Characterization of the skeletal muscle sodium ion-potassium ion-ATPase, and the acute effects of exercise on sodium ion-potassium ion-ATPase activity

Abstract

The purpose of this thesis was to characterize the Na^+-K^+-ATPase (Na,K-pump) in skeletal muscle and to measure pump activity in response to exercise. To address these objectives, Na^+-K^+-ATPase activity, content and isoform distribution were measured in muscles of various fibre types. Subsequently, Na^+-K^+-ATPase was measured following acute exercise in both rat and human. The hypothesis was that muscles of varying fibre type composition would exhibit differences in Na^+-K^+-ATPase content and isoform distributino, and these factors would indicate muscle specific differences in Na^+-K^+-ATPase activity when measured in vitro. An additional hypothesis was that the Na^+-K^+-ATPase would be altered by exercise; to result in reduced Na^+-K^+-ATPase activity measured in vivo and in vitro.

In the first study, a comparative approach assessed pump characteristics between soleus (SOL), red gastrocnemius (RG), white gastrocnemius (WG), and extensor digitorum longus (EDL) skeletal muscles of the rat. These muscles are representative of primarily slow-oxidative type I fibres (SOL), fast oxidative-glycolytic type IIA fibres (RG), fast glycolytic type IIB fibres (WG), and muscle having a mixed complement of fast fire types (EDL). Tissue was sampled at rest under anaesthetic, from a total of 38 male, 16 week old Wistar rats weighing 413 +_ 6.0 g (mean +_ SE). Na^+-K^+-ATPase activity was determined in homogenates (HOM) and isolated crude membranes (CM) for the regenerating ouabain-inhabitable hydrolytic activity assay (ATPase_ and the 3-)-methylfluorescein K^+-stimulated phosphatase (3-O-MFPase) assays in vitro. In addition, pump content was determined by [^3H]ouabain binding, and the distribution of a1, a2, a3, and B1, and B2 isoforms was determined by Western blot. Differences (P<0.05) in enzyme activity between muscles were observed in HOM (EDL>WG) and in CM (SOL>EDL=RG=WG) for the ATPase assay. For the 3-O-MFPase assay, differences (P<0.05) were also found for HOM (SOL>EDL=RG>WG) and CM (SOL=WG>RG). To determine if activity was related to pump content, differences in [^3H]ouabain maximum binding (Bmax) were observed in the order of EDL = RG > SOL = WG (P<0.05). Western blot results were similar between HOM and CM. In CM, a there was greater distribution of a1 in SOL than WG and EDL (P<0.05) with a similar relation observed for a2. The B1 was greater (P<0.05) in EDL and WG, and the B2 was greater in SOL and RG (P<0.05). The presence of the a3 isoform, not previously observed in skeletal muscle, was identified in greater proportion in WG and EDL than SOL and RG (P<0.05). The a1 distribution correlated to HOM 3-O-MFPase (r=0.79, P<0.05) CM ATPase (r=0.59, P<0.005) and CM 3-O-MFPase activity (r=0.33, P<0.05). B1 distribution was related to HOM 3-O-MFPase (r=0.61, P<0.005) and CM ATPase (r=0.42, P<0.05) activity. HOM a2 distribution correlated to ouabain binding Bmax (r=0.64, P<0.005).