© 2000 American Heart Association, Inc.
UltraRapid Communication |
iNOS Gene Expression Modulates Microvascular Responsiveness in Endotoxin-Challenged Mice
Presented in abstract form at the 32nd annual meeting of the Association for Academic Surgery, Seattle, Wash, November 19–22, 1998.
From the Departments of Anesthesiology (Anesthesiology Research Unit) (W.A.B., L.S.P., V.B., C.S.), Surgery (Cellular Injury and Adaptation Laboratory) (L.S.P., J.P.C.), and Molecular Biology and Pharmacology (W.A.B.), Washington University School of Medicine, St. Louis, Mo; and the Department of Surgery (V.E.L.), University of Virginia Health Sciences Center, Charlottesville, Va.
Correspondence to Dr Walter A. Boyle, Washington University School of Medicine, Box 8054, 660 S Euclid Ave, St. Louis, MO 63110. E-mail boylew{at}notes.wustl.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—Septic shock is characterized by vasodilation and decreased responsiveness to vasoconstrictors. Recent studies suggest this results from nitric oxide (NO) overproduction after expression of the calcium-independent isoform of NO synthase (iNOS) in smooth muscle cells. However, direct evidence linking iNOS (NOS2) expression and decreased microvascular responsiveness after septic stimuli is lacking. In the present study, we determined the effect of bacterial lipopolysaccharide (LPS, 20 mg/kg, IP) on smooth muscle contraction and endothelial relaxation in mesenteric resistance arteries from wild-type and iNOS knockout mice. Four hours after challenge with LPS or saline in vivo, concentration-dependent responses to norepinephrine (NE) and acetylcholine (NE+ACh) were measured in cannulated, pressurized vessels ex vivo. In vessels from wild-type mice, NE-induced contraction was markedly impaired after LPS, and pretreatment with the iNOS inhibitor aminoguanidine (AG) partly restored the NE contraction. In contrast, NE contraction in microvessels from iNOS knockout mice was unaffected by LPS. ACh-induced relaxation was unaffected by LPS in vessels from either genotype. These data provide direct evidence that iNOS gene expression mediates the LPS-induced decrease in microvascular responsiveness to vasoconstrictors. Moreover, the observation that AG did not fully restore NE contraction after LPS, whereas iNOS gene deficiency did, suggests that iNOS expression plays a central role in the development of the NO-independent effect of LPS on microvascular responsiveness. Finally, our data indicate that LPS or iNOS expression has little effect on endothelium-dependent relaxation, and eNOS activity does not appear to play a role in the decreased smooth muscle responsiveness after LPS in this model. The full text of this article is available at http://www.circresaha.org.
Key Words: transgenic mice • inducible nitric oxide synthase • microvascular • lipopolysaccharide • sepsis
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Evidence to date strongly suggests nitric oxide (NO) is the key mediator of the vasodilation and catecholamine-resistant hypotension that occurs in septic shock.1 2 Earlier investigations implicated NO, and it was suggested that endothelial NO release may be involved.3 However, recent studies indicate that NO production by endothelial cells is decreased in septic shock,4 5 and the vascular overproduction of NO seems to result from de novo synthesis of the calcium-independent isoform of NO synthase (iNOS or NOS2) in smooth muscle cells.6 7 8 Bacterial wall lipopolysaccharide (LPS) as well as several host factors that circulate in high concentrations in septic shock, including tumor necrosis factor (TNF-
),
interleukin-1ß, and interferon 
, either alone or in combination,
seem capable of inducing iNOS expression in vascular smooth muscle
cells.6 7 8 Moreover, pharmacological
inhibitors of iNOS attenuated both the NO
overproduction and hypotension in septic animals as well as the
catecholamine hyporesponsiveness observed in isolated
vessels.1 9 Recently, investigators have turned to genetically altered mice with null mutations of the iNOS allele (iNOS knockouts) to additionally define the importance of iNOS expression in septic shock. MacMicking et al10 reported that LPS treatment in iNOS knockouts did not elicit the marked hypotension observed in wild-type mice, and early mortality (<4 hours) was eliminated in the knockout mice. Similarly, Hollenberg et al11 demonstrated improved responsiveness of cremasteric arterioles to topically applied norepinephrine (NE) and decreased 2-day mortality in iNOS knockout mice in a fluid- and antibiotic-treated cecal ligation and puncture model of septic shock. Using echocardiography, Nicholson et al12 have additionally noted that iNOS knockout animals do not display the LPS-induced increases in cardiac output evident in wild-type controls, consistent with the absence of iNOS-dependent NO-induced vasodilation in the knockout animals. However, iNOS