1 Department of Medicine, University of Colorado Health Sciences Center, Denver 80262; 2 Department of Medicine, Baylor College of Medicine, Houston, Texas 77030; and 3 Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acute renal failure
(ARF) during sepsis is associated with increased nitric oxide (NO) and
oxygen radicals, including superoxide (O
sepsis; extracellular superoxide dismutase; lipopolysaccharide; kidney
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DURING ENDOTOXEMIA, reactive oxygen
species (ROS) have been shown to be increased in several species
(5, 16, 27). The injurious effects of ROS are attenuated,
at least in part, by antioxidants. Superoxide dismutase (SOD) is an
endogenous antioxidant which can scavenge superoxide
(O
In addition to increased generation of ROS, endotoxemia also results in
the induction of nitric oxide synthase (iNOS) and increased circulating
nitric oxide (NO) (9, 29). We hypothesized that renal
EC-SOD may be downregulated during endotoxemia, resulting in increased
O
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals. The experimental protocol was approved by the Animal Ethics Review Committee at the University of Colorado Health Sciences Center. C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Male mice aged 8-10 wk were used throughout the study. Mice were maintained on a standard rodent chow and had free access to water.
Materials. Chemicals were purchased from Sigma (St. Louis, MO) unless otherwise specified. Manganese (III) mesotetrakis(N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP) was a kind gift of Incara Pharmaceuticals (Research Triangle Park, NC).
Measurement of renal blood flow, glomerular filtration rate, and
mean arterial pressure.
The animals were anesthetized with pentobarbital sodium (60 mg/kg) and
placed on a thermostatically controlled surgical table. A tracheotomy
was performed, at which time a steady steam of 100% oxygen was blown
over the tracheal tube throughout the experiment. Catheters (custom
pulled from PE-250) were placed in the jugular vein for maintenance
infusion and the carotid artery for blood pressure determinations. The
kidney was exposed by a left subcostal incision and was dissected free
from perirenal tissue, and renal arteries were isolated for the
determination of renal blood flow (RBF) using a blood flowmeter and
probe (0.5v; Transonic Systems, Ithaca, NY) as described by Traynor
(26). Mean arterial pressure (MAP) was measured via a
carotid artery catheter connected to a Transpac IV transducer and
monitored continuously using Windaq Waveform recording software (Dataq
Instruments). An intravenous maintenance infusion of 2.25% BSA in
normal saline (NS) at a rate of 0.25 µl · g body
wt1 · min
1 was started 1 h
before experimentation. FITC-inulin (0.75%) was added to the infusion
solution for the determination of glomerular filtration rate (GFR) as
described by Lorenz et al. (13). A bladder catheter
(PE-10) was used to collect urine. Two 30-min collections of urine were
obtained under oil and weighed for volume determination. Blood for
plasma inulin determination was drawn between urine collections. FITC
in plasma and urine samples was measured using a CytoFluor plate reader
(PerSeptive Biosystems, Foster City, CA).
Western blot analysis. Whole kidney lysate was mixed with sample buffer containing 50 mM Tris, 0.5% glycerol, 0.01% bromophenol blue, and 0.75% SDS (pH 6.8). Identical amounts of protein were fractionated by a 4-15% Tris/glycine polyacrylamide gradient gel (EC-SOD determination) or 15% polyacrylamide separating gel (MnSOD and Cu/ZnSOD determinations) and transferred to a nitrocellulose membrane (Millipore, Bedford, MA). Membranes were blocked using 5% milk in TTBS [50 mM Tris, 150 mM NaCl, 0.1% Tween 20 (pH 7.5)] at room temperature for 60 min and were subsequently incubated at 4°C overnight with rabbit anti-EC-SOD antibody (1:5,000) or 1 µg/ml rabbit anti-MnSOD antibody (Upstate Biotechnology, Lake Placid, NY) and 1 µg/ml sheep anti-Cu/ZnSOD (Upstate Biotechnology). An additional 1-h incubation was performed with a secondary antibody, goat anti-rabbit IgG or donkey anti-sheep IgG, coupled to horseradish peroxidase (Amersham, Piscataway, NJ) at 1:5,000 dilution in TTBS. Detection of the protein bands was carried out using enhanced chemiluminescence (Amersham). Membranes were then stripped and blotted with rabbit anti-mouse actin (Sigma) to examine the actual loading of the proteins. Relative densitometry was measured as the ratio of the densitometry of a specific protein to that of actin.
RNA extract and RNase protection assay.
Total RNA was prepared by using TRIzol reagent (Invitrogen, Carlsbad,
CA). An RNase protection assay was performed on 2-4 µg of RNA
with the RNase Protection Assay Kit I (Torrey Pines Biolabs, Houston,
TX) according to the manufacturer's instructions. Full-length mouse
EC-SOD cDNA was obtained from ATCC (GenBank accession no. BF300486). A
268-bp SacI-PstI fragment of EC-SOD was isolated
from the full-length cDNA and inserted in the pBluescript KS II vector.
The clone was verified by DNA sequencing and linearized with
appropriate restriction enzymes. [-32P]UTP (3,000 Ci/mmol, ICN)-labeled antisense RNA probes were synthesized by an in
vitro transcription system (Promega, Madison, WI). Antisense RNA probes
were hybridized with the RNA samples at 90°C for 25 min. Unhybridized
single-strand RNA was digested by ribonuclease A/T1 (Sigma)
for 30 min. Double-strand RNA was precipitated by stop solution at
80°C for 15-30 min and centrifuged at maximum speed for 30 min. The samples were resolved by a 6% sequencing gel. The gel was
dried and exposed to X-ray film.
Measurement of plasma NO levels. Plasma NO levels were determined by measuring plasma NO2/NO3 levels using a nitrate/nitrite colorimetric assay kit (Cayman Chemical, Ann Arbor, MI).
Statistical analysis. Values are expressed as means ± SE. Multiple comparisons were assessed by ANOVA using a post hoc Newman-Keuls test. Survival analysis was analyzed by the Kaplan-Meier method. P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Normotensive endotoxemic ARF model in mice. Mice were injected intraperitoneally with 5 mg/kg LPS (Escherichia coli 026:B6, Sigma), a relatively low and nonlethal dose of LPS that permitted surgery and physiological measurements without excessive mortality. With this dose of LPS, there was no significant change in MAP (82 ± 0.8 vs. 82 ± 2.2 mmHg, n = 6, P = not significant), thus allowing measurement of renal function in the absence of hypotension.
Effect of LPS on SOD expression in the kidney.
Western blot analysis was used to examine all three isoforms
of SOD protein expression in the mice kidneys. In control mice, there
was high expression of EC-SOD in the control kidney, but the expression
was significantly decreased in mice treated with LPS at 16 h (Fig.
1A). The relative densitometry
was 2.6 ± 0.13 vs. 1.2 ± 0.05 (n = 6, P < 0.01, Fig. 1B). Two bands at ~34 and 32 kDa can be observed in both groups. These represent intact and
proteolytically processed forms of EC-SOD, respectively (4, 6). The top band (the intact form) is much stronger
than the bottom band (proteolytic form) in vehicle-treated
mice, whereas the two bands are similar in density in the kidney of
LPS-treated mice. The ratio between the two bands are 2.97 ± 0.12 in the control group vs. 1.27 ± 0.07 in the LPS-treated group
(n = 6/group, P < 0.01). When MnSOD
and Cu/ZnSOD protein expressions in the kidney were examined, there is
no difference between the control and LPS-treated group (Fig.
2, A and B).
|
|
Effect of LPS on EC-SOD mRNA expression in the kidney in mice.
To examine whether the decreased EC-SOD protein expression was
regulated at the transcriptional level, EC-SOD mRNA in the kidney was
measured using an RNase protection assay. As shown in Fig.
3, EC-SOD mRNA was decreased in the
kidney in endotoxemic mice, thus corresponding to EC-SOD protein
expression.
|
Effect of MnTE-2-PyP on GFR, RBF, and MAP in endotoxemic mice.
