Evidence for the possible involvement of protein kinase C in the activation of non-specific phospholipase A2 in human neutrophils.

In this article, we provide mass data on released fatty acids in neutrophils stimulated with A23187 in the presence and absence of phorbol 12-myristate 13-acetate (PMA). A23187 alone caused a highly selective accumulation of non-esterified arachidonic acid (AA) relative to other saturated (palmitic and stearic acids) and unsaturated fatty acids (oleic and linoleic acids). However, the absolute levels of non-esterified arachidonic acid, oleic acid and linoleic acid were significantly increased (by twofold) in response to the action of A23187 in the presence of PMA. We also noted a further twofold increase in the non-esterified arachidonic acid in the presence of BW755C, a dual inhibitor of cyclooxygenase and lipoxygenases, with no apparent change in the levels of oleic and linoleic acids and other saturated fatty acids. These results suggest that PMA potentiates not only the release of AA but also the release of oleic and linoleic acids from neutrophil phosphoglycerides. This is possibly due to the activation of non-specific phospholipase A2 (PLA2). Our data also suggest a possible role for the 5-lipoxygenase metabolites in PMA-induced synergism with regard to the release of AA through AA-specific and PMA-sensitive PLA2. Protein kinase C appears, therefore, to play a role in the regulation of both AA-specific and non-specific PLA2 in human neutrophils.

Modification of rat platelet fatty acid composition by dietary lipids of animal and vegetable origin.

There were no statistically significant differences in final body weight or in food intake among groups of rats fed for 7 wk various fats of animal origin (lard fat and cod liver oil) or vegetable origin (corn, soybean and canola oils); the fats were fed as 10% of the diet (by wt) and were of varied fatty acid composition. Nevertheless, the mean weights of the kidneys from cod liver oil-fed animals were significantly higher than those of all other dietary groups. Platelets of rats from the groups receiving the animal fat contained significantly lower levels of linoleic acid, 18:2(n-6) [a precursor of arachidonic acid, 20:4(n-6)], than did platelets from rats receiving the fat of vegetable origin. Although the soybean-, canola- and cod liver oil-fed animals received substantial quantities of (n-3) fatty acids [alpha-linolenic acid, 18:3(n-3); eicosapentaenoic acid, 20:5(n-3); and docosahexaenoic acid, 22:6(n-3)], only the platelets of the latter two groups contained detectable levels of these fatty acids along with their products of elongation/desaturation/retroconversion. Platelets of the cod liver oil-fed group contained significantly less arachidonic acid, a major precursor of eicosanoids, than did those from all other dietary groups. However, platelet arachidonic levels also varied markedly among the other dietary groups. Diet-induced fatty acid changes observed in platelets of various dietary groups may influence platelet responses, including secretion, aggregation and biosynthesis of eicosanoids.

Effect of dietary lipid on collagen- and adenosine diphosphate-induced platelet aggregation and thromboxane A2 synthesis in the rat.

Dietary lipids containing different proportions of long-chain polyunsaturated fatty acids can affect platelet thromboxane A(2) formation and aggregation. In the present work, the effects of dietary lipid, from animal and vegetable sources, on collagen- and adenosine diphosphate (ADP)-induced thromboxane A(2) (measured as thromboxane B(2)) production and aggregation in washed rat platelets were studied. In addition, plasma thromboxane B(2) levels in rats fed different dietary lipids were measured. Animals were fed 10% fat by weight as lard (LRD), corn oil, soy bean oil, canola oil (CAN), or cod liver oil (CLO) for a period of 7 weeks. Circulating thromboxane B(2) levels detected in platelet-poor plasma of the CLO-fed animals were significantly lower than those of rats fed all other dietary lipids. The platelets of CLO-fed animals synthesized significantly less thromboxane A(2) compared with those from other dietary groups following ex vivo stimulation of platelets with agonists such as collagen and ADP, with the exception of platelets from the LRD-fed animals. Ex vivo stimulation of platelets obtained from this group with collagen resulted in the synthesis of significantly greater levels of thromboxane A(2) compared with all other groups. However, aggregation responses to collagen and ADP were not significantly affected by dietary treatment, although relatively the lowest responses to these agonists were apparent in the CLO-fed and CAN-fed groups, respectively.

