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CELLULAR METABOLISM
1Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, 2Center for Sleep and Respiratory Neurobiology, and 3Department of Anesthesiology and Critical Care, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; and 4Oscillogy, Folsom, Pennsylvania
Submitted 6 October 2007 ; accepted in final form 7 February 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX A: NO CONSUMPTION...APPENDIX B: CONVERSION OF...GRANTSREFERENCES |
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-stimulated RAW 264.7 cells while simultaneously measuring NO production via an electrochemical probe. Decreased O2 availability rapidly (
30 s) and reversibly decreased NO production with an apparent KmO2 of 22 (SD 6) Torr (31 µM) and a Vmax of 4.9 (SD 0.4) nmol·min–1·10–6 cells. To explore potential mechanisms of decreased NO production during hypoxia, we investigated O2-dependent changes in iNOS protein concentration, iNOS dimerization, and cellular NO consumption. iNOS protein concentration was not affected (P = 0.895). iNOS dimerization appeared to be biphasic [6 Torr (P = 0.008) and 157 Torr (P = 0.258) >36 Torr], but it did not predict NO production. NO consumption was minimal at high O2 and NO tensions and negligible at low O2 and NO tensions. These results are consistent with O2 substrate limitation as a regulatory mechanism during brief hypoxic exposure. The rapid and reversible effects of physiological and pathophysiological O2 tensions suggest that O2 tension has the potential to regulate NO production in vivo. inducible nitric oxide synthase; substrate limitation; nitric oxide consumption
30 Torr (mixed venous O2 tension with atelectasis) to over 650 Torr (with O2 therapy) (55). Thus, macrophages must function over a wide range of physiological and pathophysiological O2 tensions, and O2 tension has the potential to regulate macrophage NO production (17, 39, 43, 45). It is currently unknown, however, whether the macrophage response to changing O2 tension is rapid enough for O2 to play a role in the regulation of NO production.
Prior studies have explored the long-term effects (18 and 24 h) of culture PO2 (partial pressure of O2) on nitrite production in LPS- and IFN-stimulated RAW 264.7 cells, but estimates of the apparent KmO2 have varied considerably. McCormick et al. (39) reported an apparent KmO2 of 10.8% (77 Torr) for the PO2 in the headspace gas. In contrast, Otto and Baumgardner (43) estimated the apparent KmO2 at the cell surface to be 14 Torr, after normalizing to iNOS activity and accounting for the O2 diffusion gradient through the media layer. This wide range of reported KmO2 may be in part due to difficulties in accurately controlling headspace (43, 49) and cellular (6, 43) PO2 in conventional cell culture.
No prior studies of macrophage NO production explored the effects of short-term exposure to different O2 tensions, primarily because of the limitations of conventional cell culture and NO analysis methods. First, diffusion of O2 through the media covering cells cultured in dishes can be slow, requiring as long as 30 min for a change in headspace PO2 to be translated to the cell surface (4, 6). Second, the sensitivity of nitrite measurement via the Griess method, which integrates NO production over the period of the experiment, is not adequate for short time periods with less NO accumulation (25, 39, 43).
Forced convection cell culture uses a continuous flow of media to deliver O2 and nutrients directly to the cell monolayer and to remove waste products (6). Because this method of cell culture overcomes the limitations of extracellular O2 diffusion, it is ideally suited for measuring the effects of rapid changes in O2 tension. In addition, the system used for the present study controls O2 tensions with an accuracy of 1 Torr, and permits rapid, direct measurement of changes in NO in the effluent using a sensitive electrochemical probe (46, 47). Thus, the first goal of our current study was to use this recently developed method to accurately define the PO2 dependence of NO production by LPS- and IFN-stimulated RAW 264.7 cells, after brief exposures to a range of physiological and hypoxic O2 tensions.
The second goal of our study was to evaluate three mechanisms that could alter NO production after brief hypoxic exposures. The oxygen atom in NO is derived from molecular O2 (33, 35). Prior cell culture studies that used long-term exposures to varying O2 tensions, and studies with isolated NO synthases, have emphasized the potential role of O2 as a rate-limiting substrate (17, 39, 43, 45). O2 has also been shown to participate in more complex interactions with the NOS enzyme than simple substrate dependence (50). These mechanisms could operate on a short enough time scale to alter NO production after brief hypoxic exposures. There are, however, several additional opportunities for changes in PO2 to rapidly influence NO production. Our goal was to evaluate three of these additional mechanisms: changes in the cellular levels of iNOS protein, changes in iNOS dimerization, and changes in cellular NO consumption. We hypothesized that 1) brief hypoxic exposures would reduce NO production by reducing iNOS protein; 2) brief hypoxic exposures would reduce NO production by reducing iNOS dimerization; and 3) brief hypoxic exposures would reduce NO production and release from the cell by increasing intracellular consumption of NO.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX A: NO CONSUMPTION...APPENDIX B: CONVERSION OF...GRANTSREFERENCES |
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(Cell Sciences, Canton, MA) in DMEM (GIBCO, Carlsbad, CA) supplemented with 5% heat-inactivated FBS (Lonza, Visp, Valais, Switzerland) and 1% antibiotic/antimycotic (penicillin, streptamycin, fungizone; Life Technologies, Gaithersburg, MD). Following stimulation, the column of cells was transferred to the forced convection cell culture system (Fig. 1).
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Measurement of effluent NO tension.
NO was detected using a 2-mm NO electrode (NOP, World Precision Instruments, Sarasota, FL) filled with a CO2-insensitive electrolyte (World Precision Instruments). To enable calibration of the NO electrode, the forced convection cell culture system was adapted to allow defined amounts of NO (input NO) to be added to the fluid stream (Fig. 1). Deionized H2O was deoxygenated via a membrane equilibrator with certified ultrahigh-purity N2 (AirGas), then equilibrated via a second membrane equilibrator with 2,000 parts per million (ppm) NO in N2 (AirGas). As in our prior report (6), function of all membrane equilibrators was tested by confirming flow independence of the measured gas partial pressure in the equilibrator effluent. Defined amounts of 2,000 ppm NO-containing deionized H2O were injected into the fluid stream using a syringe pump (Harvard Apparatus, Holliston, MA). The electrode was calibrated with input NO partial pressure (PNO) of 19, 40, 79, 160, 319, and 500 ppm at the beginning of each day. In the forced convection system, the measured 0–95% time constant for the probe was 27 s. Because of NO probe baseline drift during experiments, the NO probe baseline was measured regularly (i.e., 5-min intervals) to allow for manual baseline correction of the data.
All NO measurements were performed in JBMEM, which was designed to minimize media NO consumption while maintaining cell viability. To evaluate NO consumption by JBMEM, the forced convection system depicted in Fig. 1 was modified by inserting two lengths of fused silica (13 cm and 30 cm) between the NO input site (labeled T in Fig. 1) and the outlet valve (labeled B in Fig. 1), resulting in exposure of NO to the media for 9 and 18 s, respectively. The NO signal was recorded for each exposure duration at two input PNO (160 and 320 ppm) and at three PO2 (0, 40 and 80 Torr). PO2 dependence of NO consumption in JBMEM was further investigated after the system was restored to the configuration of Fig. 1 (i.e., 16 cm of tubing between the NO input and the inlet valve, labeled A in Fig. 1, and 10 cm of tubing between the inlet valve and the outlet valve) in the absence of cells at five input PNO (40, 79, 160, 319, and 500 ppm) and six PO2 (0, 15, 26, 38, 85, 160 Torr). To test whether JBMEM was sufficient to support cell viability, RAW 264.7 cells were seeded onto six-well plates (9.5 cm2) and cultured in a humidified incubator (room air, 37°C, 5% CO2) in either DMEM or JBMEM. Viability was measured by Trypan blue staining after 2, 6, or 18 h of culture. Cells were evaluated with and without stimulation (1 µg/ml LPS and 100 U/ml IFN initiated 18 h before seeding).
Electrophoresis and immunoblotting. Cell lysates were prepared from columns by aspirating ice-cold protease inhibitor containing hypotonic lysis buffer [10 µM phenylmethylsulfonyl fluoride (ICN Biochemical, Aurora, OH), 5 µg/ml aprotinin (Sigma), and 5 µg/ml pepstatin (Amresco, Solon, OH) in deionized H2O] through each column. Cell lysate protein concentrations were measured using the Bio-Rad DC protein assay (Hercules, CA).
