Introduction

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The renin-angiotensin system has been well recognized as an important contributor to the pathogenesis of hypertension, cardiac hypertrophy and vascular disease (Ferrario et al., 1994). Losartan is a potent nonpeptide, selective Ang II AT₁ receptor antagonist, which produces concentration-dependent inhibition of Ang II-induced vasoconstriction in vivo and in vitro and displaces ¹²⁵I-Ang II binding in radioligand binding studies (Timmermans et al., 1993; Liu, 1993; Rhaleb et al., 1991). Losartan reduces blood pressure in human hypertensive subjects, as well as in animal models of hypertension, such as renal hypertensive rats, SHR and transgenic hypertensive rats (Townsend and Ford, 1996; Wong et al., 1990c; Moriguchi et al., 1994). Acute administration of losartan is highly effective in lowering blood pressure in SHR, a genetic model of hypertension in which plasma renin is not elevated (Ohlstein et al., 1992; Cachofeiro et al., 1995). However, it has been noted that in this same hypertensive model the peptide Ang II antagonist saralasin is not as effective as losartan, and angiotensin converting enzyme inhibitors are only modestly effective after acute administration (Pals et al., 1971; Sweet et al., 1981; Ohlstein et al., 1992). Recent studies suggest that the long-lasting antihypertensive effect of losartan may not be caused solely by Ang II receptor blockade. This was illustrated by Ohlstein et al. (1992) who showed that 48 h after the administration of losartan, blood pressure was still reduced in the presence of a normal pressor response to Ang I or II. Furthermore, Cachofeiro et al. (1995) reported that nitric oxide and prostaglandin production are involved in the prolonged blood pressure-lowering effects of losartan in SHR. Losartan has also been shown to attenuate catecholamine-induced contractions in aortic rings of SHR in an endothelium-dependent manner, stimulate vasodilatory prostaglandin release in cultured cells and at high doses bind to imidazoline/guanidinium receptors in rat forebrain cardiovascular areas (Maeso et al., 1995; Jaiswal et al., 1991; Li et al., 1996).

Recent studies have shown that losartan interacts with the TxA₂/PGH₂ receptor in human platelets and inhibits the TxA₂ analog U46619-induced platelet aggregation and pulmonary hypertension in rats (Liu et al., 1992; Bertolino et al., 1994). Furthermore, TxA₂ and PGH₂ are involved in Ang II-dependent hypertension by stimulating contraction of vascular smooth muscle by a common receptor (Lin et al., 1991, 1994). In the present study, we further investigated whether losartan and its active metabolite EXP3174 interact with the TxA₂/PGH₂ receptor and stimulate production of prostaglandin and nitric oxide in isolated canine coronary arteries.

	Materials and Methods

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After approval by the Institutional Animal Care and Use Committee, 16 male dogs (15-25 kg b.wt.) were anesthetized with ketamine (15 mg/kg i.m.) and 2% halothane inhalation; the dogs were then sacrificed with sodium pentobarbital (50 mg/kg i.v.). The heart was harvested immediately and immersed on ice-cold modified Krebs-Henseleit buffer, whereas the left anterior descending coronary artery was carefully dissected free of fat and adhering connective tissues. The coronary artery was cut into 3-mm-long rings and suspended in organ chambers containing Krebs-Henseleit solution of the following composition (in mmol/l): NaCl, 118.3; KCl, 4.7; CaCl₂, 2.5; MgSO₄, 1.2; KH₂PO₄, 1.2; NaHCO₃, 25; CaNa-ethylenediaminetetraacetic acid, 0.026; and glucose, 11. The Krebs' solution was aerated with 95% O₂ and 5% CO₂ at 37°C (pH 7.4) and the rings were allowed to equilibrate for 60 min at 1g initial resting tension. Basic tension was increased individually in a step-by-step fashion until the optimal length-tension relationship was obtained by repeated exposure to 40 mmol/l KCl. In some rings, the endothelium was denuded by gentle mechanical rubbing with a stainless steel wire. Isometric tension of vascular rings was measured continuously with force-displacement transducer (FTO3, Grass Instruments Co., Quincy, MA) connected to a Grass polygraph. The integrity of functional endothelium of vascular rings was confirmed by the presence of acetylcholine-induced relaxation in preconstricted rings with 10⁸ mol/l U46619 (more than 90% relaxation at 10⁷ mol/l of acetylcholine) and absence of acetylcholine-induced relaxation in vessels after mechanical denudation of the vascular endothelium.

