Calcitonin Gene‐Related Peptide‐Binding Sites of Porcine Cardiac Muscles and Coronary Arteries: Solubilization and Characterization

Abstract

Calcitonin gene-related peptide (CGRP)-binding sites were solubilized, using digitonin, from the porcine spinal cord, atria, and coronary arteries. The specific binding of 125I-human alpha-CGRP to the solubilized binding sites was inhibited by human alpha- and beta-CGRP and by rat alpha-CGRP, but not by angiotensin II or human calcitonin. Scatchard plot analysis of saturation gave the same KD value for CGRP in the crude membrane fractions of the tissues examined. The affinity of CGRP to the binding sites was decreased by solubilization in the atria and coronary arteries, but not in the spinal cord. Affinity labeling followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed distinct molecular sizes of the specific binding sites among the tissues; 70K for the spinal cord, 70K and 90K for the coronary arteries, and 70K and 120K for the atria. These results indicate that the molecular characteristics of the specific binding sites of CGRP in the cardiovascular system are distinct from those in the central nervous system.

Calcitonin gene-related peptide and its receptors: molecular genetics, physiology, pathophysiology, and therapeutic potentials

“…Specific CGRP receptors have been found in porcine coronary arteries (M r 90,000 and 70,000) and in the atria (318). In the porcine spinal cord, only the 70,000 M r form was identified (318)(319)(320)(321). The reported M r of the CGRP-binding protein measured by autoradiography after cross-linking with bifunctional reagents varies from 13,700 to 400,000 (296, 292-301, 315-322).…”

“…A CGRP receptor with a M r of 60,000 has been found in cultured rat vascular smooth muscle cells and in bovine endothelial cells by cross-linking with disuccinamyl suberate (318,319), and with M r of 60,000, 54,000, and 17,000 after photoaffinity labeling of human cerebellar membranes (299-301). Specific CGRP receptors have been found in porcine coronary arteries (M r 90,000 and 70,000) and in the atria (318). In the porcine spinal cord, only the 70,000 M r form was identified (318)(319)(320)(321).…”

“…The guanidine nucleotide, GTP, causes a marked increase in the affinity of CGRP for its receptor, suggesting the formation of a G-protein complex (318)(319)(320)(321)(322) and that G proteins are involved in CGRP binding to its receptors and signaling of its effector system. Some authors have suggested that different responses elicited by CGRP administered by peripheral and intracerebroventricular routes may be mediated through different receptors, but there are no data to support this hypothesis.…”

The effect of monoclonal antibodies to calcitonin gene‐related peptide (CGRP) on CGRP‐induced vasodilatation in pig coronary artery rings

“…Calcitonin gene-related peptide (CGRP) is an endogenous 37 amino-acid peptide the effects of which are receptor-mediated (Sano et al, 1989) causing vasodilatation in the pig isolated coronary artery preparation (Beny et al, 1989). Although previous work has shown the peptide fragment CGRP (8-37) to be an antagonist of CGRP in rat liver plasma membranes (Chiba et al, 1989), in vivo in conscious rats (Gardiner et al, 1990) and in pig isolated arteries (Foulkes et al, 1991) little is known about the location of the active region of the peptide.…”

Identification of a protein that confers calcitonin gene-related peptide responsiveness to oocytes by using a cystic fibrosis transmembrane conductance regulator assay.

“…Protein purification strategies based on cross-linking cell extracts to CGRP followed by SDS/PAGE analysis have identified candidate proteins for the CGRP receptor and its complex, with molecular masses of 17-70 kDa (12)(13)(14)(15)(16). CGRP activation results in increased levels of intracellular cAMP in endothelial cells and aorta (17,18).…”