We have previously demonstrated specific binding sites for adrenomedullin, a novel member of the calcitonin family of peptides, in rat muscles. It is unclear whether these receptors are vascular or muscular. Receptors for the structurally similar calcitonin gene-related peptide (CGRP) are present on myocytes and might be involved in the regulation of myocyte glucose metabolism and control by motor neurons. We investigated whether adrenomedullin binding sites were present on L6 myocytes. Specific [125I]adrenomedullin binding sites were demonstrated where adrenomedullin competed with an IC50 of 0.22±0.04 nM (mean±S.E.M.) and a concentration of binding sites (Bmax) of 0.95±0.19 pmol/mg of protein (mean±S.E.M.). CGRP and the specific CGRP receptor antagonist CGRP(8–37) competed weakly at this site (IC50 > 10 and 601±298 nM respectively). Binding studies with [125I]CGRP revealed a binding site for CGRP (IC50 = 0.13±0.01 nM; Bmax = 0.83±0.10 pmol/mg of protein) where both CGRP(8–37) and adrenomedullin competed with [125I]CGRP with IC50 values of 1.15±0.12 and 8.68±0.98 nM respectively. Chemical cross-linking showed the CGRP and adrenomedullin binding site–ligand complexes to have approximate molecular masses of 82 and 76 kDa respectively. Both CGRP and adrenomedullin increased adenylate cyclase activity with similar potencies. In both cases adenylate cyclase activation was blocked by CGRP(8–37). Stimulation with 10 nM adrenomedullin or CGRP caused an increase in the percentage of total activated cellular cAMP-dependent protein kinase from 38% in resting cells to 100% and 98% respectively. Therefore in L6 cells adrenomedullin can bind to CGRP receptors, activating adenylate cyclase and cAMP-dependent protein kinase.
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August 1996
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Research Article|
August 15 1996
A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors
Hedley A COPPOCK;
Hedley A COPPOCK
1Division of Endocrinology and Metabolic Medicine, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 0NN, U.K.
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Ali A OWJI;
Ali A OWJI
1Division of Endocrinology and Metabolic Medicine, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 0NN, U.K.
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Stephen R BLOOM;
Stephen R BLOOM
1Division of Endocrinology and Metabolic Medicine, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 0NN, U.K.
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David M SMITH
David M SMITH
*
*To whom correspondence should be addressed
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Publisher: Portland Press Ltd
Received:
February 13 1996
Revision Received:
April 18 1996
Accepted:
April 26 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 318 (1): 241–245.
Article history
Received:
February 13 1996
Revision Received:
April 18 1996
Accepted:
April 26 1996
Citation
Hedley A COPPOCK, Ali A OWJI, Stephen R BLOOM, David M SMITH; A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors. Biochem J 15 August 1996; 318 (1): 241–245. doi: https://doi.org/10.1042/bj3180241
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