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Losartan Inhibits Thromboxane A2-Induced Platelet Aggregation and Vascular Constriction in Spontaneously Hypertensive Rats

Li, Ping; Ferrario, Carlos M.; Brosnihan, K. Bridget

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Journal of Cardiovascular Pharmacology: August 1998 - Volume 32 - Issue 2 - p 198-205
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Abstract

Losartan is a potent nonpeptide, selective angiotensin II (Ang II) AT1-receptor antagonist possessing no intrinsic effects. It is the first orally active AT1-receptor blocker that has been proven clinically effective in the management of hypertension (1-3). Losartan produces concentration-dependent inhibition of Ang II-induced vasoconstriction in vitro and in vivo and displaces the binding sites of Ang II in many tissues (4-6). Losartan efficiently reduces the increased blood pressure in hypertensive patients, as well as in hypertensive animal models, such as renal hypertensive rats, spontaneously hypertensive rats (SHRs), and transgenic hypertensive rats (3,4,7). Short-term administration of losartan is highly effective in reducing blood pressure in SHRs, a genetic model of hypertension in which plasma renin is not increased (8,9). However, it has been noted that in this same hypertensive model, the peptide Ang II antagonist saralasin and angiotensin-converting enzyme (ACE) inhibitors are not so effective as losartan after their short-term administration (8,10,11). These studies suggest that although the antihypertensive action of losartan is attributed mainly to its ability to antagonize AT1 receptors, additional mechanisms may be involved in this effect. In agreement with this suggestion are the findings of Grove and Speth (12), who reported that [3H]-losartan binding sites are greater in density than those of labeled Ang II in different tissue preparations, and Ohlstein et al. (8), 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. Finally, losartan may release vasodilatory NO and prostaglandins, which can contribute to its prolonged blood pressure-reducing effects in SHRs (9,13).

Recent studies from our laboratory have shown that losartan and its active metabolite EXP3174 interact with thromboxane A2/prostaglandin H2 (TxA2/PGH2) receptors in canine coronary arteries (14). These observations are consistent with earlier reports showing losartan blocking TxA2-mediated pulmonary hypertension in rats (15), vasoconstriction in rat aorta and mesenteric vessels (16), and platelet aggregation in humans (17). The adult SHR has been shown to release an endothelium-derived constricting factor (EDCF) in response to acetylcholine (ACH) stimulation, limiting the vasodilation of ACH, and actively vasoconstricting the blood vessel at higher concentrations of ACH (18-20). Since it has been suggested that TxA2 and/or PGH2 are likely candidates for this response (19,21), we investigated whether losartan could effectively block EDCF in blood vessels from aged SHRs and whether losartan and its active metabolite EXP3174 participate in TxA2/PGH2-mediated vasoconstriction and platelet aggregation of SHRs.

METHODS

Platelet aggregation

After approval by the Institutional Animal Care and Use Committee, 22 male SHRs (32-34 weeks old; body weight, 350-400 g; and systolic blood pressure, 210 ± 10 mm Hg) from the Charles River Laboratories (Wilmington, MA, U.S.A.) were anesthetized with sodium pentobarbital (50 mgkg, i.p.). Blood was directly drawn from the abdominal aorta into a tube containing sodium citrate buffer (1:9, vol/vol). The anticoagulated blood was centrifuged at 200 g for 10 min to obtain platelet-rich plasma (PRP). The platelet-aggregation method was modified from Yokoyama et al. (22). In brief, the PRP was centrifuged at 1,000 g for 20 min, and the supernatant was discarded. The platelets were suspended in washing buffer at a volume equal to the original plasma volume and centrifuged at 1,000 g for 20 min. The platelets were resuspended in a suspension buffer and adjusted to a concentration of 5 × 108 cells/ml. The aggregation of platelets was monitored with an aggregometer (Bio-Data, Philadelphia, PA, U.S.A.), and CaCl2 solution (1 mM, final concentration) was added 1 min before stimulation. All procedures were carried out at room temperature. The washing buffer contained 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 12 mM NaHCO3, 0.4 mM NaH2PO4, 0.05 mM trisodium citrate, 0.1% glucose (wt/vol), and apyrase, 0.5 U/ml. The pH was adjusted to 6.5 with HCl. The suspension buffer contained 113 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 24 mM NaHCO3, 10 mM HEPES-NaOH (pH 7.4), and 0.1% glucose.

