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Tetrahydrobiopterin Protects against Guanabenz-Mediated Inhibition of Neuronal Nitric-Oxide Synthase in Vitro and in Vivo

Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan

(Received February 22, 2006; Accepted May 22, 2006)

	Abstract

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It is established that guanabenz inhibits neuronal nitric-oxide (NO) synthase (nNOS) and causes the enhanced proteasomal degradation of nNOS in vivo. Although the time- and NADPH-dependent inhibition of nNOS has been reported in studies where guanabenz was incubated with crude cytosolic preparations of nNOS, the exact mechanism for inhibition is not known. Moreover, even less is known about how the inhibition of nNOS triggers its proteasomal degradation. In the current study, we show, with the use of purified nNOS, that guanabenz treatment leads to the oxidation of tetrahydrobiopterin and formation of a pterin-depleted nNOS, which is not able to form NO. With the use of ¹⁴C-labeled guanabenz, we were unable to detect any guanabenz metabolites or guanabenz-nNOS adducts, indicating that reactive intermediates of guanabenz probably do not play a role in the inhibition. Superoxide dismutase, however, prevents the guanabenz-mediated oxidation of tetrahydrobiopterin and inhibition of nNOS, suggesting the role of superoxide as an intermediate. Studies in rats show that administration of tetrahydrobiopterin prevents the inhibition and loss of penile nNOS due to guanabenz, indicating that the loss of tetrahydrobiopterin plays a major role in the effects of guanabenz in vivo. Our findings are consistent with the destabilization and enhanced degradation of nNOS found after tetrahydrobiopterin depletion. These studies suggest that drug-mediated destabilization and subsequent enhanced degradation of protein targets will likely be an important toxicological consideration.

We wondered how the time-dependent inhibition of nNOS renders the enzyme susceptible for degradation. Although guanabenz is well characterized with respect to degradation of nNOS in cells and in rats, relatively little is known about how guanabenz inhibits nNOS. In the current study, we chose to address this question with the use of purified nNOS in the hope of understanding what dysfunctional forms of nNOS are recognized for degradation. We found that guanabenz causes a tetrahydrobiopterin (BH₄)-deficient state of nNOS because of the oxidative destruction of the pterin that is facilitated by the presence of NADPH. The addition of BH₄ completely reactivates this dysfunctional form of the enzyme. The administration of BH₄ to rats completely protects from guanabenz-mediated inhibition of nNOS as well as the loss of nNOS protein, suggesting that the pterin deficiency plays a major role in the in vivo effects of guanabenz on nNOS.

	Materials and Methods

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Materials. Guanabenz was purchased from Research Biochemicals International (Natick, MA). Glucose 6-phosphate, glucose 6-phosphate dehydrogenase, NADP⁺, N^G-nitro-L-arginine, L-arginine, D-arginine, dihydropteridine reductase from sheep liver, calmodulin, catalase, superoxide dismutase, and NADPH were purchased from Sigma Aldrich (St. Louis, MO). Male Wistar rats were purchased from Charles River Laboratories (Wilmington, MA). (6R)-5,6,7,8-Tetrahydro-L-biopterin (BH₄) was purchased from Dr. Schirk's Laboratory (Jona, Switzerland). The affinity-purified rabbit IgG against brain NOS used for immunoblotting nNOS was from BD Biosciences Transduction Laboratories (Lexington, KY). [Benzylidene carbon ¹⁴C]-labeled guanabenz (56 mCi/mmol) was custom-synthesized by Du Pont NEN (Boston, MA). L-[¹⁴C(U)]-Arginine (330.0 mCi/mmol) and ¹²⁵I-labeled antibody against rabbit IgG were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA).

Methods. In vitro inhibition assays. For studies on the inactivation of purified nNOS, we overexpressed the enzyme in insect cells and purified the nNOS as described previously (Billecke et al., 2004). Purified nNOS (80 µg/ml) was added to a "first reaction mixture" of 40 mM potassium phosphate, pH 7.4, containing 0.2 mM CaCl₂, 2500 units/ml superoxide dismutase, 1250 units/ml catalase, 20 µg/ml pure calmodulin, 0.23 mg/ml bovine serum albumin, and an NADPH-regenerating system composed of 0.4 mM NADP⁺, 10 mM glucose 6-phosphate, and 1 unit of glucose 6-phosphate dehydrogenase/ml, expressed as final concentrations, in a total volume of 180 µl. After incubation at 30°C, aliquots (10 µl) of the first reaction mixture were transferred to an "oxyhemoglobin assay mixture" containing 200 µM CaCl₂, 250 µM L-arginine, 100 units/ml catalase, 10 µg/ml crude calmodulin, 25 µM oxyhemoglobin, and the NADPH-regenerating system, in a total volume of 180 µl of 40 mM potassium phosphate, pH 7.4. The oxyhemoglobin assay mixture was incubated at 37°C, and the rate of NO-mediated oxidation of oxyhemoglobin was monitored by measuring the absorbance at 401 to 411 nm with a microtiter plate reader (SpectraMax Plus; Molecular Devices Corp., Sunnyvale, CA). The rate was determined from the linear portion of the time-dependent changes in absorbance. In studies where endothelial NOS was used, the enzyme was overexpressed and purified as described previously (Lowe et al., 2005). The assay conditions were the same as nNOS, except that 200 µg/ml of the endothelial NOS was used in the first reaction mixture and 20-µl aliquots were taken for the oxyhemoglobin assay mixture.

SDS-resistant dimer analysis. In studies where the SDS-resistant dimer was measured, we examined the samples by low temperature SDS-PAGE (Klatt et al., 1995). nNOS forms a very tight dimer that is resistant to SDS at low temperatures. By keeping the samples on ice and running the SDS-PAGE with a cooling unit, the stable dimeric species can be visualized. In these studies, an aliquot (10 µl) of the first reaction mixture containing purified nNOS was quenched with an equal volume of sample buffer containing 5% SDS, 20% glycerol, 100 mM dithiothreitol, 200 µM L-arginine, and 0.02% bromphenol blue in 125 mM Tris-HCl, pH 6.8. The samples were kept on ice, and 10 µl of the quenched sample was loaded for analysis by 6% SDS-PAGE. Proteins were then transferred to nitrocellulose membranes (0.2 µm; Bio-Rad, Hercules, CA) and probed with 0.1% anti-nNOS. The immunoblots were then incubated a second time with ¹²⁵I-conjugated goat anti-rabbit IgGs to visualize the immunoreactive bands. The membranes were dried and exposed to X-OMAT film (Kodak, Rochester, NY) for 1 h at –80°C. The nitrocellulose bands corresponding to nNOS were excised and the radioactivity quantified by the use of a gamma counter.

HPLC analysis. The alteration of the heme prosthetic group was measured by HPLC similar to that described previously (Jianmongkol et al., 2000). HPLC was performed with the use of a Waters 600S controller, 717 plus autosampler, and 996 photodiode array detector (Waters Corp., Milford, MA). Samples were injected onto a reverse phase HPLC column (C4 Vydac, 5 µm, 0.21 x 15 cm; Grace Vydac, Hesperia, CA) equilibrated with solvent A (0.1% trifluoroacetic acid) at a flow rate of 0.3 ml/min. A linear gradient to 75 and 100% solvent B (0.1% trifluoroacetic acid in acetonitrile) was run over 30 and 5 min, respectively. Absorbance at 220 and 400 nm was monitored.

Quantification of BH₄ and BH₂. The amounts of BH₄ and 7,8-dihydrobiopterin (BH₂) in the reaction mixtures were determined by use of an HPLC fluorescence method as described by Klatt et al. (1996). The method involves oxidization of BH₄ and BH₂ to biopterin by treatment with KI/I₂ solution under acidic conditions. To give the specific amount of BH₂, the KI/I₂ oxidation is done in a basic solution where BH₄ and BH₂ are oxidized to pterin and biopterin, respectively. Specifically for oxidation under acidic conditions, a 40-µl aliquot of the first reaction mixture was treated with 10 mM I₂ and 50 mM KI in a total volume of 50 µl of 100 mM HCl for 1 h at room temperature in the dark. The solution was neutralized with 5 µl of 1.0 M NaOH, and then 5 µl of 0.2 M ascorbate was added. An aliquot (30 µl) of the resulting solution was injected onto a reverse phase HPLC column (C18 Vydac, 5 mm, 4.6 x 250 mm) equilibrated with 20 mM NaH₂PO₄, pH 3, with 5% methanol at a flow rate of 1 ml/min. The pterins were eluted with the same mobile phase and detected by fluorescence at excitation and emission wavelengths of 350 and 418 nm, respectively. The HPLC and analysis of pterins was performed with the use of a Waters systems described above and an Applied Biosystems Spectroflow 980 fluorescence detector (Applied Biosystems, Foster City, CA). To oxidize the pterins under basic conditions, the first reaction mixture was treated as above except that 100 mM NaOH replaced 100 mM HCl, and the final solution was neutralized with 5 µl of 1 M HCl.

Treatment of nNOS with [¹⁴C]guanabenz. The purified nNOS was treated as described above in a first reaction mixture, except that nNOS (1.5 µM) was treated with 50 µM guanabenz (56 mCi/mmol) for 60 min at 22°C. An aliquot (75 µl) was injected onto a reverse phase HPLC column (C4 Vydac, 5 µm, 2.1 x 150 mm) equilibrated with solvent A (0.1% trifluoroacetic acid) at a flow rate of 0.3 ml/min. A linear gradient to 75% solvent B (0.1% trifluoroacetic acid in acetonitrile) was run over 45 min, and then a linear gradient to 100% B was run over the next 5 min. The absorbance at 220 nm was monitored. The radioactivity in the eluent was measured by an on-line radiochemical detector (Radiomatic 500TR; Packard, Downers Grove, IL).

Treatment of animals, sample preparation, and activity assays. Guanabenz was dissolved in physiological saline and administrated to male Wistar rats (150–250 g) at the indicated doses by intraperitoneal injection at 9:00 AM and 6:00 PM. BH₄ was dissolved in 0.1% (w/v) ascorbic acid in physiological saline and injected in a total volume of 1 ml, 30 min before the injection of guanabenz. The controls were given the appropriate volumes of physiological saline or 0.1% (w/v) ascorbic acid in physiological saline. Rats were sacrificed by decapitation 16 h after the last injection. Whole deskinned penis was removed, washed with ice-cold physiological saline, cut into 1- to 2-mm pieces, and homogenized in 1 ml of ice-cold homogenization buffer (10 mM HEPES, pH 7.5, containing 320 mM sucrose, 100 µM EDTA, 1.5 mM dithiothreitol, 10 µg/ml trypsin inhibitor, 10 mg/ml leupeptin, 2 µg/ml aprotinin, 1 mg/ml phenylmethanesulphonyl fluoride, and 100 µM BH₄) with the use of a metal tissue mincer (SDT Tissumizer; Tekmar, Cincinnati, OH). The homogenate was centrifuged at 245,000g for 10 min at 4°C. The supernatant fraction was collected and frozen in liquid nitrogen and stored at –80°C for later analysis. Protein concentration of these samples was determined by the method of Bradford (Bio-Rad), with the use of bovine serum albumin as a standard.

The NOS activity of samples from the in vivo studies were determined by adding the supernatant fraction (0.6 mg) to a "citrulline assay mixture" containing 1 mM CaCl₂, 1 mM NADPH, 30 µM [¹⁴C]arginine (60 mCi/mmol), 100 µM BH₄, and 10 µg/ml calmodulin in a total volume of 200 µl of 40 mM potassium phosphate, pH 7.4. The assay mixture was incubated at 37°C for 10 min, and the amount of [¹⁴C]citrulline was determined as described previously (Nakatsuka et al., 1998). The formation of [¹⁴C]citrulline was linear over the 10-min period. For experiments on the in vitro inactivation of cytosolic NOS, the supernatant fraction from untreated rats was loaded onto a Sephadex G-25 M column (PD-10; Pharmacia Biotech, Piscataway, NJ) preequilibrated in 10 mM HEPES, pH 7.5, containing 320 mM sucrose, 100 µM EDTA, 1.5 mM dithiothreitol, 10 µg/ml trypsin inhibitor, 10 mg/ml leupeptin, 2 µg/ml aprotinin, and 1 mg/ml phenylmethanesulphonyl fluoride to remove endogenous arginine and excess BH₄. An aliquot (1.2 mg) of the gel-filtered fraction was placed in a "reaction mixture" containing 1 mM CaCl₂, 1 mM NADPH, 10 µg/ml calmodulin, and the desired concentration of guanabenz, in a total volume of 1 ml of 40 mM potassium phosphate, pH 7.4. Aliquots (150 µl) were taken from the reaction mixture and placed in the citrulline assay mixture, and the activity was determined as described above.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Guanabenz-mediated inactivation of purified nNOS. A, time-dependent loss of nNOS activity as a result of guanabenz on a semi-log plot. Closed squares, treated with 100 µM guanabenz; open squares, untreated. The inactivation of nNOS activity was determined with the use of the modified first reaction mixture and oxyhemoglobin assay mixture as described in Materials and Methods. The half-life for each condition was calculated by least-squares fitting of the semi-log plot. *, NOS activity was significantly (p < 0.05) lower for the guanabenz-treated sample than that for control. B, the effect of varying the concentration of guanabenz in the first reaction mixture. The amount of guanabenz is indicated, and the activities were measured after 20 min of incubation. Inset, the endothelial NOS replaced nNOS, and the effects of guanabenz were determined. *, significantly (p < 0.05) different from untreated. The values are the mean ± S.E. (n = 3).

In vitro ubiquitylation of guanabenz-treated nNOS. We used an in vitro ubiquitylation system containing fraction II that has been established to ubiquitylate nNOS by an ATP-dependent process (Kamada et al., 2005). The specific detection of nNOS-ubiquitin conjugates has also been established (Kamada et al., 2005). Fraction II was prepared from rabbit reticulocyte lysates as described previously (Hershko et al., 1983). The nNOS was treated with 100 µM guanabenz as described above, and an aliquot (160 µl) of this first reaction mixture was incubated at 37°C in a total volume of 400 µl of 50 mM Tris-HCl, pH 7.4, containing 2 mM dithiothreitol, 15 µM ubiquitin, an ATP-regenerating system (2 mM ATP, 10 mM creatine phosphate, 5 mM MgCl₂, and 10 units/ml creatine phosphokinase), and 0.4 mg/ml fraction II. An aliquot (20 µl) of the samples was quenched with 20 µl of sample buffer containing 5% SDS, 20% glycerol, 100 mM dithiothreitol, and 0.02% bromphenol blue in 125 mM Tris-HCl, pH 6.8. The samples were boiled for 5 min, and an aliquot (30 µl) was submitted to 6% SDS-PAGE (10 x 8 cm). Proteins were then transferred to nitrocellulose membranes (0.2 µm, Bio-Rad) and probed with 0.5% anti-ubiquitin (DAKO, Carpinteria, CA). The immunoblots were then incubated a second time with ¹²⁵I-conjugated goat anti-rabbit IgGs to visualize the immunoreactive bands. The membranes were dried and exposed to X-OMAT film for 1 h at –80°C. The bands corresponding to nNOS-ubiquitin were excised and the radioactivity quantified by the use of a gamma counter.

Statistical analysis. All values are reported as the mean ± standard error (S.E.). An unpaired t test was used to compare values. We considered statistical significance to be achieved at a level of p < 0.05. PRISM statistical software (GraphPad, San Diego, CA) was used for analysis of the data sets.

	Results

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Guanabenz-Mediated Inhibition of Purified nNOS. It is established that guanabenz inhibits nNOS and enhances the proteolytic turnover of nNOS protein in cells (Noguchi et al., 2000). Consistent with this, administration of guanabenz to rats decreases nNOS activity and protein (Nakatsuka et al., 1998; Dambrova et al., 2003). Moreover, the time-dependent inhibition of nNOS due to guanabenz has been characterized in in vitro studies with the use of penile cytosol (Nakatsuka et al., 1998). To better understand the mechanism of how guanabenz inhibits nNOS and causes the enhanced turnover of the enzyme, we chose to conduct studies with purified nNOS. We established here, for the first time, that guanabenz causes a time-dependent inhibition of purified nNOS (Fig. 1A, closed squares). There is a loss of activity even in the absence of guanabenz (open squares), albeit slower, representing an autoinactivation reaction. The half-life of the activity loss was 5.0 ± 1.8 and 22.1 ± 6.9 min for the guanabenz-treated and untreated samples, respectively. These values are statistically different (p < 0.05). The loss of nNOS activity beyond this autoinactivation is dependent on the concentration of guanabenz (Fig. 1B). Taken together, these results are highly similar to those found when the nNOS in penile cytosol was treated with guanabenz (Nakatsuka et al., 1998). Moreover, guanabenz did not inhibit endothelial NOS, suggesting that the action of guanabenz is selective (Fig. 1B, inset). The autoinactivation of nNOS in the absence of substrate or guanabenz is time-, calmodulin-, and NADPH-dependent (Demady et al., 2001). The autoinactivation could be due to alteration of critical amino acid residues, the prosthetic heme group, or tetrahydrobiopterin (Demady et al., 2001).

The time-dependent inhibition of activity is thought to produce a dysfunctional, altered form of nNOS that is preferentially ubiquitylated and proteasomally degraded (Noguchi et al., 2000). Recently, it was found that destabilization of the dimeric functional form of nNOS correlates with recognition for proteasomal degradation (Dunbar et al., 2004). Thus, we asked if guanabenz destabilizes nNOS dimers. As shown in Fig. 2A, the untreated nNOS exists in part as SDS-resistant dimers, which are visualized after low-temperature SDS-PAGE and subsequent immunoblotting (Klatt et al., 1995). We quantified the bands corresponding to the dimer and monomer from these studies (Fig. 2B). This assay is not a measure of the dimeric content under native conditions but is a measure of the amount of stable dimer that is not dissociated by SDS and, thus, underestimates the total dimeric content. Moreover, it is likely that the transfer efficiency of the dimer is lower than that of the monomer, further leading to the underestimation of dimeric content. Thus, we cannot determine the absolute amounts of each form, but we can determine the relative changes in dimer and monomer.

