* Department of Chemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250; Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201;
Department of Toxicology, Faculty of Pharmaceutical Science, Khon Kaen University, Khon Kaen 40002, Thailand;
Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78230; ¶ Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201; and || Center for Low Frequency EPR for In Vivo Physiology, University of Maryland, Baltimore, Maryland 21201
Received June 7, 2004; accepted June 30, 2004
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ABSTRACT |
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Key Words: nitric oxide; superoxide; NOS I; calmodulin; divalent cations; metal toxicity.
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INTRODUCTION |
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There are regulatory co-factors that dictate whether NOS I generates NO and/or O2 and H2O2. For instance, in the absence of L-arginine, O2 (Pou et al., 1999; Yoneyama et al., 2001
) and H2O2 are directly produced; the ratio of these reduction products of O2 is set by the presence of H4B (Rosen et al., 2002
). Again, the binding of L-arginine to NOS shifts electron transport from O2 to this amino acid, producing NO at the expense of O2 (Pou et al., 1999
). Another control element is calmodulin, and, in the presence of Ca2+, it binds to NOS I and enhances electron transport through the reductase domain to the heme, allowing NO and O2 to be produced [for a review, see Roman et al. (2002)
]. We have recently demonstrated that Pb2+ and Sr2+ stimulate the NOS I generation of NO and O2 by binding to and activating calmodulin (Weaver et al., 2002
). Unlike Ca2+, which is tightly regulated in the cell (Clapham, 1995
), many divalent cations are unfettered and can compete for the four Ca2+ binding sites on calmodulin. Several other divalent cations such as Ni2+, Ba2+, Cd2+, and Mn2+ have been shown to bind to and activate calmodulin (Goldstein and Ar, 1983
; Habermann et al., 1993
; Mills and Johnson, 1985
), and several of these divalent cations have been associated with NOS, although the mechanism by which these metals affected NOS is not well understood (Gupta et al., 2000
; Mittal et al., 1995
; Palumbo et al., 2001
; Perry and Marletta, 1998
; Yamazaki et al., 1995
). Activation of NOS I by divalent cations, other than Ca2+, may initiate toxicity through over-production of NO, O2, and products derived from these free radicals.
In the present study, we investigated the possible effects of several divalent metal ions, known to activate calmodulin, on NOS I-mediated catalysis in the absence and presence of L-arginine. We demonstrate that Ni2+, Ba2+, and Mn2+ can activate NOS I to metabolize L-arginine to L-citrulline and NO. In the absence of L-arginine, these cations also promote NOS I generation of O2. It is suggested that these divalent cations, by binding to calmodulin, initiate enzymatic activity. In contrast, Cd2+ did not activate NOS I to produce either NO or O2.
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MATERIALS AND METHODS |
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Purification of NOS I. NOS I was expressed and purified essentially as described in the literature (Roman et al., 1995), with the modification that the culture volume was 500 ml rather than 1000 ml. The enzyme concentration was determined by its CO-difference spectrum, as described by Roman et al. (1995)
, using an extinction coefficient of 100 mM1cm1 at
444475 nm.
NOS I activation using the [14C]L-citrulline formation assay. The activation of purified NOS I was determined by its ability to catalyze the formation of L-citrulline from L-arginine as previously reported (Weaver et al., 2002), with modifications. A cocktail solution ([14C]L-arginine (0.6 µCi/ml) in the presence of NADPH (1 mM), L-arginine (100 µM), and calmodulin (100 U/ml) in HEPES buffer (50 mM, 0.5 mM EGTA, pH 7.4) was prepared. The reaction was initiated by the addition of the cocktail solution into the reaction mixture containing purified NOS I (3.7 µg) and Ca2+, Ni2+, Ba2+, Mn2+, or Cd2+ (from 100 µM to 1 mM) to a final volume of 150 µl. The reaction mixture was incubated at room temperature for 10 min and terminated with 2 ml of stop solution (20 mM HEPES, 2 mM EDTA, pH 5.5). The product [14C]L-citrulline was separated by passing the reaction mixture through columns containing Dowex 50W-X8 cation exchange resin preactivated with sodium hydroxide (1 M), and radioactivity was counted using a scintillation counter (Model LS 6500; Beckman Coulter Inc., Fullerton, CA). Data were expressed as means and standard deviations of multiple experiments.