Vascular ring reactivity

The thoracic aorta was removed and dissected free of connective tissues on ice. The vessels were cut into 4-mm rings and mounted in organ chambers filled with modified Krebs-Henseleit buffer (composition in mM: NaCl, 118.3; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25; CaNaEDTA, 0.026; and glucose, 11). The solution was aerated with 95% O2 and 5% CO2 at 37°C (pH 7.4). In some rings, the endothelium was denuded by gentle mechanical rubbing with a stainless steel wire. Isometric tension was measured continuously by using a polygraph (Model 7; Grass Instruments Inc., Quincy, MA, U.S.A.). Basic tension was set at 1.5 g after a 60-min equilibration period. In preliminary experiments after repeated exposure to 40 mM KCl, we established that 1.5 g of tension was optimal for the aorta rings from the 32- to 34-weeks-old SHRs. The endothelial integrity was tested by using 10−7M ACH in rings preconstricted with 10−7M norepinephrine (NE). The presence of a functional endothelium was associated with >30-50% relaxation to ACH, whereas the absence of relaxation by using the same dose of ACH indicated effective removal of the endothelium.

Experimental protocol

To elicit platelet aggregation, washed platelets were exposed to the TxA2 analog U46619 (10−7−10−5M) as a control response. The platelets were then pretreated for 5 min with losartan (10−5−10−4M) and another AT1-receptor antagonist, CV11974 (10−5M), the active form of TCV-116 (23), and then U46619-induced platelet aggregation was repeated.

To determine whether losartan has an effect on the release of EDCF in aged SHR vessels, aortic rings were preconstricted with NE (10−7M), and then ACH was added to the chamber at cumulative concentrations of 10−8−10−5M. Losartan (10 μM) and the cyclooxygenase inhibitor indomethacin (Indo; 1 μM) were used to pretreat intact rings for 30 min, and the concentration-dependent responses for ACH were then produced.

To determine whether losartan interacts with the TxA2/PGH2 receptor of SHR aortic vessels, control cumulative concentration-contractile response curves for U46619 (10−10−10−6M) were generated after 1-h equilibration in intact quiescent rings. Losartan (10−6−10−5M) was used to pretreat the aorta rings for 30 min, and the concentration-response curves for U46619 were then repeated. In additional studies, the potent, selective TxA2/PGH2 receptor antagonist SQ29,548 (24) was administered for 30 min to demonstrate that the U46619 concentration-response curves could be blocked at the TxA2 receptor. To ascertain whether the interaction of losartan with U46619 is dependent on the endothelium or NO, the concentration-response curves for U46619 were performed in endothelium-denuded vessels or vascular rings pretreated with the NO synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME; 10−4M) in the absence and presence of losartan (10−6M).

To evaluate whether other nonpeptide Ang II-receptor subtype antagonists interact with the TxA2/PGH2 receptors in SHR vessels, another selective Ang II AT1-receptor antagonist CV11974, the Ang II AT2-receptor antagonist PD123319, and an active metabolite of losartan, EXP3174 (each at 10−6M concentration) were chosen. Rings were pretreated for 30 min, and then concentration-response curves for U46619 were generated. The ACE inhibitor lisinopril (10−6M) also was tested. To determine whether losartan interacts with other vasoconstrictors, concentration-response curves using NE (10−9−10−4M) and arginine vasopressin (10−10−10−6M) also were constructed in the absence and presence of losartan (10−6M) in isolated aorta rings. The antagonists tested had no effects on the basal vascular tone, and each ring was used only once for the antagonist study. A 60-min incubation was allowed between observations.