deficiency presented a survival disadvantage in that model, and, in contrast to the results of Hollenberg et al,11 treatment with fluid had negative effects on both survival and cardiac output in the knockouts. Interestingly, these effects were reversed by a platelet-activating factor inhibitor.12 Finally, Laubach et al13 recently pointed out that LPS-induced mortality of iNOS knockout mice may also relate to gender, with increased mortality in female knockout mice. In summary, studies using iNOS knockout mice indicate that the effect of iNOS deficiency on mortality seem to be influenced by the model of sepsis used (eg, LPS vs cecal ligation and puncture); gender and genetic background of the iNOS knockout animals; administration of anesthetics, fluids, or antibiotics; and potentially other factors, such as platelet-activating factor.11 12 13 14 15 Nevertheless, it is clear from these studies that iNOS expression contributes significantly to the hemodynamic compromise after septic stimuli.10 11 12 Moreover, ablation of these adverse hemodynamic responses in the knockout animals is associated with decreased mortality in some models.10 11 Indeed, the variable effects of iNOS gene deficiency on mortality may reflect the precarious balance between the negative effects of iNOS-produced NO on vascular reactivity and the positive effects of NO on the immune or inflammatory responses.14 15
Clearly, the most important changes in vascular control during septic shock occur at the microvascular level. However, few studies have evaluated the effects of septic stimuli in resistance arteries, and little is known about the direct involvement of iNOS gene product in vascular responsiveness of microvessels during septic shock. Decreased responsiveness to vasoconstricting agonists has been demonstrated in small arteries after septic stimuli, but previous studies using NOS inhibitors present a conflicting picture regarding the contribution of NO or iNOS expression to these changes.9 16 It has recently been reported that iNOS gene deficiency prevented LPS-induced hyporesponsiveness to vasoconstrictors in large (carotid) arteries,17 but the effects of iNOS gene deficiency on responses of isolated resistance arteries to septic stimuli have not been characterized. In the present study, iNOS knockout and congenic control mice were challenged with intraperitoneal injections of LPS in vivo, and the vasoconstrictor response to NE was characterized ex vivo in cannulated, pressurized mesenteric resistance arteries. We also studied the effect of LPS on the response to the endothelium-dependent vasodilator acetylcholine (ACh) and the effect of aminoguanidine (AG), a relatively specific pharmacological inhibitor of iNOS, on NE and ACh responses.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Animals and Experimental Setup
All animal experiments were conducted in accordance with institutional guidelines. Fourteen C57BL/6 wild-type (Harlan Sprague- Dawley, Indianapolis, Ind) and 14 eighth and ninth generation age- and gender-matched congenic C57BL/6 mice lacking the iNOS gene (knockouts)18 were randomized to receive Escherichia coli endotoxin (055:B5 LPS, 20 mg/kg, IP) or an equal volume of normal saline (IP). Four hours later, small distal branches of the mesenteric artery (100 to 200 µm in diameter) were isolated under halothane anesthesia and studied using methods previously described.19
Genotyping
Mouse-tail genomic DNA was isolated (QIAamp System, Qiagen), and
polymerase chain reactions (PCRs) were performed on each sample
(GeneAmp, Perkin Elmer Corp) using pairs of normal (MNO20C
[5'-GAGGAGAGAGATCCGATTTAGAGTCTTGG-3']/MNO20D
[5'-TGAAGCCATGACCTTTCGATTAGCATGG-3']) and knockout (MNO20A
[5'-ACAGCCTCAGAGTCCTTCATGAAGCACATGC-3']/NEO4 [5'CAGAAGAAC
TCGTCAA-GAAGGCGATAGAAGG-3']) primers. MNO20C/MNO20D
yielded a 400-bp product from the wild-type gene, and MNO20A/NEO4
yielded a 1200-bp product from the disrupted gene.
Consistent with disruption of the iNOS gene, DNA from iNOS
knockout animals failed to amplify with the iNOS-specific primer after
PCR in contrast to tail DNA from wild-type mice (Figure 1
).
|
Western Analysis of iNOS
Liver (100 mg) was obtained 4 hours after IP LPS or saline,
frozen in liquid nitrogen, and homogenized in 250
mmol/L sucrose, 10 mmol/L HEPES, and 1 mmol/L EDTA containing
1 µg/mL of aprotinin, leupeptin, antipain, benzamidine, chymostatin,
and pepstatin A; 5 µg/mL trypsin inhibitor; and 87
µg/mL phenylmethyl sulfonyl fluoride. A total of 500
µL of the homogenate was centrifuged at 4°C in
25 µL of 20% SDS for 15 minutes to pellet unsolubilized protein. The
supernatant was centrifuged (100 000g) at 4°C for
1 hour and stored at -70°C. Protein quantification, electrophoresis,
immunoblotting, and visualization were accomplished, as
described previously,20 using 1:1000 rabbit
polyclonal antimouse NOS2 (iNOS) antibody (Santa Cruz Biotechnology,
Inc).
