Flow cytometric kinetic assay of the activity of Na+/H+ antiporter in mammalian cells.
BACKGROUND
The Na(+)/H(+) exchanger (NHE) of mammalian cells is an integral membrane protein that extrudes H(+) ion in exchange for extracellular Na(+) and plays a crucial role in the regulation of intracellular pH (pHi). Thus, when pHi is lowered, NHE extrudes protons at a rate depending of pHi that can be expressed as pH units/s.


METHODS
To abolish the activity of other cellular pH-restoring systems, cells were incubated in bicarbonate-free Dulbecco's modified Eagle's medium buffered with HEPES. Flow cytometry was used to determine pHi with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester or 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester acetate, and the appropriate fluorescence ratios were measured. The calibration of fluorescence ratios versus pHi was established by using ionophore nigericin. The activity of NHE was calculated by a kinetic flow cytometric assay as the slope at time 0 of the best-fit curve of pHi recovery versus time after intracellular acidification with a pulse of exogenous sodium propionate.


RESULTS
The kinetic method allowed determination of the pHi-dependent activity of NHE in cell lines and primary cell cultures. NHE activity values were demonstrated to be up to 0.016 pH units/s within the pHi range of 7.3 to 6.3. The inhibition of NHE activity by the specific inhibitor ethyl isopropyl amiloride was easily detected by this method.


CONCLUSIONS
The assay conditions can be used to relate variations in pHi with the activity of NHE and provide a standardized method to compare between different cells, inhibitors, models of ischemia by acidification, and other relevant experimental or clinical situations.
Dolz
María
M
Unidad de Investigaciones Cardíacas, Departamento de Biología y Terapia Molecular y Celular, Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas, Valencia, Spain.
O'Connor
José-Enrique
JE
Lequerica
Juan L
JL
D000818:Animals; D001578:Benzopyrans; D001671:Biochemistry / Q000379:methods; D002138:Calibration; D002460:Cell Line; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007511:Ischemia; D019169:Jurkat Cells; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000166:cytology; D041681:NIH 3T3 Cells; D009284:Naphthols / Q000494:pharmacology; D012235:Rhodamines / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
D001578:Benzopyrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009284:Naphthols; D012235:Rhodamines; D017923:Sodium-Hydrogen Exchangers; C068301:carboxy-seminaphthorhodaminefluoride; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester