An ATP-driven proton pump in brush-border membranes from rat renal cortex

233

105

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.

Section: Resultsmentioning

confidence: 63%

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

Schwartz¹,

Al‐Awqati²

1985

233

105

“…However, several groups have found that luminal Na' removal does not eliminate all acidification (67). At least one group also finds a H' translocating ATPase in brush border membranes (69). This ATPase activity may be due to contamination from intracellular vesicles, many of which contain H' ATPases.…”

Section: Transepithelial Ion Transportmentioning

confidence: 99%

Proton transport and cell function.

Ives¹,

Rector²

1984

T he past five years have witnessed an explosion of information on the many and varied roles of H+ transport in cell function. H+ transport is involved in three broad areas of cell function: (a) maintenance and alteration of intracellular pH for initiation of specific cellular events, (b) generation of pH gradients in localized regions of the cell, including gradients involved in energy transduction, and (c) transepithelial ion transport. These processes each involve one or more of several H+ translocating mechanisms. The first section of this review will discuss these H+ translocating mechanisms and the second part will deal with the cellular functions controlled by H+ transport. Mechanisms of H+/OH-TransportPrimary active H+ transport Primary active H' transport mechanisms consume energy directly for the translocation of HW. This energy can be derived from redox reactions, light, or ATP. Since most of the primary active H+ translocating processes are electrogenic, their action will produce only a membrane potential difference unless there is some provision in the membrane for counter ion movement. This is usually accomplished by a simple conductive pathway through which Cl-can move in the same direction as, or cations (e.g., K+ or Na+) can move in the opposite direction to H+ transport. As will be seen below, some ofthe active H' transport mechanisms do generate primarily a membrane potential difference, while others move significant amounts of acid across membranes. To date, there are no known primary active OHor HCO-transport mechanisms.H+ translocation by the electron transport chain. In the mitochondrion, three electron transport complexes (reduced nic-

“…Bicarbonate absorption in the rat proximal convoluted tubule is mediated on the apical membrane by a Na/H antiporter and a Na-independent, amiloride-insensitive mechanism, most likely a H-translocating ATPase (1)(2)(3)(4)(5)(6)(7)(8)(9). Base generated within the cell exits across the basolateral membrane via a Na(HCO3)3 symporter (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).…”

Section: Introductionmentioning

confidence: 99%

Chronic metabolic acidosis causes an adaptation in the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter in the rat proximal convoluted tubule.

Preisig¹,

Alpern²

1988

129

115

The effect of chronic dietary acid on the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter was examined in the in vivo microperfused rat proximal tubule. Transporter activity was assayed with the epifluorescent measurement of cell pH using the intracellular, pH-sensitive fluorescent dye, (2'7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein (BCECF). BCECF was calibrated intracellularly, demonstrating similar pH-sensitivity of the dye in control and acidotic animals. In subsequent studies, lumen and peritubular capillaries were perfused to examine Na/H and Na(HCO3)3 transporter activity in the absence of contact with native fluid. The initial rate of change in cell pH (dpHi/dt) was 97, 50, and 44% faster in tubules from acidotic animals when peritubular [HCO31 was changed from 25 to 10 mM in the presence or absence of chloride, or peritubular INal was changed from 147 to 50 mM, respectively. dpHi/dt was 57% faster in tubules from acidotic animals when luminal [Nal was changed from 152 to 0 mM. Buffer capacities, measured using NH3/NIU addition, were similar in the two groups. The results demonstrate that chronic metabolic acidosis causes an adaptation in the intrinsic properties of both the apical membrane Na/H antiporter and basolateral membrane Na(HCO3)3 symporter.