| JOURNAL HOME | CME HOME | THIS MONTH | PAST ISSUES | ETOC | COLLECTIONS |
| AUTHORS | REVIEWERS | EDITORIAL BOARD | FEEDBACK | RSS | HELP |
 
|
|
|
||
|
|
Anesth Analg 2006;102:1739-1744 © 2006 International Anesthesia Research Society doi: 10.1213/01.ANE.0000219593.15109.DB
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Lidocaine has been reported to inhibit nitric oxide (NO) production in activated murine macrophages, but the role of inducible NO synthase (iNOS) in lidocaine-induced inhibition of NO has not been explored. In addition, type-2 cationic amino acid transporter (CAT-2) and guanosine triphosphate cyclohydrolase I (GTPCH) also regulate iNOS activity. The effects of lidocaine on CAT-2 and GTPCH are unknown. To explore further these effects, confluent immortalized murine macrophages (RAW264.7 cells) were incubated with lipopolysaccharide (LPS) or in combination with lidocaine (5, 50, or 500 µM) for 18 h before harvesting. We also used tetrodotoxin (TTX) and veratridine to elucidate the possible role of voltage-sensitive Na+ channel. Our data demonstrated that LPS significantly upregulated transcription of iNOS and CAT-2 but not GTPCH in stimulated macrophages. In a dose-dependent manner, lidocaine significantly attenuated the LPS-induced upregulation of iNOS and CAT-2. Conversely, lidocaine significantly increased GTPCH transcription in LPS-stimulated macrophages. The effects of TTX on iNOS, CAT-2, and GTPCH expression were comparable to those of lidocaine. In addition, veratridine significantly attenuated the effects of lidocaine and TTX. We therefore concluded that lidocaine significantly inhibits iNOS and CAT-2 and, in turn, enhances GTPCH transcription in LPS-stimulated macrophages via a mechanism that possibly involves the voltage-sensitive Na+ channel.
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Upregulation of inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) overproduction results in significant oxidative stress (1). NO may react with DNA, proteins, and lipids, which would result in cellular damage and organ dysfunction (1). Because the cytotoxic effect is nonspecific, the consequences of overproduction of NO can be detrimental (2). Therefore, a precise regulation of iNOS-dependent NO production under pathophysiological conditions is critical for the survival of host cells.
With the identification of an amino-terminal oxygenase domain that binds l-arginine and tetrahydrobiopterin (BH4) in iNOS, intracellular concentrations of l-arginine and BH4 have been identified as two crucial factors that regulate iNOS activity (3). Cellular uptake of circulating l-arginine mainly depends on the isozymes of cationic amino acid transporter (e.g., CAT-1 and CAT-2) (4). The crucial role of CAT-2 in regulating l-arginine transport was supported by the data showing that genetic ablation of CAT-2 significantly reduces NO production and l-arginine transport in activated macrophages (5). Previous data also demonstrated that the expression of CAT-1 and CAT-2 can be modulated by bacterial endotoxins and inflammatory cytokines (6). In addition to l-arginine, the crucial role of BH4 was supported by the data that binding with BH4 stabilizes iNOS structure and its active site (7). The biosynthesis of BH4 is closely regulated by guanosine triphosphate cyclohydrolase I (GTPCH) (8). Using a septic rodent model, Hattori et al. (9) demonstrated that expression of GTPCH can be modulated by bacterial toxins. These data supported the idea that expression of GTPCH also constitutes part of the crucial regulatory pathways that regulate iNOS activity.
Lidocaine, a widely used local anesthetic and a voltage-sensitive sodium (Na+) channel blocker, has been reported to attenuate cytokine-induced cell injury (10). In addition, Shiga et al. (11) also reported that lidocaine inhibits NO production in activated murine macrophages. However, the molecular mechanisms underlying these effects are poorly delineated. To elucidate further, this study was designed to investigate the effects of lidocaine and test the hypothesis that lidocaine attenuates iNOS, CAT-1, CAT-2, and GTPCH transcription in lipopolysaccharide (LPS)-stimulated murine macrophages.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
RAW264.7 cells, an immortalized murine macrophage cell line, were cultured in Dulbecco modified Eagle’s medium (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin (Life Technologies) in a humidified incubator with 5% CO2 and 95% air at a temperature of 37°C, as previously reported (12). The culture medium was changed every day for routine culture and then 1 h before each experiment. Cells at passage 8 to 13 were used for experiments and each treatment was performed when the cells were 80% confluent.
