Anesth Analg 2001;92:128-133
© 2001 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
The Effects of Lidocaine on Nitric Oxide Production from an Activated Murine Macrophage Cell Line
Makoto Shiga, MD,
Kahoru Nishina, MD,
Katsuya Mikawa, MD, and
Hidefumi Obara, MD
Departments of Anesthesiology and Intensive Care Unit, Kobe University School of Medicine, Kobe, Japan
Address correspondence and reprint requests to Katsuya Mikawa, MD, Departments of Anesthesiology and Intensive Care Unit, Kobe University School of Medicine, Kusunoki-cho 7, Chuo-ku, Kobe 650-0017, Japan.
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Abstract
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Nitric oxide (NO), overproduced by activated macrophages, is important in the pathogenesis of various diseases, including septic shock and inflammatory tissue injury, as well as antibacterial host defense mechanisms. We examined the effects of lidocaine on NO production and the expression of inducible NO synthase (iNOS) protein and messenger RNA (mRNA) in activated macrophages. Murine macrophage-like cell line RAW 264 was stimulated for 8 h with lipopolysaccharide (10 mg/mL) and interferon-
(50 U/mL) in the presence of various concentrations of lidocaine (0–500 mg/mL). NO production was assessed by measuring levels of the stable metabolites, nitrite and nitrate (NOx), in the culture medium with an automatic analyzer using the Griess reaction. Expression of iNOS mRNA in harvested RAW 264 was quantified by Northern blot analysis using mouse iNOS complementary DNA probe. Expression of iNOS protein in the cells was assessed by Western blot analysis using anti-iNOS antibody. Lidocaine dose-dependently attenuated the increase in NOx levels in response to the stimulants. The drug at any concentration failed to decrease iNOS mRNA expression in RAW 264. Lidocaine at 500 mg/mL decreased iNOS protein levels. These data suggest that lidocaine reduced NO production in activated macrophages at multiple levels after transcription. The inhibitory site appeared to vary with the dose of lidocaine.
Implications: Lidocaine dose-dependently inhibited nitric oxide production by activated macrophages, presumably at levels after transcription. The attenuating effect of lidocaine on organ injuries previously reported may be explained by the ability of the drug to suppress this inflammatory mediator.
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Introduction
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Endotoxin or certain cytokines, e.g., interferon (IFN)- induce expression of inducible nitric oxide synthase (iNOS), a Ca2+-independent form, in macrophages to produce a large amount of nitric oxide (NO) (1,2). Overproduction of NO, associated with macrophage activation, is pivotal in the pathogenesis of septic shock or inflammatory tissue injury leading to multiple organ failure, as well as in host defenses against microorganisms (2,3). Therefore, inhibition of NO overproduced via iNOS in the phagocytes may act advantageously in hyperinflammatory diseases (e.g., acute respiratory distress syndrome, multiple organ failure), and disadvantageously in infection.
iNOS expression is regulated by various drugs, including glucocorticoid (4), pentoxifylline (5), and prostaglandin E1 (6) mainly at the transcriptional level. A few anesthetics/sedatives alter NO production via iNOS (7,8). Because lidocaine is often used to treat and/or prevent ventricular arrhythmia in an intensive care unit for critically ill patients who are potential immunocompromised hosts or who may be in a hyperinflammatory state, it is important to elucidate whether the drug regulates NO production from activated macrophages. However, although lidocaine may reduce NO production via constitutive NO synthase in vascular endothelial cells (9), no information concerning the effects of lidocaine on NO hyperproduction by iNOS in macrophages is available. In several animal experiments, lidocaine has improved hemodynamic derangement and survival in septic shock (10), and attenuated endotoxin/sepsis-induced organ dysfunction (11). The successful outcome in these studies may be ascribed to the inhibitory effect of lidocaine on NO overproduction. In the current study, therefore, we examined whether lidocaine changes NO production through iNOS expression in activated macrophages. To make this assessment, we compared the expression of iNOS messenger RNA (mRNA) and protein, as well as levels of NO metabolites (nitrite plus nitrate [NOx]), using macrophage-derived cell line stimulated by lipopolysaccharide (LPS) and IFN- in the presence and absence of lidocaine.
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Methods
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The murine macrophage cell line RAW 264 was purchased from RIKEN Cell Bank (Tsukuba, Japan). Lidocaine was purchased from Sigma (St. Louis, MO). LPS (Escherichia coli 055:B5) and recombinant mouse interferon (IFN)- was purchased from Difco (Detroit, MI) and Genzyme (Cambridge, MA), respectively.
