Histochemistry of adenylate cyclase activity in the anterior segment of the eye: a methodological evaluation with biochemical background

A histochemical technique for adenylate cyclase activity suitable for both light and electron microscopy is described. This technique is based on the Sr2+ capture reaction using adenylylimidodiphosphate as the substrate. The precipitated strontium phosphate is converted to lead phosphate, which is electron-dense for electron microscopy and can also be made visible by light microscopy. Different fixatives and fixation times were tested; paraformaldehyde at a concentration of 1% for 5 min demonstrated the enzyme activity best. The enzyme was protected and-or stimulated during all stages with isoproterenol, 5-guanylylimidodiphosphate, and dimethyl sulfoxide (DMSO). Numerous capturing agents (Pb2+, Co2+, Ce2+, Ba2+, Cd2+, and Sr2+) were tested. Strontium chloride (10 mM) produced the best result, inhibiting the enzyme less than 50%. The others gave inconsistent results. By this technique, adenylate cyclase activity was localized on the cell membranes of the deep layers of the rabbit corneal epithelium, on the endothelium, and on the cell membranes of the non-pigmented epithelium in the ciliary processes. These sites correspond well with earlier autoradiographic localization of beta-adrenergic receptors in the rabbit eye.