Two groups of confluent RAW264.7 cells that were allocated to receive LPS (100 ng/mL; Escherichia coli serotype 0127:B8; Sigma-Aldrich, St. Louis, MO) or 1x phosphated buffer saline (PBS, Life Technologies) were used as positive or negative control (designated as the LPS or PBS group). To elucidate the effects of lidocaine and the possible role of voltage-sensitive Na+ channel, another 8 groups of confluent RAW264.7 cells were allocated to receive LPS plus lidocaine (5, 50, or 500 µM; Sigma-Aldrich)[designated as the LPS + L(5), LPS + L(50), or LPS + L(500) group], LPS plus tetrodotoxin (TTX), a specific voltage-sensitive Na+ channel inhibitor (13)(1 µM; Sigma-Aldrich) [designated as the LPS + TTX group], LPS plus lidocaine (5, 50, or 500 µM) plus veratridine, a voltage-sensitive Na+ channel activator (13) (50 µM, Sigma-Aldrich)[designated as the LPS + L(5) + V, LPS + L(50) + V, or LPS + L(500) + V group], or LPS plus TTX plus veratridine (designated as the LPS + TTX + V group). To control the effects of each additive, another 4 groups of confluent RAW264.7 cells were allocated to receive lidocaine (500 µM), TTX (1 µM), veratridine (50 µM), or LPS plus veratridine (designated as the Lido, TTX, Vera, or LPS + V group). In groups that received LPS plus lidocaine or LPS plus TTX, lidocaine or TTX was added immediately after LPS. In groups that received LPS plus lidocaine plus veratridine or LPS plus TTX plus veratridine, veratridine was added 5 min before LPS followed by lidocaine or TTX. According to our previous data (12), cell cultures were also treated with a NOS inhibitor, NG-Nitro l-arginine methyl ester (1 mM; Sigma-Aldrich) to minimize the inhibitory effects of NO on iNOS expression. After reacting with LPS for 18 h, cell cultures were harvested for subsequent analysis.
Total RNA was isolated from cell cultures with TRIzol Reagent (Life Technologies). RNA samples were then extracted by a phenol-chloroform technique. The RNA concentrations were quantified by measuring ultraviolet light absorbance at 260-nm wavelength. Maloney murine leukemia virus reverse transcriptase and random hexamer primers (Ready-To-Go reverse transcription polymerase chain reaction [RT-PCR] Beads; Amersham Pharmacia Biotec, Inc, Piscataway, NJ) were used to reversely transcribe all messenger RNA species to complementary DNA (cDNA). The reaction was incubated at 42°C for 30 min in a thermocycler. cDNA samples were then incubated at 95°C for 5 min to inactivate the reverse transcriptase enzyme. Separately carrying each sample through the PCR procedure without adding reverse transcriptase ensured the absence of genomic DNA contamination.
The cDNA encoding iNOS, CAT-1, CAT-2, GTPCH, and ß-actin (as an internal standard) were then amplified using PCR. The primer sequences for each of the enzymes were designed in accordance with published rat DNA sequences and obtained from our previous reports (12,14). The cycling conditions were: 35 cycles for iNOS/ß-actin and 30 cycles for GTPCH/ß-actin at 92°C for 40 s, 57°C for 40 s, 75°C for 75 s and final extension was accomplished at 55°C over 5 min. Amplification of CAT-1/ß-actin and CAT-2/ß-actin were performed using 35 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 2 min, and a final extension of products at 72°C for 7 min.
PCR-amplified samples were electrophoretically separated on 1% ethidium bromide-stained agarose gels. The Gel Documentation System (Gel Doc 2000; Bio-Rad Laboratories, Hercules, CA) was used to assay the PCR products. The cDNA band densities were quantified by using densitometric techniques with Scion Image for Windows (Scion Corp, Frederic, MD).
