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1 Department of Intensive Care, and 2 Department of Pathology, Erasme University Hospital, Free University of Brussels, B-1070 Brussels, Belgium
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Zhang, Haibo, Peter Rogiers, Nadia Smail, Ana Cabral,
Jean-Charles Preiser, Marie-Odile Peny, and Jean-Louis Vincent.
Effects of nitric oxide on blood flow distribution and
O2 extraction capabilities during
endotoxic shock. J. Appl. Physiol.
83(4): 1164-1173, 1997.
The effects of the nitric oxide (NO)
synthase inhibitor
NG-monomethyl-L-arginine
(L-NMMA) and the NO donor
3-morpholinosydnonimine (SIN-1) were tested in 18 endotoxic dogs. L-NMMA infusion
(10 mg · kg
1 · h1)
increased arterial and pulmonary artery pressures and systemic and
pulmonary vascular resistances but decreased cardiac index, left
ventricular stroke work index, and blood flow to the hepatic, portal,
mesenteric, and renal beds. SIN-1 infusion (2 µg · kg1 · min1)
increased cardiac index; left ventricular stroke work index; and
hepatic, portal, and mesenteric blood flow. It did not significantly influence arterial and pulmonary artery pressures but decreased renal
blood flow. The critical O2
delivery was similar in the L-NMMA group and in the control
group (13.3 ± 1.6 vs. 12.8 ± 3.3 ml · kg1 · min1)
but lower in the SIN-1 group (9.1 ± 1.8 ml · kg1 · min1,
both P < 0.05). The critical
O2 extraction ratio was also
higher in the SIN-1 group than in the other groups (58.7 ± 10.6 vs.
42.2 ± 7.6% in controls, P < 0.05; 43.0 ± 15.5% in
L-NMMA group,
P = not significant). We conclude that
NO is not implicated in the alterations in
O2 extraction capabilities
observed early after endotoxin administration.
cardiac output; hypotension; organ perfusion; oxygen delivery; sepsis; endothelium-derived relaxing factor; nitrite; tumor necrosis
factor- INTRODUCTION SEPTIC SHOCK, a major clinical problem with mortality
rates of up to 70%, is characterized by systemic hypotension, vascular hyporeactivity and myocardial depression. Despite the increased cardiac
output, cellular O2 utilization
may be inadequate because maldistribution of blood flow can profoundly
alter O2 availability. Regional
blood flow to the various organs may also be altered nonuniformly. In
particular, the hepatosplanchnic blood flow may decrease more than
blood flow to other regions to favor blood supply to vital organs, such
as the heart and the brain. Because hepatosplanchnic hypoxia may
contribute to the development of multiple organ failure (11), the
maintenance of sufficient hepatosplanchnic blood flow may be a
fundamental goal in septic shock.
Nitric oxide (NO) is a paracrine-acting gas enzymatically synthesized
from L-arginine. In basal
conditions, the release of NO via calcium-dependent constitutive NO
synthase (cNOS) plays an essential role in the maintenance of capillary
flow and O2 availability to the
tissues. The greater release of NO via a calcium-independent inducible
form of NO synthase (iNOS) has been implicated in the pathophysiological alterations of severe sepsis and septic shock. The
resulting overproduction of NO may induce deleterious effects, including arterial hypotension (6), vascular hyporeactivity, and
myocardial depression (5). The induction of iNOS in various cells,
including macrophages, endothelial cells, vascular smooth muscle cells,
or even myocardial cells, requires several hours to be expressed after
stimulus with endotoxin and cytokines, such as tumor necrosis
factor- Nitric oxide synthase (NOS) inhibition by the administration of
L-arginine analogs has yielded
controversial results in sepsis. Although such intervention does
reverse endotoxin- or TNF- During ischemic or hypoxic hypoxia in dogs, blockade of NOS by
NG-nitro-L-arginine methyl ester
(L-NAME) does not appear to
affect the O2 extraction ratio
(ERO2)
(15, 31, 33). During endotoxemia in dogs, Walker et al. (36) reported
that L-NAME administration decreased whole body and intestinal
O2 delivery
( Recently, we found (40) that
N-acetyl-L-cysteine
(NAC), a potent antioxidant substance that enhances endothelium-derived relaxing factor activity, improved the
O2 extraction capabilities during
endotoxic shock in dogs, as indicated by a significantly lower critical
It is intriguing, therefore, to test the hypothesis that NO-releasing
compounds may have beneficial effects in septic shock. As a NO donor we
chose 3-morpholinosydnonimine (SIN-1), the vasoactive metabolite of
molsidomine currently used as a nitrate compound in the treatment of
coronary artery disease. Also, we recently observed (41) that the
administration of SIN-1 during endotoxic shock in dogs increased
splanchnic blood flow without adverse effects on arterial pressure.
