Cytochemistry of adenylate cyclase. Quantitative analysis of the effect of cytochemical procedures on adenylate cyclase in heart tissue homogenates.

Abstract

The effects of different preparative and cytochemical procedures on adenylate cyclase (AC) activity in heart muscle homogenates were studied by quantitative analysis. We were mainly concerned with perfusion prefixation, using glutaraldehyde (GA) with and without DMSO, and with the influence of cytochemical incubation with lead ions as the capture reagent. Furthermore, we measured the direct effect on the AC activity of lead, cobalt, and strontium ions in prefixed heart homogenates. We also studied the influence of phosphatidylinositol and 2',5'-dideoxyadenosine. The following results were obtained: 1. Perfusion fixation using 2% GA buffered with cacodylate reduced the AC activity by about 20%. After the entire cytochemical procedure was finished, 20% of the original AC activity was still present. Stimulation by epinephrine, histamine and fluoride, which increased the activity of AC two or three times in our experiments, was only slightly reduced by the cytochemical treatments. 2. Lead ions (2 mM), added to the biochemical assay without chelating compounds, reduced the AC activity about 90%. 5 muM phosphatidylinositol stabilized the fluoride- and hormone-sensitive AC activity. 3. Co2+ also reduced the AC activity, though less than Pb2+. Sr2+ had no effect on the basic activity of the AC but had a slightly inhibitory effect on the hormone and fluoride stimulation. 4. 5% DMSO added to the fixative had no influence on the basic activity of the AC. However, this solvent definitely reduced the level of stimulation by fluoride and guanine nucleotide plus hormones. 5. A potential inhibitor of enzyme activity and of the hormone- and fluoride-sensitive AC was the adenosine derivative, 2',5'-dideoxyadenosine. This compound, at a concentration of 10(-3) M, inhibited all AC activity in the heart homogenates.