Comparison of the contractile effects and binding kinetics of endothelin-1 and sarafotoxin S6b in rat isolated renal artery

Abstract

1. To date, only two mammalian endothelin (ET) receptors, termed ET_A and ET_B, have been cloned, sequenced and characterized. However, several functional studies of isolated blood vessels suggest that ET-1-induced contractions may be mediated by multiple ET_A receptors. In this study, the ET_A receptors in renal arteries isolated from Wistar rats were characterized by isometric tension recording and radioligand binding techniques.

2. ET-1, sarafotoxin S6b (StxS6b) and ET-3 produced concentration-dependent contraction with similar response maxima in endothelium-denuded arteries, whereas the ET_B receptor-selective agonist StxS6c was inactive. ET-1 and StxS6b were equipotent and 30 times more potent than ET-3. This agonist profile, together with the findings that the ET_A receptor-selective antagonists, BQ-123 and FR-139317 caused concentration-dependent, rightward shifts of the concentration-effect curves to each agonist indicated that ET-1-induced contractions in rat renal artery were mediated via ET_A receptors.

3. BQ-123 and FR-139317 were both significantly more potent inhibitors of contractions induced by StxS6b or ET-3 than of responses to ET-1, raising the possibility that a component of ET-1-induced contraction was mediated through atypical, BQ-123 (or FR-139317)-insensitive ET_A receptors. However, in competition binding studies, specific [¹²⁵I]-ET-1 and [¹²⁵I]-StxS6b binding to rat renal artery sections was completely abolished by BQ-123 in a manner consistent with an action at a single site. Thus, competition binding studies did not provide any supportive evidence of the existence of a BQ-123-insensitive ET_A receptor.

4. Additional studies revealed marked differences in the kinetics of [¹²⁵I]-ET-1 and [¹²⁵I]-StxS6b binding. Following a 3 h period of association of [¹²⁵I]-ET-1 with its receptors, no significant dissociation of receptor-bound [¹²⁵I]-ET-1 was observed during a 4 h washout period. In stark contrast, dissociation studies revealed that specific [¹²⁵I]-StxS6b binding to ET_A receptors was reversible (t_0.5diss, 100 min). A series of association binding studies were also consistent with the specific binding of [¹²⁵I]-ET-1 and [¹²⁵I]-StxS6b being irreversible and reversible processes, respectively.

5. Thus, differences in BQ-123 potency against ET-1 and StxS6b-induced contractions in rat renal arteries might be due to differences in the kinetics of agonist binding, rather than due to the existence of atypical ET_A receptors.

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