The Effect of Divalent Cations on Neuronal Nitric Oxide Synthase Activity

John Weaver*,{dagger},||,1, Supatra Porasuphatana{ddagger}, Pei Tsai{dagger},||, Guan-Liang Cao{dagger}, Theodore A. Budzichowski*, Linda J. Roman§ and Gerald M. Rosen{dagger},||

* Department of Chemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250; {dagger} Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201; {ddagger} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuronal nitric oxide synthase (NOS I) is a Ca2+/calmodulin–binding enzyme that generates nitric oxide (NO•) and L-citrulline from the oxidation of L-arginine, and superoxide (O2) from the one-electron reduction of oxygen (O2). Nitric oxide in particular has been implicated in many physiological processes, including vasodilator tone, hypertension, and the development and properties of neuronal function. Unlike Ca2+, which is tightly regulated in the cell, many other divalent cations are unfettered and can compete for the four Ca2+ binding sites on calmodulin. The results presented in this article survey the effects of various divalent metal ions on NOS I–mediated catalysis. As in the case of Ca2+, we demonstrate that Ni2+, Ba2+, and Mn2+ can activate NOS I to metabolize L-arginine to L-citrulline and NO•, and afford O2 in the absence of L-arginine. In contrast, Cd2+ did not activate NOS I to produce either NO• or O2, and the combination of Ca2+ and either Cd2+, Ni2+, or Mn2+ inhibited enzyme activity. These interactions may initiate cellular toxicity by negatively affecting NOS I activity through production of NO•, O2 and products derived from these free radicals.

Key Words: nitric oxide; superoxide; NOS I; calmodulin; divalent cations; metal toxicity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuronal nitric oxide synthase (NOS I) belongs to a family of enzymes, nitric oxide synthases (NOS, EC 1.14.13.39), known to catalyze the conversion of L-arginine to nitric oxide (NO•) and L-citrulline (Moncada and Higgs, 1993Go; Nathan and Xie, 1994Go). NOS I and endothelial NOS (NOS III) are constitutive isoforms dependent on the transient influx of Ca2+ to activate calmodulin, which binds to the isoform to elicit enzyme activity. A third isoform, inducible NOS (NOS II), is a cytokine-inducible isoform independent of Ca2+ in which calmodulin is permanently bound (Nathan and Xie, 1994Go). These enzymes are composed of an N-terminal oxidase domain containing an iron protoporphyrin IX (heme), tetrahydrobiopterin (H4B) with a binding site for L-arginine, and a C-terminal reductase domain containing the flavin adenine dinucleotide (FAD) and the flavin adenine mononucleotide (FMN). The two domains are connected by a calmodulin-binding motif to which a transient influx of Ca2+ binds (Abu-Soud et al., 1994Go; Kobayash et al., 2001Go; Matsuda and Iyanagi, 1999Go; Miller et al., 1999Go). Besides producing NO•, NOS also generates superoxide (O2), the ratio of these free radicals is dependent on the concentration of L-arginine (Pou et al., 1999Go; Yoneyama et al., 2001Go). Nitric oxide from NOS I has been implicated in many physiological processes, including vasodilator tone and the development and properties of neuronal function (Moncada and Higgs, 1993Go; Roskams et al., 1994Go). In addition, NOS I has been associated with hypertension (Chrissobolis et al., 2002Go).

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., 1999Go; Yoneyama et al., 2001Go) and H2O2 are directly produced; the ratio of these reduction products of O2 is set by the presence of H4B (Rosen et al., 2002Go). 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., 1999Go). 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)Go]. 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., 2002Go). Unlike Ca2+, which is tightly regulated in the cell (Clapham, 1995Go), 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, 1983Go; Habermann et al., 1993Go; Mills and Johnson, 1985Go), 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., 2000Go; Mittal et al., 1995Go; Palumbo et al., 2001Go; Perry and Marletta, 1998Go; Yamazaki et al., 1995Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. L-arginine, NADPH, calcium chloride, nickel chloride, magnesium chloride, catalase, xanthine oxidase, ferricytochrome c (bovine), hypoxanthine, and calmodulin were obtained from Sigma-Aldrich (St. Louis, MO). Cadmium nitrate was obtained from Allied Chemical (Morristown, NJ). Potassium chloride was obtained from Mallinckrodt Baker, Inc. (Phillipsburg, NJ). Manganese chloride, potassium nitrate, and barium chloride were obtained from Fisher Scientific (Fair Lawn, NJ). Superoxide dismutase (SOD) was obtained from Roche Diagnostics (Mannheim, W-Germany). L-[U-14C]arginine monohydrochloride ([14C]L-arginine) was purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). 5-tert-Butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) was synthesized as described in the literature (Stolze et al., 2003Go; Tsai et al., 2003Go). Dowex 50W-X8 cation exchange resin was obtained from Bio-Rad (Hercules, CA). All other chemicals were used as purchased without further purification. Buffers used contain less than 0.01% of inorganic salts, including Ca2+.

