Angela Wyche

Platelet and blood vessel arachidonate metabolism and interactions.

Exogenous arachidonate addition to intact platelets, in the absence or the presence of blood vessel microsomes, results in the production of thromboxane B(2) (the stable degradation product of thromboxane A(2)) only. Prostaglandin (PG) endoperoxides are released from intact platelets only when thromboxane synthetase is inhibited. Thus, addition of exogenous arachidonate to imidazole-pretreated platelets in the presence of bovine aorta microsomes (source of prostacyclin synthetase) results predominantly in the synthesis of 6-keto-PGF(1alpha) (the stable degradation product of prostacyclin). Strips of intact aorta were removed from aspirin-treated rabbits, thus the isolated blood vessels were unable to convert endogenous or exogenous arachidonate to prostacyclin. Human platelets, with [(14)C]arachidonate-labeled phospholipids, adhered to the blood vessel segments and released some thromboxane B(2). The subsequent addition of thrombin facilitated the release of endogenous arachidonate and thromboxane, but no labeled 6-keto-PGF(1alpha) was detectable. There is therefore no direct chemical evidence of PG-endoperoxide release from human platelets during either aggregation or adhesion, which therefore precludes the possibility that blood vessels use platelet PG-endoperoxide for prostacyclin synthesis. Imidazole inhibited the thromboxane synthetase in the labeled platelets, and thereafter thrombin stimulation resulted in the release of platelet-derived, labeled PG-endoperoxides that were converted to labeled prostacyclin by the vascular prostacyclin synthetase. The latter result suggests a potential antithrombotic therapeutic benefit might be achieved using an effective thromboxane synthetase inhibitor.

Cardiac and Renal Prostaglandin I2: BIOSYNTHESIS AND BIOLOGICAL EFFECTS IN ISOLATED PERFUSED RABBIT TISSUES

Both the isolated perfused rabbit heart and kidney are capable of synthesizing prostaglandin (PG) I(2). The evidence that supports this finding includes: (a) radiochemical identification of the stable end-product of PGI(2), 6-keto-PGF(1alpha), in the venous effluent after arachidonic acid administration; (b) biological identification of the labile product in the venous effluents which causes relaxation of the bovine coronary artery assay tissue and inhibition of platelet aggregation; and (c) confirmation that arachidonic acid and its endoperoxide PGH(2), but not dihomo-gamma-linolenic acid and its endoperoxide PGH(1), serve as the precursor for the coronary vasodilator and the inhibitor of platelet aggregation. The rabbit heart and kidney are both capable of converting exogenous arachidonate into PGI(2) but the normal perfused rabbit kidney apparently primarily converts endogenous arachidonate (e.g., generated by stimulation with bradykinin, angiotensin, ATP, or ischemia) into PGE(2); while the heart converts endogenous arachidonate primarily into PGI(2). Indomethacin inhibition of the cyclo-oxygenase unmasks the continuous basal synthesis of PGI(2) by the heart, and of PGE(2) by the kidney. Cardiac PGI(2) administration causes a sharp transient reduction in coronary perfusion pressure, whereas the intracardiac injection of the PGH(2) causes an increase in coronary resistance without apparent cardiac conversion to PGI(2). The perfused heart rapidly degrades most of the exogenous endoperoxide probably into PGE(2), while exogenous PGI(2) traverses the heart without being metabolized. The coronary vasoconstriction produced by PGH(2) in the normal perfused rabbit heart suggests that the endoperoxide did not reach the PGI(2) synthetase, whereas the more lipid soluble precursor arachidonic acid (exogenous or endogenous) penetrated to the cyclooxygenase, which apparently is tightly coupled to the PGI(2) synthetase.

Manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: pharmacological basis for a therapeutic approach.

Addition of the one-, two- or three- series endoperoxide to human platelet-rich plasma tend to suppress aggregation, through the action of their respective non-enzymatic breakdown products PGE1, PGD2, or PGD3 all of which elevate cyclic AMP levels. On the other hand, these stable primary products do not arise in appreciable amounts from intrinsic endoperoxides generated from either endogenous or exogenous free fatty acids. 5,8,11,14,17-Eicosapentaenoic acid (EPA) suppresses arachidonic acid (5,8,11,14-eicosatetraenoic acid) conversion by cyclooxygenase (as well as lipoxygenase) to aggregatory metabolites in platelets. Exogenously added EPA was capable of inhibiting PRP aggregation induced either by exogenous or endogenous (released by ADP or collagen) arachidonate. The hypothetical combination of an EPA-rich diet and a thromboxane synthetase inhibitor might abolish production of the pro-aggregatory species, thromboxane A2, and enhance formation of the anti-aggregatory metabolite, prostacyclin. Whereas EPA is not detectably metabolized by platelets, dihomo-gamma-linolenic acid (8,11,14-eicosatrienoic acid) is primarily converted by cyclooxygenase and thromboxane synthetase into the inactive metabolite, 12-hydroxyheptadecadienoic (HHD) acid. Pretreatment of human platelet suspensions with the thromboxane synthetase inhibitor imidazole unmasks the aggregatory property of PGH1 and DLL which was partially compromised by the PGE1 formed. The combination of the thromboxane synthetase inhibitor and an adenylate cyclase inhibitor unmasks a complete irreversible aggregation by DLL or PGH1. The basis of a dietary strategy that replaces AA with DLL must rely on the production by the platelet of an inactive metabolite (HHD) rather than thromboxane A2.

Hormonal stimulation of arachidonic release from isolated perfused organs relationship to prostaglandin biosynthesis

The lipids of isolated Krebs perfused rabbit kidneys and hearts were labelled with [14C]arachidonic acid. Subsequent hormonal stimulation (e.g. bradykinin, ATP) of the pre-labelled tissue resulted in dose-dependent release of [14C]prostaglandins; little or no release of the precursor [14C]arachidonic acid was observed. When fatty acid-free bovine serum albumin was added to the perfusion medium as a trap for fatty acids substantial release of [14C]arachidonic acid was detected following hormonal stimulation. The release of [14C]arachidonic acid was dose-dependent and greater than 3 fold that of [14C]prostaglandin release. Indomethacin by inhibiting the cyclo-oxygenase, completely inhibited release of [14C]prostaglandins and only slightly inhibited release of [14C]arachidonic acid. These results demonstrate that in both rabbit kidney and heart much more substrate is released by hormonal stimulation than is converted to prostaglandins. This suggests that either the deacylation reaction is not tightly coupled to the prostaglandin synthetase system or that there are two deacylation mechanisms, one which is coupled to prostaglandin synthesis while the other is non-specific. It has previously been shown that prostaglandin release due to hormones such as bradykinin is transient despite continued presence of the hormone (tachyphylaxis). By utilizing albumin to trap released fatty acid, it was found that hormone-stimulated release of arachidonic acid is also transient. This directly demonstrates that tachyphylaxis occurs at a step prior to the cyclo-oxygenase.

Inactivation of vascular prostacyclin synthetase by platelet lipoxygenase products

Summary The sensitivity of prostacyclin synthetase to inactivation by hydroperoxy-fatty acids suggests that vascular prostacyclin synthesis might be modulated by the platelet lipoxygenase product 12-hydroperoxyeicosatetraenoic acid (12-HPETE). On incubation of a lipoxygenase source from lysed platelets, a prostacyclin synthetase source from bovine aortic microsomes, and arachidonic acid, rapid inactivation of prostacyclin synthetase resulted. This was reflected by failure to convert exogenous prostaglandin H2 to 6-keto prostaglandin F1α, and the inactivation was prevented by the lipoxygenase inhibitor eicosatetraynoic acid. Prostacyclin synthetase inactivation did not occur when whole platelets were used as the lipoxygenase source unless concentrations of arachidonic acid high enough to induce platelet lysis were employed. Vascular prostacyclin synthesis is thus not likely to be influenced by 12-HPETE released from platelets.

Fatty acids as sources of potential “magic bullets” for the modification of platelet and vascular function

Platelet cyclooxygenase exhibits a pronounced structural specificity whereas the lipoxygenase does not. Agonist recognition by platelets appears to be highly discriminatory. Endoperoxides apparently act on different receptors than do the thromboxanes and furthermore, thromboxane receptor recognition and/or activation must differ in blood vessels and platelets. The α-chain of the fatty acid metabolites profoundly influences receptor recognition without apparent influence of substrate affinity by the synthetic enzymes. The availability of inactive products or of partial agonists for the vascular and platelet receptors could lead to the development of selective receptor agonists and antagonists. Obviously there would be a considerable advantage in developing platelet-specific receptor analogs which do not influence smooth muscle receptors. Fatty acids which possess a Δ5 unsaturation are readily converted by a calcium-dependent, cell-free enzyme system into mono- and dihydroxy fatty acids. However, in the presence of glutathione, this enzyme system completely assembles the biologically active slow-reacting substance of anaphylaxis (now termed leukotrienes C and D). Thus, arachidonic acid (5, 8, 11, 14), eicosapentaenoic acid (5, 8, 11, 14, 17), and 20:3 (5, 8, 11; which accumulates during essential fatty acid deficiency) all are excellent substrates for the synthesis of potent biologically active leukotrienes. Eicosapentaenoic acid (EPA, 5, 8, 11, 14, 17-) can serve as a prototype for the utilization of a fatty acid as a dietary strategy for the manipulation of certain disease processes. EPA as well as other members of the ω3 fatty acid family are effective antagonists of arachidonic acid metabolism (both exogenous or endogenous) by platelet cyclooxygenase. A substitution of EPA or possibly its precursor (9, 12, 15-octadecatrienoic acid, α-linolenic acid) in the diet would be expected to lead to both an inhibition of arachidonic acid metabolism and the lowering of endogenous arachidonate in tissue (e.g., platelets) lipids. The net anticipated result would be a marked reduction in the generation of PGH2 and thromboxane A2 which cause platelet aggregation. A preliminary clinical trial supporting this hypothesis has recently appeared. However, the discovery of the ease of conversion of EPA into the bronchoconstrictor leukotrienes demands appropriate caution and additional experimentation prior to widespread dietary supplementation.

A unique cardiac cytosolic acyltransferase with preferential selectivity for fatty acids that form cyclooxygenase/lipoxygenase metabolites and reverse essential fatty acid deficiency

The rabbit heart contains a cytosolic enzyme which selectively incorporates polyunsaturated fatty acids into phosphatidylcholine. This unique acyltransferase is selective for fatty acids, thus far tested, that are substrates for cyclooxygenase or lipoxygenase (i.e., arachidonic, eicosapentaenoic, linoleic and dihomo-gamma-linoleic acids) or which reverse the symptoms of essential fatty acid deficiency (columbinic acid). On the other hand, palmitic, oleic, 5,8,11-eicosatrienoic (n-9, Mead acid), and docosatetraenoic acid (n-6, adrenic acid) were not incorporated in phospholipids by the cytosolic acyltransferase. No such fatty acid selectivity was exhibited by the cytosolic acyl-CoA synthetase or by the acyltransferase activities present in cardiac microsomes and mitochondria.

