K(+)-H+ exchange, a fundamental cell acidifier in corneal epithelium.
Rabbit corneal epithelial cells, loaded with the pH-sensitive fluorescent dye 2',7'-bis(2-carboxy-ethyl)-5(6)-carboxyfluorescein, show a profound acidification (pH 7.33 to 6.75) in HCO3(-)-free Ringer solution when exposed to the Na(+)-H+ exchange inhibitor amiloride. This indicates that the cells are under a constant acid load that is being countered by Na(+)-H+ exchange. Amiloride-induced acidification was affected neither by incubation in Cl(-)-free Ringer solution nor by hypoxia, indicating that the potential acid loaders, Cl(-)-HCO3- (or OH-) exchange or glycolytic metabolism, are not contributing to the acidification. The possibility of a H+ influx dependent on the outward K+ gradient was tested. Perfusion with a high-K+ Ringer solution (77 mM) caused Na(+)- and Cl(-)-independent alkalinizations. Membrane depolarization by gramicidin, Ba2+, Cl(-)-free Ringer solution, or ouabain all produced small (less than 0.1 pH units) acidifications, inconsistent with contribution by a membrane potential driven passive H+ influx. In Na(+)-free Ringer solution, intracellular pH (pHi) of 6.4-6.6, addition of nigericin (a 1K(+)-1H+ ionophore) produced no significant change in pHi, indicating that [K+]i/[K+]o = [H+]i/[H+]o. Both amiloride-induced acidifications and high-K(+)-induced alkalinizations were significantly stimulated by the presence of 1 mM ZnSO4 and unaffected by H2-DIDS (0.5 mM, an anion transport blocker) or 100 microM SCH28080 (K(+)-H(+)-ATPase blocker). In the absence of a demonstrable H+ conductance, it is concluded that amiloride-induced acidification and K(+)-induced pHi changes are via a carrier-mediated K(+)-H+ exchanger. In addition to pHi regulation, K(+)-H+ exchange may play a role in cell volume control.(ABSTRACT TRUNCATED AT 250 WORDS)
Bonanno
J A
JA
Morton D. Sarver Center for Cornea and Contact Lens Research, School of Optometry, University of California, Berkeley 94720.
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D003315:Cornea / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008856:Microscopy, Fluorescence; D011188:Potassium / Q000378:metabolism*; D027961:Potassium-Hydrogen Antiporters; D011817:Rabbits
D002352:Carrier Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D027961:Potassium-Hydrogen Antiporters; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D011188:Potassium