JPET Fast Forward. Published on May 23, 2006 as DOI:10 ......2006/05/23 · JPET #103713 4...

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BIOTRANSFORMATION OF GLYCERYL TRINITRATE BY RAT HEPATIC

MICROSOMAL GLUTATHIONE S-TRANSFERASE 1

Yanbin Ji and Brian M. Bennett

Department of Pharmacology and Toxicology

Faculty of Health Sciences, Queen’s University

Kingston, Ontario, Canada,

K7L 3N6.

JPET Fast Forward. Published on May 23, 2006 as DOI:10.1124/jpet.106.103713

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 23, 2006 as DOI: 10.1124/jpet.106.103713

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Running Title: Biotransformation of GTN by Microsomal GST Corresponding Author: Dr. Brian M. Bennett Department of Pharmacology and Toxicology Faculty of Health Sciences Queen’s University Kingston, Ontario, Canada K7L 3N6

Telephone: (613) 533-6473

Facsimile: (613) 533-6412 e-mail: [email protected]

Text pages: 28

Tables: 2 Figures: 5 References: 37 Abstract: 218 words Introduction: 480 words Discussion: 1628 words

Abbreviations: GTN, glyceryl trinitrate; GDN, glyceryl dinitrate; NO, nitric oxide; MGST1,

microsomal glutathione S-transferase 1; CDNB, 1-chloro-2, 4-dinitrobenzene; GSH, reduced

glutathione; DTT, dithiothreitol; NEM, N-ethylmaleimide; GSNO, S-nitrosoglutathione;

TBARS, thiobarbituric acid-like reactive substances.

Section Assignment: Cardiovascular


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ABSTRACT

Although the biotransformation of organic nitrates by the cytosolic glutathione S-

transferases (GSTs) is well known, the relative contribution of the microsomal GST (MGST1)

to nitrate biotransformation has not been described. We therefore compared the denitration of

glyceryl trinitrate (GTN) by purified rat liver MGST1 and cytosolic GSTs. Both MGST1 and

cytosolic GSTs catalyzed the denitration of glyceryl trinitrate (GTN), but the activity of

MGST1 towards GTN was 2-3 fold higher. To mimic oxidative/nitrosative stress in vitro, we

treated enzyme preparations with hydrogen peroxide, S-nitrosoglutathione and peroxynitrite.

Both oxidants and nitrating reagents increased the activity of MGST1 towards the GST

substrate, 1-chloro-2,4-dinitrobenzene (CDNB) whereas these treatments inhibited GTN

denitration by MGST1. Alkylation of the sole cysteine residue of MGST1 by N-ethylmaleimide

markedly increased enzyme activity with CDNB as substrate but decreased the rate of GTN

denitration. In aortic microsomes from GTN-tolerant animals, there was a decreased abundance

of MGST1 dimers and trimers. In hepatic microsomes from GTN-tolerant animals, GTN

biotransformation was unaltered whereas the rate of CDNB conjugation was doubled,

suggesting that chronic GTN exposure causes structural modifications to the enzyme resulting

in increased activity to certain substrates. Collectively, these data indicate that MGST1

contributes significantly to the biotransformation of GTN, and that chemical modification of the

microsomal enzyme has differential effects on the catalytic activity towards different substrates.


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Introduction

Organic nitrates such as glyceryl trinitrate (GTN) have been used clinically for over a

century in the treatment of angina pectoris and congestive heart failure. It is generally accepted

that GTN is a prodrug that requires bioactivation to form nitric oxide (NO), or a closely related

species, prior to activation of guanylyl cyclase, increased cyclic GMP, and vascular smooth

muscle relaxation. In addition to this mechanism-based biotransformation of GTN to activators

of guanylyl cyclase, the denitration of GTN in vascular and non vascular tissues to inorganic

nitrite anion (NO2- ) and its two dinitrate metabolites, glyceryl-1,2-dinitrate (1,2-GDN) and

glyceryl-1,3-dinitrate (1,3-GDN) also occurs. This is catalyzed by a number of proteins and

enzymes, including hemoglobin and myoglobin (Bennett et al., 1986), the cytochromes P450-

NADPH cytochrome P450 reductase system (McDonald and Bennett, 1990, 1993; McGuire et

al., 1998), xanthine oxidoreductase (Doel et al., 2000; Ratz et al., 2000b), old yellow enzyme

(Meah et al., 2001), aldehyde dehydrogenase 2 (Chen et al., 2002) and the glutathione S-

transferases (GSTs)(Habig et al., 1975; Tsuchida et al., 1990; Nigam et al., 1996).