Purification of NOS I. NOS I was expressed and purified essentially as described in the literature (Roman et al., 1995Go), 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)Go, using an extinction coefficient of 100 mM–1cm–1 at {Delta}{varepsilon} 444–475 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., 2002Go), 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, 1994Go). 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 mM–1cm–1 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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FIG. 4. Spin trapping of O2 from purified NOS I by BMPO. A representative plot of the first low-field peak height, arrows in the insert, of the EPR spectrum of BMPO-OOH from O2 generated from divalent cation-activated NOS I in the absence of L-arginine. This nitroxide is derived from the reaction of O2 with BMPO. Each point represents the mean ± S.D. of the % control from three independent experiments on the same preparation of purified NOS I. Insert – The EPR spectrum of BMPO-OOH was obtained 10 min after the spin trapping of O2 generated Ca2+-activated NOS I in the absence of L-arginine. The reaction system consisted of NOS I (14.2 µg), CaCl2 (500 µM), calmodulin (100 U/ml), NADPH (100 µM), and BMPO (50 mM) in phosphate buffer (50 mM, pH 7.4, 1 mM DTPA, 1 mM EGTA). Receiver gain was 5 x 104.

 
Generation of O2 by hypoxanthine/xanthine oxidase. Superoxide was generated from the action of xanthine oxidase on hypoxanthine (400 µM, final concentration) in sodium phosphate buffer (50 mM, pH 7.4, 1 mM DTPA, 1 mM EGTA; Chelexed, pH 7.4). The initial rate of O2 generation was estimated spectrophotometrically by measuring the SOD-inhibitive reduction of ferricytochrome c (80 µM) at 550 nm using an extinction coefficient of 21 mM–1 cm–1 at 550 nm (Kuthan and Ullrich, 1982Go).

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 mM–1 cm–1 at 550 nm.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of Divalent Metal Ions on Calmodulin-Dependent Activation of Purified NOS I
Several divalent cations have been shown to bind and activate calmodulin (Goldstein and Ar, 1983Go; Habermann et al., 1993Go; Mills and Johnson, 1985Go). Therefore, we investigated what effect Ba2+, Mn2+, Ni2+, and Cd2+, known to affect other calmodulin processes, may have on the activation of calmodulin-dependent NOS I. NOS I activation was estimated by measuring the amount of [14C]L-citrulline produced from L-arginine (100 µM) to which [14C]L-arginine (0.6 µCi/ml) was added in the presence of the different divalent cations, as described in Materials and Methods. Maximal activation for each divalent cation, except for Mn2+, was reached at ~500 µM (Fig. 1). Ca2+- and Ba2+-activated NOS I showed similar dose-response curves, where in up to 1 mM of each divalent cation salt there was no apparent inhibition in NOS I production of L-citrulline (Fig. 1). Of note, at maximal activation Ba2+-activated NOS I was ~70% that of Ca2+-activated NOS I. In contrast, NOS I activation by Ni2+ and Mn2+ exhibited biphasic response curves (Fig. 1) for each metal ion salt, but at concentrations up to 1 mM, NOS I activation decreased by ~90% and ~60%, respectively. At maximal response, Ni2+ and Mn2+ produced ~40% and ~70%, respectively, of L-citrulline as compared to Ca2+-stimulated NOS I (Fig. 1). Interestingly, Cd2+ demonstrated negligible NOS I activation (~9%), even at 500 µM. From these data, it was determined that 500 µM of each divalent cation would produce respective maximal activation under these experimental conditions, and this concentration was used for further experiments.



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FIG. 1. Effect of divalent cations on the formation of L-citrulline by NOS I. The formation of [14C]L-citrulline (represented as % control with respect to Ca2+-activated NOS I) from the NOS oxidation of L-arginine. NOS I activity was assayed in the reaction containing NOS I (3.7 µg), NADPH (1 mM), calmodulin (100 U/ml), [14C]L-arginine (0.6 µCi/ml), L-arginine (100 µM) and ({blacksquare}) CaCl2, ({blacktriangleup}) BaCl2, (x) MnCl2, (•) NiCl2, or ({diamondsuit}) Cd(NO3)2 (various concentrations) in HEPES buffer (50 mM, 0.5 mM EGTA, pH 7.4). Each point represents the mean ± S.D. from three independent experiments on the same preparation of purified NOS I.

 
Next, we examined the effect of various divalent cations on Ca2+-activated NOS I. The reactions contained all components described in Materials and Methods, except that the divalent cation, Cd2+, Ni2+, Ba2+, or Mn2+ (500 µM), was included along with Ca2+ (500 µM). Activation of NOS I was estimated following the formation of [14C]L-citrulline from [14C]L-arginine (0.6 µCi/ml) in the presence of L-arginine (100 µM). The combination of Ca2+ and either Cd2+, Ni2+, or Mn2+ resulted in a diminution in enzyme activation by ~90%, ~85%, and ~40%, respectively, as compared to Ca2+ alone (Fig. 2). Surprisingly, the combination of Ba2+ and Ca2+ nearly doubled NOS I response, indicating a synergistic effect of Ba2+ on Ca2+-activated NOS I.



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FIG. 2. Effect of divalent cations on the activation of NOS I in the presence of Ca2+. The formation of [14C]L-citrulline from the NOS oxidation of L-arginine. NOS I activity was assayed in the reaction containing NOS I (3.7 µg), NADPH (1 mM), calmodulin (100 U/ml), [14C]L-arginine (0.6 µCi/ml), L-arginine (100 µM), CaCl2 (500 µM) and either BaCl2 (500 µM), MnCl2 (500 µM), NiCl2 (500 µM), or Cd(NO3)2 (500 µM) in HEPES buffer (50 mM, 0.5 mM EGTA, pH 7.4). Each point represents the mean ± S.D. of the % control from three independent experiments on the same preparation of purified NOS I.

 
Initial Rate of NO• Production by Divalent Cation-Activated NOS I
While the above experiments determined the effect of various divalent cations on NOS-mediated oxidation of L-arginine to L-citrulline and, by implication, NO•, we measured the initial rate of NO• production from NOS I, activated either by Ca2+, Ni2+, Ba2+, Mn2+, or Cd2+. Initial rates of NO• generation were estimated using the oxyhemoglobin assay, as described in Materials and Methods. For these experiments, the concentration of L-arginine was set at 100 µM, whereas the concentration of Ca2+, Ni2+, Ba2+, Mn2+, or Cd2+ was fixed at 500 µM. The initial rate of NO• production catalyzed by Ca2+ or Ba2+ was approximately the same, at ~370 nmoles/min/mg protein (Table 1). In the presence of Cd2+, no measurable rate of NO• generation was observed, as compared to the control, in the absence of calmodulin.


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TABLE 1 Initial Rates of NO• Generation from NOS I Induced by Various Divalent Cations

 
A correlation between the initial rates of NO• generation and production of L-citrulline was established by converting the respective values to percent control with respect to Ca2+-activated NOS I (Table 1 and Fig. 3). The percent control values show that the initial rates of NO• production are comparable to the L-citrulline assay, each following the same trend of activation in which Mn2+ and Ni2+ produced the least amount of NO•.



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FIG. 3. A plot of NOS I activation versus ionic radii of the divalent cations. The formation of [14C]L-citrulline (•) by the [14C]L-citrulline formation assay and NO• ({blacksquare}) production by the hemoglobin method from the NOS I oxidation of L-arginine with respect to the ionic radii of each divalent cation (500 µM) (Huheey et al. 1993Go). Each point represents the mean ± S.D. of the % control with respect to Ca2+-activated NOS I of three independent experiments on the same preparation of purified NOS I.

