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Originally published In Press as doi:10.1074/jbc.M511803200 on March 9, 2006

J. Biol. Chem., Vol. 281, Issue 18, 12546-12554, May 5, 2006
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Characterization of the Mechanism of Cytochrome P450 Reductase-Cytochrome P450-mediated Nitric Oxide and Nitrosothiol Generation from Organic Nitrates*

Haitao Li1, Xiaoping Liu, Hongmei Cui, Yeong-Renn Chen, Arturo J. Cardounel, and Jay L. Zweier2

From the Center for Biomedical EPR Spectroscopy and Imaging, Davis Heart and Lung Research Institute, and the Division of Cardiovascular Medicine, Department of Internal Medicine, Ohio State University College of Medicine, Columbus, Ohio 43210-1252

Received for publication, November 2, 2005 , and in revised form, March 2, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian cytochrome P450 reductase (CPR) and cytochrome P450 (CP) play important roles in organic nitrate bioactivation; however, the mechanism by which they convert organic nitrate to NO remains unknown. Questions remain regarding the initial precursor of NO that serves to link organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of CPR-CP-mediated organic nitrate bioactivation, EPR, chemiluminescence NO analyzer, NO electrode, and immunoassay studies were performed. With rat hepatic microsomes or purified CPR, the presence of NADPH triggered organic nitrate reduction to Formula. The CPR flavin site inhibitor diphenyleneiodonium inhibited this Formula generation, whereas the CP inhibitor clotrimazole did not. However, clotrimazole greatly inhibited Formula-dependent NO generation. Therefore, CPR catalyzes organic nitrate reduction, producing nitrite, whereas CP can mediate further nitrite reduction to NO. Nitrite-dependent NO generation contributed <10% of the CPR-CP-mediated NO generation from organic nitrates; thus, Formula is not the main precursor of NO. CPR-CP-mediated NO generation was largely thiol-dependent. Studies suggested that organic nitrite (R-O-NO) was produced from organic nitrate reduction by CPR. Further reaction of organic nitrite with free or microsome-associated thiols led to NO or nitrosothiol generation and thus stimulated the activation of sGC. Thus, organic nitrite is the initial product in the process of CRP-CP-mediated organic nitrate activation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Organic nitrates (R-O-NO2) such as glyceryl trinitrate (GTN)3 and isosorbide dinitrate (ISDN) have an important role in cardiovascular therapy. Over the last decade, this role has been attributed to their enzymatic metabolism to produce nitric oxide (NO) (1-4). However, the mechanism of organic nitrate bioactivation to produce NO has not been elucidated. Numerous reports have shown that the mammalian cytochrome P450 reductase (CPR)-cytochrome P450 (CP) enzyme system plays an important role in the process of organic nitrate biotransformation (5-10). However, questions remain regarding the enzymatic mechanism by which CPR and CP transform organic nitrate to NO. The chemical process of organic nitrate conversion to NO and nitrosothiols that links organic nitrates to the activation of soluble guanylyl cyclase (sGC) remains unknown.

Mammalian CPR was first identified in 1950 (11). CPR is a flavoprotein containing both FAD and FMN (11, 12) and is situated on the endoplasmic reticulum (microsomes) (13, 14). Previous studies have shown that CPR plays important roles in organic nitrate biotransformation. The flavoprotein inhibitor diphenyleneiodonium (DPI) greatly inhibits the metabolic activation of GTN or ISDN through inhibition of CPR (8, 9, 15). However, the enzymatic mechanism of CPR-mediated organic nitrate reduction has not been elucidated. To date, no research has been done to assess NO formation in the process of CPR-mediated organic nitrate biotransformation. It is not certain whether CPR can directly catalyze the reduction of organic nitrate to produce NO or to produce nitrite anion (Formula) or other species as intermediates that may be further transformed to NO or S-nitrosothiols. Therefore, the enzymatic mechanism of NO and S-nitrosothiol generation remains unclear in the process of organic nitrate bioactivation.

CPR is the electron donor for several oxygenase enzymes, including CP, a family of hemeproteins involved in the metabolism of many drugs and dietary substances and in the synthesis of steroid hormones and other extracellular lipid signaling molecules. It has been reported that the activation of sGC by GTN can be inhibited by inhibitors of CP and suggested that at least a portion of the vascular biotransformation of GTN is mediated by CP (16-19), but other studies have shown that classical inhibitors of CP or hemeproteins have no effect on GTN-induced vasodilation (16, 20-22). Therefore, controversy remains regarding whether CP can mediate organic nitrate biotransformation to NO, and the mechanism of this biotransformation process remains unknown.

Although numerous studies have shown that the CPR-CP system plays an important role in the process of bioactivation of organic nitrate, controversy still exists over which of these two enzymes are involved and how they interact in the mechanism by which NO and nitrosothiols are generated. This information is of particular importance to understand the cellular mechanisms by which these compounds are metabolized and bioactivated in vivo as well as the process by which tolerance occurs, rendering these compounds less active as vasodilators.

To characterize the enzymatic mechanism of CPR-CP-catalyzed organic nitrate reduction and to elucidate the precise molecular mechanism of organic nitrate bioactivation, EPR, chemiluminescence NO analyzer, NO electrode, and immunoassay studies were performed under anaerobic conditions to characterize the initial product, the precursor of NO and nitrosothiols, and to determine the link between organic nitrate and sGC activation. This study demonstrates that CPR catalyzes the bioactivation of organic nitrate through reduction to form the intermediate organic nitrite, which is converted to NO and nitrosothiols in a thiol-dependent reaction.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—DPI chloride, sodium nitrite, Griess reagent, beta-NADPH, beta-NADH, cytochrome P450 2B4, NADPH P450 reductase (human recombinant), a cytochrome c reductase activity assay kit, and a protein concentration assay kit were obtained from Sigma. sGC was obtained from Alexis Biochemicals (San Diego, CA). A direct cGMP assay kit was obtained from Assay Designs (Ann Arbor, MI), and cGMP production was quantified by immunoassay according to the protocol provided by the company. Anti-cytochrome P450 reductase antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). GTN was synthesized as described (23). N-Methyl-D-glucamine dithiocarbamate (MGD) was synthesized using carbon disulfide and N-methyl-D-glucamine (24, 25). Ferrous ammonium sulfate was purchased from Aldrich (99.997%). Dulbecco's phosphate-buffered saline (PBS) was obtained from Invitrogen. Millipore Ultrafree centrifugal filters (nominal Mr limit of 10,000) were obtained from Fisher.

Preparation of Rat Liver Microsomes—Microsomal fractions from rat liver were prepared by standard differential centrifugation techniques. Liver microsomes were prepared from individual male Wistar rats (250-300 g) (8, 26). Rat liver microsomes (100,000 x g pellet) were resuspended in 50 mM potassium phosphate buffer (pH 7.0). Protein concentrations were determined by a modification of the Lowry method using the Sigma protein concentration assay kit. CPR activity was measured by following the NADPH-dependent reduction of cytochrome c using a Sigma CPR activity assay kit. Reaction rates were calculated using an extinction coefficient for ferrous cytochrome c of 21 mM-1 cm-1 at 550 nm. Microsomes (3.6 g of protein) were prepared from 10 rats (175 g of livers) and stored at -80 °C in PBS at a concentration of ~10 mg/ml protein (~1.1 ± 0.2 units/ml CPR; 1 unit will cause the reduction of 1.0 µmol of cytochrome c by NADPH/min at pH 7.0 at 37 °C).

EPR Spectroscopy—EPR measurements were performed using a Bruker EMX spectrometer with an HS resonator operating at X-band. Measurements were performed at ambient temperature with a modulation frequency of 100 kHz, a modulation amplitude of 2.5 G, and a microwave power of 20 milliwatts. NO generated from the reaction solution was purged out using argon to a vessel containing 5 ml of the Fe2+·MGD spin-trap solution as described previously (27). This setup was designed to isolate the reaction solution from the spin trap and thus avoid any possible perturbation caused by Fe2+·MGD complexes in the reaction system. Fe2+·MGD complexes were prepared by adding solid ferrous ammonium sulfate and MGD (1:5 molar ratio) to the deoxygenated (argon-purged) solution with a final concentration of 2 mM iron. Fe3+·MGD complexes were prepared by mixing ferric chloride and aerobic MGD solution (1:5 molar ratio) with a final concentration 2 mM iron. Quantitation of NO formation and trapping was performed by double integration of the observed EPR signal compared with that from the similar aqueous NO·Fe2+·MGD standard (27).

Chemiluminescence Measurements—The rate of the NO production was measured using a Sievers 270B nitric oxide analyzer interfaced through a DT2821 A/D board to a personal computer. In the analyzer, NO is reacted with ozone, forming excited-state NO2, which emits light. Mixing of reagents and separation of NO from the reaction mixture were done at controlled temperature in a glass purging vessel equipped with heating jacket. The release of NO was quantified by analysis of the digitally recorded signal from the photomultiplier tube using specially designed data acquisition and analysis software developed in our laboratory (28). After an initial 60-120 s of equilibration of flow from the purging vessel to the detector, the signal provides the rate of NO formation over time.

As described previously (29), nitrite in the solution is measured from its reduction to NO under conditions of acidic pH in the presence of glacial acetic acid and 1% KI. This method has been shown to be highly sensitive, enabling detection of nitrite down to picomole amounts. Neither GTN nor ISDN was found to generate any detectable NO under these conditions. Calibration of the magnitude of nitrite and NO production was determined from the integral of the signal over time compared with that from nitrite concentration standards (29, 30).

Electrochemical Measurements—Electrochemical measurements of NO generation under anaerobic conditions were carried out at 37 °C in a sealed electrochemical vial using a CHI 832 electrochemical detector (CH Instruments, Inc., Austin, TX) and a Clark-type NO electrode (ISO-NOP, World Precision Instruments, Sarasota, FL). A slow flow of argon gas was maintained in the space above the solution. The electrochemical detector continuously recorded the current through the working electrode, which is proportional to the NO concentration in the solution. The sensor was calibrated before and after experiments with known concentrations of NO using NO equilibrated solutions.

Assay of S-Nitrosothiol—The concentrations of S-nitrosothiol were determined from the increase in nitrite concentration after treatment of samples with HgCl2. Spectrophotometric quantitation of nitrite with Griess reagent was performed according to the protocol provided by Sigma using a Varian Cary 300 UV-visible spectrophotometer. One milliliter of sample was supplemented with 50 µl of 10 mM HgCl2 and 1 ml of Griess reagent for 10 min at room temperature. Background levels of nitrite in the samples were determined using Griess reagent without the addition of HgCl2. The standard curve was obtained with a known amount of nitrite.

Immunoassay of Guanylyl Cyclase Activity—Activation of sGC was investigated by enzyme-linked immunoassay. After incubation of microsomes (4 mg/ml protein) and GTN (10 µM) or ISDN (100 µM) with 1 ml of reaction buffer (10 ng of sGC, 5 mM EDTA, 2 mM MgCl2, 1 mM GTP, and 100 µM NADPH in 1 ml of PBS) at 37 °C under anaerobic conditions, the enzymes in the samples were removed using Millipore filters (centrifugation at 2000 x g for 10 min at 4 °C). Measurements of cGMP in the solution were performed by immunoassay using the direct cGMP assay kit according to the manufacturer's protocol. The standard curve was obtained with known amounts of cGMP.

Statistical Analysis—Values are expressed as the means ± S.D. of at least three repeated measurements, and statistical significance of difference was evaluated by Student's t test. A p value of 0.05 or less was considered to indicate statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Inorganic Nitrite Generation from Organic Nitrate Reduction—Formula has normally been assumed to be the initial product of organic nitrate metabolism and the precursor of NO that accounts for the bioactivity of organic nitrates. To investigate the mechanism and magnitude of CPR-CP-mediated organic nitrate reduction with Formula formation as well as the possible role of nitrite as an intermediate in NO production, the rates of nitrite formation derived from the organic nitrates GTN and ISDN were measured under anaerobic conditions. The reaction mixture was sampled every 5 min, and the nitrite concentration was determined by NO analyzer analysis with reduction of nitrite to NO using 1% KI under acidic conditions. In the presence of ISDN (100 µM) or GTN (10 µM) with either 100 µM NADPH or 1 mM NADH as electron donor, a large amount of nitrite was generated upon the addition of microsomes (2 mg/ml protein) (Fig. 1A). Nitrite concentrations in the reaction mixture linearly increased at first and then started to plateau with a decrease in nitrite generation rates. For either ISDN or GTN, we observed that 100 µM NADPH triggered about twice as much nitrite generation compared with 1 mM NADH as reducing substrate (Fig. 1A, trace a versus trace c and trace b versus trace d). Thus, NADPH is a much more efficient electron donor than is NADH in the process of CPR-CP-mediated organic nitrate reduction. The flavin site inhibitor DPI greatly inhibited nitrite generation (Fig. 1A, trace e), whereas the CP inhibitor clotrimazole (5 µM) or carbon monoxide showed no inhibition (data not shown). Thus, CPR is the main enzyme that catalyzes the reduction of organic nitrate to nitrite.


