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The Inhibitory Effect of KT3-671, a Nonpeptide Angiotensin-Receptor Antagonist, on Rabbit and Rat Isolated Vascular Smooth Muscles: A Possible Involvement of KATP Channels

Satake, Nobuhiro; Imanishi, Masami; Keto, Yoshihiro; Ishikawa, Makoto; Yamada, Hiroyuki; Shibata, Shoji; Tomiyama, Akira*

Journal of Cardiovascular Pharmacology: March 2000 - Volume 35 - Issue 3 - p 457-467
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The vasoinhibitory effect of KT3-671, a recently synthesized nonpeptide angiotensin II (Ang II), AT1-receptor antagonist, and the factors affecting insurmountable antagonism of Ang II were examined in rabbit and rat isolated vascular smooth muscle preparations. In rabbit and rat aortic rings, KT3-671 caused insurmountable antagonism of Ang II. In addition, KT3-671 inhibited contractile responses to angiotensin III (Ang III). In rabbit isolated smooth muscles, KT3-671 was most effective in reducing the maximal contraction induced by Ang II in the renal artery followed by the basilar artery and the aorta. In rat renal arterial rings, KT3-671 (10−5M) inhibited the concentration-response curves of prostaglandin F and STA2. In rabbit and rat aortic rings without endothelium, the insurmountable antagonisms of Ang II by KT3-671 and EXP 3174 were changed to surmountable antagonism by pretreatment with DuP 753 and KT3-671, respectively. In addition, KT3-671 abolished the inhibitory effect of CV-11974 in the rat aorta but not in the rabbit aorta. Indomethacin (10−5M) or the removal of endothelium did not affect the inhibitory effect of Ang II by CV-11974 or EXP 3174 but enhanced the insurmountable antagonism by KT3-671. ODQ (3 × 10−6M), NG-nitro-L-arginine (3 × 10−4M), 4-aminopyridine (3 × 10−3M), tetraethylammonium (TEA; 10−3M), or iberiotoxin (10−7M) did not affect the inhibitory action of KT3-671 or CV-11974. Methylene blue (3 × 10−6M), KCl (10−2M), TEA (10−2M), or BaCl2 (10−4M) changed the insurmountable antagonism by KT3-671 to surmountable antagonism and abolished the inhibitory effect of CV-11974. However, glibenclamide (3 × 10−6M) did not affect the inhibitory action of KT3-671 but reduced the insurmountable antagonism by CV-11974. These results indicate that KT3-671 is an insurmountable antagonist of Ang II in the rabbit and rat aorta. The results in the rat aorta also suggest that KATP channels may be involved in insurmountable antagonism of Ang II by KT3-671 and CV-11974.

Department of Pharmacology, University of Hawaii, School of Medicine, Honolulu, Hawaii, U.S.A.; and *Kotobuki Pharmaceutical Co. Ltd., Nagano, Japan

Received May 26, 1999; revision accepted November 15, 1999.

Address correspondence and reprint requests to Dr. S. Shibata at Department of Pharmacology, University of Hawaii, School of Medicine, Honolulu, HI 96822, U.S.A.

Present addresses: M. Imanishi, Department of Emergency and Critical Care Medicine Nara Medical University, Nara 634, Japan; Y. Keto, M. Ishikawa, and H. Yamada, Department of Applied Pharmacology, Kyoto Pharmaceutical University Kyoto 607, Japan.

The renin-angiotensin system (RAS) is one of the important elements in blood pressure regulation and electrolyte and fluid homeostasis (1). Angiotensinogen from the liver is cleaved by renin to produce angiotensin I (Ang I). Biologically inactive Ang I is cleaved by angiotensin-converting enzyme (ACE) to produce angiotensin II (Ang II). The success of ACE inhibitors such as captopril (2,3) and enalapril (4) in the treatment of hypertension and congestive heart failure has increased the interest in exploring new ways to interfere with the RAS cascade (5,6). Ang II can also be formed in vivo by the action of enzymes other than ACE (7). In addition ACE is a nonspecific protease that also is responsible for the hydrolysis of bradykinin, substance P, and enkephalins (8). The lack of specificity of ACE may account for some of the side effects such as dry cough (9) and angioedema (10) observed in the population treated with ACE inhibitors. These side effects have been attributed to bradykinin potentiation. (11-13).

