Characterization and Regulation of High Affinity Calcitonin Gene-Related Peptide Receptors in Cultured Neonatal Rat Cardiac Myocytes*

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 microM), 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.

Decreased endothelium-dependent pulmonary vasodilator effect of calcitonin gene-related peptide in hypoxic rats contrasts with increased binding sites

“…The effect of GTP-)I-S and the lack of binding inhibition by unrelated peptides further emphasizes the binding specificity in rat lung. Our results, therefore, provide evidence that 125 I-hCGRP binding sites are present in the rat lung and are regulated by guanine nucleotides, which is in agreement with similar behaviour seen with CGRP binding sites in other tissues [14,15,17].…”

“…These results suggest that 125 I-hCGRP binding sites may be sensitive to structural changes in the hCGRP molecule, in that removal or modification of the N-terminal caused a reduction in inhibitory potency, as seen with a-hCGRP and [Cys(ACM) 2,7 ]hCGRP compared to hCGRP. Similar observations have been made previously in rat brain and cultured neonatal rat cardiac myocytes [14,15]. However, in membrane preparations of rat and guinea-pig tissues, a-hCGRP has been shown to have a greater inhibitory potency relative to hCGRP for 125 I-hCGRP binding sites [8], suggesting that there may be regional or species differences in the binding affinity of 125 I-hCGRP binding sites.…”

“…Studies on rat cerebellum and myocardial cell membranes have shown that CGRP binding sites can occur in different affinity states [14,15]. However, whilst the concentration-dependent inhibition of 125 IhCGRP binding by GTP-c-S suggests that high-affinity binding sites are present in the rat lung, the near complete dissociation of 125 I-hCGRP by GTP-c-S and incomplete dissociation with hCGRP further suggest that binding sites for CGRP in rat lung may also occur in other affinity states.…”

Mechanism of action of amylin in bone

“…The relative potency of human amylin to induce cAMP production by KS-4 cells correlated with the binding affinity of this peptide for CGRP receptors in the rat brain. Chatterjee et al (1991) characterized high affinity receptors for CGRP in cultured rat cardiac myocytes. Amylin displaced the CGRP binding, but its affinity was much weaker than that of CGRP (Chatterjee et al, 1991).…”

“…Chatterjee et al (1991) characterized high affinity receptors for CGRP in cultured rat cardiac myocytes. Amylin displaced the CGRP binding, but its affinity was much weaker than that of CGRP (Chatterjee et al, 1991). Zhu et al (1991) also reported that amylin increased cAMP production by L6 myocytes via CGRP receptors.…”