The identification of an epoxy-hydroxy acid as a product from the incubation of arachidonic acid with washed blood platelets

An epoxy-hydroxy compound, 10-hydroxy-11,12-epoxy-eicosa-5,8,14-trienoic acid, has been identified as a product on incubation of arachidonic acid with washed blood platelets from human, horse, cat, dog and rabbit. Gas chromatographic - mass spectrometric (GC-MS) evidence of structure is discussed.     

The identification of trihydroxyeicosatrienoic acids as products from the incubation of arachidonic acid with washed blood platelets

Arachidonic acid is converted by washed platelets from man, horse and dog into a mixture of 8, 9, 12-trihydroxyeicosa-5, 10, 14-trienoic acid and 8, 11, 12-trihydroxyeicosa-5, 9, 14-trienoic acid (termed 8, 9, 12-THETA and 8, 11, 12-THETA respectively and THETA collectively). Gas chromatographic — mass spectrometric evidence of structure is discussed.     

Complete separation by high performance liquid chromatography of metabolites of arachidonic acid from incubation with human and rabbit platelets

Separations of all major cyclooxygenase and lipoxygenase metabolites of arachidonic acid were obtained by high performance liquid chromatography (HPLC). A C₁₈ reverse-phase column was used in ion suppression mode to separate underivatized metabolites of arachidonic acid isolated from human and rabbit platelets. The metabolites were monitored by measuring radioactivity or ultraviolet light absorption at 192 nm (absorption by double bonds). Comparisons of TLC and HPLC separations demonstrated that the HPLC separation of metabolites of [1-¹⁴C]arachidonic acid was quantitative. HPLC also resolved several minor metabolites that were not detected by scanning of TLC separations.     

Optimization of an assay for studying the effects of agents on cyclooxygenase and lipoxygenase metabolism of arachidonic acid in washed human platelets

Before one can examine the effects of substances on the metabolism of arachidonic acid (AA) by the cyclooxygenase and lipoxygenase pathways, an assay system which allows one to detect increases or decreases in both pathways in needed. In order to develop such a system, we have examined nonaggregating washed human platelets (10(8) platelets/0.5 ml) incubated for various times with 2 microCi 3H-AA and increasing concentrations of AA. T/B2, HHT, 12-HETE, and AA were extracted and separated using reverse phase-HPLC. We first calculated the mass of AA products formed with 10(-7) to 10(-4) M AA and found that the cyclooxygenase was saturated with 10(-5) M AA whereas the lipoxygenase was not saturated with 10(-4) M AA. Cyclooxygenase products were more prevalent than 12-HETE below 10(-5) M AA, while lipoxygenase products predominated at 3 x 10(-5)-10(-4) M AA. Using 3 microM AA, which does not saturate the cyclooxygenase, we examined the effect of 0.25-10 minute incubation durations on the distribution of AA metabolites and found AA product formation to increase throughout this period without completely depleting the substrate. Since substrate depletion does not occur and further metabolism could be detected for both pathways with a 5 minute incubation with 3 microM AA, these incubation parameters were chosen in order to further test the assay system. Using these parameters, we found that 10(-4) M 5-hydroxytryptamine enhanced platelet 12-HETE formation and decreased T/B2 and HHT formation, thus demonstrating the capacity of this system to simultaneously detect changes in cyclooxygenase and lipoxygenase enzyme metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of platelet activating factor by rabbit platelets. Lyso-platelet activating factor as a key intermediate with phosphatidylcholine as the source of arachidonic acid in its conversion to a tetraenoic acylated product

[3H]PAF (platelet activating factor or 1-alkyl-2-acetyl-GPC) is converted to 1-alkyl-2-lyso-GPC and 1-alkyl-2-acyl-GPC by rabbit platelets (GPC is sn-glycero-3-phosphocholine). The deacetylation reaction does not involve the transfer of the acetate of PAF to any other lipid class and added exogenous lyso-PAF readily mixes with the cellular pool of the [3H]lyso-PAF intermediate formed from [3H]PAF. [3H]1-Alkyl-2-acyl-GPC produced during the inactivation of [3H]PAF contained primarily the tetraenoic acyl species (approximately 80% of the 3H in this fraction). The source of the arachidonic acid used for the reacylation of the lyso-PAF intermediate is the diacyl species, phosphatidylcholine.     

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.     

