Occurrence of lysophosphatidic acid and its alkyl ether-linked analog in rat brain and comparison of their biological activities toward cultured neural cells.

Rat brain was found to contain substantial amounts of potent bioactive lipids lysophosphatidic acid (acyl LPA) (3.73 nmol/g tissue) and lysoplasmanic acid (alkyl LPA) (0.44 nmol/g tissue). The presence of alkyl LPA was confirmed by mild alkaline hydrolysis analysis and by gas chromatography/mass spectrometry analysis of the trimethylsilyl derivative. This is the first clear evidence of the occurrence of an alkyl LPA in nature. The predominant molecular species of acyl LPA are 18:1-, 18:0- and 16:0-containing species (46. 9, 22.5 and 18.8%, respectively). A significant amount of a 20:4-containing species (7.2%) was also detected in the acyl LPA fraction. We also confirmed that rat brain alkyl LPA consists of 16:0-, 18:0- and 18:1-containing species. Noticeably, either acyl or alkyl LPA is capable of stimulating neuroblastomaxglioma hybrid NG108-15 cells to elicit a Ca(2+) transient, the potencies being almost the same. Both acyl and alkyl LPAs also induce cell rounding upon addition to the cells. These results suggest that acyl and alkyl LPAs play important physiological roles as intercellular signaling molecules as well as the roles as metabolic intermediates in the nervous system.

Effects of suramin on human platelet aggregation and Ca2+ mobilization induced by thrombin and other agonists.

The purpose of this study was to investigate the effect of suramin, a polyanionic napthalene sulfonic acid, on human platelet aggregation and Ca2+ mobilization induced by various agonists. Our results show that suramin completely inhibited aggregation by thrombin, platelet activating factor (PAF), alkyllysophosphatidic acid (ALPA), or arachidonic acid in a concentration-dependent manner. The IC50 values of suramin for inhibition of aggregation by PAF, arachidonic acid, and thrombin were 76.7, 239, and 1.49 microg/ml, respectively. Ca2+ mobilization induced by thrombin was inhibited by suramin with an approximate IC50 value of 20 microg/ml. This concentration of suramin had no effect on PAF or oleic acid-induced Ca2+ mobilization. The mechanism by which suramin inhibits aggregation is not clear, but our results suggest that suramin inhibits the ligand-receptor interaction.

Oleic acid-induced Ca2+ mobilization in human platelets: is oleic acid an intracellular messenger?

The purpose of this study was to explore the effect of oleic acid (OA) on intracellular Ca2+ mobilization in human platelets. When applied extracellularly, OA produced a concentration dependent rise in cytosolic [Ca2+] ([Ca2+]cyt) when extracellular [Ca2+] (Ca2+]ext) was zero (presence of EGTA), suggesting that OA caused an intracellular release of Ca2+. Intracellular Ca2+ release was directly proportional to entry of OA into platelets and OA entry was indirectly proportional to [Ca2+]ext. In permeabilized platelets, OA caused the release of 45Ca2+ from ATP dependent intracellular stores. Finally, our results show that thrombin stimulated the release of [3H]OA from platelet phospholipids. The saturated fatty acids stearic and palmitic acid did not stimulate an increase in [Ca2+]cyt under these conditions, but the unsaturated fatty acid, linolenic acid produced effects similar to those of OA, suggesting specificity among fatty acids for effects on [Ca2+]cyt. Taken together, our experiments suggest that OA which has been incorporated into platelet phospholipids was released into the cytosol by thrombin stimulation. Our experiments also show that OA stimulates Ca2+ release from intracellular stores. These results support the hypothesis that OA may serve as an intracellular messenger in human platelets.

Signaling responses to alkyllysophosphatidic acid: the activation of phospholipases A2 and C and protein tyrosine phosphorylation in human platelets.

