CHARACTERIZATION OF A COMMON VIP-PACAP RECEPTOR IN HUMAN SMALL INTESTINAL EPITHELIUM

Vasoactive intestinal peptide (VIP) receptors were characterized in epithelial plasm membranes from human small intestine. Native VIP inhibited the binding of I-125-labeled VIP to jejunal membranes, and Scatchard analysis of these data was consistent with the existence of one class of receptor with a dissociation constant of 42 pM and a maximal binding of 256 fmol/mg membrane protein. VIP stimulated adenylyl cyclase activity in human jejunal membranes in the 0.01 nM-1 muM range [half-maximal effective dos = 0.7 nM]. Coupling of VIP receptors with a G(s) protein was further assessed by the ability of GTP (10(-8) to 10(-3) M) to inhibit I-125-VIP binding to membranes. I-125-VIP binding was seven to eight times higher in villus cells than in crypt cells. Finally, I-125-VIP binding was detectable throughout the small and large intestines with the highest binding in jejunum. Among the natural peptides structurally related to VIP, some inhibited I-125-VIP binding with the following order of potency: VIP = pituitary adenylate cyclase-activating peptide (PACAP)-27 = PACAP-38 > helodermin much greater than peptide histidine methionineamide = human growth hormone-releasing factor > secretin. The same order of potency of peptides for inhibiting I-125-VIP or I-125-labeled PACAP was observed, supporting that the two tracers bound to a common VIP-PACAP receptor site. This order of potency was also observed for the stimulation of adenylyl cyclase activity by these peptides. I-125-VIP was cross-linked to membranes using disuccinimidyl suberate. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, one single band of 70,000 mol wt was observed. Its labeling was completely inhibited by 1 muM VIP or PACAP-27. The same labeling pattern was observed when I-125-PACAP-27 was used as tracer. Assuming one molecule of tracer was bound per molecule of protein, a 67,000-mol wt protein was identified as a common VIP-PACAP receptor in human small intestine.