NOVEL CYCLIC PEPTIDE AGONIST OF HIGH POTENCY AND SELECTIVITY FOR THE TYPE-II VASOACTIVE-INTESTINAL-PEPTIDE RECEPTOR

Ro 25-1392 (17),Ala(19),Asp(25),Leu(26),Lys(27,28)-vasoactive intestin al peptide(cyclo 21-25)] is a cyclic peptide analog of vasoactive inte stinal peptide (VIP) that potently exerts cellular effects typical of VIP. The selectivity of Ro 25-1392 for type I (VIPR1) and type II (VIP R2) VIP receptors was investigated first in competitive binding studie s using Chinese hamster ovary cell transfectants stably expressing rec ombinant human VIPR1 and VIPR2. Nonradioactive Ro 25-1392 was as poten t a competitive inhibitor as VIP for the binding of I-125-VIP to VIPR2 transfectants (K-i = 9.6 +/- 1.0 and 16 +/- 1.7 nM, respectively; mea n +/- S.E.M., n = 4). In contrast, Ro 25-1392 had a very low affinity for VIPR1, compared with VIP, and attained a maximum of only 40% mean inhibition of binding of I-125-VIP at 1 mu M. The affinity of VIP (K-i = 3.4 +/- 1.5 nM, mean +/- S.E.M., n = 4) for binding to VIPR1 was 10 00-fold greater than that of Ro 25-1392. Ro 25-1392 evoked concurrent and concentration-dependent increases in intracellular levels of calci um and cyclic AMP (EC50 = 3.0 +/- 0.4 nM, mean +/- S.E.M., n = 4) in V IPR2 transfectants, but not in VIPR1 transfectants. The VIP receptor s pecificity of Ro 25-1392 was confirmed by preincubation of Chinese ham ster ovary transfectants with 0.1 mu M Ro 25-1392 for 18 hr at 37 degr ees C, to down-regulate each type of VIP receptor. Pretreatment of VIP R2 transfectants with Ro 25-1392 decreased B-max by a mean of 58% and VIP-induced increases in the intracellular concentration of cyclic AMP by a mean of 65%. In contrast, there was no significant change in VIP R1 transfectants after pretreatment with Ro 25-1392. Ro 25-1392 thus i s selectively recognized by VIPR2, with consequent initiation of cycli c AMP and Ca++ signals and down-regulation of VIPR2. This potent analo g of VIP may prove useful for investigations of VIPR2-mediated physiol ogical effects of VIP and exploration of the roles of VIPR2 in disease s.