ABSTRACT
In the present study we show that the synthetic peptides [4-Cl-D-Phe6,Leu17]VIP and the growth hormone releasing factor (GRF) analog [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 inhibit in a competitive manner the specific [125I]VIP binding to both rat and mouse peritoneal macrophages. In rat peritoneal macrophages, the order of potency of the different peptides, as expressed by the IC50 values was: VIP (IC50 = 1.90 +/- 0.16 nM) > [4-Cl-D-Phe6,Leu17]VIP (IC50 = 125.8 +/- 13.2 nM) > [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 (IC50 = 354.8 +/- 21.2 nM). In mouse peritoneal macrophages a similar pattern of potency was observed: VIP (IC50 = 1.58 +/- 0.12 nM) > [4-Cl-D-Phe6,Leu17]VIP (IC50 = 110.8 +/- 10.7 nM) > [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 (IC50 = 251 +/- 19.2 nM). The behavior as VIP receptor antagonists of both [4-Cl-D-Phe6,Leu17]VIP and [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 in rat and mouse peritoneal macrophages was confirmed by: (a) the shift to the right of VIP dose-stimulated cyclic AMP production curves in the presence of the two antagonists; (b) the agreement between the order of efficacy of the two peptides in competition experiments with the corresponding inhibition of cyclic AMP production; (c) the inefficiency of the two antagonists on the stimulation of cyclic AMP production by the beta-adrenoceptor agonist isoproterenol, which indicates the specificity of the interaction; (d) the synergic effect of VIP on isoproterenol-stimulated cyclic AMP production was completely abolished by [4-Cl-D-Phe6,Leu17]VIP or [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2, suggesting that both antagonists acted via specific VIP receptors. Moreover, propranolol, a beta-adrenoceptor antagonist, did not affect the VIP-stimulated cyclic AMP production and the antagonist role of [4-Cl-D-Phe6,Leu17]VIP or [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2; (e) in cross-linking experiments, the intensity of the labeling of the [125I]VIP/receptor complexes was significantly lower with the antagonists than in the control experimental situation in both mouse and rat peritoneal macrophage membranes.
Subject(s)
Macrophages, Peritoneal/drug effects , Receptors, Vasoactive Intestinal Peptide/antagonists & inhibitors , Adrenergic beta-Agonists/pharmacology , Adrenergic beta-Antagonists/pharmacology , Animals , Binding, Competitive , Cell Membrane/chemistry , Cross-Linking Reagents , Cyclic AMP/biosynthesis , In Vitro Techniques , Isoproterenol/pharmacology , Macrophages, Peritoneal/metabolism , Mice , Propranolol/pharmacology , Rats , Sermorelin/analogs & derivatives , Sermorelin/metabolism , Sermorelin/pharmacology , Vasoactive Intestinal Peptide/analogs & derivatives , Vasoactive Intestinal Peptide/chemistry , Vasoactive Intestinal Peptide/metabolism , Vasoactive Intestinal Peptide/pharmacologyABSTRACT
Receptors for VIP in mouse peritoneal macrophages (MPM) were examined using [125I]labeled VIP as ligand. The receptor binding was rapid, reversible, saturable, specific, and dependent on time, pH, temperature and cell concentration. At 15 degrees C, the stoichiometric data suggested the presence of two classes of VIP receptors with Kd values of 1.05 +/- 0.2 and 66.4 +/- 11.0 nM and binding capacities of 19.2 +/- 2.8 and 706.6 +/- 172.0 fmol VIP/10(6) cells. The interaction showed a high degree of specificity, as suggested by competition experiments with various peptides structurally related to VIP as follows: VIP > helodermin > rGRF > PHI >> secretin. Glucagon, pancreastatin, somatostatin, insulin, and octapeptide of cholecystokinin (CCK 26-33) were ineffective at concentrations as high as 1 microM. VIP was a potent and efficient stimulator of cyclic AMP production in MPM. The stimulation was observed at a concentration as low as 0.01 nM VIP. Half-maximal stimulation (ED50) was observed at 1.0 +/- 0.2 nM VIP, and maximal stimulation (three-fold above basal levels) was obtained between 0.1-1 microM. The cyclic AMP system of mouse peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP > helodermin > rGRF > PHI >> secretin. Glucagon, insulin, pancreastatin, somatostatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
