Effect of citrulline on muscle protein turnover in an in vitro model of muscle catabolism.

OBJECTIVE: Muscle net catabolism, as seen after severe trauma or sepsis or in postoperative situations, is mediated by hormones (e.g., cortisol) and proinflammatory cytokines (e.g., tumor necrosis factor alpha [TNF-α]). Specific amino acids may be able to limit this muscle mass loss. Citrulline (CIT) stimulates muscle protein synthesis in various situations, but little data exist on hypercatabolic situations and the effects on protein breakdown are unknown. Our aim was to assess the effect of CIT on protein turnover in an in vitro model of muscle hypercatabolism. METHODS: Myotubes derived from C2C12 myoblasts were treated with 150 nM dexamethasone (DEX), 10 ng/mL TNF-α, or 0.006% ethanol (as control [CON]) for 24 h. Subsequently, myotubes were incubated with or without 5 mM CIT for 6 h. Muscle protein synthesis rate was evaluated by the surface sensing of translation method and by l-[3,5-3H]tyrosine (Tyr) incorporation. The muscle protein breakdown rate was evaluated from Tyr release into culture medium. CIT action was analyzed by non-parametric Kruskal-Wallis and Mann-Whitney tests. RESULTS: CIT treatment significantly increased protein synthesis rates compared with the DEX or TNF-α group (surface sensing of translation method; DEX + CIT versus DEX; P = 0.03 and TNF-α+CIT versus TNF-α; P = 0.05) and significantly decreased protein breakdown rate in the CON and DEX groups (CON + CIT versus CON; P = 0.05 and DEX + CIT versus DEX; P = 0.05). CONCLUSIONS: CIT treatment regulated muscle protein turnover in an in vitro model of muscle net catabolism. Exploring the underlying mechanisms would also be of interest.

Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice.

Molecules that reduce the level of cyclic adenosine 5'-monophosphate (cAMP) in the platelet cytosol, such as adenosine 5'-diphosphate (ADP) secreted from dense granules, trigger platelet activation. Therefore, any change in the distribution and/or availability of cyclic nucleotides or ADP may interfere with platelet reactivity. In this study, we evaluated the role of multidrug resistance protein 4 (MRP4, or ABCC4), a nucleotide transporter, in platelet functions in vivo and in vitro by investigating MRP4-deficient mice. MRP4 deletion resulted in a slight increase in platelet count but had no impact on platelet ultrastructure. In MRP4-deficient mice, the arterial occlusion was delayed and the tail bleeding time was prolonged. In a model of platelet depletion and transfusion mimicking a platelet-specific knockout, mice injected with MRP4(-/-) platelets also showed a significant increase in blood loss compared with mice injected with wild-type platelets. Defective thrombus formation and platelet activation were confirmed in vitro by studying platelet adhesion to collagen in flow conditions, integrin αIIbß3 activation, washed platelet secretion, and aggregation induced by low concentrations of proteinase-activated receptor 4-activating peptide, U46619, or ADP. We found no role of MRP4 in ADP dense-granule storage, but MRP4 redistributed cAMP from the cytosol to dense granules, as confirmed by increased vasodilator-stimulated phosphoprotein phosphorylation in MRP4-deficient platelets. These data suggest that MRP4 promotes platelet aggregation by modulating the cAMP-protein kinase A signaling pathway, suggesting that MRP4 might serve as a target for novel antiplatelet agents.