The cells were cultured in Dulbecco’s modified Eagle’s medium with 10% heat-inactivated fetal calf serum, 2 mM L-glutamate, 50 U/mL penicillin, and 50 mg/mL streptomycin. For the experiment, the cells were harvested 105/cm2 for 24 h up to confluent, then the medium was replaced with fresh medium containing various concentrations of lidocaine (5, 50, and 500 mg/mL) and stimulants (LPS 10 mg/mL plus IFN- 50 U/mL). These concentrations of lidocaine corresponded to 1, 10, and 100 times clinical plasma concentrations. Cell toxicity, which was assessed by counting cell number with Cell Counting Kit-8® (Dojindo Laboratories, Kumamoto, Japan) 8 h after incubation, was not observed in any doses of lidocaine. After incubation of RAW 264 with the stimulants and various concentrations of lidocaine for 8 h, cell-free medium was removed for assessment of NO production by measuring NOx levels, and the harvested RAW 264 was also used to determine expression of iNOS protein by Western blotting and amount of cell protein corresponding to the number of cells. Another harvested RAW 264 mRNA was used to determine iNOS mRNA expression by Northern blotting. Concentrations of the stimulants and duration of stimulation (8 h) used in the current study were determined by observations in our preliminary experiments. Briefly, we confirmed that LPS and IFN- additively increased NO production, and both stimulants had a ceiling effect for NO production (LPS at >50 mg/mL and IFN- >100 U/mL) (data not shown). Northern blotting analysis revealed that LPS 10 mg/mL plus IFN- 50 U/mL induced the maximum expression of iNOS mRNA (data not shown). Our preliminary time-course study revealed that the peak induction of iNOS mRNA was observed at 12 h after stimulation with LPS plus IFN- (data not shown). Furthermore, stimulation with LPS plus INF- for 8 h increased accumulation of NOx to levels enough to assess the effect of lidocaine on NO production, if observed.
The NO production was assessed by measuring NOx, stable metabolites. The total of NOx was determined by an automatic analyzer (TCI-NOX 1000®; Nihon-Kasei, Tokyo, Japan) using the Griess reaction. Culture medium (0.1 mL) and 0.3 mL of 0.4 N NaOH were mixed and incubated at room temperature for 5 min. Then, 0.3 mL of 5% ZnSO4 was also added to the mixture and incubated for a further 5 min at room temperature. The mixture was centrifuged 6000 rpm for 20 min at 4°C. After this procedure to remove protein, the supernatant was applied to TCI-NOX 1000®.
The total RNA was isolated from cultured macrophages by using the TRIzol RNA isolation reagent (GIBCO BRL, Long Island, NY). Total RNA (5 mg per lane) was loaded on a 1% agarose gel in MOPS buffer containing 6% formaldehyde, and transferred to a nylon membrane (Boehringer Mannheim, Mannheim, Germany) by capillary blotting overnight after uniformity of loading was confirmed by examination of 18S and 28S ribosomal RNA bands by ethidium bromide. Then, the blot was fixed by cross-linking with ultraviolet irradiation and by baking at 80°C for 30 min. The filter was hybridized with digoxigenin-labeled complementary DNA probe overnight at 50°C. After hybridization, the filter was washed twice with 0.1x standard saline citrate (SSC; 0.15 M NaCl, 15 mM Na citrate [pH 7.0]) and 0.1% sodium dodecyl sulfate at 55°C for 15 min. The probe on the filter was detected with sheep anti-digoxigenin antibody conjugated with alkaline phosphatase. Full-length murine iNOS complementary DNA, which was a gift from Dr. Y. Kawahara (Kobe University School of Medicine, Kobe, Japan), was cloned into pGEM vector and labeled by digoxigenin using polymerase chain reaction with SP6 and T7 primers. The antibody was detected with chemiluminescence substrate CSPD® (3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo [3.3.1.1{3,7}]decan}-4-yl) phenyl phosphate) (Boehringer Mannheim). The hybrids were exposed to radiographic film.
