2) activity reverses endotoxin-induced hypotension via decreased nitric oxide production in rats

Pharmacological Research 56 (2007) 56–64

Inhibition of extracellular signal-regulated kinase (ERK1/2) activity reverses endotoxin-induced hypotension via decreased nitric oxide production in rats B. Tunctan a,∗ , B. Korkmaz a , Z.N. Dogruer b , L. Tamer b , U. Atik b , C.K. Buharalioglu a a

Department of Pharmacology, Faculty of Pharmacy, Yenisehir Campus, Mersin University, Mersin 33169, Turkey b Department of Biochemistry, Faculty of Medicine, Mersin University, Mersin, Turkey Accepted 30 March 2007

Abstract Overproduction of reactive oxygen and nitrogen species leads to oxidative stress and decreased total antioxidant capacity, which is responsible for high mortality from several inflammatory diseases such as endotoxic shock. Among reactive nitrogen species, nitric oxide (NO) produced by inducible NO synthase (iNOS) during endotoxemia is the major cause of vascular hyporeactivity, hypotension and multiple organ failure. This study was conducted to determine if mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK1/2) contributes to endotoxin-induced hypotension as well as vascular inflammation and oxidative stress via NO production. In conscious male Wistar rats, injection of endotoxin (10 mg kg−1 , i.p.) caused a decrease in mean arterial pressure (MAP) for 4 h and increased levels of nitrite in serum, aorta and mesenteric artery. These effects of endotoxin were prevented by selective inhibition of ERK1/2 phosphorylation by MAPK kinase (MEK1/2) with U0126 (5 mg kg−1 , i.p.; 1 h after endotoxin). Endotoxin also caused an increase in protein levels of phosphorylated ERK1/2 in aorta which was abolished by U0126. Selective inhibition of iNOS with phenylene-1,3-bis[ethane-2-isothiourea] dihydrobromide (1,3-PBIT) (10 mg kg−1 , i.p.; 1 h after endotoxin) did not change the endotoxin-induced increase in ERK1/2 activity. Myeloperoxidase activity was increased in aorta and decreased in mesenteric artery by endotoxin, which was reversed by U0126. Endotoxin-induced decrease in one of the products of lipid peroxidation, malonedialdehyde (MDA) was prevented by U0126 in mesenteric artery; however, U0126 caused a further decrease in the levels of MDA in aorta. These data suggest that increased phosphorylation of ERK1/2 by MEK1/2 contributes to the endotoxin-induced hypotension via NO production rat aorta and mesenteric artery. It is likely that ERK1/2 mediates the effect of endotoxin on MPO activity in a different degree in the tissues suggesting possible involvement of any mediator and/or mechanism which also causes neutrophil infiltration during inflammatory response at least in mesenteric artery. Moreover, ERK1/2 seems to be involved in the endotoxin-induced increase in total antioxidant capacity in mesenteric artery. © 2007 Elsevier Ltd. All rights reserved. Keywords: Rat; Endotoxin; Blood pressure; Aorta; Mesenteric artery; Inducible nitric oxide synthase; Extracellular signal-regulated kinase

1. Introduction Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants and/or a depletion of antioxidants. The excessive production of reactive oxygen and nitrogen species (ROS and RNS, respectively) associated with inflammation leads to oxidative stress, which is involved with the high mortality from several diseases such as endotoxic shock [1–4]. There is a considerable body of evidence for redox imbalance and oxidative stress in endotoxic shock. It has been reported

∗

Corresponding author. Tel.: +90 324 3410605; fax: +90 324 3410605. E-mail address: [email protected] (B. Tunctan).