Chemicals and drugs

Losartan and EXP3174 were generous gifts from DuPont Merck Company (Wilmington, DE, U.S.A.). PD 123319 was generously supplied by Parke-Davis Inc. (Ann Arbor, MI, U.S.A.) and CV11974 by Takeda Chemical Industries, Ltd. (Osaka, Japan). Nω-nitro-L-arginine methyl ester and SQ29,548 were purchased from Research Biochemicals International (Natick, MA, U.S.A.). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Indomethacin, CV11974, and EXP3174 were dissolved in 0.2N Na2CO3 solution and diluted with Krebs buffer. U46619 and SQ29,548 were prepared as stock in ethanol and diluted with Krebs buffer. The concentrations of drugs reported are at final concentration in chambers.

Data analysis and statistics

Vascular relaxation was expressed as percentage of isometric tension of rings preconstricted with 10−7M NE, and constriction was normalized as percentage of the maximal contraction. The concentration of drugs causing 50% of the maximal contraction (EC50) was calculated by using a nonlinear regression sigmoid curve-fitting program of PRISM (Graphpad Inc., San Diego, CA, U.S.A.). Data are expressed as mean ± SEM. (standard error of mean). One-way analysis of variance (ANOVA) followed by Newman-Keul's multiple comparisons and Student's t test for paired observations were used for statistical evaluation. A value of p < 0.05 was considered statistically significant.

RESULTS

Inhibition of losartan on U46619-induced platelet aggregation in SHRs

Figure 1A-C shows the typical aggregating responses of washed platelets stimulated with increasing doses of the thromboxane A2 agonist, U46619, and the attenuation of this response when the platelets are pretreated with losartan (10−5M;Fig. 1D-F). The average data are shown in Fig. 2. Losartan, but not the AT1-receptor antagonist, CV11974, attenuated U46619-induced platelet aggregation. At 10−4M concentration of losartan, the U46619-induced platelet-aggregation response was nearly abolished (95% inhibition, n = 2).

FIG. 1
FIG. 1:
Typical traces of concentration-dependent washed platelet aggregation induced with U46619 during control conditions (A-C) and after pretreatment with 10 μM losartan (D-F).
FIG. 2
FIG. 2:
M) before (CONTROL) and after pretreatment with losartan (10 μM) and CV11974 (10 μM). Platelets were obtained from eight rats in the control group and from five to six rats in the treated groups. *p < 0.05, **p < 0.01, compared with control).

Effect of losartan on the endothelium-dependent responses of ACH in aortic rings of adult SHRs

Figure 3 shows typical ACH-mediated vasoactive responses in NE (10−7M) preconstricted aorta rings of adult SHRs in the presence and absence of losartan (10−5M). At low concentrations (10−8−10−7M), ACH caused vasorelaxation, whereas at high concentrations (10−6−10−5M), ACH caused vasoconstriction. Removal of the endothelium abolished the ACH-induced vasodilation and vasoconstriction (data not shown), findings consistent with the release of endothelium-dependent NO and EDCF, respectively (25-27). Pretreatment with 10 μM losartan augmented the ACH-induced vasodilation and abolished the ACH-mediated vasoconstriction. The average data are shown in Fig. 4, which also demonstrates that pretreatment with the cyclooxygenase inhibitor indomethacin (1 μM) had no effect on ACH vasodilation at low concentrations (10−8−10−7M), but blocked the vasoconstriction and enhanced the vasodilation at high concentration (10−6−10−5M) ACH.

FIG. 3
FIG. 3:
Typical recordings of acetylcholine (ACH)-elicited endothelium-dependent vasodilation and vasoconstriction in norepinephrine (NE; 10−7 M) preconstricted aortic rings of adult spontaneously hypertensive rats (SHRs) before (A) (control) and after losartan (B) (10 μM) pretreatment. Losartan enhanced the vasodilation and abolished the vasoconstriction.
FIG. 4
FIG. 4:
Average acetylcholine (ACH)-induced responses during control conditions and after pretreatment with losartan (10 μM) and indomethacin (Indo, 1 μM). Control groups contain 23 rings from 10 rats and the treated groups had six to 10 rings from four to six rats.