The cell biology of fibroblast cyclooxygenase

We have prepared polyclonal antisera against sheep seminal vesicles cyclooxygenase (COX) which cross-reacted with human COX. We employed this antisera in studies with human dermal fibroblast cultures to immunoprecipitate selectively the COX enzyme. Labeling of the cells with [35S]-methionine, solubilization of cellular COX followed by its immunoprecipitation, SDS-PAGE electrophoresis and fluorography enabled us to determine directly the synthetic rate of COX protein and its modulation by the monokine interleukin-1 (IL-1). The immunoprecipitated [35S]-labeled COX, as judged from SDS-PAGE electrophoresis, has a molecular size of approximately 73,000 daltons, similar to that of native sheep COX and [3H]-acetyl COX. IL-1 stimulation of enhanced COX synthesis was time and dose dependent; as little as 0.03 units/ml of IL-1 produced significant stimulation of [35S]-labeled COX synthesis. Maximum stimulation was 3-10-fold after preincubation of the cells with IL-1 for 12-16 hours. IL-1 treatment of cells in serum-free media yielded parallel dose response curves for stimulation of PGE2 formation, cellular solubilized COX activity and synthesis of newly formed COX, suggesting that this IL-1 effect is mediated solely via induction of new COX protein synthesis. In contrast, IL-1 effect on cells incubated in the presence of fetal calf serum is more complex. Serum synergistically augments the IL-1 effect on PGE2 synthesis in intact cells but concurrently blunts IL-1 induction of COX synthesis, thus suggesting that a factor (or factors) in serum may stimulate PGE2 production by activating cellular phospholipase(s).

BIOLOGICAL AND CHEMICAL CHARACTERIZATION OF A UNIQUE ENDOGENOUS VASODILATOR PROSTAGLANDIN PRODUCED IN ISOLATED CORONARY ARTERY AND IN INTACT PERFUSED HEART

Arachidonic acid (AA) decreases coronary arterial resistance in the isolated perfused rabbit heart and relaxes bovine and human coronary artery strips. PGE 2 or PGF 2α contracts these coronary strips and does not affect coronary arterial resistance in the isolated rabbit heart. This suggested that a different arachidonate metabolite may be responsible for these responses. The endogenous phospholipids of the isolated rabbit heart and the coronary artery strips were labelled with [ 14 C ] AA. The primary metabolite of AA released by the heart and the coronary strips was a novel prostaglandin, and some PGE 2 ; no free PGH 2 could be demonstrated. The novel PG was resistant to base hydrolysis and sodium borohydoride reduction. The mobility of this metabolite in multiple chromatography systems was similar to that of 6-keto-PGF 1α . This metabolite demonstrated essentially no biologic activity. The precursor of 6-keto-PGF 1α , PGH 2 , relaxes bovine coronary artery strips, but the endoperoxide itself is rapidly degraded. A potent coronary relaxant is produced by incubating PGH 2 with microsomes from bovine coronary arteries and is present in the cardiac venous effluent following hormone or AA stimulation. The coronary relaxant degraded to the stable end-product 6-keto-PGF 1α . The biologically active component of this novel pathway of arachidonate metabolism appears to be an intermediate (PGX) between PGH 2 and the final 6-keto-PGF 1α -like-product. This intermediate proves to be a potent coronary vasodilator and a probable endogenous determinant of local vascular tone.

The induction of thromboxane synthetase as an expression of renal pathology

Abstract Arachidonic acid metabolism by intact perfused kidneys or by microsomes prepared from normal rabbit kidneys primarily results in the formation of prostaglandin (PG)E 2 . Unilateral ureter obstruction or partial occlusion of the renal vein of rabbits leads to the release of a rabbit aorta contracting substance when such isolated perfused kidneys are stimulated with vasoactive peptides (e.g. angiotensin II and bradykinin). Homogenates of those perfused surgically modified kidneys exhibit the enzymatic conversion of either arachidonic acid or PGH 2 into thromboxane B 2 . Radioimmunoassay of the renal venous effluent from the perfused ureter or renal vein obstructed kidneys demonstrated the simultaneous presence of PGE 2 , thromboxane A 2 , and prostacyclin in extraordinary amounts following peptide stimulation. On the other hand, the contralateral control kidney released low levels of PGE 2 and only trace amounts of thromboxane and PGI 2 when stimulated. Thus, in renal pathological states, arachidonate metabolism results in the simultaneous synthesis of a number of biologically active substances and their interplay must play an intimate role in the ultimate response of this tissue.