View larger version (36K): [in this window] [in a new window]   FIG. 2. Effect of guanabenz on the amount of SDS-resistant dimer of nNOS. The nNOS was treated with guanabenz as described under Materials and Methods, and the amount of the SDS-resistant dimer (nNOS dimer) and the remainder of the nNOS that runs as a monomer (nNOS monomer) were measured. A, Western blot of the reaction mixture of untreated nNOS (Untreated) or nNOS treated with guanabenz (Guanabenz) for 0, 15, 30, and 45 min. B, the bands corresponding to the nNOS dimer and monomer in A were quantified by the use of 125I-labeled goat anti-rabbit IgG. Circles, untreated; squares, guanabenz-treated. The dimeric nNOS is represented by the closed symbols and the solid lines, and the monomeric nNOS is represented by the open symbols and the dashed lines. *, significantly (p < 0.05) lower dimer for guanabenz-treated versus untreated. #, significantly (p < 0.05) higher monomer for guanabenz-treated over the untreated. C, the effect of varying the concentration of guanabenz on the amount of SDS-resistant dimer after treatment for 20 min. The bands were quantified as in B. Solid triangles, nNOS dimer; open triangles, monomeric nNOS. *, significantly (p < 0.05) lower dimer than untreated. #, significantly (p < 0.05) higher monomer than untreated. The values are the mean ± S.E. (n = 3).

As shown in Fig. 2B, we found that the SDS-resistant dimeric form of the untreated nNOS is unstable (closed circles), probably reflecting an autoinactivation reaction. Treatment with guanabenz further destabilizes the dimeric nNOS (closed squares) and gives an increase in the monomeric nNOS (open squares). As shown in Fig. 2C, the destabilization of the dimeric nNOS (closed triangles) and the formation of monomeric nNOS (open triangles) is dependent on the concentration of guanabenz. Both the time dependence and concentration dependence of the loss of dimeric nNOS reflect the loss of nNOS activity seen above. These results are entirely consistent with the notion that destabilization of the dimer by guanabenz generates some altered nNOS form that is more susceptible for proteasomal degradation. We next sought to determine how guanabenz destabilizes the nNOS dimer.

As shown in Fig. 3A, we first determined the cofactor dependence of the inhibition by treating nNOS under the indicated conditions for 20 min in the presence (open bars) or absence (closed bars) of 100 µM guanabenz. The greatest decrease in activity due to guanabenz occurs when both calmodulin and NADPH are present. Under these conditions, the L-isomer, but not the D-isomer, of arginine protects from the inhibition, suggesting an active site-directed process. These findings are similar to those found for nNOS in penile cytosol (Nakatsuka et al., 1998). However, unlike the previous observations with crude cytosol, approximately half the activity is lost even when purified nNOS is treated with guanabenz in the absence of exogenous NADPH. Even under this condition, calmodulin is necessary for guanabenz-mediated inhibition of nNOS (data not shown). We wondered why NADPH was necessary for inhibition of the crude cytosolic preparation of nNOS (Nakatsuka et al., 1998) but not for the purified enzyme. To better understand the inactivation process and why there are seemingly disparate findings, we further investigated both the mechanism of inactivation without NADPH as well as that found with NADPH. In the course of our studies, we discovered that the concentration of BH₄ in the first reaction mixture is a critical factor and that the presence of NADPH, superoxide dismutase, and catalase also has an effect on the guanabenz-mediated inhibition of nNOS. To dissect these effects, we initially investigated the effect of BH₄ on the guanabenz-mediated inhibition of nNOS in the absence of superoxide dismutase and catalase (Fig. 3B). Under these conditions, guanabenz in the presence of NADPH causes a nearly complete inactivation of nNOS, and the addition of BH₄ during the treatment has a protective effect (solid circles). Interestingly, NADPH alone has a large inhibitory effect, but in this case, the addition of even small amounts of BH₄ protects from the loss of activity (compare solid squares with open squares). Low levels of BH₄ also protect the enzyme when nNOS is treated with guanabenz in the absence of NADPH (open circles). Overall, greater inhibition of nNOS is observed when NADPH and guanabenz are present over that when NADPH is omitted (compare solid circles with open circles). This clearly demonstrates how NADPH-dependent inactivation of nNOS could be observed depending on the BH₄ concentration, and this probably explains the seemingly disparate observation made in a previous study on NADPH dependence (Nakatsuka et al., 1998).

