Varanda W A, Lacaz-Vieira F
J Membr Biol. 1979 Sep;49(3):199-233. doi: 10.1007/BF01871119.
Experiments were carried out in the isolated short-circuited skin of the toad Bufo marinus ictericus. 42K influx and efflux experiments were carried out with skins bathed on both sides by NaCl-Ringer's solution. Those fluxes showed very similar kinetics of equilibration with time and the results could be fitted by equations of a model of two intraepithelial compartments and the bathing solutions. In the steady state K influx is 3.99 +/- 0.36 nmol cm-2 hr-1 (n = 7) and efflux 3.62 +/- 0.38 nmol cm-2 hr-1 (n = 7) and are not statistically different, indicating that no net K flux is present across the epithelium. Different kinds of perturbations affecting the rates of 42K discharge into the bathing solutions were studied. Immediately after addition of amiloride (10(-4) M) to the outer solution, a sharp decline is observed in the rate of 42K discharge into the bathing solution, JK21, which falls from 3.62 +/- 0.38 nmol cm-2 hr-1 to 2.02 +/- 0.04 nmol cm-2 hr-1 (n = 7) 2 min after addition of the drug, followed by a partial recuperation with time. A complete Na by K substitution in the outer bathing solution induces a prompt and marked decline in JK21 which is similar to that induced by amiloride. Increase in the outer bathing solution Na concentration from zero Na concentration induces a nonlinear increase in JK21 and a linear relationship was observed between JK21 and short-circuit current in the range of 0 to 115 mM external Na concentration. The decline in JK21 induced by amiloride or by lowering external Na concentration was interpreted as being caused by electrical hyperpolarization of the external barrier of the epithelium induced by these procedures. Depolarization of the epithelial barriers by inner Na by K substitution in the short-circuited state (when the potential barriers are equal) drastically interfere with the rate of 42K discharge from the epithelium into the bathing solutions. Thus, transient increases are observed both in the rate of 42K discharge to the outer and to the inner bathing solutions upon depolarization of the barriers. These results indicate that at least the most important component of transepithelial K unidirectional fluxes goes through a transcellular route with a negligible paracellular component. Addition of ouabain (10(-3) M) to the inner bathing solution induces a transient rise in the rate of 42K discharge to the outer bathing solution with a peak on the order of 200% of the stationary value previous to the action of the inhibitor, followed by a return to new stationary values not statistically different from those observed previously to the effect of ouabain. The behavior of JK21 upon the effect of ouabain, as suggested by comparison with predictions from computer simulation, strongly supports the notion of a rheogenic Na pump in the inner barrier of the epithelium against the notion of a nonrheogenic 1:1 Na--K pump.
实验在海蟾蜍（Bufo marinus ictericus）离体的短路皮肤中进行。用氯化钠 - 林格氏溶液双侧浸泡皮肤进行了⁴²K流入和流出实验。这些通量显示出与时间非常相似的平衡动力学，结果可以用两个上皮内隔室和浸泡溶液的模型方程来拟合。在稳态下，K流入为3.99±0.36 nmol cm⁻² hr⁻¹（n = 7），流出为3.62±0.38 nmol cm⁻² hr⁻¹（n = 7），且无统计学差异，表明上皮细胞两侧不存在净K通量。研究了影响⁴²K释放到浸泡溶液中速率的不同类型扰动。向外部溶液中立即加入氨氯地平（10⁻⁴ M）后，观察到⁴²K释放到浸泡溶液中的速率JK₂₁急剧下降，药物加入后2分钟，JK₂₁从3.62±0.38 nmol cm⁻² hr⁻¹降至2.02±0.04 nmol cm⁻² hr⁻¹（n = 7），随后随时间部分恢复。外部浸泡溶液中完全用Na替代K会导致JK₂₁迅速且显著下降，这与氨氯地平诱导的情况相似。外部浸泡溶液中Na浓度从零浓度增加会导致JK₂₁非线性增加，并且在0至115 mM外部Na浓度范围内观察到JK₂₁与短路电流之间存在线性关系。氨氯地平或降低外部Na浓度诱导的JK₂₁下降被解释为是由这些操作引起的上皮细胞外部屏障的电超极化所致。在短路状态下（当电位屏障相等时）通过内部用Na替代K使上皮屏障去极化，会极大地干扰⁴²K从上皮细胞释放到浸泡溶液中的速率。因此，在屏障去极化时，观察到⁴²K释放到外部和内部浸泡溶液中的速率都有短暂增加。这些结果表明，至少跨上皮K单向通量的最重要成分是通过跨细胞途径进行的，而细胞旁成分可忽略不计。向内浸泡溶液中加入哇巴因（10⁻³ M）会导致⁴²K释放到外部浸泡溶液中的速率短暂升高，峰值约为抑制剂作用前稳态值的200%，随后恢复到新的稳态值，与哇巴因作用前观察到的值无统计学差异。与计算机模拟预测结果相比，哇巴因作用下JK₂₁的行为有力地支持了上皮细胞内屏障中存在生电Na泵的观点，而非非生电1:1 Na - K泵的观点。
