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. 1996 Aug 15;318(Pt 1):241–245. doi: 10.1042/bj3180241

A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors.

H A Coppock 1, A A Owji 1, S R Bloom 1, D M Smith 1
PMCID: PMC1217614  PMID: 8761478

Abstract

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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Selected References

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  1. Bhogal R., Smith D. M., Bloom S. R. Solubilization of binding sites for IAPP and CGRP from rat lung. Peptides. 1994;15(8):1383–1390. doi: 10.1016/0196-9781(94)90113-9. [DOI] [PubMed] [Google Scholar]
  2. Bhogal R., Smith D. M., Owji A. A., Bloom S. R. Binding sites for islet amyloid polypeptide in mammalian lung: species variation and effects on adenylyl cyclase. Can J Physiol Pharmacol. 1995 Jul;73(7):1030–1036. doi: 10.1139/y95-145. [DOI] [PubMed] [Google Scholar]
  3. Bhogal R., Smith D. M., Purkiss P., Bloom S. R. Molecular identification of binding sites for calcitonin gene-related peptide (CGRP) and islet amyloid polypeptide (IAPP) in mammalian lung: species variation and binding of truncated CGRP and IAPP. Endocrinology. 1993 Nov;133(5):2351–2361. doi: 10.1210/endo.133.5.8404688. [DOI] [PubMed] [Google Scholar]
  4. Changeux J. P., Duclert A., Sekine S. Calcitonin gene-related peptides and neuromuscular interactions. Ann N Y Acad Sci. 1992 Jun 30;657:361–378. doi: 10.1111/j.1749-6632.1992.tb22783.x. [DOI] [PubMed] [Google Scholar]
  5. De Souza E. B., Webster E. L., Grigoriadis D. E., Tracey D. E. Corticotropin-releasing factor (CRF) and interleukin-1 (IL-1) receptors in the brain-pituitary-immune axis. Psychopharmacol Bull. 1989;25(3):299–305. [PubMed] [Google Scholar]
  6. Eguchi S., Hirata Y., Kano H., Sato K., Watanabe Y., Watanabe T. X., Nakajima K., Sakakibara S., Marumo F. Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells. FEBS Lett. 1994 Mar 7;340(3):226–230. doi: 10.1016/0014-5793(94)80143-6. [DOI] [PubMed] [Google Scholar]
  7. Feng C. J., Kang B., Kaye A. D., Kadowitz P. J., Nossaman B. D. L-NAME modulates responses to adrenomedullin in the hindquarters vascular bed of the rat. Life Sci. 1994;55(22):PL433–PL438. doi: 10.1016/0024-3205(94)00347-5. [DOI] [PubMed] [Google Scholar]
  8. Fraker P. J., Speck J. C., Jr Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun. 1978 Feb 28;80(4):849–857. doi: 10.1016/0006-291x(78)91322-0. [DOI] [PubMed] [Google Scholar]
  9. Glass D. B., Cheng H. C., Mende-Mueller L., Reed J., Walsh D. A. Primary structural determinants essential for potent inhibition of cAMP-dependent protein kinase by inhibitory peptides corresponding to the active portion of the heat-stable inhibitor protein. J Biol Chem. 1989 May 25;264(15):8802–8810. [PubMed] [Google Scholar]
  10. Haynes J. M., Cooper M. E. Adrenomedullin and calcitonin gene-related peptide in the rat isolated kidney and in the anaesthetised rat: in vitro and in vivo effects. Eur J Pharmacol. 1995 Jun 23;280(1):91–94. doi: 10.1016/0014-2999(95)00230-i. [DOI] [PubMed] [Google Scholar]
  11. Ichiki Y., Kitamura K., Kangawa K., Kawamoto M., Matsuo H., Eto T. Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett. 1994 Jan 24;338(1):6–10. doi: 10.1016/0014-5793(94)80106-1. [DOI] [PubMed] [Google Scholar]
