Enzyme cytochemical study of rat cardiac muscle I. Adenylate cyclase and guanylate cyclase.

The cytochemical localization of adenylate cyclase and guanylate cyclase in rat cardiac muscle was demonstrated with the lead nitrate method using dimethyl sulfoxide (DMSO) (Fujimoto et al., 1981). Tissues were fixed in a mixture of 2% paraformaldehyde, 0.25% glutaraldehyde and 5% DMSO in 0.1M cacodylate buffer, pH7.4, for 30min, and then washed with 5% DMSO in 0.1M cacodylate buffer. Vibratome or Microslicer sections were incubated in the following medium for 30min at 37°C.Medium for adenylate cyclase (ACLase): 80mM Tris-maleate buffer, pH 7.4; 4mM MgSO4; 10mM NaF; 2mM theophylline; 2mM lead nitrate; 0.25M sucrose; 5% DMSO; 2.5mM levamisole; 0.5mM AMP-PNP. Medium for guanylate cyclase (GCLase): 80mM Tris-maleate buffer, pH 7.4; 3mM MnCl2; 2mM theophylline; 2mM lead nitrate; 0.25M sucrose; 5% DMSO; 2.5mM levamisole; 0.5mM GMP-PNP. The most intense reaction of ACLase was localized in the sarcolemma, sarcoplasmic reticulum and gap junction. GCLase activity was also seen in the same regions. In the unfixed isolated gap junctions, the reaction product was seen to interrupt the interspace of the junction, forming bridges between the junctional membranes. ACLase was activated by NaF and isoproterenol. Alloxan was effective as an inhibitor of ACLase. GCLase was stimulated by the in vitro acetylcholine treatment.