Altered reactivity of hemolysate-treated cultured smooth-muscle cells from rabbit basilar artery determined by digital imaging microscopy

Abstract

During culture, smooth-muscle cells obtained from rabbit basilar arteries were examined for contractile activity by means of differential interference microscopy with a video analysis system (digital imaging microscopy system). This system proved useful for observing the contraction and ultrastructural changes of the living cells. Hemolysate-treated cells showed augmented responses to 5-hydroxytryptamine and leukotriene C4, but not to KCl. This augmented response diminished gradually during the culture period. Both a phospholipase C blocking agent, 2-nitro-4-carboxyphenyl-n,n-diphenylcarbamate (NCDC), and a myosin light chain kinase blocking agent, 1-(5-chloronaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), suppressed this augmented response. Protein kinase C activity of the cells, as measured by Western blot analysis, did not increase during the period of culture with hemolysate. The results obtained suggest that hemolysate had the following effects on the cells: 1) acute but gradual contraction of the cells; 2) augmentation of cellular responses to vasoactive agents; and 3) progressive contraction and morphological alteration of the cells. Possible mechanisms by which hemolysate exerts these effects are discussed, taking into consideration the interrelationship between these effects.