Intracellular pH regulation in U-2OS human osteosarcoma cells transfected with P-glycoprotein

The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl- and Na+ gradients, both in HEPES- and HCO3-/CO2-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na+/H+ exchanger. In addition to the amiloride-sensitive Na+/H+ exchanger, another H+ extruding mechanism was detected in U-2 OS cells, the Na+-dependent HCO3-/Cl- exchanger. No significant difference in resting pH(i) and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V-max and K' values for intracellular [H+] of the Na+/H+ exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U-2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH(i) regulation in U-2 OS cells, the kinetic parameters of the Na+/H+ exchanger were altered, suggesting a functional relationship between the two membrane proteins. (C) 2002 Elsevier Science B.V. All rights reserved.