Although the GSTs are found primarily in the cytosol as homodimers or heterodimers, a

membrane-bound GST (MGST1) that accounts for up to 3% of microsomal protein also has

been identified (Morgenstern et al., 1982). The MGST1 is a member of the membrane-

associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily of

proteins, and although classified as a glutathione S-transferase, this GST isoform bears no

obvious structural resemblance (amino acid sequence, molecular weight, or immunological

properties) to the cytosolic GSTs.

Most previous studies have focused on the role of the cytosolic GSTs in the

biotransformation of GTN (Habig et al., 1975; Posadas del Rio et al., 1988; Tsuchida et al.,


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1990; Nigam et al., 1996; Kurz et al., 1993). However, the GSH-dependent biotransformation

of GTN in hepatic microsomes also occurs (Lau and Benet, 1990), suggesting a role of MGST1

in GTN biotransformation. The interpretation of microsomal biotransformation studies is

complicated by the association of certain cytosolic GST isoforms with the microsomal fraction

(Morgenstern et al., 1983; Hayes et al., 2005) and there is little information regarding the

specific role of MGST1 in GTN biotransformation. A characteristic of rat liver MGST1 is that

covalent modification (eg. alkylation by N-ethylmaleimide) of its single cysteine residue

(Cys49) results in a marked increase in enzyme activity. Oxidative modification of Cys49 also

results in increased enzyme activity, and recent data from our laboratory have demonstrated

that in addition to oxidative stress, nitrosative stress increases MGST1 activity by tyrosine

nitration of Tyr92, and to a lesser extent, by nitrosylation of Cys49 (Ji et al., 2002, 2006; Ji and

Bennett, 2003).

In the present study, we wished to compare GTN biotransformation by the microsomal

and cytosolic GSTs, to assess the effect of various modifications of MGST1 on GTN

biotransformation, and to assess whether MGST1 activity or expression are altered in GTN-

tolerant animals.


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Methods

Materials. Rat liver cytosolic GSTs, 1-chloro-2, 4-dinitrobenzene (CDNB), GSH,

dithiothreitol (DTT), CM-Sepharose, hydroxyapatite, N-ethylmaleimide (NEM), and Triton X-

100 were purchased from Sigma-Aldrich Canada Ltd (Oakville, ON, Canada). Manganese (IV)

oxide was from Aldrich (Milwaukee, WI). TRIDIL GTN injection USP (5 mg/ml) in ethanol:

propylene glycol: water (1:1:1.33) was obtained from Sabex Inc. (Boucherville, QC, Canada)

and transdermal GTN patches were obtained as Transderm-Nitro brand (0.2mg/hr) from Novartis

Pharmaceuticals (Dorval, QC, Canada). 1,2-GDN and 1,3-GDN were prepared by acid

hydrolysis of GTN and purified by thin-layer chromatography. Horseradish peroxidase-linked

goat anti-rabbit IgG was obtained from Bio-Rad Laboratories Ltd (Mississauga, ON, Canada).

Polyclonal anti-rat MGST1 antiserum was raised in rabbits by Cocalico Biologicals Inc

(Reamstown, PA) using purified rat hepatic MGST1 prepared in our laboratory.

Chemiluminescence reagents were from Kirkegaard and Perry Laboratories (Gaithersburg, MA).

RNeasy Mini Kits were from QiaGen Inc (Mississauga, ON, Canada) and QuikHyb Rapid

Hybridization from Stratagene Co. (Cedar Creek, TX,). Both Hybond-N+ membrane and α32P-

dCTP (specific activity of 6000 Ci/mmmol) were from Amersham Pharmacia Biotech Inc. (Baie

d’Urefe, QC, Canada); 18S ribosomal RNA DECA probe template was purchased from Ambion

Inc. (Austin, TX). All other chemicals were of reagent grade and were obtained from a variety of

commercial sources.

Induction of GTN-tolerance in vivo. All procedures for animal experimentation were

undertaken in accordance with the principles and guidelines of the Canadian Council on Animal

Care. GTN-tolerance was induced in male Sprague-Dawley rats (250-300g) by exposing animals

to a continuous source of GTN via the subdermal implantation of two 0.2 mg h 1 transdermal


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GTN patches (tolerant) or drug-free patches (control) for 48 h as described (Ratz et al., 2000a).

Briefly, rats were anesthetized with halothane, a 1-cm transverse incision was made in the upper

dorsal region, and the site was disinfected with 2.5% iodine. The skin was separated from the

underlying fascia by blunt dissection and two transdermal patches were inserted back-to-back

into the resulting subdermal space. The site was sutured closed and disinfected again with iodine.

At 24 h, the site was reopened and both patches were replaced, and at 48 h livers and/or aortae

were harvested.

Synthesis of GSNO and peroxynitrite. GSNO was prepared by reacting equimolar

concentrations of sodium nitrite and GSH as described previously (Ji et al., 1996). The

concentration of GSNO was determined spectrophotometrically at 334 nm (ε = 767 M-1cm-1).

Peroxynitrite was synthesized from acidified nitrite and H2O2 as described by Beckman et al

(1994) and stored at –70 °C. The concentration of peroxynitrite was determined

spectrophotometrically at 302 nm (ε = 1670 M-1cm-1) at the time of synthesis and again just prior

to use. The H2O2 contamination of peroxynitrite solutions was removed by manganese dioxide

chromatography and filtration (Beckman et al., 1994).

Measurement of microsomal lipid peroxidation. Lipid peroxidation products in hepatic

microsomes from control and GTN-tolerant rats were determined spectrophotometrically at 532

nm (ε = 1.56 x 105 M-1cm-1) as thiobarbituric acid-like reactive substances (TBARS)(Aust,

1985).

Isolation of rat liver microsomes and purification of MGST1. Rat hepatic or aortic

microsomes were prepared as described (Ji et al., 1996, MacDonald and Bennett, 1993).

Microsomes were washed twice with 100 mM Tris-HCl (pH 7.4) to decrease cytosolic

contamination and then stored at -70°C. Hepatic MGST1 was purified by hydroxyapatite and


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CM-Sepharose chromatography by the method of Morgenstern and DePierre (1983) as described

in Ji and Bennett (2003). GST activity was determined using the method of Habig et al (1974).

Samples (1.0 ml) contained 100 mM potassium phosphate, pH 6.5, 1 mM GSH and 1 mM

CDNB at 25 °C. For the assay of MGST1 or microsomal fractions, 0.5% Triton X-100 was

added to the incubation buffer.

GTN biotransformation studies. Chemical modification of cytosolic GSTs or MGST1 was

performed by incubation of enzyme in 100 mM potassium phosphate (pH 7.0) with 1.0 mM

NEM for 1 min or 2 mM peroxynitrite for 10 seconds at room temperature, and with 2 mM

GSNO for 10 min, or 5 mM H2O2 for 30 min at 37°C (Ji et al., 2002; Ji and Bennett, 2003; Aniya

and Anders, 1992). To assess GTN biotransformation, 5 µg purified cytosolic or MGST1 was

incubated with the indicated concentrations of GTN and 1 mM GSH at 37°C for 60 min in a final

volume of 0.5 ml. Some samples were pre-treated with 1 mM NEM prior to assessment of GTN

biotransformation. The GTN metabolites, 1,2-GDN and 1,3-GDN were quantitated by gas

chromatography with electron capture detection as described (McDonald and Bennett, 1990).

Denitration rates were corrected for the nonenzymatic formation of GDNs (ca 5% of total

metabolites)

SDS-PAGE and immunoblot analysis. Rat liver and aortic microsomes from control and

GTN-tolerant animals were resolved on a 15% SDS-PAGE gel under non-reducing conditions.

Proteins were transferred electrophorectically to PVDF membranes and then incubated with

specific anti-serum against rat liver MGST1 (1:5000). The immunoreactive protein bands were

visualized by enhanced chemiluminescence.

Northern analysis. Total tissue RNA was isolated from rat liver or aorta using Qiagen

RNeasy spin columns according to the manufacturer’s recommendations. Total RNA (10-20 µg)


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was resolved on 1% agarose gels containing 1.1% formaldehyde and was transferred to nylon

membranes (Hybond N+) by overnight capillary blotting. Membranes were prehybridized in

Quickhyb solution for 30 min at 68°C and then hybridized for 2 h at 68°C with cDNA probes

32P-radiolabelled with [32P]-dCTP (6000 Ci/mmol) by random priming. The 599 bp MGST1

cDNA probe was generated by an RT-PCR procedure using rat liver as the source of RNA.

Membranes were exposed to a phosphor storage screen at -70 °C for 2 hours, scanned with a

Bio-Rad Molecular Imager FX (Bio-Rad Laboratories Ltd, Mississauga, ON, Canada) and

quantitated using Corel Photo-Paint version 8 software. After quantitation of MGST1 mRNA,

membranes were stripped and reprobed with an 18S rRNA probe to correct for variations in

RNA loading.

Data Analysis. Data was analyzed by the appropriate statistical test as indicated. A p value

of 0.05 or less was considered statistically significant.


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Results

GTN biotransformation studies. Purified MGST1 and cytosolic GSTs both catalyzed the

denitration of GTN to the 1,2- and 1,3-GDN metabolites (Fig. 1, A and B) and exhibited

selectivity for the formation of 1,3-GDN. The specific activity of the MGST1 for GTN

denitration was at least 2-3 fold greater than the rat liver cytosolic GSTs, with the greatest

differences observed at low GTN concentration (1 µM). It is well documented that NEM

markedly activates MGST1, but partially inhibits cytosolic GST activity, when CDNB is used as

the second substrate. In contrast, although NEM did inhibit GTN biotransformation by the

cytosolic GSTs, it also inhibited GTN denitration by the microsomal enzyme (Fig. 1, A and B).

The derived kinetic parameters for the GTN denitration reaction mediated by MGST1 were

calculated using the data in Fig. 1 and gave values of 44 nmol/min/mg protein for Vmax and 49

µM for Km.

Effect of oxidants and nitrating reagents on microsomal and cytosolic GST activity.

Structural modifications of the MGST1 result in increased activity towards the classical GST

substrate, CDNB, and we wished to determine whether activity towards GTN was altered in a

similar manner. As seen in Table 1, treatment of the MGST1 with a variety of modifying

reagents resulted in an increase in activity towards CDNB, whereas when GTN was used as

substrate, the denitration activity of the modified enzyme was decreased (with the exception of

GSNO-treated enzyme). The most dramatic difference in activity occurred after alkylation of

Cys49 by NEM. In this case, activity towards CDNB was increased 12-fold whereas activity

towards GTN was decreased by almost 90%. For the cytosolic GSTs, all of the protein

modifying reagents inhibited cytosolic GST activity towards both CDNB and GTN, albeit to

differing degrees. Another striking difference between the two substrates was the greater specific


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activity of the cytosolic GSTs for CDNB relative to the MGST1, whereas for GTN the opposite

was true.

Hepatic microsomal and cytosolic GST activities in GTN-tolerant animals. To determine

whether chronic exposure to GTN results in changes in hepatic GST activity, we assessed

enzyme activity in subcellular fractions of the livers from naïve and GTN-tolerant animals. For

the cytosolic fraction, activity towards both CDNB and GTN was reduced by about 25% in

preparations from GTN-tolerant animals. In contrast, GTN biotransformation was unaltered in

microsomes from GTN-tolerant animals, and activity in control and tolerant microsomes was

inhibited to the same degree after treatment of microsomes with NEM (Table 2). Using CDNB as

substrate, however, enzyme activity was increased more than 2-fold in microsomes from GTN-

tolerant animals, although the maximal activation after treatment of microsomes with NEM was

the same for the two microsomal preparations. Since it has been suggested that

oxidative/nitrosative stress occurs in GTN tolerance one might predict that there would be

increased lipid peroxidation as a consequence of chronic GTN exposure. We assessed the level

of lipid peroxidation, as measured by TBARS levels, and found no difference between

microsomes from naïve and GTN-tolerant animals (0.20 ± 0.05 and 0.18 ± 0.04 nmol TBARS/

mg protein, respectively, p > 0.05, unpaired t-test, n=3).

Immunoblot and Northern analysis of MGST1. Immunoblot and Northern blot analyses

were performed to determine whether chronic exposure to GTN resulted in changes in MGST1

expression. The data in Figs. 2 and 3 indicate that the hepatic MGST1 was down-regulated at the

mRNA level in GTN-tolerant animals. There was approximately a 60% reduction in mRNA

levels, whereas the levels both the monomeric and dimeric forms of the enzyme were essentially

unchanged. We also assessed MGST1 protein in aortic microsomes and found that there was a


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reduction in the dimeric form of the enzyme in microsomes from GTN-tolerant animals and also

a reduction in the levels of MGST1 trimer. However, in contrast to the data from liver, there was

an increase in the levels of MGST1 monomer in aortic microsomes from GTN-tolerant animals.

The levels of aortic MGST1 mRNA from GTN-tolerant animals were somewhat variable, and

although the average level of expression was doubled, this increase was not statistically

significant.


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Discussion

In the present study we wished to assess the relative rate of GTN biotransformation by

MGST compared to the cytosolic GSTs and to determine whether MGST1 activity or

expression is altered in GTN-tolerant animals. As seen in Fig. 1, the rate of denitration of GTN

by MGST1 was significantly greater than that of the cytosolic GSTs, especially at lower, more

pharmacologically relevant concentrations of GTN. Thus at an initial substrate concentration of

1 µM, the rate of denitration by MGST1 was 3-fold greater than that by the cytosolic GSTs.

Both enzyme preparations exhibited regioselectivity for GTN denitration, with selective

formation of the 1,3-GDN metabolite. Treatment of the cytosolic GSTs with the alkylating

agent NEM resulted in a decrease in GTN denitration (Fig. 1 A and B) and a parallel decrease

in the enzyme activity towards CDNB (Table 1). However, in marked contrast to the increase in

MGST activity towards CDNB after alkylation of Cys49 by NEM (Table 1), the GTN

denitration activity of MGST1 activity towards GTN was inhibited markedly by NEM (Fig. 1 A

and B).

Activation of MGST1 by covalent modification of Cys49 is a characteristic that distinguishes

the microsomal GST from its cytosolic counterparts (Morgenstern and DePierre, 1983). The

MGST1 is thought to exist as a homotrimer in the native state. The trimer binds one molecule of

GSH and altered communication between subunits is believed to play a role in enzyme activation

by agents such as NEM. The formation of the thiolate consists of fast and slow components, with

rapid binding of GSH followed by slower formation of thiolate (Svensson et al, 2004).

Modification of Cys49 by NEM increases the rate of thiolate formation and increases kcat. The

activation of MGST1 by NEM with respect to CDNB conjugation is in marked contrast to the

inhibition of GTN biotransformation after alkylation of the enzyme with NEM, and suggests a


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differential interaction of the two substrates with the enzyme. In previous studies examining the

kinetic properties of the NEM-activated enzyme, it was found that an increase in the rate of

catalysis occurred only with certain substrates and that with others it was unaltered, and that this

was related to the relative reactivity of substrate (Morgenstern and DePierre, 1983, Morgenstern

et al., 1988). Although GTN is a substrate for the enzyme, the specific activity for denitration is

quite low compared to the rate of the conjugation reaction with CDNB. However, this would not

explain the observed decrease in enzyme activity after treatment with NEM.

Another possible mechanism for the inhibitory effect of NEM could be that there is a direct

interaction of the organic nitrate with the free SH groups of the enzyme resulting in denitration,

and that this interaction is prevented by alkylation of the sulfhydryl group by NEM. We

examined whether other modifications of the enzyme resulted in changes in activity towards

GTN and as seen in Table 1, treatment of the MGST1 with reagents that would alter Cys49 also

inhibited GTN denitration activity. Thus a direct interaction of GTN with Cys49 seems possible.

Tolerance to GTN after chronic exposure in vivo is associated with attenuation of the

vasodilator responses to GTN, and also decreases in the vascular biotransformation of GTN

(Ratz et al 2000a). In addition, pattern of GTN metabolite formation in the whole animal is

altered in GTN-tolerant animals (Ratz et al., 2002). To assess whether chronic GTN exposure

might alter the contribution of MGST1 to the overall biotransformation of GTN, we compared

the activity and expression of MGST1 in hepatic microsomes from naïve and GTN-tolerant

animals. The levels of MGST1 mRNA were significantly reduced in the livers of GTN-tolerant

animals, although the level of expression of MGST1 protein was unchanged (Figs. 2 and 3).

However, the levels of MGST1 protein did not correlate well with enzyme activity towards

CDNB. With respect to GTN, there was no change in the rate of metabolite formation whereas


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for CDNB conjugation, GST activity was actually increased despite the fact that MGST1 protein

expression was unchanged. This would suggest that the chronic exposure to GTN resulted in

some sort of structural or conformational change in the enzyme that resulted in an increase in

catalytic activity towards CDNB but not to GTN. An alternative explanation could be that the

expression of microsome-associated cytosolic GSTs is increased during chronic GTN exposure

and that this accounts for the increase in GST activity. It is noteworthy that the expression of the

class alpha GST present in human hepatic microsomes is increased following exposure of

cultured human hepatoblastoma (HepG2) cells to the prooxidant, hydrogen peroxide (Prabhu et

al., 2004). With respect to the cytosolic GSTs, biotransformation of GTN was decreased in the

hepatic cytosolic fraction from GTN tolerant animals, as was the enzyme activity of the cytosol

towards CDNB. A similar degree of inhibition of CDNB conjugation was found after incubation

of purified enzyme preparations with GTN (Lee and Fung, 2003).

As was observed with the purified MGST1, treatment of hepatic microsomes with NEM

markedly inhibited GTN denitration, and this occurred to a similar extent in microsomes from

control and GTN-tolerant animals. In contrast, in a previous study it was found that incubation of

hepatic microsomes with the alkyl nitrites, amyl nitrite and n-butyl nitrite, resulted in the

formation of GSNO, and that treatment of microsomes with NEM increased the rate of GSNO

formation (Ji et al., 1996). These data suggest that subtle changes in substrate structure have

profound effects on catalysis by MGST1, and that the interaction of organic nitrites and organic

nitrates with MGST1 is fundamentally different.

It was not technically feasible to generate sufficient quantities of aortic microsomes to assess

changes in MGST activity. However, we were able to assess changes in aortic MGST1

expression after chronic GTN exposure. In contrast to the findings in liver, mRNA levels were


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unchanged in aortae from GTN-tolerant animals and levels of the MGST1 monomer were

increased rather than unchanged (Fig. 4 and 5). In situ, MGST1 is thought to exist as a

homotrimer (Lundqvist et al., 1992), although on both reducing and non-reducing SDS-PAGE

gels the enzyme migrates primarily as the 17 kDa monomer. As seen in the immunoblots of

hepatic and aortic microsomes from naïve animals, immunoreactive bands are evident at

mobilities consistent with MGST1 dimers, and in the aorta as trimers as well, suggesting that a

proportion of monomers are covalently linked to one another. However, in aortic microsomes

from GTN-tolerant animals there was clear reduction in the dimeric and trimeric forms of the

enzyme as seen on SDS-PAGE gels. The functional significance of this finding is uncertain, but

the observation that the relative proportions of the enzyme in dimeric or trimeric forms is altered,

suggests that chronic exposure to GTN changes the association properties of the MGST1

monomer.

