Thromboxane/Prostaglandin Endoperoxide Receptor Activation 'Mark 0

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 contraction. 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 vasoconstriction 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. Other stock solutions were made on the day of use: (2 flufenamic acid and indomethacin were dissolved in 10% E X ~~~~~~~~~TXB2 NaHCO3, CGS12970 was dissolved in O.1N NaOH and all co 20- (7) remaining agents were prepared directly in buffered PSS. All 0 stock dilutions into PSS were made on the day of use and Dfl-n- Clo discarded at the end of the experiment. At the maximum 11 0l 9 8 7 6 5 4 added volumes, none of the solvent vehicles alone had any -log [Agonist] (M) observable effect on relaxed or NA-constricted jugular or femoral veins. Figure 1 Contractile response of canine jugular and femoral veins to the thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) analogues SQ26655 and U44069, bradykinin (BK), PGF26, TXB2 and PGD2. Statistics (a) Jugular; (b) femoral vein rings. All concentration-response curves are normalized to the average maximum SQ26655 response for each Statistical data are presented as the means + s.e.mean. Each vein. For Figures 1, 2, 4 and 5A, all values are means with vertical mean value is the average of data points from at least 3 lines showing s.e.mean and numbers in parentheses = number of rings animals. Data were compared by Student's t test for paired used. BRADYKININ-INDUCED CONTRACTION IN CANINE VEINS 463

> U44069 > BK > PGF2, > TXB2 > PGD2 for both veins. 6 5 SQ26655 and U44069 were respectively 700-1900 times and a 7. 100-200 times more potent than PGF2<,. Of the above agon- U44069 61g- _ U44069 ,/ 6 ists, only the TXA2/PGH2 analogues exceeded BK in potency 8 1:girnnirn -_ as vasoconstrictors of canine jugular and femoral vein. (1) Wash 7 1oq 8: (2) 10-8 M 1v,-~~~--6-- _ Effect ofcyclo-oxygenase inhibitors on BK-induced 10. ., SQ29548 2 _ .2 _. ,! contractions 9 10989 7 5 Canine jugular and femoral vein were tested with 2 inhibitors 6 6 of the cyclo-oxygenase pathway, indomethacin and flufenamic b 7. acid (Flower, 1974), to determine the influence of BK-evoked U44069 6 U44069 55- cyclo-oxygenase products on contraction (Figure 2). Indo- (1) Wash ,>- x maximum BK methacin (3 10- 7M) significantly reduced the (2) 10-6 M 7. ,:6 (10-6M)induced contractions to 32.5 + 8.0% (n = 11) and 19 BM13177 26.0 + 7.5% (n = 10) of control responses in jugular and 10 98 femoral vein, respectively. Flufenamic acid, a less potent cyclo- 10 9 8,- 10 98I oxygenase inhibitor, showed a similar inhibitory action at 10 9 8 10- 5M by reducing BK-induced responses in the jugular and Figure 3 Effect of thromboxane A2 (TXA2)/prostaglandin H2 to 29.1 + 8.2% = and 3.1 + 1.8% (n = 8) of (PGH2) receptor antagonists SQ29548 and BM13177 on U44069 femoral vein (n 9) concentration-response curves in canine jugular and femoral veins, as their respective control values. shown by representative tracings. Numbers indicate cumulative Neither indomethacin nor flufenamic acid at 10-5M had agonist concentrations shown as -log[agonist]. Scale at top applies any significant effect on NA (10- 5M)-induced contraction