  12. Ishizaka Y., Ishizaka Y., Tanaka M., Kitamura K., Kangawa K., Minamino N., Matsuo H., Eto T. Adrenomedullin stimulates cyclic AMP formation in rat vascular smooth muscle cells. Biochem Biophys Res Commun. 1994 Apr 15;200(1):642–646. doi: 10.1006/bbrc.1994.1496. [DOI] [PubMed] [Google Scholar]
  13. Jougasaki M., Wei C. M., Heublein D. M., Sandberg S. M., Burnett J. C., Jr Immunohistochemical localization of adrenomedullin in canine heart and aorta. Peptides. 1995;16(4):773–775. doi: 10.1016/0196-9781(95)00011-8. [DOI] [PubMed] [Google Scholar]
  14. Kapas S., Catt K. J., Clark A. J. Cloning and expression of cDNA encoding a rat adrenomedullin receptor. J Biol Chem. 1995 Oct 27;270(43):25344–25347. doi: 10.1074/jbc.270.43.25344. [DOI] [PubMed] [Google Scholar]
  15. Kitamura K., Kangawa K., Kawamoto M., Ichiki Y., Nakamura S., Matsuo H., Eto T. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun. 1993 Apr 30;192(2):553–560. doi: 10.1006/bbrc.1993.1451. [DOI] [PubMed] [Google Scholar]
  16. Krebs E. G. Nobel Lecture. Protein phosphorylation and cellular regulation I. Biosci Rep. 1993 Jun;13(3):127–142. doi: 10.1007/BF01149958. [DOI] [PubMed] [Google Scholar]
  17. Kreutter D., Orena S. J., Andrews K. M. Suppression of insulin-stimulated glucose transport in L6 myocytes by calcitonin gene-related peptide. Biochem Biophys Res Commun. 1989 Oct 16;164(1):461–467. doi: 10.1016/0006-291x(89)91742-7. [DOI] [PubMed] [Google Scholar]
  18. Kureishi Y., Kobayashi S., Nishimura J., Nakano T., Kanaide H. Adrenomedullin decreases both cytosolic Ca2+ concentration and Ca(2+)-sensitivity in pig coronary arterial smooth muscle. Biochem Biophys Res Commun. 1995 Jul 17;212(2):572–579. doi: 10.1006/bbrc.1995.2008. [DOI] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Laufer R., Changeux J. P. Calcitonin gene-related peptide elevates cyclic AMP levels in chick skeletal muscle: possible neurotrophic role for a coexisting neuronal messenger. EMBO J. 1987 Apr;6(4):901–906. doi: 10.1002/j.1460-2075.1987.tb04836.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leighton B., Cooper G. J. Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro. Nature. 1988 Oct 13;335(6191):632–635. doi: 10.1038/335632a0. [DOI] [PubMed] [Google Scholar]
  22. Lippton H., Chang J. K., Hao Q., Summer W., Hyman A. L. Adrenomedullin dilates the pulmonary vascular bed in vivo. J Appl Physiol (1985) 1994 May;76(5):2154–2156. doi: 10.1152/jappl.1994.76.5.2154. [DOI] [PubMed] [Google Scholar]
  23. Miura K., Ebara T., Okumura M., Matsuura T., Kim S., Yukimura T., Iwao H. Attenuation of adrenomedullin-induced renal vasodilatation by NG-nitro L-arginine but not glibenclamide. Br J Pharmacol. 1995 Jul;115(6):917–924. doi: 10.1111/j.1476-5381.1995.tb15898.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Murray K. J., England P. J., Lynham J. A., Mills D., Schmitz-Peiffer C., Reeves M. L. Use of a synthetic dodecapeptide (malantide) to measure the cyclic AMP-dependent protein kinase activity ratio in a variety of tissues. Biochem J. 1990 May 1;267(3):703–708. doi: 10.1042/bj2670703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nuki C., Kawasaki H., Kitamura K., Takenaga M., Kangawa K., Eto T., Wada A. Vasodilator effect of adrenomedullin and calcitonin gene-related peptide receptors in rat mesenteric vascular beds. Biochem Biophys Res Commun. 1993 Oct 15;196(1):245–251. doi: 10.1006/bbrc.1993.2241. [DOI] [PubMed] [Google Scholar]
