Minor involvement of nitric oxide during chronic endotoxemia in anesthetized pigs

Catherine M. Pastor, Antoine Hadengue, and Andreas K. Nussler

Division d'Hépatologie et de Gastroentérologie, Hôpital Cantonal Universitaire de Genève, CH 1211 Geneva, Switzerland; and Allgemein-, Viszeral-, und Transplantationschirurgie, medizinische Facultät der Humboldt-Universitat, 13353 Berlin, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To study the modifications of hepatic blood flow and hepatic function over time during endotoxemia, 10 pigs received a continuous intravenous infusion of endotoxin (Endo, 160 ng · kg-1 · h-1) over 18 h and 7 control (Ctrl) animals received a saline infusion. The involvement of nitric oxide (NO) in this endotoxic model was assessed by measuring plasma concentrations of NO-2, NO-3, and cGMP, by testing vascular reactivity to ACh, and by evaluating inducible NO synthase (NOS 2) expression in hepatic biopsies. Endotoxin induced hypotensive and normokinetic shock in association with few modifications of hepatic blood flow, and hepatic injury was observed in both groups. Endotoxin did not increase plasma concentrations of NO-2, NO-3, and cGMP. The ACh-dependent decrease of mean arterial pressure was reduced in Endo pigs, whereas a minor difference was observed between Ctrl and Endo pigs for ACh-dependent modification of hepatic perfusion. Hepatic NOS 2 mRNA was not detected in Ctrl pigs. In Endo pigs, NOS 2 protein expression was detected only in tissues surrounding the portal vein and the inferior vena cava, whereas NOS 2 mRNA was expressed in all hepatic biopsies. Thus, although endotoxemia induces NOS 2 expression in the liver, our findings show that NO involvement is lower in pigs than in rodents during endotoxemia.

liver; vascular reactivity; acetylcholine; inducible nitric oxide synthase; hepatic circulation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SEPTIC SHOCK, which remains an important cause of death in intensive care units, is characterized by hypotension, vascular hyporeactivity to vasoactive agents, myocardial dysfunction, and altered regional blood flows (4, 25). The multiple-organ dysfunction syndrome, which is defined as two or more organs failing at the same time, frequently occurs and is associated with a high mortality rate. Endotoxin, which is included in the wall of Gram-negative bacteria, is responsible for most of the abnormalities observed in sepsis and is commonly used to mimic septic shock in experimental models.

The liver is crucial in severe sepsis because it contains most of the macrophages of the body (Kupffer cells) able to clear endotoxin and bacteria that may stimulate the systemic inflammatory response. Moreover, hepatocytes synthesize the acute-phase proteins and enzymes necessary to modulate the inflammatory response. Finally, because hepatic blood flow represents 25% of cardiac output (CO), modifications of hepatic perfusion may greatly interfere with systemic hemodynamics.

Nitric oxide (NO) is one of the major mediators that induce cardiovascular abnormalities during septic shock. Under physiological conditions, the constitutive endothelial isoform of NO synthase (eNOS or NOS 3) produces low levels of NO and regulates vascular tone as well as numerous cell functions (24). In contrast, during sepsis, the inducible form of NOS (iNOS or NOS 2) produces large amounts of NO (14, 33). However, evidence exists that humans produce less NO than experimental animals during septic shock (28). NOS 2 expression has been observed mostly in rodents intraperitoneally injected with endotoxin, and few studies sought to determine protein expression in large animals. Because the induction of NOS 2 requires de novo protein synthesis of the enzyme, studies longer than 4 h are required to accurately evaluate the role of NO originating from NOS 2 in the pathogenesis of endotoxemia.

