Proton release from HeLa cells and alkalization of cytoplasm induced by diferric transferrin or ferricyanide and its inhibition by the diarylsulfonylurea antitumor drug N-(4-methylphenylsulfonyl)-N'-(4-cholorophenyl) urea (LY181984).
Proton release from HeLa cells was stimulated by an external oxidant, potassium ferricyanide, or by the growth factor diferric transferrin. This stimulated proton release was inhibited by the antitumor sulfonylurea LY181984 [N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea] over the concentration range 10 nM to 1 microM. The antitumor-inactive sulfonylurea analog LY181985 [N-(4-methylphenylsulfonyl)-N'-(phenyl)urea] was without effect at 1 microM and required 10-100 microM concentrations to inhibit proton release. Diferric transferrin-induced alkalization of the cytoplasm estimated by BCECF [2',7'-bis(2-carboxyethyl)-5,(and 6)-carboxyfluorescein] fluorescence also was inhibited by 1 microM LY181984 but not by 1 microM LY181985. The inhibited component appeared to be amiloride resistant. The proton release induced by either ferricyanide or diferric transferrin was inhibited by about 35% at a near optimal amiloride concentration of 0.2 mM or at a dimethylamiloride concentration of 0.075 mM. However, the induced proton release was inhibited further by LY181984. Conversely, when proton release was inhibited fully by LY181984 at a near optimal concentration of 10 microM (50% inhibition), increasing concentrations of amiloride or dimethylamiloride resulted in additional inhibitions of 16 and 23%, respectively. However, the inhibitions by LY181984 and the amilorides were additive, suggesting that amiloride and the sulfonylureas may act independently. Evidence for an action of the sulfonylurea in inhibiting proton efflux differently from that of the amilorides came from measurements of sodium uptake either by fluorometry or by direct measurement with 22Na+. Sodium uptake was not inhibited by either LY181984 or LY181985 in HeLa cells at concentrations of LY181984 sufficient to inhibit proton efflux by 80% or more. The results show LY181984 to be a potent inhibitor of diferric transferrin- or ferricyanide-induced proton efflux and cytoplasmic alkalization in HeLa cells and that the inhibition may involve a component of proton transport that is resistant to amiloride.
Sun
E
E
Department of Medicinal Chemistry, Purdue University, West Lafayette, IN 47907, USA.
Lawrence
J
J
Morré
D M
DM
Sun
I
I
Crane
F L
FL
MacKellar
W C
WC
Morré
D J
DJ
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002138:Calibration; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004347:Drug Interactions; D005292:Ferricyanides / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011522:Protons; D012964:Sodium / Q000493:pharmacokinetics; D013268:Stimulation, Chemical; D013453:Sulfonylurea Compounds / Q000494:pharmacology*; D014168:Transferrin / Q000037:antagonists & inhibitors / Q000494:pharmacology*
D000970:Antineoplastic Agents; D005292:Ferricyanides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011522:Protons; D013453:Sulfonylurea Compounds; D014168:Transferrin; C028283:diferric transferrin; C007931:hexacyanoferrate III; C062821:LY 181984; C039465:5-dimethylamiloride; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium