Kovbasnjuk O, Chatton J Y, Friauf W S, Spring K R
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1598, USA.
J Membr Biol. 1995 Dec;148(3):223-32. doi: 10.1007/BF00235040.
The kinetics of Na movement across the tight junctions of MDCK cells, grown on coverslips and perfused with HEPES or bicarbonate Ringer at 37 degrees C, were investigated after filling the lateral intercellular spaces (LIS) of the epithelium with SBFO, an Na-sensitive fluorescent dye. Dilution and bi-ionic potential measurements showed that MDCK cell tight junctions, although cation-selective, were poorly permeable to N-methyl-D-glucamine Cl (NMDG) but freely permeable to Li. In previous experiments in which Na was replaced by NMDG, a very slow decrease in LIS Na concentration (time constant = 4.8 min) resulted. In the present study, reduction of perfusate Na from 142 to 14 or 24 mM with Na replaced by Li caused LIS Na concentration to decrease with a time constant of 0.43 min. The time constant for Na increase of the LIS was 0.28 min, significantly shorter than that for Na decrease because of the additional component of transcellular Na influx. Ouabain eliminated the transcellular component and equalized the time constants for Na influx and efflux. These results were incorporated into a mathematical model which enabled calculation of the transcellular and paracellular Na fluxes during fluid reabsorption. Regulation of the Na permeability of individual tight junctions by protein kinase A (PKA) was evaluated by treating the monolayers with the Sp-cAMPS, a cAMP substitute, or Rp-cAMPS, a specific inhibitor of PKA. Stimulation of PKA strikingly increased tight junctional permeability while PKA inhibition diminished junctional Na permeability.
将生长在盖玻片上并于37℃用HEPES或碳酸氢盐林格液灌注的MDCK细胞的紧密连接处，用钠敏感荧光染料SBFO填充上皮细胞的细胞间侧间隙（LIS）后，研究了钠跨这些紧密连接处的动力学。稀释和双离子电位测量表明，MDCK细胞紧密连接处虽然对阳离子有选择性，但对N-甲基-D-葡萄糖胺氯（NMDG）的通透性较差，而对锂则可自由通透。在先前用NMDG替代钠的实验中，LIS钠浓度出现了非常缓慢的下降（时间常数 = 4.8分钟）。在本研究中，将灌注液中的钠从142 mM降至14或24 mM，并用锂替代钠，导致LIS钠浓度以0.43分钟的时间常数下降。LIS中钠增加的时间常数为0.28分钟，明显短于钠减少的时间常数，这是因为存在跨细胞钠内流的额外成分。哇巴因消除了跨细胞成分，并使钠内流和外流的时间常数相等。这些结果被纳入一个数学模型，该模型能够计算液体重吸收过程中的跨细胞和细胞旁钠通量。通过用cAMP替代物Sp-cAMPS或PKA特异性抑制剂Rp-cAMPS处理单层细胞，评估了蛋白激酶A（PKA）对单个紧密连接处钠通透性的调节。刺激PKA显著增加紧密连接的通透性，而抑制PKA则降低连接处的钠通透性。