The inhibition of Ang II receptor may offer a more specific approach to inhibition of the RAS. In addition, because the Ang II receptor antagonist would not affect ACE, the side effects observed during therapy with ACE inhibitors would not be expected during the therapy with the Ang II-receptor antagonist. Although potent peptide Ang II-receptor antagonists have been developed, they have limited therapeutic value because of the partial agonistic activities and lack of oral effectiveness. (14,15). Nonpeptide Ang II-receptor antagonists would be expected to lack the disadvantages of the peptide Ang II-receptor antagonists. Since the discovery of DuP 753, an orally active and nonpeptide Ang II-receptor antagonist (16), many other nonpeptide Ang II-receptor antagonists have been reported (17). 2-Propyl-8-oxo-1-[(2′-(1H-tetrazole-5-yl)biphenyl-4-yl)methyl]-4,5,6,7-tetrahydrocycloheptimidazole (KT3-671) is one of the potent nonpeptide Ang II-receptor antagonists (18,19). KT3-671 is a competitive, AT1-specific antagonist in Ang II-receptor binding assay (18,19). In the rabbit isolated aorta, low concentrations of KT3-671 shifted the concentration-contraction curve for Ang II to the right in a parallel fashion without affecting the maximal contraction (19). DuP 753, a prototype of nonpeptide Ang II-receptor antagonists, is a competitive inhibitor of Ang II-receptor binding and exhibits surmountable Ang II antagonism (10,16). EXP 3174, an active metabolite of DuP 753 (16), and CV-11974 also are competitive inhibitors of Ang II-receptor binding (16,20) but exhibit insurmountable Ang II antagonism (16,20). Many theories have been advanced in an attempt to explain the discrepancy in the mode of inhibitory action of some of the Ang II antagonists between the receptor-binding assay showing the competitive antagonism and the functional assay indicating the insurmountable antagonism.

Further evaluation of the pharmacologic properties revealed that KT3-671 is an insurmountable antagonist of the contractions induced by Ang II in the rat and rabbit aorta. Therefore we have studied the mode of Ang II antagonism by KT3-671 in comparison with other nonpeptide Ang II-receptor antagonists and also attempted to examine the factors affecting the insurmountable antagonism of Ang II by KT3-671.

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METHODS

Tissue preparations and recording of mechanical action

Thoracic aorta and renal artery were obtained from male New Zealand White rabbits weighing 2-3 kg and from male Wistar rats weighing 150-200 g. Basilar artery was obtained from male New Zealand White rabbits. Each animal was killed by rapid exsanguination under halothane anesthesia. Adhering fat and connecting tissues were removed, and ring preparations of aorta, renal artery, and basilar artery were prepared. Each preparation was placed in a 25-ml organ bath containing Krebs-Ringer solution (in mM: NaCl, 120.3; KCl, 4.8; CaCl2, 1.2; KH2PO4, 1.2; MgSO4, 1.3; NaHCO3, 24.2; glucose, 5.5) maintained at 37°C and bubbled with 95% O2 and 5% CO2. A resting tension of 2 g in the rabbit and rat aorta and renal artery and 0.5 g in the rabbit basilar artery was applied. The isometric tension was recorded through a force-displacement transducer (FT-03) connected to a six-channel Grass polygraph. Angiotensins were added cumulatively to induce contractions of the isolated vascular tissues, because we did not observe any difference in angiotensin-induced contractions between cumulative and noncumulative additions of angiotensins. The presence of endothelium was confirmed by the acetylcholine (10−6M)-induced relaxation (>80%) of the vascular tissues precontracted by phenylephrine (PE) (3 × 10−7M). The absence of endothelium was confirmed by the absence of the acetylcholine-induced relaxation.

