Patten G S, Leifert W R, Burnard S L, Head R J, McMurchie E J
Division of Human Nutrition, CSIRO, O'Halloran Hill, South Australia.
Mol Cell Biochem. 1996 Jan 26;154(2):133-41. doi: 10.1007/BF00226781.
Proton-dependent, ethylisopropylamiloride (EIPA)-sensitive Na+ uptake (Na+/H+ antiporter) studies were performed to examine if saliva, and ionophores which alter cellular electrolyte balance, could influence the activity of the cheek cell Na+/H+ antiporter. Using the standard conditions of 1 mmol/l Na+, and a 65:1 (inside:outside) proton gradient in the assay, the uniport ionophores valinomycin (K+) and gramicidin (Na+) increased EIPA-sensitive Na+ uptake by 177% (p < 0.01) and 227% (p < 0.01), respectively. The dual antiporter ionophore nigericin (K(+)-H+) increased EIPA-sensitive Na+ uptake by 654% (p < 0.01), with maximal Na+ uptake achieved by 1 min and at an ionophore concentration of 50 mumol/l, with an EC50 value 6.4 mumol/l. Pre-incubation of cheek cells with saliva or the low molecular weight (MW) components of saliva (saliva activating factors, SAF) for 2 h at 37 degrees C, also significantly stimulated EIPA-sensitive Na+ uptake. This stimulation could be mimicked by pre-incubation with 25 mmol/l KCl or K(+)-phosphate buffer. Pre-incubating cheek cells with SAF and the inclusion of 20 mumol/l nigericin in the assay, produced maximum EIPA-sensitive Na+ uptake. After pre-incubation with water, 25 mmol/l K(+)-phosphate or SAF, with nigericin in all assays, the initial rate of proton-gradient dependent, EIPA-sensitive Na+ uptake was saturable with respect to external Na+, with Km values of 0.9, 1.7, and 1.8 mmol/l, and Vmax values of 13.4, 25.8, and 31.1 nmol/mg protein/30 sec, respectively. With 20 mumol/l nigericin in the assay, Na+ uptake was inhibited by either increasing the [K+]o in the assay, with an ID50 of 3 mmol/l. These results indicate that nigericin can facilitate K+i exchange for H+o and the attending re-acidification of the cheek cell amplifies 22Na+ uptake via the Na+/H+ antiporter. The degree of stimulation of proton-dependent, EIPA-sensitive Na+ uptake is therefore dependent, in part, on the intracellular [K+]i.
进行了质子依赖性、乙基异丙基amiloride（EIPA）敏感的Na⁺摄取（Na⁺/H⁺反向转运体）研究，以检验唾液以及改变细胞电解质平衡的离子载体是否会影响颊细胞Na⁺/H⁺反向转运体的活性。在测定中使用1 mmol/L Na⁺的标准条件以及65:1（胞内:胞外）的质子梯度，单向离子载体缬氨霉素（K⁺）和短杆菌肽（Na⁺）分别使EIPA敏感的Na⁺摄取增加了177%（p < 0.01）和227%（p < 0.01）。双反向转运体离子载体尼日利亚菌素（K⁺-H⁺）使EIPA敏感的Na⁺摄取增加了654%（p < 0.01），在1分钟时达到最大Na⁺摄取，离子载体浓度为50 μmol/L，EC50值为6.4 μmol/L。将颊细胞与唾液或唾液的低分子量（MW）成分（唾液激活因子，SAF）在37℃预孵育2小时，也显著刺激了EIPA敏感的Na⁺摄取。这种刺激可以通过用25 mmol/L KCl或K⁺-磷酸盐缓冲液预孵育来模拟。将颊细胞与SAF预孵育并在测定中加入20 μmol/L尼日利亚菌素，产生了最大的EIPA敏感的Na⁺摄取。在用蒸馏水、25 mmol/L K⁺-磷酸盐或SAF预孵育后，在所有测定中加入尼日利亚菌素，质子梯度依赖性、EIPA敏感的Na⁺摄取的初始速率相对于外部Na⁺是可饱和的，Km值分别为0.9、1.7和1.8 mmol/L，Vmax值分别为13.4、25.8和31.1 nmol/mg蛋白/30秒。在测定中加入20 μmol/L尼日利亚菌素时，通过增加测定中的[K⁺]o，Na⁺摄取受到抑制，ID50为3 mmol/L。这些结果表明，尼日利亚菌素可以促进K⁺i与H⁺o交换，随之颊细胞的再酸化通过Na⁺/H⁺反向转运体放大了²²Na⁺摄取。因此，质子依赖性、EIPA敏感的Na⁺摄取的刺激程度部分取决于细胞内[K⁺]i。
