Characteristics and distribution of endogenous RFamide-related peptide-1

We have recently identified RFamide-related peptide (RFRP) gene that would encode three peptides (i,e., RFRP-1, -2, and -3) in human and bovine, and d emonstrated that synthetic RFRP-1 and -3 act as specific agonists for a G p rotein-coupled receptor OT7T022. However, molecular characteristics and tis sue distribution of endogenous RFRPs have not been determined yet. In this study, we prepared a monoclonal antibody for the C-terminal portion of rat RFRP-1. As this antibody could recognize a consensus sequence among the C-t erminal portions of rat, human, and bovine RFRP-1, we purified endogenous R FRP-1 from bovine hypothalamus on the basis of immunoreactivity to the anti body. The purified bovine endogenous RFRP-1 was found to have 35-amino-acid length that corresponds to 37-amino-acid length in human and rat. We subse quently constructed a sandwich enzyme immunoassay using the monoclonal anti body and a polyclonal antibody for the N-terminal portion of rat RFRP-1, an d analyzed the tissue distribution of endogenous RFRP-1 in rats. Significan t levels of RFRP-1 were detected only in the central nervous system, and th e highest concentration of RFRP-1 was detected in the hypothalamus. RFRP-1- positive nerve cells were detected in the rat hypothalamus by immunohistoch emical analyses using the monoclonal antibody. In culture, RFRP-1 lowered c AMP production in Chinese hamster ovary cells expressing OT7T022 and it was abolished by pre-treatment with pertussis toxin, suggesting that OT7T022 c ouples G(i)/G(o) in the signal transduction pathway. (C) 2001 Elsevier Scie nce B.V. All rights reserved.