Na,K-ATPase expression in C2C12 cells during myogenesis: Minimal contribution of α2 Isoform to Na,K transport

Abstract

Cells of the murine skeletal muscle line, C2C12, undergo differentiation from mononuclear myoblasts to multinuclear myotubes that express a number of proteins associated with striated muscle. We examined the relationship between the abundance of the mRNAs encoding the fast-twitch Ca-ATPase and the alpha isoforms of Na,K-ATPase and the subsequent expression of their respective polypeptides. Both the mRNA and protein levels of the alpha 1 isoform remained constant throughout differentiation. In contrast, the content of mRNAs encoding the alpha 2 isoform and fast-twitch Ca-ATPase increased coordinately with the abundance of their corresponding polypeptides during myotube development. Despite the dramatic increase in alpha 2 expression, estimates of in vitro Na,K-ATPase activity and assessments of in vivo transport activity suggest that alpha 2 contributes little to ionic homeostasis in C2C12 myotubes.

Chronic stimulation of acetylcholine receptors: Differential effects on Na,K-ATPase isoforms in a myogenic cell line

“…Immunoblots prepared in parallel and probed with anti-HERED, a site-directed antibody specific for a2, also displayed 100-kDa bands (Fig. 4B), consistent with the expression of a second catalytic isoform in the differentiated state (Higham et al, 1993;Orlowski and Lingrel, 1988). In contrast to al, which was unchanged, there was a significant (51%) increase in the intensity of the a2 bands in the carbachol-treated samples, relative to control (Table I).…”

“…It is not obvious, for example, why ion concentration-dependent up-regulation would effect preferentially the a 2 isoform. Indeed, exposure to sublethal concentrations of digitalis glycosides (a treatment that also induces up-regulation in many systems) has been shown to produce an increase in abundance of the mRNAs encoding both catalytic isoforms in CzClz myotubes (Higham et al, 1993). Moreover, treatment of myotubes with chlorpromazine, a blocker of the acetylcholine receptor channel, does not prevent the pumpmediated hyperpolarization caused by carbachol (Henning et al, 1993).…”

“…Two isoforms of the catalytic subunit, a1 and a2, are expressed by the murine myogenic cell line, C2CI2-a property shared with intact skeletal muscle (Orlowski and Lingrel, 1988). Although the appearance of a2 is characteristic of differentiation from myoblasts to myotubes, our earlier work indicates that the newly induced isoform contributes little to ionic homeostasis, at least under the basal conditions found in typical cultures (Higham et al, 1993). We speculated that the function of a2 might be more important under physiological conditions in which the cells are stimulated appropriately.…”

“…Lysates of myotubes were prepared, electrophoresed through sodium dodecyl sulfate-polyacrylamide gels, and electroblotted onto nitrocellulose as described (Higham et al, 1993;Pressley, 1992). The quantity of protein in each lane and the efficiency of transfer were confirmed by staining with Ponceau S. The blots were then probed with anti-NASE and anti-HERED, sitedirected rabbit polyclonal antibodies specific for the a1 and a2 isoforms, respectively (Pressley, 1992).…”

“…Growth of the myoblasts on collagen-coated culture plates and induction of myogenesis were as described (Higham et al, 1993). Briefly, myoblasts were grown in Dulbecco's modified Eagle medium (DMEM) containing 20% definedhpplemented bovine calf serum (HyClone Laboratories, Logan, UT).…”