MnTE-2-PyP (10 mg/kg) or vehicle (NS) was injected
intraperitoneally 30 min before LPS (5 mg/kg) (Fig.
4). At 16 h after LPS injection,
GFR, MAP, and RBF were examined in control mice and MnTE-2-PyP-treated
mice. There was no difference in MAP between vehicle- and
MnTE-2-PyP-treated endotoxemic mice. Sixteen hours after LPS injection,
there were significant decreases in GFR (50 ± 16 vs. 229 ± 21 µl/min, n = 8, P < 0.001) and RBF
(0.61 ± 0.10 vs. 0.86 ± 0.05 ml/min, n = 8, P < 0.05) compared with vehicle-treated controls (NS).
The decreased GFR and RBF were dramatically reversed by MnTE-2-PyP,
which brought GFR and RBF up to 182 ± 40 µl/min (n = 6, P < 0.01 vs. vehicle-treated
controls) and 1.08 ± 0.10 ml/min (n = 6, P < 0.05 vs. vehicle-treated controls), respectively. The above experiments were performed when MnTE-2-PyP was injected before LPS. Further experiments were undertaken, in which MnTE-2-PyP was administered 6 h after LPS administration. In these
experiments, GFR was partially protected (85 ± 10 vs. 37 ± 12 µl/min of vehicle-treated controls, n = 8, P < 0.05).
|
Effect of MnTE-2-PyP on mortality in endotoxemia.
A large dose of LPS (30 mg/kg) was injected intraperitoneally, and the
mortality of MnTE-2-PyP (10 mg/kg ip 30 min before LPS)- vs.
vehicle-treated mice was examined for 48 h. The 24-h survival rate
was 0% in vehicle-treated mice (n = 18), whereas it
was 50% in MnTE-2-PyP-treated mice (n = 8, P < 0.05, Fig. 5).
|
Effect of iNOS inhibition with
L-N6-(1-iminoethyl)-lysine on the protective
effect of MnTE-2-PyP during endotoxemia.
L-N6-(1-iminoethyl)-lysine
(L-NIL; 10 mg/kg ip, Alexis Biochemicals, Carlsbad, CA), a
selective inhibitor of iNOS (14), was administered 30 min
before LPS (5 mg/kg ip) either alone or with MnTE-2-PyP (10 mg/kg ip).
As shown in our previous study (9, 29), plasma NO levels
were significantly higher in endotoxemic mice compared with control
mice (227 ± 16 vs. 2.5 ± 0.4 µM, n = 6;
P < 0.01). This high plasma NO level decreased
significantly after the treatment with L-NIL (51 ± 4 vs. 227 ± 16 µM, n = 6, P < 0.01). When L-NIL was administered with MnTE-2-PyP, it
abolished the renal protective effect of MnTE-2-PyP during endotoxemia
because GFR decreased from 138 ± 8 to 49 ± 9 µl/min
(n = 4, P < 0.01, Fig.
6A) and RBF decreased from
1.05 ± 0.09 to 0.55 ± 0.08 ml/min (n = 4, P < 0.05, Fig. 6B), respectively.
|
Effect of tempol on renal function in endotoxemic mice. To examine whether the protective effect of MnTE-2-PyP was specific to superoxide scavenging, the effect of another superoxide dismutase, tempol (Calbiochem Bioscience, La Jolla, CA), was also examined during endotoxemia. LPS (E. coli 0111:B4, 2.0 mg/kg, LIST Biological Laboratories, Campbell, CA) was used in this study. This LPS compound is purer and more potent than the LPS from Sigma. The 2 (LIST) and 5 mg/kg (Sigma) dosages resulted in comparable effects on GFR and RBF. Similar to what was observed with MnTE-2-PyP, tempol significantly improved both RBF (1.21 ± 0.05 vs. 0.67 ± 0.04 ml/min, n = 4, P < 0.01) and GFR (102 ± 8 vs. 58 ± 11 µl/min, n = 7, P < 0.01). These protective effects were also reversed by L-NIL because RBF (1.21 ± 0.05 vs. 0.85 ± 0.05 ml/min, n = 4, P < 0.01) and GFR (102 ± 8 vs. 60 ± 11 µl/min, n = 4, P < 0.01) decreased significantly with the administration of L-NIL (10 mg/kg) with tempol compared with tempol alone during endotoxemia.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In several studies, sepsis has been identified as the most common cause of ARF (1, 11). Moreover, the combination of sepsis and ARF is associated with mortality as high as 60-80%. However, there is no consensus either about the pathophysiology of sepsis-related ARF or the appropriate treatment.
The present study was therefore undertaken to examine the
pathophysiology of endotoxemia-related ARF in a normotensive mouse model. In this model, the endotoxemia was associated with a progressive increase in NO (9, 25, 29, 30), an effect that does not occur in iNOS knockout mice (10). Sepsis is also known to
increase O
To focus on the potential effect of ROS in endotoxemia-related ARF, the
role of endogenous and exogenous ROS scavengers was examined. EC-SOD, a
secreted endogenous antioxidant enzyme, is the predominant form of SOD
in the vasculature and is highly expressed in mouse kidneys (8,
17, 19). In the present study, endotoxemia was associated with a
decrease in renal EC-SOD, an effect that could lead to increased
O
Further study of the role of ROS during endotoxemia was undertaken by
examining the effect of a potent exogenous antioxidant, MnTE-2-PyP, in
endotoxemia-related ARF. The antioxidant properties of this agent
include scavenging O (2, 3, 20). The administration of
this antioxidant before LPS was associated with a highly significant
improvement in both GFR and RBF during endotoxemia. Renal protection
could also be demonstrated when the antioxidant was administered 6 h after LPS. A significant decrease in mortality at 24 h was also
observed when the antioxidant was administered with an otherwise
uniformly fatal dose of LPS (30 mg/kg).
An increase in NO, secondary to decreased scavenging by ROS, could contribute to the beneficial effect of the antioxidant against the endotoxin-induced renal vasoconstriction and ARF. To examine whether some of the protective effect of the antioxidant was due to increased bioavailability of NO, the potent antioxidant was administered in combination with the specific inhibitor of iNOS. The administration of L-NIL decreased plasma NO and reversed the renal protective effect of the antioxidant on GFR and RBF, thus supporting a vascular protective effect of NO. Similar results were also observed when tempol, a chemically dissimilar superoxide dismutase, was studied.
This role of endogenous and exogenous antioxidants in modulating endotoxemia-related renal vasoconstriction by enhancing the bioavailability of NO has implications for the early phase of endotoxemia (24). The early phase of this normotensive mouse model of endotoxin-induced ARF has been shown to be associated with activation of the sympathetic and renin-angiotensin systems (29). The systemic pressor effects of these events counterbalance the systemic vasodilatory effects of NO during endotoxemia and thereby support MAP. Nevertheless, these events occur at the expense of renal vasoconstriction. This sequence of events has been supported by demonstrating a renal protective effect of acute renal denervation in this normotensive endotoxemic model of ARF (29). The present results provide further understanding of the early events that occur during endotoxin-related ARF.
The increase in NO, which results from the endotoxin-related
induction of NOS, is scavenged by the increased O has
also been implicated in this early phase of endotoxemia-related ARF (10) and therefore would be expected to contribute not
only to the induction of NOS but also to enhanced ROS activity
(7).
In summary, the early ARF in endotoxemia involves a complex sequence of events leading to renal vasoconstriction. The present results demonstrate that the predisposition to renal vasoconstriction during endotoxemia involves a downregulation of renal EC-SOD with resultant scavenging of bioavailable NO by ROS. Antioxidant treatment by chemically dissimilar compounds exhibited an impressive amelioration of the endotoxin-mediated decrease in GFR and RBF. Increased NO bioavailability appears to be involved because the beneficial effect of antioxidants was reversed by the specific inhibition of iNOS. Scavenging of renal iNOS-related NO by ROS thus appears to be an important factor in the renal vasoconstriction associated with early (16 h) endotoxemia in mice.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Institutes of Health Grants DK-52599 and P01-HL-31992.
