Eicosanoid biosynthesis and action: novel opportunities for pharmacological intervention

Novel eicosanoid biosynthetic pathways and receptors are reviewed as potential targets for pharmacological intervention. In addition to the cyclooxygenase and lipoxygenase pathways of arachidonate metabolism, a cytochrome P450-dependent monooxygenase has been identified in corneal and renal epithelial cells. Elucidation of the enzymatic pathways of thromboxane (TX) disposition and development of analytical techniques for measuring urinary metabolites have allowed a reliable assessment of TXA2 biosynthesis in health and disease, and provide a rationale for the combined use of TX-synthase inhibitors and TXA2-receptor antagonists in the setting of platelet activation. Recent evidence for a transcellular metabolism of neutrophil-derived leukotriene (LT) A4 by other human blood cells might link platelet and neutrophil activation to the occurrence of vasospastic phenomena. Prostacyclin (PG1(2)), PGE2, PGD2, TXA2/PGH2, and sulfidopeptide-LT receptors are being characterized in terms of distribution, signal-transduction mechanisms, and agonist-mediated regulation. Development of relatively selective agonists and antagonists of these receptors is providing novel therapeutic strategies for several human diseases.     

Mechanism for antiplatelet effect of onion: AA release inhibition, thromboxane A(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade

Antiplatelet actions of aqueous extract of onion were investigated in rat and human platelet. IC(50)values of onion extract for collagen-, thrombin-, arachidonic acid (AA)-induced aggregations and collagen-induced thromboxane A(2)(TXA(2)) formation were 0.17 +/- 0. 01, 0.23 + 0.03, 0.34 +/- 0.02 and 0.12 +/- 0.01 g/ml, respectively. [(3)H]-AA release induced by collagen (10 microg/ml) in rat platelet was decreased by onion compared to control (22.1 +/- 2.13 and 5.2 +/- 0.82% of total [(3)H]-AA incorporated, respectively). In fura-2 loaded platelets, the elevation of intracellular Ca(2+)concentration stimulated by collagen was inhibited by onion. Onion had no cytotoxic effect in platelet. Onion significantly inhibited TXA(2)synthase activity without influence on COX activity. Platelet aggregation induced by U46619, a stable TXA(2)mimetic, was inhibited by onion, indicating its antagonism for TXA(2)/PGH(2)receptor. These results suggest that the mechanism for antiplatelet effect of onion may, at least partly, involve AA release diminution, TXA(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade.     

Modulation of platelet aggregation by areca nut and betel leaf ingredients: roles of reactive oxygen species and cyclooxygenase

There are 2 to 6 billion betel quid (BQ) chewers in the world. Areca nut (AN), a BQ component, modulates arachidonic acid (AA) metabolism, which is crucial for platelet function. AN extract (1 and 2 mg/ml) stimulated rabbit platelet aggregation, with induction of thromboxane B2 (TXB2) production. Contrastingly, Piper betle leaf (PBL) extract inhibited AA-, collagen-, and U46619-induced platelet aggregation, and TXB2 and prostaglandin-D2 (PGD2) production. PBL extract also inhibited platelet TXB2 and PGD2 production triggered by thrombin, platelet activating factor (PAF), and adenosine diphosphate (ADP), whereas little effect on platelet aggregation was noted. Moreover, PBL is a scavenger of O2(*-) and *OH, and inhibits xanthine oxidase activity and the (*)OH-induced PUC18 DNA breaks. Deferoxamine, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and neomycin prevented AN-induced platelet aggregation and TXB2 production. Indomethacin, genistein, and PBL extract inhibited only TXB2 production, but not platelet aggregation. Catalase, superoxide dismutase, and dimethylthiourea (DMT) showed little effect on AN-induced platelet aggregation, whereas catalase and DMT inhibited the AN-induced TXB2 production. These results suggest that AN-induced platelet aggregation is associated with iron-mediated reactive oxygen species production, calcium mobilization, phospholipase C activation, and TXB2 production. PBL inhibited platelet aggregation via both its antioxidative effects and effects on TXB2 and PGD2 production. Effects of AN and PBL on platelet aggregation and AA metabolism is crucial for platelet activation in the oral mucosa and cardiovascular system in BQ chewers.     

