FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2049920954> ?p ?o ?g. }

• W2049920954 endingPage "10046" @default.
• W2049920954 startingPage "10040" @default.
• W2049920954 abstract "The expression of inducible nitric-oxide synthase (iNOS) and subsequent “high-output” nitric oxide (NO) production underlies the systemic hypotension, inadequate tissue perfusion, and organ failure associated with septic shock. Therefore, modulators of iNOS expression and activity, both endogenous and exogenous, are important in determining the magnitude and time course of this condition. We have shown previously that NO from the constitutive endothelial NOS (eNOS) is necessary to obtain maximal iNOS expression and activity following exposure of murine macrophages to lipopolysaccharide (LPS). Thus, eNOS represents an important regulator of iNOS expression in vitro. Herein, we validate this hypothesis in vivo using a murine model of sepsis. A temporal reduction in iNOS expression and activity was observed in LPS-treated eNOS knock-out (KO) mice as compared with wild-type animals; this was reflected in a more stable hemodynamic profile in eNOS KO mice during endotoxaemia. Furthermore, in human umbilical vein endothelial cells, LPS leads to the activation of eNOS through phosphoinositide 3-kinase- and Akt/protein kinase B-dependent enzyme phosphorylation. These data indicate that the pathogenesis of sepsis is characterized by an initial eNOS activation, with the resultant NO acting as a co-stimulus for the expression of iNOS, and therefore highlight a novel pro-inflammatory role for eNOS. The expression of inducible nitric-oxide synthase (iNOS) and subsequent “high-output” nitric oxide (NO) production underlies the systemic hypotension, inadequate tissue perfusion, and organ failure associated with septic shock. Therefore, modulators of iNOS expression and activity, both endogenous and exogenous, are important in determining the magnitude and time course of this condition. We have shown previously that NO from the constitutive endothelial NOS (eNOS) is necessary to obtain maximal iNOS expression and activity following exposure of murine macrophages to lipopolysaccharide (LPS). Thus, eNOS represents an important regulator of iNOS expression in vitro. Herein, we validate this hypothesis in vivo using a murine model of sepsis. A temporal reduction in iNOS expression and activity was observed in LPS-treated eNOS knock-out (KO) mice as compared with wild-type animals; this was reflected in a more stable hemodynamic profile in eNOS KO mice during endotoxaemia. Furthermore, in human umbilical vein endothelial cells, LPS leads to the activation of eNOS through phosphoinositide 3-kinase- and Akt/protein kinase B-dependent enzyme phosphorylation. These data indicate that the pathogenesis of sepsis is characterized by an initial eNOS activation, with the resultant NO acting as a co-stimulus for the expression of iNOS, and therefore highlight a novel pro-inflammatory role for eNOS. The systemic hypotension, organ failure, and morbidity that occur during bacterial sepsis are associated with the expression of inducible nitric-oxide synthase (iNOS) 1The abbreviations used are: iNOS, inducible nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; LPS, lipopolysaccharide; KO, knock-out; WT, wild-type; TNF, tumor necrosis factor; HUVEC, human umbilical vein endothelial cell; PI3K, phosphoinositide 3-kinase; MABP, mean arterial blood pressure; PBS, phosphate-buffered saline.1The abbreviations used are: iNOS, inducible nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; LPS, lipopolysaccharide; KO, knock-out; WT, wild-type; TNF, tumor necrosis factor; HUVEC, human umbilical vein endothelial cell; PI3K, phosphoinositide 3-kinase; MABP, mean arterial blood pressure; PBS, phosphate-buffered saline. and excessive production of nitric oxide (NO). This is evidenced by the increased levels of nitrite (NO–2) and nitrate (NO–3; stable metabolites of NO) measured in the plasma of septic patients (1Evans T. Carpenter A. Kinderman H. Cohen J. Circ. Shock. 1993; 41: 77-81PubMed Google Scholar, 2Gomez-Jimenez J. Salgado A. Mourelle M. Martin M.C. Segura R.M. Peracaula R. Moncada S. Crit. Care Med. 1995; 23: 253-258Crossref PubMed Scopus (197) Google Scholar), inflammation-induced iNOS expression (3Anstey N.M. Weinberg J.B. Hassanali M.Y. Mwaikambo E.D. Manyenga D. Misukonis M.A. Arnelle D.R. Hollis D. McDonald M.I. Granger D.L. J. Exp. Med. 1996; 184: 557-567Crossref PubMed Scopus (363) Google Scholar, 4Thoenes M. Forstermann U. Tracey W.R. Bleese N.M. Nussler A.K. Scholz H. Stein B. J. Mol. Cell. Cardiol. 