to all tracings. Jugular: solid lines; femoral: dashed lines. (a) Antago- (Figure 2). Indomethacin (10-sM) also had negligible effect on nism of U44069-induced contraction by 10-8 M SQ29548. (b) Antago- contractions to submaximal NA concentrations as well as on nism of U44069-induced contraction by 10-6 M BM13177. contractions induced by U44069 (data not shown). Thus indomethacin and flufenamic acid specifically blocked a major portion of the contraction to BK in jugular and femoral vein, in Figure 4, SQ29548 (10-8M) reduced tension elicited by presumably through inhibition of cyclo-oxygenase metabo- 106M BK to 14.1 + 3.9% (n = 9) and 2.1 + 1.0% (n = 8) of lism. control for jugular and femoral veins respectively. SQ29548 (10-7M) reduced the BK response to less than 10% in both veins. Antagonism of TXA2/PGH2 receptors with SQ29548 and A second selective TXA2/PGH2 receptor antagonist, BM13177 BM13177 (Stegneier et al., 1984), likewise significantly antagonized U44069- (Figure 3b) as well as BK-induced con- SQ29548 is a potent, specific TXA2/PGH2 receptor antagonist tractions (Figure 4) in both vein preparations. BM13177 in platelets and smooth muscle (Ogletree et al., 1985). In (1O- 6M) reduced tension elicited by 10- 6M BK to canine jugular and femoral vein, SQ29548 (10-8M) shifted the 40.9 + 6.4% (n = 8) and 33.0 + 7.9% (n = 7) of control in U44069 dose-response curve to the right, as shown by repre- jugular and femoral veins respectively. BM13177 (10- 5M) sentative tracings in Figure 3a, indicating antagonism at the further reductions to less than 25%. TXA2/PGH2 receptor. yielded Preincubation with SQ29548 (10-8 and 10-7M) also significantly reduced the level of BK-induced contraction. As shown

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Ino (M0 (11)3 (9)0- (8) NA (M) 10-6 10-5 - 10-6 10-5 NA(M) 10-5 10-5 BK (M) - - 10-6 10-6 _ _ 10-6 10-6 10-7 - - BK(M) - 106,, 10-6 SQ29548 (M) o-7 10-7 10-8 1- 10-6 10-5 Indo (M) 10-5 3 x BM13177 (M) _- - 10-5 10-5 FA (M) - 10-5 10-5 Figure 4 Effect of the thromboxane A2 (TXA2)/prostaglandin H2 Figure 2 Effect of the cyclo-oxygenase inhibitors indomethacin (PGH2) receptor antagonists SQ29548 and BM13177 on noradrena- (Indo) and flufenamic acid (FA) on noradrenaline (NA)- and brady- line (NA) and bradykinin (BK)-induced contractions in canine jugular kinin (BK)-induced contractions in canine jugular and femoral vein. and femoral vein. As in Figure 2, the dotted line represents the nor- The dotted line represents the normalized control response to NA or malized control response to NA or BK for each group of rings before BK for each group of rings before incubation with a given inhibitor. incubation with a given antagonist. Columns show responses to NA Columns show contractile responses to NA or BK in presence of or BK in the presence of antagonists as a % of control values. inhibitors as a % of control values. Hatched columns-jugular vein; Hatched columns-jugular vein. Solid columns- femoral vein. *In- solid columns-femoral vein. *Indicates significant difference dicates significant difference (P < 0.05) between inhibited and control (P < 0.05) between inhibited and control values. values. 464 M.O. AKSOY et al.

