Ultrastructural localization of acidic compartments in cells of isolated rabbit PCT

Abstract

Acidic intracellular compartments were identified in cells of isolated perfused rabbit proximal convoluted tubules (PCT) with the weak base, N-(3-[(2,4-dinitrophenyl)amino]propyl)-N-(3-aminopropyl)methylamine, dihydrochloride (DAMP), a congener of dinitrophenol (DNP) and immunogold staining at the ultrastructural level in Lowicryl-embedded tissue. DAMP is protonated upon entrance into an acidic compartment and binds to protein. Glutaraldehyde fixation cross-links the DAMP-protein complex to maintain the complex in its original environment. Monoclonal antibodies directed against DNP cross-react with DAMP to identify its location in tissue sections. Accumulation of colloidal gold-conjugated antibodies to the monoclonal anti-DNP antibodies, indicating the presence of an acidic environment, was found in large endocytic vacuoles (diam greater than 0.5 micron) and lysosomes, and to a lesser extent in endocytic vesicles. Qualitatively, activity greater than background was not found in other organelles, including the Golgi apparatus and the cell nuclei. The specificity of the technique was tested by treating tubules with the ionophore, monensin, to collapse the intracellular pH gradient. This treatment resulted in the complete disappearance of the specific localization of colloidal gold particles. Quantitative analysis of the number of colloidal gold particles per area of cross-sectioned cell organelle demonstrated that monensin especially affected those counts obtained over large endocytic vacuoles and lysosomes. The present ultrastructural observations, therefore, identify endocytic vesicles, endocytic vacuoles, and lysosomes as acidic intracellular compartments in isolated rabbit PCT. This technique affords the opportunity to identify the location of acidic compartments along the entire nephron at the ultrastructural level.