Characterization and regulation of high affinity calcitonin gene-related peptide receptors in cultured neonatal rat cardiac myocytes

Tapan Kumar Chatterjee, Jadine A. Moy, Rory A. Fisher, Rory A. Fisher

Research output: Contribution to journalArticle

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Abstract

Previous studies have shown that stimulation of cultured beating cardiac myocytes with calcitonin gene-related peptide (CGRP) produces increased beating frequency, increased cellular cAMP concentration, and a homologous desensitization of the cAMP-elevating action of CGRP. In the present study, the characteristics and regulation of [125I]CGRP binding sites in cultured cardiac myocytes were investigated. Binding of [125I] CGRP to membranes prepared from these cells was selective, saturable, and of high affinity. Scatchard transformation of the saturation isotherm generated a linear plot suggesting the existence of a homogeneous population of binding sites with an equilibrium binding constant of 41 ± 7 pM and maximum binding capacity of 31 ± 5 fmol/mg protein. Binding of [125I]CGRP to membranes was inhibited completely by guanosine 5’-(3-O- thio)triphosphate (250 μM), suggesting association of the binding sites with a G protein. Consistent with the saturation binding data, association kinetic studies indicated that [125I]CGRP associated with a single population of binding sites. Dissociation kinetic data, in contrast, indicated that [125I]CGRP dissociated from two affinity component sites on membranes, suggesting the existence of multiple affinity states of the G protein-coupled forms of the CGRP receptor. Nonequilibrium dissociation kinetic experiments revealed a time-dependent conversion of [125I] CGRP binding sites from a fast- to a slow-dissociating state. Desensitization of cells to CGRP by prior exposure to CGRP (10 nM) for 5 min reduced the maximal cAMP response of cells to further CGRP challenge and the number of [125I]CGRP binding sites in membranes prepared from these cells approximately 90% and 80%, respectively. These results demonstrate the existence of high affinity CGRP receptors in cardiac myocytes which appear coupled to G proteins and which undergo ligand-induced affinity alterations and desensitization-induced loss of receptor activity. The present findings also suggest the existence of multiple affinity states of the CGRP:receptor: G protein ternary complex.

Original languageEnglish (US)
Pages (from-to)2731-2738
Number of pages8
JournalEndocrinology
Volume128
Issue number6
DOIs
StatePublished - Jan 1 1991

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Calcitonin Gene-Related Peptide Receptors
Calcitonin Gene-Related Peptide
Cardiac Myocytes
Binding Sites
GTP-Binding Proteins
Cell Membrane
Guanosine 5'-O-(3-Thiotriphosphate)
Membranes
Population

ASJC Scopus subject areas

  • Endocrinology

Cite this

Characterization and regulation of high affinity calcitonin gene-related peptide receptors in cultured neonatal rat cardiac myocytes. / Chatterjee, Tapan Kumar; Moy, Jadine A.; Fisher, Rory A.; Fisher, Rory A.

In: Endocrinology, Vol. 128, No. 6, 01.01.1991, p. 2731-2738.

Research output: Contribution to journalArticle

Chatterjee, Tapan Kumar ; Moy, Jadine A. ; Fisher, Rory A. ; Fisher, Rory A. / Characterization and regulation of high affinity calcitonin gene-related peptide receptors in cultured neonatal rat cardiac myocytes. In: Endocrinology. 1991 ; Vol. 128, No. 6. pp. 2731-2738.
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abstract = "Previous studies have shown that stimulation of cultured beating cardiac myocytes with calcitonin gene-related peptide (CGRP) produces increased beating frequency, increased cellular cAMP concentration, and a homologous desensitization of the cAMP-elevating action of CGRP. In the present study, the characteristics and regulation of [125I]CGRP binding sites in cultured cardiac myocytes were investigated. Binding of [125I] CGRP to membranes prepared from these cells was selective, saturable, and of high affinity. Scatchard transformation of the saturation isotherm generated a linear plot suggesting the existence of a homogeneous population of binding sites with an equilibrium binding constant of 41 ± 7 pM and maximum binding capacity of 31 ± 5 fmol/mg protein. Binding of [125I]CGRP to membranes was inhibited completely by guanosine 5’-(3-O- thio)triphosphate (250 μM), suggesting association of the binding sites with a G protein. Consistent with the saturation binding data, association kinetic studies indicated that [125I]CGRP associated with a single population of binding sites. Dissociation kinetic data, in contrast, indicated that [125I]CGRP dissociated from two affinity component sites on membranes, suggesting the existence of multiple affinity states of the G protein-coupled forms of the CGRP receptor. Nonequilibrium dissociation kinetic experiments revealed a time-dependent conversion of [125I] CGRP binding sites from a fast- to a slow-dissociating state. Desensitization of cells to CGRP by prior exposure to CGRP (10 nM) for 5 min reduced the maximal cAMP response of cells to further CGRP challenge and the number of [125I]CGRP binding sites in membranes prepared from these cells approximately 90{\%} and 80{\%}, respectively. These results demonstrate the existence of high affinity CGRP receptors in cardiac myocytes which appear coupled to G proteins and which undergo ligand-induced affinity alterations and desensitization-induced loss of receptor activity. The present findings also suggest the existence of multiple affinity states of the CGRP:receptor: G protein ternary complex.",
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