To study the involvement of hepatic NO during endotoxemia over time in a species different from rodents, we measured plasma concentrations of NO-2, no3, and cGMP, tested the effect of ACh on mean arterial pressure (MAP) and hepatic blood flows over time, and evaluated NOS 2 expression in hepatic biopsies in anesthetized pigs infused with endotoxin from Escherichia coli (160 ng · kg-1 · min-1) over a 18-h period.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and anesthesia. Male or female minipigs (23 ± 1 kg, n = 20) were fasted with free access to water for 24 h before the experiment, premedicated, and placed in a supine position on the operating table. After the induction of anesthesia with halothane, pigs were mechanically ventilated with air and oxygen [fractional inspired O2 concentration (FIO2) = 0.4] to obtain normal arterial PCO2. After intubation, halothane was withdrawn and anesthesia was maintained with thiopental (5 mg · kg-1 · h-1) and fentanyl (10-20 µg · kg-1 · h-1). In addition, pancuronium (0.2 mg · kg-1 · h-1) was used as a skeletal muscle relaxant. The protocol was approved by the Animal Welfare Committee of the University of Geneva and the Veterinary Office and followed the Guidelines for the Care and Use of Laboratory Animals.

Surgical procedure. The right carotid artery was cannulated to measure MAP and to collect blood samples. A catheter was inserted into the right external jugular vein for fluid and drug administration. A pulmonary artery catheter (131H-7F, Baxter, Düdingen, Switzerland) was inserted through the right internal jugular vein to measure mean pulmonary arterial pressure (mmHg), central venous pressure (mmHg), pulmonary wedge pressure (mmHg), and CO (ml · min-1 · kg-1).

After a midline abdominal incision, the bladder was drained. Two flow probes were positioned around the portal vein and the hepatic artery (above the bifurcation of the common hepatic artery and the gastroduodenal artery) to determine portal vein blood flow (PVBF, ml/min) and hepatic artery blood flow (HABF, ml/min). Blood flows were measured with the ultrasound transit time flow technique (Transonic System, Ithaca, NY). To measure portal vein pressure (PVP, mmHg) and hepatic vein pressure (HVP, mmHg), catheters were inserted into the portal vein through a side branch and into the hepatic vein via the left external jugular vein, respectively. Location of the catheter tips were confirmed by direct palpation. When surgery was completed, the abdominal wall was tightly reapproximated to minimize heat loss during the experiment. Core temperature was maintained with heating lamps.

Experimental protocol. After a 2-h stabilization period, hemodynamic parameters were measured from time (t) = 0 (baseline value) over 18 h. Endotoxic (Endo, n = 13) pigs received a continuous intravenous infusion of endotoxin from E. coli (160 ng · kg-1 · min-1) over 18 h. Control (Ctrl, n = 7) animals received a saline infusion in similar volume. Animals were infused with saline (12 ml · kg-1 · h-1 during surgery and 8 ml · kg-1 · h-1 during experimental protocol) to compensate for fluid loss induced by anesthesia and surgery. After the recovery period, hemodynamic parameters were recorded every 3 h for the next 18 h.

Determination of systemic and hepatic O2 delivery and O2 consumption. Expired flow from the ventilator was directly connected to a metabolic measurement cart (Datex, Helsinki, Finland) for continuous measurements of O2 consumption (VO2, ml/min). O2 contents in the carotid artery (a), the portal vein (pv), and the hepatic vein (hv) were calculated according to the equation
O<SUB>2</SUB> content = (P<SC>o</SC><SUB>2</SUB> ⋅ 0.003) + (Hb ⋅ S<SC>o</SC><SUB>2</SUB> ⋅ 1.36)
The following equations were used
systD<SC>o</SC><SUB>2</SUB> = CO ⋅ aO<SUB>2</SUB> content

aD<SC>o</SC><SUB>2</SUB> = HABF ⋅ aO<SUB>2</SUB> content

pvD<SC>o</SC><SUB>2</SUB> = PVBF ⋅ pvO<SUB>2</SUB> content

hepD<SC>o</SC><SUB>2</SUB> = pvD<SC>o</SC><SUB>2</SUB> + aD<SC>o</SC><SUB>2</SUB>

hepV<SC>o</SC><SUB>2</SUB> = [(aO<SUB>2</SUB> content ⋅ HABF) + (pvO<SUB>2</SUB> content ⋅ PVBF)] − [hvO<SUB>2</SUB> content ⋅ (HABF + PVBF)]
where systDO2, aDO2, pvDO2, and hepDO2 are systemic, hepatic artery, portal vein, and hepatic O2 delivery, respectively, and hepVO2 is hepatic VO2.