1996; 28: 165-169Abstract Full Text PDF PubMed Scopus (88) Google Scholar), and by the ability of selective iNOS inhibitors to restore blood pressure in experimental models of sepsis and reverse hypotension in human endotoxaemia (5Wray G.M. Millar C.G. Hinds C.J. Thiemermann C. Shock. 1998; 9: 329-335Crossref PubMed Scopus (114) Google Scholar, 6Tunctan B. Uludag O. Altug S. Abacioglu N. Pharmacol. Res. 1998; 38: 405-411Crossref PubMed Scopus (66) Google Scholar, 7Petros A. Lamb G. Leone A. Moncada S. Bennett D. Vallance P. Cardiovasc. Res. 1994; 28: 34-39Crossref PubMed Scopus (454) Google Scholar, 8Petros A. Bennett D. Vallance P. Lancet. 1991; 338: 1557-1558Abstract PubMed Scopus (758) Google Scholar, 9Wright C.E. Rees D.D. Moncada S. Cardiovasc. Res. 1992; 26: 48-57Crossref PubMed Scopus (559) Google Scholar, 10Rees D.D. Monkhouse J.E. Cambridge D. Moncada S. Br. J. Pharmacol. 1998; 124: 540-546Crossref PubMed Scopus (79) Google Scholar). Inducible NOS is effectively absent under physiological conditions but is expressed in many cell types in response to pro-inflammatory cytokines and lipopolysaccharides (LPS); in accord, induction of this NOS isoform is necessary for “high output” NO production and cytostatic and cytotoxic effects that facilitate host defense (11MacMicking J. Xie Q.W. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3458) Google Scholar). While iNOS expression is essential to combat bacterial infection, the sustained overproduction of NO is deleterious to the host, as exemplified by the cardiovascular dysfunction during sepsis. Since iNOS is regulated primarily at a transcriptional level (12Xie Q.W. Kashiwabara Y. Nathan C. J. Biol. Chem. 1994; 269: 4705-4708Abstract Full Text PDF PubMed Google Scholar, 13Xie Q. Nathan C. J. Leukocyte Biol. 1994; 56: 576-582Crossref PubMed Scopus (470) Google Scholar, 14de Vera M.E. Shapiro R.A. Nussler A.K. Mudgett J.S. Simmons R.L. Morris Jr., S.M. Billiar T.R. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1054-1059Crossref PubMed Scopus (356) Google Scholar), a better understanding of the mechanisms involved in iNOS mRNA and protein expression should lead to improved treatment for endotoxaemia and other inflammatory cardiovascular disorders. In contrast to iNOS, a potential role for endothelial NOS (eNOS) in the pathogenesis of sepsis is unsubstantiated. Initial studies suggested that eNOS knock-out (KO) animals respond to endotoxin in an identical manner to wild-type (WT) littermates (15Shesely E.G. Maeda N. Kim H.S. Desai K.M. Krege J.H. Laubach V.E. Sherman P.A. Sessa W.C. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13176-13181Crossref PubMed Scopus (783) Google Scholar), and more recently, mice overexpressing eNOS were shown to express equivalent levels of iNOS mRNA and generate similar levels of plasma NO–2 and NO–3 (an index of iNOS activity) to control animals (16Julou-Schaeffer G. Gray G.A. Fleming I. Schott C. Parratt J.R. Stoclet J.C. Am. J. Physiol. 1990; 259: H1038-H1043PubMed Google Scholar) in response to LPS. However, we have recently demonstrated that eNOS acts in a pro-inflammatory manner in immune cells by facilitating iNOS expression in response to endotoxin, such that in bone marrow-derived macrophages from eNOS KO mice, iNOS expression and activity are less than 50% of WT controls (17Connelly L. Jacobs A.T. Palacios-Callender M. Moncada S. Hobbs A.J. J. Biol. Chem. 2003; 278: 26480-26487Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). This pro-inflammatory effect of eNOS-derived NO occurs, at least in part, via a modulation of the activity of the transcription factor, nuclear factor κB (NF-κB) (18Connelly L. Palacios-Callender M. Ameixa C. Moncada S. Hobbs A.J. J. Immunol. 2001; 166: 3873-3881Crossref PubMed Scopus (284) Google Scholar). Further studies give support to a potential pro-inflammatory role for eNOS. For instance, a reduction in both basal and LPS-induced tumor necrosis factor-α (TNF-α) production occurs in neonatal mouse cardiomyocytes from eNOS KO mice (19Peng T. Lu X. Lei M. Feng Q. J. Biol. Chem. 2003; 278: 8099-8105Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and in HeLa and microvascular endothelial cells, TNF-α has been shown to activate eNOS (20Barsacchi R. Perrotta C. Bulotta S. Moncada S. Borgese N. Clementi E. Mol. Pharmacol. 