HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 294/4/H1621    most recent 01008.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Calvert, T. J. Articles by Nelin, L. D. Search for Related Content PubMed PubMed Citation Articles by Calvert, T. J. Articles by Nelin, L. D.

Deficiency of mitogen-activated protein kinase phosphatase-1 results in iNOS-mediated hypotension in response to low-dose endotoxin

Centers for Perinatal Research and Gene Therapy, The Research Institute at Nationwide Children's Hospital, and the Department of Pediatrics, The Ohio State University College of Medicine, Columbus, Ohio

Submitted 30 August 2007 ; accepted in final form 11 February 2008

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Mitogen-activated protein kinase phosphatase-1 (MKP-1) is essential in limiting the proinflammatory response to lipopolysaccharide (LPS). We hypothesized that Mkp-1^–/– mice would respond to low-dose LPS with a fall in blood pressure due to augmented expression of inducible nitric oxide (NO) synthase (iNOS). To test this hypothesis, Mkp-1^–/– mice and their wild-type littermates were treated with 10 µg/kg iv LPS, and mean arterial blood pressure (MAP) and exhaled NO production (exNO) were measured. Tissues were harvested for an assessment of iNOS protein levels. Wild-type mice had no change in MAP or exNO during the experimental period, whereas Mkp-1^–/– mice had a fall (P < 0.005) in MAP [79 ± 5% of baseline (BL)] and an increase (P < 0.01) in exNO (266 ± 50% of BL) after 150 min. The tissue levels of iNOS were greater in Mkp-1^–/– than in wild-type mice. In additional experiments, 60 min after LPS treatment, Mkp-1^–/– and wild-type mice were given N-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine, and MAP and exNO were monitored for 90 min. Treatment with L-NAME prevented the LPS-induced increase in exNO and decrease in MAP but resulted in decreased exNO and elevated MAP in wild-type mice. Aminoguanidine prevented the increase in exNO and the fall in MAP caused by LPS in Mkp-1^–/– mice, without significantly affecting MAP or exNO in wild-type mice. These results demonstrate that a deficiency of MKP-1 results in an exaggerated hypotensive response to LPS mediated by augmented iNOS expression. We speculate that defects in the Mkp-1 gene may increase susceptibility for the development of septic shock.

lipopolysaccharide; septic shock; cell signaling; nitric oxide

The proinflammatory cytokines, such as TNF- and IFN-, play an important role in the induction of inducible nitric oxide (NO) synthase (iNOS) protein expression during microbial infection (14, 25). MAPKs contribute to iNOS induction in LPS-stimulated RAW264.7 cells, a macrophage cell line (7, 25). Our laboratory has recently shown that MKP-1 overexpression blunts LPS-induced iNOS protein expression in RAW264.7 cells, whereas MKP-1 deficiency leads to augmented iNOS protein production in these cells (25). Our laboratory has also recently reported that Mkp-1^–/– mice exhibit an exaggerated hypotensive response to intraperitoneal LPS challenge detected by tail-cuff systolic blood pressure measurements (36). Thus we hypothesized that MKP-1 is critical in limiting iNOS upregulation and thereby preventing or attenuating the hypotensive response following LPS stimulation. To test this hypothesis, we utilized Mkp-1^–/– mice and their wild-type littermates and measured blood pressure responses and exhaled NO production (exNO) following a challenge with low-dose LPS. We found that a deficiency in MKP-1 resulted in greater iNOS protein expression and NO production, as well as decreased blood pressure in response to low-dose LPS. The role of iNOS in LPS-induced hypotension was confirmed using pharmacological inhibitors. Our results strongly suggest that MKP-1 is critical in regulating iNOS protein production and thereby prevents septic shock during host inflammatory responses.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Mice. The generation of MKP-1 knockout mice has been described previously (10). Cryopreserved embryos of the Mkp-1 knockout mouse (–/+ and –/–) on a C57BL6/129-mixed background were kindly provided by Bristol-Myers Squibb Pharmaceutical Research Institute (Lawrenceville, NJ) and were regenerated into mice in Jackson Laboratory (Bar Harbor, ME). These mice were bred in-house to yield both wild-type and Mkp-1^–/– mice. All mice were maintained at 24°C with a relative humidity between 30% and 70% on a 12-h:12-h light-dark cycle. The mice were fed Harlan Teklad irradiated diet (Harlan Sprague-Dawley) ad libitum. All animals received humane care in accordance with the guidelines of the National Institutes of Health under a protocol approved by the Institutional Animal Care and Use Committee of the Columbus Children's Research Institute.