Inhibition of protein kinase C by H-7 potentiates the release of oleic, linoleic and arachidonic acids in A23187-stimulated human neutrophils.

This present report describes the effect of H-7, a protein kinase C inhibitor, on the release of oleic, linoleic and arachidonic acids in A23187-stimulated neutrophils. Surprisingly, the inhibitor potentiated the release of all three unsaturated fatty acids in neutrophils stimulated with A23187 alone. In contrast, released oleic acid, linoleic acid and arachidonic acid in phorbol 12-myristate 13-acetate-primed neutrophils were attenuated by 35, 47 and 33%, respectively, in the presence of H-7 (300 microM). Phorbol 12-myristate 13-acetate (PMA) had no effect on A23187-stimulated release of saturated fatty acids. Both PMA and H-7 when used alone had no effect on the release of saturated or unsaturated fatty acids. We, therefore, conclude that H-7 may have effects other than inhibiting PMA-primed responses including superoxide generation, degranulation and arachidonic acid release in human neutrophils.

Mobilization of arachidonic acid in thrombin-stimulated human platelets.

In the present work we investigated the effect of serine esterase inhibitors such as 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC) and phenylmethylsulfonyl fluoride (PMSF), as well as the effect of mepacrine on thrombin-induced mobilization of arachidonic acid (AA) in human platelets. The inhibitor NCDC (0.6 mM) completely abolished the thrombin-induced activation of phospholipase C, phospholipase A2, and transacylase enzymes, whereas the pretreatment of platelets with PMSF (2 mM) resulted in a highly selective inhibition of phospholipase A2 and transacylase activities, with no marked effect on thrombin-induced activation of phospholipase C. The thrombin-induced release of [3H]AA from phosphatidylcholine and phosphatidylinositol was reduced by 90 and 56%, respectively, in the presence of PMSF. This inhibitor also caused a parallel inhibition in the accumulation of [3H]AA (85%) with little effect on thrombin-induced formation of [3H]phosphatidic acid (5%), whereas mepacrine (0.4 mM) caused a selective inhibition of phospholipase A2 and transacylase activities with concomitant stimulation of [3H]phosphatidic acid formation in intact human platelets. These results demonstrate that NCDC and PMSF (serine esterase inhibitors) do not affect agonist-induced activation of phospholipases that mobilize arachidonic acid through a common site. Our results further demonstrate that the inhibition of [3H]AA release observed in the presence of NCDC, PMSF, and mepacrine is primarily due to their direct effects on enzyme activities, rather than due to their indirect effects through formation of complexes between inhibitors and membrane phospholipids. Based upon these results, we also conclude that the combined hydrolysis of phosphatidylcholine and phosphatidylinositol by phospholipase A2 serves as a major source for eicosanoid biosynthesis in thrombin-stimulated human platelets.

Morphology of pulmonary intravascular macrophages (PIMs) in ruminants: ultrastructural and cytochemical behavior of dense surface coat.

Recent studies have indicated that pulmonary intravascular macrophages (PIMs) are a resident cell population which in structure and function resemble mature macrophages of the mononuclear phagocyte system (MPS) in various domestic species, particularly the ruminants. The ultrastructural features of PIMs of the goat and calf lungs were studied by using vascular perfusion and direct airway instillation of fixatives. Staining with tannic acid as a component of paraformaldehyde-glutaraldehyde-based fixative revealed the presence of an electron-dense coat on the surface of the cell membrane of the PIMs. The surface coat disappeared after heparin infusion and after enzymatic digestion with lipolytic lipase, suggesting that the surface coat was predominantly lipoprotein in nature. The lipoprotein coat was organized in the form of a linear chain of spherical globules with a consistent periodicity created by the intervening translucent space between individual globules. The surface coat was separated from the outer-leaflet of the cell membrane by an empty space measuring 35-39 nm in width. PIMs possessed a significant number of coated pits and coated vesicles, the cell organelles of receptor-mediated endocytosis of lipoproteins. In concurrence with the coated pits and vesicles, microtubules, multivesicular bodies, and lipoprotein-positive vesicles were also observed. It is conceivable that PIMs are involved in lipid metabolism and are the major source of vasoactive substances, which significantly influence both the dynamics of pulmonary circulation and the surfactant turnover of the ruminant lung.