Experimental Protocol
Vessels were cannulated and equilibrated for 1 hour in
HEPES-buffered saline (mmol/L): NaCl 135,
NaHCO3 2.6,
Na2HPO4 0.34,
KH2PO4 0.44, KCl 5,
CaCl2 1.6, MgSO4 1.17, EDTA
0.025, HEPES 10, and glucose 5.5 (pH 7.35 at 35°C). An image
analysis system was used to measure vessel diameter changes at
constant transmural pressure (40 mm Hg) during application of
increasing doses of NE
(10-7 to
3x10-5 mol/L). This was
followed by addition of ACh
(10-7 to
3x10-5 mol/L) to the NE
(3x10-5
mol/L)-constricted vessels (ie, NE+ACh). Vessels were then incubated
for 60 minutes in HEPES-buffered saline containing 300 µmol/L
AG, and measurements of vascular contraction and relaxation during NE
and NE+ACh applications were repeated.
Statistics
Data for NE and ACh responses for each genotype were
compared using 2-way ANOVA (concentration/treatments: LPS, saline,
LPS+AG, saline+AG) with post hoc testing at each NE or ACh
concentration using the Bonferroni test or Student’s paired
t test (for responses before and after AG). Comparison of
responses between the iNOS knockout and wild-type mice were
analyzed using 2-way ANOVA (genotype/treatments) at
each NE or ACh concentration with post hoc Bonferroni testing, as
needed. Significance was defined as P<0.05 for all of the
tests. In this study, n indicates the number of blood vessels, and N
indicates the number of animals. Statistics were calculated using
n.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
iNOS Expression
As shown in Figure 2
, immunoblots of liver homogenates using an
antimouse iNOS polyclonal antibody displayed evidence of iNOS protein
after LPS treatment in the wild-type mice, with no evidence of
LPS-induced iNOS protein expression in the iNOS knockout animals.
|
NE-Mediated Contraction
NE produced concentration-dependent contraction of mesenteric
arterioles in vitro (Figure 3). In
vessels from wild-type mice, LPS treatment significantly reduced the
NE-induced contractions compared with those from the saline-treated
controls (Figure 3A). The maximal contraction (at 30
µmol/L NE) was reduced from 26.69±1.25% (percent decrease in
outside diameter; mean±SEM) to 9.45±1.31% in vessels from control
and LPS-treated wild-type mice, respectively. Statistical
analysis of the data from the wild-type mice (2-way ANOVA: NE
concentration/treatments) indicated significant effects of both NE
concentration and treatment (LPS, saline, LPS+AG, saline+AG) as well as
a significant interaction effect. Post hoc testing (Bonferroni test)
indicated that LPS treatment resulted in significantly decreased NE
contractions at all NE doses >1 µmol/L. Treatment with AG, a
relatively specific pharmacological inhibitor of iNOS,
resulted in partial recovery of NE contractions in the LPS-treated
group, and post hoc testing (Student’s paired t tests)
indicated significant increases in NE contractions after AG at all NE
doses 
1 µmol/L. The maximal NE contraction increased
significantly from 9.45±1.31% to 17.05±2.67% after AG in
LPS-treated vessels from wild-type mice. In vessels from the
saline-treated wild-type mice, AG also produced some small increases in
NE contractions; however, these effects were significant only at
submaximal NE concentrations, and AG had no significant effect on the
maximal NE contraction in these vessels (Figure 3A).
|
,
n=10, N=7 for each genotype) or endotoxin (
, n=11 to 12, N=7
for each genotype). Contractile responses are presented
as percent decrease (mean±SEM) in outside diameter (OD). After the
initial measurement of NE contraction (and ACh relaxation [see
below]), AG (300 µmol/L) was added to the vessel bath, and
measurements were repeated (
, 
). C, Comparison of maximal
NE-induced contractions (at 30 µmol/L NE) is shown in vessels
from each genotype and treatment group. *Significant effect of
AG treatment (Student’s paired t test). 
Significant
difference between saline- and endotoxin-treatment groups within
genotype (Bonferroni test). S indicates significant difference
between wild-type and knockout groups (Student’s t
test). P<0.05.
In contrast to the effect of LPS in wild-type mice, treatment with LPS
did not significantly affect NE-induced contractions in vessels from
iNOS knockout mice (Figure 3B
). The maximal contractions (at
30 µmol/L NE) were 26.35±2.42% and 29.79±1.56% in vessels
from saline- and LPS-treated iNOS knockout animals, respectively.
Statistical analysis (2-way ANOVA: NE concentration/treatments)
indicated only a significant NE concentration effect, with no
significant effect of treatments (LPS, saline, LPS+AG, saline+AG). AG
treatment of these vessels did not increase the NE-induced contraction
significantly, and, in fact, AG produced a small decrease in the NE
response of vessels from the LPS-treated knockout animals (Figure 3B).
