Jensen B S, Jessen F, Hoffmann E K
Institute of Biological Chemistry A, August Krogh Institute, University of Copenhagen, Denmark.
J Membr Biol. 1993 Feb;131(3):161-78. doi: 10.1007/BF02260106.
Net Cl- uptake as well as unidirectional 36Cl influx during regulatory volume increase (RVI) require external K+. Half-maximal rate of bumetanide-sensitive 36Cl uptake is attained at about 3.3 mM external K+. The bumetanide-sensitive K+ influx found during RVI is strongly dependent on both Na+ and Cl-. The bumetanide-sensitive unidirectional Na+ influx during RVI is dependent on K+ as well as on Cl-. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na+, K+ and Cl-. In the presence of ouabain and Ba+ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na+, 0.8 K+, 2.0 Cl- or approximately 1:Na, 1:K, 2:Cl. Under these circumstances the K+ and Cl- flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 +/- 0.08 and 1.82 +/- 0.15 which should be compared to the gradient for the Na+, K+, 2Cl- cotransport system at 1.75 +/- 0.24. Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K+ influx is activated. During hypotonic conditions a small bumetanide-sensitive K+ influx is observed, indicating that the cotransport system is already activated. The cotransport is activated 10-15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca2+ and activation of protein kinase C. The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K+ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na+, K+, Cl- cotransport involves both Ca2+/calmodulin and protein kinase C.
在调节性容积增加（RVI）过程中，净氯离子摄取以及单向³⁶Cl内流都需要细胞外钾离子。布美他尼敏感的³⁶Cl摄取的半数最大速率在细胞外钾离子浓度约为3.3 mM时达到。在RVI过程中发现的布美他尼敏感的钾离子内流强烈依赖于钠离子和氯离子。RVI过程中布美他尼敏感的单向钠离子内流既依赖于钾离子也依赖于氯离子。因此，艾氏腹水癌细胞在RVI过程中被激活的协同转运体似乎能转运钠离子、钾离子和氯离子。在哇巴因和钡离子存在的情况下，布美他尼敏感的净通量的化学计量比可测定为1.0个钠离子、0.8个钾离子、2.0个氯离子，即大约1:钠离子、1:钾离子、2:氯离子。在这些情况下，布美他尼敏感成分的钾离子和氯离子通量比（内流/外流）估计分别为1.34±0.08和1.82±0.15，应与钠离子、钾离子、2氯离子协同转运系统的梯度1.75±0.24进行比较。向高渗溶液中添加蔗糖会导致艾氏腹水癌细胞收缩，且无RVI迹象，而用高渗标准培养基（所有细胞外离子浓度均升高）使其收缩则会引发对原始细胞体积的RVI反应。在这两种情况下，都会激活布美他尼敏感的单向钾离子内流。在低渗条件下，可观察到少量布美他尼敏感的钾离子内流，这表明协同转运系统已经被激活。缓激肽可使协同转运激活10 - 15倍，缓激肽是一种激动剂，可刺激磷脂酶C，导致细胞内钙离子释放并激活蛋白激酶C。抗钙调蛋白药物匹莫齐特可抑制RVI过程中大部分布美他尼敏感的钾离子内流。协同转运体可被佛波酯TPA激活。这些结果表明，钠离子、钾离子、氯离子协同转运的激活涉及钙离子/钙调蛋白和蛋白激酶C。