Aggregation and/or oxygenated products of arachidonic acid are not required for collagen-induced deacylation of phosphatidylcholine in human platelets.

In the present study the effects of collagen on platelet aggregation and arachidonic acid (AA) mobilization, specifically from phosphatidylcholine (PC), were investigated in the presence and absence of BW755C, a selective inhibitor of cyclo-oxygenase and lipoxygenases. The inhibition of cyclo-oxygenase and lipoxygenase(s) by BW755C (75 microM) resulted in severe impairment in collagen-induced platelet aggregation. In the presence of BW755C, the aggregation response amounted to 14, 26, 37 and 49% of the corresponding controls (without BW755C) at 10, 25, 50 and 100 micrograms of collagen respectively. On the contrary, the amount of AA released from PC, which ranged from 3.5 to 8.6 nmol/10(9) platelets, in response to the action of collagen (10-100 micrograms) remained unaffected by the presence of BW755C. Substantial amounts of non-esterified AA were detected in the free fatty acid fractions obtained from collagen-stimulated platelets in the presence as well as in the absence of BW755C. However, the presence of BW755C caused a greater accumulation of free AA (mass) and this ranged from 4 to 16 nmol, depending upon the amount of collagen. In addition, small increases in free stearic and oleic acids were observed in collagen-stimulated platelets as compared with unstimulated platelets. The amount of AA lost from PC represented 67, 80, 49 and 52% of the free AA obtained at 10, 25, 50 and 100 micrograms of collagen respectively. Our results adhesion of platelets to collagen fibres may be responsible for much of the AA release from PC Furthermore, these results demonstrate that aggregation and/or cyclo-oxygenase/lipoxygenase metabolites are not obligatory for the release of AA from PC in collagen-stimulated human platelets.

The metabolism of arachidonic and eicosapentaenoic acids in human neutrophils stimulated by A23187 and FMLP.

A23187 stimulates the metabolism of endogenous as well as exogenous arachidonic acid (AA) and eicosapentaenolc acid (EPA) to their corresponding leukotrienes in human neutrophils. In contrast, conflicting results have been obtained concerning the effect of FMLP on the metabolism of these fatty acids. In the present study we compared the effect of A23187 and FMLP on the release and metabolism of these fatty acids in neutrophils. Stimulation of neutrophils with A23187, but not with FMLP, resulted in detectable levels of AA in the presence or absence of BW755C (a dual inhibitor of cyclooxygenase and lipoxygenase). The absolute amount of nonesterified AA in the extracts of neutrophils exposed to the agonist A23187 in the presence of BW755C was 20% higher than that obtained in the absence of BW755C, indicating that only a small fraction of the released AA was converted to lipoxygenase products. Furthermore, significant quantities of AA and EPA metabolites were detected only after treatment of neutrophils with A23187, but not with FMLP. Both A23187 and FMLP stimulated the conversion of exogenous EPA to 5-lipoxygenase products, with A23187 being somewhat more effective. In addition, significant differences were noted on the effect of EPA and DHA on the conversion of AA to its metabolites in A23187-stimulated neutrophils. Our results provide strong evidence that the amounts of eicosanoid precursors mobilized in response to FMLP are extremely small, if any, and this appears to be the likely explanation for the lack of eicosanoid detection by HPLC in FMLP-stimulated neutrophils.

Mobilization of arachidonic acid in collagen-stimulated human platelets.