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Chemicals

The following agents were used: KT3-671, DuP 753, EXP 3174, and CV-11974 (Kotobuki Seiyaku, Nagano, Japan); tetraethylammonium (TEA), methylene blue, Ang II, angiotensin III (Ang III), indomethacin, prostaglandin F (PGF), 4-aminopyridine (4-AP), and PE (Sigma, St. Louis, MO, U.S.A.); 9,11-epithio-11,12-methano-thromboxane A2 (STA2) (Ono Pharmaceutical, Osaka, Japan); glibenclamide (Upjohn Co., Kalamazoo, MI, U.S.A.); NG-nitro-L-arginine (NOARG), iberiotoxin (ibTX; RBI, Natick, MA, U.S.A.); (1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one) (ODQ; Tocris, Ballwin, MO, U.S.A.).

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Statistical analysis

Data are presented as the mean ± SEM and were analyzed using Student's t test. Differences were considered significant at p < 0.05. The pA2 value was determined according to the method described by Arunlakshana and Schild (21). The pD′2 value was calculated according to the method described by van Rossum (22).

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RESULTS

Effects of KT3-671 on ANG II- and ANG III-induced contractions in rabbit vascular smooth muscles

In rabbit aortic rings, Ang II (10−10 to 3 × 10−8M) induced contractile responses in a concentration-dependent manner (Fig. 1A). Pretreatment with KT3-671 (10−9-10−7M) for 30 min inhibited the responses to Ang II (Fig. 1A). Specifically, pretreatment by KT3-671 at 10−9, 10−8, or 10−7M shifted the concentration-response curve of Ang II to the right of the control, whereas pretreatment with KT3-671 at 10−7M also reduced the maximal contraction induced by Ang II (Fig. 1A). The apparent pA2 value for KT3-671 (10−9-10−8M) against Ang II was 9.4 ± 0.16, and the slope of the regression line was not significantly different from unity (1.0 ± 0.02; n = 5). The pD′2 value was calculated to be 6.45 ± 0.11 (Table 1).

FIG. 1

FIG. 1

TABLE 1

TABLE 1

In rabbit aortic rings, Ang III (10−9-10−5M) also induced contractile responses in a concentration-dependent manner (Fig. 1B). Pretreatment with KT3-671 (10−9-10−7M) for 30 min shifted the concentration-response curve of Ang III to the right without significantly reducing the maximal contraction (Fig. 1B). Specifically, the apparent pA2 value for KT3-671 against Ang III was 9.3 ± 0.21, and the slope of the regression line was not significantly different from unity (0.92 ± 0.06; n = 5).

The inhibitory effect of KT3-671 on the maximal contraction induced by Ang II also was compared among smooth muscle preparations isolated from the rabbit aorta, renal artery, and the basilar artery (Table 1). The pretreatment of the tissues with KT3-671 at 10−8M (10−7M in the aortic rings) inhibited the maximal response to Ang II (10−6M). Based on the pD′2 values, KT3-671 was most effective in the renal arterial rings as compared with the basilar arterial rings and the aortic rings.

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Effects of KT3-671 in rat vascular smooth muscles

In rat aortic rings, Ang II (3 × 10−9 to 10−7M) and Ang III (3 × 10−9-3 × 10−5M) induced contractile responses in a concentration-dependent manner (Fig. 2A and B). The pretreatment with KT3-671 (3 × 10−9 to 10−7M) for 30 min inhibited the responses to Ang II (Fig. 2A). Specifically, pretreatment with KT3-671 at 3 × 10−9, 10−8, or 10−7M shifted the concentration-response curve of Ang II to the right of the control and reduced the maximal contraction (Fig. 2A). The pD′2 value was calculated to be 7.43 ± 0.18 (n = 5). The pretreatment with KT3-671 (3 × 10−9 to 10−7M) for 30 min shifted the concentration-response curve of Ang III to the right (Fig. 2B). In the presence of KT3-671 at 10−7M, no plateau was reached by the highest concentration of Ang III that could be used in this study (Fig. 2B). The pA2 value for KT3-671 (3 × 10−9 and 10−8M) against Ang III was 8.62 ± 0.08, and the slope of the regression line was significantly different from unity (0.52 ± 0.10; n = 5).