The profile of biochemical responses following stimulation of human platelets with 1-alkyl-2-lyso-sn-glycero-3-phosphate (ALPA), a derivative of platelet-activating factor (PAF), was investigated. In the presence of extracellular Ca2+, ALPA evoked a dose-dependent increase and sustained elevation of the intracellular free Ca2+ concentration and stimulated the formation of phosphatidic acid. Platelets released free [3H]arachidonic and [3H]oleic acid at maximal rates between 5 and 15 s following ALPA stimulation. However, in platelets labeled with myo-[3H]inositol, ALPA induced [3H]phosphoinositide breakdown and formation of [3H]inositol phosphates with slower kinetics. Intracellular Ca2+ mobilization and the release of free fatty acids and inositol phosphates were not inhibited by pretreatment of platelets with pertussis toxin (PTX) or the PAF receptor antagonist WEB 2086. Following platelet stimulation with ALPA, tyrosine phosphorylation of proteins with apparent molecular masses of 65-95, 110-130, and 145-170 kDa was increased in a time-dependent manner, while phosphorylation of 40- to 45-kDa proteins was decreased. One of the platelet proteins phosphorylated on tyrosine residues in response to ALPA was found to be PLC-gamma1. Exogenous [3H]ALPA was metabolized primarily to [1-3H]alkyl-2,3-diacylglycerol. The metabolic conversion of [3H]ALPA involved a dephosphorylation reaction, and the formation of the dephosphorylated product, [1-3H]alkyl-monoglycerol, was detected within 5 s. These data demonstrate that an ether-linked lysophosphatidic acid can activate human platelets by a PTX-insensitive mechanism which does not involve the PAF receptor. Upon stimulation of platelets, ALPA induces the activation of phospholipases A2 and C, and tyrosine phosphorylation of several cellular proteins including PLC-gamma1. These signal transduction responses in platelets are accompanied by rapid metabolism of ALPA.

Biochemical characterization of the interaction of lipid phosphoric acids with human platelets: comparison with platelet activating factor.

A series of lipid phosphoric acids, including 1-O-alkyl-2-lyso-glycerophosphoric acid, 1-O-acyl-2-lyso-glycerophosphoric acid, hexadecylpropanediolphosphoric acid, N-acyl-2-aminoethanolphosphoric acid, sphingosine phosphoric acid, and certain homologues and analogues, were synthesized and characterized by thin-layer chromatography, fast-atom bombardment mass spectrometry, and their ability to aggregate human platelets. The presence of a receptor for these lipid phosphoric acids that is distinct from the PAF receptor is strongly suggested from experiments involving a desensitization procedure, platelet-activating factor (PAF) receptor antagonists, and inhibitors of the lipid phosphoric acids. The unique features of the interaction of these lipid phosphoric acids with platelets include: (a) evidence for a separate receptor(s) for this diverse group of synthetic compounds, (b) no requirement for stereospecificity (i.e., no glycerol backbone), and (c) a structural requirement for a long-chain hydrocarbon residue covalently bound to a phosphoric acid residue. In the interaction of these compounds with the platelet, it is mandatory that extracellular Ca2+ and ADP be present for maximum biological activity. The potential involvement of a lipid phosphoric acid receptor, which could form a component of the activation pathway associated with various lysophospholipids and analogues, such as PAF, via a phospholipase D activation, is discussed.

Isolation of a phospholipid inhibitor of platelet activating factor-induced activity from perfused rat liver: identification as phosphatidylglycerol.

An endogenous inhibitor of platelet activating factor action was isolated from perfused rat liver. It was purified by thin-layer chromatography and high-performance liquid chromatography and subjected to chemical modifications in order to identify its structure. On the basis of its fast atom bombardment-mass spectrum it was characterized as phosphatidylglycerol composed mainly of 16:0/18:1 and 16:0/20:2 fatty acyl chains ([M+H]+ at m/z 749 and 775, respectively) and very minor levels of 18:0/18:1 and 18:0/20:2. The purified compound exhibited inhibition on rabbit platelet aggregation induced by 5 x 10(-10) M platelet activating factor (PAF) at an EC50 value near 2.5 x 10(-6) M and on the serotonin secretion at an EC50 7 x 10(-6) M. Other phospholipids isolated from the liver preparations, such as phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin (diphosphatidylglycerol), and phosphatidic acid, exhibited no inhibitory activity in the concentration range from 1 x 10(-4) to 1 x 10(-7) M nor did they induce any aggregation, or lysis, of the platelets. Of importance, phosphatidylglycerol could inhibit thrombin- and ADP-induced aggregation of rabbit platelets. These results suggested a possible site of inhibition common to the signal transduction pathway of these agonists. Preliminary binding experiments showed a noncompetitive type of inhibition on PAF binding to intact rabbit platelets.

