Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Thorez pr. 44, 194223 St. Petersburg, Russia.
J Comp Physiol B. 2010 Mar;180(3):385-91. doi: 10.1007/s00360-009-0418-5.
Recently (Agalakova and Gusev in J Comp Physiol 179:443-450, 2009), we demonstrated that the activity of K-Cl cotransport (KCC) in frog red blood cells is inhibited under stimulation of protein kinase C (PKC) with phorbol ester PMA (12-myristate-13-acetate). Present work was performed to uncover possible implication of protein kinases and protein phosphatases (PPs) in the regulation of baseline and volume-dependent KCC activity in these cells. K+ influx was estimated as 86Rb uptake by the cells in isotonic or hypotonic media in the presence of ouabain, K+ efflux was determined as the difference between K+ loss by the cells incubated in parallel in isotonic or hypotonic K(+)-free Cl(-)- and NO(3)(-)-media. Swelling of the cells in hypotonic medium was accompanied by approximately 50% activation of Cl-dependent K+ influx and efflux. Protein tyrosine kinase (PTK) inhibitor genistein (0.1 mM) stably and considerably (up to 89%) suppressed both baseline and volume-dependent KCC activity in each direction. Other PTK blockers (tyrphostin 23 and quercetin) had no influence on KCC activity in frog erythrocytes. PKC inhibitor chelerythrine (20 microM) and both PP inhibitors, fluoride (5 mM) and okadaic acid (1 microM), reduced KCC activity by 25-70%. Neither basal nor swelling-activated KCC in frog erythrocytes was affected by PKC inhibitor staurosporine (1 microM). Based on the previous and present results, we can suggest that the main role in the maintenance of basal and volume-dependent KCC activity in frog erythrocytes belongs to PTKs and PPs, whereas PKC is a negative regulator of this ion system.
最近（Agalakova 和 Gusev 在 J Comp Physiol 179:443-450, 2009 中），我们证明了蛋白激酶 C（PKC）刺激佛波酯 PMA（12-肉豆蔻酸-13-醋酸盐）可抑制蛙红细胞 K-Cl 共转运体（KCC）的活性。本研究旨在揭示蛋白激酶和蛋白磷酸酶（PPs）在调节这些细胞中基础和体积依赖性 KCC 活性中的可能作用。在存在哇巴因的情况下，通过细胞摄取 86Rb 来估计 K+内流，通过在等渗或低渗 K(+)-无 Cl(-)-和 NO(3)(-)-介质中平行孵育的细胞来确定 K+外排，作为细胞 K+损失的差异。在低渗介质中细胞肿胀伴随着大约 50%的 Cl-依赖性 K+内流和外排的激活。蛋白酪氨酸激酶（PTK）抑制剂染料木黄酮（0.1 mM）稳定且显著（高达 89%）抑制了两个方向的基础和体积依赖性 KCC 活性。其他 PTK 阻滞剂（tyrphostin 23 和槲皮素）对蛙红细胞的 KCC 活性没有影响。PKC 抑制剂 Chelerythrine（20 μM）和两种 PP 抑制剂氟化物（5 mM）和 okadaic acid（1 μM）使 KCC 活性降低 25-70%。蛙红细胞的基础和肿胀激活的 KCC 均不受 PKC 抑制剂 staurosporine（1 μM）的影响。基于先前和现在的结果，我们可以假设，PTKs 和 PPs 在维持蛙红细胞的基础和体积依赖性 KCC 活性中起主要作用，而 PKC 是该离子系统的负调节剂。
