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CRITICAL CARE AND TRAUMA

Selective iNOS Inhibition Prevents Hypotension in Septic Rats While Preserving Endothelium-Dependent Vasodilation

Volker Strunk^*, Klaus Hahnenkamp^*, Maik Schneuing^*, Lars G. Fischer, MD^*, and George F. Rich, MD, PhD^*

*Department of Anesthesiology, University of Virginia Health System, Charlottesville, Virginia; and Klinik und Poliklinik für Anästhesiologie und operative Intensivmedizin, Westfälische-Wilhelms-Universität Münster, Germany

Address correspondence to George F. Rich, MD, PhD, University of Virginia Health System, Department of Anesthesiology, PO Box 800710, Charlottesville, VA 22908-0710. Address e-mail to gfr2f @virginia.edu.

	Abstract

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Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) mediates hypotension and metabolic derangements in sepsis. We hypothesized that selective iNOS-inhibition would prevent hypotension in septic rats without inhibiting endothelium-dependent vasodilation caused by the physiologically important endothelial NOS. Rats were exposed to lipopolysaccharide (LPS) for 6 h and the selective iNOS-inhibitor L-N6-(1-iminoethyl)-lysine (L-NIL), the nonselective NOS-inhibitor N^G-nitro-L-arginine methyl ester (L-NAME), or control. Mean arterial pressure (MAP) and vasodilation to acetylcholine (ACh, endothelium-dependent), sodium nitroprusside (SNP, endothelium-independent), and isoproterenol (ISO, endothelium-independent ß agonist) were determined. Exhaled NO, nitrate/nitrite-(NOx) levels, metabolic data, and immunohistochemical staining for nitrotyrosine, a tracer of peroxynitrite-formation were also determined. In control rats, L-NAME increased MAP, decreased the response to ACh, and increased the response to SNP, whereas L-NIL did not alter these variables. LPS decreased MAP by 18% ± 1%, decreased vasodilation (ACh, SNP, and ISO), increased exhaled NO, NOx, nitrotyrosine staining, and caused acidosis and hypoglycemia. L-NIL restored MAP and vasodilation (ACh, SNP, and ISO) to baseline and prevented the changes in exhaled NO, NOx, pH, and glucose levels. In contrast, L-NAME restored MAP and SNP vasodilation, but did not alter the decreased response to ACh and ISO or prevent the changes in exhaled NO and glucose levels. Finally, L-NIL but not L-NAME decreased nitrotyrosine staining in LPS rats. In conclusion, L-NIL prevents hypotension and metabolic derangements in septic rats without affecting endothelium-dependent vasodilation whereas L-NAME does not.

Implications: Sepsis causes hypotension and metabolic derangements partly because of increased nitric oxide. Selective inhibition of nitric oxide produced by the inducible nitric oxide synthase enzyme prevents hypotension and attenuates metabolic derangements while preserving the important vascular function associated with endothelium-dependent vasodilation in septic rats.

	Introduction

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Septic shock is the leading cause of death in intensive care units (1). Sepsis is manifested by hypotension, resistance to vasopressors, myocardial depression, altered organ blood flow distribution, hypoglycemia, and lactic acidosis (2). The mechanisms underlying the cardiovascular dysfunction during sepsis are complex; however, they are partially mediated by an uncontrolled production of nitric oxide (NO) (1).

NO is synthesized from L-arginine by a family of enzymes called nitric oxide synthases (NOS). The constitutive isoform of the enzyme is present in the vascular endothelium (eNOS) and releases NO in response to pulsatile flow, sheer stress, and a variety of substances including acetylcholine (ACh) (1). NO stimulates soluble guanylate cyclase (sGC), which increases cyclic 3', 5' guanosine monophosphate and leads to smooth muscle relaxation. Endothelial NOS plays a fundamental role in organ blood flow distribution and is involved in regulation of microvascular permeability and platelet, leukocyte, and endothelial interactions. In contrast, the inducible isoform of NOS (iNOS) is normally present in only very small quantities but increases greatly after induction by endotoxin or various cytokines in several cell types, including leukocytes, endothelial, and vascular smooth muscle cells, and cardiac myocytes (1). Sustained production of NO secondary to iNOS has been implicated in the prolonged smooth muscle vasodilation and poor response to vasoconstrictors in sepsis. NO derived from iNOS results in cytotoxicity (1) primarily mediated by the highly cytotoxic agent peroxynitrite (3,4), which is generated via the reaction of NO with superoxide (5).