Hepatic injury. To assess hepatic function, we measured the bile flow every 3 h (µl · min-1 · 100 g-1) and calculated the ratio between the wet and the dry weights of the liver. Hepatic cell damage was assessed by arterial concentrations of aspartate transaminase (ASAT) and alanine transaminase (ALAT), gamma -glutamyl transpeptidase (gamma -GT), alkaline phosphatase, and lactate dehydrogenase (LDH) (all in IU/l).

Vascular reactivity to ACh. ACh (10 µg/kg) was intravenously injected over 20 s, and changes in MAP, HABF, and PVBF were observed over the 200 s following the drug administration. To compare the evolution of the vascular reactivity over time, three tests (A, B, and C) were performed 5, 11, and 17 h after initial endotoxin or saline administration.

Arterial levels of NO-2, NO-3, cGMP, and 6-keto-PGF1alpha . Serum samples were assayed for the stable end products of NO oxidation (NO-2 and NO-3) using a modified procedure based on the Griess reaction as recently described (23). Briefly, samples were deproteinized by ultrafiltration (Centrisart, Sartorius, Göttingen, Germany) to eliminate high-molecular-weight particles. NO-3 was then enzymatically reduced to NO-2 using nitrate reductase, and samples were screened for total NO-2 concentrations (representing NO-2 + NO-3) with a mixture of Dapsone (4,4'-diaminodiphenyl sulfone), and N-(1-naphthyl)ethylenediamine. After incubation at room temperature, light absorption was measured at 550 nm in a microplate reader (EAR 300; SLT, Crailsheim, Germany). NO-2 concentrations were calculated from a standard NO-2 curve. Plasma concentrations of cGMP and 6-keto-PGF1alpha (a stable breakdown product of prostacyclin) were measured by radioimmunoassay technique according to the instructions provided by the manufacturer (Amersham, Little Chalfont, UK).

NOS 2 synthase expression in liver. To assess the induction of NOS 2 at various sites of the liver, biopsies were collected from the right and left lobes from tissues surrounding the great vessels (inferior vena cava and portal vein) 6 and 9 h after the start of endotoxin administration. Biopsies were collected, snap frozen, and stored at -70°C before being assayed.

Analysis of NOS 2 mRNA by RT-PCR. Total RNA was isolated and subjected to RT-PCR analysis as previously described (17). Two milligrams of total RNA were reverse transcribed into cDNA at 37°C for 60 min in 10 µl of solution containing 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 4 mg MgCl2, 1 mM each dNTP (Boehringer Mannheim), 10 mM DL-dithiothreitol, 10 U of human placental RNAse inhibitor (GIBCO BRL, Gaithersburg, MD), oligo(dT) primers (Boehringer Mannheim), and 200 U of Moloney murine leukemia virus reverse transcriptase (GIBCO BRL). The cDNA was then used to detect NOS 2 mRNA by PCR using human primers. The sequences for NOS 2 were 5'-GCCTCGCTCTGGAAAGA-3' (bases 1,425-1,441, sense) and 5'-TCCATGCAGACAACCTT-3' (bases 1,908-1,924 antisense), amplifying a 499-bp product. Equal loading of RNA was verified by beta -actin, using specific primers as follows: 5'-CGGGAACCGCTCATTGCC-3' (sense) and 5'-ACCCACACTGTGCCCATCTA-3' (antisense) amplifying a 289-bp PCR product. PCR reactions were carried out in 50 µl of solution containing 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 200 mM each dNTP, 1.5 mM MgCl2, and 1.25 U of Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT). Thirty cycles of 1 min at 96°C (denaturing), 2 min at 56°C (annealing), and 2 min at 72°C (extension) were performed in a thermal cycler (Perkin-Elmer Cetus).

