Larsson L, Clapp W L, Park C H, Cannon J K, Tisher C C
Am J Physiol. 1987 Jul;253(1 Pt 2):F95-103. doi: 10.1152/ajprenal.1987.253.1.F95.
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.
在分离灌注的兔近端曲管（PCT）细胞中，使用弱碱N-（3-[(2,4-二硝基苯基)氨基]丙基）-N-（3-氨基丙基）甲胺二盐酸盐（DAMP，二硝基苯酚（DNP）的同系物）进行鉴定，并在Lowicryl包埋组织的超微结构水平上进行免疫金染色，从而识别酸性细胞内区室。DAMP进入酸性区室后会质子化并与蛋白质结合。戊二醛固定使DAMP-蛋白质复合物交联，从而将复合物维持在其原始环境中。针对DNP的单克隆抗体与DAMP发生交叉反应，以确定其在组织切片中的位置。在大型内吞泡（直径大于0.5微米）和溶酶体中发现了与单克隆抗DNP抗体结合的胶体金缀合抗体的积累，表明存在酸性环境，在内吞小泡中的积累程度较低。定性地说，在包括高尔基体和细胞核在内的其他细胞器中未发现活性高于背景的情况。通过用离子载体莫能菌素处理肾小管以破坏细胞内pH梯度来测试该技术的特异性。这种处理导致胶体金颗粒的特异性定位完全消失。对每个横截面细胞器区域的胶体金颗粒数量进行定量分析表明，莫能菌素尤其影响在大型内吞泡和溶酶体上获得的计数。因此，目前的超微结构观察结果确定内吞小泡、内吞泡和溶酶体是分离的兔PCT中的酸性细胞内区室。该技术提供了在超微结构水平上识别整个肾单位中酸性区室位置的机会。
