Cellular Physiology Laboratory, Biomedical Department, Faculty of Health Sciences, Universidad de Antofagasta, Antofagasta, Chile.
PLoS One. 2012;7(12):e51451. doi: 10.1371/journal.pone.0051451. Epub 2012 Dec 6.
Arsenic main inorganic compound is arsenic trioxide (ATO) presented in solution mainly as arsenite. ATO increases intracellular pH (pHi), cell proliferation and tumor growth. Sodium-proton exchangers (NHEs) modulate the pHi, with NHE1 playing significant roles. Whether ATO-increased cell proliferation results from altered NHEs expression and activity is unknown. We hypothesize that ATO increases cell proliferation by altering pHi due to increased NHEs-like transport activity. Madin-Darby canine kidney (MDCK) cells grown in 5 mmol/L D-glucose-containing DMEM were exposed to ATO (0.05, 0.5 or 5 µmol/L, 0-48 hours) in the absence or presence of 5-N,N-hexamethylene amiloride (HMA, 5-100 µmol/L, NHEs inhibitor), PD-98059 (30 µmol/L, MAPK1/2 inhibitor), Gö6976 (10 µmol/L, PKCα, βI and μ inhibitor), or Schering 28080 (10 µmol/L, H(+)/K(+)ATPase inhibitor) plus concanamycin (0.1 µmol/L, V type ATPases inhibitor). Incorporation of [(3)H]thymidine was used to estimate cell proliferation, and counting cells with a hemocytometer to determine the cell number. The pHi was measured by fluorometry in 2,7-bicarboxyethyl-5,6-carboxyfluorescein loaded cells. The Na(+)-dependent HMA-sensitive NHEs-like mediated proton transport kinetics, NHE1 protein abundance in the total, cytoplasm and plasma membrane protein fractions, and phosphorylated and total p42/44 mitogen-activated protein kinases (p42/44(mapk)) were also determined. Lowest ATO (0.05 µmol/L, ~0.01 ppm) used in this study increased cell proliferation, pHi, NHEs-like transport and plasma membrane NHE1 protein abundance, effects blocked by HMA, PD-98059 or Gö6976. Cell-buffering capacity did not change by ATO. The results show that a low ATO concentration increases MDCK cells proliferation by NHEs (probably NHE1)-like transport dependent-increased pHi requiring p42/44(mapk) and PKCα, βI and/or μ activity. This finding could be crucial in diseases where uncontrolled cell growth occurs, such as tumor growth, and in circumstances where ATO, likely arsenite, is available at the drinking-water at these levels.
砷的主要无机化合物是三氧化二砷（ATO），以亚砷酸盐的形式主要存在于溶液中。ATO 会增加细胞内 pH 值（pHi）、细胞增殖和肿瘤生长。钠-质子交换器（NHEs）调节 pHi，其中 NHE1 起着重要作用。ATO 是否通过改变 NHEs 的表达和活性来增加细胞增殖尚不清楚。我们假设 ATO 通过增加 NHEs 样转运活性导致 pHi 改变，从而增加细胞增殖。在 5mmol/L D-葡萄糖含量的 DMEM 中生长的 Madin-Darby 犬肾（MDCK）细胞在无或存在 5-N，N-六亚甲基阿米洛利（HMA，5-100μmol/L，NHEs 抑制剂）、PD-98059（30μmol/L，MAPK1/2 抑制剂）、Gö6976（10μmol/L，PKCα、βI 和μ抑制剂）或 Schering 28080（10μmol/L，H(+)/K(+)ATPase 抑制剂）加康纳霉素（0.1μmol/L，V 型 ATPases 抑制剂）的情况下，暴露于 0.05、0.5 或 5μmol/L ATO（0-48 小时）。用[（3）H]胸苷掺入法估计细胞增殖，用血细胞计数器计数细胞以确定细胞数量。用 2,7-二羧乙基-5,6-羧基荧光素负载的细胞通过荧光法测量 pHi。测定了 Na(+)-依赖性 HMA 敏感的 NHEs 样介导的质子转运动力学、总蛋白、细胞质和质膜蛋白部分的 NHE1 蛋白丰度以及磷酸化和总 p42/44 丝裂原激活蛋白激酶（p42/44(mapk))。在这项研究中使用的最低 ATO（0.05μmol/L，约 0.01ppm）增加了细胞增殖、pHi、NHEs 样转运和质膜 NHE1 蛋白丰度，这些效应被 HMA、PD-98059 或 Gö6976 阻断。细胞缓冲能力不因 ATO 而改变。结果表明，低浓度的 ATO 通过 NHEs（可能是 NHE1）样转运依赖性增加 pHi 来增加 MDCK 细胞增殖，需要 p42/44(mapk)和 PKCα、βI 和/或μ 活性。这一发现可能在不受控制的细胞生长发生的疾病中至关重要，例如肿瘤生长，以及在饮用水中可能存在这些水平的 ATO（可能是亚砷酸盐）的情况下至关重要。
