Br. J. Pharmacol. (1990), 99, 461-466 I--, Macmillan Press Ltd, 1990 Mediation of bradykinin-induced contraction in canine veins via thromboxane/prostaglandin endoperoxide receptor activation 'Mark 0. Aksoy, *Concetta Harakal, *J. Bryan Smith, Gwendolyn J. Stewart & Charles R. Zerweck Thrombosis Research Center and *Dept. of Pharmacology, Temple University School of Medicine, 3400 N. Broad St., Philadelphia, PA, 19140, U.S.A. 1 Canine jugular and femoral veins were studied to determine the possible importance of thromboxane (TXA2) and prostaglandin endoperoxides (prostaglandin H2, PGH2) in mediating bradykinin(BK)- induced contraction. 2 Isolated vein rings incubated in modified Krebs solution contracted to TXA2/PGH2 analogs SQ26655 and U44069 with potency of contraction exceeding that for BK. The potency ranking for both veins was SQ26655 > U44069 > BK > PGF21i> TXB2 > PGD2. 3 The cyclo-oxygenase inhibitors indomethacin (3 x 107M) and flufenamic acid (10- 5M) reduced BK contractions without affecting those induced by noradrenaline (NA). 4 TXA2/PGH2 receptor antagonists SQ29548 (10-8 M) and BM13177 (10-6 M) strongly inhibited BK- induced tension. The action of antagonists was reversible with negligible influence on NA-elicited contrac- tion. Selective removal of endothelium had no effect on BK-induced contraction or the action of the antagonists. 5 The thromboxane synthase inhibitors dazoxiben (10-4M) and CGS 12970 (10- M) had no significant inhibitory effect on BK-induced tension. 6 These results suggest that in canine jugular and femoral vein, the action of BK is largely dependent upon stimulation of the cyclo-oxygenase pathway to produce PGH2 and possibly TXA2, which can activate a smooth muscle TXA2/PGH2 receptor to elicit vasoconstriction. Introduction vasoconstriction associated with release of TXA2; this vaso- constriction can be reduced by the thromboxane synthase Bradykinin (BK) is a potent autacoid released during trauma, inhibitor OKY1581 (Kawasaki & Needleman, 1982). anaphylactic and other types of shock, and inflammatory The potential of PGH2 as an endogenous mediator of reactions. Although it can exert a hypotensive effect in vivo agonist-induced vasoconstriction is less well known, despite its due to arteriolar dilatation, BK has a predominantly con- ability to constrict vessels (Hamberg et al., 1975b; Goldberg et strictive action in many mammalian isolated veins (Tsuru et al., 1977; Coleman et al., 1981). A recent study has suggested al., 1976; Gaudreau et al., 1981). that prostaglandin endoperoxides can mediate arachidonic Evidence for endogenous prostaglandin synthesis along the acid-induced vasoconstriction in the rat perfused kidney cyclo-oxygenase pathway associated with BK-induced con- through the TXA2/PGH2 receptor, under conditions where traction has been obtained in several vascular preparations. TXA2 synthesis is inhibited (Quilley et al., 1989). However, the net vasoactive influence of the prostaglandins In this study, we show that the contractile action of BK on synthesized varies with species and vascular bed. Thus the 2 canine isolated vein preparations proceeds primarily cyclo-oxygenase inhibitors meclofenamate and indomethacin through activation of the cyclo-oxygenase pathway and sub- reduce contraction to BK in human foetal placental arteries sequent interaction of evoked PGH2 and possibly TXA2 with (Tulenko, 1981) and bovine mesenteric vein (Wong et al., the TXA2/PGH2 receptor. 1977) respectively, implying that a cyclo-oxygenase product mediates the contraction. Yet in rabbit ear vein (Crouch et al., 1981) and canine intestinal vasculature (Greenberg & Kado- Methods witz, 1982), indomethacin either has no effect or potentiates Dissection ofcanine veins the response to BK. Though BK can stimulate production of the vasoconstrictor Femoral and external jugular veins (4 vessels per dog) were prostaglandin F2. (PGF2e) in certain mesenteric veins (Wong carefully harvested from female mongrel dogs anaesthetized et al., 1977; Limas, 1977), few BK-related studies have been with sodium pentobarbitone (Nembutal, 25mgkg-1 animal done to determine the possible mediator role of the potent weight, injected into the cephalic vein). Supporting tissue was cyclo-oxygenase products thromboxane A2 (TXA2) and the carefully removed and side branches tied off, with care taken precursor prostaglandin endoperoxides (PGH2, PGG2) to maintain blood flow and not disturb the endothelial surface (Hamberg et al., 1975a), which interact with the thromboxane/ in the main vessels. The veins were then perfused with saline prostaglandin endoperoxide (TXA2/PGH2) receptor (Kennedy solution, ligated, excised and transferred to bicarbonate- et al., 1982; Saussy et al., 1985). Endogenous TXA2 can buffered physiological saline solution (PSS) at 40C. The dogs mediate vasoconstriction to acetylcholine in rabbit pulmonary were then killed by an intravenous injection of T-61. Veins artery (Altiere et