486
Chem. Res. Toxicol. 2005, 18, 486-493
Nitric Oxide, Oxygen, and Superoxide Formation and Consumption in Macrophage Cultures Nitesh Nalwaya† and William M. Deen*,†,‡ Department of Chemical Engineering and Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received May 3, 2004
To examine the potential for generating toxic nitrogen oxides during the immune response, rates of formation and consumption of NO, O2, and O2- were measured in murine macrophagelike RAW264.7 cells. Cellular kinetic parameters for NO and O2 were obtained by monitoring their time-dependent concentrations in a closed chamber, and net cellular synthesis of O2was quantified from ferricytochrome c reduction in cultures where NO synthesis was inhibited. Also measured was the photosensitive generation of O2- in the culture media. Unactivated cells (without NO synthesis) had an O2 consumption rate of 32 ( 3 pmol s-1 (106 cells)-1, typical of mammalian cells. Also typical was that adding NO rapidly and reversibly inhibited respiration. Activated cells synthesized NO at a rate of 4.9 ( 0.6 pmol s-1 (106 cells)-1. When NO synthesis was inhibited, they consumed three times as much O2 as unactivated cells [108 ( 17 pmol s-1 (106 cells)-1]; however, O2 consumption of activated cells exposed to 1 µM NO was calculated to be comparable to that of NO-free unactivated cells. Rates of intracellular NO consumption were small, implying that enzymatic consumption does little to limit net NO synthesis by macrophages. Accounting for O2- generation in the culture media resulted in net rates of cellular O2- synthesis smaller than previously reported; the rate was 6% of NO synthesis in activated cells and was undetectable in unactivated cells.
Introduction Nitric oxide is synthesized throughout the body by constitutive NO synthases and has a number of regulatory and signaling functions that are essential for good health (1). In addition, NO is produced at relatively high rates by the inducible NO synthase (iNOS) that is expressed in macrophages during the nonspecific immune response (2, 3). The resulting local elevations in the concentrations of NO and other nitrogen oxides assist in killing pathogens but risk collateral damage to host cells. An understanding of the rates of formation of these nitrogen oxides is needed to assess the cytotoxic and genotoxic consequences of chronic infections and inflammations, including their association with certain forms of cancer (4). Central to such an understanding is the manner in which NO, O2, and O2- interact within macrophages and in the adjacent extracellular fluid, through enzymatic as well as nonenzymatic reactions. Starting with extracellular events, we note that the NO that is produced in the cytosol of a macrophage can freely diffuse out of the cell, where it may generate other nitrogen oxides via two main pathways. The first, in which NO is oxidized ultimately to NO2-, may be summarized as +2H2O
4NO + O2 f 2N2O3 98 4NO2- + 4H+
(1)
In addition to N2O3, another intermediate in the actual multistep sequence (not shown) is NO2 (5). The second pathway involves O2- generated at the outside surface * To whom correspondence should be addressed. Tel: 617-253-4535. Fax: 617-258-8224. E-mail:
[email protected]. † Department of Chemical Engineering. ‡ Biological Engineering Division.
of the macrophage by a membrane-bound NADPH oxidase (6, 7). The extremely rapid reaction of NO with O2(8, 9) leads to peroxynitrite (ONOO-) and (mainly) NO3-. In simplified form, this pathway is CO2
NO + O2- f ONOO- 98 NO3-
(2)
Not shown is the nitrosoperoxycarbonate intermediate formed during the CO2-catalyzed consumption of ONOO-, which in turn gives rise to NO2 and CO3- radicals (10). Also omitted is NO2- formation from ONOOH decomposition (11), which is minor at physiological CO2 levels. Although NO3- is the principal end product from peroxynitrite in culture media, the availability of organic substrates to react with peroxynitrite will influence the fate of the nitrogen that comes from NO. Reactions 1 and 2 will occur inside macrophages as well as outside. That is, O2 is readily available intracellularly, and although there appear to be no data specifically for macrophages, O2- is generated in most cells in both mitochondria and cytosol (12, 13). Intracellular levels of O2- are lowered by superoxide dismutase (13), which provides an O2- consumption route that competes with reaction 2. Another set of interactions among NO, O2, and O2- is due to intracellular enzymatic processes. NO has been found to competitively inhibit mitochondrial O2 consumption (respiration) in mammalian cells (14, 15). In the synthesis of NO by iNOS (or other forms of NOS), O2 is a substrate in addition to L-arginine. It has been shown also that NO binds reversibly to the heme iron in iNOS, where it can be oxidized by O2 to NO3- (16). Thus, iNOS is capable of consuming as well as producing NO. A similar consumption mechanism is provided by the NO
10.1021/tx049879c CCC: $30.25 © 2005 American Chemical Society Published on Web 02/08/2005
Nitric Oxide in Macrophage Cultures
dioxygenase (NOD) activity identified in a variety of cells (17). There may be other protein-based NO consumption mechanisms in macrophages. For example, NO can react with an oxygen-ligated, reduced metal to produce the oxidized metal and NO3-, similar to the rapid reaction of NO with oxyhemoglobin or oxymyoglobin (18). Reaction 2 provides yet another pathway for intracellular NO consumption. To the extent that any of these NO consumption mechanisms occur in macrophages, they would tend to limit the maximum achievable NO concentration. Murine macrophage-like RAW264.7 cells have been commonly used for NO toxicity studies (19-23). Net rates of NO and O2- production by those cells have been determined in certain settings (20, 24, 25). Those results and other literature data have formed the basis for models that describe intracellular and/or extracellular interactions among NO, O2, and O2- (26-30). However, key pieces of information have been missing. Rates of O2 consumption by RAW264.7 cells have not been measured, nor has their ability to consume as well as synthesize NO been examined. Moreover, it was recently shown that O2- can be generated by two common components of cell culture media, HEPES buffer and (in the presence of light) riboflavin (31). Thus, some of the O2- production that was attributed to the macrophages may have been an artifact due to O2- generation in the media. We report here a series of experiments in which O2- formation in the media was taken into account in determining the rates of synthesis of NO and O2- by RAW264.7 cells. Also quantified were cellular O2 and NO consumption and the effects of NO on respiration.
