Regulation of contraction-stimulated system a amino acid uptake in skeletal muscle: Role of vicinal sulfhydryls

Abstract

Functional vicinal sulfhydryls are essential for insulin-stimulated system A neutral amino acid uptake in the rat epitrochlearis muscle. In skeletal muscle, system A uptake is also activated by contractile activity. Therefore, the purposes of this study were to characterize the stimulation of system A activity by contractions induced by electrical stimulation in vitro, and to assess the role of vicinal sulfhydryls in this process. System A activity in the isolated epitrochlearis muscle was measured using the nonmetabolizable analogue α-(methylamino)isobutyric acid (MeAIB). Contractions increased MeAIB uptake by increasing the apparent maximal velocity (V max), with no alteration in the apparent K m . The maximal stimulatory effects of insulin and contractions on MeAIB uptake were completely additive, demonstrating that these two stimuli exert their effects via different mechanisms. Phenylarsine oxide (PAO), a vicinal sulfhydryl antagonist, at greater than 20 μmol/L inhibited basal and contraction-stimulated MeAIB uptake by approximately 50% and 70%, respectively, by decreasing V max, with no change in K m . Both inhibitory effects were completely prevented by cotreatment with the vicinal dithiol dimercaptopropanol (DMP), indicating the effects were mediated by interactions with vicinal sulfhydryls. Contraction-stimulated MeAIB uptake was rapidly (half-time, ∼7 minutes) reversed by the addition of PAO. These results (1) define conditions under which contraction-stimulated system A amino acid uptake can be studied in an isolated mammalian skeletal muscle preparation, and (2) indicate that vicinal sulfhydryls are essential for stimulation of system A activity by muscle contractions.