ETA and ETB receptors coexist on rabbit pulmonary artery vascular smooth muscle mediating contraction.

The possibility that both ETA and ETB endothelin receptor subtypes could mediate contractile activity in the same tissue was investigated in isolated, endothelium denuded rabbit pulmonary arteries. The ETB selective agonist, sarafotoxin 6c (S6c), produced potent contractile activity, equal to the non-selective ETA and ETB receptor agonist endothelin-1 (ET-1), indicating a contractile role for ETB receptors in this tissue. In addition BQ-123 (10.0 microM), the ETA selective antagonist, was only partially effective in blocking ET-1 induced contractions further indicating a contractile role for ETB receptors. However, the partial blockade by BQ-123 suggested a possible contractile role for ETA receptors. To address this possibility, ETB receptors were desensitized with a 30 minute pretreatment of S6c (0.01 microM). Under these conditions, we were able to demonstrate full ET-1 contractile activity that was now sensitive to blockade by BQ-123. The coexistence of both ETA and ETB receptors was confirmed through receptor binding experiments indicating 40/60 ratio, respectively. We conclude that 1) both ETA and ETB receptors coexist on vascular smooth muscle of rabbit pulmonary artery, 2) activation of either receptors subtype results in contraction, and 3) prolong activation of the ETB receptor subtype produces tachyphylaxis preventing further activation by S6c or ET-1.

Why big endothelin-1 lacks a vasodilator response.

The biphasic arterial blood pressure response to endothelin-1 (ET-1) results from a transient decrease, followed by a longer-lasting increase, in systemic vascular resistance. In contrast to ET-1, big endothelin-1 (bET-1) produces monophasic increases in systemic vascular resistance and arterial blood pressure. This is somewhat surprising, because bET-1 activity is reportedly due to ET-1, bET-1 being converted to ET-1 by a putative converting enzyme. In this study we tested two hypotheses that could explain the singular effect of bET-1 on the arterial vasculature: that bET-1 vasoconstriction, mediated by ETA receptors at the level of the smooth muscle, masks the release of endothelial derived vasodilators, and/or that the endothelium develops tachyphylaxis owing to prolonged activation of endothelial ETB receptors. In anesthetized rats, blockade of the vasoconstrictor activity of bET-1 with BQ-123, an ETA-receptor antagonist, did not reveal a masked bET-1 vasodilator component in the rat hindquarter. Furthermore, in the presence of bET-1 (after 3.0 nmol/kg bET-1 i.v.), low doses of ET-1 (0.03-0.3 nmol/kg) produced dose-dependent hindquarter vasodilation, indicating activation of endothelial ETB and therefore no tachyphylaxis. Collectively, these experiments suggest that i.v. administration of bET-1 results in little or no activation of endothelial ETB receptors and therefore lacks a vasodilator response.

In vitro and in vivo studies with a series of hexapeptide endothelin antagonists.

The effects of different amino acids incorporated into the 16 and 17 positions of the C-terminal hexapeptide of ET-1 were examined. Structure-activity relationships (SAR) of the ET receptor antagonists PD 142893 [Ac-(D-Dip16-L-Leu17-L-Asp-L-Ile-L-Ile-L-Trp) (D-Dip = 3,3-D-diphenylalanine)] and PD 145065 [Ac-(D-Bhg16-L-Leu17-L-Asp-L-Ile-L-Ile-L-Trp) (D-Bhg = 5H-dibenzyl[a,d]cycloheptene-10,11-dihydro-glycine)] uncovered certain requirements for high potency. The disodium salt of PD 145065 has 4.0 and 15 nM binding affinity (IC50 values) for the ETA (rabbit renal artery vascular smooth-muscle cells) and ETB receptor (rat cerebellum), respectively. The compound is also an antagonist of ET-1- and SRTX-6c-stimulated vasoconstrictor activity, with pA2 values of 6.9 (rabbit femoral artery, ETA assay) and 7.1 (rabbit pulmonary artery, ETB assay). The tripeptidic ETA antagonist FR 139317 was found to be less active in the rabbit femoral artery, with a pA2 value of 6.0, and inactive in the rabbit pulmonary artery. Substitution of acidic and basic residues at position 17 in PD 142893 and PD 145065 indicates differences in selectivity. Incorporation of bulky non-natural amino acids at position 16 has led to potent nonselective analogues, including Ac-D-Bheg16-L-Leu-L-Asp-L-Ile-L-Ile-L-Trp [D-Bheg (5H-dibenzo[a,d]cycloheptene glycine)]. The in vivo effects of single-bolus doses of selected ET antagonists on depressor and pressor responses to ET-1 in anesthetized ganglion-blocked rats were evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)