Hence, the present study investigated the effects of
L-NMMA and SIN-1 in a dog model
of endotoxic shock. In addition to global hemodynamics, we
studied regional blood flow in hepatic, portal, mesenteric, and renal
vasculatures. We also studied the effects of these interventions
on tissue O2 extraction
capabilities by progressively decreasing blood flow induced by cardiac
tamponade.
MATERIALS AND METHODS 
(TNF-) or interferon-
.
-induced hypotension (6, 38), it also
generally decreases blood flow (19, 23), so that it may worsen tissue
injury and even increase mortality rate in some experimental models of
septic shock (6, 23, 38). Several groups of investigators have also
stressed that NOS inhibition may result in hepatosplanchnic ischemia
(23).
O2)
by 50% but O2 uptake
(
O2) remained stable, so that
whole body and intestinal ERO2 values
increased above 50%. However, the effects of NO on tissue
O2 extraction capabilities during
septic shock have not been well defined. We chose to study the effects
of NG-monomethyl-L-arginine
(L-NMMA), a NOS inhibitor
presently tested in clinical trials (35), on regional blood flow
distribution and O2 extraction
capabilities during endotoxic shock.
O2
(O2 crit)
and higher critical
ERO2
(ERO2 crit) when blood flow was progressively reduced by tamponade. NAC also significantly increased cardiac index (CI) by an improvement in myocardial contractility and lowered pulmonary hypertension (40).
1 · h1
(Infusomat II pump; B. Braun, Melsungen, Germany) through the right
forepaw vein. After being endotracheally intubated with a cuffed
endotracheal tube, the dog was mechanically ventilated with room air by
using a servo ventilator (model 900B, Siemens-Elema, Solna, Sweden).
Controlled ventilation was facilitated with pancuronium bromide given
as an initial bolus of 0.15 mg/kg followed by an infusion of 0.075 mg · kg1 · h1.
Respiratory rate was set at 12 breaths/min and tidal volume was
adjusted to obtain an end-tidal PCO2
(PETCO2) between 28 and 34 Torr. These ventilatory conditions were not changed thereafter. A right
femoral arterial catheter was inserted and connected to a pressure
transducer for arterial pressure monitoring. The left forepaw vein was
cannulated for normal saline and drug infusion. A balloon-tipped
pulmonary artery catheter (model 93A-131-7-Fr; Baxter, Irvine, CA)
was inserted through the right external jugular vein under guidance of
pressure waves, as determined from a four-channel monitor (Sirecust
302A; Siemens, Erlangen, Germany). A left thoracotomy between the
fourth and fifth intercostal space was performed; bleeding was
controlled by electrocautery. Via a 2- to 3-mm incision in the anterior
pericardium, a 16-gauge polyethylene catheter (Intracath; Deseret
Medical, Sandy, UT) with multiple side holes was positioned in the
pericardial space with its tip adjacent to the diaphragmatic surface of
the left ventricle. The catheter was secured with purse-string sutures.
The pericardial cavity was drained, with replacement of 30 ml of warm
sterile saline before sealing. The thoracic cavity was then carefully
closed in three layers, and a chest tube (Trocar catheter A75, 28 Ch-40 cm, Argyle, Ireland) was placed through the seventh
intercostal space to allow gentle evacuation of the chest.
1 · min1)
was measured by the thermodilution technique (cardiac output computer,
COM-2, Baxter) by using three to five 5-ml injections of cold 5%
dextrose in ice water. Each injection was started at end inspiration. A
temperature probe was used on-line to control for variations in
injectate temperature. Regional blood flow was estimated simultaneously
in the common hepatic artery, portal vein, mesenteric artery, and
left renal artery by a previously calibrated blood flowmeter
(model T208; Transonic Systems, Ithaca, NY). Because ~90% of
common hepatic blood flow goes to the liver, and the other 10%
goes to the gastroduodenal artery in control conditions (30), one
should consider that the hepatic blood flow we obtained was an
estimation rather than an exact measurement.
Exhaled gases were directed through a mixing chamber for sampling of
expired gases to measure expired
O2 fraction
(FEO2) (P. K. Morgan, Chatham, UK).
PETCO2 was monitored
simultaneously (47210A capnometer; Hewlett-Packard, Waltham, MA). The
gas analyzers were calibrated before the experiment. Expired minute
volume (VE) was
measured with a spirometer (Haloscale Wright Respirometer; Edmonton,
London, UK).
Arterial and mixed venous blood samples were simultaneously withdrawn
for immediate determination of blood gases and lactate concentration
(ABL 500; Radiometer, Copenhagen, Denmark; lactate/glucose analyzer
2300 Stat Plus, Yellow Springs Instruments, Yellow Springs, OH). In
each sample, hemoglobin and O2
saturation were measured (OSM 3 Hemoximeter, calibrated for dog blood;
Radiometer).
O2
was calculated as the product of arterial
O2 content and CI.
O2 was determined by the
formula
|
![× {[(1 − F<SC>e</SC><SUB>O<SUB>2</SUB></SUB> − F<SC>e</SC><SUB>CO<SUB>2</SUB></SUB>)/(1 − F<SC>i</SC><SUB>O<SUB>2</SUB></SUB>)] × F<SC>i</SC><SUB>O<SUB>2</SUB></SUB> − F<SC>e</SC><SUB>O<SUB>2</SUB></SUB>}](/content/vol83/issue4/fulltext/1164/img003.gif)
|
O2/O2.
TNF- levels were measured by using murine immunoglobulin G1 antibody
to TNF- (W. Buurman). This antibody to human TNF- sees canine
TNF- in Western blot (unpublished data) and inhibits the biological
activity of canine TNF- to a lesser degree than it inhibits human
TNF-.
NO production was determined spectrophotometrically by measuring the
accumulation of both nitrite and nitrate (the latter is reduced to
nitrite) in plasma and was reported by the percentage of changes in
nitrite from the baseline. Nitrate was stoichometrically reduced to
nitrite by incubation of sample (100 µl plasma) for 2 h at 37°C,
in the presence of 0.1 U/ml nitrate reductase [NAD(P)H, nitrate
oxidoreductase (EC 1.6.6.2;
Aspergillus species) Sigma Chemical,
St. Louis, MO], 120 µM NADPH, and 5 µM flavinadenine dinucleotide
(Sigma Chemical) in a final volume of 103 µl. After nitrate had been reduced to nitrite, NADPH that interfered with the
subsequent nitrite determination was oxidized with 10 U/ml L-lactic dehydrogenase (EC
1.1.1.27, type XI, from rabbit muscle; Sigma Chemical) and 10 mM sodium
pyruvate for 30 min at 37°C in a final volume of 114 µl. Sodium
nitrate was used as a standard. Nitrite concentration in plasma was
assayed by a standard Griess reaction. Briefly, 100 µl plasma were
incubated with an equal volume of Griess reagent (1%
sulpanilamide-0.1% napthylenediamine dihydrochloride-2.5%
H3PO4)
at room temperature for 10 min. The absorbance of the chromophore
formed was determined at 540 nm by using a microtiter plate reader
(Molecular Devices, Menlo Park, CA). Sodium nitrite was used as a
standard, with control baseline plasma as a blank control reference.
Experimental protocol.
After surgical preparation, the dog was placed in supine position and
allowed to stabilize for 30 min before control measurements [baseline 1 (B1)]. Each dog received
Escherichia coli endotoxin (E. coli 055:B5 lipopolysaccharide,
no. 3120-10-7; Difco, Detroit, MI) as a slow
intravenous bolus of 2 mg/kg over 2 min. Thirty minutes later, a second
set of measurements [baseline 2 (B2)] was obtained. The dog
then received a generous saline infusion to restore and maintain
pulmonary artery occlusion pressure at baseline levels.
