AMP-activated protein kinase regulates L-arginine mediated cellular responses

BACKGROUND: Our prior study revealed the loss in short-term L-Arginine (ARG) therapeutic efficacy after continuous exposure; resulting in tolerance development, mediated by endothelial nitric oxide synthase (eNOS) down-regulation, secondary to oxidative stress and induced glucose accumulation. However, the potential factor regulating ARG cellular response is presently unknown. METHOD: Human umbilical vein endothelial cells were incubated with 100 μM ARG for 2 h in buffer (short-term or acute), or for 7 days in culture medium and challenged for 2 h in buffer (continuous or chronic), in the presence or absence of other agents. eNOS activity was determined by analyzing cellular nitrite/nitrate (NO(2)(–)/NO(3)(–)), and AMP-activated protein kinase (AMPK) activity was assayed using SAMS peptide. (13)C(6) glucose was added to medium to measure glucose uptake during cellular treatments, which were determined by LC-MS/MS. Cellular glucose was identified by o-toluidine method. Superoxide (O(2)(•–)) was identified by EPR-spin-trap, and peroxynitrite (ONOO(–)) was measured by flow-cytometer using aminophenyl fluorescein dye. RESULTS: Short-term incubation of cells with 100 μM ARG in the presence or absence of 30 μM L-N(G)-Nitroarginine methyl ester (L-NAME) or 30 μM AMPK inhibitor (compound C, CMP-C) increased cellular oxidative stress and overall glucose accumulation with no variation in glucose transporter-1 (GLUT-1), or AMPK activity from control. The increase in total NO(2)(–)/NO(3)(–) after 2 h 100 μM ARG exposure, was suppressed in cells co-incubated with 30 μM CMP-C or L-NAME. Long-term exposure of ARG with or without CMP-C or L-NAME suppressed NO(2)(–)/NO(3)(–), glucose uptake, GLUT-1, AMPK expression and activity below control, and increased overall cellular glucose, O(2)(•–) and ONOO(–). Gluconeogenesis inhibition with 30 μM 5-Chloro-2-N-2,5-dichlorobenzenesulfonamido-benzoxazole (CDB) during ARG exposure for 2 h maintained overall cellular glucose to control, but increased cellular glucose uptake. Continuous co-incubation with CDB and ARG increased NO(2)(–)/NO(3)(–), glucose uptake, GLUT-1, AMPK expression and activity, and maintained overall cellular glucose, O(2)(•–) and ONOO(–) to control conditions. CONCLUSION: The present study provides the fundamental evidence for AMPK as the primary modulator of ARG cellular responses and for regulating the mode of glucose accumulation during short-term and continuous ARG treatments.