Carbon dioxide causes exocytosis of vesicles containing H+ pumps in isolated perfused proximal and collecting tubules

Abstract

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 (~ 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°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.