Materials and Methods Reagents. Dulbecco’s modified Eagles’s medium (DMEM), DMEM with 25 mM HEPES (pH 7.4), PBS (pH 7.4), fetal bovine serum, glutamine, and penicillin/streptomycin were obtained from BioWhittaker (Walkersville, MD). Recombinant mouse interferon-γ (IFN-γ) (specific activity 2.81 × 106 U/mg, 0.33 mg/ mL) was purchased from R&D Systems (Minneapolis, MN). Trypan blue (4% in saline) and Escherichia coli lipopolysaccharide (LPS) (serotype 0127:B8) were from Sigma Aldrich, and NGmonomethyl-L-arginine (NMA) was from Calbiochem (Salt Lake City, UT). The culture medium consisted of DMEM supplemented with 584 mg/L glutamine, 10% (v/v) heat-inactivated fetal bovine serum (heated at 56 °C for 30 min prior to use), 200 U/mL penicillin, and 200 µg/mL streptomycin. Ar and 10% NO in N2 were obtained from BOC Gases (Edison, NJ). Cell Line. Macrophages from the immortalized hygromycin resistant cell line RAW264.7 (American Type Tissue Culture Collection) were grown in 100 mm Falcon polystyrene tissue culture dishes with supplemented DMEM medium and kept in a humid 5% CO2-95% air atmosphere at 37 °C. NO Consumption in Culture Media. A 50 mL septumsealed glass vial with a magnetic stir bar was used to determine the rate of NO consumption by reaction 2 in DMEM with 25 mM HEPES (pH 7.4). Experiments with PBS (pH 7.4) served as the control. After the vial was filled with the test liquid, NO was introduced by injecting a saturated solution (see below) through the septum with a syringe, to give a mixture concentration of 1 µM. The rate of NO consumption was calculated from the subsequent decay of the NO concentration, which was monitored continuously with an electrode (see below). The experiments were carried out in a warm room at 37 °C, with the temperature verified throughout each run. Because O2generation by riboflavin has been reported to be light sensitive (31), experiments were performed both in room light and in the dark. NO concentrations were measured using an electrode (World Precision Instruments, Sarasota, FL) inserted through the septum. The NO electrode was precalibrated by adding
Chem. Res. Toxicol., Vol. 18, No. 3, 2005 487
Figure 1. Cross-section of apparatus used to monitor NO and O2 concentrations in culture media. When present, the RAW264.7 cells adhered to the horizontal dish surface. The enclosed liquid volume was 8.8 mL. known amounts of NO2- to a solution with excess H2SO4 and using KI to generate equimolar amounts of NO (32). Linear calibration curves at 37 °C were obtained over the range of concentrations studied (0-5 µM). The NO electrode had a lower detection limit of 1 nM and a response time of 5 nM. This made the rate of NO consumption by reaction 2 zero-order (i.e., independent of the NO concentration). Apparatus for NO and O2 Measurements with Cells. As shown in Figure 1, a polycarbonate insert was machined so that it formed a seal with the top rim of a 60 mm Falcon polystyrene tissue culture dish. When assembled, the enclosed volume was 8.8 mL. Incorporated into the insert were the NO electrode, a fiber optic O2 sensor (Instech Laboratories, Plymouth Meeting, PA), and a motor-magnet assembly that propelled a magnetic stir bar within the closed chamber. The O2 sensor had a dynamic range of 0-2 mM and a response time of 5 s. It was calibrated using air-saturated water and a deoxygenated solution prepared by adding excess Na2SO4 to water. The sensors allowed the O2 and/or NO concentrations to be monitored continuosly in shortterm (