Determination of nitric oxide production. The initial rate of NO production by purified NOS I was estimated using the hemoglobin assay (Murphy and Noack, 1994). The reaction was initiated by the addition of NOS I (2.0 µg) to a cuvette containing HEPES buffer (50 mM, 0.5 mM EGTA, pH 7.4), oxyhemoglobin (10 µM), the divalent cation (500 µM), calmodulin (100 U/ml), NADPH (100 µM), and L-arginine (100 µM) to a final volume of 500 µl at room temperature. A UV-Vis spectrophotometer (Uvikon, Model 940, Research Instruments International, San Diego, CA) was used to monitor the conversion of oxyhemoglobin to methemoglobin during the course of the reaction. Specifically, the increase in absorbance at 401 nm was used to quantitate the reaction, using an extinction coefficient of 60 mM1cm1 at 401 nm.
Spin trapping of NOS I-generated O2 using BMPO. Spin trapping of O2 by BMPO was conducted in a reaction mixture containing, NOS I (14.2 µg), various divalent cations (500 µM), and calmodulin (100 U/ml) in phosphate buffer (50 mM, pH 7.4, 1 mM DTPA, 1 mM EGTA). The reaction was initiated by the addition of BMPO (50 mM) and NADPH (100 µM) into the reaction mixture to a final volume of 0.3 ml at room temperature. The reaction mixture was mixed, transferred into a quartz flat cell and fitted into the cavity of the EPR spectrometer. EPR spectra were continually recorded at room temperature and data shown below in Figure 4 were obtained 10 min after the initiation of the reaction. Instrument settings were as follows: microwave power, 20 mW; modulation frequency, 100 kHz; modulation amplitude, 0.5 G; sweep time, 12.5 G/min; and response time, 0.5 s. The receiver gain for each experiment is given in the legend of Figure 4.
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Reduction of ferricytochrome c by NOS I. The reduction of ferricytochrome c (80 µM) by NOS I (0.4 µg) was monitored in a reaction containing Ca2+, Ni2+, Ba2+, Mn2+, or Cd2+ (500 µM), calmodulin (100 U/ml), and NOS I in HEPES buffer (50 mM, pH 7.4, 0.5 mM EGTA) at room temperature. The reaction was initiated by the addition of NADPH (100 µM) to the reaction mixture to a final volume of 500 µl. The initial rate of ferricytochrome c reduction in the absence and presence of SOD (30 U/ml) at 550 nm was estimated spectrophotometrically using an extinction coefficient of 21 mM1 cm1 at 550 nm.
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RESULTS |
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Spin Trapping of NOS-Generated O2
NOS I produces O2 in the absence of L-arginine (Pou et al., 1992; Yoneyama et al., 2001
). Of interest is whether Ba2+, Ni2+, or Mn2+, which activate NOS I to metabolize L-arginine to L-citrulline and NO, can, in the absence of substrate, generate O2. Given that ferricytochrome c and its derivatives cannot report NOS generated O2 (Weaver et al. 2003
), we turned to spin trapping/EPR spectroscopy. For these experiments, BMPO was chosen as the spin trap, because its reaction with O2 gives a spin-trapped adduct, BMPO-OOH, that exhibits a long half-life (Rosen et al. 2002
). As depicted in Figure 4, BMPO spin trapped O2 from NOS I activated by various divalent cations. However, the EPR spectral peak height of BMPO-OOH was much greater with Ca2+ than with Ba2+, Ni2+, and Mn2+ (500 µM for each ion)-activated NOS I. These data suggested that NOS I generated considerably less O2 when activated by these metal ions than when activated by Ca2+. In the case of Mn2+, the EPR spectral peak height of BMPO-OOH was diminished by nearly 80% as compared to control (Fig. 4).
The Effect of Divalent Cations on the Rate of Ferricytochrome c Reduction by O2
To determine whether the decrease in O2 spin trapping was attributable to competition for O2 by Ba2+, Ni2+ or Mn2+, a series of control experiments were performed. A hypoxanthine/xanthine oxidase system was used as a source of O2. And the SOD-like property of each of the divalent cations (500 µM), which was dissolved in the buffer used in the experiments above, was estimated by monitoring the initial rate of ferricytochrome c reduction as compared to that observed with SOD (30 U/ml). The control was performed in the absence of these metal cations. In the absence of divalent cations, 0.613 ± 0.17 µM/min of O2 was generated, as measured by the reduction of ferricytochrome c, and in the presence of Ca2+, Ba2+, Ni2+, or Mn2+, 0.598 ± 0.17, 0.547 ± 0.18, 0.604 ± 0.15, or 0.540 ± 0.16 µM/min of O2, respectively, was observed. From these data, no significant SOD-like property was attributed to any of the divalent cations.
