u-0126 and Endotoxemia

Cardiomyocyte tumour necrosis factor α (TNF-α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)-induced cardiomyocyte TNF-α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS-induced TNF-α production in a dose-dependent manner. α₁- adrenoceptor (AR) antagonist (prazosin), but neither β₁- nor β₂-AR antagonist, abrogated the inhibitory effect of NE on LPS-stimulated TNF-α production. Furthermore, phenylephrine (PE), an α₁-AR agonist, also suppressed LPS-induced TNF-α production. NE inhibited p38 phosphorylation and NF-κB activation, but enhanced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and c-Fos expression in LPS-treated cardiomyocytes, all of which were reversed by prazosin pre-treatment. To determine whether ERK1/2 regulates c-Fos expression, p38 phosphorylation, NF-κB activation and TNF-α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c-Fos expression, p38 mitogen-activated protein kinase (MAPK) phosphorylation and TNF-α production, but not NF-κB activation in LPS-challenged cardiomyocytes. In addition, pre-treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS-induced TNF-α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c-Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF-α production and prevented LPS-provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁-AR by NE suppresses LPS-induced cardiomyocyte TNF-α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF-κB activation.

Topics: Adrenergic alpha-1 Receptor Agonists; Adrenergic alpha-1 Receptor Antagonists; Animals; Animals, Newborn; Butadienes; Endotoxemia; Enzyme Inhibitors; Gene Expression Regulation; Imidazoles; Lipopolysaccharides; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardium; Myocytes, Cardiac; NF-kappa B; Nitriles; Norepinephrine; p38 Mitogen-Activated Protein Kinases; Phenylephrine; Prazosin; Proto-Oncogene Proteins c-fos; Pyridines; Rats; Signal Transduction; Tumor Necrosis Factor-alpha

Oxidative stress and apoptosis are the states that can contribute to the pathogenesis of sepsis. In this study we aimed to investigate whether mitogen-activated protein kinase kinase 1 (MEK1)/extracellular signal-regulated kinase 1/2 (ERK1/2)/inducible nitric oxide synthase (iNOS) pathway plays a role in oxidative stress and apoptosis in endotoxemic rats. Systemic total antioxidant, SOD, GPx, and GR activities as markers of oxidative stress, and tissue caspase-3 enzyme activity as a marker of apoptosis were measured in sera and thoracic aortae of male Wistar rats sacrificed 4 h after being treated with saline (vehicle) or lipopolysaccharide (LPS) (10 mg/kg, i.p.). A decrease in total antioxidant activity and caspase-3, SOD, GPx, and GR enzyme activities was occured by LPS. These changes caused by LPS were prevented when a selective iNOS inhibitor, 1,3-PBIT (10 mg/kg, i.p.) or a selective inhibitor of ERK1/2 phosphorylation by MEK1, U0126 (5 mg/kg, i.p.) were given 1 h after administration of LPS. Our results suggest that decreased activity of MEK1/ERK1/2/iNOS pathway prevents oxidative stress by increasing systemic antioxidant enzyme activities and restores decreased caspase-3 activity in thoracic aorta in endotoxemic rat.

Topics: Animals; Antioxidants; Aorta, Thoracic; Apoptosis; Butadienes; Caspase 3; Endotoxemia; Endotoxins; Enzyme Inhibitors; Glutathione Peroxidase; Glutathione Reductase; Heart Rate; Male; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 3; Nitric Oxide Synthase Type II; Nitriles; Oxidative Stress; Rats; Rats, Wistar; Superoxide Dismutase; Thiourea

Bacterial infection of the kidney is associated with renal tubule dysfunction and dysregulation of systemic electrolyte balance. Whether bacterial molecules directly affect renal tubule transport is unknown. We examined the effects of LPS on HCO3(-) absorption in the isolated rat and mouse medullary thick ascending limb (MTAL). LPS decreased HCO3(-) absorption when added to bath or lumen. The MEK/ERK inhibitor U0126 eliminated inhibition by bath LPS but had no effect on inhibition by lumen LPS. Conversely, the mammalian target of rapamycin (mTOR) inhibitor rapamycin eliminated inhibition by lumen LPS but had no effect on inhibition by bath LPS. Inhibiting basolateral Na(+)/H(+) exchange with amiloride eliminated inhibition of HCO3(-) absorption by lumen but not bath LPS. Confocal immunofluorescence showed expression of TLR4 in basolateral and apical membrane domains. Inhibition of HCO3(-) absorption by bath and lumen LPS was eliminated in MTALs from TLR4(-/-) mice. Thus LPS inhibits HCO3(-) absorption through distinct TLR4-dependent pathways in basolateral and apical membranes. These results establish that bacterial molecules can directly impair the transport function of renal tubules, identifying a new mechanism contributing to tubule dysfunction during bacterial infection. The LPS-induced reduction in luminal acidification may contribute to Gram-negative pathogenicity by promoting bacterial adherence and growth and impairing correction of infection-induced systemic acid-base disorders.

Topics: Amiloride; Animals; Bicarbonates; Butadienes; Disease Progression; Endotoxemia; Escherichia coli Infections; Escherichia coli K12; Immunity, Innate; In Vitro Techniques; Kidney Tubules; Lipid A; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitriles; Rats; Rats, Sprague-Dawley; Serum; Signal Transduction; Sirolimus; Toll-Like Receptor 4; Urinary Tract Infections

Lipopolysaccharide (LPS) is an endotoxin causing sepsis. Studies from our laboratory revealed impaired intestinal absorption of L-leucine and D-fructose in LPS-treated rabbits. The aim of this study was to examine intestinal D-galactose transport following intravenous administration of LPS in the rabbit and to identify the cellular mechanisms driving this process. Endotoxin treatment diminished the buildup of D-galactose in intestinal tissue, the mucosal to serosal transepithelial flux of the sugar and its uptake by brush border membrane vesicles (BBMVs). Intracellular signaling pathways associated with protein kinase C (PKC), protein kinase A (PKA), p38 mitogen-activated protein kinase (p38MAPK), Jun N-terminal kinase (JNK), MAPK/extracellular signal-regulated kinases 1 and 2 (MEK1/2) and proteasome were found to be involved in this reduction in sugar uptake. Na(+)/glucose cotransporter 1 (SGLT1) protein levels in BBMVs were lower for LPS-treated animals than control animals. These findings indicate that LPS inhibits the intestinal absorption of D-galactose via a complex cellular mechanism that could involve posttranscriptional regulation of the SGLT1 transporter.

Topics: Animals; Biological Transport; Blotting, Northern; Blotting, Western; Butadienes; Endotoxemia; Galactose; Imidazoles; Intestinal Mucosa; Intestines; Lipopolysaccharides; Male; Mitogen-Activated Protein Kinases; Nitriles; Protein Kinase C; Pyridines; Rabbits; Sodium-Glucose Transporter 1