Protein Mediators Of Biotransformation

Bioactivation of nitrates both to yield NO and to yield cGMP-independent bioactivity requires reduction and oxygen atom transfer. Therefore, an enzyme or protein mediator of bioactivation requires a functional group capable of supplying electrons and/or accepting an oxygen atom; the logical candidates are metaloproteins and protein thiols and sulfides. Several proteins have been identified that are capable of mediating the denitration of GTN, including Hb, Mb, XOR, old yellow enzyme, glutathione ^-transferase (GST), CYP,

NADPH cytochrome P450 reductase (CPR), and aldehyde dehydrogenase type-2 (ALDH2) [119,134,155,181-192]. Most of these proteins were reported in the last century to mediate nitrate biotransformation, and several have been subject to intensive further study in recent years. In all cases, a problem in unambiguous interpretation of both in vivo and tissue studies, of the role of specific proteins in nitrate bioactivation, is the lack of specificity of many enzyme inhibitors.

Nitrate bioactivation (sometimes termed mechanism-based biotransformation [6]) has been intimately linked with nitrate tolerance, the phenomenon that describes the decreased vasodilator and anti-anginal efficacy of nitrates, in particular GTN, after longer term clinical administration. It is reasonably postulated that tolerance occurs when the biological apparatus responsible for reductive bioactivation is exhausted, presumably through oxidation/consumption of reducing equivalents. A role in tolerance is therefore often viewed as a requirement for the nitrate bioactivation mechanism. Denitration of GTN yields the dinitrate metabolites, glyceryl-1,2-dinitrate (1,2-GDN) and glyceryl-1,3-dinitrate (1,3-GDN); regioselectivity being demonstrated in vascular biotransformation for formation of 1,2-GDN, which is lost in tolerant tissue [119,185,189,193-200]. Thus, regioselectivity has been used as a marker in the search and validation of bioactivation mechanisms. The fascinating observation of stereoselective bioactivation of nitrates has been used less often, but has been used to support a role for CPR in nitrate bioactivation [201,202]. In this work, stereoselectivity was lost on addition of the flavoprotein inhibitor DPI. However, DPI shifted GTN dose-response curves to the right in both tolerant and non-tolerant aortic tissue arguing against a dominant role for CPR in vascular bioactivation associated with tolerance [119].

More recent work has supported a function for CPR in nitrate biotransformation [203]. Under anaerobic conditions and in the presence of both NAD(P)H and added thiol, CPR was observed to generate nitrosothiol and NO, the formation of which was inhibited by DPI. The requirement for added thiols implicates the thiol-reactive nitrite, NOGDN, as the product of CPR-mediated bioactivation, and the progenitor of NO. Inhibition of GTN metabolism by DPI is compatible with a role for flavoenzymes in nitrate denitration and possibly bioactivation. Mechanisms may be drawn for the nitrate-dependent conversion of reduced flavin to oxidized flavin in both flavin-dependent dehydrogenases and in flavin-dependent oxidases (Fig. 6). In the oxidase mechanism, the nitrate substitutes for O2, and therefore this pathway may be important in nitrate bioactivation under hypoxic conditions.

Mammalian molybdoenzymes (aldehyde oxidoreductase, sulfite oxidoreductase, and XOR) catalyze oxygen atom transfer and e- transfer: for example, a bacterial nitrate reductase molybdoenzyme is able to catalyze the 2e- reduction of NO- to NO-. The presence of flavin and iron-sulfur clusters in addition to molybdenum redox centres, presents a number of options for nitrate group reduction. Over 50 molybdoenzymes have been identified, however, only XOR has been examined for nitrate bioactivation [118,166,181,192,204-206].