Protein tyrosine phosphorylation and regulation of the receptor for platelet-activating factor in rat Kupffer cells. Effect of sodium vanadate.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC) and sodium vanadate (a phosphotyrosine phosphatase inhibitor) induced a time- and concentration-dependent increase in phosphotyrosine in several proteins and stimulated prostaglandin (PG) E2 production in cultured rat Kupffer cells. In addition, vanadate induced a decrease in the surface expression of AGEPC receptors and, as a consequence, inhibited AGEPC-stimulated PGE2 production. The vanadate-induced decrease in the surface expression of AGEPC receptors was time- and concentration-dependent and was partially prevented by genistein, a putative tyrosine kinase inhibitor. Upon removal of vanadate from the culture medium and re-incubation of cells in vanadate-free medium, the surface AGEPC receptors were restored within 7 h after the removal of vanadate. Both AGEPC- and vanadate-stimulated PGE2 formation was attenuated by genistein. Thus the present investigation demonstrates that both AGEPC and sodium vanadate stimulate tyrosine phosphorylation of cellular proteins, and vanadate induces a decrease in the number of the surface AGEPC receptors. These results suggest that protein tyrosine phosphorylation may play a role, directly or indirectly, in the regulation of surface expression of AGEPC receptors as well as in PGE2 production in response to vanadate and AGEPC.

Dual effects of oleic acid on Ca2+ mobilization and protein phosphorylation in human platelets in presence or absence of platelet activating factor.

This laboratory demonstrated earlier that oleic acid inhibited platelet activating factor (PAF)-induced aggregation and serotonin release of rabbit platelets (M. Miwa, C. Hill, R. Kumar, J. Sugatani, M. S. Olson, and D. J. Hanahan, 1987, J. Biol. Chem. 262, 527-530). More recently, we reported that oleic acid caused a decrease in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2), but did not affect the level of inositol-1,4,5-trisphosphate (IP3), in rabbit platelets (D. Nunez, J. Randon, C. Gandhi, A. Siafaka-Kapadai, M. S. Olson, and D. J. Hanahan, 1990, J. Biol. Chem. 265, 18330-18838). These results suggested that oleic acid did not stimulate phospholipase C. In contrast, PAF induced a decrease in PIP2 and an increase in PIP level and IP3. These effects were shown to be attenuated by oleic acid. In this current study, our experiments show that (a) oleic acid blocked PAF-induced rise in intracellular [Ca2+] (to provide a mechanism in agreement with our previous experiments which showed that oleic acid inhibited PAF-induced IP3 rise in platelets) and (b) oleic acid itself induced a gradual rise in [Ca2+]i, which would provide a mechanism for oleic acid-induced aggregation despite the fact that oleic acid did not cause the production of IP3 (Nunez et al., 1990). Oleic acid, in a dose-dependent manner, was shown to inhibit PAF-induced Ca2+ mobilization from intra- and extracellular sources. The inhibition was closely related to the suppressive effect of oleic acid on PAF-induced aggregation. Furthermore, oleic acid inhibited the PAF-stimulated phosphorylation of the 20- and 40-kDa proteins. At concentrations above 20 microM, oleic acid itself could induce platelet aggregation and Ca2+ mobilization, but the time sequence of these two responses in human platelets was significantly different from those obtained with PAF. Oleic acid alone, at 20 microM, caused a 1.4-fold increase in the cAMP level in platelets which was followed by a decline to a basal value at higher concentrations of this fatty acid. It seemed clear that elevation of adenylate cyclase activity was not associated with free fatty acid inhibition of platelet activation. Interestingly, both PAF and oleic acid added separately to human platelets induced protein-tyrosine phosphorylation, but oleic acid did not cause any inhibition of PAF-induced protein-tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

The human platelet as a reproducible and sensitive cell for the detection and assay of platelet-activating factor.

A new procedure for the preparation of human platelets consistently sensitive to platelet-activating factor (PAF) in the low nanomolar range has been developed. Key to the success of this approach was the addition of adenosine during the isolation phase, providing an excellent recovery of stable cells, and the inclusion of ADP in the aggregation assay, providing increased sensitivity to PAF. Examination of the binding profile of tritium-labeled PAF to these platelets in the presence or absence of ADP revealed significant difference in the Kd values but not in the number of specific binding sites. Other reagents having an influence on the reactivity and stability of the human platelets, as regards its interaction with PAF, are described.

31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase.

31P NMR spectroscopy has been utilized in conjunction with site-directed mutagenesis and phospholipid analysis to determine structural aspects of the prosthetic flavins, FAD and FMN, of NADPH-cytochrome P450 reductase. Comparisons are made among detergent-solubilized and protease (steapsin)-solubilized preparations of porcine liver reductases, showing unequivocally that the 31P NMR signals at approximately 0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phospholipids. By extraction and TLC analysis, the phospholipid contents of detergent-solubilized rat liver reductase, both tissue-purified and Escherichia coli-expressed, have been determined to reflect the membranes from which the enzyme was extracted. In addition, the cloned, wild-type NADPH-cytochrome P450 reductase exhibits an additional pair of signals downfield of the normal FAD pyrophosphate resonances reported by Otvos et al. [(1986) Biochemistry 25, 7220-7228], but these signals are not observed with tissue-purified or mutant enzyme preparations. The Tyr140----Asp140 mutant, which exhibits only 20% of wild-type activity, displays no gross changes in 31P NMR spectra. However, the Tyr178----Asp178 mutant, which has no catalytic activity and does not bind FMN, exhibits no FMN 31P NMR signal and a normal, but low intensity, pair of signals for FAD. The latter experiments, taking advantage of mutations in residues putatively on either side of the FMN isoalloxazine ring, suggest subtle to severe changes in the binding of the flavin prosthetic groups and, perhaps, cooperative interactions of flavin binding to NADPH-cytochrome P450 reductase.

Platelet-activating factor-stimulated protein tyrosine phosphorylation and eicosanoid synthesis in rat Kupffer cells. Evidence for calcium-dependent and protein kinase C-dependent and -independent pathways.

The lipid mediator platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC) has been shown to elicit several important biochemical signaling responses in mammalian cells, including polyphosphoinositide hydrolysis, arachidonic acid release/eicosanoid production, and protein tyrosine phosphorylation. In the present study, the roles of Ca2+ and protein kinase C (PKC), two signaling components of the phospholipase C pathway, in AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation, were investigated in cultured rat Kupffer cells. AGEPC at nanomolar concentrations induced an increase in intracellular calcium concentration ([Ca2+]i), stimulated membrane PKC activity, and resulted in protein tyrosine phosphorylation. The maximal increase in [Ca2+]i and membrane PKC activity in response to AGEPC were observed within 30-50 s, whereas the AGEPC-induced protein tyrosine phosphorylation reached maximal levels within 2-5 min. [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) but not 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of calcium release from intracellular compartments, nearly abolished the AGEPC-induced increase in [Ca2+]i suggesting involvement of extracellular calcium influx in this event. Both EGTA and TMB-8 abolished or inhibited AGEPC-stimulated protein tyrosine phosphorylation and eicosanoid formation, respectively. The calcium ionophore A23187 alone stimulated eicosanoid production and protein tyrosine phosphorylation with an identical pattern to that of AGEPC. Phorbol myristate acetate (PMA), an activator of PKC, which did not affect [Ca2+]i, mimicked the actions of AGEPC, stimulating eicosanoid production and promoting tyrosine phosphorylation of a set of proteins similar to those phosphorylated following AGEPC stimulation. AGEPC-enhanced tyrosine phosphorylation of some of the protein substrates and eicosanoid production were inhibited in cells "down-regulated" for PKC. Furthermore, both PMA- and AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation were attenuated or abolished by at least one of the PKC inhibitors, staurosporine, and calphostin C. Taken together, these results are consistent with the conclusions that: (a) AGEPC stimulates the phospholipase-mediated arachidonic acid release/eicosanoid synthesis cascade and protein tyrosine phosphorylation through extracellular Ca(2+)-dependent and PKC-dependent and -independent mechanism(s) and (b) the Ca(2+)-PKC interaction determines the efficacy of the AGEPC-stimulated cellular events.

Paracrine interactions between platelet-activating factor and prostaglandins in hormonally-treated human luteal phase endometrium in vitro.

