CHARACTERISTICS OF RECEPTORS FOR VIP IN RAT PERITONEAL MACROPHAGE MEMBRANES

Vasoactive intestinal peptide (VIP) receptors were investigated in rat peritoneal macrophage membranes (RPMM) using [I-125]VIP as ligand. Th e receptor binding was rapid, reversible, saturable, specific, and dep endent on time, temperature, and membrane concentration. The Scatchard analysis of binding data was consistent with the existence of two cla sses of VIP binding sites with K(d) values of 0.60 +/- 0.08 and 275 +/ - 39 nM and binding capacities of 580 +/- 71 and 72,500 +/- 810 fmol V IP/mg protein, respectively. The interaction showed a high degree of s pecificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP. Thes e pharmacological studies showed the following order of potency: VIP ( IC50 = 1 nM) > rGRF (IC50 = 13 nM) > PHI (IC50 = 421 nM) >> secretin. Glucagon, somatostatin, insulin, octapeptide of cholecystokinin [CCK(2 6-33)], and pancreastatin were ineffective at concentrations up to 1 m uM. Binding of [I-125]VIP to membranes is markedly reduced by increasi ng the ionic strength of incubation medium. Treatment of membranes wit h dithiothreitol, trypsin, and phospholipases A2 and C resulted in a l oss of the ability of these membranes to bind VIP. However, treatment with phospholipase D did not affect binding of VIP by membranes. The m olecular characterization of VIP receptors in RPMM was performed after [I-125]VIP cross-linking to membranes using the cross-linker dithiobi s (succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins revealed specific [I-125]VIP-pro tein complexes of M(r) 55,000 +/- 1700, 35,000 +/- 900, and 22,000 +/- 500.