CHARACTERIZATION OF THE Na+K+-ATPase IN ISOLATED BOVINE ARTICULAR CHONDROCYTES; MOLECULAR EVIDENCE FOR MULTIPLE α AND β ISOFORMS

Abstract

We have used isoform-specific antibodies against the Na+K+-ATPase αα1, α2 and α3) and ββ1 and β2) subunit isoforms in order to establish their specific localization in isolated bovine articular chondrocytes. Immunoblotting confirmed the presence of the α1 and α3 isoforms, although α1 expression was significantly greater than α3 as assessed by immunofluorescence confocal laser scanning microscopy and PCR. A similar approach revealed the presence of the β1 and β2 isoforms in chondrocytes, although β2 immunostaining on the plasma membrane was more punctate than β1 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 specific3H-ouabain binding. Our observations suggest that the expression of α3 and β2 is not restricted to excitable tissues as previously reported. The physiological relevance of α3 expression in chondrocytes may be related to its low affinity for intracellular Na+in an extracellular environment where Na+concentration is unusually high (260–350mm) compared to other cell types (140mm). Glycoproteins and their branched carbohydrates have been implicated in cell recognition events, thus the β2 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.