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 370C) 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.

Nongenomic stimulation of vacuolar H + -ATPases in intercalated renal tubule cells by aldosterone

“…These results demonstrate that the stimulatory effect of aldosterone on vacuolar H ϩ -ATPase activity is mainly mediated by a nongenomic pathway. Good evidence exists that trafficking of vacuolar H ϩ -ATPases plays an important role in the regulation of their activity in the plasma membrane of renal cells (1,(28)(29)(30). We tested the role of such trafficking in the aldosterone-induced stimulation of vacuolar H ϩ -ATPase activity in OMCD-intercalated cells.…”

PKA Regulates Vacuolar H+-ATPase Localization and Activity via Direct Phosphorylation of the A Subunit in Kidney Cells

“…); *, p Ͻ 0.05 versus V-ATPase A WT; n ϭ 20 -45 cells analyzed for both conditions). (20,51,58,59). Carbonic anhydrase and sAC are very abundant in kidney-intercalated cells, where sAC activation generates cAMP upon increases of CO 2 that may occur under conditions of acute respiratory acidosis (31,60).…”

Tyrosine-based Membrane Protein Sorting Signals Are Differentially Interpreted by Polarized Madin-Darby Canine Kidney and LLC-PK1 Epithelial Cells

“…It has been noted, however, that LLC-PK 1 cells morphologically and functionally resemble cells found in the proximal tubule of the nephron (48), whereas MDCK cells resemble those found in the distal nephron segments (49). Perfusion studies using fluorescein isothiocyanate dextran have shown that proximal tubule cells endocytose marker from their apical surfaces, whereas only intercalated and ADH-stimulated principal cells from the collecting duct endocytose marker from their apical membrane (50). In addition, a significantly larger percent of HA-Y543 is internalized from the apical membrane domain of LLC-PK 1 cells than from the apical membrane domain of MDCK cells (Table I).…”