Skeletal muscle atrophy is a common phenomenon during the prolonged muscle disuse caused by cast immobilization, extended aging states, bed rest, space flight, or other factors. However, the cellular mechanisms of the atrophic process are poorly understood. In this study, we investigated the involvement of mitogen-activated protein kinase (MAPK) in the expression of muscle-specific RING finger 1 (MuRF1) during atrophy of the rat gastrocnemius muscle. Histological analysis revealed that cast immobilization induced the atrophy of the gastrocnemius muscle, with diminution of muscle weight and cross-sectional area after 14 days. Cast immobilization significantly elevated the expression of MuRF1 and the phosphorylation of p38 MAPK. The starvation of L6 rat skeletal myoblasts under serum-free conditions induced the phosphorylation of p38 MAPK and the characteristics typical of cast-immobilized gastrocnemius muscle. The expression of MuRF1 was also elevated in serum-starved L6 myoblasts, but was significantly attenuated by SB203580, an inhibitor of p38 MAPK. Changes in the sizes of L6 myoblasts in response to starvation were also reversed by their transfection with MuRF1 small interfering RNA or treatment with SB203580. From these results, we suggest that the expression of MuRF1 in cast-immobilized atrophy is regulated by p38 MAPK in rat gastrocnemius muscles.
We used 2-DE and MALDI-TOF/TOF to identify proteins of vascular smooth muscle cells whose expression was or was not altered by exposure to 500 microM H2O2 for 30 min. We detected more than 800 proteins on silver-stained gels of whole protein extracts from rat aortic smooth muscle strips. Of these proteins, 135 clearly unaffected and 19 having levels altered by exposure to H2O2 were identified. Protein characterization revealed that the most prominent vascular smooth muscle proteins were those with antioxidant, cytoskeletal structure, or muscle contraction. In addition, cofilin, an isoform of the actin depolymerizing factor family, shifted to its basic site on the 2-DE gel as a result of H2O2 treatment. In Western blot analysis of proteins from A7r5 aortic smooth muscle cells, the phosphorylation, but not the expression, of cofilin was decreased by H2O2 in a dose-dependent manner. The H2O2-induced dephosphorylation of cofilin and apoptosis was inhibited by Na3VO4, an inhibitor of protein tyrosine phosphatase (PTP). These results suggest that cofilin is one of the proteins regulated by H2O2 treatment in vascular smooth muscle, and has an important role in the induction of vascular apoptosis through PTP-dependent mechanisms.
Abstract. We determined the action mechanism of cordycepin, a major bioactive component of Cordyceps militaris, on responses of rat aortic smooth muscle cells (RASMCs) and on vascular disorders, especially neointimal formation. Cordycepin inhibited platelet-derived growth factor-BB (PDGF-BB)-induced RASMCs migration and proliferation in a dose-dependent manner. However, pre-treatment with N ω -nitro-L-arginine methyl ester, a nitric oxide synthase (NOS) inhibitor, and 1,3-dipropyl-8-sulphophenylxanthine (DPSPX), an A 1 / A 2 adenosinereceptor antagonist, abolished the inhibitory role of cordycepin. Cordycepin suppressed the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and heat shock protein 27 (Hsp27), but not that of extracellular signal-regulated kinase (ERK) 1/2 in RASMCs stimulated by PDGF-BB. The production of reactive oxygen species (ROS), O 2 − and H 2 O 2 , induced by PDGF-BB was abolished by the treatment of cordycepin. Moreover, the sprout outgrowth of aortic rings by PDGF-BB was inhibited by cordycepin. In vivo neointimal formation evoked by balloon-injury was significantly attenuated by the administration of cordycepin. These results demonstrate that cordycepin may exert inhibitory effects on PDGF-BB-induced migration and proliferation via interfering with adenosine receptor-mediated NOS pathways, thus resulting in the attenuation of neointima formation. In conclusion, cordycepin may be a potent, promising anti-atherosclerosis agent.
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