 
Control experiments were also performed to ensure that the counter ions, Cl or NO3, did not contribute to the observed results (Nishimura et al., 1999Go; Schrammel et al., 1998Go). Substituting KCl (500 µM) or KNO3 (500 µM) for the divalent salts resulted in no activation of NOS I in the absence of Ca2+ or inhibition in the presence of Ca2+ (500 µM) (data not shown). It was concluded that any effect these counter ions have on NOS I activity is negligible in this study, and all observations were a consequence of the respective divalent cations. These data also further confirmed that Cd2+ does not activate NOS I.

Spin Trapping of NOS-Generated O2
NOS I produces O2 in the absence of L-arginine (Pou et al., 1992Go; Yoneyama et al., 2001Go). 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. 2003Go), 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. 2002Go). 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 Cation–Activated NOS I
As reported in the literature, ferricytochrome c is reduced by NOS I (Heinzel et al., 1992Go; Mayer et al., 1991Go; Pou et al., 1992Go; Roman et al., 2000Go; Sheta et al., 1994Go). 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., 2000Go). 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 cation–activated 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. 1992Go).


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TABLE 2 Reduction Rates of Ferricytochrome c by NOS I

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of calmodulin occurs when Ca2+ occupies all four EF-hand domains (Kern et al., 2000Go). The binding of Ca2+ to calmodulin produces a conformational change that converts the protein to an active form, which binds to NOS I, allowing electron transport through the reductase domain of NOS to the oxidase domain to facilitate the production of NO•, O2, and H2O2. Several divalent and trivalent cations besides Ca2+ can bind to calmodulin and exert cellular toxicity by altering the normal homeostasis of the Ca2+/calmodulin pathway (Habermann et al., 1993Go; Mills and Johnson, 1985Go; Ozawa et al., 1999Go). We explored the effect Ni2+, Ba2+, Cd2+, or Mn2+ has on calmodulin-dependent NOS I-generated NO• and O2.

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 cation–activated 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., 2000Go). 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., 1999Go). 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., 1993Go), 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., 1993Go; Weaver et al., 2002Go). 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., 1993Go; Ozawa et al., 1999Go).

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., 1993Go). 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 1994Go). 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., 2000Go; Mittal et al., 1995Go; Yamazaki et al., 1995Go), purified (Perry and Marletta, 1998Go), or recombinant (Palumbo et al., 2001Go) NOSs as well as NO• generated from macrophages (Tian and Lawrence, 1996Go). For instance, Ni2+ has also been shown to enhance NOS I activity in the brain and adrenal glands (Gupta et al., 2000Go), whereas it has been shown to inhibit NOS I activity in other studies (Mittal et al., 1995Go; Palumbo et al., 2001Go). Several mechanisms have been suggested for this activation and inhibition of NOS (Gupta et al., 2000Go; Palumbo et al., 2001Go), 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., 1995Go; Palumbo et al., 2001Go), 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)Go, 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, 2002Go; Gupta et al., 2000Go). 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)Go]. 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., 2002Go). Hypertension has also been reported in both laboratory and clinical studies of Ba2+ (Jacobs et al., 2002Go). Excessive Mn2+ intake can result in neurobehavioral deficits, neurological syndrome similar to chronic Parkinson's disease, and oxidative stress (Chetty et al., 2001Go; Husain et al., 2001Go). Cadmium ion has been associated with hypertension and myocarditis among its many toxic effects (Kopp et al., 1983Go; Thun et al., 1989Go). 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, 1993Go), toxic effects could be attributed to the ability of these divalent cations to affect NOS I activity as demonstrated in this paper.


    ACKNOWLEDGMENTS
 
This research was supported in part by grants from the National Institutes of Health, EB-2034 (G.M.R., J.W.), R25-GM55036 (J.W.), AG-20445 (P.T.) and GM52419, and from the Robert A. Welch Foundation, grant AQ1192 (L.J.R.).


    NOTES
 

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) 706–8184. E-mail: weaver{at}gl.umbc.edu.


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