Statistical analysis of the influence of genotype
on response to LPS treatment (2-way ANOVA: treatments/genotype)
at each NE dose indicated significant effects of both treatments (LPS,
saline, LPS+AG, saline+AG) and genotype (wild-type or knockout)
as well as significant interaction effects at several NE
concentrations, including the maximum (30 µmol/L). Post hoc
testing (Bonferroni test) indicated that the maximum NE response of
vessels from LPS-treated wild-type mice was significantly lower than
that of the knockout animals both before and after AG treatment (Figure 3C).
ACh-Mediated Relaxation
Application of the endothelium-dependent
vasodilator ACh to the maximal NE-constricted vessels induced
concentration-dependent relaxation (Figure 4). The maximum ACh relaxation (at
30 µmol/L ACh) was 55±6% and 57±8% (percent of NE
contraction) in vessels from the saline-treated wild-type and knockout
animals, respectively. Statistical analysis of the ACh
relaxation data (2-way ANOVA: ACh concentration/treatments) did reveal
significant ACh concentration and treatment (LPS, saline, LPS+AG,
saline+AG) effects in vessels from both genotypes. However,
post hoc analysis at each ACh concentration did not reveal any
significant effect of LPS treatment on ACh response in mice from either
genotype. The only significant differences identified were
significant decreases in the ACh relaxation after pretreatment with AG
(Figures 4A and 4B). Thus, although LPS treatment significantly
reduced the maximal NE contraction in the wild-type mice (see above),
there was no effect of LPS treatment on the ACh relaxation produced in
vessels from either genotype. AG reduced ACh responses in both
saline- and LPS-treated vessels from wild-type and knockout animals
(Figure 4).
|
Analysis of the ACh response as a function of genotype
(2-way ANOVA: treatments/genotype) indicated no significant
effect of genotype on maximal ACh relaxation. A significant
treatment effect (LPS, saline, LPS+AG, saline+AG) was identified by
this analysis, but post hoc testing again revealed only a
significant inhibition of ACh relaxation by AG in both
genotypes, as previously noted. There was no significant
difference in the maximal ACh-mediated relaxation between the two
genotypes treated with LPS or saline, before or after AG
(Figure 4C).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The major finding of the present study is that mesenteric microvessels from iNOS knockout mice do not display any impairment of NE-induced contraction after challenge with bacterial endotoxin (LPS). In contrast, there is marked impairment of NE contraction after LPS in vessels from wild-type animals. Consistent with these findings, the impaired NE contractions in microvessels from wild-type mice after LPS are improved after treatment with AG, a relatively specific inhibitor of iNOS. However, AG is not as effective as disruption of the iNOS gene in eliminating the effect of LPS. In addition, our data indicate that endothelial relaxation is not affected by LPS treatment in either the wild-type or iNOS knockout mice.
Exposure to LPS as well as other proinflammatory cytokines can stimulate expression of iNOS in vascular smooth muscle cells.6 7 8 However, although iNOS mRNA and iNOS protein have both been detected in blood vessels after LPS treatment, the functional significance of iNOS expression in microvessels has not been clear. Indirect approaches using pharmacological inhibitors of iNOS have suggested that iNOS expression in small blood vessels results in impaired responses to vasoconstrictors.4 21 However, the effectiveness of the NOS inhibitors at reversing the vascular hyporesponsiveness after septic stimuli has been variable, with some investigators indicating little or no effect.22 Nevertheless, our results using microvessels from iNOS knockout animals provide clear evidence that iNOS expression is critical for the development of the NE hyporesponsiveness that occurred in the mesenteric resistance vessels after exposure to LPS.
Similarly, it has been demonstrated recently that treatment with
antisense oligonucleotides to iNOS largely eliminated
the LPS-induced vascular hyporeactivity to NE in rat small mesenteric
arteries.23 Our results are also in agreement with those
from another recent report using carotid arteries from iNOS knockout
mice after challenge with LPS.17 In that study, the
carotid artery segments from the iNOS knockouts did not display
impairment of contractile responses to either
PGF2 or the thromboxane
A2 analog U-46619, whereas the responses in
artery segments from wild-type controls were significantly impaired.
The study also noted that AG restored the impaired contractile
responses of the carotid artery segments from the LPS-treated wild-type
mice.
