Characterisation and molecular identification of adrenomedullin binding sites in the rat spinal cord: a comparison with calcitonin gene-related peptide receptors.
Calcitonin gene-related peptide (CGRP) and its receptors are found in mammalian spinal cord. We show, for the first time, binding sites for the novel related peptide adrenomedullin in rat spinal cord microsomes. 125I-Adrenomedullin binding showed high affinity (KD = 0.45 +/- 0.06 nM) and sites were abundant (Bmax = 723 +/- 71 fmol/mg of protein). CGRP, amylin, and calcitonin did not compete at these sites (Ki > 10 microM). High-affinity CGRP binding sites (KD = 0.18 +/- 0.01 nM) were much less numerous (Bmax = 17.7 +/- 2.4 fmol/mg of protein) and showed competition by unlabeled adrenomedullin (Ki = 34.6 +/- 2.4 nM). Chemical cross-linking revealed a major band for 125I-adrenomedullin of M(r) = 84,400 +/- 1,200 and a minor band of M(r) = 122,000 +/- 8,700. 125I-CGRP cross-linking showed bands of lower molecular weight (M(r) = 74,500 +/- 5,000 and 61,000 +/- 2,200). Enzymic deglycosylation of the adrenomedullin binding site showed a considerable carbohydrate content. Neither adrenomedullin nor CGRP was able to increase cyclic AMP in spinal cord. Adrenomedullin mRNA was present in spinal cord, at one-third of its level in lung, and adrenomedullin immunoreactivity was present, at a low concentration (40 fmol/g of tissue). Thus, the presence of abundant binding sites and adrenomedullin mRNA and immunoreactivity anticipate an as yet undefined function for this peptide in spinal cord.
Owji
A A
AA
Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, England.
Gardiner
J V
JV
Upton
P D
PD
Mahmoodi
M
M
Ghatei
M A
MA
Bloom
S R
SR
Smith
D M
DM
D000262:Adenylyl Cyclases / Q000378:metabolism; D053607:Adrenomedullin; D000682:Amyloid / Q000494:pharmacology; D000818:Animals; D000959:Antihypertensive Agents / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002116:Calcitonin / Q000494:pharmacology; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism / Q000494:pharmacology; D003432:Cross-Linking Reagents; D019204:GTP-Binding Proteins / Q000378:metabolism; D007457:Iodine Radioisotopes; D058228:Islet Amyloid Polypeptide; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism*; D010452:Peptide Biosynthesis; D010455:Peptides / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011863:Radioimmunoassay; D051381:Rats; D058265:Receptors, Adrenomedullin; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000378:metabolism*; D018000:Receptors, Peptide; D013116:Spinal Cord / Q000378:metabolism*
D000682:Amyloid; D000959:Antihypertensive Agents; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D058228:Islet Amyloid Polypeptide; D008565:Membrane Proteins; D010455:Peptides; D012333:RNA, Messenger; D058265:Receptors, Adrenomedullin; D018015:Receptors, Calcitonin Gene-Related Peptide; D018000:Receptors, Peptide; D053607:Adrenomedullin; D002116:Calcitonin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D015740:Calcitonin Gene-Related Peptide