A little effect on the NA-induced contraction which retained 100- over 80% of its initial control tension in both veins (Figure 4). 0) Since PGF2. can interact at TXA2/PGH2 receptors, the TXA2/PGH2 antagonists might be expected to have some CD effect against the PGF20-induced contraction. SQ29548 -a0 - (10-8M) did inhibit contractions to PGF2. (10- M) in both ci veins. The extent of inhibition (38.8 + 3.4% (n = 4) for jugular and 43.6 + 6.4% (n = 4) for femoral) was substantially less 0 than that of BK-induced contraction. Influence ofremoval ofthe endothelium on BK-induced C.)x contractions E 20- To determine the extent of endothelial influence on the contractile action of BK in canine jugular and femoral vein, BK concentration-response curves were obtained from vein rings 9 before and after endothelium removal (see Methods). Removal -log [BK] (M) of endothelium had a negligible effect on the BK response in both veins except for a slight but not-significant inhibition of femoral vein tension at 10-6M and 10-5M BK (Figure 5A). 40 0 Likewise, endothelium removal had no pronounced effect on 7,, B 5 the inhibition of BK-induced contraction by SQ29548 6 a (10-8M) or BM13177 (10-6 M) in either vein (Figure 5B). BK BK 9 8 7 6 5 Thus, the endothelium appears to exert little influence on the (1) Wash contraction of either vein by BK. 76 5 (2) 10-8MO BK 9 8 7 6 5 BK 9 8 SQ29548 _t _-2 _2 Effect ofthromboxane synthase inhibitors on BK-induced contractions 1g 6 5 10 min Since a TXA2/PGH2 receptor antagonist cannot discriminate b 6 5 between vasoactive effects due to TXA2 and PGH2, 2 inhibi- (1) Wash BK 7 tors of thromboxane synthase were tested to determine BK 9 whether TXA2 alone was responsible for the mediation of BK- BK 9 (2) 10-6 M first inhibitor, dazoxiben (Randall et 7 BM13177 6 5 induced contraction. The BK 9 8 7 .!,- BK 9 8,'- -I -- al., 1981), had no significant (P < 0.05) inhibitory effects on BK (10-6M)-induced contraction in either vein at 10-SM or Figure 5 (A) Effect of endothelial removal on canine jugular and 1O-4M (Table 1). Similarly, the maximum BK-induced con- femoral vein contraction to bradykinin (BK). Control responses: was not a second jugular (0) (9); femoral (-) (11). Responses following endothelial traction reduced by CGS12970 (10-5M), removal: jugular (0) (9); femoral (El) (11). For each vein, all responses more potent thromboxane synthase inhibitor (Ambler et al., are normalized to the maximum control response to BK. See Methods 1985) (Table 1). In general, inhibition of thromboxane syn- for protocol for removal of endothelium. (B) Inhibitory effect of thase, and hence of thromboxane production, did not signifi- TXA2/PGH2 receptor antagonists 10-8M SQ29548 (a) and 10-6M cantly decrease the contractile response to BK in canine BM13177 (b) on BK-induced contraction in vein rings denuded of jugular and femoral vein. endothelium. Numbers indicate cumulative concentrations shown as -log[BK] in representative tracings. Jugular: solid lines; femoral: dashed lines. Discussion This study has shown that the major component of contrac- The inhibition by SQ29548 or BM13177 was also tion to BK in 2 canine vein preparations may proceed via reversible. Thirty min after washout of either antagonist, both stimulation of the TXA2/PGH2 receptor. The rank order of jugular and femoral veins largely regained their ability to con- agonist potency in both veins, showing the high potency of tract to BK. In contrast to their inhibition of BK, high con- TXA2/PGH2 analogues vis a vis prostanoids such as PGF20 centrations of SQ29548 (10-7M) or BM13177 (10-5M) had and PGD2 (Figure 1), resembles that for other vascular preparations shown to possess TXA2/PGH2 receptors (Coleman et al., 1981; Tore et al., 1984). This suggests the presence of Table 1 Effect of thromboxane synthase inhibitors dazoxi- TXA2/PGH2 receptors in both veins according to the classi- ben and CGS12970 on bradykinin (BK)-induced contraction fication of Kennedy et al. (1982). in canine jugular and femoral vein Two cyclo-oxygenase inhibitors, indomethacin and flufenamic acid, reduced BK-induced contractions in canine jugular % control tensiont and femoral vein (Figure 2), implying a dependence of con- Condition* Jugular Femoral traction on product(s) of the cyclo-oxygenase pathway. Indo- methacin was effective at a low concentration of 3 x 10-7M 10-M Dazoxiben 120.9 + 8.2 126.6 + 13.4 where non-specific interactions are unlikely (Flower, 1974). 