The iNOS protein was analyzed by immunoblotting with the anti-iNOS antibody. The cells (1 x 106) were lysed with 100 mL of a lysis buffer (50 mM Tris/Cl, pH 7.5, 150 mM NaCl, 1% Triton, and 1 mM phenylmethylsulfonyl fluoride) on ice for 10 min at 4°C. The lysates were then centrifuged at 15000 rpm for 10 min, and the supernatants were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7.5% gel, 15 mg protein per lane) by using buffer system of Laemmli (12). The separated proteins were electrophoretically transferred to polyvinylidene difluoride membrane by using a semi-dry gel electroblotter (Horizblot®; Atto, Tokyo, Japan). The polyvinylidene difluoride membrane was soaked for 30 min in 25 mM Tris/Cl, pH 7.4, 150 mM NaCl, 0.1% Triton, and 5% skimmed milk to block nonspecific binding. The polyvinylidene difluoride blots were then incubated with anti-iNOS antibody (PA3–030; Affinity BioReagents, Golden, CO) in 25 mM Tris/Cl, pH 7.4, 150 mM NaCl, 0.1% Triton, and 1% bovine serum albumin for 1 h, followed by horseradish peroxidase-labeled donkey antirabbit immunoglobulin (NA934; Amersham Pharmacia Biotech, Tokyo, Japan) in the same buffer for 1 h. The blots were washed extensively with 25 mM Tris/Cl, pH 7.4, 150 mM NaCl, and 0.1% Triton for 20 min. Peroxidase-labeled proteins were detected by enhanced chemiluminescence system (RPN2134; Amersham Pharmacia Biotech) followed by exposure to radiograph film.
Cell protein was determined by DC Protein Assay Kit II® (Bio-Rad, Hercules, CA), in which bovine serum albumin serves as a standard, according to the manufacturer’s protocol. Densities of the bands (corresponding to iNOS mRNA and protein, and 18S- and 28S-ribosomal RNA) on radiograph films were determined with the NIH-Image, a public domain image processing and analysis program.
Data were expressed as mean ± SEM (n = 7 for NOx, and n = 4 for mRNA and protein), and were analyzed for statistical significance by Friedman rank test followed by Dunnett test for post hoc comparison. P < 0.05 was deemed significant. Data on NOx in four LPS/IFN--treated groups were statistically reanalyzed by using an analysis of variance followed by Bonferroni test for post hoc comparison to assess dose-dependent effect of lidocaine. The sample size of the current study is sufficient to detect large differences (effect size = (µ1-µ2)/
= 1.2) in variables at a significance level of 0.05, although the power of the study is relatively weak (power = 1-ß = 0.7).
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Results
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Lidocaine reduced NOx accumulation induced by LPS plus IFN- in a dose-dependent manner although the drug per se had no effect on basal NOx levels without the stimulants (Fig. 1). There was no significant difference in band densities of ribosomal RNA between groups, indicating uniformity of gel loading conditions (Fig. 2, Table 1). In unstimulated RAW 264, iNOS mRNA was not detectable. Expression of iNOS mRNA was induced by LPS plus IFN- (Fig. 2). Lidocaine had no significant effects on LPS plus IFN- stimulated expression of iNOS mRNA (Fig. 2 & Table 1). Lidocaine at 5 or 50 mg/mL had no effect on iNOS protein levels. In contrast, the drug at 500 mg/mL inhibited expression of iNOS protein expression (Fig. 3 & Table 1).
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Table 1. Quantitation of Expression of iNOS mRNA and Protein in RAW 264 Stimulated with Lipopolysaccharide (LPS) Plus Interferon (IFN)-
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Discussion
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The iNOS excessively synthesizes NO by oxidation of the terminal guanidino-nitrogen atom of L-arginine (1–3). This enzyme is comprised of an oxygenase domain containing iron protoporphyrin IX (heme), tetrahydrobiopterin, a substrate binding site, and a reductase domain containing flavin adenine dinucleotide, flavin mononucleotide, calmodulin, and nicotinamide adenine dinucleotide phosphate binding site (13). Dimerization activates NO synthesis by enabling electrons to transfer between the reductase and oxygenase domains (13). Requirements for stable dimerization includes binding of tetrahydrobiopterin, arginine, and heme to iNOS subunit (14).