Figure 1
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  		FIGURE 1.
Time course of nitrite generation from GTN or ISDN reduction. A, measurements were performed under anaerobic conditions at 37 °C in 5 ml of PBS (pH 7.4) in the presence of microsomes (2 mg/ml protein) with ISDN (100 µM) and NADPH (100 µM) (trace a); GTN (10 µM) and NADPH (100 µM) (trace b); ISDN (100 µM) and NADH (1 mM) (trace c); GTN (10µM) and NADH (1 mM)(trace d); or GTN (10µM), NADPH (100µM), and DPI (50 µM) (trace e). B, measurements were performed as described for A in the presence of recombinant CPR (0.02 mg/ml) with ISDN (100µM) and NADPH (100µM)(trace a); GTN (10 µM) and NADPH (100µM)(trace b); or GTN (10µM), NADPH (100µM), and DPI (50µM)(trace c). The reaction mixture was sampled (0.1 ml) every 5 min, and the nitrite concentration was measured using an NO analyzer as described under "Experimental Procedures."

 


Figure 2
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  		FIGURE 2.
Kinetics of nitrite generation as a function of GTN or ISDN concentration. The initial rates of nitrite production were measured using a chemiluminescence NO analyzer as described in the legend to Fig. 1. A, effects of [GTN] on the rate of nitrite generation from microsomes (2 mg/ml protein) and NADPH (100 µM) in 5 ml of PBS (pH 7.4); B, effects of [GTN] on the rate of nitrite generation from microsomes (2 mg/ml protein) and NADH (1 mM); C, effectsof[ISDN] on the rate of nitrite generation from microsomes (2 mg/ml protein) and NADPH (100µM); D, effects of [ISDN] on the rate of nitrite generation from microsomes (2 mg/ml protein) and NADH (1 mM). For each of these graphs, the corresponding fits (solid lines) and Km and Vmax data were obtained using the Michaelis-Menten equation.

 
To further verify that CPR catalyzes the reduction of organic nitrate to nitrite and thus to confirm the enzymatic role of CPR in the process of organic nitrate biotransformation, studies were performed using purified recombinant CPR, and comparative studies of nitrite generation from GTN or ISDN reduction were carried out. In the presence of ISDN (100 µM) or GTN (10 µM) and 100 µM NADPH, following the addition of recombinant CPR (0.02 mg/ml), a large amount of nitrite was generated (Fig. 1B). The observed rate of nitrite formation with ISDN (100 µM) was about twice that with GTN (10 µM). This CPR-dependent nitrite generation was totally inhibited by the flavin site inhibitor DPI. These results demonstrate that CPR is highly effective in triggering organic nitrate reduction, leading to nitrite formation.

The concentration dependence of nitrite production from CPR in microsomes was determined as a function of GTN (1-100 µM) or ISDN (0.01-3 mM) concentration. The initial rate of nitrite formation was determined from the ratio of the increase in nitrite concentration during the first 5 min. With NADPH (100 µM) or NADH (1 mM) as reducing substrate, the initial rate of nitrite formation was determined as a function of GTN (Fig. 2, A and B) or ISDN (Fig. 2, C and D) concentration, and Michaelis-Menten kinetics were observed with a correlation coefficient ({gamma}2) of >98% (Fig. 2). The apparent Km and Vmax values are shown under each curve. The apparent Km values for GTN and ISDN are higher than typical clinical levels of organic nitrates in tissues or blood. Thus, at pharmacological levels of GTN (lower micromolar range) or ISDN (10-100 µM), the rate of organic nitrate reduction would be expected to increase linearly with the given organic nitrate dose (Fig. 2). From the observed concentration-dependent kinetics of nitrite formation from these organic nitrates, we can further consider the possible contribution of nitrite as precursor of NO in the process of organic nitrate biotransformation.


Figure 3
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  		FIGURE 3.
EPR measurement of NO generated from GTN or ISDN under anaerobic conditions in PBS (pH 7.4) at 37 °C. Trace A, ISDN (100 µM) and NADPH (100 µM); trace B, microsomes (2 mg/ml protein), ISDN (100 µM), and NADPH (100 µM); trace C, microsomes (2 mg/ml protein), GTN (10 µM), and NADPH (100 µM); trace D, microsomes (2 mg/ml protein), GTN (10 µM), and NADH (1 mM); trace E, microsomes (2 mg/ml protein), ISDN (100 µM), NADPH (100 µM), and DPI (50 µM); trace F, microsomes (2 mg/ml protein), ISDN (100 µM), NADPH (100 µM), and clotrimazole (5 µM). Reactions were performed with a 5-ml sample volume in the reaction vessel. NO was continuously purged using argon from the reaction vessel into a trap vessel containing 5 ml of 2 mM Fe2+·(MGD)2. Samples were taken from the trap vessel after 30 min, and the spectra of NO·Fe2+·(MGD)2 adducts formed are shown.

 
Nitric Oxide Generation from CPR-CP-mediated GTN or ISDN Reduction—It is well known that organic nitrates such as GTN and ISDN are prodrugs requiring metabolism to generate bioactive NO. To investigate whether CPR-CP can catalyze the reduction of these organic nitrates to NO and to quantitate their NO generation, EPR spectroscopy was applied to directly measure GTN- and ISDN-mediated NO generation under anaerobic conditions. NO generated was purged from the reaction vessel using argon to a secondary vessel containing the spin trap Fe2+·MGD. NO is paramagnetic and binds with high affinity to the water-soluble spin trap Fe2+·MGD, forming the mononitrosyl iron complex NO·Fe2+·MGD with a characteristic triplet spectrum at g = 2.04 and hyperfine splitting aN = 12.8. From the intensity of the observed spectrum, quantitative measurement of NO generation can be performed (31-33). With ISDN (100 µM) or GTN (10 µM) in the presence of NADPH (100 µM) or NADH (1 mM), no signal was seen (Fig. 3A). In the presence of NADPH (100 µM), microsomes (2 mg/ml protein) were added, and this triggered marked NO generation from 10 µM GTN with 0.21 µM NO trapped over 30 min (Fig. 3B) and from 100 µM ISDN with 0.19 µM NO trapped (Fig. 3C). With 1 mM NADH as electron donor, microsomes (2 mg/ml protein) also triggered 0.10 µM NO generation from 100 µM ISDN (Fig. 3D). The CPR flavin site inhibitor DPI (50 µM) effectively blocked microsome-mediated NO formation (Fig. 3E). In contrast, the CP inhibitor clotrimazole (5 µM) showed no significant inhibition (Fig. 3F); and similarly, carbon monoxide did not alter the observed NO generation (data not shown). This suggests that CPR is the primary microsomal enzyme that catalyzes the reduction of organic nitrate to nitrite and NO.

To further quantitate the rates of NO generation from microsomal CPR-CP-mediated GTN and ISDN reduction, studies were performed using a chemiluminescence NO analyzer. NO was purged from the solution by argon and then reacted with ozone in the analyzer to form excited-state NO2, which emits light. This method provides direct measurement of the rate of NO generation as a function of time. In the presence of NADPH (100 µM) with GTN (10 or 50 µM), the addition of microsomes (2 mg/ml protein) triggered prominent NO generation (Fig. 4A, traces a and b). In the presence of NADH (1 mM) with GTN (10 µM), the addition of microsomes (2 mg/ml protein) triggered GTN reduction to produce a relatively small amount of NO (Fig. 4A, trace c). The presence of the flavin site inhibitor DPI greatly inhibited NO generation (Fig. 4A, trace d).


Figure 4
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  		FIGURE 4.
A, measurement of the rate of NO generation from CPR-CP-catalyzed GTN reduction. Measurements were performed using a chemiluminescence NO analyzer under anaerobic conditions at 37 °C in 5 ml of PBS (pH 7.4). Trace a, rate of NO generation by microsomes (2 mg/ml protein), NADPH (100 µM), and GTN (50 µM); trace b, rate of NO generation by microsomes (2 mg/ml protein), NADPH (100 µM), and GTN (10 µM); trace c, rate of NO generation by microsomes (2 mg/ml protein), NADH (1 mM), and GTN (10 µM); trace d, rate of NO generation by microsomes (2 mg/ml protein), NADPH (100 µM), GTN (50 µM), and DPI (50 µM). B, measurement of the rate of NO generation from CPR-CP-catalyzed ISDN reduction. Measurements were performed using a chemiluminescence NO analyzer under anaerobic conditions at 37 °C in 5 ml of PBS (pH 7.4). Trace a, rate of NO generation by microsomes (2 mg/ml protein), NADPH (100 µM), and ISDN (500 µM); trace b, rate of NO generation by microsomes (2 mg/ml protein), NADPH (100 µM), and ISDN (100 µM); trace c, rate of NO generation by microsomes (2 mg/ml protein), NADH (1 mM), and ISDN (100 µM); trace d, rate of NO generation by microsomes (2 mg/ml protein), NADPH (100 µM), ISDN (500 µM), and DPI (50 µM).

 


Figure 5
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  		FIGURE 5.
Electrochemical measurement of NO generation from CPR-CP-mediated ISDN reduction. The time course of NO generation was measured using an electrochemical NO sensor under anaerobic conditions at 37 °C in 2 ml of PBS (pH 7.4). The arrow shows the time at which ISDN was added. Trace A, data obtained with ISDN (500 µM) in the presence of microsomes (2 mg/ml protein) and NADPH (100 µM); trace B, data obtained with ISDN (100 µM) in the presence of microsomes (2 mg/ml protein) and NADPH (100 µM); trace C, data obtained with ISDN (500 µM) in the presence of microsomes (2 mg/ml protein), NADPH (100 µM), and DPI (50 µM).

 
CPR-CP-mediated ISDN reduction was similar to GTN reduction. In the presence of microsomes (2 mg/ml), ISDN (100 or 500 µM) was reduced to NO with either NADPH (100 µM) (Fig. 4B, trace a or b) or NADH (1 mM) (trace c) as reducing substrate. This NO generation was totally inhibited by the flavin site inhibitor DPI (Fig. 4B, trace d).

To further confirm these observations and to quantitate the NO concentration that accumulated, NO generation was measured by electrochemical detection. Prior to the addition of microsomes, there was no detectable NO. After the addition of microsomes (2 mg/ml protein), NO generation was triggered from ISDN (500 or 100 µM) and NADPH (100 µM) (Fig. 5, traces A and B). This NO generation was inhibited by DPI (Fig. 5C).

Thus, EPR, chemiluminescence NO analyzer, and NO electrode studies demonstrate NO generation from CPR-CP-mediated organic nitrate reduction. Under similar conditions, the NO generation rate detected by EPR was similar to that detected using the chemiluminescence NO analyzer and to the initial rate detected by NO electrode analysis.


Figure 6
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  		FIGURE 6.
Measurement of the rate of NO generation from recombinant CPR-catalyzed ISDN reduction. Measurements were performed using a chemiluminescence NO analyzer under anaerobic conditions at 37 °C in 5 ml of PBS (pH 7.4). Trace A, rate of NO generation by recombinant CPR (0.02 mg/ml), NADPH (100 µM), ISDN (100 µM), and L-cysteine (5 mM); trace B, rate of NO generation by recombinant CPR (0.02 mg/ml), NADPH (100 µM), and ISDN (100 µM).

 
A comparative study of NO generation from recombinant CPR-mediated organic nitrate reduction was performed. With ISDN (100 µM)in the presence of CPR (0.02 mg/ml) and NAPDH (100 µM), no significant NO generation was seen (Fig. 6, trace B). To test whether sulfhydryl compounds play a role in CPR-mediated transformation of organic nitrates, L-cysteine (5 mM) was added, and this triggered marked NO generation from 100 µM ISDN at a rate of 12 nM min-1 (Fig. 6, trace A). With 10 µM GTN, L-cysteine (5 mM) similarly triggered NO formation at a peak rate of 18 nM min-1. The FAD site inhibitor DPI (50 µM) totally blocked this CPR-dependent NO formation (data not shown). As described above with microsomes instead of purified recombinant CPR, prominent NO generation occurred in the absence of added thiols (Figs. 3, 4, 5). This suggests that sulfhydryl groups in the microsomal proteins or enzymes may play important roles in the process of organic nitrate biotransformation to NO.

Nitric Oxide Generation from Formula Reduction—Formula has normally been assumed to be the initial product of organic nitrate metabolism and the precursor of NO. To assess the contribution of NO generation from Formula in organic nitrate biotransformation, nitrite-dependent NO generation was measured using a chemiluminescence NO analyzer. In the presence of NADPH (100 µM), the addition of 20 or 40 µM nitrite triggered NO generation at ~2.8 or ~4.5 nM min-1 from microsomes (2 mg/ml) (Fig. 7A). The rates of NO generation from CPR-CP-mediated nitrite (10-200 µM) reduction with 100 µM NADPH (trace a) and 1 mM NADH (trace b) are shown in Fig. 7B. The CP inhibitor clotrimazole (5 µM) greatly inhibited nitrite-dependent NO generation (Fig. 7B, trace c). Therefore, it is CP that catalyzed nitrite reduction to NO in microsomes. Of note, with the same amount of microsomes, the reduction of 100 µM ISDN or 10 µM GTNby100 µM NADPH produced only ~4.8 or 3.4 µM total nitrite, respectively, over 30 min (Fig. 1A, traces a and b). Nitrite-dependent NO generation kinetics (Fig. 7A) revealed that NO generation from nitrite reduction contributed <10% of the total NO generation in the process of organic nitrate biotransformation. Our results suggest that nitrite is not the primary precursor of NO in the process of microsomal CPR-CP-mediated organic nitrate biotransformation.

A comparative study of NO generation from isolated CP-mediated nitrite reduction was performed. Isolated CP 2B4 was obtained from Sigma with ~0.2 units of CPR activity/mg of protein. In the presence of 100 µM NADPH, the addition of 100 µM nitrite triggered significant NO generation at a rate of ~6 nM min-1 (Fig. 7C, trace c). NO generation from nitrite was increased with an increase in nitrite concentration (Fig. 7C, trace b) or CP concentration (trace a) in the reaction mixture. Furthermore, this NO generation derived from nitrite was greatly inhibited by the CP inhibitor cyanide (Fig. 7C, trace d).

Effects of pH on CPR-mediated Nitrite Generation and CP-mediated NO Generation—To assess organic nitrate bioactivation under different physiological or pathological conditions and to further characterize the mechanism of CPR-CP-mediated organic nitrate reduction, experiments were performed to measure the effect of pH on the magnitude of nitrite generation from CPR-mediated organic nitrate reduction and on the magnitude of NO generation from CP-mediated nitrite reduction. As shown in Table 1, CPR-mediated organic nitrate reduction was maximized at pH 7.4. However, CP-mediated nitrite reduction to NO was greatly increased under acidic conditions, whereas it was diminished under alkaline conditions.


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  		TABLE 1
Effect of pH on CPR-mediated organic nitrate reduction and CP-mediated nitrite reduction In Part A, the initial nitrite generation rates from CPR-mediated organic nitrate reduction were calculated as described in the legend to Fig. 2. Measurements were performed with microsomes (2 mg/ml protein) in the presence of GTN (10 µM) or ISDN (100 µM) with NADPH (100 µM) as reducing substrate. In Part B, NO generation rates from CP-mediated nitrite reduction were calculated as described in the legend to Fig. 7. Measurements were performed with microsomes (2 mg/ml protein) in the presence of ISDN (100 µM), GTN (10 µM), or nitrite (20 µM) with NADPH (100 µM) as reducing substrate.

 
Assay of Nitrosothiol Formation from Microsomal CPR-CP-mediated GTN or ISDN Reduction—It has been reported that thiols react with labile organic nitrite esters, as could be formed from reduction of organic nitrates, to give rise to corresponding S-nitrosothiols either chemically or enzymatically and that these S-nitrosothiols can serve in turn as precursors for NO formation (34-38). To investigate whether the initial product of CPR-CP-mediated reduction of organic nitrate is organic nitrite (R-O-NO) and whether this organic nitrite can be the nitrosothiol precursor, studies were performed to measure nitrosothiol production from microsomal CPR-CP-catalyzed organic nitrate reduction (Fig. 8). Following incubation at 37 °C under anaerobic conditions (argon), the reaction mixture was sampled at the times indicated, and nitrosothiols in the solution were detected and quantified using the Saville assay, which is based on mercury ion-mediated heterolytic cleavage of the S-NO bond. Nitrosonium ion is released, which is in turn measured by the Griess reaction. The concentrations of S-nitrosothiols were determined from the increase in the nitrite concentration induced by treatment of samples with HgCl2. In the presence of microsomes, nitrosothiol production from GTN (10 µM) increased to 3.1 µM within 30 min (Fig. 8, trace A). Similar microsomal nitrosothiol production was seen with ISDN (100 µM) (Fig. 8, trace B), with 2.6 µM levels after 30 min. The addition of the flavin site inhibitor DPI completely inhibited microsomal nitrosothiol production from ISDN (Fig. 8D) and largely blocked that from GTN (Fig. 8, trace C).


