McMurchie E J, Burnard S L, Patten G S, Lee E J, King R A, Head R J
Division of Human Nutrition, Glenthorne Laboratory, Commonwealth Scientific and Industrial Research Organization, Australia.
Am J Physiol. 1994 Jul;267(1 Pt 1):C84-93. doi: 10.1152/ajpcell.1994.267.1.C84.
Na+ transport activity was characterized in human cheek epithelial cells obtained from normotensive adult subjects. The cells were isolated using a mouth-wash procedure and assayed for Na+ uptake using a radioactive (22Na+) rapid filtration assay. Cheek cells displayed proton-dependent Na+ uptake activity that was dependent on the magnitude of the externally directed proton gradient measured using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to determine intracellular pH. Amiloride, ethylisopropylamiloride (EIPA), 5-(N,N-dimethyl)-amiloride, 5-(N-methyl-N-isobutyl)-amiloride (MIA), and 5-(N,N-hexamethylene)-amiloride (NNHA) all inhibited proton-dependent Na+ uptake, with MIA, EIPA, and NNHA being the most potent. The Michaelis constant (Km) for extracellular Na+ was 5.7 mM, while the maximum velocity for Na(+)-H+ antiporter activity was 4.3 nmol Na+.mg protein-1.30s-1. The Km for intracellular H+ was 0.17 microM, with a Hill coefficient of 0.7. Stimulation by ouabain and inhibition by bumetanide of cheek cell proton-dependent Na+ uptake indicated only relatively low activities of Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- cotransport, respectively. These results are consistent with the presence of Na(+)-H+ antiporter activity in cheek cells. Cheek cells therefore provide a convenient, relatively noninvasive source of tissue for examining Na(+)-H+ antiporter activity in human subjects.
对从血压正常的成年受试者获取的人颊上皮细胞中的钠离子转运活性进行了表征。使用漱口程序分离细胞，并使用放射性（²²Na⁺）快速过滤测定法检测钠离子摄取。颊细胞表现出质子依赖性钠离子摄取活性，该活性取决于使用荧光探针2',7'-双（羧乙基）-5（6）-羧基荧光素测量细胞内pH值来确定的外向质子梯度的大小。氨氯地平、乙基异丙基氨氯地平（EIPA）、5-（N，N-二甲基）-氨氯地平、5-（N-甲基-N-异丁基）-氨氯地平（MIA）和5-（N，N-六亚甲基）-氨氯地平（NNHA）均抑制质子依赖性钠离子摄取，其中MIA、EIPA和NNHA最为有效。细胞外钠离子的米氏常数（Km）为5.7 mM，而钠离子-氢离子反向转运体活性的最大速度为4.3 nmol Na⁺·mg蛋白质⁻¹·30秒⁻¹。细胞内氢离子的Km为0.17 μM，希尔系数为0.7。哇巴因对颊细胞质子依赖性钠离子摄取的刺激和布美他尼的抑制分别表明钠离子-钾离子-ATP酶和钠离子-钾离子-2氯离子协同转运的活性相对较低。这些结果与颊细胞中存在钠离子-氢离子反向转运体活性一致。因此，颊细胞为检查人类受试者的钠离子-氢离子反向转运体活性提供了一种方便、相对非侵入性的组织来源。