![]() |
FOOTNOTES |
---|
J. D. Crapo and B. J. Day are consultants for and hold equity in Incara Pharmaceuticals.
Address for reprint requests and other correspondence: R. W. Schrier, Univ. of Colorado Health Sciences Ctr., 4200 East 9th Ave., Box B178, Denver, CO 80262 (E-mail: robert.schrier{at}uchsc.edu).
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.
10.1152/ajprenal.00323.2002
Received 6 September 2002; accepted in final form 19 November 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, R,
and
Schrier RW.
Acute renal failure.
In: Diseases of the Kidney and Urinary Tract (7th ed.), edited by Schrier RW.. New York: Lippincott Williams & Wilkins, 2001, p. 1093-1136.
2.
Batinic-Haberle, I.
Manganese porphyrins and related compounds as mimics of superoxide dismutase.
Methods Enzymol
349:
223-233,
2002[ISI][Medline].
3.
Batinic-Haberle, I,
Benov L,
Spasojevic I,
and
Fridovich I.
The ortho effect makes manganese(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin a powerful and potentially useful superoxide dismutase mimic.
J Biol Chem
273:
24521-24528,
1998
4.
Bowler, RP,
Nicks M,
Olsen DA,
Thogersen IB,
Valnickova Z,
Hojrup P,
Franzusoff A,
Enghild JJ,
and
Crapo JD.
Furin proteolytically processes the heparin-binding region of extracellular superoxide dismutase.
J Biol Chem
277:
16505-16511,
2002
5.
Brackett, DJ,
Lai EK,
Lerner MR,
Wilson MF,
and
McCay PB.
Spin trapping of free radicals produced in vivo in heart and liver during endotoxemia.
Free Radic Res Commun
7:
315-324,
1989[ISI][Medline].
6.
Enghild, JJ,
Thogersen IB,
Oury TD,
Valnickova Z,
Hojrup P,
and
Crapo JD.
The heparin-binding domain of extracellular superoxide dismutase is proteolytically processed intracellularly during biosynthesis.
J Biol Chem
274:
14818-14822,
1999
7.
Goossens, V,
Grooten J,
De Vos K,
and
Fiers W.
Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity.
Proc Natl Acad Sci USA
92:
8115-8119,
1995[Abstract].
8.
Karlsson, K,
and
Marklund SL.
Extracellular superoxide dismutase in the vascular system of mammals.
Biochem J
255:
223-228,
1988[ISI][Medline].
9.
Knotek, M,
Esson M,
Gengaro P,
Edelstein CL,
and
Schrier RW.
Desensitization of soluble guanylate cyclase in renal cortex during endotoxemia in mice.
J Am Soc Nephrol
11:
2133-2137,
2000
10.
Knotek, M,
Rogachev B,
Wang W,
Ecder T,
Melnikov V,
Gengaro PE,
Esson M,
Edelstein CL,
Dinarello CA,
and
Schrier RW.
Endotoxemic renal failure in mice: Role of tumor necrosis factor independent of inducible nitric oxide synthase.
Kidney Int
59:
2243-2249,
2001[ISI][Medline].
11.
Liano, F,
Junco E,
Pascual J,
Madero R,
and
Verde E.
The spectrum of acute renal failure in the intensive care unit compared with that seen in other settings. The Madrid Acute Renal Failure Study Group.
Kidney Int, Suppl
66:
S16-S24,
1998[Medline].
12.
Ling, H,
Edelstein C,
Gengaro P,
Meng X,
Lucia S,
Knotek M,
Wangsiripaisan A,
Yeuxian S,
and
Schrier R.
Attenuation of renal ischemia-reperfusion injury in inducible nitric oxide synthase knockout mice.
Am J Physiol Renal Physiol
277:
F383-F390,
1999
13.
Lorenz, JN,
and
Gruenstein E.
A simple, nonradioactive method for evaluating single-nephron filtration rate using FITC-inulin.
