Introduction

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The role of NO^· in mammalian physiology is an area of intense research, which has provided insights into many biological processes, including vascular tone and host immune response. Nitric oxide is synthesized via the oxidation of L-arginine by a family of enzymes, nitric-oxide synthases (NOS; EC 1.14.13.39) (Moncada and Higgs, 1993). Nitric-oxide synthases fall into two categories: 1) the constitutive forms, NOS I and NOS III, which are dependent upon the transient influx of Ca²⁺ to activate calmodulin and which bind to NOS to elicit enzyme activity and 2) a cytokine-inducible form, NOS II, by which calmodulin is permanently bound (Marletta, 1993). Nitric oxide produced by NOS I has been implicated in many physiological processes, including long-term potentiation (LTP), a model for learning and memory (Quinn and Harris, 1995; Ko and Kelly, 1999; Jacoby et al., 2001), long-term synaptic depression (Quinn and Harris, 1995), and the development of neuronal function (Quinn and Harris, 1995).

All NOS isoforms contain two prosthetic groups: FAD and FMN, located in the C-terminal reductase domain, and iron protoporphyrin IX (heme) and tetrahydrobiopterin (H₄B), located in the N-terminal oxidase domain, which also contains a binding site for L-arginine. The FAD and FMN are involved in electron storage and delivery, accepting two electrons from NADPH and then delivering single electrons to the heme iron, where O₂ is also required to form the catalytic intermediate that oxidizes L-arginine to L-citrulline and NO^· (Lane and Gross, 1999). To activate NOS I, Ca²⁺ has to bind to calmodulin, which then allows electron transport through the enzyme, resulting in the formation of NO^· from L-arginine (Abu-Soud et al., 1994, 2000; Matsuda and Iyanagi, 1999; Miller et al., 1999; Kobayashi et al., 2001).

Calmodulin, by binding, for example, to other divalent metal ions (Goldstein and Ar, 1983; Habermann et al., 1983), may initiate cellular toxicity by altering intracellular Ca²⁺ signaling required for a number of enzymatic functions (Kern et al., 2000). For instance, NOS I-derived NO^· has been associated with neurotransmission, cAMP metabolism, protein phosphorylation, and memory (Egrie et al., 1977; Bredt et al., 1992; Sandhir and Gill, 1994). Yet, little is known as to how divalent metal ions other than Ca²⁺, such as Pb²⁺, Sr²⁺, and Cd²⁺, impact NOS activity. For instance, it has been reported that Ni²⁺ inhibits NOS I (Perry and Marletta, 1998). Such antagonism appears to be bifunctional, inhibiting the binding of L-arginine and interfering with the ability of Ca²⁺/calmodulin to transport electrons from the reductase domain to the oxidase domain (Palumbo et al., 2001). Similarly, Pb²⁺ has been shown to displace Ca²⁺ from Ca²⁺-binding proteins (Fullmer et al., 1985). The consequences of these events have, however, not been well defined. Strontium ions have also been shown to replace Ca²⁺ in many physiological processes, including the relaxation of smooth muscle by the production of NO^· (Ohashi et al., 1995; Yamazaki et al., 1995) and activation of phosphodiesterase by calmodulin (Cox et al., 1981; Hoch and Wilson, 1984). In particular, how would replacement of Ca²⁺ by other divalent metal ions alter the physiological function of various Ca²⁺-dependent cell-signaling pathways?

In the present study, we investigated the effect divalent metal ions have on NOS I-mediated oxidation of L-arginine to L-citrulline and NO^·. We demonstrate that Pb²⁺ and Sr²⁺ can activate NOS I to metabolize L-arginine to L-citrulline and NO^·, presumably by binding to calmodulin. Furthermore, in the absence of L-arginine, these divalent cations can activate NOS I to generate O, as has been demonstrated for Ca²⁺-activated NOS I (Pou et al., 1999). Biological consequences of unregulated production of NO^·, O, and other free radicals derived from these reactive species are discussed.

	Materials and Methods

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Reagents. L-Arginine, NADPH, calcium chloride, catalase, and calmodulin were obtained from Sigma-Aldrich (St. Louis, MO). Strontium chloride and lead carbonate were obtained from Mallinckrodt Baker, Inc. (Phillipsburg, NJ). Superoxide dismutase was obtained from Roche Diagnostics (Mannheim, Germany). H₄B was obtained from Schircks Laboratories (Jona, Switzerland). 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 (Zhao et al., 2001). Dowex 50W-X8 cation exchange resin was obtained from Bio-Rad (Hercules, CA). All other chemicals were used as purchased without further purification.

Synthesis of Lead Nitrate. Lead carbonate was dissolved in water until the solution became oversaturated. Concentrated nitric acid was added dropwise with stirring until the solution became clear. This reaction was allowed to stir for 1 h. Afterward, ethanol was added to make a 50:50 water/ethanol solution, and the mixture was allowed to cool at 20°C. The precipitate, lead nitrate, was filtered and allowed to dry overnight.

Purification of NOS I. NOS I was expressed and purified essentially as described by Roman et al. (1995), with the modification that the culture volume was 500 rather than 1000 ml. The enzyme concentration was determined by its CO-difference spectrum, as described in Roman et al. (1995), using an extinction coefficient of 100 mM¹ cm¹ at 444 to 475 nm.

NOS I Activity by [¹⁴C]L-Citrulline Formation Assay. The enzymatic activity of purified NOS I was determined by its ability to catalyze the formation of L-citrulline from L-arginine as previously reported (Pou et al., 1999), with modifications. The reaction mixture contained purified NOS I (4.97 µg) and cocktail solution [[¹⁴C]L-arginine (0.6 µCi/ml) in the presence of NADPH (1 mM), L-arginine (50 µM), and calmodulin (100 U/ml) in HEPES buffer (50 mM), 0.5 mM EGTA, pH 7.4]. The reaction was started by the addition of the cocktail solution into the reaction mixture containing purified NOS I and either CaCl₂, SrCl₂, or Pb(NO₃)₂ (various concentrations as presented under Results). The reaction mixture was incubated at 23°C 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 NaOH solution (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 determined using a hemoglobin assay (Murphy and Noack, 1994). The reaction was initiated by the addition of NOS I (7.38 µg) to a cuvette containing HEPES buffer (50 mM, 0.5 mM EGTA, pH 7.4), oxyhemoglobin (8 µM), CaCl₂, SrCl₂, or Pb(NO₃)₂ (0.5 mM), calmodulin (100 U/ml), NADPH (100 µM), H₄B (10 µM), and L-arginine (100 µM) to a final volume of 1 ml at 23°C. A UV-visible 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 mM¹ cm¹ at ₄₀₁.

Spin Trapping/EPR Spectroscopy. Spin trapping experiments with purified NOS I were conducted by mixing all components described in each figure legend to a final volume of 0.3 ml. The reaction mixture was then transferred to a flat quartz cell and placed into the cavity of an EPR spectrometer (model E-109; Varian Medical Systems, Inc., Palo Alto, CA). EPR spectra were recorded at room temperature after the reaction was initiated. 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 is given in each figure legend.

	Results

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Effect of Metal Ions on Calmodulin-Dependent NOS I. NOS I activity was determined by measuring the amount of [¹⁴C]L-citrulline formed during enzymatic oxidation of [¹⁴C]L-arginine in the presence of several metal ions. NOS I activity increased with increasing concentrations of Ca²⁺, as shown in Fig. 1, with a half-maximal activity at 0.1 mM. Of particular interest was the finding that Pb²⁺ in the absence of Ca²⁺ activated NOS I with a half-maximal concentration of 0.4 mM (Fig. 2).

View larger version (0K): [in this window] [in a new window]   Fig. 1.   Effect of Ca2+ on the activity of NOS I. The formation of [14C]L-citrulline from the NOS oxidation of L-arginine. NOS I activity was assayed in the reaction containing NOS I (4.97 µg), NADPH (1 mM), CaCl2 (various concentrations), calmodulin (100 U/ml), [14C]L-arginine (0.6 µCi/ml), and L-arginine (50 µM) in HEPES buffer (50 mM, 0.5 mM EGTA, pH 7.4). Each point represents the mean ± S.D. from six independent experiments on the same preparation of purified NOS I.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effect of Pb2+ on the activity of NOS I. The formation of [14C]L-citrulline from the NOS oxidation of L-arginine. NOS I activity was assayed in the reaction containing NOS I (4.97 µg, NADPH (1 mM), Pb(NO3)2 (various concentrations), calmodulin (100 U/ml), [14C]L-arginine (0.6 µCi/ml), and L-arginine (50 µM) in HEPES buffer (50 mM, 0.5 mM EGTA, pH 7.4). Each point represents the mean ± S.D. from six independent experiments on the same preparation of purified NOS I.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effect of Sr2+ on the activity of NOS I. The formation of [14C]L-citrulline from the NOS oxidation of L-arginine. NOS I activity was assayed in the reaction containing NOS I (4.97 µg), NADPH (1 mM), SrCl2 (various concentrations), calmodulin (100 U/ml), [14C]L-arginine (0.6 µCi/ml) and L-arginine (50 µM) in HEPES buffer (50 mM, 0.5 mM EGTA, pH 7.4). Each point represents the mean ± S.D. from six independent experiments on the same preparation of purified NOS I.

