In hypothyroid rat myocardium, the low-ouabain-sensitivity Na,K-ATPase activity had a KI = 10(-4) M and accounted for approximately 95% of the enzyme activity, while the high-ouabain-sensitivity activity contributed approximately 5% to the total activity, with a KI = 3 x 10(-7) M. mRNA alpha 1 was 7.2- and 5.5-fold more abundant than mRNA alpha 2 and mRNA beta, respectively, in hypothyroid ventricles while mRNA alpha 3 was undetectable. Administration of T3 increased total Na,K-ATPase activity 1.6-fold; the low-ouabain-sensitivity activity increased 1.5-fold while high-ouabain-sensitivity activity was stimulated 3.2-fold. T3 increased the number of high-affinity ouabain-binding sites 2.9-fold with no change in Kd (approximately 2 x 10(-7) M). The abundances of mRNA alpha 1, mRNA alpha 2, and mRNA beta (per unit RNA) following T3 treatment increased 3.6-, 10.6-, and 12.7-fold, respectively. The larger increments in subunit mRNA abundances than in Na,K-ATPase activity suggests the involvement of translational and/or post-translational regulatory steps in Na,K-ATPase biogenesis in response to T3. It is concluded that T3 enhances myocardial Na,K-ATPase subunit mRNA abundances and Na,K-ATPase activity, and that the expression of the high- and low-ouabain-sensitivity activities are probably a reflection of the abundances of the alpha 2 and alpha 1 isoforms, respectively. The physiological role played by the beta subunit remains uncertain.

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.

Regulation of Na,K-ATPase mRNA alpha isoform and mRNA beta expression by thyroid hormone (T3) in neonatal rat myocardium was examined. In euthyroid neonates between ages of 2 and 5 days, mRNA alpha 1, mRNA alpha 3, and mRNA beta 1 abundances were nearly constant while mRNA alpha 2 was undetectable. During the interval between postnatal days 5 and 15, mRNA alpha 3 decreased to negligible levels and mRNA alpha 2 became expressed and increased in abundance to account for approximately 20% of the mRNA alpha pool by the 15th postnatal day. To examine the effect of T3 on this developmental program, neonates were injected with 75 micrograms T3/100 g body weight or diluent alone on the second and third postnatal days and myocardial Na,K-ATPase subunit-mRNA abundances were determined on the third and fourth postnatal days. Because T3 treatment increased the RNA/DNA ratios of myocardial tissue, the subunit-mRNA abundances were normalized per unit DNA. Following 24 and 48 hr of T3 treatment, the abundances of mRNA alpha 1, mRNA alpha 3, and mRNA beta 1 increased, while mRNA alpha 2 continued to remain undetectable during the 2-day interval between the second to fourth postnatal days. It is concluded that T3 augments the abundance of Na,K-ATPase subunit mRNAs that are already being expressed in the neonatal rat myocardium. The results further suggest that T3 does not act as a "molecular switch" in the developmental expression of the mRNA alpha isoforms in rat myocardium during the first four postnatal days.