Blood pressure measurements. The animals were anesthetized with 50 mg/kg ip pentobarbital sodium. An incision was made over the trachea, which was isolated and exposed. The trachea was cannulated using pulled polyethylene (PE)-90 tubing (BD Scientific, Franklin Lakes, NJ) and secured using a 4-0 silk suture (Ethicon, Piscataway, NJ). The tracheal cannula was connected to a rodent ventilator (Hugo/Saks) at a tidal volume of 5 ml/kg body wt and a respiratory rate of 50 breaths/min. The carotid artery was then isolated, and 6-0 Prolene (Ethicon) ties were placed around it. The distal tie was secured, a 32-gauge needle was used to pierce the artery, and pulled PE-50 tubing was inserted (BD) into the carotid artery. The proximal suture was then secured. The catheter was connected to a blood pressure transducer (Columbus Instruments, Columbus, OH), and the blood pressure was continuously monitored using Cardiomax hardware and software (Columbus Instruments) and a Dell laptop personal computer. The jugular vein was then exposed and isolated, and 6-0 Prolene ties were placed around it. The distal tie was secured, and a 32-gauge needle was used to introduce pulled PE-50 tubing. The jugular venous catheter was used for the administration of fluids and medications.

Exhaled NO measurements. Exhaled NO was measured as previously described (6, 26). The animals were ventilated with a NO free room air-gas mixture supplied from a mylar balloon attached to the inhalation port of the ventilator. The tidal volume and respiratory rate were held constant throughout the experimental period. During each experimental condition, exhaled gas was collected for the last 5 min of the experimental condition into a mylar balloon attached to the ventilator exhaust port. The gas collected in the mylar balloon was analyzed using a chemiluminescence NO analyzer (Sievers, Boulder, CO). The analyzer was calibrated using a standard curve generated daily with authentic NO (1 parts per million in N₂; Matheson, Chicago, IL) mixed with NO free N₂ using precision flow meters to obtain concentrations ranging from 0 to 500 parts per billion (ppb) (6, 26). The NO detection limit was 0.5 ppb (vol/vol). The exNO rate was calculated from the minute ventilation and the expired NO concentration.

Tissue preparation. After the 150-min experimental period, the mice were euthanized with an injection of pentobarbital sodium (100 mg/kg iv). The lungs, heart, aorta, and kidneys were removed and frozen at –80°C for further analyses. The organs were homogenized, as previously described (20), in 0.8 ml of ice-cold Dulbecco's phosphate-buffered saline (pH 7.4). Samples were centrifuged at 12,000 g for 15 min, and the supernatants were collected and analyzed for total protein contents using the Bradford assay (Bio-Rad, Hercules, CA). The supernatants were stored at –80°C.

Western blot analysis. The tissue homogenates were assayed for iNOS, endothelial NOS (eNOS), neuronal NOS (nNOS), and β-actin protein by Western blot analysis as previously described (25, 35, 36). Briefly, aliquots of tissue homogenate containing equal amounts of protein were diluted 1:1 with SDS sample buffer, heated to 80°C for 15 min, and then centrifuged at 10,000 g at room temperature for 2 min. The aliquots of the supernatant were used for SDS-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride membranes and blocked overnight in phosphate-buffered saline with 0.1% Tween (PBS-T) containing 5% nonfat dried milk and 3% albumin. The membranes were then incubated with the primary antibody, iNOS (1:5,000; BD Transduction, San Diego, CA), eNOS (1:1,000; BD Transduction), or nNOS (1:500; BD Transduction) for 4 h and then washed three times with PBS-T with 1% nonfat dried milk. The membranes were then incubated with the biotinylated IgG secondary antibody (1:5,000; Vector, Burlingame, CA) for 1 h, washed, and then incubated with streptaviden-horseradish peroxidase conjugate (1:1,500; Bio-Rad) for 30 min. The protein bands were visualized using enhanced chemiluminescence (ECL reagent; Amersham Pharmacia Biotech, Piscataway, NJ) and quantified using densitometry (Sigma Gel; Jandel Scientific, San Rafael, CA). To control for protein loading, the blots were stripped using a stripping buffer containing 62.5 mM Tris·HCl (pH 6.8), 2% SDS, and 100 mM 2-β-mercaptoethanol, and the blots were reprobed for β-actin (1:10,000; Abcam, Cambridge, MA) as described above.

Statistical analysis. Values are means ± SE. One-way ANOVA was used to compare the data. Significant differences were identified using a Newman-Keuls post hoc test. For the N-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine dose-response data in wild-type mice, mean arterial blood pressure (MAP) and exNO between subsequent doses were also compared using a repeated-measures one-way ANOVA. The two regression lines in Fig. 7C were compared using covariance analysis and a t-test. SigmaStat 3.5 (Jandel Scientific) was the statistical program used to run these various tests. Differences were considered significant when P < 0.05.

View larger version (11K): [in this window] [in a new window]   Fig. 7. Selective NOS inhibition dose dependently rescued the LPS-induced fall in MAP and increase in exNO in KO mice. A: MAP in WT (n = 5; white circles) and KO (n = 5; black circles) mice in response to increasing doses of aminoguanidine. The animals were given LPS (0.01 mg/kg iv; BL), and 150 min (labeled as 0 mg/kg aminoguanidine on graph) later the dose-response experiments were started. Aminoguanidine was administered in cumulative doses to a maximum dose of 10 mg/kg iv every 15 min. *P < 0.05, KO different from WT same dose; P < 0.01, time 0 different from BL same genotype. B: exNO rates in the animals shown in A. *P < 0.05, KO different from WT mice same dose; #P < 0.05, different from previous dose same genotype; P < 0.01, time 0 different from BL same genotype. C: exNO was inversely correlated to MAP both in WT (white circles) and KO (black circles) mice treated with aminoguanidine. The dashed line is the regression line fit to the data from the WT mice (y = –0.56x + 121.5, r = –0.44, P < 0.005). The solid line is the regression line fit to the data from the KO mice (y = –0.59x + 148.8, r = –0.37, P < 0.05). The slopes of the regression lines were not different between WT and KO mice.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

View larger version (8K): [in this window] [in a new window]   Fig. 1. Inhibition of nitric oxide (NO) synthase (NOS) with N-nitro-L-arginine methyl ester (L-NAME), but not with aminoguanidine, results in increased blood pressure and decreased exhaled NO production (exNO). A: mean arterial blood pressure (MAP) in response to increasing doses of L-NAME (black circles) or aminoguanidine (gray squares) in anesthetized wild-type (WT) mice (n = 5 in each group). L-NAME (0, 2.5, 7.5, 25, and 75 mg/kg cumulative dose) or aminoguanidine (0, 0.1, 1, 10, and 100 mg/kg cumulative dose) was administered intravenously every 30 min, following a 30-min equilibration period [baseline (BL)]. B: exNO rates measured during BL or after administration of either L-NAME or aminoguanidine in these anesthetized mice. C: exNO was inversely correlated with MAP in the L-NAME-treated anesthetized WT mice. The 2 points where both exNO and MAP were measured in these 5 L-NAME-treated WT mice are plotted, and the BL point for each of the animals (100, 100) was excluded. The solid line represents the linear regression fit of the data (y = –0.67x + 140, r = –0.64, P < 0.05). *P < 0.05, different from BL; #P < 0.05, different from previous dose.

To determine whether there were differences in NO sensitivity due to MKP-1 deficiency, we instrumented Mkp-1^–/– (n = 5) and wild-type mice (n = 3) as described in METHODS, and then 0.001, 0.01, and 0.1 nmole cumulative doses of the NO donor spermineNONOate were given intravenously every 30 min while measuring MAP and exNO as described in METHODS. SpermineNONOate caused a dose-dependent decrease in MAP (Fig. 2A) and a dose-dependent increase in exNO (Fig. 2B) in both Mkp-1^–/– and wild-type mice. There were no differences in the response to the NO donor in Mkp-1^–/– and wild-type mice. Thus these data suggest that Mkp-1^–/– and wild-type mice have similar sensitivity to NO.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Mitogen-activated protein kinase phosphatase-1 (Mkp-1)–/– knockout (KO) and WT mice have similar sensitivities to NO. Instrumented WT animals were given 0.001, 0.01, and 0.1 nmol iv cumulative doses of the NO donor spermineNONOate every 30 min while measuring MAP and exNO. A: the MAP response to increasing doses of the NO donor, spermineNONOate, in KO (n = 5; black circles) and WT (n = 3; white circles) mice. SpermineNONOate caused a dose-dependent decrease in MAP, and there was no difference in the MAP response to spermineNONOate between KO and WT mice. B: the exNO response to increasing doses of the NO donor, spermineNONOate, in KO (n = 5; black circles) and WT (n = 3; white circles) mice. As expected, the NO donor spermineNONOate caused a similar dose-dependent increase in exNO in both KO and WT mice. *P < 0.05, different from BL same genotype.

To determine the role of MKP-1 in LPS-induced alterations in blood pressure and exNO, we studied wild-type (n = 6) and Mkp-1^–/– (n = 6) mice. Low-dose LPS challenge had little effect on MAP in wild-type mice (Fig. 3A). On the other hand, in Mkp-1^–/– mice, MAP was significantly lower at 120 and 150 min after treatment with low-dose LPS (Fig. 3A). Although low-dose LPS had little effect on exNO in wild-type mice (Fig. 3B), it resulted in a significantly higher exNO in Mkp-1^–/– mice beginning at 120 min and continuing at 150 min after treatment (Fig. 3B).

View larger version (9K): [in this window] [in a new window]   Fig. 3. Administration of 10 µg/kg iv lipopolysaccharide (LPS) resulted in a fall in blood pressure and an increase in exNO rate only in anesthetized KO mice. A: MAP as a percentage of baseline in WT (n = 6; gray circles) and KO (n = 6; black circles) mice. LPS was administered at time 0. There was no change in MAP during the 150-min experimental period in WT mice. The KO mice had a decrease in MAP beginning 120 min after LPS treatment. *P < 0.05, different from time 0. B: exNO rate as a percentage of baseline in the same mice shown in A. The KO mice had an increase in exNO beginning 120 min after LPS treatment. *P < 0.05, different from time 0. C: exNO was inversely correlated to MAP. The exNO (percentage of baseline) plotted against the MAP (percentage of baseline) for the measurements obtained at 120 and 150 min in the LPS-treated WT and KO mice are shown. Fitting the data with a straight line yields y = –4.3x + 562 and r = –0.613 (P < 0.002).

