Evidence that ET-1, but not ET-3 and S6b, ET(A)-receptor mediated contractions in isolated rat mesenteric arteries are modulated by co-activation of ET(B) receptors

Abstract

The effects of agonists with endothelin (ET) ET(A)-receptor activity have been analysed in relation to their interaction with ET(B) receptors in rat mesenteric arteries. ET-1, sarafotoxin 6b (S6b) and ET-3 induced large, slow-onset and sustained contractions whereas S6c induced weak transient contractions. However, following pre-contraction with U46619 and subsequent relaxation with forskolin, the effect of S6c was amplified, indicating a potential for powerful ET(B)-receptor mediated contraction. The selective ET(A)-receptor antagonist, FR139317, produced parallel rightward shifts of ET-1, S6b and ET-3 concentration-effect curves indicating that the contractions were mediated by ET(A) receptors. However, the corresponding FR139317 pK(B) values were significantly different between the agonists. As expected FR139317 had no effect on S6c responses. Pre-treatment with S6c to desensitize ET(B) receptors, increased ET-1 potency and the pK(B) value for FR139317. In contrast, neither the potency of S6b and ET-3 nor the pK(B) values for FR139317 estimated using these agonists were affected by ET(B)-receptor desensitization. Segments pre-contracted with submaximal concentrations of S6b and ET-3, but not ET-1, rapidly relaxed following wash-out or FR139317 administration. The results indicate that the small contractile response to selective ET(B) receptor activation, barely detectable under standard bioassay conditions, is greatly amplified when adenylate cyclase activity is elevated. Moreover, the response to ET(A) receptor activation by ET-1, but not ET-3 and S6b, is significantly modified by co-activation of ET(B) receptors. This interaction has a significant effect on the apparent affinity of ET(A)-receptor selective antagonists when ET-1 is used as agonist and decreases the potency of ET-1.

Figure 1

Representative experimental traces showing the contraction developed in rat mesenteric artery branch segments during cumulative administration of ET-1 (a), ET-1 pre-treated for 1 h with FR139317 (b), S6b (c), ET-3 (d), S6c (e; insert – 20×amplitude magnification) and S6c following pre-contraction with 0.1 μM U46619 and subsequent dilatation with 0.1 μM forskolin (f). Agonist concentrations are expressed as log₁₀ values.

Figure 2

Concentration-effect curves obtained on rat small mesenteric artery segments to ET-1 in the absence and presence of 0.03, 0.1, 0.3 and 1 μM FR 139317 – control (a), pre-treated with 0.3 μM S6c (b) and pre-treated with 1 μM IRL 2500 (c). Contractions are expressed as a percentage of the response to 60 mM KCl. Corresponding Schild plots are shown in panel (d). Each point represents the mean of all segments tested with error bars representing s.e.mean from 5 – 10 animals.

Figure 3

Concentration-effect curves obtained on rat small mesenteric artery segments to S6b in the absence and presence of 0.01, 0.03 and 0.1 μM FR 139317 – control (a), pre-treated with 0.3 μM S6c (b). Concentration-effect curves to ET-3 (c) in the absence and presence of 0.01 μM FR 139317 for controls and in the absence and presence of 0.01 μM FR 139317 in arteries pre-treated with 0.3 μM S6c. Contractions are expressed as a percentage of the response to 60 mM KCl. Corresponding Schild plots are shown in panel (d). The hatched line shows the extrapolated Schild plot for the interaction between ET-3 and the single concentration of FR139317 investigated. Each point represents the mean of all segments tested with error bars representing s.e.mean from five animals.

Figure 4

Representative experimental traces showing the contractions developed in rat mesenteric arterial branch segments to a single concentration (10 nM) of ET-1 and S6b followed by washout (three successive exchanges of the organ bath solution) or administration of FR139317 (1 and 0.1 μM for ET-1 and S6b, respectively).