title: Intracellular pH of tissue-cultured bovine corneal endothelial cells.
abstract: Intracellular pH (pHi) of bovine tissue-cultured corneal endothelial cells has been measured under several experimental conditions. Determinations were made on individual cells using video-imaging techniques that allowed assessment of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence at 440 and 490 nm. Each experiment had a calibration performed on a cell monolayer: this was performed using a high K(+)-nigericin solution. Resting pHi was 7.25 +/- 0.03 (n = 18) in bicarbonate solution at pH 7.4. Amiloride (1 mM) caused an acidification of approximately 0.2 U within 2 min: replacement with normal Ringer allowed a return to normal pHi after an alkali overshoot. Exposure to 20 mM NH4Cl caused alkalinization that became acidic upon washout of NH4Cl. In Na(+)-rich solution pHi returned to normal after acidification but pHi remained low in Na(+)-free solution until substituted by Na(+)-rich solution. Removal of HCO3- from the bathing solution caused a nonsignificant acidification of pHi by 0.1 U at 2 and 4 min, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 1 mM) acidified pHi by 0.14 U at 2 min and 0.24 U at 4 min. Addition of DIDS (1 mM) in a HCO3(-)-free solution had no effect on pHi. Hydrogen peroxide acidified pHi by 0.3 U at 50 microM and 1 mM. These results indicate that a Na+:H+ antiport exists that regulates pHi even at normal ambient pH in the presence of bicarbonate: this process becomes highly activated after an acid load. There is a DIDS-sensitive HCO3- movement that is probably coupled to Na+ or Cl-.
authors.affiliation: Department of Ophthalmology, Medical College of Georgia, Augusta 30912-3400.
authors.forename: T C
authors.initials: TC
authors.lastname: Chu
authors.forename: C H
authors.initials: CH
authors.lastname: Keith
authors.forename: E
authors.initials: E
authors.lastname: Yee
authors.forename: K
authors.initials: K
authors.lastname: Green
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000187:drug effects / Q000502:physiology*; D005110:Extracellular Space / Q000502:physiology; D005452:Fluoresceins; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D017923:Sodium-Hydrogen Exchangers
chemical_list: D002352:Carrier Proteins; D005452:Fluoresceins; D017923:Sodium-Hydrogen Exchangers; D000643:Ammonium Chloride; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D006861:Hydrogen Peroxide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid