphosphate (PIP2) turnover in Swiss-3T3 cells was studied. Previously [Morris, Price, Lloyd, Marshall & Hall (1989) Oncogene 4, 27-31] we demonstrated that [Val-12]p21ras activates protein kinase C within 10 min of scrape loading. Here, we show that [Val-12]p21ras inhibits bombesin and platelet-derived growth factor-stimulated PIP2 breakdown 1.5-4 h after scrape loading. This effect persisted for at least 18 h and could be mimicked in control cells by activation of protein kinase C with 12-O-tetradecanoyl 13-acetate (TPA) 15 min prior to ligand stimulation. When protein kinase C was down-regulated by chronic TPA treatment, [Val-12]p21ras was no longer able to inhibit agonist-stimulated inositol phosphate production. These results indicate that changes in inositol phosphate levels caused by ras protein are probably due to activation of protein kinase C and not to an interaction of ras with phospholipase C.     

Phorbol 12,13-dibutyrate and 1-oleyl-2-acetyldiacylglycerol stimulate inositol trisphosphate dephosphorylation in human platelets

Inositol trisphosphate (IP3) is formed in response to specific agonists that cause activation of phospholipase C and degradation of phosphatidylinositol bisphosphate. IP3 is a second messenger that releases Ca2+ from the dense tubular system to the cytosol in stimulated platelets. Our present information indicates that [3H]IP3 is dephosphorylated to [3H]inositol bisphosphate (IP2) and [3H]inositol monophosphate (IP) by human platelets treated with 0.05-0.10% Triton X-100. This dephosphorylation of [3H]IP3 to [3H]IP2 and [3H]IP is also observed when platelets are permeabilized by electrical stimulation or by 20 micrograms/ml saponin. These detergents or electropermeabilization allow IP3 to access cytosolic IP3 phosphatase. Pretreatment of intact platelets with phorbol dibutyrate and 1-oleyl-2-acetyldiacylglycerol for 30 s, at concentrations that maximally activate protein kinase C, stimulates the conversion of IP3 to IP2 and IP. This suggests a role for protein kinase C in the regulation of IP3 degradation.     

Manganese stimulates incorporation of [3H]inositol into an agonist-insensitive pool of phosphatidylinositol in brain membranes

In a particulate preparation from rat brain, manganese ions stimulate the incorporation of [3H]inositol into inositol phospholipids in a concentration-dependent manner. Incubation with CDP-diacylglycerol (0.5 mM) alone had no effect on the incorporation of [3H]inositol but potentiated the stimulatory effect of manganese. Despite the increase in [3H]inositol incorporation into phosphatidylinositol, the carbachol-induced accumulation of [3H]inositol-1-phosphate was unaltered in membranes preincubated with manganese but when coincubated with CDP-diacylglycerol the carbachol-induced accumulation of [3H]inositol-1-phosphate was increased. These data suggest that manganese stimulates the incorporation of [3H]inositol into an agonist-insensitive pool of phosphatidylinositol.     

Activation of phospholipase C in platelets by platelet activating factor and thrombin causes hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate

Despite their physicochemical and mechanistic differences platelet activating factor (or acetylglycerylether phosphorylcholine; AGEPC) and thrombin, both platelet stimulatory agents, induce phosphoinositide turnover in platelets. We therefore investigated the stimulation of the phosphoinositide phosphodiesterase by these agents and questioned whether they evoked hydrolysis of the same or different pools of phosphoinositides. [3H]Inositol-labelled rabbit platelets were challenged with thrombin and/or AGEPC under a variety of protocols, and the phospholipase C mediated production of radioactive inositol monophosphate (IP); inositol bisphosphate (IP2) and inositol trisphosphate (IP3) was used as the parameter. AGEPC (1 X 10(-9) M) caused a transient maximum (5 to 6-fold) increase in [3H]IP3 at 5 s followed by a decrease. Thrombin (2 U/ml) elicited an increase in [3H]IP3 at a much slower rate than AGEPC; 2 fold at 5 s, 5 fold at 30 s and a maximum 6 to 8-fold at 2-5 min. Compared to AGEPC, thrombin stimulated generation of [3H]IP2 and [3H]IP were severalfold higher. When thrombin and AGEPC were added together to platelets there was no evidence for an additive increase in inositol polyphosphate levels except at earlier time points where increases were submaximal. When AGEPC was added at various time intervals after thrombin pretreatment, no additional increases in [3H]IP3 were observed over that maximally seen with thrombin or AGEPC alone. In another set of experiments, submaximal increases (about 1/4 and 1/2 of maximum) in [3H]IP3 were achieved by using selected concentrations of thrombin (0.1 U and 0.3 U, respectively) and then AGEPC (1 X 10(-9) M) was added for 5 s. Once again the increase in [3H]IP3 was close to the maximal level seen with thrombin or AGEPC individually. It is concluded that thrombin and AGEPC differentially activated phosphoinositide phosphodiesterase (phospholipase C) in rabbit platelets and that the stimulation of the phospholipase C by these two stimuli causes IP3 production via hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate.     