Proteins (10 µg) were separated on a 7.5% Tris·HCl gel using SDS-PAGE or low-temperature (LT) SDS-PAGE as previously described (57), except the final β-mercaptoethanol concentration for samples subjected to LT SDS-PAGE was 0.1% (vol/vol). Proteins were transferred to polyvinylidene fluoride (Immobilon-FL 0.2 µm; Millipore, Bedford, MA) and immunoblotted for iNOS (1:1,000 to 1:2,000; NOS2 M19 sc650, Santa Cruz Biotechnology, Santa Cruz, CA) and the loading control, Raf-1 (1:200 to 1:500; Raf-1 sc227, Santa Cruz Biotechnology). Primary antibodies were immunocomplexed with IRDye 800 goat anti-rabbit (1:20,000; Rockland, Gilbertsville, PA). Proteins were detected, documented, and analyzed using an Odyssey Imaging System and software (LiCor Biosciences, Lincoln, NE).
Cellular NO consumption.
Endogenous NO production by LPS and IFN stimulated RAW 264.7 cells cultured in the forced convection system was inhibited by a 36- to 48-min exposure to 100 µM N-{[3-(aminomethyl)phenyl]methyl}-ethanimidamide, dihydrochloride (1400W; Cayman Chemical, Ann Arbor, MI) in JBMEM lacking L-arginine. Maximal NO inhibition by 1400W [86% (SD 7) of basal NO production; n = 8] was expedited by three to four periods of stopped flow for 5 min followed by 7 min of flow. Once maximal inhibition of endogenous NO production was achieved, cells were returned to JBMEM with L-arginine and were sequentially exposed to input PNO of 40, 79, 160, 319, 500, and 0 ppm in the presence of 6, 36, or 83 Torr O2. Average effluent PNO was recorded for each input PNO once steady state was achieved, and it was compared with average effluent PNO recorded on the same day for the same input PNO in the absence of cells. The difference between effluent PNO with cells and without cells for each input PNO was assumed to be due to the net result of NO consumption and residual endogenous NO production. Testing for zero-order, first-order, or higher-order dependence of NO consumption on PNO and PO2 was performed, and the data were analyzed, by considering a mass balance on the cell column:
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Our analysis assumes that NO production is independent of NO concentration, since the range of NO concentrations we studied is below the range associated with NO feedback inhibition of iNOS (1). Consumption is modeled, as a starting point, as first order in both NO and O2 (51). It is known that autoxidation is second order in NO and first order in O2 (18), but the reaction is too slow to consume NO before it reaches the electrode. Additionally, our data were calibrated to the effluent PNO detected for the five input PNO in JBMEM in the absence of cells and O2. Therefore, the detected NO consumption in our experiments is expected to be dominated by intracellular consumption reactions. The mass balance on the column of cells becomes
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NO is the NO solubility in media at 37°C (2.13 x 106 pM/Torr) (53), PNOi is the NO partial pressure in media entering the column (Torr), PNOe is the NO partial pressure in media leaving the column (Torr), kc is the consumption rate constant (pmol NO·cell–1·s–1·Torr–2), PO2 is the average oxygen partial pressure for cells in the column, PNO is the average NO partial pressure for cells in the column, f(PO2) is the functional dependence of NO production on PO2, kp is the maximal NO production for each column at high PO2 (pmol NO·cell–1·s–1), and N is the number of cells in the column. Statistical analysis. Comparison of means were tested by a one-way ANOVA for each PNO for PO2-dependent NO consumption in the absence of cells (Fig. 2), a two-way ANOVA for the effects of DMEM versus JBMEM over time on cell survival (Table 2), and a three-way ANOVA for the effects of input PNO, PO2 and exposure time on NO degradation in JBMEM using SigmaStat version 3.1. All other statistics were performed using GraphPad InStat (version 3.06 for Windows 95, GraphPad Software, San Diego, CA). Changes in NO production with repeated cycling between 0 and 36 Torr O2 (Fig. 3A) were tested by linear regression. The apparent Km and Vmax were calculated by SigmaPlot Enzyme Kinetics Module 1.1 using a Michaelis-Menten nonlinear analysis (Fig. 3C). Comparison of means for iNOS protein concentration data (Fig. 4) were tested by one-way ANOVA. Comparison of means for iNOS dimerization data (Fig. 5) were tested by pairwise t-tests with a Bonferroni correction. Testing of the NO consumption model was performed with linear regression as described in APPENDIX A.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX A: NO CONSUMPTION...APPENDIX B: CONVERSION OF...GRANTSREFERENCES |
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2; P = 0.516 and P = 0.201, respectively), indicating negligible consumption by the media for exposures <18 s. Consistent with these observations, PO2-dependent NO consumption in JBMEM was not detectable for any of the input PNO investigated with the system in its standard configuration as depicted in Fig. 1, with a 15-s transit time from the NO input to the NO electrode (Fig. 2; n = 3; 500 ppm, P = 0.152; 319 ppm, P = 0.264; 160 ppm, P = 0.370; 79 ppm, P = 0.951; 40 ppm, P = 0.468). For all subsequent experiments, NO consumption during the 3.5-s average transit time from the cells to the NO electrode was therefore considered negligible.