Western blot analysis. Tissues were homogenized in 20 mM TES, 2 mM DL-dithiothreitol, 10% glycerol, 25 mg/ml antipain, 25 mg/ml aprotinin, 25 mg/ml leupeptin, 25 mg/ml chymostatin, 50 mM phenanthroline, 10 mg/ml pepstatin A, and 100 mM phenylmethylsulfonyl fluoride and centrifuged at 100,000 g (crude cytosol) as previously described (20). Equal amounts of protein (60 µg) obtained from biopsies were separated by 7.5% SDS-PAGE and electrophoretically transferred to nitrocellulose membranes using a transblot apparatus (Pase, Lübeck, Germany). Nonspecific binding to the membrane was blocked by 5% nonfat dry milk in PBS-Tween 20 overnight at 4°C. The blots were washed twice in PBS and then incubated with an affinity-purified IgG polyclonal rabbit-anti-mouse antibody (1:1,000, Transduction Laboratories, Lexington, KY). This antibody has been chosen because it cross-reacts with the porcine NOS 2 isoform (13). As positive control we used isolated pig hepatocytes exposed over 9 h to rh-interferon-gamma (rh-IFN-gamma , 200 U/ml), rh-tumor necrosis factor-alpha (rh-TNF-alpha , 1,000 U/ml), rh-interleukin-1beta (rh-IL-1beta , 15 U/ml), endotoxin (10 µg/ml), and 1 mM L-arginine. All culture additives were purchased from Sigma (Deideshoffen, Germany). After incubation with the first antibody, membranes were washed (5 times) with PBS-Tween-20 and incubated with the second antibody (1:1,000; goat-anti-rabbit IgG, conjugated with horseradish peroxidase, Amersham) for 1 h at room temperature. After the incubation, membranes were washed with PBS-Tween-20, developed with 10 ml of a 1:1 mixture of solutions 1 and 2 of the ECL detection system (Amersham), and exposed to a film.

Statistical analysis. Data are expressed as means ± SE. Data were analyzed using one-way and two-way ANOVA for repeated measures with Bonferroni test for within-group comparison over time when appropriate. Significance was established at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hemodynamic parameters in Ctrl and Endo pigs. All animals survived in the Ctrl group, whereas three pigs died in the Endo group after 3, 9, and 12 h of endotoxemia, respectively. Data from these three pigs were not included in the Endo group (Table 1). In the Ctrl group, MAP decreased after t = 12 h, whereas CO remained steady over time. In contrast, in the Endo group, MAP rapidly decreased at t = 3 h and remained low until the end of the experimental protocol, whereas the only significant decrease in CO was observed at t = 6 h. Heart rate increased from t = 3 h to t = 18 h in the Endo group but remained steady in the Ctrl group. HABF and PVBF were not modified over time in either group.

                              
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Table 1.   Systemic and hepatic hemodynamic parameters in control and endotoxic pigs

Systemic and hepatic parameters of oxygenation in Ctrl and Endo pigs. sysDO2 and VO2 remained steady over time in the two groups (Table 2). In Ctrl pigs, pvDO2 and aDO2 did not change over time. Infusion of endotoxin did not modify pvDO2 and aDO2. However, hepDO2/sysDO2 increased at t = 18 h in Ctrl pigs and did not change in Endo pigs.

                              
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Table 2.   Systemic and hepatic parameters of oxygenation in Ctrl and Endo pigs

Biological parameters in Ctrl and Endo pigs. Endotoxin infusion induced metabolic acidosis that started at t = 3 h and peaked at t = 18 h (Table 3). Furthermore, we observed a significant increase of arterial lactate concentration by 18 h. In Ctrl pigs arterial pH and HCO-3 remained within normal concentrations, whereas arterial lactate concentrations decreased over time. Hemoconcentration was observed only from t = 3 h to t = 9 h in the Endo group. Platelet count decreased over time in both groups, but the decrease was higher in the Endo than in the Ctrl group. Leukocyte count remained steady in the Ctrl group, but leukopenia was severe in the Endo group at t = 6 h, with a partial recovery over time. The PaO2-to-FIO2 ratio was unchanged in the Ctrl group over time. In Endo pigs, the ratio slightly dropped at t = 6 h after the start of endotoxin infusion and remained low until the end of the protocol. Temperature remained steady in both groups over time.

                              
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Table 3.   Biological parameters in arterial plasma from Ctrl and Endo pigs

Hepatic function in Ctrl and Endo pigs. Bile flow decreased similarly in the groups over time (Table 4). The ratio between the wet and dry weights of the liver was also similar in Ctrl (3.61 ± 0.14) and Endo (3.88 ± 0.09) groups. ALAT and alkaline phosphatase remained steady in the Ctrl group, whereas gamma -GT and LDH decreased and ASAT increased. In the Endo group, ASAT also increased and the increase was higher in this group than in the Ctrl group. LDH and alkaline phosphatase showed a twofold increase over time, whereas, similar to the Ctrl group, gamma -GT decreased.

                              
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Table 4.   Hepatic functions in Ctrl and Endo pigs

Plasma concentrations of NO-2, NO-3, cGMP, and 6-keto-PGF1alpha . We found that NO-2 + NO-3 and cGMP did not change over time in Ctrl and Endo pigs (Table 5). In contrast, 6-keto-PGF1alpha remained steady in Ctrl pigs and significantly increased in Endo pigs over time.

                              
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Table 5.   NO-2 + NO-3, cGMP, and 6-keto-PGF1alpha in arterial plasma from Ctrl and Endo pigs

MAP and hepatic blood flow changes after ACh injection. To test NO-dependent vascular reactivity, we compared the MAP response to ACh in Ctrl and Endo pigs 5 (test A), 11 (test B), and 17 (test C) h after initial endotoxin or saline administration (Fig. 1). MAP decreased after ACh injection, and the decrease remained steady over time in both groups. However, the decrease was significantly lower in the Endo group (A: -14 ± 2 mmHg; B: -17 ± 3 mmHg; C: -12 ± 2 mmHg) than in the Ctrl group (A: -31 ± 3 mmHg; B: -31 ± 4 mmHg; C: -26 ± 3 mmHg). HABF increased after ACh injection. The HABF increase diminished over time, but the impaired vascular response was similar in both groups. In contrast, the PVBF decrease after ACh was constant over time in Ctrl pigs but significantly diminished in Endo pigs 5 h after the start of endotoxin infusion.


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Fig. 1.   Hepatic artery blood flow (HABF; A) and portal vein blood flow (PVBF; B) after ACh injection (10 µg/kg). Tests were performed 5 h (test A), 11 h (test B), and 17 h (test C) after initial endotoxin or saline administration. Endotoxic pigs (filled bars, n = 10) were infused with endotoxin (160 ng · kg-1 · min-1) and control pigs (open bars, n = 7) were infused with saline. *dagger P < 0.05 vs. control pigs during test A.

NOS 2 mRNA and protein expression in liver biopsies. After the injection of endotoxin, NOS 2 mRNA is expressed between 2 and 6 h (20). No NOS 2 mRNA was detected in Ctrl pigs at t = 6 h (Fig. 2). In contrast, mRNA scored positive in the right and left lobes of the liver as well as in tissues surrounding the inferior vena cava and the portal vein. Similar results were observed at t = 9 h.


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Fig. 2.   Nitric oxide synthase (NOS) 2 mRNA expression in hepatic biopsies collected from control (lanes 1 and 2) and endotoxic (lanes 3-9) pigs. Biopsies were collected around portal vein (lane 3), inferior vena cava (lane 4), right superior lobe (lanes 5 and 6), left superior lobe (lanes 7 and 8), and right inferior lobe (lane 9). Biopsies were collected 6 h after start of saline or endotoxin infusion. MW, molecular weight.

At t = 9 h, the NOS 2 protein was not detected in Ctrl pigs (Fig. 3). In one Endo pig, the protein was expressed only in tissues surrounding the portal vein and the inferior vena cava, whereas in other hepatic regions no protein could be detected. The porcine NOS 2 isoform also scored positive in hepatocytes exposed to cytokines and endotoxin. Similar results were found in two other Endo pigs at t = 9 h, whereas at t = 6 h the NOS 2 expression was much weaker (Table 6). Thus, although mRNA was found in all hepatic regions, the protein was expressed only in the regions surrounding the great vessels.


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Fig. 3.   NOS 2 protein expression in hepatic biopsies collected from 1 Ctrl and 1 Endo pig. Positive control (PC) shows protein expression in porcine hepatocytes incubated with endotoxin and cytokines [rh-interferon-gamma (200 U/ml), rh-tumor necrosis factor-alpha (1,000 U/ml), rh-interleukin-1beta (15 U/ml)] over 9 h. Biopsies were collected around portal vein (lane 3), inferior vena cava (lane 4), right superior lobe (lanes 5 and 6), left superior lobe (lanes 7 and 8), and right inferior lobe (lane 9) 9 h after start of endotoxin [lipopolysaccharide (LPS)] administration or saline infusion (Ctrl).


                              
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Table 6.   NOS 2 protein expression in porcine liver biopsies


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In anesthetized pigs, continuous infusion of endotoxin over 18 h induced a hypotensive and normokinetic shock in association with minor modifications of hepatic blood flows and O2 delivery to the liver. Metabolic acidosis and pulmonary dysfunction occurred gradually in Endo pigs. Minor hepatic injury was observed in both groups. Endotoxin infusion did not significantly increase plasma concentrations of NO-2 + NO-3 and cGMP. Moreover, the MAP decrease after ACh injection was lower in Endo than in Ctrl pigs. The HABF increase after ACh gradually decreased over time, whereas the PVBF decrease remained steady over time in both groups. Endotoxin did not modify the hepatic responses to ACh, except at t = 5 h, when the PVBF decrease was lower in Endo than in Ctrl pigs. NOS 2 mRNA was not detected in hepatic biopsies collected from Ctrl pigs. Although NOS 2 mRNA was present in various hepatic segments collected from Endo pigs, NOS 2 protein expression was detected only in tissues surrounding the portal vein and the inferior vena cava. In contrast, plasma concentrations of prostacyclin greatly increased over time. Thus, although endotoxemia induces NOS 2 expression in the liver, NO involvement remained minor in this model of sepsis.

Endotoxic model. Numerous porcine models of endotoxemia have been described in the literature, but dose and length of infusion varied from study to study. For example, Hasibeder et al. (15) infused 2 µg/kg of endotoxin over 20 min, Cohn et al. (7) infused 250 µg/kg over 20 min, Breslow et al. (5) infused 5 µg/kg over 40 min, and Klemm et al. (18) infused 15 µg/kg over 3 h. In all studies, except that published by Weingand et al. (36), pigs were anesthetized. The mortality rate in these experimental models varied from 38% to 58% and was mostly the consequence of severe pulmonary hypertension. In our study, the mortality rate was lower because 13 pigs were infused with endotoxin and only 3 pigs died before the end of the experiment. As observed in the present study, most of the porcine models of sepsis described hypotensive and normo- or hypokinetic shock with pulmonary hypertension (30, 35). This model was also characterized by hemoconcentration, decreased platelet count, leukopenia, and metabolic acidosis. The PaO2-to-FIO2 ratio decreased significantly over time in Endo pigs but remained steady in Ctrl pigs. Interestingly, surgery and long-term anesthesia also modified hemodynamic and biological parameters in Ctrl pigs: MAP decreased whereas CO remained constant, and platelet count decreased whereas leukocyte count, hematocrit, pH, and plasma lactate concentrations did not change over time. In contrast to rodents, in which endotoxin is mainly injected intraperitoneally, large animals received endotoxin as a bolus or a continuous infusion. Because endotoxin might be detoxified by extrahepatic macrophages when it is injected intravenously, the total amount of endotoxin reaching the liver may be lower in pigs than the amount found in rodents in which endotoxin is intraperitoneally injected. This fact might explain the difference in NOS 2 expression between pigs and rodents during endotoxemia. Moreover, bacterial translocation that might occur during endotoxemia was more likely to reach the systemic circulation through the mesenteric lymph node than through the portal vein (22, 34).

Hepatic flow and function during endotoxemia. In the present study, hepatic perfusion was not significantly modified over time in both groups. Recently, in anesthetized pigs, Saetre et al. (30) found a severe hepatic hypoperfusion within 3 h of endotoxin infusion that was reversed by the selective iNOS inhibitor aminoethyl-isothiourea. Concomitantly, hepatic DO2 decreased, whereas hepatic VO2 was maintained constant by the hepatic O2 extraction increase (30). Minor and transient hepatic hypoperfusion were also observed after a single bolus injection of endotoxin by Maeda et al. (21). In a porcine model of peritonitis, Rasmussen et al. (29) found that hepatic VO2 is limited by decreased hepatic DO2 associated with lactic acidosis and hepatic lactate release, suggesting that hepatic ischemia occurred in this model.

The decrease of bile flow and the ratio between the wet and dry weights of the liver were similar in these groups. Additionally, minor hepatic injury was also observed in both Endo and Ctrl groups. The alteration of hepatic tests after surgery in anesthetized pigs infused with saline has already been described (21).

NO-2 and NO-3 concentrations in arterial plasma. Among the several methods available to estimate the release of NO, the determination of plasma concentrations of the stable NO end products, i.e., NO-2 and NO-3 (or NOx) was commonly used. Plasma NOx concentrations in control large animals were <5 µM (11, 18, 38), but Santak et al. (32) measured higher NOx concentrations as observed in the present study. After a bolus injection of endotoxin, a minimal increase of arterial NOx (<12 µM) was measured (11, 18, 38) and this increase was much lower than the increase measured in rodents (20). In healthy humans, plasma NOx concentrations were between 20 and 30 µM and reached up to 150 µM during septic shock (23, 28). Moreover, human hepatocytes (26) and macrophages (2) produced less NO than the same cell types isolated from rodents. Consequently, evidence exists that in septic conditions, plasma NOx concentrations were higher in rodents than in humans, whereas the NOx concentrations were much lower in large animals than in rodents. Furthermore, plasma NOx concentrations observed in anesthetized pigs and dogs might not reliably reflect overall NO production. In endotoxic pigs, Santak et al. (32) showed that the absence of NOx increase did not reflect the increase of NO production measured by the 15NaNO3 kinetic method. Similarly to our study, baseline plasma NOx concentrations did not differ 9 h after an endotoxin bolus, whereas the direct measurement of NO production increased significantly over time. Moreover, measurements of plasma NOx might have led to errors in estimating the true amount of NO formation when renal function and/or extracellular volume were altered (37). These findings made it difficult to correlate hemodynamic changes during endotoxemia with NOx concentrations in plasma (11). Finally, because atrial natriuretic factor (ANF) also acted on the guanylate cyclase and because ANF concentration in serum was increased by endotoxin, cGMP measurements did not solely reflect the production of NO but might also be the consequence of an increased production of ANF (1).

NO release after ACh injection. An alternative way to detect overall NO production is to study the vascular response to ACh injection, which induces endothelium relaxation through the release of NO. Accordingly, ACh injection decreased MAP in the two experimental groups. This effect could be fully reversed by NOS inhibitors (8). In Endo pigs, the MAP decrease after ACh injection was lower than that observed in Ctrl pigs, showing that NO release from the endothelium was impaired during endotoxemia. Interestingly, the MAP response to ACh remained steady in Ctrl and Endo pigs over time and the HABF responses to ACh decreased in both groups. In contrast, the PVBF decrease (which reflects decreased blood flow coming from splanchnic organs) did not change over time. Moreover, the PVBF response during test A was lower in Endo pigs than in Ctrl pigs.

