Mairbäurl H, Hoffman J F
Department of Cellular and Molecular Physiology, Yale University Medical School, New Haven, Connecticut 06510.
J Gen Physiol. 1992 May;99(5):721-46. doi: 10.1085/jgp.99.5.721.
This study is concerned with the relationship between the Na/K/Cl cotransport system and the steady-state volume (MCV) of red blood cells. Cotransport rate was determined in unfractionated and density-separated red cells of different MCV from different donors to see whether cotransport differences contribute to the difference in the distribution of MCVs. Cotransport, studied in cells at their original MCVs, was determined as the bumetanide (10 microM)-sensitive 22Na efflux in the presence of ouabain (50 microM) after adjusting cellular Na (Nai) and Ki to achieve near maximal transport rates. This condition was chosen to rule out MCV-related differences in Nai and Ki that might contribute to differences in the net chemical driving force for cotransport. We found that in both unfractionated and density-separated red cells the cotransport rate was inversely correlated with MCV. MCV was correlated directly with red cell 2,3-diphosphoglycerate (DPG), whereas total red cell Mg was only slightly elevated in cells with high MCV. Thus intracellular free Mg (Mgifree) is evidently lower in red cells with high 2,3-DPG (i.e., high MCV) and vice versa. Results from flux measurements at their original MCVs, after altering Mgifree with the ionophore A23187, indicated a high Mgi sensitivity of cotransport: depletion of Mgifree inhibited and an elevation of Mgifree increased the cotransport rate. The apparent K0.5 for Mgifree was approximately 0.4 mM. Maximizing Mgifree at optimum Nai and Ki minimized the differences in cotransport rates among the different donors. It is concluded that the relative cotransport rate is regulated for cells in the steady state at their original cell volume, not by the number of copies of the cotransporter but by differences in Mgifree. The interindividual differences in Mgifree, determined primarily by differences in the 2,3-DPG content, are responsible for the differences in the relative cotransport activity that results in an inverse relationship with in vivo differences in MCV. Indirect evidence indicates that the relative cotransport rate, as indexed by Mgifree, is determined by the phosphorylated level of the cotransport system.
本研究关注钠/钾/氯共转运系统与红细胞稳态体积（平均红细胞体积，MCV）之间的关系。在来自不同供体的具有不同MCV的未分级及密度分离的红细胞中测定共转运速率，以观察共转运差异是否导致MCV分布的差异。在细胞处于其原始MCV时研究共转运，通过在哇巴因（50μM）存在下将细胞内钠（Nai）和钾（Ki）调整至接近最大转运速率后，测定布美他尼（10μM）敏感的22Na外流来确定共转运。选择该条件以排除可能导致共转运净化学驱动力差异的Nai和Ki中与MCV相关的差异。我们发现，在未分级及密度分离的红细胞中，共转运速率均与MCV呈负相关。MCV与红细胞2,3 - 二磷酸甘油酸（DPG）直接相关，而在高MCV的细胞中，红细胞总镁含量仅略有升高。因此，在高2,3 - DPG（即高MCV）的红细胞中，细胞内游离镁（Mgifree）明显较低，反之亦然。在用离子载体A23187改变Mgifree后，在其原始MCV下进行通量测量的结果表明共转运对镁离子具有高敏感性：Mgifree的耗尽会抑制共转运，而Mgifree的升高则会增加共转运速率。Mgifree的表观K0.5约为0.4 mM。在最佳Nai和Ki条件下使Mgifree最大化可最小化不同供体之间共转运速率的差异。得出的结论是，在稳态下细胞处于其原始细胞体积时，相对共转运速率的调节不是通过共转运体的拷贝数，而是通过Mgifree的差异。Mgifree的个体间差异主要由2,3 - DPG含量的差异决定，这些差异导致相对共转运活性的差异，从而与体内MCV的差异呈负相关。间接证据表明，以Mgifree为指标的相对共转运速率由共转运系统的磷酸化水平决定。