FIG. 2

FIG. 2

In rat renal arteries, PGF (10−7 to 5 × 10−5M;Fig. 3A) and STA2 (10−9-10−7M;Fig. 3B) induced contractile responses in a concentration-dependent manner. Pretreatment of the aortic rings with KT3-671 at 10−5M, but not 10−6M, inhibited the responses induced by PGF(Fig. 3A) and STA2(Fig. 3B).

FIG. 3

FIG. 3

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Interactions among KT3-671, DuP 753, EXP 3174, and CV-11974 in the rabbit aorta without endothelium

In the rabbit aorta without endothelium, KT3-671 (10−7M) shifted the concentration-response curve of Ang II to the right and reduced the maximal contraction (Fig. 4A). The removal of endothelium increased the maximal contraction induced by Ang II (3 × 10−8M) (131. 2 ± 4.1% of the control). DuP 753 (10−7M) also shifted the concentration-response curve of Ang II to the right (Fig. 4A). In the presence of DuP 753 (10−7M), KT3-671 (10−7M) neither inhibited the maximal contraction induced by Ang II (Fig. 4A) nor further shifted the concentration-response curve of Ang II (Fig. 4A). In the rabbit aorta without endothelium, KT3-671 (10−8M) shifted the concentration-response curve of Ang II to the right (Fig. 4B). EXP 3174 (10−9M) inhibited the maximal contraction induced by Ang II (Fig. 4B). However, in the presence of KT3-671 (10−8M), EXP 3174 (10−9M) did not further affect the responses to Ang II (Fig. 4B). The pretreatment with CV-11974 (10−10M) also inhibited the maximal contraction induced by Ang II (Fig. 4C). In the presence of KT3-671 (10−8M), CV-11974 (10−10M) still caused the inhibition of the maximal contraction induced by Ang II (Fig. 4C).

FIG. 4

FIG. 4

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Interactions among KT3-671, DuP 753, EXP 3174, and CV-11974 in the rat aorta without endothelium

KT3-671 (10−8M) shifted the concentration-response curve of Ang II to the right and reduced the maximal contraction in the rat aorta without endothelium (Fig. 5A). The removal of endothelium increased the maximal contraction induced by Ang II (10−7M; 151.2 ± 6.1% of the control). DuP 753 (10−8M) also shifted the concentration-response curve of Ang II to the right without inhibiting the maximal response to Ang II (Fig. 5A). In the rat aorta pretreated with DuP 753 (10−8M), KT3-671 (10−8M) shifted the concentration-response curve of Ang II to the right without significantly affecting the maximal contraction induced by Ang II (Fig. 5A). KT3-671 (3 × 10−9M) shifted the concentration-response curve of Ang II to the right without affecting the maximal contraction (Fig. 5B). EXP3174 (10−9M) shifted the concentration-response curve of Ang II to the right and also reduced the maximal contraction (Fig. 5B). However, in the presence of KT3-671 (3 × 10−9M), EXP 3174 (10−9M) did not significantly affect the response to Ang II (Fig. 5B). CV-11974 (10−10M) inhibited the maximal contraction induced by Ang II (Fig. 5C). KT3-671 (10−8M) shifted the concentration-response curve of Ang II and reduced the maximal response to Ang II (Fig. 5C). In the presence of KT3-671 (10−8M), CV-11974 (10−10M) did not significantly affect the response to Ang II (Fig. 5C).

