Intracellular pH regulation in U-2 OS 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 HCO(3)(-)/CO(2)-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 HCO(3)(-)/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 U2-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.
Porcelli
Anna Maria
AM
Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Italy.
Scotlandi
Katia
K
Strammiello
Rosaria
R
Gislimberti
Gabriella
G
Baldini
Nicola
N
Rugolo
Michela
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics; D000584:Amiloride; D002021:Buffers; D027963:Chloride-Bicarbonate Antiporters / Q000378:metabolism; D015536:Down-Regulation; D005452:Fluoresceins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D012516:Osteosarcoma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D017693:Sodium Bicarbonate; D012965:Sodium Chloride; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002021:Buffers; D027963:Chloride-Bicarbonate Antiporters; D005452:Fluoresceins; D020033:Protein Isoforms; D017923:Sodium-Hydrogen Exchangers; D012965:Sodium Chloride; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D017693:Sodium Bicarbonate