As noted above, it is evident from our data and other previous reports that treatment with NOS inhibitors does not result in complete recovery of the vascular hyporesponsiveness after LPS or other septic stimuli.9 17 22 Indeed, this is of particular interest, because the hyporesponsiveness after LPS is completely eliminated by disruption of the iNOS gene or pretreatment with iNOS antisense oligonucleotides.17 23 One potential explanation for the inability of AG to fully block the vascular effects of iNOS expression, in our study, is incomplete inhibition of iNOS activity. As an inhibitor of iNOS activity, AG is approximately equipotent with other nonspecific NOS inhibitors, with published IC50 values of 5 to 50 µmol/L for inhibition of iNOS-induced nitrite accumulation, arginine to citrulline conversion, or NO production using a cGMP reporter system.24 25 At the 300-µmol/L concentration used in our study, AG would be predicted to inhibit iNOS activity by more than 90%.24 26 Duration of AG pretreatment can also be a factor, although with the dose used (300 µmol/L), steady-state conditions would be expected by the end of the 60-minute preincubation period.27 Thus, although incomplete blockade of iNOS activity by AG cannot be excluded entirely, available data suggest that this is not likely to account for the fact that AG produced only 50% recovery of the NE responsiveness in our study.
Alternatively, iNOS-produced NO may initiate some process that, once
developed, is no longer dependent on NO or sensitive to acutely
administered NOS inhibitors. Several recent studies
indicate that NO and the same septic stimuli that induce iNOS
expression also stimulate expression of inducible isoforms of
cyclooxygenase (COX-2)28 29 30 31 and heme
oxygenase (HO-1)32 33 34 in vascular tissue.
Moreover, vasodilatory products of COX-2 seem to contribute to
vasodilation in septic animals and man,17 35 and HO-1 is
the major cellular source of carbon monoxide (CO), which mimics many of
the actions of NO, including activation of guanylate
cyclase and vascular relaxation.36 Thus, the
iNOS-dependent but AG-insensitive vascular hyporesponsiveness after
septic stimuli observed in this and previous studies could arise from
NO-mediated increased COX-2 and HO-1 expression. Recent studies also
indicate that iNOS expression may stimulate cellular production
of TNF-,37 which may also contribute to NOS
inhibitor–insensitive vascular relaxation in septic
animals.38 39 40 MacMicking et al10 reported no
difference in TNF- levels between wild-type and iNOS knockout mice
after LPS. However, the genetic background and responses to LPS of
those mice and the ones used in our study have been noted
already.18 Indeed, several factors may play a role in the
NOS inhibitor–insensitive component of the LPS-induced
vascular hyporeactivity, and the list of potential mechanisms
presented here is not exhaustive.41 42 43 However,
our results and the other available data suggest that iNOS expression
may be at the origin of the vascular hyporesponsiveness after septic or
inflammatory stimuli.
Expression or activity of the constitutive endothelial NO synthase (eNOS or NOS3) has also been suggested to play some role in hypotension and vascular hyporesponsiveness after LPS treatment. In particular, some authors have demonstrated that the hypotension that occurs immediately after LPS injection may be related to increased eNOS activity.3 43 However, other recent studies indicate that eNOS activity is reduced after septic stimuli,4 44 and impaired endothelium-dependent relaxation occurs during septic shock.5 45 Additional data have also been presented indicating that the continuous generation of NO after induction of iNOS expression by inflammatory mediators results in downregulation of eNOS expression and decreased eNOS-generated NO production by endothelial cells.8 46 Because the LPS-induced vascular hyporesponsiveness in our study was completely eliminated in the iNOS knockout animals, our data do not support a role for increased eNOS activity in the vascular hyporesponsiveness after treatment with LPS. In addition, because endothelium-dependent relaxation by ACh is well-known to be eNOS-dependent,47 the lack of effect of LPS treatment or genotype on ACh responses suggests that LPS treatment or iNOS expression did not modulate eNOS expression or activity. Thus, a change in eNOS activity does not seem to play any role in the vascular hyporesponsiveness after LPS in this model. Interestingly, some recent data suggest that septic stimuli result in greater impairment of endothelium-dependent relaxation in large vessels48 than in resistance arterioles,49 suggesting that vessel size or bed may contribute to differences in the effects of septic stimuli on eNOS activity. Finally, we noted that AG produced significant inhibition of ACh relaxation. AG is known to be a relatively selective inhibitor of iNOS, but with reported IC50 values of 150 to 850 µmol/L for inhibition of eNOS or bNOS activity,24 25 26 some inhibition of eNOS would be expected at the AG concentration (300 µmol/L) used in our study. Given the well-described biological actions of AG in vascular tissue and other systems,50 another explanation for the effects of AG on ACh relaxation seems unlikely. It is possible, therefore, that a small portion of the recovery of NE responsiveness in the wild-type animals after AG was related to AG inhibition of eNOS. However, given the similarity of endothelium-dependent relaxation in the two genotypes and lack of effect of AG in the knockout animals, it seems unlikely that inhibition of eNOS activity contributed significantly to the recovery of LPS-induced NE hyporesponsiveness after AG in the wild-type mice.
