The effects of nitric oxide synthesis on the Na+ ,K+-ATPase activity in guinea pig kidney exposed to lipopolysaccharides

Endotoxins (lipopolysaccharides; LPS) are known to cause multiple organ failure, including renal dysfunction. LPS triggers the synthesis and release of cytokines and the vasodilator nitric oxide (NO.). A major contributor to the increase in NO(.)production is LPS-stimulated expression of inducible nitric oxide synthase (iNOS). This occurs in vasculature and most organs including the kidney. During endotoxemia, NO. and superoxide react spontaneously to form the potent and versatile oxidant peroxynitrite (ONOO- stop) and the formation of 3-nitrotyrosine (nTyr)-protein adducts is a reliable biomarker of ONOO- generation. Therefore, the present study was aimed at investigating the role of endogenous nitric oxide in regulating Na+,K+-ATPase activity in the kidney, and at investigating the possible contribution of reactive nitrogen species (RNS) by measuring of iNOS activity. In addition, the present study was aimed at investigating the relationship between nTyr formation with iNOS and Na+,K+-ATPase activities. Previously in our study, nTyr was not detectable in kidney of normal control animals but was detected markedly in LPS exposed animals. In this study, kidney Na+, K+-ATPase activity were maximally inhibited 6 h after LPS injection (P:0.000) and LPS treatment significantly increased iNOS activity of kidney (P:0.000). The regression analysis revealed a very close correlation between Na+, K+-ATPase activity and nTyr levels of LPS treated animals (r = -0.868, P = 0.001). Na+, K+-ATPase activity were also negatively correlated with iNOS activity (r = -0.877, P = 0.001) in inflamed kidney. These data suggest that NO. and ONOO. contribute to the development of oxidant injury. Furthermore, the source of NO. may be iNOS. iNOS are expressed by the kidney, and their activity may increase following LPS administration. In addition, NO. and ONOO- formation inhibited Na+, K+-ATPase activity. This results also have strongly suggested that bacterial LPS disturbs activity of membrane Na+, K+-ATPase that may be an important component leading to the pathological consequences such as renal dysfunction in which the production of RNS are increased as in the case of LPS challenge

Effect of vitamin A pretreatment on Escherichia coli-induced lipid peroxidation and level of 3-nitrotyrosine in kidney of guinea pig

In the present study, we report the effect of vitamin A (Vit A, retinol palpitate) on kidney lipid peroxidation and 3-nitrotyrosine (3-NT) levels induced after Escherichia coli administration to guinea pigs. Vit A was administrated intraperitoneally (i.p.) to guinea pigs at a dose 15,000 IU/kg per day for 7 days prior to E. coli injection. On day 8, the animals were injected i.p. with E. coli dosed at 12 x10(9) colony forming units per kilogram. Kidneys were collected 6 h after administration of E. coli. Malondialdehyde (MDA) as a lipid peroxidation product, and 3-NT levels were measured by reverse phase high-performance liquid chromatography. There was a significant increase in MDA and 3-NT levels in lipopolysaccaharide-induced group (p < 0.001). 3-NT was not detectable in kidney of normal control animals. However, Vit A administration prior to E. coli injection prevented 3-NT formation but did not prevent the rice in MDA level of kidney (p < 0.001). Vit A alone did not alter the MDA level in the kidney of the control group

Effects of melatonin on 3-nitrotyrosine formation and energy charge ratio in guinea pig kidney in LPS-induced stress

The aim of this study was to evaluate the effects of Escherichia coli-derived lipopolysaccharide on guinea pig kidney by measuring the energy charge ratio and 3-nitrotyrosine levels. In addition the possible protective role of melatonin against lipopolysaccharide-mediated peroxynitrite formation and energy depletion of kidney was determined. Guinea pigs were either pretreated with melatonin or saline (for the control) followed by intraperitoneal administration of E. coli. Six hours after the administration of E. coli, guinea pig kidney ATP, ADP, AMP and 3-nitrotyrosine levels were measured by reverse-phase high performance liquid chromatography. There was a significant increase in the formation of 3-nitrotyrosine and decrease in energy charge in the endotoxin-induced group. However melatonin administration prevented 3-nitrotyrosine formation while failing to prevent or restore changes in the energy charge ratio of the kidney. Copyright (c) 2004 John Wiley & Sons, Ltd

The effects of peroxynitrite on erythrocytes

Endotoxin-induced peroxynitrite formation has been demonstrated in plasma. The aim of this study is to evaluate whether this has an effect on erythrocytes. For this purpose erythrocyte 3-nitrotyrosine (3-NT) level, Na+-K+ ATPase and glutathione peroxidase activities were measured both in vivo and in vitro. In vivo peroxynitrite formation was induced in rats, by intraperitoneal Escherichia coli (E.coli) injection. Erythrocytes were directly incubated with peroxynitrite in the in vitro experiment. 3-NT levels were measured by reverse-phase HPLC, glutathione peroxidase, and Na+-K+ ATPase activities were measured by spectrophoto-metric techniques. There was a marked increase in the 3-NT levels in both experiments. However, glutathione peroxidase activity was significantly increased in in vivo experiments, while decreasing in in vitro conditions. Although Na+-K+ ATPase activities were significantly reduced by peroxynitrite in vitro, Na+-K+ ATPase activities were similar in control and E.coli-injected rat erythrocytes. Although nitrating effect of peroxynitrite does not seem to be preventable by endogenous antioxidants, this effect of peroxynitrite may not endanger erythrocytes if the oxidative damage of peroxy, nitrite is prevented