1043-6618/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2007.03.006

that endotoxemia increases markers of oxidative stress and tissue injury in different animal models and in humans. This effect of endotoxin has been found to be correlated with decreased total antioxidant potential and blood levels of several antioxidants such as ␣-tocopherol, retinol, vitamin E, vitamin C and ␤-carotene in humans with sepsis and septic shock [1–4]. It has also been reported that although total antioxidant capacity was decreased in patients with sepsis, it was increased in patients with septic shock [5]. Increased xanthine oxidase, superoxide dismutase and glutathione peroxidase activity has also been reported in patients with sepsis suggesting increased production of ROS [6]. However, malondialdehyde (MDA) (an index for lipid peroxidation) levels were also increased, suggesting that the elevations of these antioxidant enzymes were not so effective as to prevent

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cellular damage. Among RNS, nitric oxide (NO) derived from inducible NO synthase (iNOS) is considered to be the major cause for systemic hypotension, vascular hyporeactivity, multiple organ failure and high mortality rate that is associated with septic shock [4,7]. Although the role of NO is controversial in tissue injury with oxidative DNA damage in endotoxemia, most of the clinical trials demonstrate that iNOS inhibitors may serve as a potentially effective pharmacological agents in alleviating endotoxin-induced decrease in antioxidant capacity and tissue injuries [1,4,7]. Many tissues, such as vascular smooth muscle cells (VSMC) and endothelial cells, express extracellular signal-regulated kinase (ERK1/2) [8,9]. ERK1/2 is activated by phosphorylation on both threonine and tyrosine residues through an upstream kinase, mitogen-activated protein kinase (MAPK) kinase (MEK1/2). Although ERK activation is generally involved in the regulation of gene expression via phosphorylation of transcription factors, protein synthesis and the activation of cytoplasmic and membrane proteins [10], this pathway has also shown to play an important role in vascular smooth muscle contraction under calcium-dependent and -independent conditions [8,11]. It has been demonstrated that MEK1/2-ERK1/2 pathway is involved in the vasoconstriction induced by Gprotein-coupled agonists, such as norepinephrine, angiotensin II or 5-hydroxytryptamine [12,13]. Involvement of MAPKs including ERK1/2 has also been reported in the endotoxininduced cellular responses, such as the increased activity of iNOS [14–17]. It has been demonstrated that endotoxin activates ERK1/2, as well as inhibitor of ␬B (I␬B) degradation and ensuing nuclear factor (NF)-␬B (NF-␬B) activation in rat VSMC [18]. Subsequent activation of NF-␬B by ERK1/2 has shown to increase iNOS protein expression and activity [19,20]. These observations suggest the involvement of MEK1/2-ERK1/2 pathway in the vascular hyporeactivity to vasoconstrictor agents and decreased blood pressure in endotoxemic rats. Although inhibitors of p38 MAPK have shown to be beneficial in the treatment of inflammatory diseases, including sepsis and septic shock, in animals [14,17,21–23] and humans [24–27], there is no study in the literature reporting the importance of MEK1/ERK1/2 pathway on the NO-related events in the pathogenesis of these diseases. We have previously shown that increased ERK1/2 activity contributes to the endotoxin-induced hyporeactivity to norepinephrine via induction of iNOS activity in rat isolated aorta [28]. Moreover, increased NO production by iNOS 4 h after endotoxin injection causes hypotension associated with cardiac and aortic inflammation in conscious rats, and NOderived from heart, aorta and mesenteric artery contributes to these effects of endotoxin [29–32]. Furthermore, we have shown that endotoxemia-induced increase in NO production suppresses lipid peroxidation in cardiac and vascular tissue and selective inhibition of iNOS with 1,3-PBIT restores tissue antioxidant capacity and consequently MAP presumably due to decreased levels of antioxidant molecules or activities of antioxidant enzyme systems [29,30]. In this study, we tested the hypothesis that ERK1/2 contributes to the endotoxininduced hypotension as well as vascular inflammation and