View larger version (21K): [in this window] [in a new window]   FIG. 3. Effect of substrate, NADPH, tetrahydrobiopterin, and calmodulin on the guanabenz-mediated inactivation of nNOS. A, the effect of calmodulin (CAM), NADPH, L-arginine (L-Arg), and D-arginine (D-Arg) on the extent of inhibition of nNOS after treatment for 20 min with 100 µM guanabenz (open bars) or untreated (solid bars). B, the protective effect of BH4 on the inhibition of nNOS caused by guanabenz when superoxide dismutase and catalase were omitted from the first reaction mixture described under Materials and Methods. nNOS was either treated with 100 µM guanabenz (circles) or untreated (squares). Open symbols, NADPH was omitted from the first reaction mixture; closed symbols, NADPH was present in the first reaction mixture. C, the protective effect of BH4 on the extent of inhibition of purified nNOS caused by 100 µM guanabenz (closed triangles), 10 µM NG-nitro-L-arginine (X), or untreated (open triangles) in the first reaction mixture. This mixture contained superoxide dismutase, catalase, and NADPH. The values are the mean ± S.E. (n = 3). *, significantly (p < 0.05) lower activity for treated versus untreated.

As shown in Fig. 3C, BH₄ completely protects the enzyme from inactivation when nNOS is treated with guanabenz and NADPH in the presence of superoxide dismutase and catalase (compare solid triangles with open triangles). In addition, the presence of superoxide dismutase and catalase protects against the autoinactivation of nNOS that occurs in the presence of NADPH. As a control, we show that BH₄ does not affect the inhibition of nNOS because of N^G-nitro-L-arginine, a slowly reversible active site-directed inhibitor (X). It is noteworthy that N^G-nitro-L-arginine stabilizes the SDS-resistant dimeric form of nNOS (Dunbar et al., 2004). Thus, guanabenz seems to inhibit nNOS by a process that is antagonized by BH₄. We will show below that a BH₄-deficient enzyme is formed.

Tetrahydrobiopterin Depletion As a Mechanism for Guanabenz-Mediated Inhibition of Purified nNOS. Initially, we investigated the guanabenz-mediated inhibition of nNOS in the absence of NADPH. As shown in Fig. 4A, the calmodulin- and guanabenz-dependent loss of activity occurs concomitantly with the loss of BH₄. Approximately 60% of the loss of BH₄ is accounted for by the formation of BH₂. As shown in Fig. 4B, the activity loss seen when nNOS is incubated with calmodulin and guanabenz in the absence of NADPH is completely reversed by the addition of BH₄ in the oxyhemoglobin assay mixture. A concentration of 0.1 µM BH₄ is sufficient for complete reversal, consistent with the approximate loss of BH₄. The restoration of activity is rapid, since there is no incubation step with BH₄ before the activity is measured. The restoration is nearly complete, suggesting that the depletion of BH₄ is the major mechanism for the activity loss under these conditions. It is noteworthy that in previous studies with the cytosolic fraction containing nNOS (Nakatsuka et al., 1998), BH₄ was present in the assay mixture; therefore, the NADPH-independent inhibition of nNOS by guanabenz would have been obscured. There is also a loss of BH₄ after inhibition of nNOS in the presence of NADPH (Fig. 4C). The addition of dihydropteridine reductase, which reduces BH₂ to BH₄, abolishes the inhibition of nNOS as a result of guanabenz (Fig. 4C), indicating that the oxidation of BH₄ to BH₂ and the subsequent formation of a BH₄-deficient nNOS is the cause of the nNOS inhibition. Consistent with this finding, BH₄ completely reverses the inhibition of nNOS even when NADPH is present (Fig. 4D). This is similar to that found above for the inhibition when NADPH was omitted. Thus, under all conditions examined here, BH₄ deficiency is the major mechanism of nNOS inhibition due to guanabenz.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Guanabenz causes the loss of nNOS activity and tetrahydrobiopterin. A, guanabenz causes the loss of BH4. The nNOS was treated with 100 µM guanabenz for 20 min in the first reaction mixture as indicated under Materials and Methods, except that NADPH was omitted. The amount of BH4 present in the first reaction mixture was measured by HPLC and compared with the nNOS activity. The presence of calmodulin and guanabenz are as indicated. B, the activity loss due to guanabenz is reversed by the addition of BH4 to the oxyhemoglobin assay mixture. The activity was measured by the oxyhemoglobin assay containing the indicated amounts of BH4. Closed squares, nNOS treated with guanabenz; open squares, untreated nNOS. C, the conditions were as in A, except that NADPH was present in the first reaction mixture. All samples contained calmodulin, and some samples contained 10 units/ml dihydropteridine reductase (DHPR) as indicated. D, the conditions were as in B, except that NADPH was present in the first reaction mixture. The values are the mean ± S.E. (n = 3). *, significantly (p < 0.05) lower values for guanabenz-treated versus untreated.

