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 PGF2α 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 F2α in coronary arteries (34). Further, KT3-671, only at a high concentration, inhibited the contractions induced by prostaglandin F2α 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:© 2000 Lippincott Williams & Wilkins, Inc.
KT3-671; Rabbit; Rat; Vascular smooth muscle; Angiotensin II; Insurmountable antagonist; KATP channels