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, no significant effect of media (P = 0.364) or time (P = 0.894) was detected.
Effect of O2 tension on effluent PNO.
Steady-state NO release by LPS- and IFN-stimulated RAW 264.7 cells exposed to 36 Torr O2 was 3.08 (SD 1.14) nmol·min–1·10–6 cells (n = 5). Unstimulated cells did not produce detectable NO (data not shown). Exposure of the cells to 0 Torr O2 decreased effluent PNO within 30 s to an undetectable amount (Fig. 3A). Similarly, within 30 s of reexposure to 36 Torr O2, effluent PNO was greater than or equal to the initial measured concentration. Repeated cycling between 0 Torr (2 min) and 36 Torr O2 (3 min) consistently produced these rapid changes in effluent PNO, with a cumulative 10% increase in effluent PNO over a period of 40 min (P < 0.0001; n = 2). Unstimulated cells subjected to identical cycling patterns between 0 Torr and 36 Torr O2 for 40 min did not produce detectable NO (data not shown).
Exposure of LPS- and IFN-stimulated RAW 264.7 cells to a range of O2 tensions elicited corresponding changes in effluent PNO, which predominantly followed a Michaelis-Menten kinetic model (Fig. 3B). A nonlinear analysis computed an apparent KmO2 of 22 (SD 6) Torr and Vmax of 4.9 (SD 0.4) nmol·min–1·10–6 cells (n = 5; R2 = 0.80). A slight deviation from a smooth monotonic function is apparent between 6 and 36 Torr O2 (Fig. 3C).
Effect of O2 tension on iNOS.
O2 tension did not influence iNOS protein concentration (Fig. 4; n 4; P = 0.895), but it did influence the bands thought to contain iNOS dimers (Fig. 5; n = 3). In the Western blot derived from the partially denaturing gel, three bands were present: a band corresponding to the expected monomer molecular mass (130 kDa) (16), a band corresponding to the expected dimer molecular mass (260 kDa), and an unexpected band of much higher molecular mass (500 kDa). Compared with 36 Torr, the ratio of the 260-kDa band to the 130- kDa band was significantly increased in lysates from samples at 6 Torr (P = 0.003). A similar finding was observed for the ratio of the 500-kDa band to the 130-kDa band (P = 0.008). The ratios also appeared to increase in lysates from samples at 157 Torr, but the increase was not statistically significant (260 kDa ratio P = 0.258; 500 kDa ratio P = 0.129).
Effect of NO and O2 tension on cellular NO consumption.
