Inducible nitric oxide synthase (iNOS) is expressed in many different cell types and tissues/organs when exposed to inducing factors such as cytokines, microbes, or microbial products (e.g., bacterial lipopolysaccharide [LPS]) (1). The induction of iNOS thus results in nitric oxide (NO) overproduction, which not only initiates inflammatory responses, but also poses oxidative stress and causes cell and tissue damage (2). Furthermore, NO overproduction has also been shown to induce necrosis or apoptosis (3).
An early increase of catecholamines (e.g., epinephrine and norepinephrine) during septic shock has been reported (4). In addition to their effects on circulatory and immune systems, catecholamines are also reported to play an important role in modulating inflammatory responses (5). Therefore, it would be interesting, and of profound clinical importance, to understand the interaction between NO and catecholamines during sepsis.
Cellular uptake of L-arginine, the sole substrate for iNOS, has been identified as a crucial regulatory mechanism involved in the formation of NO by iNOS (6). Cellular uptake of L-arginine requires the specialized transmembrane transport system y+ (7). This system y+ activity is encoded by the cat-1 and cat-2 (cationic amino acid transporter) genes, with complimentary (c)DNA clones denoted as CAT-1 and CAT-2 (8). The CAT-2 transporter has a higher substrate affinity than CAT-1 (9). A previous study demonstrated that the contribution of CAT-1 to overall system y+ activity is negligible (<3%) (10). Evidence that sustained NO production in macrophages requires CAT-2 clearly demonstrates the importance of this transporter (11).
In addition to CAT-2, two alternative spliced transcripts of CAT-2 have been identified: low-affinity CAT-2A (12) and high affinity CAT-2B (10). We recently reported that low-affinity CAT-2A expression in the rat kidney is constitutive and unaffected by LPS stimulation (13). Our work, together with that of another group (14), suggests that the role of CAT-2A in sepsis is negligible. In contrast, Stevens et al. (10) demonstrated that induced NO biosynthesis depends on induced high-affinity CAT-2B. In the aforementioned report, we found that LPS co-induces the expression of iNOS, CAT-2, and CAT-2B in LPS-stimulated rat kidney (13). These data further support the idea that CAT-2 and CAT-2B are crucial in regulating iNOS activity during sepsis.
In a recent report, Chi et al. (15) demonstrated that catecholamines enhance iNOS expression and resultant NO biosynthesis in LPS-stimulated macrophages. As mentioned previously, CAT-2 and CAT-2B are crucial to the regulation of iNOS activity. However, whether catecholamines exert similar effects on CAT-2 and CAT-2B in stimulated macrophages remains unstudied. To explore further, we therefore conducted this cell culture study with the hypothesis that catecholamines upregulate CAT-2 and CAT-2B expression in LPS-stimulated murine macrophages. The effects of catecholamines on other members of CAT isozymes, including CAT-1 and CAT-2A, were also evaluated.
Immortalized murine macrophages (RAW264.7 cells) were plated in cell culture dishes (60 × 15 mm) and grown in Dulbecco modified Eagle's medium (DMEM; Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Life Technologies). RAW264.7 cells were incubated in a humidified chamber at 37°C in a mixture of 95% air and 5% CO2. Bacterial LPS (100 ng/mL; Escherichia coli serotype 0127:B8; Sigma-Aldrich, St. Louis, MO) was used to induce enzyme induction, NO formation, and L-arginine transport. For messenger (m)RNA analysis, cell cultures were treated with a NOS inhibitor, NG-Nitro L-arginine methyl ester (1 mM; Sigma-Aldrich). For NO analysis, cell cultures were incubated in a phenol red-free culture medium (Leibovitz L-15 medium; Life Technologies) without NG-Nitro L-arginine methyl ester.