To further examine the effects of low-dose LPS on NO production in these mice, the protein levels in the aorta, heart, kidney, liver, and lungs for iNOS, eNOS, and nNOS 150 min after vehicle or LPS treatment were determined. In the vehicle-treated mice, there were no readily detectable levels of iNOS protein in any organ studied in either the Mkp-1^–/– or wild-type animals (Fig. 4A). The levels of iNOS protein from the aorta, heart, and lungs were significantly greater in the LPS-treated Mkp-1^–/– than in the LPS-treated wild-type mice (Fig. 4B). There was no significant difference in iNOS protein levels in the kidneys between the groups, and interestingly, the liver levels of iNOS protein were actually lower in the LPS-treated Mkp-1^–/– than in the LPS-treated wild-type mice (Fig. 4B). To determine whether other NOS isoforms may be involved in the responses we saw, we measured eNOS and nNOS protein levels in these same organs. In animals treated with vehicle, there were no differences in the levels of eNOS protein between Mkp-1^–/– and wild-type mice, except that the wild-type mice had greater eNOS protein levels than did Mkp-1^–/– mice in the kidneys (eNOS/β-actin, 1.33 ± 0.24 wild-type vs. 0.50 ± 0.14 Mkp-1^–/– mice, P < 0.05). In Mkp-1^–/– mice treated with LPS, there were significantly greater eNOS protein levels in the aorta than in that of the LPS-treated wild-type animals, and there were no other statistically significant differences between LPS-treated Mkp-1^–/– and wild-type mice (Fig. 4C). In animals treated with vehicle, there were no differences between Mkp-1^–/– and wild-type mice in nNOS protein levels in any of the organs studied. In mice treated with LPS, the nNOS levels were lower than those in vehicle-treated animals in the aorta, kidney, liver, and lungs (Fig. 4D). Wild-type mice treated with LPS had significantly greater kidney nNOS protein levels than did LPS-treated Mkp-1^–/– mice, whereas LPS-treated Mkp-1^–/– mice had significantly greater lung nNOS protein levels than did LPS-treated wild-type mice (Fig. 4D). These results are consistent with the premise that iNOS is the major NOS isoform contributing to the LPS-induced decrease in MAP and increase in exNO following low-dose LPS in the Mkp-1^–/– mice.

View larger version (17K): [in this window] [in a new window]   Fig. 4. LPS treatment resulted in significantly greater aorta, lung, and heart levels of inducible NOS (iNOS) protein in KO than in WT mice. A: representative Western blots for iNOS, endothelial NOS (eNOS), neuronal NOS (nNOS), and β-actin from aortic tissue from WT and KO mice with or without LPS treatment. B: the mean densities for iNOS protein in the aorta, heart, kidney, liver, and lungs of WT (n = 5–9; gray columns) and KO (n = 5–9; black columns) mice 150 min after LPS treatment. The iNOS protein densities were normalized to the β-actin protein densities. In both WT and KO mice not treated with LPS, iNOS protein levels were undetectable using our Western blot analysis protocol. C: the mean densities for eNOS protein in the aorta, heart, kidney, liver, and lungs of WT (n = 3; gray columns) and KO (n = 3; black columns) mice. Since eNOS protein levels were detectable in mice not treated with LPS, the data in this graph are shown relative to the eNOS protein levels from the same genotype not given LPS, such that 1 represents no change due to LPS treatment and a value <1 represents a decrease due to LPS treatment, whereas a value >1 represents an increase due to LPS treatment. D: the mean densities for nNOS protein in the aorta, heart, kidney, liver, and lungs of WT (n = 3; gray columns) and KO (n = 3; black columns) mice. Since nNOS protein levels were detectable in mice not treated with LPS, the data in this graph are shown relative to the nNOS protein levels from the same genotype not given LPS, as in Fig. 4C. *P < 0.05, KO different from WT mice in the same organ.

View larger version (11K): [in this window] [in a new window]   Fig. 5. Nonselective NOS inhibition prevented the LPS-induced fall in MAP and increase in exNO in KO mice, although it resulted in hypertension and decreased exNO in WT mice. A: MAP in WT (n = 5; gray circles) and KO (n = 5; black circles) mice following LPS (0.01 mg/kg iv) administration at time 0 and L-NAME (7.5 mg/kg iv) administration at 60 min. *P < 0.01, different from time 0 same genotype; #P < 0.05, KO different from WT mice at same time point. B: exNO rates in the same mice shown in A. *P < 0.01, different from time 0 same genotype.

