Fitz J G, Persico M, Scharschmidt B F
Department of Medicine, University of California School of Medicine, San Francisco 94143.
Am J Physiol. 1989 Mar;256(3 Pt 1):G491-500. doi: 10.1152/ajpgi.1989.256.3.G491.
Recent observations suggest that hepatocytes exhibit basolateral electrogenic Na+-coupled HCO3- transport. In these studies, we have further investigated this transport mechanism in primary culture of rat hepatocytes using intracellular microelectrodes to measure membrane potential difference (PD) and the pH-sensitive fluorochrome 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to measure intracellular pH (pHi). In balanced media containing 25 mM HCO3-, PD averaged -32.1 +/- 0.6 (SE) mV and pHi averaged 7.22 +/- 0.03. PD became more negative (hyperpolarized) when extracellular [HCO3-] was increased and less negative (depolarized) when extracellular HCO3- was decreased. Acute replacement of extracellular Na+ by choline also resulted in membrane depolarization of 18.0 +/- 1.6 mV, suggesting net transfer of negative charge. This decrease in PD upon Na+ removal was HCO3- -dependent, amiloride insensitive, and inhibited by the disulfonic stilbene 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). PD also decreased upon acute exposure to SITS. The degree of depolarization seen with removal of Na+ or HCO3- correlated directly with resting PD (r = 0.81 and 0.95, respectively), suggesting a voltage-dependent mechanism. Removal of extracellular Na+ also decreased pHi to 7.06 +/- 0.02, and this acidification was decreased in the absence of HCO3- or in the presence of SITS or amiloride. These studies provide direct evidence for electrogenic Na+-coupled HCO3- transport in rat hepatocytes. Further, they suggest that it represents a major pathway for conductive movement of Na+ across the membrane and that it contributes, along with Na+-H+ exchange, to the intracellular acidification observed upon removal of extracellular Na+.(ABSTRACT TRUNCATED AT 250 WORDS)
最近的观察结果表明，肝细胞表现出基底外侧电生性Na⁺偶联HCO₃⁻转运。在这些研究中，我们使用细胞内微电极测量膜电位差（PD），并使用对pH敏感的荧光染料2'，7'-双（羧乙基）-5（6）-羧基荧光素测量细胞内pH（pHi），进一步研究了大鼠肝细胞原代培养中的这种转运机制。在含有25 mM HCO₃⁻的平衡培养基中，PD平均为-32.1±0.6（SE）mV，pHi平均为7.22±0.03。当细胞外[HCO₃⁻]增加时，PD变得更负（超极化），而当细胞外HCO₃⁻减少时，PD变得不那么负（去极化）。用胆碱急性替代细胞外Na⁺也导致膜去极化18.0±1.6 mV，表明负电荷的净转移。Na⁺去除后PD的这种降低是HCO₃⁻依赖性的，对氨氯地平不敏感，并被二磺酸芪4-乙酰氨基-4'-异硫氰酸芪-2,2'-二磺酸（SITS）抑制。急性暴露于SITS时PD也降低。去除Na⁺或HCO₃⁻时观察到的去极化程度与静息PD直接相关（r分别为0.81和0.95），提示存在电压依赖性机制。去除细胞外Na⁺也使pHi降至7.06±0.02，并且在没有HCO₃⁻或存在SITS或氨氯地平的情况下，这种酸化作用减弱。这些研究为大鼠肝细胞中电生性Na⁺偶联HCO₃⁻转运提供了直接证据。此外，它们表明这代表了Na⁺跨膜传导运动的主要途径，并且它与Na⁺-H⁺交换一起，导致去除细胞外Na⁺后观察到的细胞内酸化。（摘要截短于250字）