Confluent cells were then randomized to 1 of the 14 groups. Each group contained six culture dishes (n = 6). Among them, two groups of cell cultures that received either phosphated buffer saline (Life Technologies) or LPS served as negative or positive control, respectively. To elucidate the effects of catecholamines, another 2 groups of cell cultures that received LPS plus either epinephrine (5 × 10−6 M; Sigma-Aldrich) or norepinephrine (5 × 10−6 M; Sigma-Aldrich) were used. A previous study clearly demonstrated that the catecholamine enhancement of NO biosynthesis could be attenuated by propranolol and dexamethasone (15). To determine whether the effects of catecholamines on enzyme expression (if any) could be attenuated by propranolol or dexamethasone, another 4 groups of cell cultures were used. These 4 groups of cell cultures thus received 1-h pretreatment of either propranolol (5 × 10−6 M; Sigma-Aldrich) or dexamethasone (1 × 10−6 M; Sigma-Aldrich), followed by LPS plus either epinephrine or norepinephrine. In addition, 2 more groups of cell cultures that received 1-h pretreatment with either propranolol (5 × 10−6 M) or dexamethasone (1 × 10−6 M), followed by LPS, were included to further determine the effects of propranolol and dexamethasone. Finally, 4 more groups of cell cultures exposed to epinephrine, norepinephrine, dexamethasone, or propranolol were used to control the effects of the additives. The concentration of each additive was based on a previously published article (15). Furthermore, according to our published data (16), we chose to harvest the cultures after exposing them to LPS for 18 h or a comparable duration in groups without LPS.
Harvested culture media were analyzed for the concentration of stable NO metabolites, nitrite (NO2–), and nitrate (NO3–) using chemiluminescence to determine the NO concentration of each sample. The protocols for NO measurement were adapted from a published article (17). The sample content of NO was quantified using a Sievers 280 NO analyzer (Sievers, Boulder, CO) within 1 h after the experiment was terminated. In short, the reaction chamber of the equipment was filled with 8 mL of glacial acetic acid containing 100 mg of potassium iodide at room temperature to reduce nitrites to NO. A 50-μL sample was injected into the reaction chamber, and a nitrogen stream carried the resulting NO gas to a cell in which the specific chemiluminescence generated by the NO-ozone reaction was detected by a photomultiplier. Calibration of the equipment was performed daily using standards of 10–1000 μM of sodium nitrite. The sensitivity of the equipment allows for a detection threshold of 0.1–0.3 μM of NO. Background buffer readings were subtracted to determine NO release. We chose to integrate NO release for 4 min because this period accounted for >90% of the NO peak.
L-arginine transport was determined by measuring L-[3H]arginine uptake. Murine macrophages were cultured for 18 h in the absence or presence of test substances. Then the cellular uptake of L-arginine was determined using protocols modified from a previously published article (18). In brief, murine macrophages were incubated at 37°C for 2 min in uptake solutions (137 mM of NaCl, 5.4 mM of KCl, 2.8 mM of CaCl2, 1.2 mM of MgSO4, 10.0 mM of HEPES, and 10.0 mM of Tris, with a pH value of 7.4) supplemented with 0.1 mM of L-arginine containing 1.0 μCi/mL of L-[3H]arginine. Uptake was stopped with ice-cold stop solution (137 mM of NaCl, 14 mM of Tris, and 14 mM of HCl, with a pH value of 7.4). Cells were then lysed in 0.5 mL of Tris-Triton (0.1%) solution followed by determination of the cellular radioactivity and protein content using the DC Protein Assay (Bio-Rad Laboratories, Hercules, CA).
Total RNA was extracted from cell cultures using TRIzol Reagent (Life Technologies). RNA samples were extracted by a phenol-chloroform technique. The RNA concentrations were then determined by ultraviolet light absorbance using a 260-nm wavelength. Maloney murine leukemia virus reverse transcriptase and random hexamer primers (Ready-To-Go Reverse Transcriptase Polymerase Chain Reaction [RT-PCR] Beads; Amersham Pharmacia Biotec, Inc, Piscataway, NJ) were used to reverse transcribe all mRNA species to cDNA. The reaction was incubated at 42°C for 30 min in a thermocycler. Separately carrying each sample through the PCR procedure without adding reverse transcriptase ensured the absence of genomic DNA contamination.