Stimulation of platelets with collagen results in the mobilization of arachidonic acid (AA) from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). In this study the effect of aspirin, indomethacin, BW755C and prostaglandin H2 (PGH2) on labelled AA release in response to varied concentrations of collagen was investigated. Our results indicate that aspirin (0.56 mM) and indomethacin (5.6 microM) not only inhibited the collagen-mediated formation of cyclo-oxygenase metabolites, but also caused a significant reduction in the accumulation of free labelled AA and 12-hydroxyeicosatetraenoic acid (12-HETE) (21-64%). Aspirin and indomethacin also inhibited the release of [3H]AA from PC (37-75%) and PI (33-63%). The inhibition of AA release caused by aspirin was reversed partially by PGH2 (1 microM). In contrast, a smaller/no inhibition of collagen-stimulated labelled AA and 12-HETE accumulation (0-11%) and of collagen-stimulated AA loss from PC and PI was observed in the presence of BW755C. The results obtained in the presence of aspirin, indomethacin and BW755C at lower concentrations of collagen further demonstrate that AA release from PI (45-61% inhibition at 10 micrograms of collagen), but not from PC, was affected by the inhibition of cyclo-oxygenase. The results obtained on the effect of PGH2 further support that deacylation of phospholipids occurs independently of cyclo-oxygenase metabolites, particularly at higher concentrations of collagen. These results also demonstrate that aspirin and indomethacin, but not BW755C, cause a direct inhibition of collagen-induced [3H]AA liberation from PC as well as from PI. We also conclude that the diacylglycerol lipase pathway is a minor, but important, route for AA release from PI in collagen-stimulated human platelets. The mechanisms underlying the regulation of AA release by collagen in the absence of cyclo-oxygenase metabolites are not clear.

The inhibition of arachidonic acid mobilization in human platelets by R59 022, a diacylglycerol kinase inhibitor.

R59 022 (6-[2-[4-[(4-fluorophenyl)phenylmethylene]-1- piperidinyl]ethyl]-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one) has been suggested as an inhibitor of diacylglycerol kinase in erythrocyte membranes and intact platelets. In the present study, we have investigated the effects of this drug on arachidonic acid mobilization occurring in response to thrombin in intact human platelets. Our results indicate that release of arachidonic acid from membrane phospholipids such as phosphatidylcholine and phosphatidylinositol was severely impaired by R59 022 and the extent of inhibition amounted to 77% and 84%, respectively, as compared to controls. This resulted in a dramatic decrease in the accumulation of free arachidonic acid (labeled/unlabeled) and the percent inhibition of free arachidonic acid accumulation amounted to 80-90% as compared to controls. Furthermore, the drug caused a significant accumulation of thrombin-induced diacylglycerol (labeled) without affecting the formation of labeled phosphatidic acid (PA). We found no significant changes in the radioactivity of either phosphatidylethanolamine or phosphatidylserine following stimulation with thrombin in the presence or absence of R59 022. We conclude that the observed inhibition of thrombin-induced arachidonic acid mobilization by R59 022 may be due to its effects on the activities of diacylglycerol lipase/phospholipase A2. In addition, the failure of further stimulation of thrombin-induced PA by R59 022 may indicate that PA-specific phospholipase A2 is either not involved in the release of arachidonic acid or is not a major source for arachidonic acid release in thrombin-stimulated human platelets. These findings may prove to be important when this drug is used as a selective inhibitor of diacylglycerol kinase.

[3H]phosphatidic acid formed in response to FMLP is not inhibited by R59 022, a diacylglycerol kinase inhibitor.

R59 022 has been suggested to function as a selective inhibitor of diacylglycerol kinase in platelets and erythrocyte membranes. In the present study we have studied the effect of this drug on [3H]diacylglycerol and [3H]phosphatidic acid formed in response to FMLP in human neutrophils. Our results indicate that R59 022 (50 microM) itself (without any stimulus) caused a significant hydrolysis of [3H]phosphatidylinositol (6-7%), which resulted in an accumulation of [3H]diacylglycerol and [3H]phosphatidic acid. On the other hand, R59 022 at lower concentrations (10 microM) exhibited a biphasic response on the time-dependent formation of [3H]phosphatidic acid in response to FMLP. [3H]phosphatidic acid formed at 30 sec and 60 sec after stimulation with FMLP was neither inhibited nor stimulated whereas the amount of [3H]phosphatidic acid formed at 2 min and 3 min was significantly higher than that obtained with FMLP alone. Our results demonstrate that the increased formation of diacylglycerol and phosphatidic acid in response to FMLP in the presence of R59 022 is likely due to the activation of phospholipase C and/or D rather than the inhibition of DG kinase. We therefore conclude that R59 022 is relatively nonspecific and can affect several other enzymes involved in the agonist-stimulated turnover of phospholipids.