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oxidative stress via NO production in aorta and mesenteric artery. 2. Materials and methods 2.1. Animals Male Wistar rats weighing 200–300 g were fed with standard chow. They were synchronized by maintenance of controlled environmental conditions throughout the duration of the experiments. The circadian rhythmicity of the animals were entrained by a standardized 12 h light and 12 h dark. All animal experiments were carried out according to the proposal of the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by ethics committee of Mersin University School of Medicine. 2.2. Materials Endotoxin (Escherichia coli lipopolysaccharide, 0111:B4), phenylene-1,3-bis[ethane-2-isothiourea] dihydrobromide (1,3PBIT), sodium nitrite, bovine serum albumin (BSA) and Bradford reagent were purchased from Sigma Chemical Co. (St. Louis, U.S.A.). [1,4-Diamino-2,3-dicyano-1,4-bis(2aminophenylthio)butadiene] (U0126) was obtained from Tocris (Bristol, UK). Other chemicals were obtained from Merck (Darmstadt, Germany). U0126 was dissolved in dimethylsulfoxide (71%, v/v). The other chemicals were dissolved in saline. 2.3. Endotoxic shock model Endotoxic shock was induced as described by Tunctan et al. [31]. Briefly, conscious rats received either endotoxin (10 mg kg−1 , i.p., sublethal dose) or saline (4 ml kg−1 , i.p.) at time 0 and mean arterial blood pressure (MAP) and heart rate were measured using the tail-cuff method at 0, 1, 2, 3 and 4 h. Separate groups of rats were treated with endotoxin alone or in combination with 1,3-PBIT, a highly selective iNOS inhibitor [33] (10 mg kg−1 , i.p.), or U0126, a selective inhibitor of ERK1/2 phosphorylation by MEK1/2 [34] (5 mg kg−1 , i.p.), at 1 h after injection of saline or endotoxin. Rats were sacrificed 4 h after endotoxin challenge, and the blood, aorta and mesenteric artery were collected. Sera were obtained from blood samples by centrifugation at 18,000 rpm for 15 min at 4 ◦ C and stored at −20 ◦ C until analyzed for the measurement of nitrite levels. The tissues were homogenized in ice-cold buffer (1 ml) (mM: HEPES 20 [pH 7.5], ␤-glycerophosphate 20, sodium pyrophosphate 20, sodium orthovanadate 0.2, EDTA 2, sodium fluoride 20, benzamidine 10, dithiothreitol 1, leupeptin 20 and aprotinin 10) [31]. Cell debris was removed by centrifugation 18,000 rpm for 15 min at 4 ◦ C followed by sonication for 15 s on ice with 50 ␮l ice-cold Tris (50 mM, pH 8.0) and KCl (0.5 M). The samples were centrifuged at 18,000 rpm for 15 min at 4 ◦ C and then supernatants were removed and stored at −20 ◦ C until analyzed for the measurement of myeloperoxidase (MPO) activity and

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total protein, nitrite, phoshorylated ERK1/2 protein and malondialdehyde (MDA) levels. 2.4. Measurement of tissue protein content The total protein content in the tissue homogenates was determined according to Coomassie blue method using BSA for standard [31]. Briefly, Bradford reagent (200 ␮l) were added to the mixture of tissue homogenate (5 ␮l) and distilled water (795 ␮l). Samples (100 ␮l) were then pipetted into 96 well microtiter plates and absorbance was measured at 620 nm with a microplate reader (Organo Teknika Microwell System, Holland). Linear regression analysis was used to calculate the protein amount in the tissue homogenates from the standard calibration curves of BSA. 2.5. Measurement of serum and tissue nitrite levels The concentrations of nitrite in serum and tissue homogenates were measured by using the diazotization method based on the Griess reaction, which is an indirect assay for NO production [35]. Briefly, samples (50 ␮l) were pipetted into 96 well microtiter plates and an equal volume of Griess reagent (1% sulphanylamide (25 ␮l) and 0.1% N-1-naphtylethylenediamine dihydrochloride (25 ␮l) in 2.5% ortophosphoric acid) was added to each well. After incubation for 10 min at room temperature, absorbance was measured at 540 nm with a microplate reader (Organo Teknika Microwell System, Holland). Linear regression analysis was used to calculate the nitrite concentrations in the sera and the tissue homogenates from the standard calibration curves of sodium nitrite. Serum and tissue nitrite levels were expressed as ␮M or mmol mg−1 protein−1 , respectively.

2.7. Measurement of tissue MPO activity MPO is a haem-containing enzyme within the azurophil granules of neutrophils and MPO activity was measured as a simple quantitative method of detecting leukosequestration. The determination of MPO activity as an index of neutrophil infiltration in tissue homogenates depends on the fact that oxidized hidrogen peroxide reduces o-dianisidine [36]. Reduced o-dianisidine was measured at 410 nm by spectrophotometer. One unit of MPO activity was defined as that degrading 1 ␮mol of hydrogen peroxide to water per minute at 25 ◦ C. Tissue MPO activity was expressed as U mg−1 protein−1 . 2.8. Measurement of MDA levels in tissues As an index of lipid peroxidation, the levels of MDA in tissue homogenates were determined by thiobarbituric acid reaction according to Yagi [37]. The method depends on the measurement of the pink color produced by interaction of the barbituric acid with MDA caused by lipid peroxidation. Linear regression analysis was used to calculate the MDA levels in the tissue homogenates from the standard calibration curves of 1,1,3,3-tetraethoxypropane. Tissue MDA levels were expressed as mmol mg−1 protein−1 . 2.9. Statistical analysis All data were expressed as the mean ± S.E.M; n refers to the number of animals used. Data were analyzed by one-way ANOVA followed by Student–Newman–Keuls test for multiple comparisons and Student’s t or Mann–Whitney U tests when necessary. P value of <0.05 is considered statistically significant. 3. Results

2.6. Measurement of phosphorylated ERK1/2 protein levels in tissues Total amount of phosphorylated ERK1/2 was measured using the BioSource International Inc. ERK1/2 [pTpY185/187] ELISA kit (BioSource International Inc., Camarillo, CA, USA) according to the manufacturer’s instructions. This kit is designed to detect and quantify the level of both dual-phosphorylated ERK2 at threonine 185 and tyrosine 187 and ERK1 at threonine 202 and tyrosine 204. A monoclonal antibody specific for ERK1/2 (both phosphorylated and unphosphorylated) has been coated onto the walls of microtitre wells. Samples are pipetted into these wells, and the ERK1/2 antigens bind to the antibodies. After washing, an antibody specific for phosphorylated ERK1/2 is added and binds to the immobilized ERK1/2 proteins in the wells. Then a horseradish peroxidase-labeled anti-rabbit IgG is added, binding to the second antibody. Finally, a substrate solution is added, which the enzyme uses to produce color. The intensity of the color is proportional to the amount of phosphorylated ERK1/2 in the sample. This is measured with a microtitre plate reader at 450 nm. Phosphorylated ERK1/2 protein levels were calculated as U ml−1 mg protein−1 and expressed as percent of control.

3.1. Decreased MAP in response to endotoxin is mediated by ERK1/2-induced NO production In order to evaluate the contribution of ERK1/2 to the endotoxin-induced hypotension via vascular NO production, a selective inhibitor of ERK1/2 phosphorylation by MEK1/2, U0126, was injected to the animals alone or in combination with endotoxin. Injection of endotoxin to rats caused a decrease in MAP (Fig. 1). U0126 prevented the endotoxin-induced decrease in MAP, while it alone had no effect on MAP (Fig. 1). The endotoxin-induced decrease in MAP was also associated with an increase in the levels of nitrite in serum (Fig. 2A), aorta (Fig. 2B) and mesenteric artery (Fig. 2C). Endotoxin-induced increase in systemic (Fig. 2A) and vascular (Fig. 2B and C) nitrite production was blunted by the administration of U0126. U0126 had no effect on basal serum (Fig. 2A) and vascular (Fig. 2B and C) nitrite levels. Moreover, endotoxin caused an increase in the protein levels of phosphorylated ERK1/2 in aorta (Fig. 3A) and mesenteric artery (Fig. 3B), which was prevented by U0126. U0126 alone had no effect on the vascular ERK1/2 activity (Fig. 3). On the other hand, endotoxin-induced increase in ERK1/2 activity was not significantly affected by 1,3-

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Fig. 1. Effect of U0126 (selective inhibitor of ERK1/2 phosphorylation by MAPK kinase) on mean arterial pressure (MAP) 4 h after saline (vehicle) (4 ml kg−1 , i.p.) or endotoxin (ET) (10 mg kg−1 , i.p.) injection in conscious rats. U0126 (5 mg kg−1 , i.p.) was given at 1 h after ET injection. Values are expressed as means ± SEM from six to eight rats per treatment group. * P < 0.05 vs. saline-treated group (vehicle). + P < 0.05 vs. ET-treated group.

