Orlov S N, Kuznetsov S R, Kolosova I A, Aksentsev S L, Konev S V
Laboratory of Physical Chemistry of Biomembranes, Faculty of Biology, Lomonosov State University, Vorobyovy Gory, Moscow, 119899, Russia.
Ross Fiziol Zh Im I M Sechenova. 1997 May-Jun;83(5-6):119-47.
This study examines the effect of heat-induced cytoskeleton transitions and phosphoprotein phosphatase inhibitors on the activity of shrinkage-induced Na+, K+, 2Cl- cotransport and Na+/H+ exchange in rat erythrocytes and swelling-induced K+, Cl- cotransport in human and rat blood cells. Preincubation of human and rat erythrocytes at 49 degrees C drastically activated K+, Cl- cotransport and completely (rat) or partly (human) abolished its volume-dependent regulation. The same procedure did not affect basal activity of Na+, K+, 2Cl- cotransport but completely abolished its activation by shrinkage thus suggesting the involvement of a thermosensitive element of cytoskeleton network in the volume-dependent regulation of cotransporters. Both the shrinkage- and electrochemical proton gradient-induced Na+/H+ exchange was inhibited by the heat treatment to the same extent (50-70%), thus indicating the different signaling pathways involved in the activation of Na+, K+, 2Cl- cotransport and Na+/H+ exchange by cell shrinkage. This suggestion is in accordance with data on the different kinetics of volume-dependent activation and inactivation of these carriers as well as on their sensitivity to medium osmolality. Both swelling- and heat-induced increments of K+, Cl- cotransport activity were diminished by inhibitors of phosphoprotein phosphatases (okadaic acid and calyculin). In rat erythrocytes these compounds potentiate shrinkage-induced Na+/H+ exchange. On the contrary, neither basal nor shrinkage-induced Na+, K+, 2Cl- cotransport was affected by these compounds. Our results indicate a key role of cytoskeleton network in volume-dependent activation of K+, Cl- and Na+, K+, 2Cl- cotransport and the involvement of protein phosphorylation-dephosphorylation cycle in regulation of the activity of K+, Cl- cotransport and Na+/H+ exchange.
本研究考察了热诱导的细胞骨架转变和磷蛋白磷酸酶抑制剂对大鼠红细胞中收缩诱导的Na⁺、K⁺、2Cl⁻协同转运及Na⁺/H⁺交换活性，以及人和大鼠血细胞中肿胀诱导的K⁺、Cl⁻协同转运的影响。人和大鼠红细胞在49℃预孵育可显著激活K⁺、Cl⁻协同转运，并完全（大鼠）或部分（人）消除其对体积的依赖性调节。相同处理不影响Na⁺、K⁺、2Cl⁻协同转运的基础活性，但完全消除其由收缩引起的激活，这表明细胞骨架网络的热敏元件参与了协同转运体对体积的依赖性调节。热处理对收缩和电化学质子梯度诱导的Na⁺/H⁺交换的抑制程度相同（50 - 70%），这表明细胞收缩激活Na⁺、K⁺、2Cl⁻协同转运和Na⁺/H⁺交换涉及不同的信号通路。这一推测与这些载体对体积依赖性激活和失活的不同动力学以及它们对介质渗透压的敏感性的数据一致。磷蛋白磷酸酶抑制剂（冈田酸和花萼海绵诱癌素）可减弱肿胀和热诱导的K⁺、Cl⁻协同转运活性增加。在大鼠红细胞中，这些化合物增强收缩诱导的Na⁺/H⁺交换。相反，这些化合物对基础的或收缩诱导的Na⁺、K⁺、2Cl⁻协同转运均无影响。我们的结果表明细胞骨架网络在K⁺、Cl⁻和Na⁺、K⁺、2Cl⁻协同转运对体积的依赖性激活中起关键作用，并且蛋白磷酸化 - 去磷酸化循环参与K⁺、Cl⁻协同转运和Na⁺/H⁺交换活性的调节。
