In adipocytes, insulin stimulates glucose transport primarily by promoting the translocation of GLUT4 to the plasma membrane. Requirements for Ca2+/ calmodulin during insulin-stimulated GLUT4 translocation have been demonstrated; however, the mechanism of action of Ca2+ in this process is unknown. Recently, myosin II, whose function in non-muscle cells is primarily regulated by phosphorylation of its regulatory light chain by the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), was implicated in insulin-stimulated GLUT4 translocation. The present studies in 3T3- F442A adipocytes demonstrate the novel finding that insulin significantly increases phosphorylation of the myosin II RLC in a Ca2+-dependent manner. In addition, ML-7, a selective inhibitor of MLCK, as well as inhibitors of myosin II, such as blebbistatin and 2,3-butanedione monoxime, block insulin- stimulated GLUT4 translocation and subsequent glucose transport. Our studies suggest that MLCK may be a regulatory target of Ca2+/calmodulin and may play an important role in insulin-stimulated glucose transport in adipocytes.
Protein Transport/drug effects ; Phosphorylation ; Naphthalenes/pharmacology ; Myosin-Light-Chain Kinase/antagonists & inhibitors/*metabolism ; Myosin Type II/*metabolism ; Mice ; Insulin/*pharmacology ; Glucose Transporter Type 4/*metabolism ; Enzyme Inhibitors/pharmacology ; Dose-Response Relationship, Drug ; Calmodulin/antagonists & inhibitors/physiology ; Azepines/pharmacology ; Animals ; Adipocytes/cytology/*drug effects/metabolism ; 3T3 Cells

OBJECTIVETo determine the role of asymmetric dimethylarginine (ADMA) in acute lung injury induced by cerebral ischemia/reperfusion (I/R) injury in rats.

METHODSAdult male SD rats were randomly divided into 4 groups, namely the sham-operated group (S), cerebral I/R model group, ADMA+I/R group, and dimethylarginine dimethylaminohydrolase (DDAH)+I/R group. In the latter 3 groups, acute lung injury was induced by left middle cerebral artery occlusion for 120 min. After a 24-h reperfusion, the rats were sacrificed and the activities of nitric oxide synthase (NOS) and contents of nitric oxide (NO) were measured using reductase and colorimetric assay. The mRNA and protein expressions of protein kinase C (PKC) and myosin light chain kinase (MLCK) in the lung tissues were detected with RT-PCR and Western blotting, respectively. The contents of ADMA in the bronchoalveolar lavage fluid (BALF) and blood flowing into and out of the lungs were measured by ELISA.

RESULTSCerebral I/R injury caused significantly elevated ADMA levels in the BALF and blood flowing into the lungs, and obviously lowered the NO concentration and NOS activity in the lung tissues (P<0.05). Following cerebral I/R injury, MLCK and PKC mRNA and protein expressions were significantly upregualted in the lung tissues (P<0.05). Exogenous DDAH obviously decreased the levels of ADMA in the BALF and blood flowing into the lungs, increased NO concentration and NOS activity, and down-regulated MLCK and PKC mRNA and protein expressions in lung tissues of rats with cerebral I/R injury (P<0.05).

CONCLUSIONADMA contributes to the development of acute lung injury following cerebral I/R injury in rats by upregulating MLCK and PKC expression. ADMA may serve as a novel therapeutic biomarker and a potential therapeutic target for acute lung injury induced by cerebral I/R injury.

Acute Lung Injury ; etiology ; physiopathology ; Animals ; Arginine ; analogs & derivatives ; metabolism ; pharmacology ; Brain Ischemia ; complications ; Male ; Myosin-Light-Chain Kinase ; genetics ; metabolism ; Nitric Oxide Synthase ; antagonists & inhibitors ; Protein Kinase C ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; complications ; physiopathology ; Up-Regulation ; drug effects

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47 PHOX. Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47 PHOX may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.
Animals ; Cell Line ; Cell Membrane ; Cytosol ; Enzyme Inhibitors/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Macrophage-1 Antigen/pharmacology ; Macrophages/drug effects/*metabolism/ultrastructure ; Mice ; Myosin-Light-Chain Kinase/metabolism ; Opsonin Proteins/blood/*metabolism ; *Phagocytosis ; Protein Transport ; Protein-Serine-Threonine Kinases/metabolism ; Research Support, Non-U.S. Gov't ; *Signal Transduction ; Superoxides/*metabolism ; Tetradecanoylphorbol Acetate/pharmacology ; Zymosan/*blood ; p38 Mitogen-Activated Protein Kinases/metabolism ; rhoA GTP-Binding Protein/antagonists & inhibitors/*metabolism