Na+-K+-ATPase in rat skeletal muscle: content, isoform, and activity characteristics

Abstract

The purpose of this study was to investigate the hypothesis that muscle Na+-K+-ATPase activity is directly related to Na+-K+-ATPase content and the content of the α2-catalytic isoform in muscles of different fiber-type composition. To investigate this hypothesis, tissue was sampled from soleus (Sol), red gastrocnemius (RG), white gastrocnemius (WG), and extensor digitorum longus (EDL) muscles at rest from 38 male Wistar rats weighing 413 ± 6.0 g (mean ± SE). Na+-K+-ATPase activity was determined in homogenates (Hom) and isolated crude membranes (CM) by the regenerating ouabain-inhibitable hydrolytic activity assay (ATPase) and the 3- O-methylfluorescein K+-stimulated phosphatase (3- O-MFPase) assay in vitro. In addition, Na+-K+-ATPase content (Bmax) and the distribution of α1-, α2-, β1-, and β2-isoforms were determined by [3H]ouabain binding and Western blot, respectively. For the ATPase assay, differences ( P < 0.05) in enzyme activity between muscles were observed in Hom (EDL > WG) and in CM (Sol > EDL = WG). For the 3- O-MFPase assay, differences ( P < 0.05) were also found for Hom (Sol > RG = EDL > WG) and CM (Sol = WG > RG). For Bmax, differences in the order of RG = EDL > Sol = WG ( P < 0.05) were observed. Isoform distribution was similar between Hom and CM and indicated in CM, a greater density ( P < 0.05) of α1in Sol than WG and EDL ( P < 0.05), but more equal distribution of α2between muscles. The β1was greater ( P < 0.05) in Sol and RG, and the β2was greater in EDL and WG ( P < 0.05). Over all muscles, the correlation ( r) between Hom 3- O-MFPase and Bmaxwas 0.45 ( P < 0.05) and between Hom α2and Bmax, 0.59 ( P < 0.05). The α1distribution correlated to Hom 3- O-MFPase ( r = 0.79, P < 0.05) CM ATPase ( r = 0.69, P < 0.005) and CM 3- O-MFPase activity ( r = 0.32, P < 0.05). The α2distribution was not correlated with any of the Na+-K+-ATPase activity measurements. The results indicate generally poor relationships between activity and total pump content and α2isoform content of the Na+-K+-ATPase. Several factors, including the type of preparation and the type of assay, appear important in this regard.