CYTOCHEMICAL DEMONSTRATION OF ADENYLATE-CYCLASE IN CARDIAC-MUSCLE EFFECT OF DIMETHYL-SULFOXIDE

Adenylate cyclase (AC) activity was evaluated after perfusion fixation of rat and dog myocardium with 4% paraformaldehyde (PFA), 2% glutaraldehyde (GA) or a combination of both, in cacodylate buffer. Dimethyl sulfoxide (DMSO) was added to the fixatives and its effect on the preservation of cell organelles and enzyme activity was determined. Adenylate cyclase activity was preserved best after fixation with 4% paraformaldehyde but this fixative did not provide for optimal maintenance of structure. Prefixation with 2% glutaraldehyde and 5% dimethyl sulfoxide provided the most effective preservation of both structural and enzymatic integrity. Precipitation of lead diphosphoimide was the morphologic indicator of sites of adenylate cyclase activity. The most intense precipitate was in the lumen of junctional sarcoplasmic reticulum in close contact with T-tubules and in subsarcolemmal cisternae. Evidence of activity was also seen on the intracellular aspect of the sarcolemmal membrane and in the nexus segment of the intercalated discs. Alloxan was effective as an inhibitor of adenylate cyclase activity only if the concentration of the activating substance sodium fluoride (NaF) was 20 mM or lower.