Data were expressed as mean ± sd. To determine the inter-group differences, one way analysis of variance was used. The Tukey test was used for multiple comparisons. The significance level was set at 0.05. A commercial software package (SigmaStat for Windows, SPSS Science, Chicago, IL, USA) was used for data analysis.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
We performed 3 independent analyses for each sample to determine the mean mRNA data of each harvested sample. Our data revealed that iNOS and CAT-2 mRNA concentrations in the PBS, Lido, Vera, or TTX group were small (Figs. 1 and 2). As expected, iNOS and CAT-2 mRNA concentrations in the LPS group were significantly larger than those in the PBS group (both P < 0.001, Figs. 1 and 2). Although larger than those in the PBS group, concentrations of iNOS and CAT-2 mRNA in the LPS + L(500), LPS + L(50), and LPS + L(5) groups were significantly smaller than those in the LPS group (iNOS: all P < 0.001; CAT-2: P < 0.001, P = 0.007 and 0.002, respectively). In addition, iNOS and CAT-2 mRNA concentrations in the LPS + L(500) group were significantly smaller than those in the LPS + L(50) and LPS + L(5) groups (all P < 0.001). Similarly, mRNA concentrations of iNOS in the LPS + L(50) group were significantly smaller than those in the LPS + L(5) group. However, CAT-2 mRNA concentrations of these two groups were comparable. Our data further revealed that mRNA concentrations of CAT-2 in. the LPS + L(500) group were significantly smaller than those in the LPS + L(500) + V group (P < 0.001), whereas iNOS mRNA concentrations in these two groups were comparable. We also found that iNOS mRNA concentrations in the LPS + L(50) group were significantly smaller than those in the LPS + L(50) + V group (P < 0.001), whereas CAT-2 mRNA concentrations in these two groups were comparable. Concentrations of iNOS and CAT-2 mRNA in the LPS + L(5) group were significantly smaller than those in the LPS + L(5) + V group (iNOS: P < 0.001; CAT-2: P = 0.027). Concentrations of iNOS and CAT-2 mRNA, however, were similar between the LPS + V group and the LPS group.
|
P < 0.05 the LPS + Lido (500) or LPS + Lido (50) group compared with the LPS + Lido (5) group; 
P < 0.05 the LPS + Lido (500) group compared with the LPS + Lido (50) group; ¶ P < 0.05 the LPS + Lido (50) group compared with the LPS + Lido (50) + Vera group, the LPS + Lido (5) group compared with the LPS + Lido (5) + Vera group, or the LPS + TTX group compared with the LPS +TTX + Vera group.
|
Our data also showed that concentrations of iNOS and CAT-2 mRNA in the LPS + TTX and LPS + TTX + V groups were significantly smaller than those in the LPS group (iNOS: P < 0.001 and P = 0.0011; CAT-2: P < 0.001 and P = 0.004, respectively) (Figs. 1 and 2). In addition, concentrations of iNOS and CAT-2 mRNA in the LPS + TTX group were significantly smaller than those in the LPS + TTX + V groups (iNOS: P = 0.018; CAT-2: P = 0.023, respectively).
In contrast to iNOS and CAT-2, CAT-1 mRNA concentrations in the 14 groups of cell cultures were comparable (Fig. 3). Our data also revealed that mRNA concentrations of GTPCH were comparable in the PBS, Lido, Vera, and TTX groups (Fig. 4). GTPCH mRNA concentrations in the LPS and LPS + V groups were similar to those in the PBS group. To our surprise, our data revealed that GTPCH mRNA concentrations in the LPS + L(500), LPS + L(50), and LPS + L(5) groups were significantly larger than those in the LPS group (all P < 0.001). In addition, GTPCH mRNA concentrations in the LPS + L(50) and LPS + L(5) groups were comparable and both were significantly larger than those in the LPS + L(500) group (both P < 0.001). We also found that GTPCH mRNA concentrations in the LPS + L(500) group were significantly larger than those in the LPS + L(500) + V group (P = 0.034). Similarities were also observed between the LPS + L(50) and LPS + L(50) + V groups as well as the LPS + L(5) and LPS + L(5) + V groups. In addition, GTPCH mRNA concentrations in the LPS + TTX and LPS + TTX + V groups were significantly larger than those in the LPS group (P < 0.001 and P = 0.003, respectively). Furthermore, GTPCH mRNA concentrations in the LPS + TTX group were significantly larger than those in the LPS + TTX + V group (P = 0.017).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Data from this study provide the first evidence and confirmed part of our hypothesis that lidocaine significantly inhibits the transcription of iNOS and CAT-2 in LPS-stimulated murine macrophages. Furthermore, our data demonstrated that the observed inhibitory effects of lidocaine may involve the voltage-sensitive Na+ channel. This is the first report to highlight the involvement of the voltage-sensitive Na+ channel in regulating iNOS and CAT-2 transcription and, thus, warrants further investigation.