Superoxide Dismutase Prevents the Guanabenz-Mediated Loss of nNOS Activity and Tetrahydrobiopterin. A more detailed analysis of the dependence on catalase and SOD was performed (Fig. 5). As shown in Fig. 5A, superoxide dismutase protects against the loss of activity with nearly complete protection at 1000 units/ml (solid circles). Catalase at concentrations up to 400 units/ml has no effect (solid triangles). In a previous study, 100 units/ml catalase completely protected nNOS from the oxidative inactivation caused by agmatine (Demady et al., 2001). Superoxide dismutase alone is nearly as effective as superoxide dismutase in combination with catalase (solid squares). Thus, it seems that the nNOS-mediated superoxide formation is mainly responsible for the activity loss. Consistent with these findings, superoxide dismutase alone could completely protect against the guanabenz-mediated loss of BH₄ (Fig. 5B).

View larger version (17K): [in this window] [in a new window]   FIG. 5. Effect of superoxide dismutase on the guanabenz-mediated loss of nNOS activity and tetrahydrobiopterin. The nNOS was treated with guanabenz (100 µM) for 20 min, and the nNOS activity and pterin were measured as described under Materials and Methods. A, nNOS activity. The amount of superoxide dismutase (SOD) or catalase (CAT) or both were varied in the reaction mixtures treated with guanabenz. Closed squares, a combination of SOD and CAT were added; closed circles, SOD was added; closed triangles, CAT was added. As a control, SOD and CAT were added to a reaction mixture not treated with guanabenz (open squares). B, tetrahydrobiopterin. The 100 µM guanabenz (G), 2500 units/ml SOD, or 1 mM NADPH was omitted from the reaction mixtures as indicated. The values are the mean ± S.E. (n = 3). *, significantly (p < 0.05) lower values relative to untreated.

View larger version (30K): [in this window] [in a new window]   FIG. 6. HPLC profile of nNOS treated with 14C-labeled guanabenz in the absence or presence of calmodulin. The nNOS was treated with radiolabeled guanabenz as described under Materials and Methods. A, nNOS treated with guanabenz in the absence of calmodulin. B, nNOS treated with guanabenz in the presence of calmodulin. The residual activity was 85 and 38% for the untreated and guanabenz-treated samples, respectively. G, guanabenz; NOS, nNOS; CAM, calmodulin.

View larger version (19K): [in this window] [in a new window]   FIG. 7. Tetrahydrobiopterin protects against guanabenz-mediated inactivation and loss of penile nNOS in rats. A, the effect of guanabenz (5 mg/kg/day) and BH4 (200 mg/kg/day) on penile NOS activity (solid bars) and penile nNOS protein (open bars) after treatment of rats for 4 days. The treatment of the rats and the measurement of activity by the citrulline assay are as described under Materials and Methods. *, significantly (p < 0.05) lower values relative to untreated. B, dose response of BH4 on the penile nNOS activity under conditions of A. *, significantly (p < 0.05) higher activity relative to untreated. C, treatment of penile cytosol in vitro with guanabenz and BH4. The inactivation of nNOS activity was determined with the use of the reaction mixture and citrulline assay mixture as described under Materials and Methods. The reaction mixture was incubated for 15 min in the presence of the following: closed circles, 100 µM guanabenz; open circles, 5 µM NG-nitro-L-arginine; x, untreated. The indicated amounts of BH4 were added to the reaction mixture. The values are the mean ± S.E. (n = 3). *, significantly (p < 0.05) lower activity relative to untreated.

In Vitro Ubiquitylation of Guanabenz-Treated nNOS. As shown in Fig. 8, top panel, there is a ubiquitin conjugate that is readily visualized with anti-ubiquitin in the 160-kDa region. The identity of this band as a nNOS-ubiquitin conjugate has been previously established (Bender et al., 2000; Kamada et al., 2005). The band corresponding to nNOS-ubiquitin was quantified and plotted (Fig. 8, bottom panel). There is an increase in the nNOS-ubiquitin conjugates found for guanabenz-treated nNOS (lane 2) over that for untreated nNOS (lane 1). When MG132, an inhibitor of the proteasome, is not present, then the nNOS-ubiquitin conjugate due to guanabenz is greatly reduced (lane 4).