10-4M Dazoxiben 90.4 ± 14.4 90.3 + 16.7 Neither inhibitor affected NA-induced 10-5M CGS12970 113.4 + 10.0 130.5 + 13.5 contractions, indicating the absence of a general effect on calcium mobilization at the Each value is the mean ± s.e.mean of data from at least 3 concentrations used. animals. n = 5-6. SQ29548 is a TXA2/PGH2 receptor antagonist which selec- * Each ring was initially assayed with 106 M BK alone, tively binds to TXA2/PGH2 receptors (Mihara et al., 1989) relaxed, then incubated with inhibitor and reassayed with and inhibits TXA2 analogue-induced vascular contraction BK. t Values represent BK-induced tension in the presence (Ogletree et al., 1985; Darius et al., 1985; Mihara et al., 1989). of inhibitor expressed as % of tension to BK alone according It effectively blocked the BK-induced contraction in canine to the formula: jugular and femoral veins at concentrations as low as 10-8M was likewise (TensionBK+inhibitor/TensionBK alon.) 100. (Figure 4). Inhibition of BK-induced contraction BRADYKININ-INDUCED CONTRACTION IN CANINE VEINS 465 demonstrated with the TXA2/PGH2 receptor antagonist effect on the longer term BK-induced contraction in either BM13177, which at 3 x 10-6 to 3 x 10-'M inhibits the vein (Figure 5A), though a slight reduction in the maximum TXA2-mimetic U46619 in rabbit aorta (Stegmeier et al., 1984) femoral response suggests the marginal influence of an and rat perfused kidney (Quilley et al., 1989). At 10- 6 M, endothelium-derived vasoconstrictor. The action of the BM13177 reduced the BK-induced contraction by over 60%; TXA2/PGH2 antagonists (Figure SB) was also unaffected by at 10-SM, inhibition exceeded 80% (Figure 4). These inhibi- endothelium removal, indicating that their principal site of tory effects of SQ29548 and BM13177 were reversible and action is on smooth muscle. Thus, the endothelium contrib- apparently specific, implying that contraction in both veins utes little to the steady-state contractile action of BK or the due to activation of the BK receptor is largely mediated effect of the TXA2/PGH2 antagonists. through the TXA2/PGH2 receptor, presumably by PGH2 In contrast to the pronounced antagonism of BK-induced and/or TXA2. contraction by SQ29548 and BM13177, high levels of the SQ29548 also moderately antagonized the venous contrac- thromboxane synthase inhibitors dazoxiben and CGS12970 tion induced by PGF2.. The possibility cannot be rig- had no significant inhibitory effect (Table 1). One possible orously excluded that a component of this antagonism occurs explanation is that, in situ, PGH2 itself may be capable of at PGF2<,, as opposed to TXA2/PGH2 receptors. Nonetheless, contracting both veins. PGH2 has a longer half-life than the agonist potency rankings in Figure 1 imply that TXA2 (5min vs 32s at 370C and pH 7.4) (Hamberg et al., TXA2/PGH2 rather than PGF2. receptors predominate in 1975a), and is a potent vasoconstrictor (Hamberg et al., these preparations and mediate the action of PGF2,. Radioli- 1975b; Goldberg et al., 1977; Coleman et al., 1981). The con- gand studies in pig aorta have shown that binding of [3H]- tractile action of accumulated PGH2 resulting from inhibition U44619 to the TXA2/PGH2 receptor can be inhibited by of TXA2 synthesis could therefore mask any reduction in con- PGF2. (Mihara et al., 1989). This interaction of PGF2. with traction due to reduced TXA2. Under these conditions, a TXA2/PGH2 receptors can account for the SQ29548 antago- thromboxane synthase inhibitor might not yield a decrease in nism of PGF2. in canine jugular and femoral vein. the final contractile response to BK even if TXA2 production Previous studies have shown stimulation of PGF2. synthe- were significantly reduced. It could therefore be concluded sis by BK in mesenteric veins (Wong et al., 1977; Limas, 1977), that both TXA2 and PGH2 normally mediated the contrac- suggesting a possible role for PGF2a in the mediation of BK- tion to BK. Alternatively, the thromboxane synthase inhibi- induced contraction. However, in vascular preparations con- tors might be ineffectual because both veins may normally taining TXA2/PGH2 receptors, PGF2. is significantly less have negligible thromboxane synthase activity during BK- potent than PGH2 and TXA2 (Hamberg et al., 1975b; induced contraction. In this case, only PGH2 would be the Coleman et al., 1981), the PGH2 analogue U44069 (Tore et al., principal mediator of the contraction. 