Shiga et al. (11) reported that lidocaine dose-dependently inhibits NO production but not iNOS expression in macrophages stimulated with a combination of LPS and interferon (INF)-
. Previous data indicated that macrophages treated with a combination of LPS and INF- would have a five- to sixfold larger amount of NO and even higher iNOS expression (both mRNA and protein) than those treated with LPS alone (15). Judging from these data, we speculated that the contradicting data regarding lidocaine’s effects on NO production and iNOS expression reported by Shiga et al. may be attributable, at least in part, to the inhibitory effects of lidocaine, which were not potent enough to counteract the synergistic effects of LPS and INF- on iNOS induction but were potent enough to reduce NO production. To avoid this possibility, we chose to treat macrophages with LPS alone to allow lidocaine to exhibit its inhibitory effects on iNOS expression. Data from this present study supported our speculation and further confirmed that lidocaine has significant inhibitory effects, on iNOS transcription. These data also help to clarify the mechanisms that underline the antiinflammatory capacity of lidocaine.
In keeping with previous data (12), this study confirmed that LPS significantly induces iNOS and CAT-2 transcription in murine macrophages. On exposure to LPS, membrane surface receptors, especially toll-like receptor 4, in macrophages were activated (16). Activation of toll-like receptor 4 increases the production of reactive oxygen species, which causes an activation of nuclear factor (NF)-
B, a crucial factor for maximal transcription of a wide array of proinflammatory molecules, including iNOS and CAT-2 (17,18). This signaling cascade is suppressed by antioxidant compounds (17). Though commonly known as a local anesthetic, lidocaine is also a potent antioxidant that scavenges hydroxyl radicals and singlet oxygen (19). We therefore speculate that lidocaine, as an antioxidant, may act through inhibiting NF-B activation to reduce iNOS and CAT-2 transcription in activated macrophages.
There is no direct evidence to indicate the existence of a voltage-sensitive Na+ channel in macrophages. However, previous data that activation of microglia (i.e., brain macrophages) in experimental autoimmune encephalomyelitis significantly increased Na+ channel expression whereas TTX, a specific voltage-sensitive Na+ channel blocker, significantly reduced the activity of activated microglia (20) suggested the existence of a voltage-sensitive Na+ channel in brain macrophages and the possible role of Na+ channel on regulating activation of brain macrophages. Data from this study further revealed that lidocaine as well as TTX had significantly inhibitory effects on iNOS and CAT-2 transcription. In addition, these inhibitory effects could be attenuated by veratridine, a voltage-sensitive Na+ channel activator. These data suggest the existence of a voltage-sensitive Na+ channel in macrophages and support the concept that a voltage-sensitive Na+ channel is involved in lidocaine-induced inhibition of iNOS and CAT-2 transcription. No data are available regarding the interaction between voltage-sensitive Na+ channel and NF-B. However, data clearly demonstrated the interaction between a voltage-sensitive Na+ channel and p38 mitogen-activated protein kinase (MAPK), one crucial pathway that modulates LPS-induced iNOS, CAT-2, and NF-B activation (21,22). Judging from these data, we speculate that lidocaine may also act through inhibiting the Na+ channel and, consequently, the p38 MAPK pathway to exert its inhibitory effects on iNOS and CAT-2 transcription. More studies are needed before further conclusion can be reached.