Determination of NO^· Production by NOS I. The rate of NOS I generation of NO^· was estimated spectrophotometrically using the oxyhemoglobin method as described under Materials and Methods. NOS I production of NO^· was observed with all three metal ions investigated, using concentrations that maximize production of L-citrulline. The rate of NO^· production catalyzed by the competent NOS I in the presence of Pb²⁺ was considerably less than that observed with Ca²⁺ (Table 1). However, the rate of NO^· production catalyzed by the addition of Sr²⁺ was similar to that observed with Ca²⁺ (Table 1). As a control, Pb(NO₃)₂ (0.5 mM) was added to a competent NOS I in the absence of L-arginine to assure that the source of NO^· was from NOS-dependent oxidation of L-arginine and not from NO. In this case, NO^· was not detected using the oxyhemoglobin assay. To ensure that Pb(NO₃)₂ does not interfere with the oxidation of oxyhemoglobin or react with NO^·, the reaction between NO^· and oxyhemoglobin in the absence and presence of Pb(NO₃)₂ was observed at 576 and 401 nm (Murphy and Noack, 1994). There was no change in the absorbance between these two experimental conditions (data not shown).

Spin Trapping of NOS-Generated O. We have previously demonstrated that a competent NOS I produces O in the absence of L-arginine and that the site of free radical production is the heme (Pou et al., 1999). Therefore, we investigated whether the substitution of various divalent cations in place of Ca²⁺ would likewise result in O production. As depicted in Fig. 4, O was spin trapped by BMPO, independent of the divalent metal ion. Of particular interest was the finding that Ca²⁺, Pb²⁺, and Sr²⁺ were similarly efficient in the NOS-dependent production of O.

View larger version (0K): [in this window] [in a new window]   Fig. 4.   Spin trapping of O from purified NOS I by BMPO. Typical EPR spectra for the reaction of BMPO with of O-generated NOS I in the absence of L-arginine. A, the reaction system consisted of NOS I (14.91 µg), CaCl2 (0.5 mM), calmodulin (100 U/ml), NADPH (1 mM), and BMPO (50 mM) in phosphate buffer (50 mM, pH 7.4) and 1 mM each diethylenetriaminepentaacetic acid and EGTA. B, same conditions as in A except that Pb(NO3)2 (0.5 mM) was added in place of CaCl2. C, same conditions as in A except that SrCl2 (0.5 mM) was added in place of CaCl2. EPR spectra were recorded 10 min after initiation of the reaction. Receiver gain was 10 × 104.

	Discussion

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Calmodulin contains four EF-hand domains, each of which is capable of binding Ca²⁺. Full activation of calmodulin occurs when Ca²⁺ occupies all four sites (Kern et al., 2000). The binding of Ca²⁺ to calmodulin produces a conformational change that converts the protein to an active form. Calmodulin binds to NOS I, allowing electron transport through the reductase domain of NOS to the oxidase domain and the oxidation of L-arginine to L-citrulline and NO^·. Data from other laboratories demonstrate that Pb²⁺ binds to calmodulin with a higher affinity than it does to Ca²⁺ and promotes the activation of calmodulin (Habermann et al., 1983; Bressler and Goldstein, 1991). More recent studies have shown that Pb²⁺ exhibits a biphasic effect on calmodulin, i.e., Pb²⁺ is able to activate calmodulin at low concentrations, whereas at high concentrations of Pb²⁺, the activity of the target enzyme activated by calmodulin binding is markedly decreased (Sandhir and Gill, 1994). Studies have also shown Sr²⁺ to be an activator of calmodulin with respect to phosphodiesterase activity but with a potency lower than that of Pb²⁺ or Ca²⁺ (Habermann et al., 1983). As NOS I is Ca²⁺/calmodulin-dependent, we explored the effect of various divalent cations on NOS I activity.

The results presented in this study indicate that Pb²⁺ and Sr²⁺ can act as surrogates for Ca²⁺ in the activation of NOS I via calmodulin binding. The results exhibit a biphasic effect for Pb²⁺ in the absence of Ca²⁺. Of note, inhibition of NOS I was not observed with combinations of Ca²⁺ (0.125 and 0.15 mM) and Pb²⁺ (0.25-1.0 mM), but repression of enzyme activity was observed using Pb²⁺ concentrations above 0.5 mM in the absence of Ca²⁺ or in the presence of 0.1 mM Ca²⁺. However, we demonstrate that in the absence of Ca²⁺, Sr²⁺ can activate NOS I in a similar dose-dependent fashion. Within the concentration range studied using Sr²⁺, there was no inhibition in NOS I activity.