RT-generated cDNA encoding iNOS, CAT-1, CAT-2, CAT-2A, CAT-2B, and β-actin (as an internal standard) was then amplified using PCR. The primer sequences for each of the enzymes were designed in accordance with published rat DNA sequences and had been reported in our previous work (19). Amplification of mRNA for iNOS/β-actin detection was performed using 35 cycles of 92°C for 40 s, 57°C for 40 s, 75°C for 75 s, and a final primer extension at 55°C for 5 min. Amplification of CAT-1/β-actin, CAT-2/β-actin, and CAT-2A/β-actin was performed using 35 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 2 min, and a final extension using the temperature of 72°C for 7 min. Amplification of CAT-2B/β-actin was performed using 32 cycles at 94°C for 30 s, 58°C for 45 s, and 72°C for 60 s. Primer extension was accomplished over a 5-min period at 56°C.
All PCR products were electrophoretically separated on 1% agarose gel and stained with ethidium bromide. The Gel Documentation System (Gel Doc 2000; Bio-Rad) 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).
To determine between-group differences in iNOS, CAT-1, CAT-2, CAT-2A, and CAT-2B expression, one-way analysis of variance was used. To define the relationship between measurements, regression analyses were performed. All data are presented as mean ± sd. The significance level was set at 0.05. A commercial software package (SigmaStat for Windows; SPSS Science, Chicago, IL) was used for data analysis.
We performed three independent analyses for each sample to determine the mean NO production and L-arginine transport data of each harvested sample. The mean value and the standard deviation of each group (presented as mean ± sd) were then calculated from the six mean values. Our data revealed that basal NO production and L-arginine transport in unstimulated macrophages were low. In contrast, LPS exposure significantly increased NO production and L-arginine transport in stimulated macrophages, because our data revealed an approximately 8-fold increase in NO concentrations and 3-fold increases in L-arginine uptake in macrophages treated with LPS (both P < 0.001; Fig. 1). Furthermore, this LPS-induced NO production and L-arginine transport could be enhanced by catecholamines; we found an approximately 30% increase in NO concentrations and a 20% increase in L-arginine uptake in macrophages treated with LPS plus either epinephrine or norepinephrine as compared with those in macrophages treated with LPS alone (P = 0.003, 0.015, 0.017, and 0.023, respectively; Fig. 1). Catecholamine-enhanced NO production and L-arginine transport in LPS-stimulated macrophages were significantly attenuated by propranolol (P = 0.026, 0.022, 0.014, and 0.033, respectively; Fig. 1). In addition, dexamethasone pretreatment completely inhibited both the LPS-induced NO production and L-arginine transport and the further enhancement induced by catecholamines in stimulated macrophages (Fig. 1). Our data also revealed that none of the additives, including epinephrine, norepinephrine, dexamethasone, and propranolol, had significant effects on NO production and L-arginine transport in unstimulated macrophages (data not shown).
We also performed three independent analyses for each sample to determine the mean mRNA data of each harvested sample. The mean value and the standard deviation of each group (presented as mean ± sd) were then calculated from the six mean values. Densitometric evaluation revealed that iNOS, CAT-2, and CAT-2B mRNA concentrations in unstimulated macrophages remained almost undetectable, whereas LPS significantly increased iNOS, CAT-2, and CAT-2B mRNA concentrations in stimulated macrophages (all P < 0.001; Figs. 2–4). Similar to NO and L-arginine data, we found that this LPS-induced iNOS transcription could be further enhanced by epinephrine and norepinephrine (P = 0.021 and 0.036, respectively; Fig. 2). However, unlike iNOS, neither epinephrine nor norepinephrine had significant effects on LPS-induced CAT-2 or CAT-2B transcription (Figs. 3 and 4). The epinephrine- and norepinephrine-induced iNOS transcription enhancement could be attenuated by propranolol (P = 0.019 and 0.028, respectively; Fig. 2). In addition, dexamethasone significantly attenuated the induction of iNOS, CAT-2, and CAT-2B transcription in stimulated macrophages that were exposed to LPS alone or LPS plus catecholamines (all P < 0.05; Figs. 2–4).