  26. Owji A. A., Smith D. M., Coppock H. A., Morgan D. G., Bhogal R., Ghatei M. A., Bloom S. R. An abundant and specific binding site for the novel vasodilator adrenomedullin in the rat. Endocrinology. 1995 May;136(5):2127–2134. doi: 10.1210/endo.136.5.7720662. [DOI] [PubMed] [Google Scholar]
  27. Poyner D. R., Andrew D. P., Brown D., Bose C., Hanley M. R. Pharmacological characterization of a receptor for calcitonin gene-related peptide on rat, L6 myocytes. Br J Pharmacol. 1992 Feb;105(2):441–447. doi: 10.1111/j.1476-5381.1992.tb14272.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Poyner D. R. Calcitonin gene-related peptide: multiple actions, multiple receptors. Pharmacol Ther. 1992;56(1):23–51. doi: 10.1016/0163-7258(92)90036-y. [DOI] [PubMed] [Google Scholar]
  29. Rink T. J., Beaumont K., Koda J., Young A. Structure and biology of amylin. Trends Pharmacol Sci. 1993 Apr;14(4):113–118. doi: 10.1016/0165-6147(93)90081-t. [DOI] [PubMed] [Google Scholar]
  30. Sakata J., Shimokubo T., Kitamura K., Nakamura S., Kangawa K., Matsuo H., Eto T. Molecular cloning and biological activities of rat adrenomedullin, a hypotensive peptide. Biochem Biophys Res Commun. 1993 Sep 15;195(2):921–927. doi: 10.1006/bbrc.1993.2132. [DOI] [PubMed] [Google Scholar]
  31. Salomon Y., Londos C., Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem. 1974 Apr;58(2):541–548. doi: 10.1016/0003-2697(74)90222-x. [DOI] [PubMed] [Google Scholar]
  32. Shimekake Y., Nagata K., Ohta S., Kambayashi Y., Teraoka H., Kitamura K., Eto T., Kangawa K., Matsuo H. Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells. J Biol Chem. 1995 Mar 3;270(9):4412–4417. doi: 10.1074/jbc.270.9.4412. [DOI] [PubMed] [Google Scholar]
  33. Sugo S., Minamino N., Kangawa K., Miyamoto K., Kitamura K., Sakata J., Eto T., Matsuo H. Endothelial cells actively synthesize and secrete adrenomedullin. Biochem Biophys Res Commun. 1994 Jun 30;201(3):1160–1166. doi: 10.1006/bbrc.1994.1827. [DOI] [PubMed] [Google Scholar]
  34. Sugo S., Minamino N., Shoji H., Kangawa K., Kitamura K., Eto T., Matsuo H. Production and secretion of adrenomedullin from vascular smooth muscle cells: augmented production by tumor necrosis factor-alpha. Biochem Biophys Res Commun. 1994 Aug 30;203(1):719–726. doi: 10.1006/bbrc.1994.2241. [DOI] [PubMed] [Google Scholar]
  35. Walsh D. A., Glass D. B., Mitchell R. D. Substrate diversity of the cAMP-dependent protein kinase: regulation based upon multiple binding interactions. Curr Opin Cell Biol. 1992 Apr;4(2):241–251. doi: 10.1016/0955-0674(92)90039-f. [DOI] [PubMed] [Google Scholar]
  36. Withers D. J., Coppock H. A., Seufferlein T., Smith D. M., Bloom S. R., Rozengurt E. Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells. FEBS Lett. 1996 Jan 2;378(1):83–87. doi: 10.1016/0014-5793(95)01427-6. [DOI] [PubMed] [Google Scholar]
  37. Zimmermann U., Fischer J. A., Muff R. Adrenomedullin and calcitonin gene-related peptide interact with the same receptor in cultured human neuroblastoma SK-N-MC cells. Peptides. 1995;16(3):421–424. doi: 10.1016/0196-9781(94)00195-c. [DOI] [PubMed] [Google Scholar]

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