Contraction of cultured vascular smooth muscle cells induced by “Ca2+ paradox”.

Abstract

Contractilities of smooth muscle cells (SMCs) in primary culture and subculture were examined by using the technique of Ca2+ paradox, namely, Ca2+-repletion (l.OmM Ca2+) after incubation with Ca2+-free 0.1mM EGTA medium for 10 min. Aortic SMCs from male Wistar rats were cultured by the method of Chamely et al. with a minor modification. Direct immunofluores-cent staining of smooth muscle actin revealed positive staining both in primary culture and successive subcultures of SMCs, while that of smooth muscle myosin showed positive staining only in primary culture and in the second passage, but negative staining in the further passages of SMCs. The degrees of contraction were assessed by measuring the changes in the area of SMCs, assuming the value prior to the experiment to be 100%. SMCs in primary culture did not contract at all during the Ca2+-free period. After 30 min of Ca2+-repletion, area of SMCs decreased to 46.7±18.0% (mean+S.D.). The changes were reversible, namely, the cells regained their normal original shapes and cell areas (100%) 120 min after Ca2+-repletion. No contraction during Ca2+-repletion was observed for 3rd and the later passages of SMCs. It was suggested that subcultured (over 2nd passage) vascular SMCs have no contractile activity, which was strongly attributable to their loss of myosin.