View larger version (11K): [in this window] [in a new window]   Fig. 6. Selective NOS inhibition prevented the LPS-induced fall in MAP and increase in exNO in KO mice and had little effect on MAP or exNO in WT mice. A: MAP in WT (n = 6; gray circles) and KO (n = 6; black circles) mice following LPS (0.01 mg/kg iv) administration at time 0 and aminoguanidine (AG; 10 mg/kg iv) administration at 60 min. There were no differences either within groups or between groups. B: exNO rates in the same mice shown in A. *P < 0.05, 90 and 120 min different from time 0 only in the WT mice.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The main findings of this study were 1) exhaled NO inversely correlated with MAP in wild-type mice given L-NAME; 2) low-dose LPS resulted in a fall in MAP and an increase in exNO only in the Mkp-1^–/– mice, which was associated with an increase in iNOS protein levels; 3) L-NAME prevented the LPS-induced fall in MAP and the increase in exNO in Mkp-1^–/– mice and caused an increase in MAP and a decrease in exNO in wild-type mice; 4) aminoguanidine prevented the LPS-induced fall in MAP and increase in exNO in Mkp-1^–/– mice, without significantly affecting MAP in wild-type mice; and 5) aminoguanidine actually reversed the LPS-induced fall in MAP and the increase in exNO in Mkp-1^–/– mice after the drop in MAP had occurred. Taken together, these findings demonstrate that Mkp-1^–/– mice respond to low-dose LPS with an increase in iNOS protein levels and NO production, which resulted in a fall in MAP, whereas wild-type mice are relatively unaffected by the same dose of LPS. These findings are consistent with our hypothesis and demonstrate the central role of MKP-1 in preventing iNOS overexpression in response to LPS.

In this study, we found that changes in the measured exNO rates inversely correlated with changes in the measured MAP. The exNO has been used as a measure of pulmonary NO production both experimentally (1, 2, 6, 26) and clinically (30). However, the finding that exNO correlates with changes in systemic blood pressure suggests that the measurement of exNO can be a marker of systemically relevant NO production. Interestingly, Pedoto et al. (28) found that in rats given LPS, the exNO rates did not correlate with MAP, despite the finding that MAP decreased and exNO increased with LPS treatment. However, in a subsequent study the same group (27) found that the increase in exNO was temporally related to the fall in blood pressure in LPS-treated rats. A temporal relationship between changes in exNO and MAP has also been described in rabbits given nitroglycerin (2) or a cyclooxygenase inhibitor (1). A recent case report found an inverse relationship between exNO and MAP in a patient with hepatopulmonary syndrome treated with either curcumin or terlipressin (4). The advantage of exNO measurements as an index of NO production is that exNO can be determined in real time, whereas other measures of NO production require invasive sampling and later processing, i.e., plasma nitrite/nitrate or tissue NOS activities.

The fall in MAP was temporally correlated with the increase in exNO in the Mkp-1^–/– mice given low-dose LPS. This suggests that the iNOS induction by intravenous LPS was involved in the fall in MAP. Further support for a role for iNOS protein induction comes from the immunoblot data, which demonstrates greater levels of iNOS protein in the aorta, heart, and lungs from Mkp-1^–/– than in those from wild-type mice. The site of iNOS protein induction is consistent with a study by Kan et al. (16) in which BALB/c mice had a significant increase in iNOS mRNA and protein in the lungs and heart after the administration of 5 mg/kg LPS intraperitoneally. The central role of iNOS in the hemodynamic alterations seen with LPS treatment has been described. For example, MacMicking et al. (19) found that wild-type mice had a 65% decrease in MAP 210 min after 1 mg/kg LPS administration, whereas the same dose of LPS had no significant effect on MAP in iNOS^–/– mice. Hallemeesch et al. (13) found that conscious iNOS^–/– mice had no decrease in blood pressure following 10 mg/kg ip LPS, whereas wild-type mice had a significant fall in blood pressure. Likewise, Carnio et al. (5) also found that iNOS^–/– mice, unlike wild-type mice, did not have a fall in MAP following LPS. Interestingly, although iNOS^–/– mice are protected from septic shock, they are not protected from inflammatory injury or mortality. In fact, iNOS^–/– mice are more susceptible to bacterial and viral pathogens than are wild-type mice (19, 22), demonstrating the critical role of early iNOS upregulation in host defense following infection. In this regard, one might speculate that MKP-1-deficient mice would initially fight off infection more effectively with the upregulation of iNOS but would then be highly likely to develop septic shock due to iNOS overproduction.

The role of iNOS in the fall in MAP following low-dose LPS is further supported by the data using the nonspecific NOS inhibitor L-NAME. When L-NAME was given 60 min after the administration of LPS, there was no significant fall in MAP or exNO at either 120 or 150 min after an LPS administration in Mkp-1^–/– mice. Interestingly, there was a significant increase in MAP starting 30 min after L-NAME and a significant fall in exNO starting 60 min after L-NAME in the wild-type mice. A hypertensive response to L-NAME in wild-type rodents has been well documented.

The putative iNOS-specific inhibitor aminoguanidine given 60 min after LPS also prevented the fall in MAP and increase in exNO in Mkp-1^–/– mice. In Wistar rats, aminoguanidine in one study (33) prevented LPS-induced hypotension, whereas in another study (24) it had little effect on LPS-induced hypotension. In mice, aminoguanidine has been shown to decrease plasma nitrates and improve mortality following LPS treatment (15, 31). In our study, aminoguanidine given to hypotensive Mkp-1^–/– mice (i.e., 150 min after LPS challenge) restored MAP and exNO values, a finding that is consistent with our hypothesis and demonstrates the importance of iNOS induction in the hypotensive response following LPS challenge. Although a study of a double knockout mouse, wherein both MKP-1 and iNOS are knocked out (Mkp-1^–/–iNOS^–/–), may give a more definitive answer than using putative-specific pharmacological inhibitors for iNOS, those studies are beyond the scope of this article. It should be noted that when aminoguanidine was given to wild-type mice after 150 min (when the LPS-induced response was established in the Mkp-1^–/– mice), there was a clear trend (although it did not reach statistical significance) toward a dose-dependent increase in MAP and there was a decrease in exNO. Indeed, when all of the individual exNO versus MAP points for the wild-type animals studied with increasing doses of aminoguanidine after LPS challenge were plotted (Fig. 7C), there was a significant inverse correlation between exNO and MAP. We did find detectable iNOS protein in the wild-type mice, although in a smaller quantity than in the Mkp-1^–/– mice. This finding is consistent with the notion that in the wild-type mice treated with low-dose LPS, there was a role for iNOS induction in the regulation of systemic blood pressure and exNO, although it was smaller than that seen in the Mkp-1^–/– mice.

