THE ENTIRE VASOACTIVE INTESTINAL POLYPEPTIDE MOLECULE IS REQUIRED FOR THE ACTIVATION OF THE VASOACTIVE INTESTINAL POLYPEPTIDE RECEPTOR - FUNCTIONAL AND BINDING-STUDIES ON OPOSSUM INTERNAL ANAL-SPHINCTER SMOOTH-MUSCLE

Because no significant information exists regarding the structure-activity of vasoactive intestinal polypeptide (VIP) to gut smooth muscle, we performed functional studies in vitro on opossum internal anal sphincter (IAS) smooth muscle strips and supplemented them with binding studies to assess the ability of VIP, its fragments and analogs to inhibit [I-125]VIP binding to IAS smooth muscle membranes. Binding of radiolabeled VIP to its receptor was specific, saturable and time- and temperature-dependent. Of all the substances tested, VIP was the most potent in causing a fall in the resting tension of the IAS and inhibiting [I-125]VIP binding. VIP 2-28, VIP 10-28 and the putative VIP antagonists (1 x 10(-6) M) [4Cl-D-Phe6,Leu 17]VIP (VIP analog) and (N-Ac-Tyr1, D-Phe 2)-growth hormone-releasing factor [GRF] (1 -29)-NH2 (GRF analog) caused significant inhibition of [I-125]VIP binding, but had only minimal effect on the resting tension of the IAS. VIP 9-18 and VIP 1-12 had neither any significant effect nor inhibition of receptor binding. The rank order of potencies for inhibition of binding was VIP > VIP analog > VIP 10-28 = VIP 2-28 > GRF analog > peptide histidine isoleucine > VIP 9-18. The IC50 values for VIP, VIP analog, VIP 10-28, VIP 2-28, GRF analog and peptide histidine isoleucine were 9.6 x 10(-9), 1.6 x 10(-7), 5.5 x 10(-7) 6.2 x 10(-7), 1.2 x 10(-6) and 1.2 x 10(-5) M, respectively. Conclusion: the full action of VIP is critically dependent upon the integrity of the entire VIP molecule. However, only the C-terminal part of the molecule is needed for binding to the receptor. These studies provide previously unknown information on selective VIP receptor antagonists and VIP receptor characterization.