al., 1986) and canine cerebral artery were stored with daily changes of PSS and were found to give (Shirahase et al., 1987), and to arachidonic acid in rabbit pul- consistent responses over a 3 day period. monary artery (Salzman et al., 1980) and intact rat kidney (Sakr & Dunham, 1982). In the perfused hydronephrotic In vitro setup and procedure rabbit kidney (a pathophysiological model), BK yields renal Circular ring segments 2.5 mm wide were cut perpendicular to 1 Author for correspondence. the vessel axis and were mounted with metal hooks and 462 M.O. AKSOY et al. jeweler's chains in individual tissue baths. Each bath was observations. Statistical significance was considered to occur equipped with (1) a micrometer gauge to measure and manip- when P < 0.05. ED50 values were derived by linear inter- ulate tissue length and (2) a Statham isometric force trans- polation of the normalized dose-response curves for a given ducer. This latter was connected through a signal amplifier agonist. and multiplexer to a Kipp and Zonen strip chart recorder. The mounted tissues were incubated in the baths with 50 ml volumes of PSS bubbled with 95% 02 - 5% CO2 at 370C, Results pH 7.4. The composition of the PSS was (mM): NaCl 127.4, KCl 4.7, MgSO4 7H20 1.2, KH2PO4 1.2, NaHCO3 16.0, Contractile responses ofcanine veins CaCl2 * 2H20 2.5, EDTA 0.04 and glucose 10.0. Each mounted vein segment was stretched in 0.4g increments at As shown in Figure 1, TXA2/PGH2 analogs SQ26655 and 15min intervals over a 90min period to a stable preload of U44069, along with PGF2a and BK, strongly contracted both 0.8g. vein preparations. The maximum responses for the latter 3 Concentration-response curves were obtained by the cumu- agonists were at least 55% of the maximum SQ26655 response lative addition of aliquots of agonist solutions. In a typical which was 3.77 + 0.40g (n = 15) for jugular and 2.17 + 0.22 g experiment, vein rings were assayed with BK to obtain a (n = 15) for femoral vein. In comparison, the maximum NA reproducible control response, then incubated with an antago- (10- M) response was 2.38 + 0.21 g (n = 40) for jugular, and nist or inhibitor for 30 min before re-assay with the agonist. 3.11 + 0.20 g (n = 37) for femoral vein. The ED50 values for the agonists in Figure 1 were: jugular-SQ26655: Removal ofendothelium 0.22 + 0.01 nM (n = 15), U44069: 1.70 + 0.03 nM (n = 8), BK: 17 + IM (n = 35), PGF2g: 159 + 5nM (n = 12); femoral- In some experiments, the endothelium was selectively removed SQ26655: 0.26 + 0.01 nm (n = 15), U44069: 2.23 + 0.05 nM from mounted jugular and femoral vein rings. The flattened (n = 10), BK: 55 + 2nM (n = 22), PGF2a,,: 501 + SnM(n = 17). end of a small wooden dowel was carefully rubbed several TXB2 contracted the veins only at concentrations exceeding times over the entire luminal surface of the ring which was 1O-6 M. PGD2 (10- 5 M) yielded negligible contractions in both then rinsed in PSS. Anatomically adjacent rings with undis- veins. In order of potency, these agents were ranked: SQ26655 turbed endothelium were used as controls. The presence or absence of endothelium was determined by scanning electron microscopy (SEM). After a set of experi- a ments, some vein rings were fixed in 1% glutaraldehyde while 120- mounted in the tissue baths, then removed for dehydration a) and critical-point drying. Samples were gold-coated and CD o 100- examined by SEM, which confirmed that the physical denu- a dation was effective in specifically removing endothelium. 0 SQ26655t LO 80- (15) LO Drugs used CD 60 Agonists used in this study were: noradrenaline-HCI (NA), (I) U444069 E prostaglandin D2 (PGD2) and thromboxane B2 (TXB2) (all E 40 / 4( from Sigma Chemical Co.); bradykinin (BK, Boehringer- E PG)F2,, TXB32 Ingelheim); prostaglandin F2g (dinoprost tromethamine, PGF2a; Upjohn Co.); U44069 ((15S)-hydro-9a,1la-(epoxyme- E 20 - PGD2 thano) prosta-5Z, 13E-dienoic acid; donated by Upjohn Co.) S ~~~~~~~~2(9) and SQ26655 ((lS-(la,2b(5Z),3a(1E,3s*),4a))-7-(3-(3-hydroxy-1- octenyl) - 7 - oxabicyclo(2.2.1)hept - 2 - yl)- 5 - heptenoic acid; 1 10 9 8 7 6 5 4 donated by Dr Ogletree, Squibb Research Inst.). Inhibitors -log [Agonist] (M) and antagonists included: indomethacin and flufenamic acid (Sigma Chemical Co.); SQ29548 ((1S4la,2b(5Z),3b,4a))-7-(3- ((2 - ((phenylamino)carbonyl) - hydrazino)methyl) - 7 - oxabicyclo b (2.2.1)hept-2-yl)-5-heptenoic acid; donated by Squibb 120 Research Inst.); BM13177 ((4-(2-benzenesulphonamido)-ethyl)- a) phenoxyacetic acid; donated by Dr Lefer, Thomas Jefferson 0 100- School of Medicine); CGS12970 (3-methyl-2(3-pyridyl)-1- a from Dr indoleoctanoic acid; gift Watthey, Ciba-Geigy, Inc.) o 80s SQ26655 and dazoxiben-HCl (4-(-2-(LH-imidazol-1-yl)ethoxy) benzoic O (15)U acid hydrochloride; donated by Dr Urquilla, Pfizer, Inc.). CD a 60 (0) Stock solutions of SQ26655, U44069, PGD2, TXB2, CO PGF21, SQ29548 and BM13177 were prepared in ethanol and stored E BK :3 40 (2 (17) at - 20°C.