Effects of losartan on U46619-induced contraction in aorta rings

Increasing cumulative doses of U46619 resulted in concentration-dependent contractions in aorta rings (Fig. 5A). Pretreatment with losartan shifted the concentration-response curve of U46619 to the right in a dose-dependent manner without changes in the maximal constriction. The EC50 of U46619 (15.84 ± 2.02 nM) was changed 2.5- and 7.6-fold by preincubation with 1 and 10 μM of losartan, respectively (p < 0.001 as compared with control; Fig. 5A; Table 1). Losartan at a concentration of 100 μM almost abolished U46619-induced contractile response in aortic rings (data not shown). The potent, selective TxA2/PGH2-receptor antagonist SQ29,548 at a concentration of 0.1 μM markedly shifted the concentration-response curves of U46619 to the right [EC50, 15.84 ± 2.02 nM (control) vs. 175 ± 5.93 nM (SQ29,548, 0.1 μM), respectively] without significantly changing the maximal contraction (Fig. 5B). Pretreatment with 1 μM SQ29,548 nearly abolished the contractile responses of U46619 (data not shown). In endothelium-denuded and L-NAME (10−4M)-treated aortic rings, losartan at 1 μM also significantly shifted the concentration-response curves to the right without a change of maximal contraction (EC50, 17.04 ± 2.15 vs. 44.03 ± 6.89 nM; 12.87 ± 1.53 vs. 44.75 ± 7.11 nM, respectively; p < 0.01, as compared with control; Fig. 6A and B).

FIG. 5
FIG. 5:
Average cumulative dose-response curves to U46619 (10−10−10−6 M) during control conditions and in the presence of 1 and 10 μM losartan (Los) (A). The selective thromboxane A2/prostaglandin H2 (TxA2/PGH2) antagonist SQ29,548 (SQ, 0.1 μM) markedly shifted concentration-response curves of U46619 to the right (B). Control groups contain 20 rings from 12 rats and the treated groups had eight to 12 rings from four to six rats.
TABLE 1
TABLE 1:
Effects of antagonists and ACE inhibitor on the U46619-induced contraction in aortic vessels of SHR
FIG. 6
FIG. 6:
Average concentration-response curves of U46619 in endothelium-denuded (A) and N ϑ-nitro-L-arginine methyl ester (L-NAME)-treated (B) aorta rings in the absence and presence of 1 μM losartan. Control and the treated groups each had eight to 10 rings from five to six rats.

Selectivity of nonpeptide Ang II antagonists on the vascular 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 μM [EC50, 15.84 ± 2.02 nM (control) vs. 17.36 ± 3.33 nM (CV11974) and 14.46 ± 1.54 nM (PD123319); p > 0.05 as compared with control; Fig. 7A). In contrast, 1 μM EXP3174, an active metabolite of losartan at the AT1 receptor, significantly shifted the concentration-response curve of U46619 to the right (EC50, 15.84 ± 2.02 vs. 55.52 ± 6.98 nM; p < 0.001 control vs. EXP3174). In addition, EXP3174 was more potent than losartan (EC50, 38.28 ± 2.89 vs. 55.52 ± 6.98 nM, losartan vs. EXP3174; p < 0.05). None of the nonpeptide Ang II-receptor antagonists changed the maximal concentration response to U46619 (Table 1). In addition, the ACE inhibitor lisinopril at 1 μM did not affect the U46619-induced contraction of aorta vessels (EC50, 15.84 ± 2.02 vs. 20.93 ± 3.04 nM; p > 0.05, control vs. lisinopril; Fig. 7B; Table 1).

FIG. 7
FIG. 7:
1-receptor antagonists [CV11974 (CV), Losartan (Los), EXP3174 (EXP)], an AT2-receptor antagonist PD123319 (PD) (A) and an angiotensin-converting enzyme (ACE) inhibitor lisinopril (Lis; B). Control groups as described in Fig. 5 and treated group had eight to 10 rings from four to six rats.

