Towards a standard method to demonstrate adenylate cyclase activity at the electron microscopical level

The ventral epidermis of the frog Rana fuscigula is a typical tight epithelium which acts as a functional syncytium in the active transepithelial transport of sodium ions. Transport across this epithelium is regulated by cyclic adenosine monophosphate (cAMP). This study was undertaken to formulate an optimal protocol for the localization, within this epithelium, of adenylate cyclase; the enzyme involved in cAMP synthesis. The ventral epithelium of R. fuscigula was collagenase treated and processed using five different fixation/incubation protocols. The components of a basal incubating medium were modified by changing the localizing agent, adding adenylate cyclase stimulators and inhibitors of other enzymes. Control incubations undertaken included a) leaving the substrate out, b) prior heat inactivation of the enzyme, c) specific blockers and d) incubation for alkaline phosphatase as an alternative enzyme. The samples were then processed for electron microscopy. Localization of adenylate cyclase was best obtained, when fixing the tissue after incubation for 30 min at 37 degrees C. The medium that gave the best and most consistent localization contained magnesium chloride; as a required ion, theophylline, dithiothreitol, ouabain, levamisole; as enzyme inhibitors, forskolin; as a stimulator of adenylate cyclase, lead nitrate; as the capture agent and column purified adenylyl imidodiphosphate; as the substrate.