A third set of measurements [baseline 3 (B3)] was obtained
30 min thereafter. Dogs were then randomly divided into three groups, receiving saline infusion at 20 ml · kg1 · h1
either alone (endotoxin, n = 6 dogs),
or in combination with L-NMMA
(endotoxin + L-NMMA;
n = 6 dogs), or in combination with SIN-1 (endotoxin + SIN-1; n = 6),
respectively. In the endotoxin + L-NMMA group,
L-NMMA
(L-NMMA acetate salt;
Calbiochem, La Jolla, CA) was continuously infused at 10 mg · kg1 · h1
(15 mg/ml solution). In the endotoxin + SIN-1 group, SIN-1 (Corvaton; Therabel Research, Brussels, Belgium) was continuously infused at 2 µg · kg1 · min1
(0.25 mg/ml solution). The SIN-1 solution was protected from light
during infusion.
Thirty minutes after the initial infusion of either
L-NMMA or SIN-1, a fourth set of
measurements [baseline 4 (B4)] was made. Cardiac tamponade
was then induced by repeated bolus injections of warm normal saline
heated to 37°C into the pericardial sac. The amount of saline
injected was 30 ml for the first two injections, then 10 ml until
O2 started to fall
from baseline levels, and finally 2-5 ml until the mean
arterial pressure (MAP) fell by 80% from baseline. The experiment was
then ended.
After each injection, a time interval of 20 min was permitted to reach
a steady state, characterized by a stable
FEO2, PETCO2, arterial pressure,
and heart rate before the next measurements were obtained. During the
study, core temperature was kept constant at its initial level with
warming lamps and a heating blanket.
After the experiment was completed, dogs were killed with potassium
chloride, and biopsies were immediately taken from the liver (left
lobe), small intestine (30 cm from flexura duodenojejunalis), and left
kidney. Tissue blocks were fixed overnight in 4% Formalin. Sections
were stained with hematoxilin and eosin. Pathological examination was
performed (M.-O. Peny).
Statistics.
The
O2 crit
was determined in each animal by dual-line regression from a plot of
O2 vs.
O2.
For each plot, linear regression by best fit was used to calculate
straight lines for the O2 supply dependency and independency. The point of intersection of their regression lines defined the
O2 crit
and the corresponding critical O2 (29).
ERO2 crit
was calculated as the ratio of
O2 to O2
at
O2 crit.
A two-way analysis of variance [for intrapericardial pressure
(IPP) and group] followed by a Dunnett's test was used for
statistical analysis. The difference in slopes of
O2/O2 was tested by the analysis of covariance. A
P value <0.05 was considered
statistically significant. All values are expressed as means ± SD
unless otherwise indicated.
RESULTS
, Endotoxin; 
,
endotoxin + L-NMMA; 
,
endotoxin + SIN-1. * P < 0.05 vs. endotoxin; 
P < 0.05 vs. endotoxin + SIN-1.
, Endotoxin; , endotoxin + L-NMMA; , endotoxin + SIN-1.
§ P < 0.05 vs. endotoxin + L-NMMA.
, endotoxin; , endotoxin + L-NMMA; , endotoxin + SIN-1.
h, hepatic artery
blood flow; B: p,
portal venous blood flow; C:
m, mesenteric artery blood flow;
D: r, renal artery
blood flow. B1-B4, as in Fig. 1. , Endotoxin; , endotoxin + L-NMMA; , endotoxin + SIN-1.
* P < 0.05 vs. endotoxin;
§ P < 0.05 vs.
endotoxin + L-NMMA; P < 0.05 vs. endotoxin + SIN-1.
Figures 5-7 show the
O2/O2
relationship in individual dogs during progressive cardiac tamponade in
each of the three groups.
O2) and
O2 delivery
(O2)
in control animals. Critical
O2
(O2 crit)
and critical O2 extraction ratio
(ERO2 crit)
are included in each plot. A-F: data
for dogs 1-6 in
group. Lines are dual-regression lines.
O2 and
O2
in L-NMMA-treated animals.
O2 crit
and
ERO2 crit
are included in each plot. A-F: data
for dogs 1-6 in group. Lines are
dual regression lines.
O2 and
O2in
SIN-1-treated animals.
O2 crit
and
ERO2 crit
are included in each plot. A-F: data
for dogs 1-6 in group. Lines are
dual regression lines.
Effects of endotoxin + L-NMMA. L-NMMA administration after endotoxin significantly increased MAP compared with the control group. When blood flow was reduced by cardiac tamponade, L-NMMA-treated animals had a higher pulmonary arterial pressure (Ppa), but a lower CI, SI, and LVSWI than the SIN-1 group (Figs. 1 and 2). Both SVR and PVR were greater in the L-NMMA-treated group than in the other groups (Fig. 3). L-NMMA did not significantly influence hepatic artery blood flow but decreased blood flow to portal and mesenteric vasculature compared with the other groups (Fig. 4). L-NMMA had no significant effect on either
O2 crit
or
ERO2 crit
(Figs. 5, 6, 7).
Effects of endotoxin + SIN-1.
SIN-1 administration had no significant influence on pressures but
significantly increased CI and LVSWI compared with
L-NMMA-treated group (Figs. 1
and 2). SIN-1 increased blood flow to the hepatosplanchnic vasculature
but decreased blood flow to the renal vasculature (Fig. 4). During
tamponade, SIN-1 delayed the occurrence of supply dependency, so that
the
O2 crit
was significantly lower in the SIN-1-treated group (9.1 ± 1.8 ml · kg1 · min1)
than in the other groups (13.3 ± 1.6 ml · kg1 · min1
in the L-NMMA-treated group and
12.8 ± 3.3 ml · kg1 · min1
in the control group, both P < 0.05), and the
ERO2 crit
was higher in the SIN-1-treated group (58.7 ± 10.6%) than in the
other groups [43.0 ± 15.5% in the
L-NMMA,
P = not significant (NS); 42.2 ± 7.6% in the control group, P < 0.05]. The slopes were identical in all three groups during the
supply-independent phase, whereas the slope was significantly steeper
in the SIN-1-treated group than in the
L-NMMA-treated group during the
supply-dependent phase (P < 0.05).
There were no significant differences in heart rate, cardiac filling
pressures, and hematocrit level between any paired groups. Plasma
lactate concentrations were identical in all three groups (data not
shown).
The amount of normal saline infusion was identical in the three groups
of animals (4.2 ± 0.8 vs. 3.5 ± 0.5 liters in endotoxin alone
vs. endotoxin + L-NMMA and vs.
3.7 ± 0.5 liters in endotoxin + SIN-1 group;
P = NS for both comparisons). In the
three groups, hematocrit decreased from 39-40 to 29-33%.
Plasma nitrite and nitrate levels were somewhat lower in the
L-NMMA-treated than in the
control animals at 240 min. Nitrite and nitrate levels were higher in
the SIN-1-treated animals than in the other groups at 180 min and
higher than the L-NMMA group at
240 min. The L-NMMA-treated
group had higher TNF- levels than the other groups at 150, 180, and
240 min (Fig. 8).
and nitrate/nitrite concentrations in 3 groups over
time. * P < 0.05 vs.
endotoxin; § P < 0.05 vs.
endotoxin + L-NMMA;
P < 0.05 vs. endotoxin + SIN-1.
Pathological examination of the liver biopsies showed a decreased number of polymorphonuclear cells, located either in the portal spaces or in the sinusoids, in both L-NMMA-treated and SIN-1-treated animals compared with the control group. The sinusoidal or centrolobular stasis and steatosis were less expressed in the SIN-1-treated group than the other groups. These observations may suggest less inflammatory response in the SIN-1-treated animals than in the other groups of animals. Pathological examination of the small intestine and the kidney revealed only minor alterations, characterized by a vascular congestion, but there were no significant differences between the control and either L-NMMA-treated or SIN-1-treated animals.
DISCUSSION
Although an increased production of NO in sepsis has been well demonstrated both in animals and humans (8, 28, 38), the beneficial effects of L-arginine analogs such as L-NMMA in septic shock have been much debated. These NOS inhibitors consistently increase arterial pressure and SVR, but may reduce tissue blood flow and alter organ function (20). The present study investigated the effects of a NOS inhibitor and a NO donor on regional blood flow and O2 extraction capabilities in experimental endotoxic shock. We used a large dog model to have easy access to global and regional blood flow measurements. Also, the administration of endotoxin followed by generous fluid infusion in this model results in an initial hyperdynamic form of endotoxic shock. We applied cardiac tamponade in the second phase of the experiments to study the O2-extraction capabilities of the animals.
The timing of interventions may be of great importance. The rapid (5-30 min) development of hypotension in response to endotoxin in vitro and in vivo may be mediated by an enhanced release of mediators such as kinins, prostacyclin, or atrial natriuretic factor (13, 28), whereas the iNOS release contributing to the hypotension may take several hours to occur (27). It is, therefore, reasonable to hypothesize that early inhibition of NOS may produce greater vasoconstriction than needed, resulting in tissue hypoperfusion. On the other hand, the activity of the cNOS may be depressed during the early phase of endotoxemia, resulting in impaired endothelium-dependent vasodilation (24), suggesting that early administration of a NO donor may be beneficial to preserve tissue perfusion. We started L-NMMA or SIN-1 infusion 1 h after endotoxin challenge to study the effects of L-NMMA preventing excessive iNOS and the effects of SIN-1 maintaining cNOS during early endotoxic shock. The increase in plasma nitrite/nitrate levels 180 min after endotoxin administration suggests that iNOS was significantly expressed. We recently observed in the same model that tissue iNOS was expressed in the heart, lung, liver, and small intestine 3 h after endotoxemia (unpublished data).
As a NOS inhibitor, we used
L-NMMA, which has been widely
used in experimental studies and has been tested in humans (12). Because only one dose of the compound could be administered, we selected a moderate dose of 10 mg · kg1 · h1
to avoid excessive vasoconstricting effects. Higher doses, especially those exceeding 30 mg/kg may have harmful hemodynamic effects and may
even increase mortality rates (38). The dose of 10 mg/kg has been shown
to be effective and well tolerated in humans with hyperdynamic septic
shock (12).
L-NMMA increased arterial pressure and SVR but reduced CI and SI. Similar observations have been reported previously (6, 20, 22), although some of them involved hypodynamic models in which the fluid status might have been inadequate for endotoxic shock. In the second phase of the study, when tamponade was induced, the effects of L-NMMA on arterial pressure were not sustained. This observation was at least in part related to a more profound myocardial depression, as shown by a lower LVSWI for a similar degree of ventricular filling as in the other animals. Although an excessive release of NO has been implicated in the development of sepsis-related myocardial depression (5), the effects of NOS inhibition on the endotoxin-induced myocardial depression are still controversial. The studies reporting that NOS inhibitors may improve myocardial contractility usually have involved isolated myocardiums (5), but other studies involving entire organisms failed to observe such effects (16, 35). NG-nitro-L-arginine could even alter myocardial function in endotoxic shock in rats (16). Treatment with aminoguanidine, a selective iNOS inhibitor, reduced plasma nitrite and nitrate but did not affect cardiac depression (14). Results of a preliminary clinical trial showed that L-NMMA treatment failed to increase ventricular stroke work despite the frequent addition of dobutamine to L-NMMA administration (35). Taken together, these observations bring into question any beneficial effect of NOS inhibition on myocardial function in septic shock.
The vasoconstricting effects of L-NMMA on the pulmonary circulation were more dramatic than on the systemic circulation. In the present model, with only moderate pulmonary hypertension, L-NMMA dramatically increased Ppa and PVR. Other studies have also emphasized that NOS inhibitors can potentiate endotoxin-induced pulmonary hypertension in endotoxic shock (20). The increase in Ppa induced by NOS inhibition may represent a major limitation to clinical use of NOS inhibitors.
The administration of L-NMMA
decreased blood flow to the hepatosplanchnic circulation. Our
observations are consistent with a number of recent studies
underscoring that NOS inhibitors can significantly
exacerbate regional vasoconstriction and ischemia (1, 22, 23, 25, 38).
In endotoxic rats, Mulder et al. (22) demonstrated that NOS inhibition
increased organ vascular resistance in the splanchnic vasculature
during the first hour of endotoxic shock. In endotoxic pigs, Ayuse et
al. (1) showed that NOS inhibition could alter local control of liver
blood flow and markedly increase resistance to venous return across the
liver. In endotoxic rabbits, Pastor and Payen (26) reported that
N
-nitro-L-arginine significantly
reduced portal vein and hepatic artery blood flows. Walker et al. (36)
showed that L-NAME reduced gut
blood flow by increasing gut vascular resistance in endotoxic dogs. In
most of these studies, NOS inhibitors were administered during early
endotoxemia or as a pretreatment, suggesting that NO plays an important
role in regional hemodynamic effects and that NOS inhibition may be
deleterious in acute endotoxemia. Other studies (3, 22) showed that NOS
inhibition enhanced both macroscopic and histological intestinal and
liver damage, but these effects were not always attributed to a reduced
blood flow in this region.