1984) and the TXA2 mimetic U44619 (Mihara et al., 1989). In blood platelets, the synthase inhibitor 1-carboxyheptyl Similarly, PGF2. as a vasoconstrictor of canine jugular and imidazole reduces TXA2 production without affecting arachi- femoral vein is 102_103 times less potent than the donic acid-induced platelet aggregation, implying that PGH2 TXA2/PGH2 analogues U44069 or SQ26655. Hence, rela- is a potential mediator of aggregation (Grimm et al., 1981). In tively massive amounts of PGF2, would be required to equal the vasculature, a recent study on rat perfused kidney has TXA2 or PGH2 in the potentiation of venous contraction to shown that the thromboxane synthase inhibitor CGS-13080 BK. Although PGF2. may be synthesized in BK-stimulated does not inhibit arachidonic acid-induced vasoconstriction, canine jugular and femoral veins, its low potency compared to despite the demonstrated dependence of this contraction on SQ26655, U44069 and BK makes it an unlikely mediator of TXA2/PGH2 receptor activation. The authors concluded that contraction to BK in either vein. Mediation via PGD2 is also PGH2 can mediate the vasoconstriction in conjunction with unlikely in view of the negligible contractile response of either TXA2 (Quilley et al., 1989). In situ, PGH2 may similarly func- vein to PGD2 (Figure 1). tion as a mediator of constriction to BK in certain vessels, Unlike their antagonism of U44069, inhibition of BK- either in conjunction with TXA2 or when TXA2 production is induced contraction by SQ29548 and BM13177 appeared to negligible. be non-competitive (see Figure 5B). It could be argued that an In this study, BK-induced contraction in 2 anatomically excess of BK might generate sufficiently large amounts of distinct canine veins can apparently be mediated through the endogenous TXA2 and/or PGH2 agonist to compete with the TXA2/PGH2 receptor. The applicability of this mechanism to antagonists at the TXA2/PGH2 receptor and yield competi- other vessels remains to be determined. There may be ana- tive inhibition. However, the higher potency of SQ26655 and tomical and species variability in the mediator processes U44069 versus BK (Figure 1) suggests that the maximum BK underlying BK-induced contraction due to differences in pros- response is mediated by relatively small amounts of the taglandin receptor type and number, as well as in the subtle endogenous agonists. Under these conditions, saturation of balance of evoked cyclo-oxygenase products between potent the BK receptor (with 10 5M BK) is unlikely to generate suffi- vasoconstrictors (TXA2 and PGH2) and vasodilators (PGI2). cient endogenous agonists to counteract the inhibitory effect These differences would also influence the possible importance of the antagonists at the TXA2/PGH2 receptor. This would of PGH2 as a mediator itself. Clearly, in situ, the role of account for the apparent non-competitive inhibition of BK- TXA2/PGH2 receptors in the mediation of vascular smooth induced contraction. J muscle contraction to BK and other agonists is an area which In response to varied pharmacological stimuli, endothelium deserves further study. can produce vasoactive metabolites such as PGI2, TXA2 and endothelium-derived relaxing factor (Shirahasi et al., 1987; Gryglewski et al., 1988). Therefore, it was important to estab- We wish to acknowledge the help and generosity of the following lish the degree to which the contractile action of BK in canine people: Patricia De Feo and Dwight Fonda produced scanning elec- jugular and femoral vein was modulated by endothelial tron micrographs of tissue samples. Dr Woodrow Wendling provided a BASIC program for pharmacological calculations. Nancy Guy and factors. Though these veins show moderate endothelium- Sujata Hanjura furnished valuable technical assistance in several dependent relaxation to BK if preconstricted with NA, the experiments. The following companies provided gifts of drugs used in relaxation is transient with tension regaining or exceeding pre- these studies: Ciba-Geigy, Pfizer, Squibb and Upjohn. This work was constriction levels within 1 min (Aksoy et al., 1984). In this funded by NIH Grant HL30980. C.R.Z. was supported by NIH study, removal of endothelium had no significant (P < 0.05) National Research Service Award HL07248. 466 M.O. AKSOY et al.

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