Differential effects of 15-HPETE on arachidonic acid metabolism in collagen-stimulated human platelets.

The 15-hydroperoxyeicosatetraenoic acid (15-HPETE) has been shown to affect platelet aggregation induced by collagen, arachidonic acid (AA), and PGH2-analogue. Furthermore, it also inhibits the platelet cyclooxygenase and lipoxygenase enzymes, and prostacyclin synthase. The present study was designed to test the effect of 15-HPETE on the mobilization of endogenous AA in collagen-stimulated human platelets. For this purpose, human platelets pretreated with BW755C (a dual inhibitor of cyclooxygenase and lipoxygenase) were stimulated with collagen in the presence of varied concentrations of 15-HPETE. We observed a significant inhibition of oxygenases at all concentrations of 15-HPETE. In contrast, our results indicate that 15-HPETE at lower concentrations (10 microM and 30 microM) significantly stimulated the collagen-induced release of AA from phospholipid sources. Although higher concentrations of 15-HPETE (50 microM and 100 microM) caused some inhibition of AA accumulation in the free fatty acid fraction (25% and 60%), the degree of inhibition was significantly lower than the inhibition observed for the oxygenases (65% and 88% for cyclooxygenase and 77% and 94% for lipoxygenase respectively). These results provide support that hydroperoxides also regulate phospholipases presumably by a different mechanism, which may be important in the detoxification of phospholipid peroxides.

Quantitative loss of individual eicosapentaenoyl-relative to arachidonoyl-containing phospholipids in thrombin-stimulated human platelets.

The thrombin-dependent losses of eicosapentaenoate (EPA) from the various phospholipids of platelets derived from human subjects ingesting a fish lipid concentrate (MaxEPA) were quantitatively assessed and studied in relation to arachidonate (AA). The net loss of AA and EPA from the total phospholipid, phosphatidylcholine (PC) + phosphatidylethanolamine (PE) + phosphatidylserine (PS) + phosphatidylinositol (PI) (loss from phosphatidylinositol minus accumulated phosphatidate), amounted to 44.4 and 7.3 nmol/2 x 10(9) platelets (mean values, n = 4 subjects), respectively, in response to thrombin (2 units/ml). The phosphatidylcholine, phosphatidylethanolamine (including alkenylacyl), phosphatidylserine, and phosphatidylinositol contributed 46, 17, less than 5, and 33%, respectively, of the AA loss; in contrast to these distributions, the corresponding phospholipid contributions to the net loss of EPA were 71, 27, less than 1, and less than 2%, respectively. Furthermore, the inhibition of AA- and EPA-phospholipid degradation by trifluoperazine indicated that almost all of the release of EPA occurs from PC and PE (greater than 95% of total EPA loss) upon thrombin stimulation and is mediated predominantly via phospholipase A2 activity with almost no contribution from PI. Similarities in the molar ratios of AA/EPA in the PC (3.9) or PE (3.7) which were degraded with those in the corresponding phospholipids from resting platelets suggested no marked selectivity by the phospholipase A2 in intact thrombin-stimulated human platelets in the hydrolysis of AA-PC (or AA-PE) versus EPA-PC (or EPA-PE). Quantitation of the newly released free AA and EPA was determined in the presence of BW755C, a dual cyclooxygenase/lipoxygenase inhibitor which was found not to influence the degradation of individual AA- and EPA-containing phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Ingestion of fish oil or a derived n-3 fatty acid concentrate containing eicosapentaenoic acid (EPA) affects fatty acid compositions of individual phospholipids of rat brain, sciatic nerve and retina.