Experimental protocol. Control cumulative concentration-contractile response curves for TxA₂ analog U46619 (10¹⁰-3 × 10⁶ mol/l) were generated after 1 h equilibration in intact quiescent rings. Losartan (10⁸-10⁵ mol/l) was used to pretreat the coronary artery rings for 30 min, and the concentration-response curves for U46619 were then repeated. To determine whether losartan interacts with other vasoconstrictors, concentration-response curves with PDGF (10 and 20 ng/ml) and KCl (10-80 mmol/l) were also constructed in the absence and presence of losartan (10⁶ mol/l) in isolated coronary vascular rings. Phenylephrine, arginine vasopressin and PGF₂ (each at concentrations ranging from 10¹⁰ to 10⁶ mol/l) were also tested. In addition, to compare the potency and selectivity of losartan on TxA₂ receptor in coronary arteries, the potent, selective TxA₂/PGH₂ receptor antagonist SQ29548 (Ogletree et al., 1985) was used to pretreat the tissues for 30 min, and then concentration-response curves for U46619 were determined.

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Chemicals. Losartan and EXP3174 were generous gifts from DuPont/Merck Company (Wilmington, DE). PD 123319 was generously supplied by Parke-Davis Inc. (Ann Arbor, MI) and CV-11974 was from Takeda Chemical Industries, LTD. (Osaka, Japan). L-NAME and SQ29548 were purchased from Research Biochemicals International (Natick, MA). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Indomethacin and CV-11974 were dissolved in 0.2 N Na₂CO₃ solution and diluted with Krebs' buffer. U46619 was prepared as stock in ethanol and diluted with Krebs' buffer. The concentrations of drugs reported are at final concentration in organ chambers.

Data and statistical analysis. The concentration of U46619 and losartan causing 50% of the maximal contraction (EC₅₀) and the maximal relaxation (IC₅₀) were calculated with use of a nonlinear regression sigmoid curve fitting program of PRISM (Graphpad Inc., San Diego, CA). The apparent dissociation constant (K_B) was calculated with the equation K_B = [B] 46 ([A]/[A] 1)¹, where [B] is the concentration of the antagonist and [A] and [A] are the EC₅₀ values obtained in each artery before and after the addition of antagonist, respectively (Corriu et al., 1995). Data were expressed as mean ± S.E.M. One-way analysis of variance followed by Newman-Keuls multiple comparisons and Student's t test for paired observations was used for statistical evaluation. P < .05 was considered statistically significant.

	Results

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Effects of losartan on U46619-induced contraction in coronary artery rings. The TxA₂ analog, U46619, caused concentration-dependent contractions in canine coronary artery rings. Figure 1 illustrates a typical response in which addition of 10⁶ M losartan reversed the contraction of U46619 and pretreatment with losartan shifted the concentration-response curve of U46619 to the right in a concentration-dependent manner without a change in the maximum constriction. The EC₅₀ of U46619 (10.2 ± 0.9 nmol/l) was shifted 3- and 13-fold by preincubation with 1 and 10 µmol/l of losartan, respectively (P < .001 as compared with control) (fig. 2A, table 1). The apparent dissociation constant K_B averaged 0.5 ± 0.1 and 0.8 ± 0.1 µmol/l in the presence of 1 and 10 µmol/l of losartan, respectively. Losartan at 10⁴ M concentration completely reversed the constriction of 10 nM U46619 (Li, P., Ferrario, C. M. and Brosnihan, K. B., unpublished observations ). The potent, selective TxA₂/PGH₂ receptor antagonist SQ29548 markedly shifted the concentration-response curves of U46619 to the right [EC₅₀, 10.6 ± 0.9 (control) vs. 199.1 ± 17.7 and 617.3 ± 63.8 nmol/l at 0.01 and 0.1 µmol/l of SQ29548, respectively] without changing the maximum contraction (fig. 2B). Pretreatment with 1 µmol/l of SQ29548 abolished the contractile responses of U46619 at the concentrations tested (Li, P., Ferrario, C. M. and Brosnihan, K. B., unpublished observations). The potency of antagonism of 0.1 µmol/l SQ29548 was 31-fold greater than losartan at equimolar concentrations [EC₅₀, 19.7 ± 2.8 (losartan) vs. 617.3 ± 63.8 (SQ29548) nmol/l).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Typical dose-response contractions of U46619 during control conditions and after pretreatment with 106 mol/l losartan. Figure in upper left-hand corner shows a contractile response to 108 mol/l U46619, which is reversed by the addition of losartan (106 mol/l).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   (A) Average dose-response contraction curve to U46619 during control conditions (n = 12 dogs) and after pretreatment with increasing concentrations of losartan (108-105 mol/l) (n = 4-6 dogs). (B) Average dose-response contraction curve to U46619 during control conditions (n = 12 dogs) and after pretreatment with increasing concentrations of SQ29548 (108 and 107 mol/l) (n = 4-6 dogs). Values are mean ± S.E.M.

