Distribution and characterization of vasoactive intestinal polypeptide binding in canine lower esophageal sphincter
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BACKGROUND: Vasoactive intestinal polypeptide (VIP) may be a nonadrenergic, noncholinergic inhibitory transmitter in the lower esophageal sphincter (LES). There is no biochemical evidence of VIP receptors in the LES. METHODS: Using membranes from canine LES, VIP receptor distribution and characterization were analyzed by radioligand binding and cross-linking experiments. RESULTS: High densities of saturable VIP receptors were found (maximum bound [Bmax], 539.2 fmol/mg in the synaptosome-enriched fraction [P2] and 732.7 fmol/mg in the smooth muscle, plasma membrane-enriched fraction [Mic II]), with high affinity for 125I-VIP (dissociation constant [Kd], 1.38 nmol/L in P2 and 1.40 nmol/L in Mic II). Competition binding studies suggested the presence of two binding sites, a high-affinity (inhibitor constant [Ki1], 0.064 nmol/L) and a low-affinity (Ki2, 2.68 nmol/L) binding site in P2 membranes, but only one binding site (Ki, 1.18 nmol/L) in Mic II membranes. Guanosine triphosphate-gamma-s pretreatment eliminated high-affinity binding in P2 membranes by conversion to binding sites of lower affinity (Ki, 2.82 nmol/L). Studies with a cross-linking agent identified VIP receptors in synaptosomal and smooth muscle plasma membrane fractions; a single polypeptide of approximately 60 kilodaltons was found in each membrane. CONCLUSIONS: Specific VIP receptors exist in both synaptosomal and smooth muscle plasma membrane of canine LES.
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