Receptors for Vasoactive Intestinal Polypeptide on Crude Smooth Muscle Membranes from Porcine Uterus*

Abstract

Vasoactive intestinal polypeptide (VIP) is a neuropeptide that causes relaxation of the myometrium. Receptors for VIP were studied on crude smooth muscle membranes (20,000 X g pellet) from porcine myometrial homogenate. The binding of 70 pM ¹²⁵I-labeled VIP could be accounted for by 80% receptor bound and 20% nondisplaceable by 1 /M VIP. The receptor binding was dependent on temperature and pH, with optimum at 20 C and pH 7.4. At 37 C, the binding was low and transient due to rapid degradation of VIP and of the receptor. Association of ¹²⁵I-labeled VIP at 20 C showed steady state after about 60 min. Dissociation of [¹²⁵I]iodo-VIP from the receptors by addition of 1 μ VIP showed a time course that was biexponential, with 30% in a rapid and 70% in a slow dissociating state. A calculation of K_d as the ratio between the rate constants of dissociation and association gave values of 0.23 and 9.5 nM. At steady state, the binding of [¹²⁵I]iodo-VIP was displaced by unlabeled VIP, with half-maximum at a concentration of 3 nM. The Scatchard plot was curvilinear with an upward concavity, suggesting that two classes of noninteracting receptors with K_dS of 0.4 and 10 nM are present in amounts of 0.2 and 1.5 pmol/mg membrane protein, respectively. Alternatively, the receptor binding may be influenced by negatively cooperative interactions. Secretin was 10² times, somatostatin 5 × 10² times, and glucagon more than 104 times less potent than VIP. Substance P, ritodrine chloride, leu-enkephalin and met-enkephalin, neurotensin, oxytocin, and prostaglandin-F₂α and prostaglandin-E₂ and no effect in concentrations up to 20 μM. The presence of specific receptors for VIP in myometrial membranes supports the hypothesis that VIP has a physiological role as smooth muscle relaxant in the uterus.