STRUCTURE-ACTIVITY RELATIONSHIP OF ADRENOMEDULLIN, A NOVEL VASODILATORY PEPTIDE, IN CULTURED RAT VASCULAR SMOOTH-MUSCLE CELLS

Vascular smooth muscle cells (VSMC) from rat aorta possess specific re ceptors for a novel potent vasorelaxant peptide, adrenomedullin (AM). To elucidate its receptor coupling to guanine nucleotide-binding stimu latory protein and the structural requirement of the AM molecule to it s vascular receptors, we have studied the effects of guanine nucleotid es on [I-125]human (h) AM binding and adenylate cyclase activity in cu ltured rat VSMC, and the effects of various synthetic hAM analogs on [ I-125]hAM binding and the cAMP response. Guanosine 5'-O-(3-thiotriphos phate) dose dependently inhibited [I-125]hAM binding to rat VSMC membr anes, hAM stimulated adenylate cyclase activity, and its effect was ad ditive with GTP. hAM-induced cAMP formation was abrogated by pretreatm ent with cholera toxin, but not by that with pertussis toxin. Intact h AM-(1-52)-NH2 and N-terminal truncated derivatives [hAM-(13-52)-NH2, h AM-(16-52)-NH2] almost equally inhibited I-125]hAM binding and stimula ted cAMP formation, whereas removal of C-terminal Tyr(52) residue [hAM -(1-51)-NH2] remarkably decreased receptor-binding activity and the cA MP response. The effects of hAM-(1-52)-OH, hAM-(1-51)-OH, and a linear hAM analog ([carbamoylmethyl-Cys(16,21)]hAM-NH2) were far less potent on receptor binding and the cAMP response than that of hAM-(1-52)-NH2 . The C-terminal fragment [hAM-(33-52)-NH2] and the N-terminal fragmen t [hAM-(1-10)-OH] had neither receptor-binding nor adenylate cyclase a ctivity, hAM-(22-52)-NH2 had no agonistic effect, but showed an antago nistic effect on the hAM-induced cAMP response. These data suggest tha t vascular AM receptors are functionally coupled to adenylate cyclase via guanine nucleotide-binding stimulatory protein. Studies of the str ucture-activity relationship of hAM revealed that the cyclic structure formed by the disulfide bridge and amidation of the C-terminal residu e of the AM molecule are critical for receptor binding and subsequent cAMP generation and suggest that the C-terminal fragment hAM-(22-52)-N H2 may be an antagonist for vascular AM receptors.