SIMPLE METHOD TO DETERMINE ADENYLATE-CYCLASE ACTIVITY IN ISOLATED SINGLE NEPHRON SEGMENTS BY RADIOIMMUNOASSAY FOR SUCCINYL ADENOSINE 3',5'-CYCLIC-MONOPHOSPHATE

Torikai, S. and Imai, M. A Simple Method to Determine Adenylate Cyclase Activity in Isolated Single Nephron Segments by Radioimmunoassay for Succinyl Adenosine 3',5'-Cyclic Monophosphate. Tohoku J. exp. Med., 1979, 129 (1), 9199 - 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°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.-adenylate cyclase; cAMP; radioimmunoassay; parathyroid hormone; vasopressin Imbert (1975a) have developed a sophisticated and extremely sensitive method to determine adenylate cyclase activity in single fragments of renal tubules. A series of extensive studies by the same group clearly demonstrated that this method provided a useful tool to explore sites of the nephron segments responsive to various hormones not only in rabbits (Chabardes 1975a, b, 1976; Imbert 1975b; Morel 1976) but also in other species including rats (Imbert-Teboul 1978; Morel 1978), mice, and human (Morel 1978). The original method utilized 32P-ATP of high specific activity as a substrate of adenylate cyclase and measured generated adenosine 3',5'-cyclic monophophate (cAMP) after elution through aluminium microcolumn. In order to simplify the procedure, we have attempted to utilize radioimmunoassay of succinylated cAMP (Honma 1977) for measuring adenylate cyclase activity in single renal tubules. © 1979, Tohoku University Medical Press. All rights reserved.