FIG. 5

FIG. 5

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Effects of endothelium removal and indomethacin on insurmountable antagonism by KT3-671, CV-11974, and EXP 3174 in the rat aorta

The pretreatment with KT3-671 (10−8M), CV-11974 (10−9M), or EXP 3174 (10−9M) inhibited the contractile responses to Ang II (Fig. 6A-C). The removal of endothelium increased the maximal contraction induced by Ang II (10−7M) (144.5 ± 5.2% of the control). In the absence of endothelium, the inhibitory effect of KT3-671 (10−8M), but not CV-11974 (10−9M) or EXP 3172 (10−9M), on the contraction induced by Ang II was significantly greater than that in the presence of endothelium (Fig. 6A-C). In the rat aorta pretreated with indomethacin (3 × 10−6M), which did not significantly affect the maximal contraction induced by Ang II (90.9 ± 6.8% of the control), the inhibitory effect of KT3-671 (10−8M), but not EXP 3174 (10−9M), on the Ang II-induced contraction also was significantly greater than that in the absence of indomethacin (10−5M;Fig. 7A and B).

FIG. 6

FIG. 6

FIG. 7

FIG. 7

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Effects of various treatments on insurmountable antagonism by KT3-671 and CV-11974 in the rat aorta

The pretreatment with methylene blue (3 × 10−6M), KCl (10−2M), TEA (10−2M), or BaCl2 (10−4M) increased the maximal contraction induced by Ang II (10−7M; methylene blue, 250.0 ± 10.2%; KCl, 184.4 ± 7.1%; TEA, 337.0 ± 10.3%; and BaCl2, 211.2 ± 9.3% of the control). In the presence of methylene blue (3 × 10−6M), KCl (10−2M), or TEA (10−2M), the concentration-response curve of Ang II was less shifted to the right by KT3-671 (5 × 10−8M), and the reduction of the maximal contraction induced by KT3-671 was eliminated (Figs. 8A, 9A, 10A). In addition, in the presence of methylene blue (3 × 10−6M), KCl (10−2M), or TEA (10−2M), CV-11974 (10−9M) did not significantly inhibit the Ang II-induced contractions (Fig. 8B, 9B, 10B). In the presence of BaCl2 (10−4M), KT3-671 (3 × 10−8M) shifted the concentration-response curve of Ang II to the right and only slightly reduced the maximal contraction (Fig. 11A). The inhibitory effect of KT3-671 (3 × 10−8M) in the presence of BaCl2 (10−4M) almost disappeared (Fig. 11A). In the presence of BaCl2 (10−4M), CV-11974 (10−9M) only slightly but significantly inhibited the maximal contraction (Fig. 11B). The inhibitory effect of CV-11974 (10−9M) in the presence of BaCl2 (10−4M) almost disappeared (Fig. 11B). The pretreatment with glibenclamide (3 × 10−6M) increased the maximal contraction induced by Ang II (182.1 ± 6.3% of the control). However, in the presence of glibenclamide (3 × 10−6M), KT3-671 (3 × 10−8M) and CV-11974 (10−9M) still significantly inhibited the Ang II-induced maximal contraction (Fig. 12A and B). The inhibitory effect of CV-11974 (10−9M) in the presence of glibenclamide (3 × 10−6M) was significantly less than that in the absence of glibenclamide (Fig. 12B). The inhibitory effect of KT3-671 (3 × 10−8M) was not affected by glibenclamide (3 × 10−6M;Fig. 12A). The pretreatment with NOARG (3 × 10−4M) or TEA (10−3M), but not ODQ (10−5M), 4-AP (3 × 10−3M), or ibTX (10−7M), increased the maximal contraction induced by Ang II (10−7M; NOARG, 139.1 ± 5.1%; TEA, 166.9 ± 6.2%; ODQ, 80.5 ± 10.3%; 4-AP, 92.4 ± 7.1%; ibTX, 83.6 ± 9.8% of the control; n = 6). However, the pretreatment with these inhibitors did not affect the inhibitory effect of KT3-671 (3 × 10−8M) or CV-11974 (10−9M; data not shown).

