Characterization of the Na+, K(+)-ATPase in isolated bovine articular chondrocytes; molecular evidence for multiple alpha and beta isoforms.
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.
Mobasheri
A
A
University Laboratory of Physiology, University of Oxford, U.K.
Errington
R J
RJ
Golding
S
S
Hall
A C
AC
Urban
J P
JP
D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000201:enzymology*; D002417:Cattle; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D007527:Isoenzymes; D000254:Sodium-Potassium-Exchanging ATPase