Osamu Hiroshima

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 α‐CGRP to the solubilized binding sites was inhibited by human α‐ and β‐CGRP and by rat α‐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.

Positive inotropic effects and receptors of calcitonin gene-related peptide (CGRP) in porcine ventricular muscles.

Calcitonin gene-related peptide (CGRP) in porcine ventricular muscles. positive inotropic effects in the isolated, electrically driven false tendon of the porcine heart. Specific CGRP-binding sites were present in solubilized membrane fractions; the dissociation constant (Kd) and the maximum binding (Bmax) were 50.4 pM and 180 fmol/mg protein, respectively. SDS-PAGE analysis of CGRP-binding sites revealed the molecular mass of 70 K and 120 K. Few CGRP-like immunoreactive nerves were present in the ventricular muscle layer. These results indicate that CGRP activates specific receptor sites on the ventricular muscles and causes positive inotropic responses. CGRP receptors in ventricles are likely to be activated by circulating CGRP.

Solubilization and Characterization of Calcitonin Gene‐Related Peptide Binding Site from Porcine Spinal Cord

The binding site for calcitonin gene‐related peptide (CGRP) was solubilized with 3‐[(3‐cholamidopropyl) dimethylammonio]‐1‐propane sulfonate (CHAPS) in an active form from porcine spinal cord. l25I‐labeled human α‐CGRP (125I‐CGRP) binding to the solubilized protein was determined by filration using a GF/B glass filter. The maximal binding activity (˜60% of the crude membrane fraction) was obtained with 5 mM CHAPS. 125I‐CGRP binding to the solubilized protein was of high affinity, saturability, and high specificity, having KD and Bmax values of 3.69 pM and 338 fmol/mg of protein, respectively. The binding activity was eluted in a single peak with a molecular mass of 400,000 daltons by gel filtration on TSK gel G4000SW. These results suggest that the solubilized protein may be responsible for the specific binding site.

Discovery and development of aryl-fused imidazole-based angiotensin II antagonists

Abstract A phenolic benzofuran derivative 1 was identified as an angiotensin II receptor antagonist by random screening. Structural modifications led to a novel series of N-(4-hydroxyphenylmethyl)benzimidazoles and imidazo[4,5-b]pyridines, some of which inhibited angiotensin II-induced contractions in rabbit aortic strips with pA2 values of nearly 9. The related biphenylmethyl derivative E4177 showed potent and long-lasting activity in vivo.