In summary, we have used iNOS knockout mice to demonstrate the critical role of iNOS expression in the catecholamine hyporesponsiveness that develops in mesenteric microvessels after treatment with endotoxin. Our results suggest that iNOS-induced NO production is responsible for the development of both NOS inhibitor–sensitive and NOS inhibitor–insensitive effects of LPS on microvascular reactivity. Finally, our data indicate that endothelial function is well preserved after LPS treatment and that changes in eNOS activity play little role in LPS-induced vascular hyporesponsiveness in this model.
|
Acknowledgments |
|---|
This study was supported by grants from the National Institutes of Health (GM55849 to W.A.B) and the Society of Critical Care Medicine (Founders Grant for Training in Critical Care Research to J.P.C).
Received August 1, 2000; revision received September 6, 2000; accepted September 7, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Rees DD. Role of nitric oxide in the vascular dysfunction of septic shock. Biochem Soc Trans. 1995;23:1025–1029.[Medline] [Order article via Infotrieve]
2.
Cobb JP, Danner RL. Nitric oxide and septic shock.
JAMA. 1996;275:1192–1196.
3. Szabo C, Mitchell JA, Thiemermann C, Vane JR. Nitric oxide-mediated hyporeactivity to noradrenaline precedes the induction of nitric oxide synthase in endotoxin shock. Br J Pharmacol. 1993;108:786–792.[Medline] [Order article via Infotrieve]
4.
Hom GJ, Grant SK, Wolfe G, Bach TJ, MacIntyre DE,
Hutchinson NI. Lipopolysaccharide-induced hypotension and
vascular hyporeactivity in the rat: tissue analysis of nitric
oxide synthase mRNA and protein expression in the presence and absence
of dexamethasone,
NG-monomethyl-L-arginine or
indomethacin. J Pharmacol Exp Ther. 1995;272:452–459.
5. Zhou M, Wang P, Chaudry IH. Endothelial nitric oxide synthase is downregulated during hyperdynamic sepsis. Biochim Biophys Acta. 1997;1335:182–190.[Medline] [Order article via Infotrieve]
6. Sirsjo A, Soderkvist P, Sundqvist T, Carlsson M, Ost M, Gidlof A. Different induction mechanisms of mRNA for inducible nitric oxide synthase in rat smooth muscle cells in culture and in aortic strips. FEBS Lett. 1994;338:191–196.[Medline] [Order article via Infotrieve]
7. Griffiths MJ, Liu S, Curzen NP, Messent M, Evans TW. In vivo treatment with endotoxin induces nitric oxide synthase in rat main pulmonary artery. Am J Physiol. 1995;268(3 pt 1):L509–L518.
8.
Kessler P, Bauersachs J, Busse R, Schini-Kerth VB.
Inhibition of inducible nitric oxide synthase restores
endothelium-dependent relaxations in proinflammatory
mediator-induced blood vessels. Arterioscler Thromb Vasc
Biol. 1997;17:1746–1755.
9. Mitolo-Chieppa D, Serio M, Potenza MA, Montagnani M, Mansi G, Pece S, Jirillo E, Stoclet JC. Hyporeactivity of mesenteric vascular bed in endotoxin-treated rats. Eur J Pharmacol. 1996;309:175–182.[Medline] [Order article via Infotrieve]
10. MacMicking JD, Nathan C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, Stevens K, Xie QW, Sokol K, Hutchinson N, Homard C, Mudgett JS. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell. 1995;81:641–650.[Medline] [Order article via Infotrieve]
11.
Hollenberg SM, Broussard M, Osman J, Parrillo JE.
Increased microvascular reactivity and improved mortality in septic
mice lacking inducible nitric oxide synthase. Circ Res. 2000;86:774–778.
12. Nicholson SC, Hahn RT, Grobmyer SR, Brause JE, Hafner A, Potter S, Devereux RB, Nathan CF. Echocardiographic and survival studies in mice undergoing endotoxic shock: effects of genetic ablation of inducible nitric oxide synthase and pharmacologic antagonism of platelet-activating factor. J Surg Res. 1999;86:198–205.[Medline] [Order article via Infotrieve]
13. Laubach VE, Foley PL, Shockey KS, Tribble CG, Kron IL. Protective roles of nitric oxide and testosterone in endotoxemia: evidence from NOS-2-deficient mice. Am J Physiol. 1998;275(6 pt 2):H2211–H2218.
14. Gross SS, Kilbourn RG, Griffith OW. NO in septic shock: good, bad or ugly? Learning from iNOS knockouts. Trends Microbiol.. 1996;4:47–49.[Medline] [Order article via Infotrieve]
15. Nathan C. Inducible nitric oxide synthase: what difference does it make? J Clin Invest. 1997;100:2417–2423.[Medline] [Order article via Infotrieve]
16. Hollenberg SM, Cunnion RE, Zimmerberg J. Nitric oxide synthase inhibition reverses arteriolar hyporesponsiveness to catecholamines in septic rats. Am J Physiol. 1993;264(2 pt 2):H660–H663.
