Clerici C, Couette S, Loiseau A, Herman P, Amiel C
Department of Physiology, Faculté de Médecine Xavier Bichat, Paris, France.
J Membr Biol. 1995 Oct;147(3):295-304. doi: 10.1007/BF00234527.
We have investigated the presence of Na-K-Cl cotransport in alveolar type II cells using uptake of 86Rb. Several data support the presence of a Na-K-Cl cotransport in these cells. First, a large fraction of ouabain-resistant 86Rb uptake was inhibited by bumetanide and furosemide. Second, bumetanide-sensitive 86Rb uptake required the presence of Na+ and Cl- in the incubation medium; dependency on extracellular Na+ and K+ was hyperbolic, with a Km of 14.6 mM and 8.3 mM, respectively, while dependency on extracellular Cl- was sigmoidal, which suggests a 1:1:2 stoichiometry. Third, a fraction of amiloride-insensitive 22Na influx was deeply inhibited by bumetanide. 22Na influx was dependent on the presence of extracellular K+ and Cl-. Since Na-K-Cl activity dramatically decreased with time in culture, further characterization of the cotransport on polarized cells could not be performed. The phorbol ester PMA inhibited Na-K-Cl cotransport in a time- and concentration-dependent manner. This inhibition was mimicked by oleoylacetylglycerol, dioctanoylglycerol, and the diacylglycerol kinase inhibitor R59022, and was reversed by an antagonist of PKC, staurosporine. Since the Na-K-Cl cotransport has been reported to be involved in cell volume regulation, we investigated its modulation by changes in extracellular osmolarity. Na-K-Cl activity was increased after a two-step procedure: swelling in hypotonic medium followed by shrinking in hypertonic medium. Under these conditions, cotransport activity increased whenever PKC activity was up- or downregulated, which suggests that the cell volume-induced modulation of the cotransport is independent from the PKC activity. Though we were not able to determine the polarity of the cotransport, it may also be involved in the absorptive function of alveolar type II cells, and would provide an alternate pathway for sodium entry.
我们利用⁸⁶Rb摄取研究了Ⅱ型肺泡细胞中钠 - 钾 - 氯共转运体的存在情况。多项数据支持这些细胞中存在钠 - 钾 - 氯共转运体。首先，布美他尼和呋塞米抑制了大部分哇巴因抵抗性的⁸⁶Rb摄取。其次，布美他尼敏感的⁸⁶Rb摄取需要孵育培养基中存在Na⁺和Cl⁻；对细胞外Na⁺和K⁺的依赖性呈双曲线关系，Km分别为14.6 mM和8.3 mM，而对细胞外Cl⁻的依赖性呈S形，这表明其化学计量比为1:1:2。第三，布美他尼深度抑制了一部分对阿米洛利不敏感的²²Na内流。²²Na内流依赖于细胞外K⁺和Cl⁻的存在。由于培养过程中钠 - 钾 - 氯活性随时间显著降低，因此无法对极化细胞上的共转运体进行进一步表征。佛波酯PMA以时间和浓度依赖性方式抑制钠 - 钾 - 氯共转运体。这种抑制作用可被油酰乙酰甘油、二辛酰甘油和二酰基甘油激酶抑制剂R59022模拟，并被蛋白激酶C拮抗剂星形孢菌素逆转。由于据报道钠 - 钾 - 氯共转运体参与细胞体积调节，我们研究了细胞外渗透压变化对其的调节作用。经过两步处理后钠 - 钾 - 氯活性增加：在低渗培养基中肿胀，随后在高渗培养基中收缩。在这些条件下，无论蛋白激酶C活性上调还是下调，共转运体活性都会增加，这表明细胞体积诱导的共转运体调节与蛋白激酶C活性无关。尽管我们无法确定共转运体的极性，但它可能也参与Ⅱ型肺泡细胞的吸收功能，并为钠进入提供一条替代途径。
