Chloride dependent intracellular pH effects of external ATP in cultured human non-pigmented ciliary body epithelium.
OBJECTIVE
To examine the effects of extracellular adenosine 5-triphosphate (ATP) on intracellular pH ([pH](i)) in cultured human non-pigmented ciliary body epithelium (HNPE).


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Cullinane
A B
AB
Wellcome Trust Cellular Physiology Research Unit, Department of Physiology, National University of Ireland, Cork, Ireland. abcullinane@hotmail.com
Coca-Prados
M
M
Harvey
B J
BJ
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D027963:Chloride-Bicarbonate Antiporters / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D002924:Ciliary Body / Q000166:cytology*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018942:Macrolides; D008856:Microscopy, Fluorescence; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D011983:Receptors, Purinergic / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013498:Suramin / Q000494:pharmacology; D015854:Up-Regulation
D000900:Anti-Bacterial Agents; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D018942:Macrolides; D011983:Receptors, Purinergic; C057620:bafilomycin A; D013498:Suramin; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D002118:Calcium