Nonselective NOS inhibition attenuates hypotension (6,7) and restores the response to vasoconstrictors (8), but also causes organ damage and inflammation (6,9). Detrimental effects of nonselective NOS inhibition include loss of local blood flow control (7), decreased cardiac output (7), augmented cellular infiltrate (9), and thrombosis formation (6) secondary to inhibition of the physiological important eNOS. Wright et al. (7) reported a shortened survival time after nonselective NOS-inhibition in animal studies.

We previously demonstrated that selective iNOS inhibition restores vasoreactivity without altering endothelium-dependent pulmonary vasodilation in isolated rat lungs (10). Therefore, we hypothesized that selective iNOS inhibition would prevent systemic hypotension in sepsis while preserving eNOS function as determined by endothelium NO-dependent vasodilation. To evaluate this hypothesis, we used an in vivo lipopolysaccharide (LPS)-induced septic rat model to determine the effects of L-N6-(1-iminoethyl)-lysine (L-NIL), a selective iNOS inhibitor, which has a 28-fold selectivity toward iNOS (11) and no effect on baseline mean arterial pressure (MAP) (12). The effects of L-NIL were compared to N^G-nitro-L-arginine methyl ester (L-NAME) a nonselective NOS inhibitor (11), and controls. We determined MAP, the response to endothelium-dependent and independent vasodilators, and the exhaled NO and plasma nitrate/nitrite (NOx) levels in septic and control rats. Finally, we determined the immunohistochemical staining for nitrotyrosine, which is a marker of peroxynitrite formation (4,13).

	Methods

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This study was approved by the Animal Research Committee at the University of Virginia. Male Harlan Sprague-Dawley rats (450–550g) were randomly divided into six groups (n = 5 in each group). Groups 1–3 were injected intraperitoneally with 1 mL of saline concurrent with 1 mL of saline (Group 1), L-NIL 4 mg/kg (Group 2), or L-NAME 5 mg/kg (Group 3). Groups 4–6 received 1 mL of Salmonella typhimurium endotoxin (LPS, 15 mg/kg) concurrent with 1 mL of saline (Group 4), L-NIL 4 mg/kg (Group 5), or L-NAME 5 mg/kg (Group 6).

Six hours later, the rats were anesthetized with halothane followed by sodium pentobarbital (65 mg/kg, intraperitoneally). The rats were placed in a supine position, and a tracheotomy was performed by using a 17-gauge blunted cannula. The rats were ventilated with 100% oxygen by using a rodent ventilator (Harvard Apparatus, Holliston, MA) at a tidal volume of 9 mL/kg and a respiratory rate of 60 breaths/min. A 800-mL sample of exhaled air was collected in a plastic bag for measurements of exhaled NO.

The right femoral artery was cannulated with polyethylene-50 tubing connected to a pressure transducer (Sorenson Transpac; Abbott Laboratories, North Chicago, IL). The arterial pressure was displayed on a monitor (Datascope 2001A; Datascope, Montvale, NJ) and recorded on a polygraph (Grass Model 7; Grass Instruments, Quincy, MA). The right femoral vein was cannulated with polyethylene-50 tubing for the administration of drugs. On completion of the surgical procedure, cardiovascular variables were allowed to stabilize for 10–15 min. MAP was recorded and an arterial blood sample (1 mL) was divided for blood gas analysis or frozen at -20°C for measurements of NOx. The response to vasodilators was determined, after which the rats were killed with an overdose of sodium pentobarbital. A midline laparotomy was performed, and the thoracic aorta was removed and prepared for immunohistochemistry.

Exhaled NO, NOx Assay, and Arterial Blood Gas Analysis (ABG)
Exhaled air was analyzed for NO using a chemiluminescent Nitric Oxide Analyzer (NOA^TM 280; Sievers Instruments, Boulder, CO). Levels of reactive nitrogen intermediates (NOx) were quantified by using the nitrate/nitrite colorimetric assay (Cayman Chemical Company, Ann Harbor, MI). The NOx sample was incubated with an enzyme cofactor mixture for 3 h. Afterward, Griess Reagent was added, and absorbance was measured at 540 nm by using a standard plate reader. ABGs were analyzed with a Chiron model 865 ABG-analyzer (Chiron Diagnostics, Norwood, MA).

MAP and Response to Vasodilators
After measurement of baseline MAP, the responses to vasodilators were determined. Vasodilators tested were the endothelium NO-dependent vasodilator ACh (0.03 µg), the endothelium-independent NO donor sodium nitroprusside (SNP, 7.5 µg) and the endothelium-independent ß adrenoreceptor agonist isoproterenol (ISO, 30 µg). Before each subsequent IV injection, MAP was allowed to return to the baseline level and stabilize for 5 min. Each drug was diluted in 0.8 mL saline, which itself had no effect on pressure. Responses to ACh, SNP, and ISO are expressed in percent (differences in MAP divided by baseline MAP).

Immunohistochemistry and Imaging
The thoracic aorta was immersed in 4% paraformaldehyde in 0.1 M phosphate buffered saline at a pH of 7.4 for 90 min, dehydrated with alcohol, and embedded in paraffin. Four µm sections were cut and mounted onto slides (Snowcoat X-tra^TM; Surgipath Medical Instruments, Richmond, IL). Fixed tissue was deparaffinized with xylene, rehydrated, boiled in 0.01 M citric buffer at a pH of 6.0 for 20 min, and treated with 0.3% H₂O₂ in methanol for 30 min. After blocking nonspecific binding with 3% normal goat serum in phosphate buffered saline containing 3% bovine serum albumin and 0.3% Triton X-100, sections were incubated with a polyclonal nitrotyrosine antibody (5 µg/mL; Upstate Biotechnology, Lake Placid, NY). Labeling was confirmed with a Vectastain^TM ABC Kit (Vector Laboratories, Burlingham, CA), and immunoprecipitation achieved with 3,3'-diaminobenzidine (SIGMA FAST^TM DAB Tablet Sets, Sigma, St. Louis, MO). Sections were counterstained with Hematoxylin I (Richard-Allan Scientific, Kalamazoo, MI). Photomicrographs were taken with a digital camera (Pixera Corporation, Los Gatos, CA) attached to a microscope (Bunton Instrument Company, Rockville, MA) and imported into a software program (Adobe Systems, Mountain View, CA).

Chemicals, Calculations, and Statistical Analysis
L-NIL was obtained from Alexis Corporation (San Diego, CA). All other chemicals were obtained from Sigma Chemicals (St. Louis, MO) and diluted in saline. The concentrations were based on pilot dose-response studies or on previous work from our laboratory (9).

Exhaled NO and NOx data were analyzed by using the Kruskal-Wallis analysis of variance on ranks, followed by the Dunn’s Method for multiple comparisons. All other data were analyzed with one-factor analysis of variance and compared with the Student Newman-Keuls post hoc test. A P < 0.05 was considered significant. All data were presented as the mean ± SEM.

	Results

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Survival rate, Exhaled NO, and NOx Concentrations
Thirty of 31 rats survived until the conclusion of the experiments. One rat, which had received LPS and L-NAME, died 240 min after the injection. There was no statistical difference in survival among the groups.

In control rats, L-NIL and L-NAME had no effect on exhaled NO (1.5 ± 0.5 ppb). LPS significantly increased exhaled NO to 163 ± 18 pbb, which was prevented by L-NIL but not L-NAME ( Fig. 1). In control rats, L-NIL and L-NAME did not alter NOx. LPS resulted in a four-fold increase in the NOx-levels, which was prevented by L-NIL ( Fig. 2). Although L-NAME significantly attenuated the increase in NOx in LPS rats, it did not restore NOx back to the control level.

View larger version (14K): [in this window] [in a new window]   Figure 1. Exhaled NO (ppb) in rats 6 h after the intraperitoneal injection of saline (control), saline plus L-N6-(1-iminoethyl)-lysine (L-NIL), saline plus NG-nitro-L-arginine methyl ester (L-NAME), lipopolysaccharide (LPS) alone, LPS plus L-NIL, or LPS plus L-NAME. Data are expressed as mean ± SEM *P < 0.01, different from control. #P < 0.01, different from LPS alone.

View larger version (16K): [in this window] [in a new window]   Figure 2. Plasma nitrate/nitrite concentration (NOx, µM) in rats 6 h after the intraperitoneal injection of saline (control), saline plus L-N6-(1-iminoethyl)-lysine (L-NIL), saline plus NG-nitro-L-arginine methyl ester (L-NAME), lipopolysaccharide (LPS) alone, LPS plus L-NIL, or LPS plus L-NAME. Data are expressed as mean ± SEM *P < 0.01 different from control. #P < 0.01, different from LPS alone.

View larger version (16K): [in this window] [in a new window]   Figure 3. Baseline mean arterial pressure (MAP) in rats 6 h after the intraperitoneal injection of saline (control), saline plus L-N6-(1-iminoethyl)-lysine (L-NIL), saline plus NG-nitro-L-arginine methyl ester (L-NAME), lipopolysaccharide (LPS) alone, LPS plus L-NIL, or LPS plus L-NAME. Data are expressed as mean ± SEM *P < 0.01, different from control. #P < 0.01, different from LPS alone.

View larger version (27K): [in this window] [in a new window]   Figure 4. Effects of IV injection of acetylcholine (ACh, 0.03 µg), sodium nitroprusside (SNP, 7.5 µg), and isoproterenol (ISO, 30 µg) in rats 6 h after the intraperitoneal injection of saline (control), saline plus L-N6-(1-iminoethyl)-lysine (L-NIL), saline plus NG-nitro-L-arginine methyl ester (L-NAME), lipopolysaccharide (LPS) alone, LPS plus L-NIL, or LPS plus L-NAME. Data are expressed as mean ± SEM and presented as the percent decrease in mean arterial pressure (MAP) from baseline values. *P < 0.01, different from control. +P < 0.01, more than control. #P < 0.01, different from LPS alone.

View larger version (114K): [in this window] [in a new window]   Figure 5. Representative nitrotyrosine-immunostaining photomicrographs of rat aorta 6 h after the intraperitoneal injection of saline (control), lipopolysaccharide (LPS) alone, LPS plus L-N6-(1-iminoethyl)-lysine (L-NIL), LPS plus NG-nitro-L-arginine methyl ester (L-NAME). Positive staining was evident in the LPS rats, but virtually absent in the control rats. L-NAME slightly attenuated staining whereas L-NIL markedly reduced the staining pattern in LPS rats.

	Discussion

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Exhaled NO was significantly increased in LPS rats, which is consistent with previous reports (9,14). The source of exhaled NO is mainly from activated leukocytes (1,14) resulting from the induction of iNOS. Because L-NIL is a potent and relatively selective inhibitor of iNOS, it is not surprising that L-NIL prevented the increase in exhaled NO associated with LPS. In our study L-NAME did not alter exhaled NO although Aaron et al. (9) showed that the increase in exhaled NO was reversible by L-NAME and Stitt et al. (14) found that L-NAME significantly attenuated, but did not prevent the increase in exhaled NO. The doses of L-NAME these investigators used were 12- to 20-fold larger than the dose we used which suggests that L-NAME inhibits iNOS but only at very large doses.

Concentrations of NOx, the stable metabolic end products of NO, are increased during human sepsis and may correlate with circulating endotoxin levels (15). In agreement with our study, Schwartz et al. (6) also showed that NOx-levels are increased in rats exposed to LPS. Horton et al. (16) demonstrated that the levels of NOx are secondary to a time-dependent induction of iNOS. Our study demonstrates that the increase in NOx can be prevented by L-NIL, which further indicates that L-NIL effectively blocks iNOS. Schwartz et al. (6) reported that L-NAME prevented the increase in NOx levels in sepsis, whereas we found that L-NAME did not. This is probably the result of the smaller dose of L-NAME we used as Salvemini et al. (12) demonstrated a dose-dependent inhibition of NOx formation after L-NAME.

The metabolic consequences associated with sepsis include hypoglycemia and lactic acidosis secondary to impaired gluconeogenesis, glycogenolysis, and inhibition of cellular respiration (2). Horton et al. (16) demonstrated in vitro that the inhibition of glucose synthesis correlates well with the induction of iNOS. Our data indicate that L-NIL prevents the hypoglycemia, acidosis, and decrease in HCO₃^- associated with LPS, whereas L-NAME only partially corrected these metabolic derangements. The PaCO₂ was not different among the groups in our study, indicating that the LPS-induced acidosis reflects metabolic rather than respiratory changes. These results may be important because in human sepsis (17) metabolic changes reflected by lactate levels are predictable of survival.

In control rats L-NIL had no effect on MAP, which agrees with studies by Salvemini et al. (12). This is important because it indicates that the dose of L-NIL that we used does not alter eNOS. In contrast, L-NAME significantly increased the baseline MAP which suggests that eNOS was blocked. Interestingly, L-NAME and L-NIL were equally effective in restoring the LPS-induced decrease in MAP back to control levels. These results suggest that L-NAME restores MAP in sepsis via eNOS inhibition in resistance vessels, whereas L-NIL prevents the hypotensive effects of LPS by inhibiting the increase in iNOS.

In control rats, L-NAME significantly decreased endothelium NO-dependent vasodilation to Ach, which is a direct result of eNOS blockade (18). L-NAME also increased the vasodilation to SNP, which Moncada et al. (19) suggested is caused by a specific supersensitivity of the sGC after the removal of basal NO release. In control rats, L-NIL had no effect on ACh or SNP vasodilation indicating that L-NIL does not inhibit eNOS.

In our study, L-NAME did not significantly block iNOS, as assessed by exhaled NO and NOx. However, it decreased the physiologically important eNOS as assessed by MAP and the responses to the endothelium-dependent vasodilators. Larger doses of L-NAME might be able to inhibit iNOS, but would still inhibit eNOS. The observation that L-NAME detrimentally blocks eNOS is underscored by clinical studies demonstrating that nonselective NOS inhibitors ameliorate hypotension, but also alter organ blood flow and decrease oxygen delivery and cardiac output in sepsis (20,21). A recent multicenter study investigating nonselective NOS inhibitors in septic patients, was terminated because of the detrimental effects in the treatment group (1). These observations, coupled with data demonstrating resistance against endotoxin-induced hypotension and death in iNOS knockout mice (22,23) suggest that selective inhibition of iNOS may be a more viable therapeutic strategy in sepsis.

LPS decreased the vasodilation to ACH, which is in agreement with previous reports (6,24). The continuous production of NO by iNOS is likely responsible for the decreased response to ACh during sepsis (24). Possible explanations for why increased iNOS decreases endothelium-dependent vasodilation include downregulation of eNOS as a result of decreased mRNA levels (25), functional inhibition by negative feedback of NO on eNOS (26), lack of substrate (L-arginine) availability (24), or direct endothelial injury (10). In our study, L-NIL restored the vasodilation to ACh in septic rats, thereby supporting the theory that iNOS is responsible for the decreased vasodilation to ACh in sepsis.

Consistent with previous reports (6), LPS decreased the response to SNP, indicating altered endothelium-independent vasodilation. Papapetropoulus et al. (27) showed that the decreased response to SNP is partly mediated by a decrease in sGC, as a mechanism to offset the large increase in iNOS (1). This proposed mechanism is consistent with our results, because L-NIL restored the SNP vasodilation. In contrast, Umans et al. (28) demonstrated that endotoxin decreased endothelium-dependent but not endothelium-independent vasodilation which suggests a receptor-mediated mechanism. The LPS-induced decrease in vasodilation to the endothelium-independent ß adrenoreceptor agonist ISO observed in our studies agrees with previous reports (29) and may result from a desensitization of adenylate cyclase stimulation (30). Our observation that L-NIL prevented the decreased response to ISO in LPS rats implies a role for iNOS in the hyporesponsiveness to ß adrenoreceptor agonists. Silverman et al. (30) showed that in human sepsis the myocardium also suffers from a desensitization of ß adrenoreceptor function. Because septic patients are dependent on oxygen delivery to prevent lactic acidosis, a functional ß adrenergic system is critical, which suggests that the septic myocardium might also benefit from L-NIL.

There is substantial evidence that the effects of prolonged NO overproduction are partially mediated by the highly cytotoxic agent peroxynitrite. Geng et al. (31) demonstrated that iNOS induction leads to blockade of mitochondrial respiration and increased lactate production, which is mainly mediated by peroxynitrite (32). Szabó et al. (32) demonstrated that peroxynitrite causes DNA strand breakage with the subsequent suicidal activation of poly-adenosine triphosphate ribosyltransferase, depleting the already decreased adenosine triphosphate- and nicotinamide adenine dinucleotide resources. Peroxynitrite impairs cellular function and causes cell death in vitro (3) and in vivo (4). Moreover, Szabó et al. (32) showed that, during endotoxemia, poly-adenosine triphosphate ribosyltransferase inhibition improves vascular reactivity and prolongs survival time.

To evaluate the role of peroxynitrite, we assessed by immunohistochemistry the localization of nitrotyrosine, a specific marker for peroxynitrite formation (4,13) in thoracic aorta. Consistent with observations of Szabó et al.(13), we found that LPS causes a substantial increase in peroxynitrite levels in the vicinity of large blood vessels. In addition to the diffuse staining, we observed that tyrosine nitration was particularly noticeable in endothelial cells and the internal luminal layers of the muscularis. Our observation that L-NIL but not L-NAME prevents tyrosine nitration suggests that iNOS is the major source of peroxynitrite.

In conclusion, L-NIL restored MAP and vasoreactivity in septic rats to control levels while preserving endothelium-dependent vasodilation. L-NIL also prevented the increase in exhaled NO, abolished the formation of NOx and peroxynitrite, and corrected the metabolic derangements in LPS rats. In contrast, L-NAME blocked eNOS and only partially restored metabolic derangements. This study indicates that L-NIL may prevent the excessive production of NO by iNOS while preserving the physiologically important eNOS.

	Footnotes

 
Presented in part at the 1999 Annual Meeting of the American Society of Anesthesiologists in Dallas, TX, October 9–13, 1999.

	References

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