Stromal cells and epithelial glands were separated after enzymic digestion of specimens obtained from 27 women at hysterectomy or endometrial biopsy during the luteal phase, and then cultured to confluence in vitro. PGE release into the culture medium (mean +/- s.e.m.: ng/mg protein/24 h) from gland cell cultures was not changed by oestradiol (17.6 +/- 1.3 for control and 25.5 +/- 2.8 for oestradiol, respectively). However, in the presence of oestradiol, PAF (5 ng/ml) significantly elevated PGE release to 44.2 +/- 5.8. No stimulation was observed in the presence of progesterone. Stromal cell medium had no effect on PGE release in gland cell cultures. PGE release was always much lower in stromal cell cultures than in glands (control: 4.7 +/- 0.6). PAF stimulated PGE release in the presence of oestradiol in these cells also; gland cell medium was without effect. In co-cultures of glandular and stromal cells, PGE release was more similar to that seen in gland cell cultures, with PAF being stimulatory under the influence of oestradiol. PGF release into the medium from the same gland cell cultures was significantly elevated by hormonal treatment, being greatest (62.0 +/- 11.3) with oestradiol alone, and was strongly inhibited in all wells by addition of PAF and stromal cell medium. In stromal cell cultures without hormonal addition, PGF levels (15.0 +/- 2.4) were similar to those seen in glands (18.1 +/- 3.1), and no stimulation was achieved by oestradiol (29.6 +/- 5.9). PAF was inhibitory on PGF release, while gland cell medium was without effect. Co-cultures gave PGF values generally similar to those of stromal cells; oestradiol was again stimulatory (55.0 +/- 9.3). PAF was significantly inhibitory in the presence of oestradiol. PAF (mean +/- s.e.m.: pmol/mg protein/24 h using a platelet serotonin release assay) in stromal cells was significantly increased from control [M199 alone] (0.31 +/- 0.12) by progesterone (1.00 +/- 0.17). Addition of PGE-2 (7.5 ng/ml) to progesterone-treated wells further increased PAF concentration (5.34 +/- 0.09), but was without effect in wells receiving oestradiol alone. Wells exposed to both hormones exhibited an intermediate response. Similar results were obtained with addition of gland cell culture medium, presumably due to its endogenous PGE content. In co-cultures, PAF concentrations were significantly elevated by progesterone alone (4.78 +/- 0.78) or when combined with oestradiol (2.38 +/- 0.51), but not by oestradiol alone. Treatment with PGE-2 caused no additional stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Novel route to preparation of high purity lysoplasmenylethanolamine.

A rapid and simple method has been developed for the preparation of highly purified lysoplasmenylethanolamine. The starting material, a phosphatidylethanolamine (PE) sample that contained a mixture of the 1, 2 diacyl- and 1-O-alkenyl-2-acyl forms was subjected to mild alkaline methanolysis for 20 min at room temperature. Addition of chloroform and water with vigorous mixing, but without acidification at this point, led to a preferential retention of the lysoplasmenylethanolamine in the alkaline aqueous phase and complete separation of the methyl esters into the chloroform phase. Neutralization of the alkaline phase with dilute acetic acid, followed by addition of chloroform, allowed recovery of the lysoplasmenylethanolamine in the chloroform phase in very high yields (75-80% based on vinyl ether content of starting material). On the other hand, a preparation of cholineglycerophospholipids enriched in plasmenylcholine, treated in exactly the same manner, gave a lysoplasmenylcholine that was not retained in the alkaline phase, but partitioned primarily into the chloroform-rich phase together with the methyl esters. Characterization of the purified lysoplasmenylethanolamine was achieved by thin-layer chromatography and compositional analysis. In addition, fast atom bombardment mass spectral analysis of the intact lysoplasmenylethanolamine together with gas chromatography-mass spectrometry of the dimethyl acetals derived from the 1-O-alkenyl chains allowed further proof of the structure and an assessment of the purity of this compound.

Regulation of platelet-activating factor receptor and platelet-activating factor receptor-mediated biological responses by cAMP in rat Kupffer cells.

Treatment of cultured Kupffer cells with the beta-adrenergic agonist isoproterenol (10 microM) for a short period of time (30 min) attenuated the subsequent platelet-activating factor (PAF)-induced arachidonic acid release and cyclooxygenase-derived eicosanoid (e.g. thromboxane B2 and prostaglandin E2) production. This effect of isoproterenol was highly specific since the alpha-adrenergic agonist phenylephrine and the beta-adrenergic antagonist propranolol had no effect on the stimulatory effect of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC). The inhibitory effect of isoproterenol on the AGEPC-induced arachidonic acid release was demonstrated through the use of a specific beta-adrenergic subtype agonist and antagonist to be mediated by beta 2-adrenergic receptors on Kupffer cells. These inhibitory effects of isoproterenol can be mimicked by dibutyryl cAMP but not by dibutyryl cGMP, suggesting that a cAMP-dependent mechanism is likely involved in the regulatory action of isoproterenol. Ligand binding studies indicated that short term (i.e. 30 min) treatment of the cultured Kupffer cells with either isoproterenol or dibutyryl cAMP had no effect on the specific [3H]PAF binding. However, long term incubation (9-24 h) with dibutyryl cAMP caused down-regulation of the PAF receptors in rat Kupffer cells. Forskolin (0.1 mM), an adenylyl cyclase activator, down-regulated the surface expression of the AGEPC receptors more rapidly, decreasing the specific [3H]AGEPC binding by approximately 40% within 2 h. The receptor regulatory effect of dibutyryl cAMP and forskolin was time- and concentration-dependent. These observations suggest that a cAMP-dependent mechanism coupled with beta 2-adrenergic receptors may have important regulatory effects on the PAF receptor and post-receptor signal transducing mechanisms for PAF in hepatic Kupffer cells.