Am J Physiol Renal Physiol
276:
F172-F177,
1999
14.
Moore, WM,
Webber RK,
Jerome GM,
Tjoeng FS,
Misko TP,
and
Currie MG.
L-N6-(1-iminoethyl)lysine: a selective inhibitor of inducible nitric oxide synthase.
J Med Chem
37:
3886-3888,
1994[ISI][Medline].
15.
Noiri, E,
Peresleni T,
Bahou WF,
and
Goligorsky MS.
In vivo targeting of inducible NO synthase with oligodeoxynucleotides protects rat kidney against ischemia.
J Clin Invest
97:
2377-2383,
1996
16.
Novelli, GP.
Role of free radicals in septic shock.
J Physiol Pharmacol
48:
517-527,
1997[ISI][Medline].
17.
Ookawara, T,
Imazeki N,
Matsubara O,
Kizaki T,
Oh-Ishi S,
Nakao C,
Sato Y,
and
Ohno H.
Tissue distribution of immunoreactive mouse extracellular superoxide dismutase.
Am J Physiol Cell Physiol
275:
C840-C847,
1998[Abstract].
18.
Oury, TD,
Day BJ,
and
Crapo JD.
Extracellular superoxide dismutase: a regulator of nitric oxide bioavailability.
Lab Invest
75:
617-636,
1996[ISI][Medline].
19.
Oury, TD,
Day BJ,
and
Crapo JD.
Extracellular superoxide dismutase in vessels and airways of humans and baboons.
Free Radic Biol Med
20:
957-965,
1996[ISI][Medline].
20.
Patel, M,
and
Day BJ.
Metalloporphyrin class of therapeutic catalytic antioxidants.
Trends Pharmacol Sci
20:
359-364,
1999[ISI][Medline].
21.
Peresleni, T,
Noiri E,
Bahou WF,
and
Goligorsky MS.
Antisense oligodeoxynucleotides to inducible NO synthase rescue epithelial cells from oxidative stress injury.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F971-F977,
1996
22.
Radi, R,
Beckman JS,
Bush KM,
and
Freeman BA.
Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.
J Biol Chem
266:
4244-4250,
1991
23.
Radi, R,
Beckman JS,
Bush KM,
and
Freeman BA.
Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide.
Arch Biochem Biophys
288:
481-487,
1991[ISI][Medline].
24.
Schrier, RW.
Cancer therapy and renal injury.
J Clin Invest
110:
743-745,
2002
25.
Schwartz, D,
Mendonca M,
Schwartz I,
Xia Y,
Satriano J,
Wilson CB,
and
Blantz RC.
Inhibition of constitutive nitric oxide synthase (NOS) by nitric oxide generated by inducible NOS after lipopolysaccharide administration provokes renal dysfunction in rats.
J Clin Invest
100:
439-448,
1997
26.
Traynor, TR,
and
Schnermann J.
Renin-angiotensin system dependence of renal hemodynamics in mice.
J Am Soc Nephrol
10, Suppl11:
S184-S188,
1999[ISI][Medline].
27.
Vespasiano, MC,
Lewandoski JR,
and
Zimmerman JJ.
Longitudinal analysis of neutrophil superoxide anion generation in patients with septic shock.
Crit Care Med
21:
666-672,
1993[ISI][Medline].
28.
Yu, L,
Gengaro PE,
Niederberger M,
Burke TJ,
and
Schrier RW.
Nitric oxide: a mediator in rat tubular hypoxia/reoxygenation injury.
Proc Natl Acad Sci
91:
1691-1695,
1994[Abstract].
29.
Wang, W,
Falk SA,
Jittikanont S,
Gengaro PE,
Edelstein CL,
and
Schrier RW.
Protective effect of renal denervation on normotensive endotoxemia-induced acute renal failure in mice.
Am J Physiol Renal Physiol
283:
F583-F597,
2002
30.
Wright, CE,
Rees DD,
and
Moncada S.
Protective and pathological roles of nitric oxide in endotoxin shock.
Cardiovasc Res
26:
48-57,
1992[ISI][Medline].