Testing the "redirection hypothesis" of prostaglandin metabolism in the kidney

Furosemide increases the synthesis of two major renal eicosanoids, prostacyclin (PGI2) and thromboxane A2 (TXA2), by stimulating the release of arachidonic acid which in turn is metabolized to PGG2/PGH2, then to PGI2 and TXA2. PGI2 may mediate, in part, the early increment in plasma renin activity (PRA) after furosemide. We hypothesized that thromboxane synthetase inhibition should direct prostaglandin endoperoxide metabolism toward PGI2, thereby enhancing the effects of furosemide on renin release. Furosemide (2.0 mg . kg-1 i.v.) was injected into Sprague-Dawley rats pretreated either with vehicle or with U-63,557A (a thromboxane synthetase inhibitor, 2 mg/kg-1 followed by 2 mg/kg-1 X hr-1). Urinary 6ketoPGF1 alpha and thromboxane B2 (TXB2), reflecting renal synthesis of PGI2 and TXA2, as well as PRA and serum TXB2, were measured. Serum TXB2 was reduced by 96% after U-63,557A. U-63,557A did not affect the basal PRA. Furosemide increased PRA in both vehicle and U63,557A treated rats. However, the PRA-increment at 10, 20 and 40 min following furosemide administration was greater in U-63,557A-treated rats than in vehicle-treated rats and urine 6ketoPGF1 alpha excretion rates were increased. These effects of thromboxane synthesis inhibition are consistent with a redirection of renal PG synthesis toward PGI2 and further suggest that such redirection can be physiologically relevant.     

Selective inhibition of arachidonic acid metabolite release from human lung tissue by antiinflammatory steroids

The effects of antiinflammatory steroids on arachidonic acid metabolite release from human lung fragments were analyzed. Incubation of lung fragments for 24 hr with 10(-6) M dexamethasone inhibited the net release of the prostacyclin metabolite 6-keto-PGF1 alpha, PGE2, and PGF2 alpha from lung fragments stimulated with anti-IgE but failed to inhibit the anti-IgE-induced release of PGD2, TXB2, and iLTC4. The IC50 of dexamethasone for inhibition of both spontaneous and anti-IgE-induced 6-keto-PGF1 alpha release was approximately 2 X 10(-8) M, and a 6-hr preincubation with the drug was required for 50% inhibition of prostaglandin release. Other agents were tested for activity in stimulating arachidonic acid metabolite release from human lung fragments. FMLP (fmet-leu-phe) stimulated the release of all metabolites tested (6-keto-PGF1 alpha, PGD2, PGE2, PGF2 alpha, TXB2, iLTC4); platelet-activating factor (PAF), but not lysoPAF, stimulated the release of PGD2, TXB2, and iLTC4. In contrast to the case with anti-IgE, where dexamethasone failed to inhibit net PGD2 and TXB2 release, the steroid inhibited the release of these metabolites stimulated by both FMLP and PAF. The steroid inhibited iLTC4 release induced by the highest concentration of PAF (10(-6)M) but did not inhibit iLTC4 release stimulated by either 10(-7) M PAF, FMLP, or anti-IgE. Because neither FMLP nor PAF caused the release of PGD2 or TXB2 from purified human lung mast cells, and because they also failed to induce histamine release from lung fragments, it is suggested that these stimuli produce PGD2 and TXB2 release in lung fragments through an action on a cell distinct from the mast cell. This suggestion is supported by the selective inhibition of the release of these arachidonic acid metabolites by dexamethasone. We suggest that the inhibitory action of steroids on arachidonic acid metabolite in human lung fragments contributes to their therapeutic efficacy in pulmonary diseases.     

Identification of a putative thromboxane A2/prostaglandin H2 receptor in human platelet membranes

The binding of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist (9,11-dimethylmethano-11, 12-methano-16-(3-aza-15 alpha beta-omega-tetranor-TXA2) ([125I]PTA-OH) to membranes prepared from human platelets was characterized. [125I]PTA-OH binding to membranes from human platelets was saturable, displaceable, and dependent on protein concentration. Scatchard analysis of equilibrium binding carried out at 30 degrees C revealed one class of binding sites with a Kd of 30 +/- 4 nM and a Bmax of 1.8 +/- 0.3 pmol/mg of protein (n = 5). Kinetic analysis of the binding of [125I]PTA-OH at 0 degrees C yielded a k1 of 1.35 X 10(6) M-1 min-1 and a k-1 of 0.032 min-1, Kd = k-1/k1 = 24 nM. The potencies of a series of TXA2/PGH2 antagonists as inhibitors of [125I]PTA-OH binding was correlated with their potencies as inhibitors of platelet aggregation induced by the TXA2/PGH2 mimetic, U46619 (1 microM) (r = 0.93, p less than 0.01). A series of TXA2/PGH2 mimetics also displaced [125I]PTA-OH from its binding site, and their potencies as inhibitors of [125I]PTA-OH binding were correlated with their potencies as stimulators of platelet aggregation (r = 0.91, p less than 0.05). The IC50 values for displacement of [125I]PTA-OH by PGF2 alpha, PGD2, and the stable PGI2 analog Iloprost were greater than 25 microM, suggesting that [125I]PTA-OH does not bind to other known platelet prostaglandin receptors. These data are consistent with the notion that this binding site may represent the platelet TXA2/PGH2 receptor.     

Metabolism of prostaglandin endoperoxide by microsomes from cat lung

It has been reported that the prostaglandin (PG) precursor, arachidonic acid, produces divergent hemodynamic responses in the feline pulmonary vascular bed. However, the pattern of arachidonic acid products formed in the lung of this species is unknown. In order to determine the type and activity of terminal enzymes in the lung, prostaglandin biosynthesis by microsomes from cat lung was studied using the prostaglandin endoperoxide, PGH2, as a substrate. The major products of incubations of PGH2 with microsomes were thromboxane (TX) B2 (the major metabolite of TXA2), 6-keto-PGF1 alpha (the breakdown product of PGI2) and 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). Formation of TXB2 was markedly reduced by imidazole. Tranylcypromine decreased the formation of TXB2 and HHT and inhibited the formation of 6-keto-PGF1 alpha. At low PGH2 concentrations, equal production of TXB2 and 6-keto-PGF1 alpha was observed. However, as PGH2 concentration increased, 6-keto-PGF1 alpha production approached early saturation while TXB2 production increased in a linear fashion. These results suggest that enzymatic formation of TXA2 and PGI2 is a function of substrate availability in the lung. These findings provide a possible explanation for the divergent hemodynamic responses to arachidonic acid infusions at high and low concentrations in the feline pulmonary vascular bed.     

Isoelectric and mass characterization of human platelet thromboxane A2/prostaglandin H2 receptors

To further characterize the human thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor, preparative isoelectric focusing (IEF) was performed on solubilized platelet membranes. TXA2/PGH2 receptors, assayed by specific binding of the TXA2/PGH2 antagonist [125I]PTA-OH, were electrofocused at pH 5.6. Scatchard analysis of IEF fraction pH 5.6 revealed a 180-fold concentration of TXA2/PGH2 receptors (Bmax = 3650 +/- 228 pM/mg focused, 19 +/- 4 pM/mg unfocused) with no change in binding affinity (Kd = 47 +/- 7 nM focused, 36 +/- 14 nM unfocused). SDS-polyacrylamide gel electrophoresis of photoaffinity-labelled electrofocused receptors revealed concentration of specifically labelled proteins having molecular masses of 49,000 and 27,000 Daltons. These results suggest that the human platelet TXA2/PGH2 receptor has a pI of 5.6, molecular mass of 49,000 Daltons, and may exist as a dimer. Preparative IEF should prove useful in the eventual purification of this receptor.     

A common binding site for primary prostanoids in vascular smooth muscles: a definitive discrimination of the binding for thromboxane A2/prostaglandin H2 receptor agonist from its antagonist

Differences in binding characteristics between agonists and antagonists for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were examined in rat cultured vascular smooth muscle cells (VSMC). Scatchard analysis indicated the existence of two binding sites for the TXA2/PGH2 agonist, whereas a single class of recognition sites for the receptor antagonists were observed with approximately the same maximum binding capacity (Bmax) as a high-affinity binding site of the agonist. Weak binding inhibition by approx. 100 nM of primary prostanoids (PGE1, PGF2 alpha and PGD2) was detected only with the TXA2/PGH2 agonist, and not with the antagonist. Primary prostanoids as well as TXA2/PGH2 agonists (U46619 and STA2) suppressed the [3H]PGF2 alpha and [3H]PGE1 binding with almost the same potency, whereas TXA2/PGH2 antagonists (S-145, SQ29,548 and ONO3708) did not. The Bmax value of the binding sites was roughly identical in PGF2 alpha, PGE1 and a low-affinity binding site of U46619. These results suggest the existence of two binding sites for TXA2/PGH2 in VSMC, i.e., a high-affinity binding site corresponding to that of the TXA2/PGH2 antagonists and a low-affinity binding site in common with primary prostanoids.     

The biological role of thromboxane A2 in the process of hemostasis and thrombosis; pharmacology and perspectives of therapeutical use of thromboxane synthetase inhibitors and receptor PGH2/TXA2 antagonists

The biological role of thromboxane A2 in the process of hemostasis and thrombosis; pharmacology and perspectives of the therapeutical use of thromboxane synthetase inhibitors and receptor PGH2/TXA2 antagonists. Acta physiol. pol., 1985, 36 (3): 153-164. The biology of thromboxane A2 and pharmacology of drugs that selectively inhibit generation and action of this eicosanoid are reviewed. Author's opinion on therapeutical perspectives for thromboxane synthetase inhibitors and receptor PGH2/TXA2 antagonists is also presented.     

The affinities of prostaglandin H2 and thromboxane A2 for their receptor are similar in washed human platelets

Both thromboxane A2 (TXA2) and its precursor prostaglandin H2 (PGH2) are labile and share a common receptor. The affinities of these two compounds for their putative common receptor are unknown. We compared the potencies of TXA2 and PGH2 to aggregate human platelets and bind to the TXA2/PGH2 receptor. TXA2 was more potent than PGH2 in initiating aggregation in platelet-rich plasma, EC50 of 66 +/- 15 nM and 2.5 +/- 1.3 microM, respectively. In washed platelets, however, PGH2 was more potent than TXA2 with EC50 values of 45 +/- 2 nM and 163 +/- 21 nM, respectively. The affinity of these two compounds in washed platelets was determined in radioligand competition binding assays employing [125I]-PTA-OH. The Kd values for PGH2 and TXA2 were 43 nM and 125 nM, respectively. The results demonstrate that the affinity of PGH2 for the platelet TXA2/PGH2 receptor is greater than previously thought. The data raise the possibility that PGH2 may significantly contribute to the responses attributed to TXA2 in vivo.     

Inhibition of GSH-dependent PGH2 isomerase in mammary adenocarcinoma cells by allicin

We have reported that allicin, a constituent of garlic oil, has no effect on the activities of platelet cyclooxygenase or thromboxane synthase, or vascular PGI2 synthase. The effect of allicin on glutathione (GSH) dependent PGH2 to PGE2 isomerase is unknown. We therefore studied the effect of allicin on PGE2 biosynthesis in a murine mammary adenocarcinoma cell line (No 4526). Intact or sonicated cells were incubated with either 14C-arachidonic acid (AA) or 14C-PGH2, respectively. Following metabolism, products were extracted, separated by TLC and analyzed by radiochromatographic scan. PGE2 was predominantly formed with minimal amounts of PGF2 alpha and PGD2. Formation of 6-keto-PGF1 alpha or TXB2 was not detected indicating the absence of TXA2 and PGI2 synthase activity. Indomethacin and ibuprofen inhibited the PGE2 formation (p less than 0.05). The enzymatic PGE2 formation in sonicates was blocked by depletion of the cellular non-protein thiols by buthionine sulfoximine and was shown to be dependent on GSH. Allicin, over the range of 10-1000 microM, inhibited the formation of PGE2 in cells exposed to 2.0 microM 14C-AA for 20 min. and in sonicated cells incubated with 20.0 microM 14C-PGH2 for 2 min (p less than 0.05). Allicin did not alter cyclooxygenase-mediated oxygen utilization in ram seminal vessicle microsomes, suggesting that allicin selectively inhibits the GSH-dependent PGH2 to PGE2 isomerase in this adenocarcinoma cell line.     