XOR has been frequently used as a source of O2- in vitro and therefore in aerobic solution may function as an NO scavenger. However, a body of work by Harrison's group showed that in anaerobic solutions, XOR mediated the reduction of organic and inorganic nitrates and nitrites and gave NO as product [118,166,181,192,206]. XOR was reported to mediate the NADH or xanthine dependent: (a) 2e- reduction of nitrates to NO2- at the flavin-site; (b) 1e- reduction of NO2- to NO at the Mo site; and (c) 1e- reduction of nitrites to NO at the flavin site. GTN, ISDN, and ISMN, were observed to be substrates for reduction under anaerobic conditions, generating a low flux of NO, but XOR was inactivated in the process. A more recent study of the XOR-mediated reduction of GTN and ISDN confirmed aspects of this work, but importantly, NO was not detectable as a product without the addition of thiols or ascorbate [207]. The thiol dependence and detection of nitrosothiols argue for XOR bioactivation of nitrates to nitrites, followed by non-enzymatic trapping by thiols to generate a nitrosothiol that itself generates NO. It was remarked that the maximum rate of NO production observed was less than 10% of that of nitrite hydrolysis to NO-. These observations are in accordance with the rapid trapping of nitrites by thiols and the rapid hydrolysis of NOGDN, the nitrite intermediate formed from GTN, which was first reported in 1994 [173]. XOR itself catalyzes NO2 reduction to NO at the Mo site [166,206], however, a leading role for XOR in vascular nitrate bioactivation is not supported by observations that the XOR inhibitor, allopurinol, has no influence on tissue relaxation [119]. A role for XOR in ischemia-reperfusion injury has often been cited and the ability of XOR to generate cytoprotective levels of NO and nitrosothiols in ischemia may be relevant.

Haemoproteins represent prime candidates for mediating nitrate bioactivation, because nitrates, nitrites, and related molecules have been demonstrated to coordinate with and be reduced at Fe-haem sites: (a) GTN reacts at the ferrous-haem of haemoglobin and myoglobin to give GDN; and (b) GTN is rapidly consumed by deoxyHb to give NO- [134,208]. CYP isoforms have often been proposed to mediate GTN bioactivation to NO, but it has recently been reported that the reduction of NO- to NO by CYP is a minor contributor to GTN bioactivation [203]. Mixed metaloprotein-sulfhydryl pathways have been proposed [9]. Doyle reported Hb-mediated reduction of nitrites to NO, including via a mixed haem-sulfhydryl pathway involving nitrosation of the Hb-^-93 cysteine thiol residue [117]. Amixed haemoprotein-sulfhydryl pathway would involve initial reduction to a nitrite, however to date, only NO2- production from Hb-mediated reactions of GTN has been reported.

The high affinity NO receptor, sGC, responsible for mediating the cGMP-dependent bioac-tivity of NO, is itself a ferrous haemoprotein [209,210]. Containing 10-24 cysteines per subunit, the various dimeric isoforms of sGC are also cysteine rich, suggesting possible sulfhydryl or mixed pathways of reaction of GTN with sGC itself. Given the oxidative chemistry of nitrates, it would be surprising if sGC was not reactive towards nitrates, especially since high concentrations of nitrates are required for sGC activation in vitro. Furthermore, even at these high concentrations, nitrates do not activate sGC in vitro unless cysteine or certain other thiols are added. In incubations with sGC, the flux of NO generated from simple reaction of GTN with cysteine is too low to account for activation of sGC by NO, unless the flux of NO is increased by reaction with sGC or other proteins in such incubations [120]. In the presence of cysteine, low potency, partial agonism of sGC activity is observed in response to GTN. The concentration-response curve for sGC activation by a true NO-donor NONOate was shifted to the right after preincubation of sGC with GTN. Furthermore at higher concentration, GTN significantly decreased the efficacy of the NO donor [120]. Inhibition of sGC, via haem or thiol oxidation, are likely inhibitory mechanisms for GTN, which is capable of rapid oxidation of deoxy-Hb to met-Hb [134], and oxidation of protein thiols [211,212]. Modification of cysteine residues of the subunit of sGC has been shown to inhibit NO-dependent activation of sGC [213]. Spectroscopic studies of sGC activation by GTN concluded that there was no evidence for a Fe2+-sGC-NO complex, but that there was substantial evidence for Fe-haem oxidation [144].