Reduction of Ferricytochrome c by Divalent CationActivated NOS I
As reported in the literature, ferricytochrome c is reduced by NOS I (Heinzel et al., 1992; Mayer et al., 1991
; Pou et al., 1992
; Roman et al., 2000
; Sheta et al., 1994
). This enzymatic reduction occurs in the absence and presence of calmodulin, although the reduction in the absence of calmodulin is 10-fold less than in the presence of calmodulin when NOS I is activated using Ca2+/calmodulin (Roman et al., 2000
). Therefore, experiments were conducted to determine what effect Ba2+, Mn2+, Ni2+, and Cd2+ had on the rate of NOS I reduction of ferricytochrome c (Table 2). Each divalent cationactivated NOS I, except Cd2+, was able to reduce ferricytochrome c at a rate comparable to that of Ca2+. Also, each cation-activated NOS I followed the trend of maximal activation similar to that seen in previous experiments. Of note, the rates of ferricytochrome c reduction in the presence of Cd2+-activated NOS I, with and without calmodulin, were the same, confirming that the activation of NOS I as measured by the L-citrulline assay by Cd2+ is negligible. In each case, whether calmodulin was present or not, SOD (30 U/ml) did not inhibit the reduction of ferricytochrome c. These data demonstrate that this reduction was not a consequence of O2, but resulted from direct reduction by NOS I, as shown previously (Pou et al. 1992
).
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DISCUSSION |
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The results described in this article indicate that Ni2+, Ba2+, and Mn2+ can activate NOS I, producing NO, and O2 (Figs. 1 and 3 and Table 1). In the presence of Ca2+, formation of L-citrulline from L-arginine was inhibited with the inclusion of Ni2+, Cd2+, or Mn2+ (Fig. 2). The increase in L-citrulline formation seen with the simultaneous inclusion of Ca2+ and Ba2+ was surprising. We are exploring this synergistic phenomenon in more detail. Of note, although the trend of activation is the same, the percent control values for NO generation and L-citrulline production differed by 10% for Ni2+ and Mn2+, with the values for Ba2+ differing by
20%. Because the trend of activation remained the same independent of the assay, these differences were disregarded.
In the absence of L-arginine, each divalent cationactivated NOS I was shown to reduce ferricytochrome c at rates comparable to Ca2+-activated NOS I (Table 2). Similarly, ferricytochrome c reduction by NOS I in the absence of calmodulin was 10-fold less than in its presence (Table 2), confirming an earlier report (Roman et al., 2000
). This finding supports the notion that the activity associated with these divalent cations is a result of these metal ions binding to calmodulin.
Depending on the divalent cation used to activate NOS, differing amounts of O2 were generated. This observation was similar to that seen with L-citrulline and NO production (Figs. 3 and 4). However, the disparity in NOS production of O2, NO cannot be overlooked. Several control experiments verified that the disparity in the EPR spectral peak heights correlated with variable amounts of NOS-generated O2, and that these differing EPR spectral peak heights were not attributed to other reactions taking place in the reaction mixture, such as variable SOD-like activity of the metal ions or an enhanced rate of BMPO-OOH decomposition of the BMPO-OOH in the presence of the various divalent cations.
We offer several possible explanations for the differences in L-citrulline, NO, and O2 production by these metal ions. It has been suggested that cations with a specific range of radii are required to induce the necessary conformational change for calmodulin activity (Ozawa et al., 1999). Therefore, a plot of NOS I activation versus ionic radii was developed (Fig. 3). This plot confirms that the initial rates of NO generation and total L-citrulline production follow the same trend, and that a specific range of radii is necessary for maximal activation with calmodulin. Divalent cations smaller or larger than Ca2+ exhibit decreases in activation. In contrast, no activation was observed using Cd2+ with ionic radii of 1.09 Å (Huheey et al., 1993
), and previous work observed that Sr2+-activated NOS I was similar to Ca2+-activated NOS I, although Sr2+(1.32 Å) has a similar ionic radius to Pb2+ (1.33 Å), which showed minimal activation, (Huheey et al., 1993
; Weaver et al., 2002
). These findings suggest that other factors are involved for calmodulin activation of NOS I. In addition, Cd2+ has been shown to activate other calmodulin-dependent processes, although NOS I activation was not observed in these studies (Habermann et al., 1993
; Ozawa et al., 1999
).
A review of hard-soft acid-base (HSAB) concepts may also provide insight into the activation of NOS I by these various divalent cations (Huheey et al., 1993). Metal ions most frequently bind to donor ligands according to preferences dictated by HSAB. Mainly, hard metals prefer hard ligands, and soft metals prefer soft ligands. Calmodulin binds to Ca2+ via side-chain carboxylates, alcohol, or carboxamide oxygen, or via backbone carbonyl groups (Lippard and Berg 1994
). These ligands are classified as hard ligands and prefer to bind to hard metals such as Ca2+, Ba2+, and Mn2+, and they do not bind favorably with Ni2+, classified as a soft metal. The decrease in NOS I activation by Ni2+ supports this theory, whereas Mn2+ and Ba2+ exhibited near maximal activation compared to Ca2+. However, the biphasic curves observed with Mn2+ and Ni2+ suggest that still other factors are involved.
Other studies have supported our observations in which divalent cations can promote or inhibit NOS activity from homogenate (Gupta et al., 2000; Mittal et al., 1995
; Yamazaki et al., 1995
), purified (Perry and Marletta, 1998
), or recombinant (Palumbo et al., 2001
) NOSs as well as NO generated from macrophages (Tian and Lawrence, 1996
). For instance, Ni2+ has also been shown to enhance NOS I activity in the brain and adrenal glands (Gupta et al., 2000
), whereas it has been shown to inhibit NOS I activity in other studies (Mittal et al., 1995
; Palumbo et al., 2001
). Several mechanisms have been suggested for this activation and inhibition of NOS (Gupta et al., 2000
; Palumbo et al., 2001
), but the results shown here suggest that Ni2+ and other divalent cations can bind to calmodulin in a manner similar to Ca2+, thereby activating NOS directly. This finding appears to be concentration specific, as higher concentrations of several metals ions did not promote activation (Fig. 1). At higher concentrations of these divalent cations, further alterations in calmodulin conformation may prevent further activation. Our results are also in agreement with studies that show that high concentrations of specific metals ions in the presence of Ca2+ promote NOS inhibition (Mittal et al., 1995
; Palumbo et al., 2001
), possibly by further altering calmodulin, as mentioned above. Cd2+ appears not to produce the desired conformational change of calmodulin for NOS activation in the absence of Ca2+, and it may only promote changes in calmodulin not suitable for NOS activation, as only inhibition was observed.
As described by Gupta et al. (2000), Ni2+ can be accumulated in the brain after exposure. Although it is unclear whether the concentration accumulated in the brain would be sufficient to apply to our results, it is noteworthy. Among the many toxic effects of Ni2+ in biological systems is the ability of this divalent cation to affect Ca2+ homeostasis, produce low but measurable levels of other free radicals, and participate with NO in Ni2+-induced hyperglycemia (Denkhaus and Salnikow, 2002
; Gupta et al., 2000
). Also, NOS I is present in non-neuronal cell types in addition to the brain and other neuronal cells types [for a review, see Förstermann et al. (1998)
]. Accumulation of Ni2+ or other divalent cations in such tissues may elicit the response observed in this study. Of note, these results reflect stimulation or inhibition from the total concentration of metal salts added. The free ion concentrations would need to be determined to correlate these findings to possible concentrations of these metal ions in vivo.
The toxic effects of Ba2+ include gastrointestinal, cardiovascular, respiratory, and neuromuscular symptoms, and it directly affects vascular, cardiac, and gastrointestinal smooth muscle (Jacobs et al., 2002). Hypertension has also been reported in both laboratory and clinical studies of Ba2+ (Jacobs et al., 2002
). Excessive Mn2+ intake can result in neurobehavioral deficits, neurological syndrome similar to chronic Parkinson's disease, and oxidative stress (Chetty et al., 2001
; Husain et al., 2001
). Cadmium ion has been associated with hypertension and myocarditis among its many toxic effects (Kopp et al., 1983
; Thun et al., 1989
). We suggest that toxicity related to Cd2+ is the result of its ability to inhibit NOS I catalysis, whereas other metal ions may exert toxicity by either producing or inhibiting free radical production dependent on the concentration of metal ion present.
Results reported here should be interpreted with caution because the free ion concentration under the conditions presented is not known. It also cannot be stated that these divalent cations reach concentrations sufficient for calmodulin activation in biological systems as described in this paper. These results merely suggest a model by which several divalent cations may affect NOS I activity. Because NO is involved in many physiological processes (Moncada and Higgs, 1993), toxic effects could be attributed to the ability of these divalent cations to affect NOS I activity as demonstrated in this paper.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 725 W. Lombard Street, Baltimore, MD 21201. Fax: (410) 7068184. E-mail: weaver{at}gl.umbc.edu.
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