17. Gunnett CA, Chu Y, Heistad DD, Loihl A, Faraci FM. Vascular effects of LPS in mice deficient in expression of the gene for inducible nitric oxide synthase. Am J Physiol. 1998;275(2 pt 2):H416–H421.
18.
Laubach VE, Shesely EG, Smithies O, Sherman PA. Mice
lacking inducible nitric oxide synthase are not resistant to
lipopolysaccharide-induced death. Proc Natl Acad Sci
U S A. 1995;92:10688–10692.
19. Boyle WA III, Maher GM. Endothelium-independent vasoconstricting and vasodilating actions of halothane on rat mesenteric resistance blood vessels. Anesthesiology. 1995;82:221–225.[Medline] [Order article via Infotrieve]
20. DeMeester SL, Qiu Y, Buchman TG, Hotchkiss RS, Dunnigan K, Karl IE, Cobb JP. Nitric oxide inhibits stress-induced endothelial cell apoptosis. Crit Care Med. 1998;26:1500–1509.[Medline] [Order article via Infotrieve]
21. Bigaud M, Julou-Schaeffer G, Parratt JR, Stoclet JC. Endotoxin-induced impairment of vascular smooth muscle contractions elicited by different mechanisms. Eur J Pharmacol. 1990;190:185–192.[Medline] [Order article via Infotrieve]
22. Vromen A, Arkovitz MS, Zingarelli B, Salzman AL, Garcia VF, Szabo C. Low-level expression and limited role for the inducible isoform of nitric oxide synthase in the vascular hyporeactivity and mortality associated with cecal ligation and puncture in the rat. Shock. 1996;6:248–253.[Medline] [Order article via Infotrieve]
23. Hoque AM, Papapetropoulos A, Venema RC, Catravas JD, Fuchs LC. Effects of antisense oligonucleotide to iNOS on hemodynamic and vascular changes induced by LPS. Am J Physiol. 1998;275(3 pt 2):H1078–H1083.
24. Misko TP, Moore WM, Kasten TP, Nickols GA, Corbett JA, Tilton RG, McDaniel ML, Williamson JR, Currie MG. Selective inhibition of the inducible nitric oxide synthase by aminoguanidine. Eur J Pharmacol. 1993;233:119–125.[Medline] [Order article via Infotrieve]
25. Corbett JA, McDaniel ML. Selective inhibition of inducible nitric oxide synthase by aminoguanidine. Methods Enzymol. 1996;268:398–408.[Medline] [Order article via Infotrieve]
26. Wolff DJ, Lubeskie A. Aminoguanidine is an isoform-selective, mechanism-based inactivator of nitric oxide synthase. Arch Biochem Biophys. 1995;316:290–301.[Medline] [Order article via Infotrieve]
27. Bryk R, Wolff DJ. Mechanism of inducible nitric oxide synthase inactivation by aminoguanidine and L-N6-(1-iminoethyl)lysine. Biochemistry. 1998;37:4844–4852.[Medline] [Order article via Infotrieve]
28. Salvemini D, Settle SL, Masferrer JL, Seibert K, Currie MG, Needleman P. Regulation of prostaglandin production by nitric oxide: an in vivo analysis. Br J Pharmacol. 1995;114:1171–1178.[Medline] [Order article via Infotrieve]
29. Inoue T, Fukuo K, Morimoto S, Koh E, Ogihara T. Nitric oxide mediates interleukin-1-induced prostaglandin E2 production by vascular smooth muscle cells. Biochem Biophys Res Commun. 1993;194:420–424.[Medline] [Order article via Infotrieve]
30.
Salvemini D, Misko TP, Masferrer JL, Seibert K, Currie
MG, Needleman P. Nitric oxide activates
cyclooxygenase enzymes. Proc Natl Acad Sci
U S A. 1993;90:7240–7244.
31. Bishop-Bailey D, Larkin SW, Warner TD, Chen G, Mitchell JA. Characterization of the induction of nitric oxide synthase and cyclo-oxygenase in rat aorta in organ culture. Br J Pharmacol. 1997;121:125–133.[Medline] [Order article via Infotrieve]
32. Hartsfield CL, Alam J, Cook JL, Choi AM. Regulation of heme oxygenase-1 gene expression in vascular smooth muscle cells by nitric oxide. Am J Physiol. 1997;273(5 pt 1):L980–L988.
33.
Durante W, Kroll MH, Christodoulides N, Peyton KJ,
Schafer AI. Nitric oxide induces heme oxygenase-1 gene
expression and carbon monoxide production in vascular smooth
muscle cells. Circ Res. 1997;80:557–564.