Our data, consistent with previous data (23), confirmed that CAT-1 is constitutively expressed in activated macrophages and LPS, lidocaine, veratridine, or TTX have no significant effects on its transcription. In contrast, CAT-2 transcription in activated macrophages is subject to the regulation of LPS, lidocaine, veratridine, and TTX. These data support the idea that CAT-2, but not CAT-1, plays a crucial role in mediating l-arginine transport in activated macrophages. Our data, together with previous data (5), further suggest that therapies aiming at regulating CAT-2 expression should be an attractive therapeutic alternative against sepsis.
Though Hattori et al. demonstrated that GTPCH expression is significantly upregulated in lung, heart, and kidney of rats treated with LPS (9), Sakai et al. (24) reported that GTPCH transcription in macrophages is constitutive. Data from this study that GTPCH mRNA concentrations in macrophages are high and not affected by LPS seem to support the data reported by Sakai et al. (24). Though BH4 is required for NO production in cytokine-stimulated macrophages, Sakai et al. (24) indicated that only a small fraction of the BH4 pool in macrophages is used to produce fully active NOS. This helps to explain why GTPCH expression, as noted in this study, is not affected by LPS in macrophages. Our data further demonstrated that lidocaine increases GTPCH mRNA concentrations in LPS-stimulated macrophage. Judging from the fact that GTPCH is constitutively expressed in macrophages, we speculate that this observed increase in GTPCH mRNA concentrations may be the result of decreased GTPCH mRNA degradation rather than increased transcription induction by lidocaine. In addition, binding with BH4 stabilizes iNOS structure and its active site (7). We, thus, speculate that lidocaine may inhibit the bioavailability of BH4 for iNOS. We further speculate that this inhibition of BH4 bioavailability, if any, may lead to a compensatory increase in GTPCH mRNA concentrations.
It has been demonstrated that GTPCH expression is regulated by NF-B (25). As aforementioned, our iNOS and CAT-2 data support the idea that lidocaine, as an antioxidant, may act through inhibiting NF-B activation to reduce iNOS and CAT-2 transcription in activated macrophages. Therefore, it is unlikely that this lidocaine-induced increase of GTPCH mRNA concentrations in activated macrophages involves NF-B. However, our data did suggest that the lidocaine-induced increase in GTPCH mRNA concentrations may involve the voltage-sensitive Na+ channel. More studies are needed before further conclusions can be reached.
In summary, lidocaine inhibits iNOS and CAT-2 and, in turn, enhances GTPCH transcription in LPS-stimulated murine macrophages through mechanisms that may involve voltage-sensitive Na+ channel.
|
Footnotes |
|---|
Accepted for publication February 15, 2006.
Supported, in part, by a grant from Mackay Memorial Hospital (MMH 9572) to Dr. Huang.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Nussler AK, Billiar TR. Inflammation, immunoregulation, and inducible nitric oxide synthase. J Leukoc Biol 1993;54:171–8.[Abstract]
- Szabo C. Alterations in nitric oxide production in various forms of circulatory shock. New Horiz 1995;3:2–32.[Medline]
- Andrew PJ, Mayer B. Enzymatic function of nitric oxide synthases. Cardiovasc Res 1999;43:521–31.
[Abstract/Free Full Text] - Baydoun AR, Bogle RG, Pearson JD, Mann GE. Arginine uptake and metabolism in cultured murine macrophages. Agents Actions 1993;38:C127–9.
- Nicholson B, Manner CK, Kleeman J, MacLeod CL. Sustained nitric oxide production in macrophages requires the arginine transporter CAT2. J Biol Chem 2001;276:15881–5.
[Abstract/Free Full Text] - Schwartz D, Schwartz IF, Gnessin E, et al. Differential regulation of glomerular arginine transporters (CAT-1 and CAT-2) in lipopolysaccharide-treated rats. Am J Physiol Renal Physiol 2003;284:F788–95.
[Abstract/Free Full Text] - Klatt P, Schmid M, Leopold E, et al. The pteridine binding site of brain nitric oxide synthase. Tetrahydrobiopterin binding kinetics, specificity, and allosteric interaction with the substrate domain. J Biol Chem 1994;269:13861–6.
[Abstract/Free Full Text] - Thony B, Auerbach G, Blau N. Tetrahydrobiopterin biosynthesis, regeneration and functions. Biochem J 2000;347:1–16.