In contrast to CAT-2 and CAT-2B, our data revealed that both CAT-1 and CAT-2A mRNA concentrations in unstimulated macrophages were large, and LPS exposure had no significant effects on CAT-1 or CAT-2A transcription (Figs. 5 and 6). Interestingly, epinephrine resulted in a 25% increase in the transcription of CAT-1 and a 105% increase in CAT-2A in LPS-stimulated macrophages (P = 0.026 and 0.018, respectively; Figs. 5 and 6). However, norepinephrine significantly enhanced the CAT-1 (approximately 60% increase; P = 0.021; Fig. 5) but not CAT-2A mRNA concentrations in LPS-stimulated macrophages. Our data further revealed that this catecholamine-enhanced CAT-1 and CAT-2A transcription could be attenuated by dexamethasone as well as propranolol in LPS-stimulated macrophages (all P < 0.05; Figs. 5 and 6). We also found that neither dexamethasone nor propranolol had a significant effect on CAT-1 and CAT-2A transcription in macrophages that were exposed to LPS (Figs. 5 and 6).
Our data further revealed that none of the additives, including epinephrine, norepinephrine, dexamethasone, and propranolol, had significant effects on iNOS, CAT-1, CAT-2, CAT-2A, and CAT-2B transcription in unstimulated macrophages (data not shown).
Data from this study clearly demonstrated that catecholamines significantly enhanced LPS-induced NO biosynthesis and iNOS expression in LPS-stimulated murine macrophages. Furthermore, these effects could be significantly attenuated by propranolol and dexamethasone. These data are consistent with those previously published (15).
The complicated interaction between NO and catecholamines during the inflammation process has been reported. For instance, NO may exacerbate hypotension at a time when endotoxemia depletes the peripheral catecholamines' stores (20) or depresses the β-adrenergic-stimulated calcium release from the sarcoplasmic reticulum in myocytes (21). Our data, together with those reported by Chi et al. (15), demonstrated that catecholamines may enhance induced NO production in murine macrophages. In contrast, catecholamines had been reported to decrease NO production by cytokine-stimulated hepatocytes (22). Judging from the above-mentioned data, we speculate that the modulating effect of catecholamines on NO production is tissue-specific. Therefore, future therapies aiming at regulating catecholamines and NO production during sepsis should be adjusted accordingly.
Previously published data clearly demonstrated that endotoxin would significantly increase NO biosynthesis and L-arginine transport in stimulated macrophages (18). Using the LPS-stimulated murine macrophages model in the present study, we observed similar results. Previously published data also indicated that catecholamines could significantly enhance NO biosynthesis in activated macrophages (15). Our study not only confirms previous data, but also provides the first evidence to demonstrate that the LPS-induced L-arginine transport increases could also be further enhanced by catecholamines. Our data clearly suggest an interaction between L-arginine transport and catecholamines in stimulated macrophages and therefore warrant further investigation.
As mentioned previously, the family of CAT isozymes, including CAT-1, CAT-2, CAT-2A, and CAT-2B, are required for transmembrane transportation of L-arginine (10,12). Unlike CAT-1, which is expressed nearly ubiquitously, the expression of CAT-2, CAT-2A, and CAT-2B is restricted to a more limited number of tissues and cell types (9). Data from the present study demonstrate that LPS significantly upregulates the expression of iNOS, CAT-2, and CAT-2B as well as L-arginine transport and NO production in stimulated macrophages. Our data, together with those previously published (11,13,14), support the idea that CAT-2 and CAT-2B are crucial in regulating iNOS activity during sepsis.
