Research paper
Expression of VIP receptors in mouse peritoneal macrophages: Functional and molecular characterization

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Abstract

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°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/106 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 μM. 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 μM. 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 μM. To characterize the molecular mass of VIP receptors, [125I]VIP was covalently bound to membranes from MPM using the cross-linker dithiobis(succinimidyl propionate) (DTSP); sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized membranes proteins revealed the presence of a major specific componentwith an Mr value of 55 300 ± 1100 as estimated in denaturing conditions. Other proteins with Mr values of 34 500 ± 700, 23 900 ± 500 and 18 500 ± 500 also were labeled. Taken together, these results demonstrate the presence of specific and functional VIP receptors in MPM and further support the concept of VIP as a neuroimmunoregulatory peptide.

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