Inhibition of oxidative-stress-induced platelet aggregation by androgen at physiological levels via its receptor is associated with the reduction of thromboxane A2 release from platelets

BACKGROUND: Platelets play a crucial role in the development of arterial thrombosis and other pathophysiologies leading to clinical ischemic events. Defective regulation of platelet activation/aggregation is a predominant cause for arterial thrombosis. The purposes of our study are to assess the effect of androgen at physiological concentration via its receptor on oxidative-stress-induced platelet aggregation and to further elucidate the possible mechanism. METHODS AND RESULTS: Plasma dihydrotestosterone (DHT) was determined by ELISA using a commercially available kit. Platelet aggregometer was used to measure platelet aggregation. The contents of thromboxane B(2) (TXB(2)) were assayed with radio-immunoassay. Our results showed that addition of DHT (2 nM) significantly inhibited platelet aggregation induced by hydrogen peroxide (H(2)O(2)) (10 mM, 25 mM) in PRP diluted with Tyrode's buffer. Moreover, H(2)O(2)-induced platelet aggregation decreased in sham-operated rats. However, H(2)O(2)-induced platelet aggregation significantly increased in castrated rats. Replacement of DHT inhibited H(2)O(2)-induced platelet aggregation in castrated rats. After PRP was pretreated with flutamide, H(2)O(2)-induced platelet aggregation increased in castrated rats again. Presence of DHT (2 nM) obviously inhibited H(2)O(2)-induced thromboxane A(2) (TXA(2)) release in castrated rats. Pretreatment of DHT and flutamide increased H(2)O(2)-stimulated TXA(2) release from platelet in castrated rats again. Castration caused a significant reduction in plasma testosterone and DHT levels, whereas DHT replaced at a dose of 0.25 mg/rat restored the circulating DHT to physiological levels, without being altered by treatment with flutamide. The plasma TXB(2) increased in castrated rats as compared with that in sham-operated rats. Replacement with DHT reduced plasma TXB(2) contents in castrated rats. However, flutamide supplementation increased plasma contents of TXB(2) in castrated rats again. CONCLUSION: Androgen at physiological doses via its receptor inhibits oxidative-stress-induced platelet aggregation, which is associated with the reduction of TXA(2) release from platelets.     

Islet amyloid polypeptide inhibits glucagon release and exerts a dual action on insulin release from isolated islets

We have studied the influence of a wide concentration range of islet amyloid polypeptide (IAPP) on both glucagon and insulin release stimulated by various types of secretagogues. In an islet incubation medium devoid of glucose, the rate of glucagon release being high, we observed a marked suppressive action by low concentrations of IAPP, 10(-10) and 10(-8) M, on glucagon release. Similarly, glucagon release stimulated by L-arginine, the cholinergic agonist carbachol, or the phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX), an activator of the cyclic AMP system, was inhibited by IAPP in the 10(-10) and 10(-8) M concentration range. Moreover, basal glucagon release at 7 and 10 mM glucose was suppressed by IAPP. In contrast, IAPP exerted a dual action on insulin release. Hence, low concentrations of IAPP brought about a modest increase of basal insulin secretion at 7 mM glucose and also of insulin release stimulated by carbachol. High concentrations of IAPP, however, inhibited insulin release stimulated by glucose (10 and 16.7 mM), IBMX, carbachol and L-arginine. In conclusion, our data suggest that IAPP has complex effects on islet hormone secretion serving as an inhibitor of glucagon release and having a dual action on insulin secretion exerting mainly a negative feedback on stimulated and a positive feedback on basal insulin release.     