Following 1400W inhibition of endogenous NO production (Fig. 6A), LPS- and IFN-stimulated RAW 264.7 cells at 6, 36, or 83 Torr O2 were exposed to five input PNO (Fig. 6B). Data are presented in Fig. 6C as the ratio of effluent PNO with cells to effluent PNO without cells. Net cellular NO consumption, as indicated by a ratio <1, was evident at input PNO of 160, 319, and 500 ppm in PO2 of 36 and 83 Torr. Calculations based on known autoxidation rate constants (18, 36) estimated that autoxidation within the cells could account for a maximum of 3% of the measured NO consumption. Cellular consumption was negligible at a PO2 of 6 Torr, regardless of the input PNO. Net cellular NO production resulted in a ratio >1 for the lower two input PNO (40 and 79 ppm) delivered in the lower two PO2 (36 and 83 Torr). The amount of NO production was consistent with the residual cellular NO that was not inhibited by 1400W. Immediately following 1400W treatment, mean cellular NO production was 14% (SD 7) (n = 8) of initial NO production. By the end of the experiment, cellular production increased to 24% (SD 6) (n = 7) of initial NO production. The relationship between net cellular NO consumption and PNO was most consistent with first-order kinetics. NO consumption also correlated positively with PO2 in a first-order-dependent manner at all PNO. The overall consumption constant (kc) for the model was 0.038 pmol NO·s–1·10–6 cells·Torr–2 (12.7 mmol NO·s–1·10–6 cells·M–2). Details of the consumption model are presented in APPENDIX A.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX A: NO CONSUMPTION...APPENDIX B: CONVERSION OF...GRANTSREFERENCES |
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Our measured apparent KmO2 is within the range of values reported previously for long-term exposures in macrophage cell culture (39, 43), and for the isolated iNOS enzyme (1, 17, 45). It is also well within the range of PO2 that would be required for O2 tension to regulate NO production in vivo, as has been suggested previously (1, 17, 39, 43, 45). Our study additionally demonstrated, however, that precisely controlled changes in extracellular O2 tension altered NO production by intact isolated cells within seconds and that this effect was immediately reversible. A slow response, or an irreversible response, would have argued against any role for the regulation of NO production by PO2 changes in intact cells. Instead, the effect on cellular NO production was rapid and reversible, further supporting a significant regulatory role for O2 tension in vivo.
Several studies have investigated PO2 dependence of NO production in intact tissues and in vivo. NO production has been shown to be rapidly decreased by hypoxia when the primary enzyme responsible for NO production was thought to be endothelial NOS (23, 31), iNOS (17), or neuronal NOS (59). O2-mediated intracellular kinetics and regulatory mechanisms, however, are difficult to evaluate in vivo and in tissue models because of the complications of tissue structure and O2 delivery dynamics. Tissues are by definition composed of several different cell types, with the potential for expression of several different NOS isoforms, and it is often difficult to unequivocally define which isoform is primarily responsible for producing the measured NO. For example, in bronchial airways, each isoform is expressed in different cells (10, 17) and in different regions of the same cells (10, 58). This could have important effects on total NO production, because the KmO2 for each isoform varies markedly in isolated enzyme studies (1, 45). Although tissue and in vivo studies are not directly comparable to our cell culture study, they do support the concept that NO production can be rapidly regulated by changes in O2 tension in vivo.
We are not aware of any prior cell culture studies of NO production during brief exposure to hypoxia for the direct comparison with our results. Two prior studies, however, investigated the effects of long-term exposure (18 h) to multiple O2 tensions on nitrite production by RAW 264.7 cells concurrently stimulated with LPS and IFN (39, 43). McCormick et al. (39) measured cellular nitrite production following 24 h of exposure to various headspace O2 tensions ranging from 1% to 21% (7 to 150 Torr). The decrease in nitrite production at low O2 tensions was well described by a hyperbolic curve fit, and the apparent KmO2 for the headspace gas was 10.8% (77 Torr) (39). Otto and Baumgardner (43) measured cellular nitrite production following 18 h of exposure to various headspace O2 tensions ranging from 1 to 677 Torr. Nitrite production decreased with decreasing O2 tension throughout the entire range. iNOS activity in cell lysates, defined by citrulline production in room air at 25°C, also showed substantial dependence on cellular PO2 before lysis, suggesting an effect of O2 tension on specific activity and/or the amount of active iNOS. After normalizing nitrite production for changes in iNOS activity, and accounting for the O2 diffusion gradient from the headspace gas to the cell surface, their estimate for the apparent KmO2 at the cell surface was 14 Torr (43). Studies of long-term exposures to different O2 tensions are not strictly comparable to the short-term exposures of the current study because of the many factors that could change slowly over time. For example, O2-dependent changes in the transcription and translation of iNOS (3, 28, 39–41, 43), as well as of other relevant proteins [e.g., mediators of arginine metabolism (37)], would be expected to take several hours (3) and could substantially influence NO production in long-term exposures, yet have minimal impact in short-term exposures.