34.
Yet SF, Pellacani A, Patterson C, Tan L, Folta SC,
Foster L, Lee WS, Hsieh CM, Perrella MA. Induction of heme
oxygenase-1 expression in vascular smooth muscle cells: a
link to endotoxic shock. J Biol Chem. 1997;272:4295–4301.
35.
Stoclet JC, Martinez MC, Ohlmann P, Chasserot S, Schott
C, Kleschyov AL, Schneider F, Andriantsitohaina R. Induction of nitric
oxide synthase and dual effects of nitric oxide and
cyclooxygenase products in regulation of
arterial contraction in human septic shock.
Circulation. 1999;100:107–112.
36. Sammut IA, Foresti R, Clark JE, Exon DJ, Vesely MJ, Sarathchandra P, Green CJ, Motterlini R. Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase-1. Br J Pharmacol. 1998;125:1437–1444.[Medline] [Order article via Infotrieve]
37.
Sanchez de Miguel L, de Frutos T, Gonzalez-Fernandez F,
del Pozo V, Lahoz C, Jimenez A, Rico L, Garcia R, Aceituno E, Millas I,
Gomez J, Farre J, Casado S, Lopez-Farre A. Aspirin inhibits inducible
nitric oxide synthase expression and tumour necrosis factor-
release by cultured smooth muscle cells. Eur J Clin
Invest. 1999;29:93–99.[Medline]
[Order article via Infotrieve]
38.
Baudry N, Vicaut E. Role of nitric oxide in effects of
tumor necrosis factor- on microcirculation in rat. J
Appl Physiol. 1993;75:2392–2399.
39.
Naziri W, Joshua IG. Role of tumor necrosis
factor- in small intestinal microcirculation. Am
Surg. 1998;64:203–209.[Medline]
[Order article via Infotrieve]
40.
Gardiner SM, Kemp PA, March JE, Woolley J, Bennett T.
The influence of antibodies to TNF- and IL-1ß on haemodynamic
responses to the cytokines, and to lipopolysaccharide,
in conscious rats. Br J Pharmacol. 1998;125:1543–1550.[Medline]
[Order article via Infotrieve]
41.
Carcillo JA, Litten RZ, Suba EA, Roth BL. Alterations
in rat aortic 1-adrenoceptors and
1-adrenergic stimulated
phosphoinositide hydrolysis in
intraperitoneal sepsis. Circ Shock. 1988;26:331–339.[Medline]
[Order article via Infotrieve]
42. Martinez MC, Muller B, Stoclet JC, Andriantsitohaina R. Alteration by lipopolysaccharide of the relationship between intracellular calcium levels and contraction in rat mesenteric artery. Br J Pharmacol. 1996;118:2118–2122.
43. Yoshikawa D, Shiga T, Saito S, Morita T, Imai T, Goto F. Platelet-activating factor receptor antagonist attenuates endotoxin-induced vascular hyporeactivity in the pithed rat. Eur J Pharmacol. 1998;342:241–245.[Medline] [Order article via Infotrieve]
44.
Graier WF, Myers PR, Rubin LJ, Adams HR, Parker
JL. Escherichia coli endotoxin inhibits agonist-mediated
cytosolic Ca2+ mobilization and nitric oxide
biosynthesis in cultured endothelial cells. Circ
Res. 1994;75:659–668.
45.
Peters TS, Lewis SJ. Lipopolysaccharide
inhibits acetylcholine- and nitric oxide-mediated vasodilation in vivo.
J Pharmacol Exp Ther. 1996;279:918–925.
46.
de Frutos T, de Miguel LS, Garcia-Duran M,
Gonzalez-Fernandez F, Rodriguez-Feo JA, Monton M, Guerra J, Farre J,
Casado S, Lopez-Farre A. NO from smooth muscle cells decreases NOS
expression in endothelial cells: role of TNF-.
Am J Physiol. 1999;277(4 pt 2):H1317–H1325.
47. Rubanyi GM. The role of endothelium in cardiovascular homeostasis and diseases. J Cardiovasc Pharmacol. 1993;22(suppl 4):S1–S14.
48. Young JS, Headrick JP, Berne RM. Endothelial-dependent and -independent responses in the thoracic aorta during endotoxic shock. Circ Shock. 1991;35:25–30.[Medline] [Order article via Infotrieve]
49. Baker CH, Sutton ET. Arteriolar endothelium-dependent vasodilation occurs during endotoxin shock. Am J Physiol. 1993;264(4 pt 2):H1118–H1123.
50. Nilsson BO. Biological effects of aminoguanidine: an update. Inflamm Res. 1999;48:509–515.[Medline] [Order article via Infotrieve]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||