2003; 63: 886-895Crossref PubMed Scopus (67) Google Scholar, 21Kawanaka H. Jones M.K. Szabo I.L. Baatar D. Pai R. Tsugawa K. Sugimachi K. Sarfeh I.J. Tarnawski A.S. Hepatology. 2002; 35: 393-402Crossref PubMed Scopus (40) Google Scholar). These studies not only demonstrate the importance of eNOS for the up-regulation of pro-inflammatory protein expression but also illustrate the auto-regulation of NOS expression by NO, since iNOS-derived NO is known to feedback and inhibit the expression and activity of eNOS (22Chauhan S.D. Seggara G. Vo P.A. MacAllister R.J. Hobbs A.J. Ahluwalia A. FASEB J. 2003; 17: 773-775Crossref PubMed Scopus (114) Google Scholar, 23Buga G.M. Griscavage J.M. Rogers N.E. Ignarro L.J. Circ. Res. 1993; 73: 808-812Crossref PubMed Scopus (325) Google Scholar). In combination, these data give rise to the hypothesis that eNOS plays a key role in the expression of iNOS and the pathogenesis of sepsis. In accord, in the present study we have employed eNOS KO mice to examine the role of this NOS isoform in an in vivo conscious animal model of sepsis (10Rees D.D. Monkhouse J.E. Cambridge D. Moncada S. Br. J. Pharmacol. 1998; 124: 540-546Crossref PubMed Scopus (79) Google Scholar). Moreover, we have used human umbilical vein endothelial cells (HUVECs) and murine bone marrow-derived macrophages (BMDMØs) (from WT and eNOS KO mice) to dissect the mechanisms underlying LPS-stimulated eNOS activity. Herein, we demonstrate that eNOS plays an important role in facilitating iNOS expression in vivo during endotoxaemia with a marked reduction in iNOS protein observed in the liver, lung, heart, and aorta of eNOS KO mice as compared with WT, in addition to an accompanying reduction in plasma levels of NO–2 and NO–3. The impaired iNOS expression and activity are accompanied by a more stable hemodynamic profile and reduced mortality in response to LPS. We also show that in HUVECs and BMDMØs, LPS activates eNOS via a phosphoinositide 3-kinase (PI3K)- and Akt/protein kinase B-dependent mechanism. Reagents—Deguelin was purchased from Alexis (Nottingham, UK). 3-Isobutyl-1-methylxanthine and LY294002 were purchased from Calbiochem (Nottingham, UK). All drugs were resuspended in dimethyl sulfoxide (Me2SO) such that the final concentration of Me2SO in culture did not exceed 0.001%. Salmonella typhosa LPS was purchased from Sigma. All other reagents were obtained from Sigma unless stated otherwise. LPS Treatment and Tissue Homogenization—S. typhosa LPS (12.5 mg/kg, intravenously) was administered to 6–8-week-old male WT (C57/BL6; 15–25 g) and eNOS KO mice via the tail vein. After the appropriate time interval the animals were euthanized and tissues and/or blood collected. Tissues were snap frozen in liquid nitrogen and stored at –80 °C before transfer into whole-cell homogenization buffer (50 mm Tris-HCl, 150 mm NaCl, 1% Triton X-100, 2 mm EDTA, 8 mm EGTA, and 1 μg/ml benzamidine, leupeptin, antipain, and aprotinin). Tissues were homogenized, centrifuged (13,793 × g, 4 °C, 15 min), and the supernatants retained for subsequent analysis. Blood samples were centrifuged (13,397 × g, room temperature, 5 min) and plasma retained. Plasma samples were then spun for a further 30 min through Microcon centrifugal filter devices (YM3, 3000-Da cutoff; Millipore, Watford, UK) before assaying. Measurement of Mean Arterial Blood Pressure—Mice (WT and eNOS KO; male; 15–25 g) were anesthetized briefly with isolflurane (2%). A cannula (internal diameter: 0.28 mm, outer diameter: 0.61 mm) was implanted in the carotid artery and jugular vein and tunnelled subcutaneously to emerge at the nape of the neck. The cannulae were connected to a swivel/tether system secured to the mouse using four silk sutures. This enabled the mouse, on recovery, to have unimpeded movement around the cage with free access to food and water. Both lines were flushed continuously with 0.1 ml/h heparinized-saline (1:1,000). Following recovery from surgery, mean arterial blood pressure (MABP) was measured (P23 XL transducer, Viggo-Spectramed, Oxnard, CA) and recorded onto a precalibrated PowerLab system (ADInstuments, Castle Hill, New South Wales, Australia), as described previously (10Rees D.D. Monkhouse J.E. Cambridge D. Moncada S. Br. J. Pharmacol. 