The effects of NOS inhibition on renal blood flow have been less well studied. Our observation of a reduced renal blood flow is in keeping with those of Spain et al. (32), indicating that NOS inhibition can further constrict renal vasculature and decrease interlobular artery flow during sepsis. However, another recent study (2) reported that L-NMMA did not reduce renal blood flow in a sheep model of hyperdynamic sepsis.
We chose SIN-1, the vasoactive metabolite of molsidomide, as a NO
donor. SIN-1 spontaneously decomposes into NO and the stable metabolite
N-3-morpholinoiminoacetonitrile
(SIN-1C). Although SIN-1 at high doses may simultaneously generate NO
and superoxide anion (17), it is an effective agent in the treatment of
coronary artery disease and has effects at least as powerful as
nitroglycerin. Also, Pastor et al. (25) recently demonstrated that
SIN-1 administration in the early phase of endotoxic shock in rabbits
can preserve systemic and hepatic perfusion while preventing lactic
acidosis. We chose a dose of 2 µg · kg1 · min · 1
of SIN-1, because the improvement in myocardial function and splanchnic
blood flow were most evident with this dose in previous experiments on
a similar canine model of fluid-resuscitated endotoxic shock (41).
Although the model used was characterized by a low vascular resistance, SIN-1 had no deleterious effect on arterial pressure. The adequate fluid loading of the animal was probably a prerequisite of such good cardiovascular tolerance, as SIN-1 may otherwise reduce venous return to the heart by its effect of venous dilation. The lack of hypotension was also related to a significant increase in CI associated with an improvement in cardiac function, as reflected by a greater LVSWI despite identical cardiac filling pressures. Such an improvement in cardiac performance after SIN-1 administration in endotoxic shock has been previously observed (41), but the exact mechanism is unclear. It may be related to a protective effect of SIN-1 on endothelial cell function or to NO-independent mechanisms related to preserved cytosolic Ca2+ levels. A reduced Ppa could also be involved.
SIN-1 administration selectively increased hepatic artery blood flow, compared with other groups, and maintained portal and mesenteric blood flow. Mulder et al. (22) showed that splanchnic blood flow is critically dependent on NO during the first hour of endotoxemia in rats. Boughton-Smith et al. (4) reported that exogenous supplementation of NO by S-nitroso-N-acetylpenicillamine administration could preserve gut blood flow and attenuate endotoxin-induced macroscopic jejunal damage in the rat. In a rabbit endotoxic shock model, Pastor et al. (25) recently demonstrated that pretreatment with SIN-1 could maintain portal vein blood flow at control level and significantly increase hepatic artery blood flow without any further effect on MAP. These studies suggest that NO release is essential to maintain splanchnic blood flow in the early phase of endotoxic shock. Whether these beneficial effects of SIN-1 are also present late in the course of endotoxic shock requires further study. On the contrary, SIN-1 administration decreased blood flow to the renal bed at moderate IPP compared with controls, an effect which was unexpected.
When O2 supply becomes limited,
the release of NO may contribute to the recruitment of unperfused
capillaries and the increase in capillary density (7). However,
previous studies, including those of healthy animals, demonstrated that
NO inhibitors did not influence O2
extraction capabilities when blood flow was acutely reduced (15, 33,
37). During hypoxic hypoxia or ischemia in dogs,
L-NAME did not affect
ERO2 (31,
33). NG-nitro-L-arginine influenced
neither
O2 crit
nor maximum
ERO2 of the
diaphragm during reductions in
O2
by hemorrhage in dogs (37). Recently, we observed (39) that the
infusion of the NO donor sodium nitroprusside did not influence
O2 crit
and ERO2 crit
when blood flow was acutely reduced by cardiac tamponade in dogs. Thus
it appears that, in basal conditions, neither increasing exogenous NO
nor blocking its release can significantly influence the
O2 extraction capabilities.