The effect of feeding redfish (Sebastes marinus or mantella) oil or a derived n-3 fatty acid concentrate containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the fatty acid compositions of individual phospholipids in selected neural tissues was studied in growing male rats. Control animals were given sunflower oil in the diet for the 5-wk feeding trial. Lipid analyses revealed that EPA (20:5n-3) became significantly enriched in all phospholipid fractions (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol) in the tissues studied (brain, retina and sciatic nerve) in the two n-3 fatty acid dietary groups relative to controls. Corresponding changes were also found in the 22:5n-3 contents of these tissues, whereas little or no significant elevation in DHA (22:6n-3) was found. In contrast, the percentages by weight of the n-6 fatty acids including 18:2n-6, 20:4n-6 (arachidonic acid, AA), 22:4n-6 and 22:5n-6 were generally lower in the various phospholipids/tissues of the animals given fish oil or the n-3 fatty acid concentrate; the levels of 22:5n-6 and 22:4n-6 were markedly affected in this regard. These results indicate that dietary n-3 fatty acids (as EPA plus DHA) can greatly affect the fatty acid compositions of the various membrane phospholipids in nervous tissues within a relatively short time. These biochemical alterations may be important for functional changes including altered membrane fluidity, cellular responses, ion transport and the biosyntheses of AA- and EPA-derived prostaglandins and leukotrienes.

Thrombin-induced transfer of arachidonic acid in human platelets is not inhibited by trifluoperazine.

Human platelets have been shown to contain a Ca++- and CoA-independent transacylase enzyme that catalyzes the transfer of arachidonic acid from phosphatidylcholine (PC) to lysoplasmenylethanolamine. It has been suggested that this route may represent a major source for released arachidonic acid in stimulated platelets. In this study, we have shown using arachidonic-labelled human platelets that the thrombin-induced activation of a transacylase reaction was not affected by concentrations of trifluoperazine (TFP) (15 micrograms/2 X 10(9) cells) which abolished the accumulation of free [3H]arachidonic acid in the presence of the cyclooxygenase/lipoxygenase inhibitor BW755C. TFP, at this concentration failed to block the hydrolysis of phosphatidylcholine (PC) completely and had no effect on the increased radioactivity seen in total phosphatidylethanolamine (PE) (160% of control after 4 min of incubation). These results suggest that the transacylase pathway activated in response to thrombin is not likely dependent on calcium. As TFP blocks effectively both the accumulation of free [3H]arachidonic acid and the mass of arachidonic acid without affecting the transfer of this fatty acid from PC to PE in thrombin-stimulated human platelets, it is very unlikely that the transacylation pathway represents a major source of release arachidonic acid. Based on these findings, we conclude that the above pathway may be primarily involved in the turnover of plasmenylethanolamine lipids in stimulated human platelets.

Diacylglycerol lipase pathway is a minor source of released arachidonic acid in thrombin-stimulated human platelets.

It has been postulated that the diacylglycerol lipase pathway is a predominant source of the free arachidonic acid which is released from phospholipids upon the exposure of human platelets to thrombin. The amount of released arachidonic acid and other fatty acids in thrombin-stimulated platelets was determined in the presence of BW755C, the cyclooxygenase/lipoxygenase inhibitor, and in relation to phosphatidylinositol degradation and phosphatidic acid formation. A stearic acid:arachidonic acid molar ratio approaching unity would be expected in the free fatty acid fraction if the latter pathway were a major source of released arachidonic acid. Our results indicate that the diacylglycerol lipase pathway contributes a maximum of 3-4 nmol of arachidonic acid/2 X 10(9) platelets or 12-15% of the total arachidonic acid released (25.8 nmol/2 X 10(9) platelets) upon exposure to thrombin (2 units/ml) for 4 min. Trifluoperazine inhibited most of the thrombin-dependent free arachidonic acid release but only 15% of the absolute loss of arachidonic acid from phosphatidylinositol. Therefore, we conclude that the diacylglycerol lipase pathway represents only a minor source of the free arachidonic acid that is released upon thrombin stimulation of human platelets.

The incorporation of [3H]glycerol and 1-[14C]acyl-sn-glycero-3-phosphocholine into different molecular species of phosphatidylcholine in human platelets.

