Abstract
Ro 25–1392 [Ac-Glu8,OCH3-Tyr10,Lys12,Nle17,Ala19,Asp25,Leu26,- Lys27,28-vasoactive intestinal peptide(cyclo 21–25)] is a cyclic peptide analog of vasoactive intestinal peptide (VIP) that potently exerts cellular effects typical of VIP. The selectivity of Ro 25–1392 for type I (VIPR1) and type II (VIPR2) VIP receptors was investigated first in competitive binding studies using Chinese hamster ovary cell transfectants stably expressing recombinant human VIPR1 and VIPR2. Nonradioactive Ro 25–1392 was as potent a competitive inhibitor as VIP for the binding of 125I-VIP to VIPR2 transfectants (K i = 9.6 ± 1.0 and 16 ± 1.7 nM, respectively; mean ± 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 of125I-VIP at 1 μM. The affinity of VIP (K i = 3.4 ± 1.5 nM, mean ± S.E.M., n = 4) for binding to VIPR1 was 1000-fold greater than that of Ro 25–1392. Ro 25–1392 evoked concurrent and concentration-dependent increases in intracellular levels of calcium and cyclic AMP (EC50 = 3.0 ± 0.4 nM, mean ± S.E.M., n = 4) in VIPR2 transfectants, but not in VIPR1 transfectants. The VIP receptor specificity of Ro 25–1392 was confirmed by preincubation of Chinese hamster ovary transfectants with 0.1 μM Ro 25–1392 for 18 hr at 37°C, to down-regulate each type of VIP receptor. Pretreatment of VIPR2 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 VIPR1 transfectants after pretreatment with Ro 25–1392. Ro 25–1392 thus is selectively recognized by VIPR2, with consequent initiation of cyclic AMP and Ca++ signals and down-regulation of VIPR2. This potent analog of VIP may prove useful for investigations of VIPR2-mediated physiological effects of VIP and exploration of the roles of VIPR2 in diseases.
Footnotes
-
Send reprint requests to: Edward J. Goetzl, M.D., Professor of Medicine and Microbiology, University of California, UB8B, Box 0711, 533 Parnassus, San Francisco, CA 94143-0711.
-
↵1 This research was supported by Grants AI29912 and AI34570 (E.J.G.) from the National Institutes of Health.
- Abbreviations:
- [Ca++]i
- intracellular concentration of Ca++
- [cAMP]i
- intracellular concentration of cyclic AMP
- CHO
- Chinese hamster ovary
- VIP
- vasoactive intestinal peptide
- VIPR1
- type I vasoactive intestinal peptide receptor(s)
- VIPR2
- type II vasoactive intestinal peptide receptor(s)
- Received September 30, 1996.
- Accepted January 8, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|