FIG. 8

FIG. 8

FIG. 9

FIG. 9

FIG. 10

FIG. 10

FIG. 11

FIG. 11

FIG. 12

FIG. 12

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DISCUSSION

In this study, vasoinhibitory effects of KT3-671, a recently synthesized nonpeptide Ang II (AT1 selective) receptor antagonist (18) were investigated in rabbit and rat isolated vascular smooth muscles. KT3-671 has been shown to interact reversibly and competitively with AT1 receptors in rat liver membranes (19). In rabbit and rat aortic rings, KT3-671 caused shifts to the right of concentration-response curves for Ang II and reduced the maximal response, indicating insurmountable antagonism. Similarly, SR 47436 competitively inhibited AT1 receptors in rat liver membranes but caused parallel shifts to the right of Ang II contractile response curves without total recovery of the maximal response (23). However, this pattern of Ang II inhibition by KT3-671 is different from that of CV-11974 or EXP 3174, an active metabolite of DuP 753, which competitively inhibited AT1 receptors in the binding assay but noncompetitively inhibited Ang II-induced contractions of rabbit aorta (16,20).

In rabbit renal and basilar arterial rings, KT3-671 caused nonparallel shifts to the right of concentration-response curve for Ang II and reduced the maximal response to Ang II, indicating insurmountable antagonism of Ang II. Based on the pD′2 values, KT3-671 is most effective in reducing the maximal response to Ang II in the rabbit renal artery as compared with the basilar artery and the aorta.

Ang III, a heptapeptide metabolite of Ang II, also plays an important role in the control of blood pressure (24,25). In addition, Ang III is reported to elicit chemotactic activity of human polymorphonuclear neutrophils (PMNs), suggesting that Ang III is a chemoattractant for PMN, having an important role in inflammation.(26)

In pithed rats and rabbit aorta, the contractile response to Ang III is inhibited by EXP 3174 and CV-11974 in a surmountable manner (16,27), whereas saralasin in conscious rats inhibits only the response to Ang II in a surmountable manner without inhibiting the response to Ang III (28). In addition, 1-Sar-8-Cys(Me)-Ang II is reported to exhibit insurmountable Ang II antagonism and surmountable Ang III antagonism in rabbit aorta (29). Our study indicates that KT3-671 at 10−9−10−7M in the rabbit aortic rings and at 3 × 10−9 and 10−8M in the rat aortic rings behaves like a surmountable antagonist of Ang III, whereas KT3-671 at similar concentrations exhibits insurmountable antagonism of Ang II. Even though our study does not assure that KT3-671 at higher concentrations is a surmountable antagonist of Ang III, these results are in agreement with the previous reports (16,27,28), suggesting that Ang II receptors may be different from Ang III receptors in the rabbit and rat aorta.

It has been reported that insurmountable antagonism can be observed with competitive antagonists that are irreversibly or pseudoirreversibly associated with receptors (30). However, it has been pointed out that even though both DuP 753 and EXP 3892 are slowly dissociating antagonists, only EXP 3892 produced insurmountable antagonism (31), indicating that pseudoirreversible antagonism may not be the explanation for the insurmountable antagonism. Ang II also stimulates the synthesis of vasoactive substance such as prostaglandins and endothelium-derived relaxing factor (32). Therefore the vasoconstrictor effect of Ang II may represent a net effect of vasoconstriction and vasodilatation. Consequently, it is conceivable that KT3-671 blocks the Ang II receptors mediating vasoconstriction more than those mediating synthesis of vasorelaxing substances, with a resultant insurmountable antagonism. However, the removal of endothelium does not affect the contractile response to Ang II in rabbit aorta (33). Because insurmountable antagonism by KT3-671 was observed in rabbit aortic ring, the endothelium-derived relaxing factor may not be involved in the insurmountable antagonism of Ang II by KT3-671 in rabbit aorta. It has been reported that CV-11974, an insurmountable antagonist, does not affect the contraction induced by PGF in rabbit aortic strips (27). In addition, EXP 3174, an insurmountable antagonist, was reported to inhibit the contraction induced by U46619, a thromboxane A2-analogue, in canine coronary arteries (34). Losartan, a surmountable antagonist, inhibits the contraction induced by prostaglandin F in coronary arteries (34). Further, KT3-671, only at a high concentration, inhibited the contractions induced by prostaglandin F and STA2, a stable analogue of thromboxane A2, in the rat renal arteries. These results may suggest that the vasoactive metabolites of arachidonic acid through the cyclooxygenase pathway do not play a major role in the insurmountable antagonism of Ang II. This is in agreement with the results in the rat aorta that indomethacin, an inhibitor of cyclooxygenase, which did not affect the Ang II-induced contraction, did not affect the inhibitory effect of EXP 3174, indicating that the stimulation of the synthesis of vasoactive substances such as prostaglandins may not play a major role in the insurmountable antagonism of Ang II by EXP 3174. In addition, the removal of endothelium, which increased the Ang II-induced contractions, failed to affect the inhibitory effect of EXP 3174, also indicating the absence of involvement of endothelium-derived relaxing factor in the insurmountable antagonism by EXP 3174.

