Micromethodology for measuring ATPase activity in renal tubules: mineralocorticoid influence.
The ATPase activity of rabbit isolated renal tubule segments was measured using a microtechnique in which the hydrolysis of ATP was enzymatically coupled to the appearance of an alkali-converted, highly fluorescent form of nicotinamide adenine dinucleotide. The methods are simple, reproducible, and have a high sensitivity in which picomole quantities of hydrolyzed ATP can readily be measured. Several methods for permeabilizing the cell membranes for measurement of Na+-K+-ATPase activity were evaluated, including osmotic (distilled water or 300 mM imidazole) and temperature (freezing) shock and addition of the nonionic detergent octylglucoside. An octylglucoside concentration of 0.5% was found to cause a maximum activation of the Na+-K+-ATPase and was comparable with that observed when tubules were permeabilized by exposure to distilled water and freezing. Incubation of tubules in 300 mM imidazole was less effective in permeabilizing the cell membranes. In all subsequent studies, the cells were permeabilized by exposure to distilled water and freezing as done by others. The methods were used to assay for the basal levels of Na+-K+-ATPase in the superficial proximal convoluted tubule, the superficial proximal straight tubule, and the cortical collecting tubule and were found to average 44.9 +/- 6.3, 26.4 +/- 2.4, and 11.8 +/- 2.2 pmol ADP X mm-1 X min-1, respectively. Furthermore, elevation of plasma mineralocorticoids by daily injections of deoxycorticosterone acetate (2 mg X kg-1 X day-1) for 4-15 days caused a doubling in the Na+-K+-ATPase activity of the cortical collecting duct, confirming the results of others. The methods presented can easily be adapted for microanalysis of other ATPases.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D002463:Cell Membrane Permeability; D003900:Desoxycorticosterone / Q000494:pharmacology; D004848:Epithelium / Q000201:enzymology; D005260:Female; D005470:Fluorometry / Q000379:methods; D005615:Freezing; D005960:Glucosides / Q000494:pharmacology; D006868:Hydrolysis; D007093:Imidazoles / Q000494:pharmacology; D007684:Kidney Tubules / Q000201:enzymology*; D008901:Mineralocorticoids / Q000502:physiology*; D009243:NAD / Q000378:metabolism; D009997:Osmotic Pressure; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013268:Stimulation, Chemical; D013997:Time Factors
D005960:Glucosides; D007093:Imidazoles; D008901:Mineralocorticoids; D009243:NAD; C018619:octyl-beta-D-glucoside; D003900:Desoxycorticosterone; D000244:Adenosine Diphosphate; D000254:Sodium-Potassium-Exchanging ATPase