Impaired Na+,K+-ATPase activity as a mechanism of reactive nitrogen species-induced cytotoxicity in guinea pig liver exposed to lipopolysaccharides

In animal models of endotoxin, the excess production of NO and the reactive nitrogen species (RNS), are potent oxidant and nitrating agents, lead to lipid peroxidation, apoptosis, tissue dysfunction and injury and inactivate enzymes in many cell types. Although liver functions are well known to deteriorate following bacterial infection, the underlying specific mechanism(s) remain a matter of considerable debate. Therefore, the aim of the present study was to determine the in vivo effect of bacterial lipopolysaccharides (LPS) on Na+, K+-ATPase activity of guinea pig liver, and to investigate the possible contribution of RNS by measuring of iNOS activity and 3-nitrotyrosine (nTyr) levels. Liver Na+, K+-ATPase activity were maximally inhibited 6 h after LPS injection (p < 0.001). nTyr was not detectable in liver of normal control animals, but was detected markedly in LPS exposed animals. LPS treatment significantly increased iNOS activity of liver (p < 0.001). The regression analysis revealed a very close correlation between Na+, K+-ATPase activity and nTyr levels of LPS treated animals (r = -0.863, p < 0.001). Na+, K+-ATPase activity were also negatively correlated with iNOS activity (r = -0.823, p < 0.003) in inflamed tissues. Our results have strongly suggested that bacterial LPS disturbs activity of membrane Na+, K+-ATPase that may be an important component leading to the pathological consequences such as hepatocyte cell loss and dysfunction in which the production of RNS are increased as in the case of LPS challenge

The effect of Escherichia coli-derived lipopolysaccharides on plasma levels of malondialdehyde and 3-nitrotyrosine

The aim of this study was to determine the effect of Escherichia coli (E.coli)-derived lipopolysaccharide on rat plasma low density lipoprotein (LDL), malondialdehyde and 3-nitrotyrosine levels (an indicator of protein nitration). Six hours after intraperitoneal administration of E.coli, plasma LDL was measured electrophoretically and malondialdehyde level was measured by spectrophotometric method. Plasma malondialdehyde was significantly (p <0.001) elevated in E.coli-injected rats (4.97 +/- 1.33; n=10) in comparison to control animals (1.83 +/- 0.5; n=10). In addition, plasma 3-nitrotyrosine level, determined by reverse-phase HPLC, was also increased in the infected group (2.84 +/- 1.17 to 0.22 +/- 0.13; n=10). This increase was statistically significant (p <0.001). An increased level of oxidation of lipids and 3-nitrotyrosine was observed as a result of free radical-mediated damage in plasma. In conclusion, asymptomatic infections may increase the risk of atherosclerosis by inducing free radical formation and a consequent increase in the oxidation of LDL

The effect of Escherichia coli-derived lipopolysaccharides on plasma levels of malondialdehyde and 3-nitrotyrosine

The aim of this study was to determine the effect of Escherichia coli (E.coli)-derived lipopolysaccharide on rat plasma low density lipoprotein (LDL), malondialdehyde and 3-nitrotyrosine levels (an indicator of protein nitration). Six hours after intraperitoneal administration of E.coli, plasma LDL was measured electrophoretically and malondialdehyde level was measured by spectrophotometric method. Plasma malondialdehyde was significantly (p <0.001) elevated in E.coli-injected rats (4.97 +/- 1.33; n=10) in comparison to control animals (1.83 +/- 0.5; n=10). In addition, plasma 3-nitrotyrosine level, determined by reverse-phase HPLC, was also increased in the infected group (2.84 +/- 1.17 to 0.22 +/- 0.13; n=10). This increase was statistically significant (p <0.001). An increased level of oxidation of lipids and 3-nitrotyrosine was observed as a result of free radical-mediated damage in plasma. In conclusion, asymptomatic infections may increase the risk of atherosclerosis by inducing free radical formation and a consequent increase in the oxidation of LDL

The effect of desferrioxamine on peroxynitrite-induced oxidative damage in erythrocytes

The aim of this stud), was to investigate the effect of desferrioxamine on peroxynitrite-mediated damage in erythrocytes by measuring the 3-nitrotyrosine level and glutathione peroxidase and Na+-K+ ATPase activities in vitro. 3-Nitrotyrosine levels were determined by HPLC; gluthione peroxidase and Na+-K+ ATPase activities were measured by spectrophotometry. Peroxynitrite increased the 3-nitrotyrosine level but decreased both enzyme activities. In the presence of desferrioxamine,glutathione peroxidase activity was increased with a decrease in the 3-nitrotyrosine level. Desferrioxamine was found to possess an important antioxidant activity as assessed in an in vitro system, reducing protein nitration, restorating enzyme activities and maintaining erythrocyte membrane integrity. Copyright (C) 2004 John Wiley Sons, Ltd