- Hattori Y, Oka M, Kasai K, et al. Lipopolysaccharide treatment in vivo induces tissue expression of GTP cyclohydrolase I mRNA. FEBS Lett 1995;368:336–8.[Medline]
- de Klaver MJ, Buckingham MG, Rich GF. Lidocaine attenuates cytokine-induced cell injury in endothelial and vascular smooth muscle cells. Anesth Analg 2003;97:465–70.
[Abstract/Free Full Text] - Shiga M, Nishina K, Mikawa K, Obara H. The effects of lidocaine on nitric oxide production from an activated murine macrophage cell line. Anesth Analg 2001;92:128–33.
[Abstract/Free Full Text] - Lin WC, Tsai PS, Huang CJ. Catecholamines’ enhancement of inducible nitric oxide synthase-induced nitric oxide biosynthesis involves CAT-1 and CAT-2A. Anesth Analg 2005;101:226–32.
[Abstract/Free Full Text] - Talvenheimo JA, Tamkun MM, Catterall WA. Reconstitution of neurotoxin-stimulated sodium transport by the voltage-sensitive sodium channel purified from rat brain. J Biol Chem 1982;257:11868–71.
[Abstract/Free Full Text] - Huang CJ, Stevens BR, Nielsen RB, et al. Interleukin-10 inhibition of nitric oxide biosynthesis involves suppression of CAT-2 transcription. Nitric Oxide 2002;6:79–84.[Web of Science][Medline]
- Noda T, Amano F. Differences in nitric oxide synthase activity in a macrophage-like cell line, RAW264.7 cells, treated with lipopolysaccharide (LPS) in the presence or absence of interferon-gamma (IFN-gamma): possible heterogeneity of iNOS activity. J Biochem (Tokyo) 1997;121:38–46.
[Abstract/Free Full Text] - Chow JC, Young DW, Golenbock DT, et al. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem 1999;274:10689–92.
[Abstract/Free Full Text] - Asehnoune K, Strassheim D, Mitra S, et al. Involvement of reactive oxygen species in Toll-like receptor 4-dependent activation of NF-kappa B. J Immunol 2004;172:2522–9.
[Abstract/Free Full Text] - Hammermann R, Dreissig MD, Mossner J, et al. Nuclear factor-kappa B mediates simultaneous induction of inducible nitric-oxide synthase and Up-regulation of the cationic amino acid transporter CAT-2B in rat alveolar macrophages. Mol Pharmacol 2000;58:1294–302.
- Das KC, Misra HP. Lidocaine: a hydroxyl radical scavenger and singlet oxygen quencher. Mol Cell Biochem 1992;115:179–85.[Web of Science][Medline]
- Craner MJ, Damarjian TG, Liu S, et al. Sodium channels contribute to microglia/macrophage activation and function in EAE and MS. Glia 2005;49:220–9.[Web of Science][Medline]
- Chen C, Chen YH, Lin WW. Involvement of p38 mitogen-activated protein kinase in lipopolysaccharide-induced iNOS and COX-2 expression in J774 macrophages. Immunology 1999;97:124–9.[Web of Science][Medline]
- Caivano M. Role of MAP kinase cascades in inducing arginine transporters and nitric oxide synthetase in RAW264 macrophages. FEBS Lett 1998;429:249–53.[Web of Science][Medline]
- Schwartz D, Schwartz IF, Gnessin E, et al. Differential regulation of glomerular arginine transporters (CAT-1 and CAT-2) in lipopolysaccharide-treated rats. Am J Physiol Renal Physiol 2003;284:F788–95.
[Abstract/Free Full Text] - Sakai N, Kaufman S, Milstein S. Tetrahydrobiopterin is required for cytokine-induced nitric oxide production in a murine macrophage cell line (RAW 264). Mol Pharmacol 1993;43:6–10.[Abstract]
- Hattori Y, Nakanishi N, Kasai K. Role of nuclear factor kappa B in cytokine-induced nitric oxide and tetrahydrobiopterin synthesis in rat neonatal cardiac myocytes. J Mol Cell Cardiol 1997;29:1585–92.[Web of Science][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