Although NOS I oxidation of L-arginine was shown to afford L-citrulline, independent verification that Pb²⁺ and Sr²⁺ generated NO^· was performed. The oxyhemoglobin assay for NO^· revealed that NOS I activated either by Ca²⁺, Pb²⁺, or Sr²⁺ generates NO^· during the metabolism of L-arginine (Table 1). A 2-fold decrease in the initial rate of NO^· production by Pb²⁺-activated NOS I was observed compared with Ca²⁺- and Sr²⁺-activated NOS I, which parallels the formation of L-citrulline from L-arginine (Figs. 1-3). The reason for this observation, although noteworthy, is unknown and is currently being investigated, as our primary focus was to ensure that NO^· was produced. Similarly, Pb²⁺ and Sr²⁺ activated NOS I in the absence of L-arginine to generate O (Fig. 4) as previously shown using Ca²⁺-stimulated NOS I (Pou et al., 1999). Again, these experiments were performed merely to confirm that O was produced in the presence of the various metal ions.

There are reports that show NO^· to be a retrograde messenger, mediating LTP in the hippocampus, initiating long-term synaptic depression, and impacting early brain development (Quinn and Harris, 1995). Additional studies have found that environmental toxicants may exert their toxicity through modulation of NO^· production (Blazka et al., 1994; Chamulitrat et al., 1995; Laskin, 1996). Lead ions are one of the more common environmental heavy metal ion toxicants. Although the mechanism of its toxicity remains unclear, the primary site of lead toxicity is the central nervous system (Sandhir and Gill, 1994). Exposure to Pb²⁺ is associated with neurobehavioral and psychological alterations, including the inhibition of LTP (Shukla and Singhal, 1984; Quinn and Harris, 1995). Although no in vivo data on Pb²⁺ toxicity associated with NO^·-induced LTP have been studied, the ability of Pb²⁺ to activate NOS and generate NO^· cannot be overlooked.

Intracellular Ca²⁺ in cells is tightly controlled through Ca²⁺ channels, thereby regulating NOS I activity (Clapham, 1995). Independent studies have shown that the permeability of these channels is at least 10 times more accessible to Pb²⁺ than Ca²⁺ and Sr²⁺ (Simons and Pocock, 1987). Therefore, since these metal ions can activate calmodulin-dependent enzymes (Habermann et al., 1983; Sandhir and Gill, 1994), one of the important mechanisms that limit NOS I activity is no longer available. This may result in uncontrolled generation of NO^· and O. The average resting intracellular free Ca²⁺ concentration in most cells is about 50 to 100 nM, high enough for at least partial activation of some calmodulin-sensitive enzymes, such as the plasma membrane Ca²⁺-ATPase (Kern et al., 2000). If this concentration of Ca²⁺ were sufficient to partially activate NOS I, the presence of trace amounts of Pb²⁺ would result in increased activation of this enzyme. Finally, Pb²⁺ may affect a wide variety of physiological functions by altering cellular concentrations of NO^·.

The standard elevated blood lead level to cause lead poisoning for adults set by the Center for Disease Control and Prevention is 25 µg/dl (or 1.25 µM) of whole blood, whereas this level is considerably lower for children at 10 µg/dl (or 0.5 µM) of blood (Center for Disease Control and Prevention, 1997). The exposure to high levels of Pb²⁺ from the environment can exert toxic effects on the central nervous system and can be fatal. Low levels of exposure can result in persistent central nervous system impairments, such as learning disabilities, as well as developmental and behavioral problems (Finkelstein et al., 1998). One can hypothesize that Pb²⁺ in the central nervous system can activate NOS I by binding to calmodulin, uninfluenced by the cellular flux of Ca²⁺. This, of course, may result in a variety of toxic events that are often associated with the presence of free radicals (Sandhir and Gill, 1994). Enhanced NOS I activity, following exposure to Pb²⁺, might lead to spontaneous neurotransmitter release, as NO^· is known as a neurotransmitter in the brain and periphery associated with the release of other neurotransmitters (Cooper et al., 1984; Bredt and Synder, 1989; Garthwaite et al., 1989).

The present study demonstrates that Pb²⁺ and Sr²⁺ activate NOS I to produce NO^· and O by substituting for Ca²⁺ in the activation of calmodulin. The ability of these metal ions to efficiently stimulate NOS I may provide some insight into the mechanism of divalent cation-induced toxicity.