Figure 7
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  		FIGURE 7.
Measurement of the rate of NO generation from nitrite reduction. Measurements were performed using a chemiluminescence NO analyzer under anaerobic conditions at 37 °C in 5 ml of PBS (pH 7.4) after incubation of microsomes (2 mg/ml protein) or isolated CP (0.05-0.1 mg/ml) with nitrite (10-200 µM). A, time course of NO generation from microsomes (2 mg/ml protein) after incubation with 20 µM nitrite (trace a) or 40 µM nitrite (trace b) using 100 µM NADPH as electron donor. B, NO generation from microsomes (2 mg/ml protein) as a function of nitrite (10-200 µM) with 100 µM NADPH (trace a), 1 mM NADH (trace b), or 100 µM NADPH and 5 µM clotrimazole (trace c). C, NO generation from the isolated CP 2B4 isoform (with ~0.2 units/ml CPR) with 0.1 mg/ml CP, 200 µM nitrite, and 100 µM NADPH (trace a); 0.05 mg/ml CP, 200 µM nitrite, and 100 µM NADPH (trace b); 0.05 mg/ml CP, 100 µM nitrite, and 100 µM NADPH (trace c); or 0.1 mg/ml CP, 200 µM nitrite, 100 µM NADPH, and 5 mM sodium cyanide (trace d).

 
Detection of CPR-mediated Organic Nitrite Formation—Organic nitrites (R-O-NO) such as amyl nitrite and butyl nitrite are widely used vasodilator drugs. Because the results from our studies suggested that R-O-NO formed by CPR is the primary precursor of NO and nitrosothiols in the process of microsomal organic nitrate (R-O-NO2) biotransformation, experiments were performed in an effort to directly detect R-O-NO formation from purified recombinant CPR. The fast hydrolysis of R-O-NO in aqueous solutions with nitrite generation makes it technically very difficult to directly measure R-O-NO production by standard analytical methods. We have worked to solve this problem and have developed an EPR approach for detection of R-O-NO. We observed that the spin trap iron·MGD can be used to distinguish R-O-NO from NO with this based on the redox state of the iron.


Figure 8
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  		FIGURE 8.
Nitrosothiol production from GTN or ISDN reduction under anaerobic conditions in 5 ml of PBS (pH 7.4) at 37 °C. Trace A, microsomes (4 mg/ml protein), GTN (10 µM), and NADPH (100 µM); trace B, microsomes (4 mg/ml protein), ISDN (100 µM), and NADPH (100 µM); trace C, microsomes (4 mg/ml protein), GTN (10 µM), NADPH (100 µM), and DPI (50 µM); trace D, microsomes (4 mg/ml protein), ISDN (100 µM), NADPH (100 µM), and DPI (50 µM). The reaction mixture was sampled at 10, 15, 20, 25, and 30 min. Nitrosothiols were detected and quantified as described under "Experimental Procedures."

 


Figure 9
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  		FIGURE 9.
EPR detection of organic nitrite formation from recombinant CPR under anaerobic conditions in 5 ml of PBS (pH 7.4) at 37 °C. Trace A, amyl nitrite (10 µM) and Fe2+·(MGD)2 (2 mM); trace B, amyl nitrite (10 µM) and Fe3+·(MGD)2 (2 mM); trace C, CPR (0.02 mg/ml protein), ISDN (100 µM), NADPH (100 µM), and Fe2+·(MGD)2 (2 mM); trace D, CPR (0.02 mg/ml protein), ISDN (100 µM), NADPH (100 µM), DPI (50 µM), and Fe2+·(MGD)2 (2 mM); trace E, CPR (0.02 mg/ml protein), ISDN (100 µM), NADPH (100 µM), and Fe3+·(MGD)2 (2 mM). Samples for traces A and B were taken from the reaction buffer immediately after mixing. Samples for traces C-E were taken from the reaction buffer after 30 min of mixing, and the spectra of NO·Fe2+·(MGD)2 adducts formed are shown.

 
With the addition of NO, both Fe2+·MGD and Fe3+·MGD give rise to the triplet NO·Fe2+·MGD signal; however, with Fe2+·MGD, the stoichiometry is 1, whereas with Fe3+·MGD, it is 0.5 (39). Under anaerobic conditions with Fe2+·MGD (2 mM) in PBS (pH 7.4), organic nitrites such as amyl nitrite (10 µM) and butyl nitrite (10 µM) immediately reacted to form prominent NO·Fe2+·MGD complexes (~6.6 µM) with a triplet EPR signal (g = 2.04, aN = 12.7) (Fig. 9, trace A); but when Fe3+·MGD was used, no signal was seen (Fig. 9, trace B). By EPR spectroscopy using iron·MGD, we directly detected organic nitrite (R-O-NO) from recombinant CPR-mediated organic nitrate reduction. Using Fe2+·MGD, we observed a large NO·Fe2+·MGD signal (~3.4 µM) with CPR (0.02 mg/ml), ISDN (100 µM), and NADPH (100 µM) (Fig. 9, trace C). Under the same conditions, except for the presence of the flavin inhibitor DPI, this signal was almost totally quenched (Fig. 9, trace D). However, using Fe3+·MGD instead of Fe2+·MGD as spin trap, we could not detect a signal (Fig. 9, trace E). Thus, these results suggest that organic nitrite is the initial product in the process of CPR-mediated organic nitrate reduction and is the precursor of NO and nitrosothiols.

Effect of CPR-CP-mediated GTN or ISDN Reduction on sGC Activity—To determine whether microsomal CPR-CP-mediated organic nitrate biotransformation can induce sGC activation, enzyme-linked immunoassays were performed to determine the cGMP concentrations in the reaction mixture. After incubation of microsomes (2 mg/ml protein) with GTN (10 µM) or ISDN (100 µM) in reaction buffer under anaerobic conditions for 10 min, measurements of the sGC product cGMP were performed. Without microsomal protein and with NADPH as CPR-reducing substrate, no cGMP could be detected, suggesting that sGC was not activated (Fig. 10, bar A). Under similar conditions with the addition of microsomes (2 mg/ml protein), a large amount of cGMP was produced from CPR-CP-mediated reduction of GTN (Fig. 10, bar B) or ISDN (bar C). Using the flavin site-specific inhibitor DPI, CPR-CP-mediated sGC activation by organic nitrate was largely abolished (>95%) (Fig. 10, bar D). When the CP inhibitor clotrimazole was used, no significant inhibition was seen (Fig. 10, bar E), further confirming that CPR rather than CP is of critical importance in organic nitrate activation.


