Sun E, Lawrence J, Morré D M, Sun I, Crane F L, MacKellar W C, Morré D J
Department of Medicinal Chemistry, Purdue University, West Lafayette, IN 47907, USA.
Biochem Pharmacol. 1995 Oct 26;50(9):1461-8. doi: 10.1016/0006-2952(95)02050-0.
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.
外部氧化剂铁氰化钾或生长因子二价铁转铁蛋白可刺激HeLa细胞释放质子。在10 nM至1 microM的浓度范围内，抗肿瘤磺酰脲LY181984 [N-(4-甲基苯基磺酰基)-N'-(4-氯苯基)脲]可抑制这种刺激引起的质子释放。抗肿瘤无活性的磺酰脲类似物LY181985 [N-(4-甲基苯基磺酰基)-N'-(苯基)脲]在1 microM时无作用，需要10 - 100 microM的浓度才能抑制质子释放。用BCECF [2',7'-双(2-羧乙基)-5,(和6)-羧基荧光素]荧光估计的二价铁转铁蛋白诱导的细胞质碱化也受到1 microM LY181984的抑制，但不受1 microM LY181985的抑制。被抑制的成分似乎对氨氯地平有抗性。在接近最佳的氨氯地平浓度0.2 mM或二甲基氨氯地平浓度0.075 mM时，铁氰化钾或二价铁转铁蛋白诱导的质子释放被抑制约35%。然而，LY181984可进一步抑制诱导的质子释放。相反，当质子释放在接近最佳浓度10 microM（50%抑制）时被LY181984完全抑制，增加氨氯地平或二甲基氨氯地平的浓度分别导致额外16%和23%的抑制。然而，LY181984和氨氯地平的抑制作用是相加的，这表明氨氯地平和磺酰脲可能独立起作用。磺酰脲抑制质子外流的作用与氨氯地平不同的证据来自通过荧光法或用22Na+直接测量钠摄取的实验。在足以抑制质子外流80%或更多的LY181984浓度下，HeLa细胞中的钠摄取不受LY181984或LY181985 的抑制。结果表明LY181984是HeLa细胞中二价铁转铁蛋白或铁氰化钾诱导的质子外流和细胞质碱化的有效抑制剂，并且这种抑制可能涉及质子转运中对氨氯地平有抗性的一个成分。
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