Thromboxane A2 analogue (U-46619) stimulates vascular PGI2 synthesis

European Journal of Pharmacology, 107 (1985) 259-262

259

Elsevier

Short communication THROMBOXANE

A 2 A N A L O G U E 0U-46619) S T I M U L A T E S VASCULAR PGI 2 S Y N T H E S I S

J A M I E Y. JEREMY, D I M I T R I P. M I K H A I L I D I S and PARESH D A N D O N A *

Metabolic Unit, Department of Chemical Pathology and Human Metabolism, Royal Free Hospital and School of Medicine, Pond Street, London, NW3, U.K. Received 29 October 1984, accepted 29 October 1984

J.Y. JEREMY, D.P. MIKHAILIDIS and P. DANDONA, Thromboxane A 2 analogue (U-46619) stimulates vascular PGI., synthesis, European J. Pharmacol. 107 (1985) 259-262. Since platelet release reaction products (e.g. serotonin, ADP) stimulate prostacyclin (PGI 2) release in vitro, we have investigated whether thromboxane A 2 (TXA2) also has a similar effect. An analogue, U-46619, was used for the experiments, since TXA 2 is extremely unstable. U-46619 stimulated rat aortic PGI 2 release; this stimulation was abolished by (a) EDTA and (b) verapamil. We conclude that TXA 2 is a calcium-dependent stimulator of PGI 2 release; this property may be relevant to the limitation of platelet aggregates in vivo and to vascular injury. Prostacyclin

Thromboxane A 2

U-46619

Verapamil

1. Introduction

2. Materials and methods

Platelets release adenosine diphosphate (ADP), adenosine triphosphate (ATP), 5-hydroxytryptamine (5HT), endoperoxides and the prostanoid, thromboxane A 2 (TXA2) during the process of aggregation (Vermylen et al., 1983). ADP, ATP and 5HT have been shown to stimulate in vitro prostacyclin (PGI2) synthesis by vascular tissue (Coughlin et al., 1984; Boeynaems and Galand, 1983). It has also been suggested that the endoperoxides (PGG2; PGH2) released by the activated platelets can be used by the vascular epithelium as substrates for PGI 2 synthesis (Vermylen et al., 1983). There are, however, no data available hitherto on the ability of TXA 2 to stimulate PGI 2 release by blood vessels. Since TXA 2 is very unstable in buffer solutions (Coleman et al., 1981) it is difficult to investigate the properties of this prostanoid in vitro. We have therefore studied the effect of a synthetic, stable, TXA 2 analogue, U46619 (Coleman et al., 1981), on PGI 2 synthesis by rat aortic tissue in vitro.

Male Sprague Dawley rats (200 g) were decapitated, their thoracic aortae excised, fatty and connective tissue removed and placed in Dulbecco's minimum essential medium (MEM; GibCo, NY, U.S.A.), which was pregassed to pH 7.4 with 95% 0 2 : 5 % CO 2 (Jeremy et al., 1984; Mikhailidis et al., 1983). The aortae were cut into 1 mm rings with a scalpel blade on a teflon block. Since cutting itself releases large amounts of PGI 2 (Jeremy et al., 1984), the rings were pooled and incubated for 6 periods of 20 min in MEM and the medium renewed at the end of each incubation period (Boeynaems and Galand, 1983). After 120 min of such incubations, which 'exhausted' the aortic rings (Jeremy et al., 1984), the tings were incubated in octuplicate (15 mg tissue per incubate) either in 1 ml of MEM alone, in MEM with varying doses of U-46619 (see fig. 1), or in MEM with a combination of 10 - 6 mol/1 U-46619 and varying doses of verapamil (see fig. 2). Verapamil alone was also evaluated (in MEM) at the doses shown in fig. 2. Control experiments consisted of MEM only incubates. Verapamil (HC1) was purchased from

* To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.

260

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Fig. 1. Dose response curve of U-46619 on rat aortic 6-oxoPGF1, ~ release (e); + 10 m M E D T A (A). Statistical analysis ( M a n n - W h i t n e y test): (a) control vs. all U-46619 concentrations greater t h a n and i n c l u d i n g 10 ~ m o l / l in n o r m a l K R B : P < 0.002; (b) control vs. all U-46619 c o n c e n t r a t i o n s in E D T A + K R B : P = not significant.

Sigma Chemical Company (Poole, Dorset, U.K.). The TXA 2 analogue U-46619 was a gift from Upjohn Company (Kalamazoo, MI, U.S.A.). Stock solutions of drugs were made up fresh, prior to experiments, in demineralised, distilled water and diluted subsequently in MEM. The effect of U46619 was also assessed in MEM containing 10 mmol/1 EDTA, the tissue having been preincubated for 120 min in MEM containing 10 mmol/1 EDTA. After 1 h incubation at 37°C in a shaking water bath, the incubation tubes were centrifuged and the supernatant collected, diluted with Tris-gelatin buffer (pH 7.4) and stored at - 7 0 ° C before estimation of 6-oxo-PGFl~ (the stable, spontaneous metabolite of PGI2) concentration (Jeremy et al., 1984; Mikhailidis et al., 1983). Radioimmunoassay for 6-oxo-PGFt~ was carried out as previously described (Jeremy et al., 1983). Antisera against 6-oxo-PGFl~ were purchased from Cappel Laboratories (West Chester, PA, U.S.A.). [3H]6-oxo-PGFI~ was purchased from New England Nuclear (Boston, MA, U.S.A.) and unlabelled 6-oxo-PGFl~ was a gift of Upjohn (Kalamazoo, MI, U.S.A.).

24

3. Results

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The TXA 2 analogue, U-46619, stimulated PGI 2 synthesis by rat aortae in a dose-dependent manner (EDs0 = 10 -7 mol/1; fig. 1). Verapamil inhibited U-46619-stimulated PGI 2 synthesis in a dose-dependent manner (IDs0 = 3 × 10 -5 mol/1; fig. 2). U-46619 did not stimulate PGI 2 release in the EDTA-containing MEM incubation (fig. 1). Verapamil alone at the doses shown in fig. 2 did not stimulate PGI 2 release in KRB incubates.

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-log [ Verapamil] (mol/I) Fig. 2. Dose response curve of v e r a p a m i l on U-46619 stimul a t e d (10 6 t o o l / l ) rat aortic 6 - o x o - P G F ] , release ( m e a n ± S . D . , n = 6). Statistical analysis ( M a n n - W h i t n e y test): (a) C o n t r o l vs. 3 × 10 6 m o l / 1 verapamil: P = not significant; (b) C o n t r o l vs. 3 × 1 0 5 m o l / 1 v e r a p a m i l a n d all c o n c e n t r a t i o n s a b o v e this: P < 0.002.

4. Discussion

Our results show that the TXA 2 analogue, U46619, stimulates in vitro PGI 2 synthesis by rat aortae. Significant stimulation was seen at concentrations of analogue comparable to the TXA 2 concentrations liberated by human platelets following in vitro aggregation (Mikhailidis et al., 1983).

261

The observations that (1) verapamil blocks U46619-stimulated PGI 2 synthesis by aortic rings, and (2) U-46619 is unable to stimulate PGI 2 synthesis when aortic rings are pre-incubated in EDTA-containing buffer, suggest that U-46619 stimulates PGI 2 synthesis through a calcium-dependent mechanism. An increase in intracellular Ca 2÷ is known to stimulate phospholipase A 2 (Brotherton and Hoak, 1982). This effect may therefore be responsible for the observed increase in PGI 2 release, since phospholipase A 2 liberates arachidonic acid, the precursor of PGI 2, from phospholipids in the membrane (Vermylen et al., 1983; Brotherton and Hoak, 1982). Since smooth muscle contraction is a calciumdependent mechanism (Somylo and Somylo, 1968), simultaneous stimulation of PGI 2 (a vasodilator) synthesis may play a part in the mechanism involved in the subsequent relaxation phase. This would account for the observation that inhibition of the cycloxygenase enzyme system (and therefore PGI 2 synthesis) enhances agonist-induced vasocontraction (Hadhazy et al., 1983). It is also of interest that both ADP and ATP which are released from activated platelets (Vermylen et al., 1983) have been shown to increase vascular PGI 2 synthesis (Boenyaems and Galand, 1983) and to induce vasodilation (Vermylen et al., 1983), although the mechanisms involved remain unclear. It has hitherto been believed that aggregating platelets release endoperoxides (PGG2; PGH2), unstable precursors of both TXA 2 and PGI2, and that these endoperoxides are taken up by the endothelial cells which convert them to PGI 2 (Vermylen et al., 1983). The stimulation of vascular PGI 2 synthesis by aggregating platelets was thus attributed to a process involving "substrate transfer". Our experiments show, however, that the stable TXA 2 analogue, U-46619, stimulates PGI 2 synthesis through a direct action on vascular tissue. Since this TXA 2 analogue also possesses endoperoxide-like activity (Coleman et al., 1981) distinct from TXA 2, it is possible that P G G 2 / P G H 2 may directly stimulate PGI 2 synthesis. The biological significance of the stimulation of PGI 2 synthesis by TXA 2 and the other products of the platelet release reaction (endoperoxides;