1998; 124: 540-546Crossref PubMed Scopus (79) Google Scholar). Twenty-four hours later, LPS (12.5 mg/kg) was administered via the jugular vein, and the MABP was monitored over an 18-h period. HUVEC Culture—HUVECs (Promocell, Heidelberg, Germany) were cultured in endothelial cell basal medium supplemented with 0.4% endothelial cell growth supplement/heparin, 2% fetal bovine serum, 0.1 ng/ml epidermal growth factor, 1 μg/ml hydrocortisone, 1 ng/ml basic fibroblast factor, 50 ng/ml amphotericin B, and 50 μg/ml gentamicin (all from Promocell). Cells were passaged by trypsinization with cells of passage 5 and below used for experimentation. For phosphorylation experiments, cells were seeded in 10 cm culture dishes and incubated at 37 °C in a humidified incubator containing 5% CO2 in air until ∼90% confluent. At 4 h before stimulation (to minimize basal Akt and eNOS phosphorylation), medium was removed and replaced with endothelial cell basal medium supplemented with 0.1% fetal bovine serum, 1 μg/ml hydrocortisone, 50 ng/ml amphotericin B, and 50 μg/ml gentamicin (all from Promocell). After activation with LPS (1 μg/ml) for appropriate times, the medium was removed, and cells were washed with PBS before addition of phospho-homogenization buffer (10 mm Tris, 50 mm NaCl, 30 mm NaPPi, 2 mm EDTA, 50 mm NaF, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4 and 1 μg/ml benzamidine, leupeptin, antipain, and aprotinin). Homogenates were scraped into 1.5-ml tubes and then centrifuged (13,793 × g, 5 min, 4 °C) and supernatants retained for subsequent analysis. Bone Marrow-derived Macrophage Culture—BMDMØs were extracted and cultured as described previously (17Connelly L. Jacobs A.T. Palacios-Callender M. Moncada S. Hobbs A.J. J. Biol. Chem. 2003; 278: 26480-26487Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). For Akt phosphorylation experiments, cells were seeded into 6-well culture plates (4 × 106 cells/well) in 2 ml of medium (RPMI 1640 medium with 25 mm HEPES supplemented with 10% New Zealand fetal bovine serum, 2 mm glutamine, 100 units/ml streptomycin, and 100 μg/ml penicillin; all from Invitrogen, Paisley, UK) and incubated overnight at 37 °C in a humidified incubator containing 5% CO2 in air. After activation with LPS (100 ng/ml) for appropriate times, the medium was removed, and cells were washed with PBS before addition of phospho-homogenization buffer (as described above). For experiments to study the effect of deguelin or LY294002 on Akt phosphorylation, cells were preincubated with deguelin (1 μm), LY294002 (10 μm), or Me2SO (control) for 30 min before addition of LPS (100 ng/ml). Homogenates were scraped into 1.5-ml tubes and then centrifuged (13,793 × g, 5 min, 4 °C) and the supernatants retained for subsequent analysis. For experiments to study the effect of deguelin or LY294002 on iNOS expression, cells were seeded into 12-well plates (1 × 106 cells/well) in 1 ml of medium and incubated overnight at 37 °C in a humidified incubator containing 5% CO2 in air. Cells were preincubated with deguelin (1 μm), LY294002 (10 μm), or Me2SO (control) for 30 min before addition of LPS (100 ng/ml). After 9 h medium was removed, and cells were washed with PBS before addition of whole-cell homogenization buffer (as above). Homogenates were scraped into 1.5-ml tubes and then centrifuged (13,793 × g, 5 min, 4 °C) and the supernatants retained for subsequent analysis. Western Blot Analysis—Protein concentrations were determined by BCA Protein Assay (Pierce). Equal volumes of protein were subjected to 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The proteins were transferred to nitrocellulose membranes with a semidry blotter (Amersham Biosciences, Buckinghamshire, UK) at 0.8 mA/cm2 for 60 min. The membranes were then incubated with shaking in 5% milk in wash buffer (PBS, 0.1% Tween 20) for 1 h at room temperature. The membranes were then incubated overnight at 4 °C, with gentle shaking with primary antibody (αiNOS, BD Biosciences, αAkt, αphospho-Akt (Ser473), αeNOS, αphospho-eNOS (Ser1177), all from Cell Signaling) diluted 1:2,000 (iNOS) or 1:500 (all other antibodies) in 1% milk in wash buffer. The membrane was washed six times (5 min/wash) and then incubated, with gentle shaking for 1 h at room temperature, with horseradish peroxidase-conjugated αrabbit IgG (Dako, Glostrup, Denmark) diluted 1:1,000 (tissue and HUVEC) or 1:2,000 (BMDMØs) in 1% milk in wash buffer. The membrane was washed as described before, and proteins were visualized using enhanced chemiluminescence (Amersham Biosciences). Bands were quantified by densitometry (NIH image). Cyclic GMP Assay—BMDMØs were seeded into 6-well culture plates (4 × 106 cells/well) in 2 ml of medium and incubated overnight at 37 °C in a humidified incubator containing 5% CO2 in air. Medium was aspirated and replaced with medium containing 1 mm 3-isobutyl-1-methylxanthine and cells preincubated with deguelin (1 μm), LY294002 (10 μm), or Me2SO (control) for 30 min. Cells were stimulated with LPS (100 ng/ml) for 30 min at which point the medium was aspirated and intracellular cGMP concentrations measured using a commercially available enzyme immunoassay (Amersham Biosciences) according to the manufacturer's instructions. Nitrite and Nitrate Measurement—Plasma samples were analyzed for NO2− and NO3− using chemiluminescence as described previously (24Ignarro L.J. Fukuto J.M. Griscavage J.M. Rogers N.E. Byrns R.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8103-8107Crossref PubMed Scopus (750) Google Scholar). Briefly, samples and standards containing NO2− and NO3− were first reduced to NO, which was then quantified after reaction with ozone using a nitric oxide analyzer (NOA 280, Sievers). To determine total NO2− and NO3− concentrations, collectively termed “NOx,” samples were added to 0.1 m vanadium (III) chloride in 1 m hydrochloric acid refluxing at 90 °C under nitrogen. Data Analysis—All statistical analysis was performed using Graph Pad Prism (Graph Pad Software Inc., San Diego, CA). Densitometric analyses were performed using NIH Image. All data are plotted graphically as mean values with vertical bars representing standard error of the mean (S.E.). A Student's t test was used to assess differences between individual experimental conditions. Analysis of variance was used to compare temporal changes in MABP in WT and eNOS KO animals. A probability (p) value of <0.05 was taken as an appropriate level of significance. Hemodynamic Profile in WT and eNOS KO Mice in Response to LPS—The administration of LPS (12.5 mg/kg) led to a biphasic effect on the MABP of WT mice (n = 6). There was an immediate but transient drop in MABP following administration of LPS (∼10 mm Hg) that recovered to basal levels after 4–5 h. At this time point, the MABP fell again and continued to decline for the duration of the experiment (18 h) or until the animal died (two out of six; Fig. 1). In contrast, in eNOS KO mice the blood pressure remained stable immediately after injection of LPS, and while at 6 h the MABP began to fall slightly, this only deviated from basal levels by <20% (Fig. 1). Plasma NOx Accumulation in Response to LPS—Resting plasma [NOx] were higher in WT compared with eNOS KO animals, as would be expected in animals lacking eNOS-derived NO. Administration of LPS (12.5 mg/kg) resulted in an increase in plasma [NOx] in both WT and eNOS KO mice (Fig. 2). The increases in plasma [NOx] closely mirrored the sustained fall in MABP observed in WT animals. However, the plasma NOx levels in eNOS KOs were significantly reduced in comparison with WT animals, such that at 12 h there was an approximate 50% reduction in plasma [NOx] in eNOS KO animals (Fig. 2). Effect of eNOS Deletion on iNOS Expression in Response to LPS—There was no detectable iNOS expression in tissues from control mice either WT or eNOS KO (saline-treated). Administration of LPS (12.5 mg/kg) led to a time-dependent increase in the expression of iNOS protein in the liver, lung, heart, and aorta of WT and eNOS KO mice (Fig. 3). However, in tissues from eNOS KO mice there was a significantly lower level of iNOS protein expression after treatment with LPS, with the maximum differential effect observed between 9 and 12 h (Fig. 3). The peak iNOS protein levels were 69.1 ± 10.5% in the liver, 51.3 ± 22.9% in the lung, 52.6 ± 30.0% in the heart, and 58.1 ± 1.8% in the aorta of those observed in WT animals (p < 0.05 for each). Akt and eNOS Phosphorylation in Response to LPS in HUVECs—Since our in vivo investigations had revealed a key role for eNOS in facilitating iNOS expression, we investigated whether