The hemodynamic and exNO findings demonstrate the central role that MKP-1 plays in limiting iNOS protein production following LPS stimulation. These results are compatible with our previous studies wherein 1.5 mg/kg ip LPS resulted in greater plasma nitrite/nitrate levels and lower tail-cuff systolic blood pressure in Mkp-1^–/– than in wild-type mice (36). MKP-1 prefers JNK and p38 as substrates, and in cultured macrophages, MKP-1 peaks 60 min after LPS stimulation and correlates with JNK and p38 dephosphorylation (8, 29, 35). Deficiency in MKP-1 results in greater and/or more sustained p38 and JNK activities due to less efficient dephosphorylation of these kinases (8, 35). Interestingly, Kan et al. (16) found that the LPS-induced iNOS mRNA induction in the lungs of mice was inhibited by pretreatment with the p38 inhibitor SB-203580. Moreover, it has recently been reported that LPS stimulates iNOS expression via the activation of NF-B in RAW264.7 macrophages (9) and that p38 activation is involved in this signaling pathway (10). Thus alterations in p38 and JNK phosphorylation are likely to be directly responsible for the observed increases in iNOS protein levels in Mkp-1^–/– mice, possible through a process that also involves NF-B signaling.

Interestingly, the levels of eNOS protein rose significantly after LPS treatment in the aorta of the Mkp-1^–/– mice, whereas eNOS protein levels did not change significantly in the aorta of wild-type mice. Our findings in wild-type mice are consistent with previous studies examining eNOS protein levels after LPS challenge (11, 12). The role of the MAPK in alterations in eNOS protein levels has not been extensively studied. It has been found in cell culture models that stimuli that upregulate MAPK activation also increase eNOS protein levels (17, 32). Our data suggest that in the presence of the normal phosphatase activity of MKP-1, there is no increase in aortic eNOS levels following LPS; however, when the normal phosphatase activity of MKP-1 is removed, there are elevated levels of aortic eNOS protein following LPS challenge, although further studies are necessary to elucidate the exact role of MKP-1 in eNOS induction due to LPS challenge. However, the increased levels of eNOS protein may contribute to the LPS-induced fall in MAP and increase in exNO seen in the MKP-1-deficient mice after LPS challenge. The data from the experiments where aminoguanidine was given 150 min after LPS (Fig. 7) support this concept, since although the aminoguanidine did reverse the fall in MAP and the increase in exNO in the Mkp-1^–/– mice, the MAP was lower and exNO was greater in the Mkp-1^–/– than in the wild-type mice.

In conclusion, we found that the fall in MAP in Mkp-1^–/– mice following low-dose endotoxin administration was due to iNOS induction, resulting in increased NO production. Although there are many protein phosphatases that can dephosphorylate p38 and JNK, MKP-1 is clearly the primary phosphatase responsible for the attenuation of these kinases. A critical physiological function of MKP-1 during gram-negative bacterial infection is to limit iNOS induction following endotoxin exposure, thereby preventing the development of septic shock. Thus MKP-1 may represent a novel target to be explored for the development of new therapies for septic shock. Furthermore, we speculate that polymorphisms in the Mkp-1 gene may underlie the susceptibility of some patients to develop septic shock following gram-negative sepsis.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was supported by National Heart, Lung, and Blood Institute Grant HL-075261 (to L. D. Nelin) and the National Institute of Allergy and Infectious Diseases Grants AI-057798 and AI-068956 (to Y. Liu).