The molecular species of phosphatidylcholine which are formed by de novo synthesis and by the acylation of 1-acyl-sn-glycero-3-phosphocholine were characterized and compared in human platelets. For this purpose, intact human platelets were incubated in the presence of [3H]glycerol or 1-[14C]palmitoyl-sn-glycero-3-phosphocholine and the newly formed radioactive phosphatidylcholine was isolated by thin-layer chromatography. The labelled phosphatidylcholines were converted to their 1,2-diacylglycerol acetate derivatives and fractionated into their various chemical classes (saturates, monoenes, dienes, trienes, tetraenes, and greater than tetraenes) by argentation thin-layer chromatography. Regardless of the precursor used, the radioactivity distributions among the various classes did not correspond closely to that of the mass. The highest percentage of the radioactivity incorporated from [3H]glycerol was found in the saturates (25% of total), followed closely by the tetraenes (21%) and monoenes (18%), with lesser amounts in the dienes (15%), pentaenes plus hexaenes (14%), and trienes (7%). These results indicate that the de novo pathway is capable of substantial synthesis of tetraenoic (1-acyl 2-arachidonoyl) phosphatidylcholine in human platelets in contrast to previous observations in rat liver. In close agreement with work in rat liver, 59% of the radioactivity in the [14C]phosphatidylcholine derived from 1-[14C]palmitoyl-sn-glycero-3-phosphocholine was found in the tetraenoic species. The present results, together with the existence of phospholipase A2 and acyltransferase activity in platelets, support the potential importance of a deacylation-acylation cycle enrichment of human platelet phosphatidylcholine in arachidonoyl species.

Relative degradation of different molecular species of phosphatidylcholine in thrombin-stimulated human platelets.

The relative degradation of the various molecular species of [3H]phosphatidylcholine in response to thrombin was studied in human platelets following prelabeling with [3H]glycerol and compared to results obtained following labeling with [14C]oleic, [14C]linoleic, or [14C]arachidonic acids. This was of interest since previous work using radioactive fatty acids had led to the conclusion that the 1-acyl-2-arachidonoyl species of phosphatidylcholine is exclusively hydrolyzed in thrombin-stimulated platelets. Within 90 s, the thrombin-dependent release of [14C]arachidonic acid from phosphatidylcholine amounted to 25% but only 3 and 6% for oleic and linoleic acids, respectively, in general agreement with previous work. However, for [3H]glycerol-labeled phosphatidylcholine, all molecular species (saturates, monoenes, dienes, trienes, tetraenes, and greater than tetraenes) were subject to significant hydrolysis in the presence of thrombin within 90 s, ranging from 12-24% across the various classes. Furthermore, the degradation of the tetraenoic species (1-acyl-2-arachidonoyl) of [3H]phosphatidylcholine was found to be only 1.5 and 1.4 times that for the monoenoic (predominantly 1-acyl-2-oleoyl) and dienoic (predominantly 1-acyl-2-linoleoyl) species, respectively. A much heavier proportional labeling of plasma membrane relative to whole platelet phosphatidylcholine was observed with [3H]glycerol as compared to [14C] oleate or [14C]arachidonate. These results indicate that the 1-acyl-2-arachidonoyl species of phosphatidylcholine are not exclusively degraded by phospholipase A2 activity in thrombin-stimulated platelets and suggest that the differential compartmentation of molecular species of phosphatidylcholine according to their metabolic origins can influence their apparent susceptibility to hydrolysis.

Degradation of different molecular species of phosphatidylinositol in thrombin-stimulated human platelets.

The relative degradation of the various molecular species of phosphatidylinositol in response to thrombin was studied in human platelets. For this purpose, platelets were prelabeled with [2-3H]glycerol and the loss of radioactivity from the saturated, monoenoic, dienoic, trienoic, tetraenoic, and greater than tetraenoic [3H]phosphatidylinositols was determined after conversion to their 1,2-diacylglycerol acetate derivatives and fractionation by argentation thin layer chromatography. Within 90 s, when the thrombin-dependent degradation of total [3H] phosphatidylinositol amounted to 49.5%, the percentage loss of radioactivity from the tetraenoic (1-stearoyl 2-arachidonoyl) species and greater than tetraenes was significantly greater than that for the other molecular classes and approximately twice that for the monoenes. Furthermore, the extent of degradation tended to decrease with decreasing unsaturation of the phosphatidylinositols in intact platelets. These results indicate that the thrombin-dependent hydrolysis of phosphatidylinositol in intact human platelets exhibits a preferential degradation of 1-acyl (predominantly stearoyl plus oleoyl) 2-arachidonoyl species relative to other molecular species which may possibly be of importance in platelet aggregation.