Characteristics of receptors for VIP in rat peritoneal macrophage membranes.
Vasoactive intestinal peptide (VIP) receptors were investigated in rat peritoneal macrophage membranes (RPMM) using [125I]VIP as ligand. The receptor binding was rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. The Scatchard analysis of binding data was consistent with the existence of two classes of VIP binding sites with Kd values of 0.60 +/- 0.08 and 275 +/- 39 nM and binding capacities of 580 +/- 71 and 72,500 +/- 810 fmol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP. These 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(26-33)], and pancreastatin were ineffective at concentrations up to 1 microM. Binding of [125I]VIP to membranes is markedly reduced by increasing the ionic strength of incubation medium. Treatment of membranes with dithiothreitol, trypsin, and phospholipases A2 and C resulted in a loss of the ability of these membranes to bind VIP. However, treatment with phospholipase D did not affect binding of VIP by membranes. The molecular characterization of VIP receptors in RPMM was performed after [125I]VIP cross-linking to membranes using the cross-linker dithiobis (succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins revealed specific [125I]VIP-protein complexes of M(r) 55,000 +/- 1700, 35,000 +/- 900, and 22,000 +/- 500.
Calvo
J R
JR
Department of Medical Biochemistry and Molecular Biology, School of Medicine, University of Sevilla, Spain.
Guerrero
J M
JM
López-Gonzalez
M A
MA
Osuna
C
C
Segura
J J
JJ
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D053379:Chromogranin A; D004229:Dithiothreitol / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D009479:Neuropeptides / Q000378:metabolism; D010187:Pancreatic Hormones / Q000378:metabolism; D010740:Phospholipases / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D018005:Receptors, Vasoactive Intestinal Peptide / Q000302:isolation & purification / Q000378:metabolism*; D012492:Salts / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*
D053379:Chromogranin A; D007328:Insulin; D009479:Neuropeptides; D010187:Pancreatic Hormones; D018005:Receptors, Vasoactive Intestinal Peptide; D012492:Salts; C050836:pancreastatin; D014660:Vasoactive Intestinal Peptide; D010740:Phospholipases; D014357:Trypsin; D004229:Dithiothreitol