Simultaneous measurement of intracellular calcium and tension in vascular smooth muscle: validation of the everted ring preparation

Abstract

This study details and validates a method that facilitates the eversion of vascular smooth muscle, a preparation employed in in vitro Ca2+ fluorometric assays. Vascular segments of porcine coronary artery, approximately 2 cm in length, were sutured to portions of polyethylene tubing inserted into the lumen of the vessel. After being secured and stabilized by the tubing, the vessel was easily everted while immersed in physiological buffer. Intracellular calcium concentrations ([Ca2+]i) and tension were measured simultaneously in everted rings denuded of the endothelium. In these preparations, increases in tension generated by KCl and prostaglandin F2 alpha (PGF2 alpha) were accompanied by increases in [Ca2+]i, as measured by fura-2 fluorescence. Isoproterenol (ISO) and sodium nitroprusside (SNP) elicited reductions in muscle tension as well as [Ca2+]i in both KCl- and PGF2 alpha-contracted rings. Comparison of the responsiveness of everted and uneverted coronary artery rings demonstrated that, while fura-2 fluorescence in uneverted rings was negligible, the magnitudes of contraction of both preparations to KCl or PGF2 alpha were similar. The relaxant responses to ISO and SNP were also similar in the everted and uneverted rings contracted with KCl or PGF2 alpha. The data suggest that the procedure employed in everting vascular segments maintains the integrity of the smooth muscle, thus making it a suitable model for the simultaneous measurement of [Ca2+]i and tension.