Thromboxane A2 in blood vessel walls and its physiological significance: Relevance to thrombosis and hypertension

It has been thought that blood vessels apart from the umbilical artery produce little or no thromboxane (TX) A2. However selective inhibitors of TXA2 biosynthesis have substantial effects on vessel physiology, suggesting that small amounts of TXA2 may be important in regulating function. This indirect evidence is now supported by direct measurements of TXB2 (the produce of TXA2 conversion) using both gas chromatography-mass spectrometry (GCMS) and radioimmunoassays. At least four independent laboratories have now demonstrated TXB2 production by various blood vessels. These studies suggest that vessel wall TXA2 is present in amounts more than adequate to exert biological actions on both vascular reactivity and on platelets. This may require re-evaluation and revision of present concepts of hypertension and thrombosis.     

Platelet activating factor stimulates cyclo-oxygenase activity in guinea pig eosinophils. Concerted biosynthesis of thromboxane A2 and E-series prostaglandins

The effect of platelet activating factor (PAF) on the generation of cyclo-oxygenase-derived arachidonic acid metabolites was examined on purified eosinophils harvested from the peritoneal cavity of male guinea pigs. PAF produced a concentration-dependent increase in the amount of immunoreactive thromboxane B2 (TXB2) and PGE1/E2 released from these inflammatory cells at a relative molar ratio of 30:1. The EC50 of PAF was 20 to 40 nM and maximum stimulation (4.5-fold) of both prostanoids occurred at 1 microM PAF. The ability of PAF to generate TXA2 was rapid (t 1/2 = 9 s), transient (40 s), noncytotoxic, and noncompetitively antagonized by the PAF-receptor blocking drug, WEB 2086. On an equimolar (100 nM) basis, PAF was significantly more effective than C5a, fMLP, and PMA at stimulating TXB2 release but markedly less potent than the calcium ionophore, calcimycin. Pretreatment of eosinophils with the cyclo-oxygenase inhibitor flurbiprofen (8 microM for 5 min) abolished the ability of PAF to promote both TXB2 and PGE1/E2 release. Likewise, dazmegrel (50 microM for 5 min), a selective inhibitor of thromboxane synthetase, abolished PAF-stimulated TXB2 release but markedly augmented the elaboration of PGE1/E2. Inhibition of cyclo-oxygenase with flurbiprofen affected neither the ability of PAF to elevate the intracellular calcium ion concentration (measured by fura-2 fluorescence) nor its appetency to generate superoxide anions at any PAF concentration examined. It is concluded that activation of guinea pig eosinophils by PAF is receptor-mediated and independent of the concomitant generation of cyclo-oxygenase-derived excitatory prostanoids. Inasmuch as TXA2 may contribute to the pathogenesis of bronchial hyperreactivity, then these data implicate the eosinophil as a potential source of this lipid mediator.     

On the mechanism of prostacyclin and thromboxane A2 biosynthesis

The present research describes studies which address the mechanism of prostacyclin (PGI2) and thromboxane A2 (TXA2) biosynthesis. In addition to prostaglandin H1 (PGH1), PGG2, PGH2, and PGH3, also 8-iso-PGH2, 13(S)-hydroxy-PGH2, and 15-keto-PGH2 were applied to determine the substrate specificities and kinetics of prostacyclin and thromboxane synthase in more detail. Human platelet thromboxane synthase converted PGH1, 8-iso-PGH2, 13(S)-hydroxy-PGH2 and 15-keto-PGH2 into the corresponding heptadecanoic acid (C17) plus malondialdehyde, whereas the thromboxane derivative was formed only from PGG2, PGH2, and PGH3 together with the corresponding C17 metabolite and malondialdehyde in a 1:1:1 ratio. In contrast, PGG2, PGH2, 13(S)-hydroxy-PGH2, 15-keto-PGH2 and PGH3 were almost completely isomerized to the corresponding prostacyclin derivative by bovine aortic prostacyclin synthase, whereas PGH1 and 8-iso-PGH2 only produced the corresponding C17 hydroxy acid plus malondialdehyde. Isotope-labeling experiments with [5,6,8,9,11,12,14,15-2H]PGH2 revealed complete retention of label and no isotope effect in the course of thromboxane biosynthesis, but the loss of one 2H atom at C-6 with an isotope effect of 1.20 during PGI2 formation. Prostacyclin and thromboxane synthase bind both 9,11-epoxymethano-PGF2 alpha and 11,9-epoxymethano-PGF2 alpha at the heme iron, but according to their difference spectra in opposite ways with respect to the 9- and 11-position. In agreement with published model studies, a cage radical mechanism is proposed for both enzymes according to which the initial radical process is terminated through oxidation of carbon-centered radicals by the iron-sulfur catalytic site, followed by ionic rearrangement to PGI2 or TXA2. Various Fe(III) model compounds as well as liver microsomes or cytochrome P-450CAM can also form small amounts of PGI2 and TXA2, but mainly yield 12(S)-hydroxy-5,8,10-heptadecatrienoic acid plus malondialdehyde probably by a radical fragmentation pathway.     