Selectivity of nonpeptide Ang II antagonists on the TxA₂/PGH₂ receptor. Pretreatment with the Ang II AT₁ receptor antagonist CV11974, the active form of TCV-116, or the AT₂ antagonist PD123319 for 30 min. did not change the concentration-dependent response curves to U46619 at 1 µmol/l [EC₅₀, 10.6 ± 0.9 (control) vs.12.6 ± 1.7 (CV11974) and 12.9 ± 0.7 (PD123319) nmol/l, P > .05 as compared with control) (fig. 3, table 2). In contrast, 1 µmol/l EXP3174, the active metabolite of losartan at the AT₁ receptor, significantly shifted the concentration-response curve of U46619 to the right (P < .001 as compared with control). In addition, EXP3174 was more potent than losartan (EC₅₀, 33.4 ± 4.6 vs. 66.9 ± 9.2 nmol/l, losartan vs. EXP3174, P < .01). None of the nonpeptide Ang II receptor antagonists changed the maximum constriction response to U46619 (table 2). In addition, the nonselective peptide Ang II receptor antagonist Sar¹Thr⁸-Ang II at 1 µmol/l did not affect the U46619-induced contraction of coronary arteries.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Average dose-response contraction curve to U46619 during control (n = 12 dogs) and after pretreatment with the Ang II AT1 receptor antagonist CV11974, the AT2 antagonist PD123319, the active metabolite of losartan EXP3174 and the nonselective peptide Ang II receptor antagonist Sar1Thr8-Ang II. All Ang II antagonists were used at 106 mol/l concentration. (n = 3-6 dogs). Values are mean ± S.E.M.

Specificity of losartan for vasoconstrictor-induced contraction in coronary arteries. Losartan pretreatment (1 µmol/l) did not affect the coronary vasoconstrictor responses to either PDGF (fig. 4) or the smooth muscle cell depolarizing agent KCl (fig. 5). Phenylephrine and arginine vasopressin showed minimal contractile responses in canine coronary arteries, and losartan did not change these contractile effects. However, in preliminary studies, losartan (10⁵ mol/l) blocked the TxA₂/PGH₂ receptor agonist PGF₂-induced dose-dependent vasoconstriction (EC₅₀, 23.4 ± 3.4 vs. 205.1 ± 23.5 nmol/l; maximal contraction, 5.0 ± 0.2 vs. 4.9 ± 0.3 g without and with losartan, n = 2).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Pretreatment with losartan (106 mol/l) did not affect the coronary constrictor responses to PDGF (10 and 20 ng/ml). Values are mean ± S.E.M. Studies were conducted in four dogs.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Pretreatment with losartan (106 mol/l) did not affect the coronary constrictor response to KCl (10- 80 mmol/l). Maximal contraction was 5.69 ± 0.26 vs. 6.1 ± 0.28 g, control vs. losartan treated. Values are mean ± S.E.M. Studies were conducted in six dogs.

Effects of losartan on the production of vasoactive prostaglandins and nitric oxide. Copreincubation of 10 µmol/l of the cyclo-oxygenase inhibitor indomethacin with 1 µmol/l of losartan for 30 min did not significantly shift the concentration-response curve of U46619 as compared with pretreatment with 1 µmol/l of losartan alone (fig. 6A). On the other hand, U46619-preconstricted rings (10 nmol/l) were dilated by losartan in a dose-dependent manner. Neither removal of the endothelium nor pretreatment with NO synthase inhibitor, L-NAME (10⁴ mol/l), abolished the relaxation induced by losartan (fig. 6B). There were no differences in the IC₅₀ of losartan-induced vasodilation [1.4 ± 0.2 (intact) vs. 1.3 ± .4 (denuded) and 1.1 ± .0.2 (L-NAME treated) µmol/l, P > .05]. On the other hand, in intact coronary rings preconstricted with 40 mmol/l KCl, losartan (10¹⁰-10⁵ mol/l) had no vasodilatory effects on the vascular rings (Li, P., Ferrario, C. M. and Brosnihan, K. B., unpublished observations).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   (A) Effect of copretreatment with losartan and indomethacin on U46619 dose-response curve. There is no difference in coronary constrictor responses to U46619 (1010-106 mol/l) when copretreated with losartan (106 mol/l) and the cyclo-oxygenease inhibitor indomethacin (105 mol/l) as compared with pretreatment with losartan (106 mol/l) alone. (B) Effect of endothelium denudation (denuded ED) or treatment with L-NAME on losartan dilation of coronary vessels preconstricted with 10 nM U46619. Studies were conducted in six to eight dogs. Values are mean ± S.E.M.

