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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.

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.

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 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)

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.

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.

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.

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)

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.

Neutral lipids, glycolipids, and phospholipids (1.3%, 0.25%, and 0.10% of seed weight) were isolated from the total lipids (chloroform-methanol) of finger millet seeds(Eleusine coracana), and four sterol-containing lipids further isolated from neutral and glycolipids by preparative column and thin layer chromatography. On seed weight, these comprised: free sterols (S) 0.091%, sterol esters (SE) 0.013%, sterol glycosides (SG) 0.025%, acyl sterol glycosides (ASG) 0.020%, and total 0.149%. The major fatty acids, totaling 85-90%, were the same in both esterified sterols, but the proportions varied: 16:0, 18:1, and 18:2 comprising 24, 49, and 17% in SE (calculated iodine value 75) and 43, 36, and 7% in ASG (calculated iodine value 46). All four sterol lipids contained 80-84% of β-sitosterol, the remainder being stigmasterol. The only sugar in SG and ASG was D-glucose. It is deduced that the major representative species are: SE, β-sitosterol oleate/palmitate; SG, β-D-glucopyranosyl-(l → 3)-β-sitosterol; and ASG, 6-0-palmitoyl-β-D-glucopyranosyl-(l → 3)-β-sitosterol.