To our surprise, our study revealed that catecholamines did not enhance the expression of CAT-2 and CAT-2B in LPS-stimulated macrophages. This study was originally designed to investigate the putative enhancement effects of catecholamines on the family of CAT-2 isozymes. We chose to harvest the cultures after 18 hours of exposure to LPS because, in our previous study (16), we observed that the expression of CAT-2 mRNA was significantly induced in RAW264.7 cells after 18 hours of exposure to LPS. We did not, however, harvest the cell cultures at different time points after LPS exposure to determine the time course of the catecholamines' enhancement effect on the family of CAT-2 isozymes. Thus, there is a possibility that catecholamines did exert an enhancement effect on CAT-2 and CAT-2B but that the effect, if any, peaked before the cultures' harvest time. In addition, we only used a single dose of each catecholamine in this study. Therefore, data from this study are not sufficient to determine the possible dose-response characteristics of catecholamines with regard to CAT-2 isozymes. Because these data are essential for assessing the cellular pathways that regulate catecholamines' effects on CAT isozymes, we will perform follow-up studies to explore further.
Our study also revealed that LPS had no effects on the expression of CAT-1 and CAT-2A in macrophages, which together with previous studies (10,13,14), confirms that CAT-1 and CAT-2A expression is constitutive. However, our study demonstrated that the expression of CAT-1 and CAT-2A, although constitutively expressed, can be enhanced by stimuli such as catecholamines in activated macrophages. Because we did not use techniques that specifically inhibit the expression or the function of CAT-1 and CAT-2A, the effects of this CAT-1 and CAT-2A expression enhancement remain unanswered in this study. However, judging from these data, we speculate that catecholamine-enhanced L-arginine transport may be mediated by CAT-1 and CAT-2A but not CAT-2 and CAT-2B in activated macrophages. This speculation is supported by our finding that propranolol, a selective antagonist of the β-adrenergic receptors, significantly attenuated catecholamine-enhanced CAT-1 and CAT-2A expression as well as L-arginine transport, whereas the expression of CAT-2 and CAT-2B remained unaffected. That catecholamines may act through a β-adrenoceptor mechanism to modulate NO production is well documented (15,21). Our study further indicates that catecholamines might exert their enhancement effects on L-arginine transport also through a β-adrenoceptor-mediated mechanism. To explore further, future studies could use techniques that specifically block the expression of CAT-1 or CAT-2A, such as using small interfering RNA or inhibiting the function of CAT-1 or CAT-2A with antibodies or specific inhibitors.
The crucial role of the nuclear factor-κB (NF-κB) for maximal transcription of a wide array of proinflammatory molecules, including iNOS, cycloxygenase-2, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α, has been well documented (23). Using an LPS-stimulated rodent model, we reported that NF-κB also involves the expression of CAT-2 and CAT-2B in rat lungs (13). Our data, demonstrating that the induction of iNOS, CAT-2, and CAT-2B, as well as NO production and L-arginine transport in LPS-stimulated macrophages, could be significantly attenuated by dexamethasone, a potent NF-κB inhibitor, further support the idea that NF-κB plays a crucial role in modulating iNOS-mediated NO biosynthesis as well as CAT-2– and CAT-2B–mediated L-arginine transport in activated macrophages. In addition, our data further demonstrate that dexamethasone significantly inhibited catecholamine-enhanced CAT-1 and CAT-2A expression, as well as NO production and L-arginine transport, in LPS-stimulated macrophages. Judging from these findings, we further speculate that NF-κB may also be involved in this catecholamine-enhanced CAT-1 and CAT-2A expression and L-arginine transport in activated macrophages. More studies are required before further conclusions can be reached.
In summary, this study provides the first evidence that L-arginine transport in LPS-stimulated murine macrophages could be further enhanced by catecholamines. Furthermore, this catecholamine-enhanced L-arginine transport might involve CAT-1 and CAT-2A but not CAT-2 or CAT-2B in stimulated macrophages.