In the rat aorta, endothelium-derived relaxing factor also may not play a significant role in the insurmountable antagonism caused by KT3-671 or CV-11974, because ODQ, an inhibitor of guanylyl cyclase (35), or NOARG, an inhibitor of nitric oxide synthase (36), which increased Ang II-induced contraction, did not affect the inhibitory effect of KT3-671 or CV-11974. However, the inhibition of Ang II by KT3-671 was increased by indomethacin or the removal of endothelium, possibly suggesting that the insurmountable antagonism of Ang II by KT3-671 is partly affected by endothelium, but not nitric oxide, or metabolites of arachidonic acids.

These results also indicate that, even though the receptor-protection experiments in the rat aorta suggest a similarity in the mechanism of insurmountable antagonism among KT3-671, EXP 3174, and CV-11974, these inhibitors behave differently in the presence of indomethacin or in the absence of endothelium. Previous findings suggested that Ang II-receptor activation stimulates the activity of phospholipases C, D, and A2 or the release of nitric oxide (32,37-39). Because arachidonic acid is metabolized in endothelium, the effect of the removal of endothelium on the insurmountable antagonism by KT3-671 also may be related to arachidonic acid. Therefore one of the simple explanations for these results is that KT3-671 may inhibit the effects of vasorelaxant metabolites of arachidonic acid in endothelium-intact tissues, thereby increasing the Ang II-induced contraction as compared with endothelium-denuded or indomethacin-treated tissues. Further study is necessary to clarify the relation between the insurmountable antagonism by KT3-671 and vasorelaxant metabolites of arachidonic acid.

de Chaffoy de Courcelles et al. (40) proposed a receptor-transducer coupling model to explain the insurmountable antagonism. According to this model, a surmountable antagonist such as DuP 753 would induce conformational changes by binding to a binding site located outside of the plasma membrane, resulting in a reduction of the affinity of the other binding site located at the inner side of the membrane for the coupling factor of the receptor. An insurmountable antagonist such as KT3-671 would diminish the binding capacity for the coupling factor. In rabbit and rat aortic rings, the receptor-protection experiments showed that the presence of DuP 753 protected against the insurmountable antagonism by KT3-671. In addition, a low concentration of KT3-671, which did not reduce the maximal contraction by Ang II, protected against the inhibitory effect by EXP 3174. These results support the hypothesis that KT3-671, DuP 753, and EXP 3174 compete for a common site.

Robertson et al. (41) has described a two-state receptor model to explain the insurmountable antagonism of some AT1-receptor antagonists. Based on the model, an antagonist, such as Dup 753, will cause competitive antagonism because of equal affinity for the two interconvertible states of the receptor, R (active) and R′ (inactive). Insurmountable antagonists, such as KT3-671 and EXP 3174, may have higher affinity for R′ than for R, resulting in a new equilibrium between R and R′, in which fewer R are available to bind the agonist. Depending on the basal R/R′ ratio, this can result in a reduction in the maximal achievable agonist response (41). In addition, an insurmountable antagonist, such as CV-11974, may have much greater affinity for R′ than it has for R, resulting in the curve depression without visible rightward displacement. In our study, coincubation of an insurmountable antagonist, such as KT3-671 or EXP 3174 in the rabbit aorta or CV-11974 in the rat aorta, with Dup 753 or KT3-671 in the rabbit aorta or KT3-671 in the rat aorta caused an enhancement of the maximal response to Ang II with a rightward shift of the concentration-response curve. These results could be explained by the two-state receptor model described by Robertson et al. (41). However, the similar experiments in the aorta from different animals (rats instead of rabbits or vice versa) resulted only in the enhancement of the maximal response to Ang II without the rightward shift or the rightward shift without the enhancement of maximal response. These results indicate that other considerations may be necessary to explain all the results in our study.