Three prior studies have investigated the apparent KmO2 for isolated iNOS. Using a steady-state kinetics approach, Rengasamy and Johns (45) measured citrulline production at various PO2 by iNOS within a RAW 264.7 cell lysate. In their system, O2 tension was rigidly controlled in the headspace gas by use of continuous gas flows, and the reaction mixture was constantly stirred to minimize diffusion gradients. They reported an apparent KmO2 for iNOS of 6.3 µM, for a solution temperature of 37°C (45). Using a rapid equilibrium kinetics approach, Abu-Soud et al. (1) and Dweik et al. (17) studied the effects of O2 tension on purified recombinant mouse iNOS by measuring the rate of NADPH oxidation spectroscopically, in a closed system at 25°C. They reported an apparent KmO2 of 130 µM (1) and 135 µM (17). When the NO produced was scavenged with oxyhemoglobin, however, the measured KmO2 was reduced approximately fourfold to 42 µM (1). The difference between these values was shown to be due to direct feedback inhibition of iNOS by NO, an effect that has been demonstrated for all three NOS isoforms (50).
In our experiments using forced convection cell culture, the flowing media continuously removed NO as it was produced, thereby minimizing NO accumulation. Our results, therefore, are most comparable to the isolated enzyme experiments that either continuously removed NO with flowing headspace gas in an open system [Rengasamy and Johns; KmO2 6.3 µM (45)] or scavenged NO with oxyhemoglobin in a closed system [Abu-Soud et al.; KmO2 42 µM (1)]. The apparent KmO2 we measured for intact cells was 22 (SD 6) Torr [31 µM based on an Ostwald solubility coefficient of 0.0271 ml O2 BTP/ml water-atm at 37°C (54)]. Unlike the isolated enzyme, within intact cells, several mechanisms in addition to substrate dependence and product feedback inhibition could acutely alter NO production after a change in PO2. We investigated three potential mechanisms: changes in iNOS protein levels, iNOS dimerization, and cellular NO consumption.
iNOS protein levels were not influenced by brief hypoxic exposures. Hypoxia has been shown to induce increased expression of iNOS mRNA and protein via hypoxia-inducible factor 1-dependent regulation (28, 40, 41). Acute changes in PO2, however, would not be expected to acutely increase iNOS protein production because transcription and translation have been shown to take up to 6 h to change after an appropriate stimulus (3). To our knowledge, the effect of hypoxia on iNOS degradation has not been investigated. The iNOS half-life in room air, however, was 1.6 h in several cell types (32). Our results showing that brief hypoxic exposures have little impact on iNOS protein are consistent with these previous studies.
iNOS dimerization was influenced by brief exposure to various O2 tensions but surprisingly did not correlate with changes in NO production. The changes in dimerization appeared to be biphasic (6 Torr and 157 Torr > 36 Torr) and were consistent for the 260-kDa band, the expected size for iNOS dimers (16), and for the 500-kDa band, an undefined iNOS-containing protein complex. Our data for NO production as a function of cellular O2 tension (Fig. 3C) and data from a prior study on iNOS activity as a function of O2 tension (43) show deviations from a smooth monotonic function in that range of PO2, which may in part be related to the biphasic changes we observed in dimerization. Decreased NO production despite a large increase in iNOS dimerization during hypoxia could be due to O2 substrate limitation, limitation of another substrate or cofactor during hypoxia, and/or the generation of inactive dimers.
Cellular NO consumption was negligible at all but the highest PO2 and PNO, with an overall consumption constant of 0.038 pmol NO·s–1·10–6 cells·Torr–2 (12.7 mmol NO·s–1·10–6 cells·M NO–1·M O2–1). There are many possible intracellular reactants that can directly consume NO, and, correspondingly, there are many possible reaction kinetics for cellular NO consumption (22, 34, 44). Our data are most consistent with first-order dependence in NO and O2, most similar to the findings of Thomas et al. (51). Our consumption rates are at the low end of the reported range for various cell types (0.050 to 1.61 pmol NO·s–1·10–6 cells·Torr–2) (20, 51), but they are consistent with a previous report of LPS- and IFN-stimulated RAW 264.7 cells [0.011 pmol NO·s–1·10–6 cells·Torr–2 (42); see APPENDIX B for conversion of consumption constants to comparable units].