	ACKNOWLEDGMENTS

 
We thank Bristol-Myers Squibb Pharmaceutical Research Institute for providing the Mkp-1 knockout mice.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. D. Nelin, The Research Institute at Nationwide Children's Hospital, 700 Children's Dr. W203, Columbus, OH 43205 (e-mail: Leif.Nelin{at}nationwidechildrens.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Adding LC, Agvald P, Andersson LI, Jonzon B, Hoogstraate J, Gustafsson LE. Direct gas measurements indicate that the novel cyclooxygenase inhibitor AZD3582 is an effective nitric oxide donor in vivo. Br J Pharmacol 145: 679–687, 2005.[CrossRef][Web of Science][Medline]
2. Agvald P, Hammer L, Gustafsson LE. Nitroglycerin-patch induced tolerance is associated with reduced ability of nitroglycerin to increase exhaled nitric oxide. Vascul Pharmacol 43: 449–457, 2005.[CrossRef][Web of Science][Medline]
3. Albiger B, Dahlberg S, Henriques-Normark B, Normark S. Role of the innate immune system in host defense against bacterial infections: focus on the Toll-like receptors. J Intern Med 261: 511–528, 2007.[CrossRef][Web of Science][Medline]
4. Almeida JA, Riordan SM, Liu J, Galhenage S, Kim R, Bihari D, Wegner EA, Cranney GB, Thomas PS. Deleterious effect of nitric oxide inhibition in chronic hepatopulmonary syndrome. Eur J Gastroenterol Hepatol 19: 341–346, 2007.[CrossRef][Medline]
5. Carnio EC, Stabile AM, Batalhao ME, Silva JS, Antunes-Rodrigues J, Branco LGS, Magder S. Vasopressin release during endotoxaemic shock in mice lacking inducible nitric oxide synthase. Eur J Appl Physiol 450: 390–394, 2005.
6. Carter BW, Chicoine LG, Nelin LD. L-Lysine decreases nitric oxide production and increases vascular resistance in lungs isolated from lipopolysaccharide treated neonatal pigs. Pediatr Res 55: 979–987, 2004.[CrossRef][Web of Science][Medline]
7. Chan ED, Riches DWH. IFN- + LPS induction of iNOS is modulated by ERK, JNK/SAPK, and p38^mapk in a mouse macrophage cell line. Am J Physiol Cell Physiol 280: C441–C450, 2001.[Abstract/Free Full Text]
8. Chen P, Li J, Barnes J, Kokkonen GC, Lee JC, Liu Y. Restraint of proinflammatory cytokine biosynthesis by mitogen-activated protein kinase phosphatase-1 in lipopolysaccharide-stimulated macrophages. J Immunol 169: 6408–6416, 2002.[Abstract/Free Full Text]
9. Deng WG, Wu KK. Regulation of inducible nitric oxide synthase expression by p300 and p50 acetylation. J Immunol 171: 6581–6588, 2003.[Abstract/Free Full Text]
10. Dorfman K, Carrasco D, Gruda M, Ryan C, Lira SA, Bravo R. Disruption of the erp/mkp-1 gene does not affect mouse development: normal MAP kinase activity in ERP/MKP-1-deficient fibroblasts. Oncogene 13: 925–931, 1996.[Web of Science][Medline]
11. Garrean S, Gao XP, Brovkovych V, Shimizu J, Zhao YY, Vogel SM, Malik AB. Caveolin-1 regulates NF-kappaB activation and lung inflammatory response to sepsis induced by lipopolysaccharide. J Immunol 177: 4853–4860, 2006.[Abstract/Free Full Text]
12. Gupta A, Sharma AC. Despite minimal hemodynamic alterations endotoxemia modulates NOS and p38-MAPK phosphorylation via metalloendopeptidases. Mol Cell Biochem 265: 47–56, 2004.[CrossRef][Web of Science][Medline]
13. Hallemeesch MM, Janssen BJA, de Jonge WJ, Soeters PB, Lamers WH, Deutz NEP. NO production by cNOS and iNOS reflects blood pressure changes in LPS-challenged mice. Am J Physiol Endocrinol Metab 285: E871–E875, 2003.[Abstract/Free Full Text]
14. Hesse M, Modolell M, La Flamme AC, Schito M, Fuentes JM, Cheever AW, Pearce EJ, Wynn TA. Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism. J Immunol 167: 6533–6544, 2001.[Abstract/Free Full Text]
15. Iskit AB, Guc MO. The timing of endothelin and nitric oxide inhibition affects survival in a mice model of septic shock. Eur J Pharmacol 414: 281–287, 2001.[CrossRef][Web of Science][Medline]
16. Kan W, Zhao K, Jiang Y, Yan W, Huang Q, Wang J, Qin Q, Huang X, Wang S. 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 signal transduction of inducible nitric oxide synthase expression. Shock 21: 281–287, 2004.[CrossRef][Web of Science][Medline]
17. Klinge CM, Blankenship KA, Risinger KE, Bhatnagar S, Noisin EL, Sumanasekera WK, Zhao L, Brey DM, Keynton RS. Resveratrol and estradiol rapidly activate MAPK signaling through estrogen receptors alpha and beta in endothelial cells. J Biol Chem 280: 7460–7468, 2005.[Abstract/Free Full Text]
18. Liu Y, Shepherd EG, Nelin LD. MAP kinase phosphatases—regulating the immune response. Nature Rev Immunol 7: 202–212, 2007.[CrossRef][Web of Science][Medline]