	Discussion

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Losartan, the nonpeptide Ang II AT₁ antagonist, inhibits TxA₂ analog U46619-induced contractions of canine coronary arteries without changing the maximal contractile response. These findings are consistent with losartan acting as a competitive antagonist of the TxA₂/PGH₂ receptor in canine coronary arteries. The active metabolite of losartan, EXP3174, also antagonized the TxA₂/PGH₂ receptor-mediated contractions in coronary arteries. In contrast, two other AT₁ receptor antagonists (CV11974 and Sarthran) and an AT₂ antagonist (PD123319) did not interact with the TxA₂/PGH₂ receptor in coronary vessels. The significant shift in the dose-response curve produced by losartan was not mediated by release of either prostaglandins or nitric oxide from the coronary arteries, because the responses were not altered by preincubation with inhibitors of cyclo-oxygenase or nitric oxide synthase. These data suggest that both losartan and its active metabolite EXP3174 bind to both the AT₁ and the TxA₂/PGH₂ receptors, a finding that calls attention to a separate and possibly complementary action of losartan potassium on proatherogenic events which result from platelet aggregation and plaque rupture (Berliner et al., 1995).

In contrast to saralasin, the first characterized Ang II peptide antagonist (Pettinger et al., 1975), losartan is an orally active, nonpeptide AT₁ antagonist possessing no intrinsic agonist effects. Losartan potassium blocks most of the known Ang II-induced vasoconstrictor, dipsogenic, aldosterone and catecholamine stimulatory responses (Timmermans et al., 1995; Chiu et al., 1991). In addition, losartan possesses significant antihypertensive activity in most species studied (Wong et al., 1990a; Ohlstein et al., 1992; Moriguchi et al., 1994). Preclinical research showed that losartan reverses cardiac hypertrophy and vascular hyperplasia (Dostal and Baker, 1992; Dahlof, 1993). EXP3174, the in vivo active metabolite of losartan, is approximately 10- to 15-fold more potent than losartan and has a longer plasma half-life (Wong et al., 1990b). Evidence suggests that EXP3174 makes a major contribution to the long-lasting blood pressure-lowering effects of losartan (Wong et al., 1990b; Tallant and Ferrario, 1996).

Several studies indicate that Ang II blockade may not fully account for the long-term antihypertensive and antiproliferative actions of losartan. Ohlstein et al.(1992) reported a recovery of the pressor responses to both Ang I and Ang II during chronic therapy with losartan. More recent studies indicate that several non-Ang II-related actions of losartan may be involved in its prolonged blood pressure-lowering effects. Those studies suggested that losartan may stimulate the production of vasodilator prostaglandins and nitric oxide (Jaiswal et al., 1991; Cachofeiro et al., 1995), interact with alpha-1 receptor (Maeso et al., 1995), and block the tachykinin (Picard et al., 1995), imidazoline/guanidinium (Li et al., 1996) and TxA₂ receptors (Liu et al., 1992; Bertolino et al., 1994; Corriu et al., 1995). In agreement with those observations, we showed that the blocking actions of losartan on the TxA₂ receptor were quite specific, because administration of either the cyclo-oxygenase inhibitor, indomethacin, or a nitric oxide synthase inhibitor did not eliminate the antagonistic effect of losartan on the U46619-induced vasoconstriction. These results suggest that the competitive inhibitory effects of losartan on the TxA₂ receptor are not mediated by stimulation of vasodilators, prostaglandins or nitric oxide. Furthermore, losartan did not inhibit coronary artery vasoconstriction induced with PDGF, KCl, phenylephrine and vasopressin. In the rat brain neither losartan nor EXP3174 at 10 µmol/l were shown to be potent antagonists at the imidazoline/guanidinium receptor sites (Li et al., 1996). Taken together, these findings are consistent with a specific effect of losartan on the TxA₂/PGH₂ receptor as reported in previous studies (Liu et al., 1992; Bertolino et al., 1994; Corriu et al., 1995).

