Sharabani-Yosef O, Bak A, Langzam L, Lui Z, Nir U, Braiman L, Sweadner K J, Sampson S R
Otto Meyerhoff Center and Health Sciences Research Center, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
J Cell Physiol. 1999 Aug;180(2):236-44. doi: 10.1002/(SICI)1097-4652(199908)180:2<236::AID-JCP11>3.0.CO;2-W.
Studies from this laboratory have shown that the physiological expression of the Na+/K+ pump in primary cultures of rat skeletal muscle increases with development. The molecular mechanisms underlying these changes are not known. Therefore, we have examined the expression of alpha and beta subunits of the Na+/K+ pump at both the protein and mRNA levels during myogenesis of primary skeletal muscle cell cultures obtained from newborn rats. Protein isoforms were identified by Western blotting techniques with specific monoclonal and polyclonal antibodies and subunit mRNA was studied with specific cDNA probes. Freshly isolated skeletal muscle from newborn rats expressed both alpha1 and alpha2 protein subunits. From day 1 after plating, primary cultures expressed only the alpha1 protein isoform. In contrast, both beta1 and beta2 isoforms were expressed in freshly isolated muscle and in primary cultures, with beta1 expression being stronger in both preparations. Studies on RNA expression showed that mRNA for alpha1, alpha2, beta1, and beta2 isoforms was identified both in freshly isolated muscle and after plating of cells in culture. These findings indicate that the lack of alpha2 protein expression in primary muscle cell cultures reflects a form of posttranscriptional regulation. There did not appear to be a quantitative difference in isoform expression as a function of age or of fusion in spite of developmental increases in Na+/K+ pump activity and its dependence on cell fusion. The lack of expression of the alpha2 subunit isoform suggests that the developmental changes in physiological expression of the Na+/K+ pump in primary cultures of skeletal muscle may be attributable either to the changes in activity of the alpha1 subunit or to differential activities of alphabeta complexes involving either of the beta subunits.
本实验室的研究表明，大鼠骨骼肌原代培养物中Na+/K+泵的生理表达随发育而增加。这些变化背后的分子机制尚不清楚。因此，我们检测了从新生大鼠获得的原代骨骼肌细胞培养物在成肌过程中，Na+/K+泵α和β亚基在蛋白质和mRNA水平的表达。通过使用特异性单克隆和多克隆抗体的蛋白质印迹技术鉴定蛋白质异构体，并用特异性cDNA探针研究亚基mRNA。新生大鼠新鲜分离的骨骼肌表达α1和α2蛋白质亚基。接种后第1天，原代培养物仅表达α1蛋白质异构体。相比之下，β1和β2异构体在新鲜分离的肌肉和原代培养物中均有表达，且在两种制剂中β1的表达更强。RNA表达研究表明，α1、α2、β1和β2异构体的mRNA在新鲜分离的肌肉和细胞接种培养后均能被鉴定出来。这些发现表明，原代肌肉细胞培养物中α2蛋白表达的缺乏反映了一种转录后调控形式。尽管Na+/K+泵活性及其对细胞融合的依赖性随发育增加，但异构体表达似乎没有随年龄或融合而出现定量差异。α2亚基异构体表达的缺乏表明，骨骼肌原代培养物中Na+/K+泵生理表达的发育变化可能归因于α1亚基活性的变化，或涉及任一β亚基的αβ复合物的不同活性。