19. MacMicking JD, Nathan C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, Stevens K, Xie Q, Sokol K, Hutchinson N, Chen H, Mudgett JS. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81: 641–650, 1995.[CrossRef][Web of Science][Medline]
20. Malleske DT, Rogers LK, Velluci SM, Young TL, Park MS, Long DW, Welty SE, Smith CV, Nelin LD. Hyperoxia increases hepatic arginase expression and ornithine production in mice. Toxicol Appl Pharmacol 215: 109–117, 2006.[CrossRef][Web of Science][Medline]
21. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 348: 1546–1554, 2003.[Abstract/Free Full Text]
22. Mashimo H, Goyal RK. Lessons from genetically engineered animals models IV. Nitric oxide synthase gene knockout mice. Am J Physiol Gastrointest Liver Physiol 277: G745–G750, 1999.[Abstract/Free Full Text]
23. Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol 1: 135–145, 2001.[CrossRef][Medline]
24. Metcalf K, Jungersten L, Lisander B. Effective inhibition of nitric oxide production by aminoguanidine does not reverse hypotension in endotoxaemic rats. Acta Anaesthesiol Scand 46: 17–23, 2002.[CrossRef][Web of Science][Medline]
25. Nelin LD, Wang X, Zhao Q, Chicoine LG, Young TL, Hatch DM, English BK, Liu Y. MKP-1 switches arginine metabolism from nitric oxide synthase to arginase following endotoxin challenge. Am J Physiol Cell Physiol 293: C632–C640, 2007.[Abstract/Free Full Text]
26. Nelin LD, Thomas CJ, Dawson CA. The effect of hypoxia on nitric oxide production in the neonatal pig lung. Am J Physiol Heart Circ Physiol 271: H8–H14, 1996.[Abstract/Free Full Text]
27. Pedoto A, Nandi J, Yang Z, Wang J, Bosco G, Oler A, Hakim TS, Camporesi EM. Beneficial effect of hyperbaric oxygen pretreatment on lipopolysaccharide-induced shock in rats. Clin Exp Pharmacol Physiol 30: 482–488, 2003.[CrossRef][Web of Science][Medline]
28. Pedoto A, Wang J, Tassiopoulos AK, Hakim TS, Yang Z, Camporesi EM. Hypotension during septic shock does not correlate with exhaled nitric oxide in anesthetized rat. Shock 17: 427–432, 2002.[CrossRef][Web of Science][Medline]
29. Shepherd EG, Zhao Q, Welty SE, Hansen TN, Smith CV, Liu Y. The function of mitogen-activated protein kinase phosphatase-1 in peptidoglycan-stimulated macrophages. J Biol Chem 279: 54023–54031, 2004.[Abstract/Free Full Text]
30. Taylor DR, Pijnenburg MW, Smith AD, De Jongste JC. Exhaled nitric oxide measurements: clinical application and interpretation. Thorax 61: 817–827, 2006.[Abstract/Free Full Text]
31. Tunctan B, Uludag O, Altug S, Abacioglu N. Effects of nitric oxide synthase inhibition in lipopolysaccharide-induced sepsis in mice. Pharmacol Rev 38: 405–411, 1998.[CrossRef]
32. Uchiba M, Okajima K, Oike Y, Ito Y, Fukudome K, Isobe H, Suda T. Activated protein C induces endothelial cell proliferation by mitogen-activated protein kinase activation in vitro and angiogenesis in vivo. Circ Res 95: 34–41, 2004.[Abstract/Free Full Text]
33. Wu CC, Chen SJ, Szabo C, Thiemermann C, Vane JR. Aminoguanidine attenuates the delayed circulatory failure and improves survival in rodent models of endotoxic shock. Br J Pharmacol 114: 1666–1672, 1995.[Web of Science][Medline]
34. Zanotti S, Kumar A, Kumar A. Cytokine modulation in sepsis and septic shock. Expert Opin Investig Drugs 11: 1061–1075, 2002.[CrossRef][Web of Science][Medline]
35. Zhao Q, Shepherd EG, Manson ME, Nelin LD, Sorokin A, Liu Y. The role of mitogen-activated protein kinase phosphatase-1 in the response of alveolar macrophages to lipopolysaccharides: attenuation of proinflammatory cytokine biosynthesis via feedback control of p38. J Biol Chem 280: 8101–8108, 2005.[Abstract/Free Full Text]
36. Zhao Q, Wang X, Nelin LD, Yao Y, Matta R, Manson ME, Baliga RS, Meng X, Smith CV, Bauer JA, Chang CH, Liu Y. MAP kinase phosphatase-1 controls innate immune responses and suppresses endotoxic shock. J Exp Med 203: 131–140, 2006.[Abstract/Free Full Text]

This article has been cited by other articles:

				X. Wang, Q. Zhao, R. Matta, X. Meng, X. Liu, C.-G. Liu, L. D. Nelin, and Y. Liu Inducible Nitric-oxide Synthase Expression Is Regulated by Mitogen-activated Protein Kinase Phosphatase-1 J. Biol. Chem., October 2, 2009; 284(40): 27123 - 27134. [Abstract] [Full Text] [PDF]

				C. M. Kinney, U. M. Chandrasekharan, L. Yang, J. Shen, M. Kinter, M. S. McDermott, and P. E. DiCorleto Histone H3 as a novel substrate for MAP kinase phosphatase-1 Am J Physiol Cell Physiol, February 1, 2009; 296(2): C242 - C249. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 294/4/H1621    most recent 01008.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Calvert, T. J. Articles by Nelin, L. D. Search for Related Content PubMed PubMed Citation Articles by Calvert, T. J. Articles by Nelin, L. D.