PBIT in aorta (endotoxin-treated group: 124 ± 3.47% of control, n = 6; endotoxin + 1,3-PBIT-treated group: 121 ± 2.33% of control, n = 6) and mesenteric artery (endotoxin-treated group: 122 ± 13.69% of control, n = 5; endotoxin + 1,3-PBIT-treated group: 96 ± 4.94% of control, n = 4). 1,3-PBIT alone had no effect on the basal ERK1/2 activity in mesenteric artery (salinetreated group: 100 ± 2.68% of control, n = 4; 1,3-PBIT-treated group: 108 ± 5.58% of control, n = 6). However, the enzyme activity was further increased by 1,3-PBIT in aorta (salinetreated group: 100 ± 1.65% of control, n = 6; 1,3-PBIT-treated group: 158 ± 7.01% of control, n = 6). 3.2. Vascular inflammation in response to endotoxin is mediated by ERK1/2-induced NO production To investigate possible contribution of ERK1/2 to the endotoxin-induced changes in neutrophil infiltration via vascular NO production, MPO activity was measured in the tissues from the U0126-treated endotoxemic animals. Myeloperoxidase activity was increased in aorta (Fig. 4A) and decreased in mesenteric artery (Fig. 4B) by endotoxin which was reversed by U0126. U0126 alone had no effect on the tissue MPO activity (Fig. 4). 3.3. Oxidant stress in response to endotoxin is mediated by ERK1/2 in mesenteric artery In order to determine whether increased ERK1/2 activity contributes to the endotoxin-induced decrease in oxidative stress via vascular NO production, one of the products of lipid peroxidation, MDA, was measured in the tissues from the U0126-treated endotoxemic rats. Endotoxin-induced decrease in the levels of MDA was prevented by U0126 in mesenteric artery (Fig. 5B);

Fig. 2. Effect of U0126 (selective inhibitor of ERK1/2 phosphorylation by MAPK kinase) on changes in serum (A), aorta (B) and mesenteric artery (C) nitrite levels 4 h after saline (vehicle) (4 ml kg−1 , i.p.) or endotoxin (ET) (10 mg kg−1 , i.p.) injection in conscious rats. U0126 (5 mg kg−1 , i.p.) was given at 1 h after ET injection. Values are expressed as means ± SEM from 4 to 10 rats per treatment group. * P < 0.05 vs. saline-treated group (vehicle). + P < 0.05 vs. ET-treated group. • P < 0.05 vs. U0126-treated group.

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Fig. 3. Effect of U0126 (selective inhibitor of ERK1/2 phosphorylation by MAPK kinase) on changes in aorta (A) and mesenteric artery (B) ERK1/2 activity 4 h after saline (vehicle) (4 ml kg−1 , i.p.) or endotoxin (ET) (10 mg kg−1 , i.p.) injection in conscious rats. U0126 (5 mg kg−1 , i.p.) was given at 1 h after ET injection. Values are expressed as means ± SEM from four to seven rats per treatment group. * P < 0.05 vs. saline-treated group (vehicle). + P < 0.05 vs. ET-treated group.

however, it caused a further decrease in the levels of MDA in aorta (Fig. 5A). U0126 had no effect on the basal levels of MDA in aorta (Fig. 5A); however, it caused an increase in lipid peroxidation in mesenteric artery (Fig. 5B). 4. Discussion This study demonstrates that increased systemic NO production, at least, in aorta and mesenteric artery 4 h after endotoxin injection causes hypotension associated with aortic inflammation in conscious rats. More importantly, we show for the first

Fig. 4. Effect of U0126 (selective inhibitor of ERK1/2 phosphorylation by MAPK kinase) on changes in aorta (A) and mesenteric artery (B) myeloperoxidase (MPO) activity 4 h after saline (vehicle) (4 ml kg−1 , i.p.) or endotoxin (ET) (10 mg kg−1 , i.p.) injection in conscious rats. U0126 (5 mg kg−1 , i.p.) was given at 1 h after ET injection. Values are expressed as means ± SEM from four to nine rats per treatment group. * P < 0.05 vs. saline-treated group (vehicle). + P < 0.05 vs. ET-treated group. • P < 0.05 vs. U0126-treated group.