NOS 2 detection in hepatic biopsies. Finally, detection of NOS in these models of endotoxic shock is an additional way to determine the involvement of NO. Three different isoforms of NOS have been characterized. Two of them are constitutive (NOS 1 and NOS 3) and produce low levels of NO. NO released from the constitutive NOS isoforms functions as a cell signaling molecule in different physiological processes, such as neurotransmission, regulation of vascular tone, and platelet aggregation. The iNOS isoform (NOS 2) produces large amounts of NO and is detected during various inflammatory events. Induction of NOS 2 in vivo in response to endotoxin was observed in numerous tissues including the liver (6, 9, 19, 20, 31). In the liver, NOS 2 was induced in hepatocytes, Kupffer cells, endothelial cells, and stellate cells during endotoxemia (20). Although numerous studies found NOS 2 expression in various tissues collected from rodents injected with endotoxin, the protein has not yet been detected in large animals infused with endotoxin. The present study clearly provides evidence for NOS 2 mRNA expression in various hepatic locations of the liver, whereas NOS 2 protein expression was found only in the hepatic tissues surrounding the great vessels. The difference between NOS 2 mRNA and protein expression might indicate some problems with regard to the specificity of the antibody used to detect NOS 2 protein. In the present study we used an affinity-purified IgG polyclonal rabbit-anti-mouse antibody that scored positively with NOS 2 protein (Fig. 3) and has been demonstrated to cross-react with porcine NOS 2 (13). Although we tried to precipitate the NOS 2 protein from the sample that scored negatively, we were unable to detect any NOS 2 protein.

Because NOS 2 has not been cloned in pigs, we used human-specific NOS 2 primers. This approach was successful in porcine platelets (3). This result was not surprising because, across species, each isoform is well conserved (>80%) (12). However, although a homology of >80% was observed among the different NOS 2 sequences, other differences, such as the partial calcium dependence of human NOS 2 or the cofactor dependence between human and murine NOS 2, have been described (26). To what extent these differences are important in the detection of porcine NOS 2 is unknown. Finally, the fact that NOS 2 mRNA was expressed in some hepatic locations where NOS 2 protein was undetectable has already been demonstrated in porcine platelets (3).

6-keto-PGF1alpha concentrations in arterial plasma. In contrast to plasma NOx concentrations, which did not increase significantly over time, the plasma concentrations of 6-keto-PGF1alpha (stable breakdown product of prostacyclin) increased in Endo pigs and remained steady in Ctrl pigs. Prostacyclin is a mediator of inflammation synthesized from arachidonic acid through the cyclooxygenase pathway. Increased release of 6-keto-PGF1alpha was also observed in numerous experimental models of sepsis (10, 16, 27).

In summary, infusion of endotoxin resulted in hypotensive and normokinetic shock in association with minor modifications of hepatic perfusion over a 18-h experimental period. The ACh-dependent decrease of MAP was reduced in Endo pigs, whereas a minor difference was observed between Ctrl and Endo pigs for ACh-dependent modification of hepatic perfusion. Endotoxin did not modify the arterial concentrations of NOx and cGMP. However, in hepatic biopsies, NOS 2 protein was expressed 6 and 9 h after the start of endotoxin injection and localized around the great vessels. Thus, although endotoxemia induces NOS 2 expression in the liver, NO involvement remains minor in this model of sepsis.


    ACKNOWLEDGEMENTS

We thank Elisabeth Bernouilli, Jennifer Hantson, Manuel Jorge-Costa, Sylvie Roulet, and Aiguo Wang for excellent technical assistance.


    FOOTNOTES

This work was supported by the Fond National Suisse de la Recherche Scientifique (nos. 3200.45985.95 and 3200.53941.98) (C. M. Pastor).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C. M. Pastor, Div. d'hépatologie et de gastroentérologie, Hôpital Cantonal Universitaire de Genève, 24 Rue Micheli-du-Crest, CH 1211 Geneva 14, Switzerland. (E-mail: Catherine.Pastor{at}medecine.unige.ch).

Received 22 February 1999; accepted in final form 4 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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