Using the data in Table 1 we derived a kcat/ Km value for GTN of 754 M-1·S-1 for MGST1 and

263 M-1·S-1 for the cytosolic GST mixture, about a 3-fold difference. In comparison, the kcat/Km

value for rat hepatic cytochromes P450-mediated GTN biotransformation in hepatic microsomes

under aerobic and anaerobic conditions is 390 M-1·S-1and 14,000 M-1·S-1, respectively

(McDonald and Bennett, 1990), and that for purified recombinant human ALDH2 (Li et al.,

2006) is 1390 M-1·S-1. Thus the catalytic efficiencies of four enzymes known to biotransform

GTN are within a 5-fold range of each other (with the exception of cytochromes P450 under

anaerobic conditions).

With respect to the overall quantitative contribution of these enzymes to GTN

biotransformation, this also would be significantly influenced by the relative abundance of

different biotransformation enzymes in a particular tissue. Microsomal protein accounts for about


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20% of the protein in rat hepatocytes (DePierre and Dallner 1975) and MGST1 comprises about

3% of microsomal protein (Morgenstern et al., 1982). On the other hand, the cytosolic GSTs

make up about 5% of the cytosolic protein, and thus if one takes into account the higher activity

but lower abundance of the MGST1 versus the lower activity and higher abundance of the

cytosolic GSTs, the relative contributions would be within a two-fold range of each other. The

cytochromes P450 content of microsomes is about double that of MGST1 and the kcat/Km value

(under aerobic conditions) is about half, and therefore their relative contribution would be about

the same as MGST1. However, since GTN biotransformation by cytochromes P450 is markedly

greater under anaerobic conditions, the relative contribution of cytochromes P450 would be

expected to be sensitive to changes in the intracellular O2 concentration. Mitochondrial protein

accounts for about 7% of rat hepatocyte protein (Colbeau et al. 1971), and although ALDH2 has

a relatively high kcat/Km value, its abundance would be low because of its mitochondrial location.

There are other factors that complicate extrapolation of enzyme reaction rates to the in vivo

situation. The lipophilic nature of GTN would predict its preferential subcellular distribution to

membranes, and thus better access to membrane-bound biotransformation enzymes, and in

addition, the rate of GTN biotransformation is markedly reduced in broken cell preparations

compared to that in intact cells (Bennett et al., 1994). With respect to the functional significance

of MGST1-mediated GTN biotransformation, we have found that in a stably transfected cell line

that overexpresses rat MGST1, there was an increase in GTN-induced cGMP formation

compared to wild type cells, suggesting that the MGST1 can mediate the mechanism-based

biotransformation of GTN to an activator of guanylyl cyclase (unpublished observations).

In summary, we have demonstrated that MGST1 can catalyze the denitration of GTN, and

that it does so with greater efficiency than its cytosolic counterparts. Chemical modification of


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MGST1 has differential effects on the catalytic activity towards different substrates, and chronic

GTN exposure appears to cause structural modifications to the enzyme that while having no

effect on the biotransformation of GTN, result in increased activity towards other MGST1

substrates.


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Acknowledgments

The authors wish to thank Mrs. Diane Anderson for technical assistance.


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Footnotes

This work was supported by grant MOP 37873 from the Canadian Institutes of Health Research.

Send reprint requests to: Dr. Brian M. Bennett, Department of Pharmacology and Toxicology, Queen’s University, Kingston, Ontario, Canada K7L 3N6. E-mail: [email protected]


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Legends for Figures

Fig 1. Denitration of GTN catalyzed by GSTs. The indicated concentrations of GTN were

incubated aerobically with 1.0 mM GSH and 5 µg purified hepatic MGST1 or a mixture of

hepatic cytosolic GSTs, at 37 ºC for 60 minutes. Some samples were pre-treated with 1 mM

NEM at room temperature for 1 min. 1,3-GDN (A) and 1,2-GDN (B) were determined by gas

chromatography. Data points represent the mean ± SEM (n=4-6). NEM significantly decreased

the rate of 1,3-GDN and 1,2-GDN formation by both cytosolic GSTs and MGST1 at all GTN

concentrations, with the exception of 1,3-GDN formation at 50 µM GTN with MGST1 (p< .05,

Student’s t-test).

Fig 2. Immunoblot analysis of hepatic MGST1 from control and GTN-tolerant rats.

Purified MGST1 (10 ng) or hepatic microsomal protein (2 µg) from four control and four GTN-

tolerant rats were resolved on a 15% SDS-PAGE gel under non-reducing conditions, transferred

to PVDF membranes, and probed with a rabbit polyclonal antibody to rat MGST1.

Fig 3. Northern blot analysis of rat liver MGST1. Northern analysis of rat hepatic RNA (10

µg total RNA per lane) from three control and three GTN-tolerant animals. The signal ratio of

MGST1/18S rRNA was reduced by about 60% in livers from GTN-tolerant animals (p<0.05,

Student’s t-test for unpaired data).

Fig 4. Immunoblot analysis of aortic MGST1 from control and GTN-tolerant rats. Purified

MGST1 (0.12 µg) or aortic microsomal protein (30 µg) from four control and four GTN-tolerant


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rats were resolved on a 15% SDS-PAGE gel under non-reducing conditions, transferred to PVDF

membranes, and probed with a rabbit polyclonal antibody to rat MGST1.

Fig 5. Northern blot analysis of rat aortic MGST1. Northern analysis of rat aortic RNA (20

µg total RNA per lane) from four control and three GTN-tolerant animals. The signal ratio of

MGST1/18S rRNA was about 2-fold higher in aortae from GTN-tolerant animals but this was

not significantly different to control.


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TABLE 1. Comparison of rat liver microsomal and cytosolic GST activities towards CDNB and

GTN. Enzyme activities of purified microsomal and cytosolic GSTs towards CDNB (1.0 mM,

n=5) or GTN (1.0 µM, n=3) were assessed after modification by various reagents as described in

“Methods”. The GSH concentration was 1.0 mM in all assays. Data are the mean ± SD of 3-5

determinations of the purified enzyme preparations. enzyme activity (µmol/min/mg protein) modifying reagents modification CDNB GTN (× 10-4) cytosolic GSTs: non-treated 2 mM GSNO 5 mM H2O2 2 mM ONOO- 1 mM NEM MGST1: non-treated 2 mM GSNO 5 mM H2O2 2 mM ONOO- 1 mM NEM

N/A S-nitrosylation S-oxidation, S-glutathiolation S-oxidation, tyrosine nitration alkylation N/A S-nitrosylation S-oxidation, S-glutathiolation S-oxidation, tyrosine nitration alkylation

21 ± 3 10 ± 2 16 ± 3 2.5 ± 0.7 12 ± 1 1.0 ± 0.2 1.8 ± 0.1 2.8 ± 0.4 5.1 ± 0.5 11 ± 2

3.4 ± 0.4 1.5 ± 0.1 0.61 ± 0.07 0.51 ± 0.030.82 ± 0.2

8.8.7 ± 0.6 8.9 ± 0.7 2.8 ± 0.9 3.9 ± 0.5 1.0 ± 0.02

All values were significantly different to non-treated (p<0.05, one way ANOVA and Dunnett’s

multiple comparisons post-hoc test), except for GSNO treatment of MGST1.


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TABLE 2. Comparison of hepatic GST activities towards CDNB (1.0 mM) and GTN (1.0 µM)

in cytosolic and microsomal fractions from three control and three GTN-tolerant rats. Enzyme

activities towards CDNB and GTN were assessed using 1.0 mM GSH. Data are the mean ± SD.

enzyme activity (µmol/min/mg protein) CDNB GTN (x 10-6) Cytosol: Control 0.82 ± 0.01 17 ± 1 Tolerant 0.66 ± 0.02a 12 ± 1a Microsomes: Control 0.043 ± 0.006 34 ± 3 Control +NEM 0.174 ± 0.004b 2.5 ± 0.5b Tolerant 0.091 ± 0.005b 31 ± 2 Tolerant +NEM 0.171 ± 0.007b 3.8 ± 0.6b

a p<0.05 versus control, unpaired Student’s t-test. b p<0.01 versus control, one way ANOVA and

Newman-Keuls post-hoc test.


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