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 receptors for a novel potent vasorelaxant peptide, adrenomedullin (AM). To elucidate its receptor coupling to guanine nucleotide-binding stimulatory protein and the structural requirement of the AM molecule to its vascular receptors, we have studied the effects of guanine nucleotides on [125I]human (h) AM binding and adenylate cyclase activity in cultured rat VSMC, and the effects of various synthetic hAM analogs on [125I]hAM binding and the cAMP response. Guanosine 5'-O-(3-thiotriphosphate) dose dependently inhibited [125I]hAM binding to rat VSMC membranes. hAM stimulated adenylate cyclase activity, and its effect was additive with GTP. hAM-induced cAMP formation was abrogated by pretreatment with cholera toxin, but not by that with pertussis toxin. Intact hAM-(1-52)-NH2 and N-terminal truncated derivatives [hAM-(13-52)-NH2, hAM-(16-52)-NH2] almost equally inhibited [125I]hAM binding and stimulated cAMP formation, whereas removal of C-terminal Tyr52 residue [hAM-(1-51)-NH2] remarkably decreased receptor-binding activity and the cAMP response. The effects of hAM-(1-52)-OH, hAM-(1-51)-OH, and a linear hAM analog ([carbamoylmethyl-Cys16,21]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 fragment [hAM-(1-10)-OH] had neither receptor-binding nor adenylate cyclase activity. hAM-(22-52)-NH2 had no agonistic effect, but showed an antagonistic effect on the hAM-induced cAMP response. These data suggest that vascular AM receptors are functionally coupled to adenylate cyclase via guanine nucleotide-binding stimulatory protein. Studies of the structure-activity relationship of hAM revealed that the cyclic structure formed by the disulfide bridge and amidation of the C-terminal residue of the AM molecule are critical for receptor binding and subsequent cAMP generation and suggest that the C-terminal fragment hAM-(22-52)-NH2 may be an antagonist for vascular AM receptors.
Eguchi
S
S
Second Department of Internal Medicine, Tokyo Medical and Dental University, Japan.
Hirata
Y
Y
Iwasaki
H
H
Sato
K
K
Watanabe
T X
TX
Inui
T
T
Nakajima
K
K
Sakakibara
S
S
Marumo
F
F
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D053607:Adrenomedullin; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D037342:Pertussis Toxin; D051381:Rats; D017208:Rats, Wistar; D013329:Structure-Activity Relationship; D014665:Vasodilator Agents / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D010455:Peptides; D014665:Vasodilator Agents; D010566:Virulence Factors, Bordetella; D053607:Adrenomedullin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002772:Cholera Toxin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D000262:Adenylyl Cyclases