In summary, we used a novel forced convection cell culture system to precisely regulate cellular O2 tensions in the range of <1 Torr to 157 Torr. In an LPS- and IFN-stimulated macrophage cell line, decreases in cellular PO2 reduced NO production within seconds, an effect that was immediately reversible with restoration of the original PO2. The apparent KmO2 for this oxygen dependence was 22 (SD 6) Torr (31 µM). The changes in NO production were not explained by the effects of cell PO2 on iNOS protein levels, iNOS dimerization, or consumption of NO. The rapid effects of cellular PO2 on macrophage NO production are consistent with regulation of NO production by O2 substrate limitation. The apparent KmO2 in intact cells and the kinetics of the PO2 dependence suggest that O2 substrate limitation could play a dynamic role in the regulation of NO production by iNOS in vivo.
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APPENDIX A: NO CONSUMPTION MODEL |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX A: NO CONSUMPTION...APPENDIX B: CONVERSION OF...GRANTSREFERENCES |
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NO(PNOi – PNOe) was plotted against PNO (Fig. S1; the online version of this article contains supplemental data) to assess for linear dependence that would indicate that first-order kinetics in NO are appropriate. The negative intercept on this plot is production, i.e., intercept = –f(PO2)kpN, which could be of any functional form (for example the Michaelis-Menten fit in Fig. 3). The only assumption required about production for this analysis of NO consumption is that the production is independent of PNO.
For the experiments at higher PO2 (36 and 83 Torr) in Fig. S1, a linear fit is clearly adequate, and the slopes were significantly different from zero. At lower PO2, as the slope of this relationship approaches zero, the power to detect a slope significantly different from zero is reduced. As expected, the trend for a linear relationship did not result in a slope significantly different from zero (P 0.122) for the PO2 = 6 Torr data sets.
For each column, the best-fit slope (b1) of the QNO(PNOi – PNOe) versus PNO data was divided by PO2 and plotted against PO2 (Fig. S2). First-order dependence in PO2 predicts that b1/PO2 should be independent of PO2. The data of Fig. S2 are consistent with a constant b1/PO2 that is independent of PO2, confirmed by a best-fit regression slope not significantly different from zero (P = 0.422).
The best estimate of the overall consumption constant kcN was estimated from a weighted average of the b1/PO2 values in Fig. S2 that accounts for the fact that the confidence in parameter estimates is increased at higher PO2. The weighted average assigned weights in direct proportion to PO2. The resulting best estimate for kcN was 0.0186 pmol NO·s–1·Torr–2.
Finally, cell number for these experiments was estimated from representative measurements of total protein after lysis of cells from the columns, combined with a previously established relationship between cell number and protein for RAW 264.7 cells (43):
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The overall NO consumption rate constant, normalized to cell number, is
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APPENDIX B: CONVERSION OF kc UNITS FOR COMPARISON WITH PREVIOUS STUDIES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX A: NO CONSUMPTION...APPENDIX B: CONVERSION OF...GRANTSREFERENCES |
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Nalwaya and Deen (42) reported NO consumption data for stimulated RAW 264.7 cells. NO consumption was treated as first order in NO and zero order in O2, with a rate constant of 0.6 s–1. They measured NO consumption over a range of PO2. Taking as an approximation a PO2 in the middle of this range at 100 Torr, with an NO solubility as above, and with their estimate of cell volume of 8.8 x 10–13 l/cell, the equivalent rate constant is 0.011 pmol·s–1·10–6 cells·Torr–2.
Gardner et al. (20) reported NO consumption data for several cell types. NO consumption values, in compatible units, ranged from 0.050 to 0.52 pmol·s–1·10–6 cells·Torr–2.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX A: NO CONSUMPTION...APPENDIX B: CONVERSION OF...GRANTSREFERENCES |
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX A: NO CONSUMPTION...APPENDIX B: CONVERSION OF...GRANTSREFERENCES |
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