5HT; ADP; ATP) is not clear. It is possible that the enhanced secretion of PGI 2 in response to the products of the platelet release reaction is the normal physiological response which limits the extension of thrombi. It is also relevant that vascular trauma which triggers platelet aggregation and adhesion is also a potent stimulus for PGI 2 secretion (Jeremy et al., 1984). These findings are in agreement with the observation that circulating concentrations of PGI 2 and its metabolites, at rest, are extremely low (Blair et al., 1984). The model also predicts that circulating/local PGI 2 levels may increase significantly in response to endothelial/vascular trauma mediated either by mechanical injury or through deposition of platelet aggregates or both.

Acknowledgements The Authors are grateful to Upjohn Company for the generous gift of U-46619 and to Pamela Dale for secretarial assistance.

References Blair, I.A., S.E. Barrow, K.A. Waddell, P.J. Lewis and C.T. Dollery, 1982, Prostacyclin is not a circulating hormone in man, Prostaglandins 23,579. Boeynaems, V.M. and N. Galand, 1983, Stimulation of vascular prostacyctin synthesis by extracellular ADP and ATP, Biochem. Biophys. Res. Commun. 112, 290. Brotherton, A.F.A. and J.C. Hoak, 1982, Role of Ca 2+ and cyclic AMP in the regulation of the production of prostacylin by the vascular endothelium, Proc. Natl. Acad. Sci. U.S.A. 79, 495. Coleman, R.A., P.P.A. Humphrey, I. Kennedy, G.P. Levy and P. Lumley, 1981, Comparison of the actions of U-46619, a prostaglandin H2-analogue, with those of prostaglandin H 2 and thromboxane A 2 on some isolated smooth muscle preparations, Br. J. Pharmacol. 73, 773. Coughlin, S.R., M.A. Moskowitz and I. Levine, 1984, Identification of a serotonin type 2 receptor linked to prostacyclin synthesis in vascular smooth muscle cells, Biochem. Pharmacol. 33,692. Hadhazy, P., L. Nagy, F. Jahasz, B. Malomvolgyi and K. Magyar, 1983, Effects of indomethacin and prostaglandin 12 and E 2 on the tone of human isolated mesenteric arteries, European J. Pharmacol. 91,477. Jeremy, J.Y., D.P. Mikhailidis and P. Dandona, 1984, Vascular trauma and prostacyclin release, Microcirc. Endothel. Lymph. (in press).

262 Mikhailidis, D.P., J.Y. Jeremy, M.A. Barradas, N. Green and P. Dandona, 1983, Effect of ethanol on vascular prostacyclin (prostaglandin I2) synthesis, platelet aggregation and platelet thromboxane release, Br. Med. J. 287, 1495. Somylo, A.P. and A.V. Somylo, 1968, Vascular smooth muscle, Pharmacol. Rev. 20, 197.

Vermylen, J., P.N. Badenhorst, H. Deckmyn and T.J. Arnou, 1983, Normal mechanisms of platelet function, in: Platelet Disorders - Clinics in Haematology, Vol. 12, eds. L.A. Harker and T.S. Zimmerman (W.B. Saunders Co. Ltd., London) p. 107.