View larger version (27K): [in this window] [in a new window]   FIG. 8. Guanabenz-treated nNOS is labilized for ubiquitylation in an in vitro system containing fraction II. Purified nNOS was treated with 100 µM guanabenz (G) and then placed in a reaction mixture containing ubiquitin, ATP, and fraction II. The generation and detection of the nNOS-ubiquitin conjugates are as described under Materials and Methods. The values are the mean ± S.E. (n = 3). *, significantly (p < 0.05) higher nNOS-ubiquitin (Ub-nNOS) conjugates relative to untreated.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Superoxide dismutase, but not catalase, prevents the loss of BH₄ from purified nNOS treated with guanabenz, indicating that nNOS-derived superoxide is responsible for the loss of BH₄. The reaction of superoxide with tetrahydrobiopterin has been previously reported and shown to form BH₂ (Vasquez-Vivar et al., 2001). Dihydropteridine reductase, which reduces BH₂ to BH₄, completely protects against the guanabenz-mediated inhibition of nNOS activity and loss of BH₄, strongly suggesting that BH₄ is oxidized to BH₂. However, under in vivo conditions where L-arginine is present, it seems that other reactive metabolites such as peroxynitrite, which can form by the reaction of superoxide with NO, are more likely the actual agents responsible for BH₄ oxidation (Milstien and Katusic, 1999; Laursen et al., 2001; Kuzkaya et al., 2003).

In the case of nNOS, BH₄ is known to stabilize the native dimeric state of the enzyme, and thus the oxidation of the BH₄ would destabilize the dimeric form, consistent with our findings. This may be important since destabilization of the dimer has recently been shown to lead to the ubiquitylation of nNOS in vitro (Dunbar et al., 2004) and in cells (Kamada et al., 2005). This notion is furthered by the finding that stabilization of the dimeric form of nNOS by N^G-nitro-L-arginine or 7-nitroindazole protects nNOS from proteasomal degradation (Dunbar et al., 2004).

Although dimer stabilization plays an important role, the actual signal or recognition site for degradation is not clear. The trigger may be due to exposure of a site that is normally hidden in the active dimeric form of nNOS, a general unfolding of nNOS after perturbation of the BH₄ site, or exposure of hydrophobic residues in the heme-active site cleft (Peng et al., 2004). A recent report on the structure of a loose dimer of NOS with a partially exposed active center and destabilized subdomains is entirely consistent with this view (Pant and Crane, 2005). The cellular factors that recognize the dysfunctional altered nNOS are also not known. In this respect, we have recently found that CHIP (C terminus of Hsc-70 interacting protein), a chaperone-assisted E3 ubiquitin ligase, ubiquitylates nNOS in cells, as well as in an in vitro system containing purified E1 ubiquitin-activating enzyme, an E2 conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system (Peng et al., 2004). The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by, and possibly requires, its interaction with nNOS-bound hsp70. This raises the possibility that hsp70 directly mediates protein triage decisions by recognition of destabilized nNOS and recruiting ubiquitin ligase machinery that involves CHIP. It remains to be determined if the guanabenz-treated nNOS is preferentially recognized by the hsp70-based chaperones in this manner. However, we did demonstrate that guanabenz-inactivated nNOS was labilized for ubiquitylation in an in vitro system that contains a crude preparation of reticulocyte proteins, including hsp70.

The molecular mechanism by which dysfunctional forms of nNOS are recognized must be a fundamental biological process that maintains the quality of the nNOS protein in cells. We describe here how guanabenz may perturb this regulatory process to cause a prolonged decrease in nNOS activity and protein levels. The xenobiotic-mediated redox regulation of BH₄ may be important in understanding how chemicals inhibit and cause the loss of nNOS in vivo. The interactions of drug molecules in protein quality control will likely be an important pharmacological and toxicological consideration in the development of safer and more effective drugs.

	Footnotes

 
This work was supported in part by the National Institutes of Health Grants GM077430 and ES08365. E.R.L. is a trainee under Pharmacological Sciences Training Program GM07767 from the National Institutes of Health. A.Y.D. is a trainee under Minority Supplement to ES08365.

A.Y.D. and G.J.J. contributed equally to this work.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.009951.

ABBREVIATIONS: NOS, nitric-oxide synthase; nNOS, neuronal nitric oxide-synthase; BH₄, (6R)-5,6,7,8-tetrahydro-L-biopterin (tetrahydrobiopterin); NO, nitric oxide; PAGE, polyacrylamide gel electrophoresis; HPLC, high-performance liquid chromatography; BH₂, 7,8-dihydrobiopterin; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; KI, potassium iodide; CHIP, C terminus of Hsc-70 interacting protein; SOD, superoxide dismutase; CAT, catalase.

¹ Current affiliation: Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.

² Current affiliation: Department of OB/GYN, Okayama University Medical School, Okayama City, Japan.

³ Current affiliation: The Dow Chemical Company, Midland, Michigan.

Address correspondence to: Dr. Yoichi Osawa, Department of Pharmacology, University of Michigan Medical School, 1301 Medical Science Research Building III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0632. E-mail: osawa{at}umich.edu

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