1. Forstermann U, Closs EI, Pollock JS, et al. Nitric oxide synthase isozymes: characterization, purification, molecular cloning, and functions. Hypertension 1994;23:1121–31.
2. Hierholzer C, Harbrecht B, Menezes JM, et al. Essential role of induced nitric oxide in the initiation of the inflammatory response after hemorrhagic shock. J Exp Med 1998;187:917–28.
3. Smith JD, McLean SD, Nakayama DK. Nitric oxide causes apoptosis in pulmonary vascular smooth muscle cells. J Surg Res 1998;79:121–7.
4. Benedict CR, Rose JA. Arterial norepinephrine changes in patients with septic shock. Circ Shock 1992;38:165–72.
5. Li CY, Tsai CS, Hsu PC, et al. Dobutamine modulates lipopolysaccharide-induced macrophage inflammatory protein-1alpha and interleukin-8 production in human monocytes. Anesth Analg 2003;97:210–5.
6. Mori M, Gotoh T. Regulation of nitric oxide production by arginine metabolic enzymes. Biochem Biophys Res Commun 2000;275:715–9.
7. Baydoun AR, Wileman SM, Wheeler-Jones CP, et al. Transmembrane signalling mechanisms regulating expression of cationic amino acid transporters and inducible nitric oxide synthase in rat vascular smooth muscle cells. Biochem J 1999;344:265–72.
8. Kavanaugh MP, Wang H, Zhang Z, et al. Control of cationic amino acid transport and retroviral receptor functions in a membrane protein family. J Biol Chem 1994;269:15445–50.
9. Closs EI. Expression, regulation and function of carrier proteins for cationic amino acids. Curr Opin Nephrol Hypertens 2002;11:99–107.
10. Stevens BR, Kakuda DK, Yu K, et al. Induced nitric oxide synthesis is dependent on induced alternatively spliced CAT-2 encoding L-arginine transport in brain astrocytes. J Biol Chem 1996;271:24017–22.
11. 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.
12. Hattori Y, Kasai K, Gross SS. Cationic amino acid transporter gene expression in cultured vascular smooth muscle cells and in rats. Am J Physiol 1999;276:H2020–8.
13. Yang S, Huang CJ, Tsai PS, et al. Renal transcription of high-affinity type-2 cationic amino acid transporter is up-regulated in LPS-stimulated rodents. Acta Anaesthesiol Scand 2004;48:308–16.
14. 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.
15. Chi DS, Qui M, Krishnaswamy G, et al. Regulation of nitric oxide production from macrophages by lipopolysaccharide and catecholamines. Nitric Oxide 2003;8:127–32.
16. 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.
17. Boric MP, Figueroa XF, Donoso MV, et al. Rise in endothelium-derived NO after stimulation of rat perivascular sympathetic mesenteric nerves. Am J Physiol 1999;277:H1027–35.
18. Kakuda DK, Sweet MJ, MacLeod CL, et al. CAT2-mediated L-arginine transport and nitric oxide production in activated macrophages. Biochem J 1999;340:549–53.
19. Huang CJ, Tsai PS, Lu YT, et al. NF-kappaB involvement in the induction of high affinity CAT-2 in lipopolysaccharide-stimulated rat lungs. Acta Anaesthesiol Scand 2004;48:992–1002.
20. Wang Y, Steinsland OS, Nelson SH. A role for nitric oxide in endotoxin-induced depletion of the peripheral catecholamine stores. Shock 2000;13:145–51.
21. Ziolo MT, Katoh H, Bers DM. Expression of inducible nitric oxide synthase depresses beta-adrenergic-stimulated calcium release from the sarcoplasmic reticulum in intact ventricular myocytes. Circulation 2001;104:2961–6.
22. Collins JL, Vodovotz Y, Yoneyama T, et al. Catecholamines decrease nitric oxide production by cytokine-stimulated hepatocytes. Surgery 2001;130:256–64.
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23. Blackwell TS, Christman JW. The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol 1997;17:3–9.