Mobasheri A, Errington R J, Golding S, Hall A C, Urban J P
University Laboratory of Physiology, University of Oxford, U.K.
Cell Biol Int. 1997 Apr;21(4):201-12. doi: 10.1006/cbir.1997.0137.
We have used isoform-specific antibodies against the Na+, K(+)-ATPase alpha (alpha 1, alpha 2 and alpha 3) and beta (beta 1 and beta 2) subunit isoforms in order to establish their specific localization in isolated bovine articular chondrocytes. Immunoblotting confirmed the presence of the alpha 1 and alpha 3 isoforms, although alpha 1 expression was significantly greater than alpha 3 as assessed by immunofluorescence confocal laser scanning microscopy and PCR. A similar approach revealed the presence of the beta 1 and beta 2 isoforms in chondrocytes, although beta 2 immunostaining on the plasma membrane was more punctate than beta 1 which in contrast predominated in a subcellular compartment. The plasma membrane abundance of the Na+, K(+)-ATPase was found to be sensitive to the extracellular ionic concentration and long-term elevation of extracellular Na+ concentration significantly upregulated Na+, K(+)-ATPase density as measured by specific 3H-ouabain binding. Our observations suggest that the expression of alpha 3 and beta 2 is not restricted to excitable tissues as previously reported. The physiological relevance of alpha 3 expression in chondrocytes may be related to its low affinity for intracellular Na+ in an extracellular environment where Na+ concentration is unusually high (260-350 mM) compared to other cell types (140 mM). Glycoproteins and their branched carbohydrates have been implicated in cell recognition events, thus the beta 2 subunit glycoprotein may allow the chondrocyte to detect changes in its extracellular environment by physically interacting with components of the cellular cytoskeleton and matrix macromolecules.
我们使用了针对Na⁺,K⁺-ATP酶α（α1、α2和α3）和β（β1和β2）亚基异构体的亚型特异性抗体，以确定它们在分离的牛关节软骨细胞中的特定定位。免疫印迹证实了α1和α3异构体的存在，尽管通过免疫荧光共聚焦激光扫描显微镜和PCR评估，α1的表达明显高于α3。类似的方法揭示了软骨细胞中β1和β2异构体的存在，尽管质膜上β2的免疫染色比β1更呈点状，相比之下，β1在亚细胞区室中占主导地位。发现Na⁺,K⁺-ATP酶的质膜丰度对细胞外离子浓度敏感，通过特异性³H-哇巴因结合测量，细胞外Na⁺浓度的长期升高显著上调了Na⁺,K⁺-ATP酶密度。我们的观察结果表明，α3和β2的表达并不像先前报道的那样局限于可兴奋组织。软骨细胞中α3表达的生理相关性可能与其在细胞外环境中对细胞内Na⁺的低亲和力有关，与其他细胞类型（140 mM）相比，该细胞外环境中Na⁺浓度异常高（260 - 350 mM）。糖蛋白及其分支碳水化合物与细胞识别事件有关，因此β2亚基糖蛋白可能通过与细胞骨架和基质大分子的成分进行物理相互作用，使软骨细胞能够检测其细胞外环境的变化。