the PI3K/Akt pathway was responsible for activation of eNOS in response to LPS in HUVECs. HUVECs were incubated in basal medium without growth factors for 4 h (to minimize basal Akt and eNOS phosphorylation) followed by stimulation with LPS (1 μg/ml) for 60 min and Akt phosphorylation assessed by Western blot. LPS caused a transient Akt phosphorylation that peaked at ∼15 min and returned to basal levels within 60 min (Fig. 4). Since Akt was phosphorylated in response to LPS stimulation, the downstream phosphorylation of eNOS was also studied under the same conditions. After 4-h incubation in basal medium, cells were stimulated with LPS (1 μg/ml) for 60 min and eNOS phosphorylation assessed by Western blot. In a pattern that mirrored Akt activation, eNOS was transiently phosphorylated after LPS treatment with peak levels of phosphorylation occurring between 15 and 30 min after activation (Fig. 5). Akt and eNOS Phosphorylation in Response to LPS in Murine BMDMØs—Since HUVECs do not express iNOS in response to inflammatory stimuli in vitro, and so that it was possible to exploit KO technology, we used BMDMØs to demonstrate a link between LPS-induced PI3K/Akt and eNOS phosphorylation with iNOS expression. Cells were treated with LPS (100 ng/ml) and phosphorylation of Akt monitored over 60 min by Western blot. A transient increase in phosphorylated Akt was observed in response to LPS treatment, with a peak effect observed between 15 and 30 min (Fig. 6). Since BMDMØs express very low levels of eNOS (some 100-fold less than endothelial cells (17Connelly L. Jacobs A.T. Palacios-Callender M. Moncada S. Hobbs A.J. J. Biol. Chem. 2003; 278: 26480-26487Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar)), it was not possible to determine whether eNOS phosphorylation occurred as a result of LPS-stimulated Akt activation in these cells. However, a similar pattern of Akt phosphorylation was observed in eNOS KO cells in response to LPS (data not shown) confirming that the activation of Akt occurs upstream of eNOS. To investigate whether the phosphorylation and activation of Akt was linked with eNOS activation and the expression of iNOS, WT and eNOS KO BMDMØs were activated with LPS (100 ng/ml) for 9 h in the presence and absence of the PI3K/Akt inhibitors deguelin (1 μm) (25Chun K.H. Kosmeder J.W. Sun S. Pezzuto J.M. Lotan R. Hong W.K. Lee H.Y. J. Natl. Cancer Inst. 2003; 95: 291-302Crossref PubMed Scopus (251) Google Scholar) or LY294002 (10 μm) and iNOS expression determined (NOx accumulation in the culture medium was below detection limits of the chemiluminescence system at this time point; data not shown). In the presence of deguelin or LY294002, iNOS expression in WT cells was 32.4 ± 8.2% (Fig. 7) and 64.9 ± 5.4% (Fig. 7) of the expression levels observed with LPS only (control). However, in eNOS KO cells, both deguelin and LY294002 failed to cause a significant reduction in LPS-stimulated iNOS expression (Fig. 7). Indeed, in the presence of deguelin and LY294002, iNOS expression in response to LPS was essentially identical in cells from WT and eNOS KO mice (Fig. 7), confirming the importance of eNOS-derived NO to iNOS expression. Effect of Deguelin and LY294002 on Akt Phosphorylation in Response to LPS in WT and eNOS KO BMDMØs—To confirm that the effects of deguelin and LY294002 on iNOS expression were due specifically to inhibition of Akt activity, BMDMØs were activated with LPS (100 ng/ml) in the presence and absence of deguelin (1 μm) or LY294002 (10 μm) and Akt phosphorylation determined by Western blot after 15 min. In the presence of deguelin, Akt phosphorylation was reduced to 60.5 ± 10.9% of the level observed with LPS only (control) in WT cells and 35.9 ± 11.3% of control levels in eNOS KO cells (Fig. 8). Accordingly, in the presence of LY294002, Akt phosphorylation was reduced to 49.0 ± 10.3% of the level observed with LPS only (control) in WT cells and 32.3 ± 6.5% of control levels in eNOS KO cells (Fig. 8). Thus, while both deguelin and LY294002 have an inhibitory effect on Akt phosphorylation in both WT and eNOS KO, this was only manifested as an inhibition of iNOS activity in cells from WT animals. Effect of Deguelin and LY294002 on cGMP Accumulation in Response to LPS in WT and eNOS KO BMDMØs—Since the concentrations of NO–2 and NO–3 generated by BMDMØ eNOS are too low to be detected by chemiluminescence analysis, we measured cGMP accumulation as an index of eNOS activity to further demonstrate that stimulation of the PI3K/Akt pathway underlies eNOS activation by LPS. We have previously demonstrated that stimulation of BMDMØs with LPS (100 ng/ml) leads to an accumulation of intracellular cGMP in WT but not eNOS KO cells (17Connelly L. Jacobs A.T. Palacios-Callender M. Moncada S. Hobbs A.J. J. Biol. Chem. 2003; 278: 26480-26487Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). In this case, cGMP accumulation in response to LPS stimulation was measured in the presence or absence of the PI3K/Akt inhibitors deguelin (1 μm) and LY294002 (10 μm). The stimulation of cells with LPS led to an ∼40% increase in intracellular cGMP concentration as compared with background (Fig. 9); this increase was reversed in the presence of deguelin (1 μm) or LY294002 (10 μm; Fig. 9). This study demonstrates that eNOS-derived NO plays a key role in facilitating iNOS expression in LPS-induced endotoxaemia in mice in vivo. In eNOS KO animals, the systemic hypotension and mortality associated with sepsis were markedly reduced, and this was mirrored by a reduction in iNOS expression in the heart, lung, liver, and aorta and a significantly smaller production of NO (as assessed by NOx accumulation). Furthermore, the activation of eNOS by LPS to enable maximal iNOS expression is triggered by the PI3K/Akt pathway, as has been identified previously to couple shear stress to NO production by the endothelium (26Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3022) Google Scholar, 27Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2214) Google Scholar). These observations extend our previous finding that eNOS-derived NO is necessary to achieve peak iNOS expression in LPS-treated murine bone marrow-derived macrophages (17Connelly L. Jacobs A.T. Palacios-Callender M. Moncada S. Hobbs A.J. J. Biol. Chem. 2003; 278: 26480-26487Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and suggest that this constitutive NOS isoform plays an important role in the pathogenesis of sepsis. Moreover, the findings assign a potential pro-inflammatory role to eNOS, an enzyme that has been classically considered to act in an anti-inflammatory manner via NO-mediated inhibition of smooth muscle proliferation, leukocyte recruitment, and platelet aggregation (28Moncada S. Ann. N. Y. Acad. Sci. 1997; 811: 60-67Crossref PubMed Scopus (299) Google Scholar). To determine whether eNOS is important for the hemodynamic changes (and consequent aberrations in tissue perfusion) observed during septic shock, conscious WT and eNOS KO animals were exposed to LPS for 18 h. During this time, WT animals displayed a rapid, transient reduction in systemic blood pressure (within 1 h) that recovered almost entirely by 5 h and then dropped steadily throughout the remainder of the investigation. In marked contrast, the eNOS KO animals did not show an initial hypotensive response to LPS administration, and while systemic blood pressure in these animals waned over the length of the experiment, this was considerably more stable than that observed in WT animals and did not deviate by more than ∼20% from baseline values. These data establish two important principles with respect to eNOS activity in response to LPS. First, endotoxin causes an immediate up-regulation of eNOS activity, which is manifested as a hypotensive effect that is both rapid in onset and short-lived; such a hypotensive effect of LPS has been demonstrated previously in WT animals (10Rees" @default.
• W2049920954 created "2016-06-24" @default.
• W2049920954 creator A5012678680 @default.
• W2049920954 creator A5057189590 @default.
• W2049920954 creator A5063653314 @default.
• W2049920954 date "2005-03-01" @default.
• W2049920954 modified "2023-10-12" @default.
• W2049920954 title "Resistance to Endotoxic Shock in Endothelial Nitric-oxide Synthase (eNOS) Knock-out Mice" @default.
• W2049920954 cites W1591881963 @default.
• W2049920954 cites W1607892649 @default.
• W2049920954 cites W1678800844 @default.
• W2049920954 cites W1966120931 @default.
• W2049920954 cites W1972724464 @default.
• W2049920954 cites W2003369052 @default.
• W2049920954 cites W2008293086 @default.
• W2049920954 cites W2011670951 @default.
• W2049920954 cites W2012100682 @default.
• W2049920954 cites W2022568707 @default.