time that endotoxemia-induced increase in phosphorylation of ERK1/2 by MEK1/2 increases NO production in aorta and mesenteric artery. We found that administration of a selective inhibitor of ERK1/2 activity, U0126, reverses the fall in MAP as well as increased NO production in the vascular tissues. Moreover, ERK1/2 seems to have a role in the effect of endotoxin on MPO activity in the tissues with a possible involvement of any mediator and/or mechanism which also contributes to neutrophil infiltration during inflammatory response at least in mesenteric artery. These data also demonstrate that ERK1/2 is involved in the endotoxin-induced increase in total antioxidant capacity in mesenteric artery. Expression of iNOS protein and mRNA expression approximately 3 h after systemic endotoxin administration to animals

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Fig. 5. Effect of U0126 (selective inhibitor of ERK1/2 phosphorylation by MAPK kinase) on changes in aorta (A) and mesenteric artery (B) malondialdehyde (MDA) levels 4 h after saline (vehicle) (4 ml kg−1 , i.p.) or endotoxin (ET) (10 mg kg−1 , i.p.) injection in conscious rats. U0126 (5 mg kg−1 , i.p.) was given at 1 h after ET injection. Values are expressed as means ± SEM from four to seven rats per treatment group. * P < 0.05 vs. saline-treated group (vehicle). + P < 0.05 vs. ET-treated group. • P < 0.05 vs. U0126-treated group.

has been reported in several tissues, including several blood vessels, heart, kidney and lung associated with significant increase in serum/plasma and tissue nitrite levels and decrease in MAP [7,38–40]. We have previously demonstrated that selective iNOS inhibitor, 1,3-PBIT, prevents the endotoxin-induced decrease in MAP as well as systemic and tissue NO production [29–32]. In the present study, administration of endotoxin to rats decreased MAP at 4 h which was also associated with an increase in the levels of nitrite in serum, aorta and mesenteric artery that was prevented after selective inhibition of ERK1/2 activity with U0126. There are conflicting results concerning the effect of

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ERK1/2 on vascular NO production. Yamakawa et al. [18] have shown that inhibition of ERK1/2 phosphorylation does not prevent the endotoxin-induced degradation of I␬B and activation of NF␬-B in rat aortic smooth muscle cell culture. The results from the other studies in rat VSMC indicate that U0126 and PD98059 prevent the interleukin-1␤-induced increase in NF␬B activity and expression and activity of iNOS [41]. There are also contradictory reports demonstrating both activation [41–44] or inactivation [45] of ERK1/2 by NO in VSMC. In the present study, the results with 1,3-PBIT that endotoxin-induced increase in ERK1/2 activity do not change in these tissues suggest that iNOS-derived NO has no effect on the phosphorylation of ERK1/2 by MEK1/2. On the other hand, we have previously shown that increased ERK1/2 activity contributes to the endotoxin-induced hyporeactivity to norepinephrine via overproduction of NO in rat isolated aorta [28]. 1,3-PBIT alone also caused an increase in the basal ERK1/2 activity in aorta without any change in the systemic and aortic NO production [30]. Based on the results of previous studies and our present findings, it can be suggested that endotoxemia-induced increase in phosphorylation of ERK1/2 by MEK1/2 increases NO production in vascular tissues. Moreover, selective inhibition of ERK1/2 activity reverses the fall in MAP as well as increased systemic and vascular NO production. Since 1,3-PBIT has been reported to inhibit constitutive NOS isoforms in a less degree (Ki values for human iNOS, endothelial NOS and neuronal NOS enzymes are 0.047, 9 and 0.25 ␮M, respectively) [33], the data with 1,3-PBIT alone also suggest that NO supresses ERK1/2 activity under basal conditions [45]. There are contradictory reports in the literature concerning role of NO as an anti-inflammatory or proinflammatory agent [7,38,39]. We have previously demonstrated that selective iNOS inhibitor, 1,3-PBIT, reverses the changes in the endotoxin-induced neutrophil infiltration (as an index for the development of inflammation) in vascular tissue [29,30]. In the present study, we found that endotoxin caused an increased levels of nitrite in serum and aorta as well as increased aortic MPO activity prevented by the selective inhibitor of ERK1/2 phosphorylation, U0126. However, increased levels of nitrite in mesenteric artery were associated with decreased MPO activity also reversed by U0126. These results suggest that NO overproduced in aortic tissue acts as an pro-inflammatory mediator in this endotoxemia model in rat. On the other hand, endotoxininduced decrease in MPO activity in mesenteric artery suggests that NO acts as an anti-inflammatory mediator to protect small resistance arteries which are responsible to maintain blood pressure under pathological conditions from detrimental effects of endotoxin. In conclusion, it can be suggested that ERK1/2 contributes to the pro-inflammatory and anti-inflammatory properties of NO in conduit and small resistance arteries. Moreover, any mediator (e.g., ROS or prostaglandins) and/or mechanism (e.g., p38 MAPK) which causes neutrophil infiltration during inflammatory response [3,10] might also contribute to the effect of endotoxin on MPO activity at least in mesenteric artery. It is well known that oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants and/or a depletion of antioxidants [4]. There is an increasing evidence that oxidative stress influences the MAPK signaling pathways

