EXPRESSION OF VIP RECEPTORS IN MOUSE PERITONEAL-MACROPHAGES - FUNCTIONAL AND MOLECULAR CHARACTERIZATION

Receptors for VIP in mouse peritoneal macrophages (MPM) were examined using [I-125]labeled VIP as ligand. The receptor binding was rapid, re versible, saturable, specific, and dependent on time, pH, temperature and cell concentration. At 15 degrees C, the stoichiometric data sugge sted the presence of two classes of VIP receptors with K-d values of 1 .05 +/- 0.2 and 66.4 +/- 11.0 nM binding capacities of 19.2 +/- 2.8 an d 706.6 +/- 172.0 fmol VIP/10(6) cells. The interaction showed a high degree of specificity, suggested by competition experiments with vario us peptides structurally related to VIP as follows: VIP > helodermin > rGRF > PHI >> secretin. Glucagon, pancreastatin, somatostatin, insuli n, and octapeptide of cholecystokinin (CCK 26-33) were ineffective at concentrations as high as 1 mu M. VIP was a potent and efficient stimu lator of cyclic AMP production in MPM. The stimulation was observed at a concentration as low as 0.01 nM VIP. Half-maximal stimulation (ED(5 0)) was observed at 1.0 +/- 0.2 nM VIP, and maximal stimulation (three -fold above basal levels) was obtained between 0.1-1 mu M. The cyclic AMP system of mouse peritoneal macrophages showed a high specificity f or VIP. The cyclic 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 mu M. To characterize the m olecular mass of VIP receptors, [I-125]VIP was covalently bound to mem branes from MPM using the cross-linker dithiobis(succinimidyl propiona te) (DTSP); sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized membranes proteins revealed the presence of a major specific component with an M(r), value of 55 300 +/- 1100 as estimate d in denaturing conditions. Other proteins with M(r) values of 34 500 +/- 700, 23 900 +/- 500 and 18 500 +/- 500 also were labeled. Taken to gether, these results demonstrate the presence of specific and functio nal VIP receptors in MPM and further support the concept of VIP as a n euroimmunoregulatory peptide.