A photoaffinity label for the thromboxane A2/prostaglandin H2 receptor in human blood platelets

A photoactive iodoarylazide derivative (I-APA-PhN3) of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13-azaprostanoic acid is evaluated. Upon photoactivation, the compound was found to inhibit specifically and irreversibly human platelet aggregation induced by the TXA2/PGH2 mimetic U46619. In receptor-binding studies using [3H]U46619, I-APA-PhN3 exhibited an IC50 of 300 nM for inhibition of U46619 binding. Photoactivation of I-APA-PhN3 resulted in an irreversible 58% reduction in specific binding of U46619. This compound and its corresponding ratio-iodinated form will prove to be useful tools for the isolation and purification of the TXA2/PGH2-binding protein in human platelets.     

Binding of an [125I] labelled thromboxane A2/prostaglandin H2 receptor agonist to baboon platelets

To characterize the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor on baboon platelets the binding of [125I]BOP was studied. [125I]BOP bound to washed baboon platelets in a saturable manner. Scatchard analysis of binding isotherms revealed a Kd of 1.12 +/- 0.08 nM and a binding capacity of 54 +/- 5 fmoles/10(8) platelets (326 sites/platelet). Several TXA2/PGH2 agonists and antagonists displaced [125I]BOP from its baboon platelet binding site with a rank order of potency similar to human platelets: I-BOP greater than SQ29548 greater than U46619 = I-PTA-OH greater than PTA-OH. I-BOP aggregated washed baboon platelets with an EC50 of 10 +/- 4 nM. The results indicate that [125I]BOP binds to the TXA2/PGH2 receptor on baboon platelets and that this receptor is similar to its human counterpart.     

Metabolism of the endocannabinoids, 2-arachidonylglycerol and anandamide,into prostaglandin,thromboxane, and prostacyclin glycerol esters and ethanolamides

Cyclooxygenase-2 (COX-2) action on the endocannabinoids, 2-arachidonylglycerol (2-AG) and anandamide (AEA), generates prostaglandin glycerol esters (PG-G) and ethanolamides (PG-EA), respectively. The diversity of PG-Gs and PG-EAs that can be formed enzymatically following COX-2 oxygenation of endocannabinoids was examined in cellular and subcellular systems. In cellular systems, glycerol esters and ethanolamides of PGE(2), PGD(2), and PGF(2alpha) were major products of the endocannabinoid-derived COX-2 products, PGH(2)-G and PGH(2)-EA. The sequential action of purified COX-2 and thromboxane synthase on AEA and 2-AG provided thromboxane A(2) ethanolamide and glycerol ester, respectively. Similarly, bovine prostacyclin synthase catalyzed the isomerization of the intermediate endoperoxides, PGH(2)-G and PGH(2)-EA, to the corresponding prostacyclin derivatives. Quantification of the efficiency of prostaglandin and thromboxane synthase-directed endoperoxide isomerization demonstrated that PGE, PGD, and PGI synthases catalyze the isomerization of PGH(2)-G at rates approaching those observed with PGH(2). In contrast, thromboxane synthase was far more efficient at catalyzing PGH(2) isomerization than at catalyzing the isomerization of PGH(2)-G. These results define the in vitro diversity of endocannabinoid-derived prostanoids and will permit focused investigations into their production and potential biological actions in vivo.