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[46]. ROS and RNI produced during oxidative stress have been shown to induce the activation of MEK1/2 and ERK1/2 pathway, and selective inhibitors of this pathway have shown to block oxidative stress-induced ERK1/2 activation. These molecules are also well known to affect proteins, such as NF␬-B, protein kinase B, ERK, big mitogen-activated protein kinase 1, cAMP response element-binding protein, and the epidermal growth factor receptor, in a manner which is thought to have a positive role in cell survival or proliferation. Although increased activity of antioxidant enzymes such as xanthine oxidase, superoxide dismutase and glutathione peroxidase has been reported in patients with sepsis, MDA levels have also shown to be increased, suggesting that the elevations of these antioxidant enzymes were not so effective as to prevent cellular damage [5]. Moreover, Mirochnitchenko et al. [47] have been reported that transgenic mice overexpressing two major forms of human glutathione peroxidases (GPs), intra- and extracellular GP, are able to modulate host response during endotoxemic conditions. They also showed that these animals had a decreased hypotension and increased survival rate after administration of a high dosage of endotoxin. We have previously shown that selective iNOS inhibitor, 1,3-PBIT, prevents the endotoxin-induced decrease in oxidative stress in heart, aorta and mesenteric artery [29,30]. In the present study, endotoxin-induced increase in the levels of nitrite in serum, aorta and mesenteric artery was also associated with decreased MDA levels in these tissues. Selective inhibitor of ERK1/2 phosphorylation, U0126, improved the total antioxidant capacity of mesenteric artery, but not of aorta. Moreover, U0126 alone caused an increase in basal lipid peroxidation in mesenteric artery. Although there are conflicting reports showing the effect of NO on lipid peroxidation and oxidative stress [48–51] our results are consistent with the observations that NO behaves as a potent antioxidant. There are several reports indicate that systemic administration of inhibitors of lipid peroxidation, anthocyanins [52] potent antioxidants such as ascorbic acid [53] and melatonin [54], a dual vitamin E-like antioxidant and inhibitor of nuclear factor-␬B, IRFI 042 [55], ethyl pyruvate [56], U-83836E [57], an alpha/beta-adrenoceptor and serotonergic receptor blocker, eugenosedin-A [53] and tyrosine kinase inhibitor, genistein [58], results in improved hemodynamic status and reversal of endotoxic shock. The protective effects of these agents have been attributed to their antioxidant properties and also inhibition of cytokine and/or NO overproduction. Based on the our findings and previous studies, therefore, it can be concluded that ERK1/2 contributes to the endotoxin-induced increase in total antioxidant capacity and consequently decreases lipid peroxidation via increased NO production in mesenteric artery. Furthermore, the data with U0126 alone also suggest that MEK1/2 and ERK1/2 pathway contributes to the maintenance of oxidant/antioxidant balance under basal conditions [46]. ROS (e.g., hydroxyl radical or hydrogen peroxide) and RNI (e.g., NO or peroxynitrite) have been reported to stimulate MEK1/2 and ERK1/2 pathway [46], and activated ERK1/2 phosphorylates numerous substrates in all cellular compartments, including various membrane proteins, nuclear substrates, several MAPK-activated protein kinases, phospholipases, and transcription factors [59]. Therefore, increased production of ROS and/or