Two distinct pathways of platelet-activating factor-induced hydrolysis of phosphoinositides in primary cultures of rat Kupffer cells.

Stimulation of rat Kupffer cells in primary culture with platelet-activating factor (PAF) caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate with a concomitant increase in the levels of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,4-bisphosphate. This phospholipase C-mediated hydrolysis of polyphosphoinositides was independent of extracellular Ca2+ but was inhibited by the intracellular Ca2+ antagonist TMB-8. A second slower response to PAF was characterized by deacylation of PI leading to the accumulation of glycerophosphoinositol (GPI). PAF-induced GPI synthesis was not inhibited by TMB-8. These effects of PAF were accompanied by initial transient mobilization of Ca2+ from intracellular stores followed by a rather slow influx of Ca2+ from the extracellular medium. PAF-stimulated deacylation and phosphodiesteric hydrolysis of inositol lipids were differentially affected by cholera toxin and pertussis toxin. Pretreatment of the Kupffer cells with either of these toxins caused inhibition of phospholipase C activity. Pertussis toxin also inhibited PAF-stimulated deacylation. However, cholera toxin itself stimulated GPI release and addition of PAF to the cholera toxin-treated cells caused a further increase in GPI release. Phorbol ester inhibited PAF-induced phosphodiesteric hydrolysis of phosphoinositides, but not deacylation. PAF-induced metabolism of phosphoinositides was inhibited by the PAF antagonist, U66985. These results suggest that PAF-induced phosphodiesteric hydrolysis and deacylation of inositol phospholipids are regulated via distinct mechanisms involving activation of separate G-proteins in rat Kupffer cells. Also the regulation of phosphoinositide metabolism by Ca2+ mobilization from two separate Ca2+ pools is indicated by this study.

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)

Regulation of platelet-activating factor receptor and PAF receptor-mediated arachidonic acid release by protein kinase C activation in rat Kupffer cells.

Phorbol 12-myristate 13-acetate (PMA), a potent protein kinase C activator, caused down-regulation of receptors for platelet-activating factor (AGEPC) on the plasma membrane of rat Kupffer cells (40-50% reduction) but had a relatively minor effect on the binding affinity of the receptors for AGEPC (Kd = 0.30 nM vs 0.56 nM) when incubated with the cells for a short period of time (30-60 min). As a consequence, the AGEPC receptor-mediated arachidonic acid release was attenuated. The PMA-induced down-regulation of AGEPC receptors was concentration-dependent, specific, and transient (the maximal effect was observed at about 1 h and the level of specific [3H]AGEPC binding gradually returned to the control level within 8.5 h and even higher than the control level at 24 h after addition of PMA). Upon removing PMA from the culture medium, more than half of the lost receptors were replaced within 1 h at 37 degrees C and the recovery process appeared to be independent of protein synthesis. The ability of PMA to down-regulate the AGEPC receptors was lost in cells "down-regulated" for protein kinase C, suggesting that the receptor-regulatory effect of PMA is protein kinase C-dependent. Protein kinase C appeared to be involved in the AGEPC-induced arachidonic acid release since 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride, a protein kinase C inhibitor, attenuated the stimulatory effect of AGEPC in this system. In addition, AGEPC-induced [3H]arachidonic acid release was inhibited significantly in cells down-regulated for protein kinase C. The present study thus demonstrates that protein kinase C has dual actions in the regulation of AGEPC-mediated events, i.e., a positive forward action, regulating AGEPC-stimulated arachidonic acid release, and a negative action, which inactivates or down-regulates AGEPC receptors.