In agreement with many previous reports (6,15), we have confirmed that stimulation with LPS plus IFN- mutually enhanced production of NO from RAW 264. We have also shown that lidocaine dose-dependently inhibited the NO production induced by these stimulants, as assessed by NOx accumulation in the culture media. We emphasize that the amount of iNOS protein was drastically decreased by lidocaine at concentrations 100 times larger than those found in plasma (toxic concentrations), although the protein expression was not affected by the drug at clinically relevant concentrations and at 10 times these concentrations. However, lidocaine at any dose failed to inhibit expression for iNOS mRNA. The precise mechanism underlying inhibition of NO production by lidocaine remains to be elucidated. Nevertheless, our findings suggest posttranscriptional mechanisms. Possible targets of lidocaine-induced inhibition are proposed. There seems to be a difference in sites where lidocaine decreased NO production between doses of the drug used. Lidocaine at the largest concentration is thought to have reduced NO production, in part, by inhibiting translation of iNOS mRNA into functional protein, reducing stability of iNOS mRNA, and/or accelerating degradation of already translated iNOS protein. In contrast, the smaller doses of lidocaine may exert an inhibitory effect on NO production by decreasing iNOS activity. The largest dose of lidocaine may also have acted through this mechanism. From our findings, we are unable to elucidate the precise molecular mechanism responsible for impairment of iNOS activity. Lidocaine may have reduced iNOS activity by inhibiting dimerization of iNOS or decreasing stability of the dimerized enzyme. Although lidocaine is thought to have scavenged NO released from RAW 264, lidocaine possesses NO scavenging capacity (16). Cyclic adenosine monophosphate-elevating agents increase protein and activity of iNOS (17). Because lidocaine decreases cyclic adenosine monophosphate levels (18), the anesthetic is likely to inhibit NO production by reducing the second messenger. In addition, iNOS expression in macrophages is regulated by calcium (19). Because lidocaine decreases intracellular calcium ion levels in smooth muscle (20), the effect of the drug on calcium mobilization in macrophages needs to be clarified.
The cytotoxic effect of NO is involved in the pathogenesis of tissue injury leading to organ dysfunction. The mechanism of NO-induced cytotoxicity includes 1) DNA damage, 2) inhibition of mitochondrial respiration, 3) inactivation of sulfhydryl-enzyme, and 4) damage of cell membrane as a result of peroxynitrite and hydroxyl radicals, which are produced from NO and oxygen free radicals (1,2). Several lines of evidence suggest that iNOS-induced hyperproduction of NO, a potent vasodilator, also contributes to the pathogenesis of cardiovascular derangement in septic shock (2–4). In several animal experiments, lidocaine improved hemodynamic variables and survival in septic shock (10), and attenuated endotoxin/sepsis-induced organ injury (11). The successful and satisfactory outcome may be ascribed, in part, to lidocaine-induced suppression of NO overproduced from macrophages. NO produced by smooth muscle cells rather than macrophages is thought to contribute mainly to hemodynamic derangement in sepsis. An animal study has demonstrated that an excessive amount of NO production via iNOS is responsible for cerebrovascular disturbances and traumatic brain injury (21).
Lidocaine improves acute neurological and motor function after brain damage in animals (22). The neuroprotective action of lidocaine may be attributable, in part, to the ability of the drug to suppress NO hyperproduction. However, because the current study was in vitro experiments using an immortalized cell line and maximal stimulation, we are unable to simply extrapolate our ex vivo data to the in vivo animal experiments or human research. Further in vivo studies are required to assess iNOS expression in native macrophages from lidocaine-treated animals as well as plasma NOx levels in those receiving the drug. Ketamine inhibits NO production by activated macrophages (7), and expression of iNOS protein (23). This inhibitory effect of ketamine is speculated to be attributed to reduction of tumor necrosis factor-
release as well as interference of calcium mobilization. In a previous report, lidocaine failed to decrease endotoxin-induced tumor necrosis factor- release (24). Unlike ketamine, direct peripheral vasodilation and myocardial depression induced by lidocaine may counteract pressor effects because of inhibition of NO production. Use of glucocorticoids (inhibitors of iNOS mRNA transcription) in sepsis or endotoxemia may increase the severity of the disease.
NO overproduced by iNOS serves as a double-edged sword. The unfavorable aspects of lidocaine-induced impairment of NO production include possible increased susceptibility to infections. IV infusion of lidocaine caused death in five of six rabbits with peritonitis induced by Staphylococcus aureus, whereas only one of six noninfused animals died (25). Although NO production was not determined in this study, lidocaine may have reduced NO release from activated macrophages. Thus, the use of lidocaine may be most appropriate in settings of sterile inflammation (e.g., systemic inflammatory response syndrome).
In conclusion, we have shown that lidocaine dose-dependently inhibited NO production by activated RAW 264, although the drug had no effect on iNOS mRNA expression. Only a toxic dose (at concentrations 100-fold larger than the clinical plasma concentration) of lidocaine decreased iNOS protein expression. These data suggest that lidocaine has multiple mechanisms (posttranscriptional processing level) responsible for suppression of NO production from the activated macrophages. We must emphasize that our experiment is an in vitro study, and that in vivo conclusions cannot be drawn at this time, particularly because nonclinical doses were used. The exact mechanism underlying inhibition of NO production by lidocaine via iNOS deserves further study.
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Accepted for publication September 1, 2000.
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Abstract
Introduction