Two different AT1-receptor subtypes (AT1A and AT1B) have been described by Inagami et al. (42) and Zhou et al. (43). In rabbit aortic rings, the receptor-protection experiment also shows that KT3-671 at a low concentration did not protect against the insurmountable antagonism by CV-11974 and that CV-11974 further shifted the Ang II concentration-response curve to the right in the presence of KT3-671. This result is in agreement with the hypothesis that there may be at least two AT1-receptor subtypes. However, in rat aortic rings, KT3-671 abolished the inhibitory action of CV-11974, suggesting that KT3-671 shares more similar Ang II-binding sites with CV-11974 in the rat aorta than in the rabbit aorta.

A possible mechanism for the insurmountable antagonism of Ang II by KT3-671 and CV-11974 was further investigated. Methylene blue, an inhibitor of guanylyl cyclase and nitric oxide synthase (44), prevented the insurmountable antagonism by KT3-671 and abolished the inhibitory action of CV-11974. However, it is not likely that the endothelium-derived relaxing factor is involved in the insurmountable antagonism, because the inhibition of nitric oxide synthase by NOARG or guanylyl cyclase by ODQ did not affect the insurmountable antagonism. The reason for the effect of methylene blue on the insurmountable antagonism by KT3-671 and CV-11974 cannot be ascertained from our study, and further studies are necessary for clarification.

A possible involvement of K channels in the insurmountable antagonism of Ang II by KT3-671 and CV-11974 is suggested by the result that the pretreatment of the rat aorta with KCl eliminated the insurmountable antagonism. Voltage-dependent K channels (Kv) are most likely not involved in the insurmountable antagonism by KT3-671 and CV-11974, because a high concentration (3 × 10−3M) of 4-AP, an inhibitor of Kv channels, did not have any effect on the insurmountable antagonism. Ca2+-dependent K channels (KCa) also may not be involved in the insurmountable antagonism, because a lower concentration of TEA (10−3M) and ibTX, which inhibit KCa channels, failed to affect the inhibitory actions of KT3-671 and CV-11974. A higher concentration (10−2M) of TEA, which inhibits ATP-sensitive K channels (KATP), completely eliminated the insurmountable antagonism, suggesting a possibility that KATP channels may be involved in the insurmountable antagonism of Ang II by KT3-671 and CV-11974. This is supported by the results that a lower concentration (10−4M) of Ba2+, which inhibits KATP channels, also markedly reduced the insurmountable antagonism by KT3-671 and CV-11974. However, KT3-671 and CV-11974, to a lesser degree, still exhibited the insurmountable antagonism against Ang II in the presence of glibenclamide, an inhibitor of KATP channels. It was recently reported that glibenclamide caused relaxations of smooth muscles by activating the release of endothelium-derived relaxing factor (45). Therefore it is possible that the effect of glibenclamide on the insurmountable antagonism may have been opposed by the relaxing effect of glibenclamide. These results taken together suggest that the activation of KATP channels may play a major role in the insurmountable antagonism by KT3-671 and CV-11974. Further studies are clearly necessary to determine the precise nature of the relation between the possible activation of KATP channels by Ang II and the insurmountable antagonism by KT3-671 and CV-11974.

In conclusion, KT3-671 is an insurmountable antagonist of Ang II in the rabbit and rat aorta. In addition, in the rat aorta, KATP channels may be involved in insurmountable antagonism of Ang II by KT3-671 and CV-11974.

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Keywords:

KT3-671; Rabbit; Rat; Vascular smooth muscle; Angiotensin II; Insurmountable antagonist; KATP channels

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