RNI (except for NO) might contribute to the effect of U0126 on the endotoxin-induced decrease in oxidative stress in aortic tissue. In summary, this is the first in vivo study to provide evidence that inhibition of ERK1/2 activity opposes the fall in MAP and the increase in NO production in rats treated with endotoxin. Our results suggest that treatment with the selective ERK1/2 inhibitors could improve the cardiovascular function associated with inflammation and decreased antioxidant capacity in patients during septic shock. Further studies with ERK1/2 inhibitors in experimental models of endotoxemia could provide a novel approach to treat hypotension in septic shock. Consideration of the effect of MEK1/2 and ERK1/2 pathway on iNOS activity may also help in the design of new therapeutic strategies to patients with endotoxemia. Acknowledgment This work was supported by the Research Foundation of Mersin University (Project Code No.: BAP ECZF EMB (BT) 2004-3). References [1] Victor VM, Rocha M, De la Fuente M. Immune cells: free radicals and antioxidant in sepsis. Int Immunopharmacol 2004;4:327–47. [2] Cadenas S, Cadenas AM. Fighting the stranger-antioxidant protection against endotoxin toxicity. Toxicology 2002;18:45–63. [3] Cuzzocrea S, Riley DP, Caputi AP, Salvemini D. Antioxidant therapy: a new pharmacological approach in shock, inflammation, and ischemia/reperfusion injury. Pharmacol Rev 2001;53:135–59. [4] Salvemini D, Cuzzocrea S. Oxidative stress in septic shock and disseminated intravascular coagulation. Free Radic Biol Med 2002;33:1173– 85. [5] Pascual C, Karzai W, Meier-Hellmann A, Oberhoffer M, Horn A, Bredle D, et al. Total plasma antioxidant capacity is not always decreased in sepsis. Crit Care Med 1998;26:705–9. [6] Batra S, Kumar R, Seema M, Kapoor AK, Ray G. Alterations in antioxidant status during neonatal sepsis. Ann Trop Paediatr 2000;20:27–33. [7] Tunctan B, Altug S. The use of nitric oxide synthase inhibitors in inflammatory diseases: a novel class of anti-inflammatory agents. Curr Med Chem: Anti-Inflammatory & Anti-Allergy Agents 2004;3:271–301. [8] Dessy C, Kim I, Sougnez CL, Laporte R, Morgan KG. A role for MAP kinase diffentiated smooth muscle contraction evoked by ␣-adrenoceptor stimulation. Am J Physiol 1998;275:C1081–6. [9] Hoefen RJ, Berk BC. The role of MAP kinases in endothelial activation. Vasc Pharmacol 2002;38:271–3. [10] Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002;298:1911–2. [11] Kwon S, Lee WJ, Fang LH, Kim B, Ahn HY. Mitogen-activated protein kinases partially regulate endothelin-1-induced contractions through a myosin light chain phosphorylation-independent pathway. J Vet Med Sci 2003;65:225–30. [12] Hague C, Gonzalez-Cabrera PJ, Jeffries WB, Abel PW. Relationship between alpha(1)-adrenergic receptor-induced contraction and extracellular signal-regulated kinase activation in the bovine inferior alveolar artery. J Pharmacol Exp Ther 2002;303:403–11. [13] Watts SW. 5-Hydroxytryptamine-induced potentiation of endothelin-1- and norepinephrine-induced contraction is mitogen-activated protein kinase pathway dependent. Hypertension 2000;35:244–8. [14] Kan W, Zhao KS, Jiang Y, Yan W, Huang Q, Wang J, et al. Lung, spleen, and kidney are the major places for inducible nitric oxide synthase expression in endotoxic shock: role of p38 mitogen-activated protein kinase in

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[15]

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