Intracellular pH regulation in basal corneal epithelial cells measured in corneal explants: characterization of Na/H exchange.
Intracellular pH (pHi) was measured in basal corneal epithelial cells from fresh corneal explants using the pH sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The overlying superficial and wing cells were removed by mechanical scraping to expose basal cells attached to their basal lamina. Tissue pieces with attached, dye-loaded basal cells were mounted in a microscope-stage-perfusion chamber which allowed rapid changes of Ringer's bathing solutions while measuring BCECF fluorescence. In NaCl-Ringer's (bicarbonate free). pHo 7.40, resting cell pHi was 7.34 +/- 0.03 (+/- S.E.M., n = 31). Buffering capacity measured by NH4Cl treatment was 31 mM pH at pHi 7.34 and increased with decreasing pHi. Recovery from 20 mM NH4Cl-induced acid loads was dependent on the presence of Na and inhibited by 1 mM amiloride. Adding amiloride to resting cells caused a slow, reversible acidification (0.04 pH units min-1). These results indicate the presence of Na:H exchange, its role in responding to acid loads and in maintaining resting cell pHi. Activation of Na:H by Nao showed simple saturation kinetics, with Km = 44 mM. Net proton efflux via Na:H exchange increased with decreasing pHi and was enhanced by depleting cells of Nai, suggesting roles for both pHi and Nai in control of Na:H activation.
Bonanno
J A
JA
Department of Physiology-Anatomy, University of California, Berkeley 94720.
Machen
T E
TE
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D003315:Cornea / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers