A simple method to determine adenylate cyclase activity in isolated single nephron segments by radioimmunoassay for...

A simple method to determine adenylate cyclase activity in isolated single nephron segments is described. Segments of the proximal convoluted tubule or the cortical collecting tubule were isolated from rabbit kidney slices pretreated with collagenase. After the tubule membranes were made permeable by adding hypotonic medium and freezing-thawing, each sample was incubated at 30 degrees C for 30 min in a medium containing ATP and theophylline. Generated cAMP was succinylated and served for radioimmunoassay. Addition of the incubation medium did not interfere the radioimmunoassay. Recovery of added cAMP was 96%. In the proximal convoluted tubule, either 8 mM NaF or 1 U/ml parathyroid hormone (PTH) markedly stimulated adenylate cyclase activity, but 1 mU/ml arginine vasopressin (AVP) did not. By contrast, in the cortical collecting tubule, either 8 mM NaF or 1 mU/ML AVP markedly stimulated adenylate cyclase activity, but 1 U/ml PTH did not. These data imply that this method is sensitive enough to detect either specific or nonspecific response of adenylate cyclase activity in single nephron segments.