Specificity of losartan for vasoconstrictor-induced contraction in aorta

Losartan pretreatment (1 μM) did not affect either NE (10−9−10−4M; EC50, 13.22 ± 2.09 vs. 14.76 ± 2.76 nM, control vs. losartan; Fig. 8A) or arginine vasopressin (10−10−10−6M) induced dose-dependent vasoconstrictor responses (EC50, 10.24 ± 0.56 vs. 12.92 ± 0.31 nM, control vs. losartan; Fig. 8B). In the presence of losartan pretreatment, the maximal contraction was unchanged for both vasoconstrictors [1.23 ± 0.13 vs. 1.15 ± 0.08 g, control vs. losartan (NE); 1.68 ± 0.23 vs. 1.59 ± 0.08 g, control vs. losartan (AVP), respectively].

FIG. 8
FIG. 8:
Average concentration-response curves to norepinephrine (A) and arginine vasopressin (B) in the absence and presence of 1 μM Losartan (Los). Control group had four to eight rings from four to six rats.

DISCUSSION

We report that losartan, the nonpeptide Ang II AT1 antagonist, inhibits TxA2 analog U46619-induced platelet aggregation and vascular contractions of SHRs without changing the maximal contractile response. These findings are consistent with losartan acting as a competitive antagonist of TxA2/PGH2 receptors in platelets and arterial vessels. Moreover, we showed in aorta rings of the adult SHRs that losartan can enhance ACH-induced vasodilation and block ACH-induced, EDCF-mediated vasoconstriction, actions attributed to TxA2/PGH2(18,19). The active metabolite of losartan, EXP3174, more potently blocked TxA2/PGH2 receptor-mediated contractions in aortic tissue as compared with losartan. In contrast, the nonpeptide Ang II AT1 antagonist, CV11974, did not interact with the TxA2/PGH2 receptor in platelets and arterial vessels of SHRs. In addition, neither PD123319, a nonpeptide Ang II AT2 antagonist, nor lisinopril, an ACE inhibitor, affected TxA2/PGH2 receptor-mediated contractile responses in arterial vessels. These data suggest that losartan, in addition to antagonizing Ang II-induced vasoconstriction, may block the TxA2/PGH2 receptor in blood vessels and platelets and thus participate in the prolonged blood pressure-reducing effects and antiatherogenic actions of losartan.

Losartan is an orally active, nonpeptide AT1-receptor antagonist, without intrinsic agonist effects. It blocks Ang II-induced vasoconstriction, dipsogenic responses, and aldosterone and catecholamine secretion (4,28). In addition, losartan reduces high blood pressure in most species studied (1,7,8,29). EXP3174, the in vivo active, hepatic metabolite of losartan after oral administration, is ∼10- to 15-fold more potent than losartan for antagonizing AT1 receptors and has a longer plasma half-life. EXP3174 contributes to the long-term antihypertensive effect of losartan (30,31). Our study demonstrated that in rat vascular rings of SHRs, EXP3174 is a more potent antagonist than losartan for U46619-induced contraction. These studies are in agreement with our previous findings in canine coronary rings (14). Thus antagonism of TxA2 receptors by EXP3174 may also contribute to the longer-lasting blood pressure-reducing effects of losartan.

Recent studies indicated that the pharmacologic actions of losartan may not be solely related to angiotensin-receptor antagonism. Ohlstein et al. (8) demonstrated that the antihypertensive action of losartan was present in the presence of normal pressor responses to Ang I and II during recovery from losartan treatment. More recent studies indicated that several non-Ang II-related actions of losartan may be involved in its prolonged blood pressure-reducing effects. Thus studies suggested that losartan may stimulate the production of vasodilator prostaglandins and NO (9,13), interact with an α1 receptor (32), and block TxA2/PGH2 receptors (15-17). In agreement with the previous studies from our laboratories (14), we found that losartan blocks the TxA2/PGH2 receptor of aorta of SHRs in a dose-dependent manner. The blocking actions of losartan on the TxA2/PGH2 receptor were quite specific, because removal of endothelium and administration of NO synthase inhibitor did not affect the antagonist effects of losartan on U46619-induced vasoconstriction. These results suggest that the effects of losartan on the TxA2/PGH2 receptor in vessels are not mediated by products released from the endothelium, and specifically, NO release is not involved. Similarly, losartan did not inhibit vasoconstriction induced with NE and vasopressin. These findings are consistent with the specificity of losartan for the TxA2/PGH2 receptor, as reported in previous studies (14-17).