Several heterocyclic small molecules are able to activate sGC in an NO-independent manner; for example YC-1, that is thought to bind to an allosteric site on sGC [214]. These activators often have modest efficacy, but substantially increase potency and efficacy of the poor sGC activator CO by inhibiting dissociation of the Fe2+-sGC-CO complex [215,216]. YC-1 potentiated the activation of sGC by GTN (in the presence of cysteine) to the same activity levels seen for NO and true NO-donors [120]. The magnitude of the potentiation of GTN activity byYC-1 suggests that GTN activation of sGC could be subject to an endogenous allosteric potentiator. YC-1 has been shown to potentiate GTN-induced tissue relaxation in both naive and tolerant aortic tissue [217]. This phenomenon may be general to nitrates since cGMP accumulation in PC12 cells in response to a hybrid nitrate drug was reported to be profoundly amplified by YC-1 [218].

ALDH2, a major NAD-dependent Phase II metabolic enzyme, essential for alcohol clearance via acetaldehyde dehydrogenation, contains 3 cysteines at the active site and is a candidate for sulfhydryl-dependent nitrate bioactivation. ALDH2 was reported in 1985 to mediate biotransformation of both GTN and ISDN [190], and in 1994 the inactivation of ALDH2 by ISDN and ISMN denitration was further studied [191,219]. ALDH2 activity in hepatic mitochondria shows significant regioselectivity towards 1,2-GDN formation from GTN, which is lost in mitochondria from tolerant tissue [152,155]. Therefore ALDH2 fulfils one of the traditional criteria for an enzyme mediating GTN bioactivation and contributing to nitrate tolerance. In support of this, cyanamide was shown to attenuate the increase in coronary blood flow and hypotension produced by GTN in anaesthesized dogs; an effect ascribed to ALDH2 inhibition [220,221]. Elegant studies by Stamler and co-workers have supported a role for ALDH2 in nitrate bioactivation, for example, aortic tissue from ALDH2 null mice was less responsive to relaxation induced by GTN (< 1 ^M), although relaxation induced by GTN (>1 ^M) and by ISDN was insensitive to the deletion of ALDH2 [222].

Nitrate tolerance can be simulated in animal models by continuous delivery of GTN, in preference to repetitive bolus administration, however, whether this completely models the human clinical condition is problematic [223]. In animal models of GTN tolerance, ALDH2 inhibitors induce a rightward shift in the GTN dose-response curve for tissue relaxation [155], however, rightward shifts were also observed in tolerant tissue that should not be susceptible to inhibitors if inhibition of ALDH2 is the cause of nitrate tolerance [152]. The last observation prompted the conclusion that ALDH2 is able to contribute to nitrate biotransformation, but there is no clear evidence to suggest that ALDH2 provides the primary pathway of bioactivation, nor does attenuation of ALDH2 represent a primary cause of nitrate tolerance.

It is useful to move discussion directly to human studies, since nitrate tolerance is a human clinical phenomenon. The ALDH2*2 allele has a prevalence up to 50% in certain Asian populations, notably Japanese and Chinese, resulting in compromised alcohol clearance and associated presentations. This Glu504Lys point mutation results in 6% enzyme activity in heterozygotes and 0% in homozygotes through perturbed NAD+ binding and Cys319 activation at the active site [224]. Subjects possessing this allele would be predicted to be poorly responsive to GTN and not to manifest symptoms of nitrate tolerance. In Chinese subjects genotyped for ALDH2, the presence of the allele was observed to decrease the responsiveness to sublingual GTN from 86% to 58% [225]. A second study measured forearm blood flow in response to GTN in a group of genotyped East Asians and additionally in a group of subjects administered the ALDH2 inhibitor disulfiram or placebo [226]. The results were internally consistent, in that the response to GTN was blunted by disulfiram and in the ALDH2*2 population, but by less than a factor of two. Both human studies concluded that several different enzymes are involved in the bioactivation of GTN in addition to ALDH2. It was noted that the literature did not indicate clinical abnormalities with respect to GTN in populations with high ALDH2*2 incidences, although compensatory mechanisms may be in place.

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