bafilomycin-a and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

To examine the effects of extracellular adenosine 5-triphosphate (ATP) on intracellular pH ([pH](i)) in cultured human non-pigmented ciliary body epithelium (HNPE).. Intracellular pH was measured using spectrofluorescence video microscopy in isolated HNPE cells loaded with the cell-permeable acetoxymethyl ester form of the fluorescent probe BCECF.. In 5%CO(2)/HCO(3)(-) buffered Ringer's the resting [pH](i) was 7.25 +/- 0.006 (mean +/- SEM). Application of 10 microM ATP significantly decreased [pH](i) to 7.00 +/- 0.007 (P < 10(-5), n = 14). In the presence of 1 mM suramin, a P(2) receptor inhibitor, this process was significantly blocked. This [pH](i) effect required the presence of Cl(-) and was significantly inhibited by 0.1 mM diisothiocyanatostilbene-2-2'-disulfonic acid or acetazolamide (500 microM), indicating the involvement of a Cl(-)/HCO(3)( +) exchange mechanism. This response exhibited little dependence on external Na(+) and remained unaffected by the addition of the Na(+)/H( +) exchanger inhibitor amiloride (1 mM). Clamping intracellular calcium levels by incubation in the cell permeable calcium chelator, the acetoxymethyl ester form of BAPTA (100 microM) in low extracellular calcium solution (pCa9) did not affect the ATP-induced [pH](i) signal. In addition, the vacuolar H(+)-ATPase (V-ATPase) inhibitor, bafilomycin A(1) (1 microM), failed to alter the [pH](i) transient.. We have demonstrated that extracellular ATP leads to a sustained increase in [H(+)](i) in HNPE cells via a purinergic receptor activated pathway which is independent of the intracellular calcium signaling system. This study demonstrates that the ATP induced [pH]( i) transient is mediated through an upregulation in Cl(-)/HCO( 3)(-) exchange across the plasmamembrane in HNPE cells.

Topics: Adenosine Triphosphate; Anti-Bacterial Agents; Calcium; Cells, Cultured; Chloride-Bicarbonate Antiporters; Chlorides; Ciliary Body; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Macrolides; Microscopy, Fluorescence; Pigment Epithelium of Eye; Proton-Translocating ATPases; Receptors, Purinergic; Spectrometry, Fluorescence; Suramin; Up-Regulation

Intercalated cells (ICs) from kidney collecting ducts contain proton-transporting ATPases (H(+)-ATPases) whose plasma membrane expression is regulated under a variety of conditions. It has been shown that net proton secretion occurs in the distal nephron from chronically K(+)-depleted rats and that upregulation of tubular H(+)- ATPase is involved in this process. However, regulation of this protein at the level of individual cells has not so far been examined. In the present study, H(+)-ATPase activity was determined in individually identified ICs from control and chronically K(+)-depleted rats (9-14 days on a low-K(+) diet) by monitoring K(+)- and Na(+)-independent H(+) extrusion rates after an acute acid load. Split-open rat cortical collecting tubules were loaded with the intracellular pH (pH(i)) indicator 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and pH(i) was determined by using ratiometric fluorescence imaging. The rate of pH(i) recovery in ICs in response to an acute acid load, a measure of plasma membrane H(+)-ATPase activity, was increased after K(+) depletion to almost three times that of controls. Furthermore, the lag time before the start of pH(i) recovery after the cells were maximally acidified fell from 93.5 +/- 13.7 s in controls to 24.5 +/- 2.1 s in K(+)-depleted rats. In all ICs tested, Na(+)- and K(+)-independent pH(i) recovery was abolished in the presence of bafilomycin (100 nM), an inhibitor of the H(+)-ATPase. Analysis of the cell-to-cell variability in the rate of pH(i) recovery reveals a change in the distribution of membrane-bound proton pumps in the IC population of cortical collecting duct from K(+)-depleted rats. Immunocytochemical analysis of collecting ducts from control and K(+)-depleted rats showed that K(+)-depletion increased the number of ICs with tight apical H(+)ATPase staining and decreased the number of cells with diffuse or basolateral H(+)-ATPase staining. Taken together, these data indicate that chronic K(+) depletion induces a marked increase in plasma membrane H(+)ATPase activity in individual ICs.

Topics: Acid-Base Equilibrium; Ammonium Chloride; Animals; Anti-Bacterial Agents; Electrolytes; Female; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Immunohistochemistry; Kidney Tubules, Collecting; Macrolides; Male; Potassium, Dietary; Proton Pump Inhibitors; Proton Pumps; Rats; Rats, Sprague-Dawley; Sodium, Dietary; Water-Electrolyte Balance