Mongin A A, Aksentsev S L, Orlov S N, Kvacheva Z B, Mezen N I, Fedulov A S, Konev S V
Institute of Photobiology, Belarussian Academy of Sciences, Minsk, Belarus,
Biochim Biophys Acta. 1996 Dec 4;1285(2):229-36. doi: 10.1016/s0005-2736(96)00165-4.
Swelling of C6 glioma cells in hypotonic medium (180 mOsm) results in two- to three-fold activation of K+ (86Rb+) influx suppressed by 10 microM bumetanide. Bumetanide-sensitive transport of 86Rb+ is dependent on extracellular K+, Na+ and Cl- both in iso-osmotic conditions and under hypo-osmotic shock, supporting the notion that it is mediated by Na+,K+,2Cl- cotransport. Inhibitors of protein kinase C (10 microM polymyxin B and l microM staurosporine) had no significant effect on basal cotransport but reduced its hypotonic stimulation by 70-80%. Similar results were obtained with calmodulin antagonist R24571 (10 microM), indicating Ca2+/calmodulin-dependence of the process. Influence of polymyxin B and R24571 was not additive. Swelling-activated Na+,K+,2Cl- cotransport was also suppressed by protein kinase C activator PMA (l microM). By contrast, preincubation of cells with inhibitors of protein phosphatases (100 microM vanadate, 5 mM fluoride and 0.5 microM okadaic acid) activated greatly the bumetanide-sensitive 86Rb+ uptake in isotonic conditions, while a subsequent hypotonic swelling led to smaller or no increment. These results indicate the involvement of Ca2+/calmodulin-dependent staurosporine/polymyxin B-sensitive protein kinase other than protein kinase C in swelling-induced activation of Na+,K+,2Cl- cotransport in glial cells.
在低渗培养基（180 mOsm）中，C6胶质瘤细胞肿胀会导致K⁺（⁸⁶Rb⁺）内流激活两到三倍，该过程被10 μM布美他尼抑制。在等渗条件和低渗休克下，布美他尼敏感的⁸⁶Rb⁺转运依赖于细胞外K⁺、Na⁺和Cl⁻，这支持了其由Na⁺,K⁺,2Cl⁻共转运介导的观点。蛋白激酶C抑制剂（10 μM多粘菌素B和1 μM星形孢菌素）对基础共转运无显著影响，但使其低渗刺激降低了70 - 80%。钙调蛋白拮抗剂R24571（10 μM）也得到了类似结果，表明该过程依赖Ca²⁺/钙调蛋白。多粘菌素B和R24571的影响并非相加性的。肿胀激活的Na⁺,K⁺,2Cl⁻共转运也被蛋白激酶C激活剂PMA（1 μM）抑制。相比之下，用蛋白磷酸酶抑制剂（100 μM钒酸盐、5 mM氟化物和0.5 μM冈田酸）预孵育细胞，在等渗条件下极大地激活了布美他尼敏感的⁸⁶Rb⁺摄取，而随后的低渗肿胀导致的增加较小或无增加。这些结果表明，除蛋白激酶C外，Ca²⁺/钙调蛋白依赖性的星形孢菌素/多粘菌素B敏感蛋白激酶参与了胶质细胞中肿胀诱导的Na⁺,K⁺,2Cl⁻共转运激活。
