Characterization of VIP receptor-effector system antagonists in rat and mouse peritoneal macrophages

Abstract

In the present study we show that the synthetic peptides [4-Cl- d-Phe 6,Leu 17]VIP and the growth hormone releasing factor (GRF) analog [Ac-Tyr 1, d-Phe 2]GRF-(1–29)-NH 2 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 IC 50 values was: VIP (IC 50=1.90±0.16 nM)>[4-Cl- d-Phe 6,Leu 17]VIP (IC 50=125.8±13.2 nM)>[Ac-Tyr 1, d-Phe 2]GRF-(1–29)-NH 2 (IC 50=354.8±21.2 nM). In mouse peritoneal macrophages a similar pattern of potency was observed: VIP (IC 50=1.58±0.12 nM)>[4-Cl- d-Phe 6,Leu 17]VIP (IC 50=110.8±10.7 nM)>[Ac-Tyr 1, d-Phe 2]GRF-(1–29)-NH 2 (IC 50=251±19.2 nM). The behavior as VIP receptor antagonists of both [4-Cl- d-Phe 6,Leu 17]VIP and [Ac-Tyr 1, d-Phe 2]GRF-(1–29)-NH 2 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 β-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-Phe 6,Leu 17]VIP or [Ac-Tyr 1, d-Phe 2]GRF-(1–29) -NH 2, suggesting that both antagonists acted via specific VIP receptors. Moreover, propranolol, a β-adrenoceptor antagonist, did not affect the VIP-stimulated cyclic AMP production and the antagonist role of [4-Cl- d-Phe 6,Leu 17]VIP or [Ac-Tyr 1, d-Phe 2]GRF-(1–29)-NH 2; (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.