Rat skeletal muscle in culture expresses the ?1 but not the ?2 protein subunit isoform of the Na+/K+ pump

Abstract

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.

Accumulation of βm, a structural member of X,K-ATPase β-subunit family, in nuclear envelopes of perinatal myocytes

“…Obviously, ␤ m expression is induced by signals that occur after primary myogenesis. Regulation of some other X,K-ATPase subunits is also not identical in vitro and in vivo (46).…”

Oleandrin-mediated inhibition of human tumor cell proliferation: Importance of Na,K-ATPase α subunits as drug targets

“…The biological expression patterns of the various Na,KATPase isoforms are subject to developmental and hormonal cues that become altered either during in vitro culturing or during the development of disease (37)(38)(39)(40)(41)(42). Arystarkhova and Sweadner reported that in rat heart, there was a postnatal switch in the expression of Na,K-ATPase isoforms.…”

“…It seems that the expression of Na,K-ATPase isoforms is posttranscriptionally regulated. The freshly isolated skeletal muscle from newborn rats expresses both α1 and α2, whereas primary cells from this tissue 1 day after culturing express only the α1 isoform (38). In consideration of the fact that expression of Na,K-ATPase may be regulated by hormone or other growth factors, we have tested the expression of α3 in the tumor tissues derived from Panc-1 tumor-bearing mice.…”

Stabilization of the α2 Isoform of Na,K-ATPase by Mutations in a Phospholipid Binding Pocket

“…10), a number of other observations in the literature indicate rapid turnover and probably instability of ␣2 in physiological and pathophysiological situations. For example, rat skeletal muscle cells, in culture, express ␣1 (and ␤1 and ␤2) but not ␣2, whereas the native muscle expresses both ␣1 and ␣2 (and ␤1 and ␤2) (59). Similarly, it has been found that in skeletal muscles of K ϩ -deprived rats, ␣2 and ␤2 proteins, but not ␣1 and ␤1, are strongly suppressed (60).…”