• W2049920954 cites W2024283446 @default.
• W2049920954 cites W2024389091 @default.
• W2049920954 cites W2024786586 @default.
• W2049920954 cites W2028354465 @default.
• W2049920954 cites W2029583951 @default.
• W2049920954 cites W2030815090 @default.
• W2049920954 cites W2033379487 @default.
• W2049920954 cites W2039406268 @default.
• W2049920954 cites W2041034845 @default.
• W2049920954 cites W2042452396 @default.
• W2049920954 cites W2046093469 @default.
• W2049920954 cites W2049406640 @default.
• W2049920954 cites W2053289392 @default.
• W2049920954 cites W2054463113 @default.
• W2049920954 cites W2074460964 @default.
• W2049920954 cites W2078890177 @default.
• W2049920954 cites W2091499945 @default.
• W2049920954 cites W2093994279 @default.
• W2049920954 cites W2097208307 @default.
• W2049920954 cites W2101392591 @default.
• W2049920954 cites W2110748980 @default.
• W2049920954 cites W2115929723 @default.
• W2049920954 cites W2121858640 @default.
• W2049920954 cites W2129250847 @default.
• W2049920954 cites W2132575805 @default.
• W2049920954 cites W2149200346 @default.
• W2049920954 cites W2160042367 @default.
• W2049920954 cites W2161266035 @default.
• W2049920954 doi "https://doi.org/10.1074/jbc.m411991200" @default.
• W2049920954 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15647265" @default.
• W2049920954 hasPublicationYear "2005" @default.
• W2049920954 type Work @default.
• W2049920954 sameAs 2049920954 @default.
• W2049920954 citedByCount "145" @default.
• W2049920954 countsByYear W20499209542012 @default.
• W2049920954 countsByYear W20499209542013 @default.
• W2049920954 countsByYear W20499209542014 @default.
• W2049920954 countsByYear W20499209542015 @default.
• W2049920954 countsByYear W20499209542016 @default.
• W2049920954 countsByYear W20499209542017 @default.
• W2049920954 countsByYear W20499209542018 @default.
• W2049920954 countsByYear W20499209542019 @default.
• W2049920954 countsByYear W20499209542021 @default.
• W2049920954 countsByYear W20499209542022 @default.
• W2049920954 countsByYear W20499209542023 @default.
• W2049920954 crossrefType "journal-article" @default.
• W2049920954 hasAuthorship W2049920954A5012678680 @default.
• W2049920954 hasAuthorship W2049920954A5057189590 @default.
• W2049920954 hasAuthorship W2049920954A5063653314 @default.
• W2049920954 hasBestOaLocation W20499209541 @default.
• W2049920954 hasConcept C104317684 @default.
• W2049920954 hasConcept C112243037 @default.
• W2049920954 hasConcept C126322002 @default.
• W2049920954 hasConcept C134018914 @default.
• W2049920954 hasConcept C181199279 @default.
• W2049920954 hasConcept C182704531 @default.
• W2049920954 hasConcept C185592680 @default.
• W2049920954 hasConcept C2777622882 @default.
• W2049920954 hasConcept C2778326061 @default.
• W2049920954 hasConcept C2781300812 @default.
• W2049920954 hasConcept C2910236225 @default.
• W2049920954 hasConcept C2992676626 @default.
• W2049920954 hasConcept C519581460 @default.
• W2049920954 hasConcept C55493867 @default.
• W2049920954 hasConcept C71924100 @default.
• W2049920954 hasConcept C86803240 @default.
• W2049920954 hasConceptScore W2049920954C104317684 @default.
• W2049920954 hasConceptScore W2049920954C112243037 @default.
• W2049920954 hasConceptScore W2049920954C126322002 @default.
• W2049920954 hasConceptScore W2049920954C134018914 @default.
• W2049920954 hasConceptScore W2049920954C181199279 @default.
• W2049920954 hasConceptScore W2049920954C182704531 @default.
• W2049920954 hasConceptScore W2049920954C185592680 @default.
• W2049920954 hasConceptScore W2049920954C2777622882 @default.
• W2049920954 hasConceptScore W2049920954C2778326061 @default.
• W2049920954 hasConceptScore W2049920954C2781300812 @default.
• W2049920954 hasConceptScore W2049920954C2910236225 @default.
• W2049920954 hasConceptScore W2049920954C2992676626 @default.
• W2049920954 hasConceptScore W2049920954C519581460 @default.
• W2049920954 hasConceptScore W2049920954C55493867 @default.

• next