Selective induction of high-ouabain-affinity isoform of Na⁺-K⁺-ATPase by thyroid hormone

The administration of thyroid hormone is known to result in an induction of the Na⁺-K⁺-adenosinetriphosphatase (Na⁺-K⁺-ATPase) in rat skeletal muscle and other thyroid hormone-responsive tissue. Since the Na⁺-K⁺-ATPase in a variety of mammalian tissues has recently been reported to exist in at least two forms distinguishable by differing affinities for the inhibitory cardiac glycoside ouabain, we have studied the effects of 3,3',5-triiodo-L-thyronine (T₃) treatment on these two forms of the enzyme in rat diaphragm. The inhibition of Na⁺-K⁺-ATPase activity in a crude membrane fraction by varying concentrations of ouabain conformed to a biphasic pattern consistent with the presence of two distinct isoforms with inhibition constants (K₁S) for ouabain of ~10^-7 and 10^-4 M, respectively. Treatment of hypothyroid rats with T₃ (50 μg/100 g body wt on 3 alternate days) nearly tripled that portion of the Na⁺-K⁺-ATPase activity corresponding to the high-ouabain-affinity form (increased by 178 ± 24%), whereas the enzyme activity corresponding to the low-ouabain-affinity form was only slightly changed (increased by 20 ± 5%). Measurement of the specific binding of [³H]ouabain to these membranes confirmed the presence of a class of high-affinity ouabain binding sites with a dissociation constant (K(d)) of slightly less than 10^-7 M, whose maximal binding capacity was increased by T₃ treatment by 185%. The calculated catalytic turnover associated with the high-affinity site was 70-80 molecules ATP hydrolyzed·site^-1·s^-1 and was unchanged by T₃ treatment. Binding studies in unfractionated homogenates of diaphragm similarly demonstrated the presence of high-affinity sites whose maximal binding capacity was comparably increased by T₃ treatment. Quantitation of both the high- and low-ouabain-affinity forms of the Na⁺-K⁺-ATPase by ouabain-dependent phosphorylation from [³²P]orthophosphate confirmed that T₃ treatment markedly increased the number of high-affinity sites while having little effect on the number of low-affinity sites. T₃ thus preferentially induces the form of the Na⁺-K⁺-ATPase having a high affinity for ouabain in rat skeletal muscle, and in contrast has at most only a small effect on the level of the low-affinity form of the enzyme. These observations provide, to our knowledge, the first demonstration that these two forms of the Na⁺-K⁺-ATPase are subject to selective hormonal induction.