The role of vicinal sulfhydryls in the stimulation by insulin of system A amino acid uptake in mammalian skeletal muscle was investigated. Neutral amino acid uptake via system A carriers was assessed using the nonmetabolizable analogue alpha-(methylamino)isobutyric acid (MeAIB). Phenylarsine oxide (PAO), a trivalent arsenical that interacts with vicinal sulfhydryls, at 40 microM inhibited basal and insulin-stimulated (2 mU/ml) MeAIB uptake in rat epitrochlearis muscles by approximately 50% and approximately 80%, respectively. No significant changes in the ATP level or in the lactate-to-pyruvate ratio were observed. Both inhibitory effects were completely preventable by coincubation with dimercaptopropanol, a vicinal dithiol, indicating the effects were mediated specifically by interactions with vicinal sulfhydryls. Stimulation of MeAIB uptake by the insulin-mimicker vanadate (10 mM) or by insulin-like growth factor I (IGF-I, 20 nM) was also inhibited by 80-90% by PAO. Kinetic analysis showed that PAO decreased the apparent Vmax for basal and insulin-stimulated MeAIB uptake without altering the apparent Km. MeAIB uptake already maximally stimulated by insulin was rapidly (half-time = approximately 10 min) reversed by the addition of PAO so that the rate of MeAIB uptake was the same as in muscles incubated throughout with insulin and PAO. These results implicate a major role for vicinal sulfhydryls in the stimulation by insulin of amino acid uptake via system A carriers in skeletal muscle and suggest that the site of action of PAO on this system is distal to the insulin receptor, possibly at the carrier molecule itself.
