Schwartz G J, Al-Awqati Q
J Clin Invest. 1985 May;75(5):1638-44. doi: 10.1172/JCI111871.
In the turtle bladder it has recently been shown that CO2 stimulates H+ secretion, at least in part, by causing fusion of vesicles enriched in H+ pumps with the luminal plasma membrane. To test for the presence of this mechanism in the kidney we perfused collecting ducts and proximal straight tubules on the stage of an inverted epifluorescence microscope with fluorescein isothiocyanate dextran (70,000 mol wt) in CO2-free medium. After washout we noted punctate fluorescence in endocytic vesicles in some collecting ducts and in all proximal straight tubule cells. More cells took up fluorescent dextran in outer medullary than in cortical collecting ducts. Using the pH dependence of the excitation spectrum of fluorescein we found the pH of the vesicles to be acid (approximately pH 6). Addition of proton ionophores increased vesicular pH by 0.6 +/- 0.1 U, suggesting that the acidity of the vesicles was caused by H+ pumps. CO2 added to the medium (25 mmHg, pH 7.6 at 37 degrees C) reduced fluorescence intensity by 24 +/- 5% in cortical collecting ducts, 27 +/- 5% in medullary collecting ducts, and 25 +/- 5% in proximal straight tubules. Since this effect was prevented by the prior addition of colchicine to the bath, we believe that CO2 caused a decrease in cytoplasmic fluorescence by stimulating exocytotic fusion of the vesicles and thereby secretion of fluorescent dextran. This exocytotic fusion also occurred when tubules that were loaded with fluorescent dextran at a pCO2 of 37 mmHg were exposed isohydrically to a pCO2 of 114 mmHg; the mean decrease was 53 +/- 4%. We conclude that some cells in the collecting ducts and all cells in the proximal straight tubule incorporate fluorescent dextran into the apical cytoplasmic vesicles and acidify them with H+ pumps. CO2 causes fusion of these vesicles with the luminal membrane, but whether CO2 stimulates H+ secretion by increasing the number of functioning H+ pumps remains to be determined.
最近研究表明，在龟膀胱中，二氧化碳至少部分地通过促使富含氢离子泵的囊泡与管腔质膜融合来刺激氢离子分泌。为了检测肾脏中是否存在这种机制，我们在倒置落射荧光显微镜载物台上，用异硫氰酸荧光素葡聚糖（分子量70,000）灌注无二氧化碳培养基中的集合管和近端直小管。冲洗后，我们在一些集合管和所有近端直小管细胞的内吞囊泡中观察到点状荧光。外髓质集合管中摄取荧光葡聚糖的细胞比皮质集合管中的更多。利用荧光素激发光谱的pH依赖性，我们发现囊泡的pH值呈酸性（约pH 6）。添加质子离子载体使囊泡pH值升高0.6±0.1单位，这表明囊泡的酸性是由氢离子泵引起的。向培养基中添加二氧化碳（25 mmHg，37℃时pH 7.6）使皮质集合管中的荧光强度降低24±5%，髓质集合管中降低27±5%，近端直小管中降低25±5%。由于预先向浴液中添加秋水仙碱可阻止这种效应，我们认为二氧化碳通过刺激囊泡的胞吐融合从而分泌荧光葡聚糖，导致细胞质荧光降低。当在37 mmHg的二氧化碳分压下加载荧光葡聚糖的小管等渗暴露于114 mmHg的二氧化碳分压时，也会发生这种胞吐融合；荧光强度平均降低53±4%。我们得出结论，集合管中的一些细胞和近端直小管中的所有细胞将荧光葡聚糖纳入顶端细胞质囊泡并用氢离子泵使其酸化。二氧化碳导致这些囊泡与管腔膜融合，但二氧化碳是否通过增加有功能的氢离子泵数量来刺激氢离子分泌仍有待确定。
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