Figure 10
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  		FIGURE 10.
sGC activation by CPR-CP-mediated GTN or ISDN reduction. The reaction buffer contained 1 ml of PBS (pH 7.4) with 10% bovine serum albumin, 5 mM EDTA, 2 mM MgCl2, 10 ng of sGC, and 1 mM GTP. Bar A, GTN (10 µM) and NADPH (100 µM); bar B, microsomes (2 mg/ml protein), GTN (10 µM), and NADPH (100 µM); bar C, microsomes (2 mg/ml protein), ISDN (100 µM), and NADPH (100 µM); bar D, microsomes (2 mg/ml protein), GTN (10 µM), NADPH (100 µM), and DPI (50 µM); bar E, microsomes (2 mg/ml protein), ISDN (100 µM), NADPH (100 µM), and clotrimazole (5 µM). Incubation was carried out for 10 min in the reaction buffer. Activation of sGC was determined from the measurements of cGMP.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Numerous studies have demonstrated that the CPR-CP system plays important roles in organic nitrate bioactivation (5, 8-10, 16, 26, 40, 41). However, the precise enzymatic mechanisms have not been elucidated. Questions remain concerning the initial product, the precursor of NO, and the link between organic nitrate and activation of sGC. To investigate the mechanism of CPR-CP-mediated organic nitrate reduction and thus to further elucidate the precise molecular mechanism of bioactivation of organic nitrate, we performed a series of studies using EPR, chemiluminescence NO analyzer, immunoassay, and NO electrode techniques to measure the magnitude of various products (Formula, NO, and R-S-NO) derived from microsomal CPR-CP and their effects on activation of sGC.

We observed that both NADPH and NADH could act as electron donors to support CPR-CP-mediated GTN/ISDN reduction to produce nitrite (Fig. 1A). Compared with NADH, NADPH is a much more efficient reducing substrate, as would be anticipated based on the known substrate affinity of CPR (42). Our data show that the addition of the flavin inhibitor DPI almost completely inhibited nitrite production from organic nitrate. However, the addition of the CP inhibitors clotrimazole and carbon monoxide did not inhibit this nitrite generation. These results demonstrate that the flavin enzyme CPR (rather than the hemeprotein CP) is the main enzyme that catalyzes the reduction of organic nitrate to nitrite. Purified recombinant CPR also showed a similar capability to catalyze the reduction of organic nitrate to nitrite, and this reduction was also inhibited by DPI (Fig. 1B).

The rate of nitrite formation was determined as a function of GTN or ISDN concentration with NADPH or NADH as reducing substrate (Fig. 2). At pharmacological doses of GTN (low micromolar) or ISDN (10-100 µM), the rate of nitrite formation was seen to increase linearly with an increase in GTN or ISDN concentration.

It is well known that organic nitrates such as GTN and ISDN are prodrugs requiring metabolism to generate bioactive NO. Therefore, we performed a series of studies using EPR, chemiluminescence NO analyzer, and NO electrode techniques to measure the magnitude and kinetics of NO formation generated from CPR-CP-mediated organic nitrate reduction. The data obtained using each of the three methods confirmed that CPR-CP does reduce GTN or ISDN to NO under anaerobic conditions. We observed that both NADPH and NADH could act as electron donors to support this CPR-CP-mediated organic nitrate reduction. The flavin modifier DPI inhibited nitrite and NO generation, whereas the CP inhibitors clotrimazole and carbon monoxide showed no significant inhibition of organic nitrate reduction, suggesting that CPR is the primary enzyme that catalyzes GTN or ISDN reduction to nitrite and NO.

It was assumed previously that Formula production is the first step in the process of organic nitrate bioactivation to form NO (3). To assess the contribution of NO generation from Formula in the process of CPR-CP-mediated organic nitrate reduction, nitrite-dependent NO generation was measured with a chemiluminescence NO analyzer. Our data show that with a high dose of GTN (10 µM) or ISDN (100 µM), ~3.4 or 4.8 µM nitrite, respectively, was generated within 30 min, whereas NO was generated at a rate of ~7.1 or 6.1 nM min-1. Under the same conditions, nitrite-dependent NO generation could account for NO production of only <1 nM min-1. The time course of NO generation showed that the NO generation rate did not increase with the accumulation of nitrite in the solution. Instead, it reached a maximum within 5 min and then slowly decreased (Fig. 4). Thus, both the time course and magnitude of NO generation revealed that nitrite is not the primary precursor of NO in the process of microsomal CPR-CP-mediated organic nitrate biotransformation.

Previous studies have shown that nitrite can accept electrons from CP of liver microsomes (43, 44). EPR spectral analysis further confirmed nitrite reduction by detecting EPR signals of complexes formed by NO and the heme iron of CP (45). The NO·heme(III) complex generated from CP and nitrite is a labile form of the NO·heme complex. NO can be released as free NO or retrapped, forming a relatively stable NO·heme(II) complex. In the presence of excessive nitrite, significant NO generation from CP was detected (Fig. 7), suggesting that CP-mediated nitrite reduction can be a source of NO.

Besides microsomal CPR, cytosolic xanthine oxidoreductase (46, 47) and mitochondrial aldehyde dehydrogenase (48) have also been reported to be able to catalyze GTN or ISDN reduction to produce nitrite. Data obtained in whole tissue, isolated mitochondria, cytosolic fractions, and microsomes have shown that clinically used GTN levels cannot produce sufficient Formula as an intermediate of NO to exhibit vasodilatory effects (49-53).4 However, long-term administration of high doses of organic nitrates can greatly elevate nitrate anion (Formula) or Formula concentrations in tissues (54), and these can be an important sources of NO during ischemia, especially under acidotic conditions (27, 29, 55-58).

It is well known that sulfhydryl compounds are needed in GTN activation and that the repeated administration of GTN causes sulfhydryl depletion and consequent tolerance to further vasodilatation. In this study, we have shown that the presence of L-cysteine triggered significant NO generation from recombinant CPR-mediated reduction of GTN/ISDN, whereas no detectable NO was generated without the addition of thiols (Fig. 6, trace A). With microsomal CPR, external thiols were not required for NO generation, as the sulfhydryl groups in the microsomal proteins may serve to reduce organic nitrite to NO (Figs. 3, 4, 5).