The structural requirements necessary for antagonism of the TxA₂/PGH₂ receptor are different than those involved in antagonism of the AT₁ receptor, because we demonstrated that losartan, but not another AT₁ receptor antagonist, CV 11974, or the nonselective angiotensin II antagonist Sar¹Thr⁸-Ang II were effective in blocking U46619-induced vasoconstrictor responses. In agreement with other studies demonstrating that EXP3174 is more potent than losartan at the AT₁ receptor (Timmermans et al., 1993; Wong et al., 1990b), our study did prove that EXP3174 is a more potent antagonist than losartan for U46619-induced contraction of canine coronary artery rings. These findings are consistent with studies which demonstrated that the metabolite acts as a selective, noncompetitive receptor antagonist which dissociates from its receptor slowly and is more potent than losartan (Sachinidis et al., 1993; Wong et al., 1990b). With use of rat denuded aorta and small mesentery vessels, Corriu et al. (1995) showed that losartan had an inhibiting effect on U46619-induced contractile response. In their studies, however, EXP3174 at 3, 10, and 30 µmol/l was without effect, which suggests that the structural requirements for this antagonistic action may be different for the TxA₂/PGH₂ receptor in the rat.

Both losartan and EXP3174 have a benzylimidazole moiety, with EXP3174 differing from losartan only by its being a diacidic metabolite of losartan. CV11974 is also a biphenyl tetrazole; however, the imidazole ring is fused to another heterocyclic ring which possesses a carboxylic acid at position 7. Our studies suggest that the extra phenyl ring of CV11974 either reduces or prevents the binding of this AT₁ antagonist at the TxA₂/PGH₂ receptor. Thus, our studies indicate that differences in the structure-activity of AT₁ receptor antagonists determine the ability of biphenyl tetrazole to bind to the TxA₂ receptor.

Recent studies have linked TxA₂ and PGH₂ to mechanisms associated with renin-dependent and angiotensin-induced hypertension. The selective antagonist of the TxA₂/PGH₂ receptor SQ29548 was reported to elicit a 20 mm Hg decrease in blood pressure in rats with either aortic coarctation-induced hypertension or Ang II-induced hypertension (Lin et al., 1991; Mistry and Nasjletti, 1988). In these experiments, administration of an angiotensin antagonist reduced blood pressure by 60 mm Hg. This suggests that in high renin models of hypertension, TxA₂ contributes about 30% to the elevation of blood pressure. An increase in the renal excretion of TxA₂ and its production in blood vessels was found in several models of hypertension, including renal hypertension and SHR (Lin et al., 1994; Konieczkowski et al., 1983). In addition, it has been shown that TxA₂ and PGH₂ are endothelium-derived contracting factors and that these factors contribute to the evolution of hypertension in SHR (Dai et al., 1992; Kato et al., 1990).

In our study, losartan blocked the TxA₂/PGH₂ receptor of canine coronary arteries as a competitive antagonist with K_B values similar to those determined by Corriu et al. (1995) in rat aorta and mesenteric arteries. This indicates that the apparent affinity of losartan for the TxA₂ receptor was approximately 1000-fold lower than that for the AT₁ receptor (Rhaleb et al., 1991). Our studies agree with observations by Liu and colleagues (1992), who reported that losartan was a competitive ligand at human platelet TxA₂ receptor with a K_d value of 9.6 µM. An effect of losartan on TxA₂ receptor cannot be completely excluded after in vivo administration of a large concentration (Wong et al., 1990a; Ohlstein et al., 1992; Cachofeiro et al., 1995; DeGraaff et al., 1993). In the rat circulation, the concentration of losartan was estimated to reach approximately 250 µmol/l after a 10 mg/kg i.v. injection (Liu et al., 1992; Corriu et al., 1995). In humans, the concentration of losartan and its active metabolite EXP3174 was 250-550 ng/ml and 500-800 ng/ml (approximately 1 µmol/l) after an oral therapeutic dose of losartan (80-120 mg oral dose) (Munafo et al., 1992). From both the rat and the human studies, we estimate that the circulating values obtained by others after losartan administration are consistent with the concentration range of the binding and functional inhibition constants at the TxA₂/PGH₂ receptor. These findings suggest that the antagonistic effect of losartan and its active metabolite EXP3174 on the vascular TxA₂/PGH₂ receptor may contribute to the prolonged blood pressure reduction during long-term antihypertensive treatment. Thus losartan with its actions on the TxA₂/PGH₂ receptor may have therapeutic advantages over a more selective AT₁ angiotensin receptor antagonist in preventing the vasoconstrictor and platelet aggregating actions of TxA₂.