Recent studies linked TxA2 and PGH2 to mechanisms associated with renin-dependent and angiotensin-induced hypertension. Selective antagonists of TxA2/PGH2 receptors were reported to reduce the blood pressure in rats with aortic coarctation-induced hypertension at early stages and in rats with Ang II-induced hypertension (33-36). Augmentation of TxA2 metabolite production in the urine and blood vessels was found in several models of hypertension, including renal hypertension and SHRs (21,37). In addition, it was shown that EDCF is released from the aorta of adult SHRs and WKY rats, that it is a cyclooxygenase product (19,25), and that TxA2/PGH2-receptor antagonists block ACH-induced contraction (18,19). These findings taken together would suggest that TxA2/PGH2 are the most likely candidates to account for EDCF, but this is not without controversy (38,39). The observation in our study that indomethacin blocked EDCF and augmented the ACH-induced vasodilation is consistent with the cyclooxygenase dependence of EDCF. Furthermore, although we did not directly determine whether EDCF is TxA2/PGH2, our demonstration of the specificity of losartan as an antagonist of U46619-induced contraction in aortic rings of SHRs lends further support that EDCF mediates its responses through a TxA2/PGH2 receptor, which is capable of being blocked by losartan.

Our experimental studies suggest that losartan may not only counteract the pathologic actions of Ang II on the evolution of arterial hypertension but also may have an additional and separate action in preventing platelet-aggregating actions of TxA2/PGH2. The parallel demonstration that this property of losartan is not shared by CV1994, another newly introduced Ang II antagonist, which is the active form of candesartan, may confer losartan with an additional and important role in cardiovascular disease management. The findings that losartan competitively antagonizes U46619-induced platelet aggregation were first reported by using human platelets (17). We were able to demonstrate that losartan blocked platelet aggregation from SHRs in response to U46619 in a concentration-dependent manner. Inhibition of platelet aggregation by losartan may have important therapeutically desirable effects of preventing adhesion to components of the vascular subendothelium.

An important question in these studies is whether losartan with dual antagonistic actions functions physiologically at TxA2/PGH2 receptors, as well as at the AT1 receptor. In the rat, the circulating levels of losartan are 250 μM after a 10 mg/kg i.v. injection (16,17). Based on the affinity of losartan for TxA2 receptors in platelets with a Kd value of 9.6 μM(17) and in canine coronary arteries with a Kd value of 1 μM(14), it appears that the concentration of drug in the circulation would be sufficient to compete at the TxA2/PGH2 receptor in vivo. Furthermore, because the metabolite of losartan, EXP3174, is even more potent than losartan at the TxA2/PGH2 receptor in rat blood vessels, our studies suggest that in vivo EXP3174, which reaches plasma levels equivalent to those of losartan within minutes but has a longer half-life (40), may exert more substantial effects than losartan at the TxA2/PGH2 vascular receptor.

In conclusion, the important role of the new class of Ang II antagonists in the treatment of arterial hypertension was amply demonstrated by the overwhelming therapeutic effectiveness that losartan had in patients with mild to moderate forms of essential hypertension (41,42). The studies described suggest that an important and independent protective action of losartan is that it can prevent TxA2/PGH2 receptor-mediated increases in vasoconstriction and platelet aggregation. These new findings suggest that blockade of the vascular TxA2/PGH2 receptor by losartan may extend the long-term antihypertensive actions of this agent by preventing the vasoconstrictor and thrombotic actions of prostanoid activation pathways in hypertension and coronary artery disease.

Acknowledgment: We thank Mr. Alan Berrier for his kind assistance in this study. This work was published as an abstract in Hypertension and supported in part by grants from the National Heart, Lung, Blood Institute P01 HL 51952 and the Merck Company Inc.

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Keywords:

Angiotensin II antagonist; Losartan; Prostaglandins; Thromboxane A2; Endothelium-derived contracting factor; Platelet; Endothelium; Aorta, SHR

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