In this study, we observed that, in addition to generating NO, CPR-CP-mediated organic nitrate reduction also resulted in nitrosothiol production. This CPR-CP-dependent nitrosothiol production was inhibited in the presence of the FAD site inhibitor DPI (Fig. 8). These results suggest that organic nitrite (R-O-NO) is the initial product in the process of CPR-CP-mediated organic nitrate reduction and is the precursor of both NO and nitrosothiols. Under hydrophilic conditions, most of the R-O-NO would quickly hydrolyze to produce Formula. Thus, the lipophilic microsomal proteins that contain thiols can be more reactive to R-O-NO than are thiols in hydrophilic solution. NO and nitrosothiols would be expected to be major products of the reaction of R-O-NO with sulfhydryl compounds either chemically formed (34) or enzymatically catalyzed by glutathione S-transferase (36, 37, 59).

Activation of sGC by organic nitrate reduction was investigated by enzyme-linked immunoassay. The flavin-binding inhibitor DPI inhibited organic nitrate reduction and sGC activation, indicating that organic nitrate reduction occurs at the flavin site. The CP inhibitor clotrimazole showed no inhibition of sGC activation (Fig. 10). Our results show that CPR is the primary enzyme that plays a crucial role in the process of organic nitrate bioactivation.

Reactions 1, 2, 3, 4, 5 define the steps in the proposed reaction mechanism of CPR-CP-mediated biotransformation of organic nitrate.

Formula 1REACTION 1

Formula 2REACTION 2

Formula 3REACTION 3

Formula 4REACTION 4

Formula 5REACTION 5

Organic nitrite (R-O-NO) is the initial product in the process of CPR-mediated organic nitrate biotransformation and is the precursor of NO and nitrosothiol. In the absence of thiols, organic nitrite undergoes hydrolysis to form nitrite. However, in the presence of thiols, either NO or nitrosothiols can be formed. Thus, R-O-NO is the link between organic nitrates and activation of sGC.

From our data, it is possible to estimate the magnitude of organic nitrate-derived NO formation that can be generated from CPR-CP in the liver or other tissues. We observed with 2 mg/ml microsomal protein that CPR-CP catalyzed reduction of 10 µM GTN with generation of NO at 6.4 nM min-1 (Fig. 4A, trace b). Using differential centrifugation, we isolated ~3.6 g of microsomal protein from 175 g of rat liver tissue. Thus, the tissue concentration of microsomal proteins was ~10-fold higher than the amount used in our assays. Assuming a ratio of intracellular water to wet weight of 0.43 (60), we estimate that the levels of CPR-CP in hepatocytes are sufficient to catalyze 10 µM GTN reduction to generate NO at ~150 nM min-1 in the liver. With pure CPR (20 µg/ml), we measured a rate of NO generation of 18 nM min-1 from 10 µM GTN or 900 nM mg-1 min-1. It has been reported that CPR levels are as high as 0.5-1% of the total microsomal protein (8), so one can estimate levels of 0.43 mg/g of cell water, and this would give rise to NO production of ~200-400 nM min-1. Because CPR-CP levels in cardiovascular tissues, including the aorta and heart, are ~5-10-fold lower than those in the liver, we estimate that the rate of CPR-CP-derived NO production could be 20-80 nM min-1 in these tissues.

In addition to CPR, we have previously observed that other flavoenzymes such as xanthine oxidase also catalyze reduction of organic nitrate to organic nitrite and that this organic nitrite can be further reduced by sulfhydryl compounds, leading to NO or nitrosothiol formation (47). In the presence of sulfhydryl compounds, pharmacological levels of GTN (10-25 µM) in the heart could generate NO at 6-12 nM min-1 from xanthine oxidase-mediated reduction under anaerobic conditions (47). Of note, exogenous sulfhydryl compounds are required for NO generation from cytosolic xanthine oxidase-mediated organic nitrate reduction and also from purified CPR, whereas NO generation from microsomal CPR-mediated organic nitrate reduction does not require exogenous thiols. The lipophilic microsomal proteins that contain thiols can be more reactive to R-O-NO compared with thiols in hydrophilic solution. A number of different flavoproteins are likely involved in the in vivo activation of organic nitrates to form NO. The association of CPR with other microsomal proteins and the microsomal lipid compartment potentiates its role in converting organic nitrates to NO. Clearly, the oxygen tension, availability of substrates for the enzymes, sulfhydryl groups or compounds, and pH play important roles in regulating organic nitrate biotransformation.

Overall, this study has demonstrated that CPR catalyzes GTN and ISDN to their respective organic nitrites (R-O-NO). These organic nitrites then react chemically or enzymatically with thiols or other reducing substrates to form nitrosothiols or NO. Nitrite anion (Formula 5)is the hydrolysis product of organic nitrite. CP can mediate further nitrite reduction to NO; however, this nitrite-dependent NO contributes only a small faction of the total CPR-CP-mediated organic nitrate biotransformation to NO, so Formula 5 is not the main precursor of NO. Thus, in the process of microsomal CPR-CP-mediated organic nitrate bioactivation, organic nitrite is the critical link between organic nitrate and activation of sGC.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL63744, HL65608, and HL38324 (to J. L. Z.) and by Ohio Valley Affiliate of the American Heart Association Grant BGIA-0565249B (to H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence may be addressed: 110G Davis Heart and Lung Research Inst., 473 West 12th Ave., Columbus, OH 43210-1252. Tel.: 614-247-7857; Fax: 614-247-7845. E-mail: haitao.li{at}osumc.edu. 2 To whom correspondence may be addressed: 110G Davis Heart and Lung Research Inst., 473 West 12th Ave., Columbus, OH 43210-1252. Tel.: 614-247-7857; Fax: 614-247-7845; E-mail: jay.zweier{at}osumc.edu.

3 The abbreviations used are: GTN, glyceryl trinitrate; ISDN, isosorbide dinitrate; CPR, cytochrome P450 reductase; CP, cytochrome P450; sGC, soluble guanylyl cyclase; DPI, diphenyleneiodonium; MGD, N-methyl-D-glucamine dithiocarbamate; PBS, phosphate-buffered saline. Back

4 H. Li, H. Cui, and J. L. Zweier, unpublished data. Back



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 DISCUSSION
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