Specific cytochemical localization of sodium-potassium dependent adenosine-triphosphatase (Na -K -atp-ASE) activity in cortical renal tubules of rat kidney

RESUMO

Utilizid para-nitrophenil-phosphate as an unspecific substrate cytochemical methods have described for and electron microscopi localizarion of Na -K -ATP-ase activity (ATP-ase-A). However, when applied to renal tubules, these methods did not clearly reveal the quantitative and quantitative distribution of the enzime. In an attempt to improve this method, we evaluated shorter fixation times, using freh paraformaldehyde after immersion of the renal tissue in an isosmotic sucrose tris buffer solution and utilized ATP as the specific substrate for the ATP-ase. The results indicated good preservation of the tissue, and under these conditions ATP was able to penetrate of the cells allowing a reliable identification at the ultrastructural level, of the ATP-ase-A, associated with subcellular structures for with the enzymatic reaction was positive. The activity observed suggest that the basolateral plasma membranes of proximal and distal renal tubules are the major sites of sites of Na -K -ATP-ase localization. However, there was also significant hydrolitic activity detectived on the surface (brush border) of proximal convoluted tubules, that disappeared with the elimination of the Na and K from the incubation medium. In both cases enzymatic activities were insensitive to 10 mM Ouabain in the medium