We investigated the effects of NO on
O2 extraction capabilities during
endotoxic shock. L-NMMA at a
dose of 10 mg · kg1 · h · 1
did not influence tissue O2
extraction capabilities, because neither
O2 crit
nor
ERO2 crit
was significantly altered. These observations are consistent with those
reported by Schumacker et al. (31), who demonstrated that
L-NAME administration did not
reverse the O2 extraction
impairment seen during endotoxemia, either in whole body or in isolated
intestine. Signals other than NO, such as hydrogen ion, calcium ion,
and ATP products are largely related to capillary recruitment, vascular
tone, and endothelial function. Although NOS inhibitors may restore
tissue perfusion pressure, they may also decrease capillary density,
induce capillary leak (24), activate platelet aggregation, and promote
leukocyte adhesion in postcapillary venules (18).
On the other hand, the NO donor SIN-1 increased the tissue
O2 extraction capabilities during
endotoxic shock, as reflected by a decrease in
O2 crit
and an increase in
ERO2 crit.
NO donors may promote the metabolic vasodilation of terminal arterioles governing flow through nutritive capillaries. However, if this were the
only mechanism, L-NMMA might be
expected to alter O2 extraction,
and this was not the case. SIN-1, like NO, can exert important
antiinflammatory effects mediated by the inhibition of TNF- (17),
oxygen free radicals (21), platelet-activating factor (3), or
thromboxane A2 (3), and these
effects may have played a role in the improved
O2 extraction capabilities.
To study the interaction between NO and TNF-, we determined TNF-
levels and found they increased after
L-NMMA and SIN-1 administration.
Other studies have shown that NOS inhibitors can enhance endotoxin- or
bacteria-induced TNF- production in experimental sepsis, both in
vitro and in vivo (10), suggesting a regulatory feedback mechanism by
which NO inhibits its own TNF--mediated production. Another recent
study, however, showed that NO can increase TNF- production from
human neutrophils independently of guanosine 3
,5-cyclic
monophosphate stimulated by endotoxin in vitro (34). Kumins et al. (17)
more recently showed that the NO donor molsidomine, which acts only
after it is converted by the liver to SIN-1, attenuated TNF-,
interleukin-1, and interleukin-6 production in endotoxic mice. The
higher TNF- release in the L-NMMA group may contribute to
the detrimental global and regional hemodynamics. A sustained release
of TNF- caused by NOS inhibition was associated with an increased
toxicity of bacteria in mice (10). Although SIN-1-treated animals had
higher nitrite and nitrate concentrations than the other groups, they
had a pattern of TNF- levels similar to the endotoxin-alone group,
showing that TNF- can be released by both NO-dependent and
NO-independent mechanisms (9).
In conclusion, the present study shows that although the NOS inhibitor L-NMMA could transiently reverse endotoxin-induced hypotension, it decreased hepatosplanchnic blood flow. These observations, made when L-NMMA was given before iNOS was expressed, stress that L-NMMA should not be administered before iNOS is induced. L-NMMA did not influence O2 extraction capabilities, suggesting that the loss in O2 extraction capabilities observed early after endotoxemia is not caused by a NO-induced loss of vascular reactivity. On the other hand, the administration of the NO donor SIN-1 could significantly increase global and hepatosplanchnic blood flow without deleterious effects on arterial pressure. SIN-1 could also improve myocardial function and increase tissue O2 extraction capabilities early during endotoxic shock.
ACKNOWLEDGEMENTS
We thank Dr. Wim A. Buurman (Department of Surgery, Immunology
Laboratory, Rijksuniversiteit Limburg, Maastricht, The Netherlands) for
the TNF- determinations, Dr. Bernard Vray (Department of Immunology,
Faculty of Medicine, Free University of Brussels, Belgium) for the
nitrite/nitrate measurement, and Therabel Research for supplying SIN-1.
FOOTNOTES
Address for reprint requests: J.-L. Vincent, Dept. of Intensive Care, Erasme Univ. Hospital, Route de Lennik 808, B-1070 Brussels, Belgium (E-mail:jlvincen{at}resulb.ulb.ac.be).
Received 14 January 1997; accepted in final form 2 June 1997.
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