Argentation thin layer chromatography of arachidonic acid metabolites isolated from human platelets

No thin layer chromatographic system in the literature offers adequate resolution of arachidonic acid and its major metabolites from platelets. We have found that argentation thin layer chromatography gives baseline separation of these compounds. Use of argentation chromatography results in decreased counting efficiency with time when quantitation is done by liquid scintillation methods. Addition of sodium chloride eliminates this complication and gives recoveries comparable to or better than those obtained using nonargented silica gel alone.     

Mobilization of arachidonic acid from phosphatidylethanolamine fraction to phosphatidylcholine fraction in platelets

Rabbit platelets rapidly incorporated methyl groups of [³H] methionine to phosphatidylcholine (PC). Rabbit platelets also incorporated [³H]choline to PC, but the rate of incorporation was far lower than that of [³H]methionine. Further fractionation of labeled PC revealed that a considerable amount of arachidonyl PC was synthesized via the N-methylation pathway. Thrombin stimulation resulted in a release of arachidonic acid from PC, and not from phosphatidylethanolamine (PE). These observations suggest that the N-methylation pathway plays an important role in the intracellular mobilization of arachidonic acid from the PE fraction to the PC fraction, this fraction being more sensitive to the hydrolysis with phospholipase A₂ during platelet activation.     

Isolation of a new lipoxygenase metabolite of arachidonic acid, 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid from human platelets

Washed human platelets incubated with 1-¹⁴C -arachidonic acid (1mM) produced a new metabolite which migrated on thin layer chromatography close to thromboxane B₂, but which was identified by mass spectrometry as a trihydroxy fatty acid. The mass spectrum was consistent with the assigned structure, 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid (THETE). Platelet THETE synthesis from arachidonate was not inhibited by preincubation with aspirin or indomethacin but was blocked by 5,8,11,14-eicosatetraynoic acid. Therefore, THETE appears to arise via the platelet lipoxygenase pathway rather than via the prostaglandin cyclooxygenase. Two proposed structures, including a novel dihydro-hydroxy-pyran cyclic intermediate, which could give rise to THETE are presented.     

Aspirin inhibits Ca2t+-stimulated fatty acid release from human washed platelets

Ca²⁺ at 2mM concentration stimulates the release of saturated and unsaturated fatty acids from intact washed platelets incubated at 37°C with stirring. Aspirin at a concentration of 0.4mM inhibits both cyclo-oxygenase activity and fatty acid efflux induced by Cat+. Thus, in intact washed platelets, aspirin reduces formation of cyclo-oxygenase products by direct inhibition of the enzyme and by reducing the availability of precursor arachidonate.     

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 stimulation of arachidonic acid metabolism in human platelets by hydrodynamic stresses

Even though shear-induced platelet activation and aggregation have been studied for about 20 years, there remains some controversy concerning the arachidonic acid metabolites formed during stress activation and the role of thromboxane A2 in shear-induced platelet aggregation. In this study, platelets were labelled with [1-14C]arachidonic acid to follow the metabolism of arachidonic acid in stimulated platelets using HPLC and scintillation counting. Platelets activated by thrombin formed principally thromboxane A2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). In contrast, for platelets activated by shear--though arachidonic acid metabolism was stimulated--only 12-HETE was formed and essentially no cyclooxygenase metabolites were detected. This indicates that physical forces may initiate a different pathway for eicosanoid metabolism than most commonly used chemical stimuli and perhaps also implies that regulation of the cyclooxygenase activity may be a secondary level of regulation in eicosanoid metabolism.     

Isolation and identification of ethisolide as an antibiotic product from Penicillium capsulatum

Summary Ethisolide has been isolated from cultures of Penicillium capsulatum, and its structure determined by means of infrared, mass spectrometry, ¹H- and ¹³C nuclear magnetic resonance. Antibiotic activity against a number of microorganisms is reported. Correspondence to: J. Atienza     

Purification of guanylate cyclase from human platelets and effect of arachidonic acid peroxide.

Guanylate cyclase of human platelets was separated from cyclic nucleotide and GTP hydrolytic activities with a 104-fold purification over the homogenate. The purified guanylate cyclase preparation requires neither the GTP regenerating system nor cyclic GMP but is stimulated by about 2-fold by 2.5 mM cyclic GMP. The molecular weight of the enzyme was estimated as 180,000 and the Km value for GTP was 95 μM. Arachidonic acid peroxide stimulated the purified enzyme by increasing maximum velocity without changing Km value.