An early transient decrease in phosphatidylinositol 4,5-bisphosphate upon stimulation of rabbit platelets with acetylglycerylether phosphorylcholine (platelet activating factor)

Washed rabbit platelets labeled with [3H]inositol were stimulated with AGEPC (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) (5 X 10(-10) M) for various time periods. Within 5 s of the mixing of these platelets with AGEPC, an approximately 25% decrease in the [3H]TPI (phosphatidylinositol 4,5-bisphosphate) was evident; immediately thereafter the radioactivity in TPI increased. These labeled platelets treated with various concentrations of AGEPC for only 5 s indicated a characteristic dose-related decrease in [3H]TPI. Radioactivity in phosphatidylinositol 4-phosphate also appeared to increase after AGEPC-induced stimulation of platelets. Interestingly, within 15 s a 15 to 20% decrease in [3H]PI (phosphatidylinositol) and an increase in [3H]lysoPI was observed. However, [3H]lysoPI could be related only to one-third of the decrease in [3H]PI. LysoGEPC (lyso-1-O-alkyl-sn-glyceryl-3-phosphorylcholine), which is ineffective in the activation of platelets, was unable to cause any changes in the phosphoinositides. The fact that the status of TPI was influenced in a time- and dose-dependent manner and the rapidity with which these changes take place suggest that this inositol phospholipid may be associated closely with the early processes which accompany the interaction of AGEPC with platelets.     

Luteinizing hormone increases inositol trisphosphate and cytosolic free Ca2+ in isolated bovine luteal cells

The present studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases intracellular cAMP, also increases "second messengers" derived from inositol phospholipid hydrolysis in isolated bovine luteal cells. In luteal cells prelabeled with 32PO4, LH provoked increases in labeling of phosphatidic acid, phosphatidylinositol, and polyphosphatidylinositol (PIP). No reductions in 32P-prelabeled PIP and PIP2 were observed in LH-treated cells. In luteal cells prelabeled with myo-[2-3H]inositol, LH provoked rapid (10-30 s) and sustained (up to 60 min) increases in the levels of inositol mono-, bis-, and trisphosphates (IP, IP2, and IP3, respectively. IP3 was formed more rapidly than IP2 or IP following LH treatment. In addition, LH increased (50%) levels of [3H]inositol phospholipids in 30-min incubations. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH. Maximal increases in IP3 occurred at 1-10 micrograms/ml of LH. Similar temporal and dose-response relationships were observed for LH-stimulated IP3 and cAMP accumulation. However, exogenous cAMP (8-bromo-cAMP, 5 mM) and forskolin (10 microM) had no effect on inositol phosphate synthesis. The initial (1 min) effects of LH on IP3 and cAMP were independent of extracellular calcium concentrations, whereas the sustained (5 min) effect of LH on IP3, but not cAMP, was dependent on a source of extracellular calcium. LH-stimulated progesterone synthesis was also dependent on the presence of extracellular calcium. LH induced rapid and concentration-dependent increases in [Ca2+]i as measured by Quin 2 fluorescence. The LH-induced increases in [Ca2+]i were maximal within 30 s (approximately 2-fold) and remained elevated for at least 10 min. In Ca2+-free media containing 2 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, LH was still able to increase [Ca2+]i, but the increase was slightly less in magnitude and of shorter duration (2-4 min). These findings demonstrate that LH can rapidly raise levels of IP3 and [Ca2+]i, as well as, cAMP in bovine luteal cells. These findings suggest that at least two second messenger systems exist to mediate the action of LH in the corpus luteum.