FEBS 20196
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`FEBS Letters 427 (1998) 225^228
`
`Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite
`to nitric oxide under hypoxic conditions
`
`Timothy M. Millara, Cli¡ R. Stevensa;*, Nigel Benjaminc, Robert Eisenthalb,
`Roger Harrisonb, David R. Blakea
`
`aBone and Joint Research Group, Department of Postgraduate Medicine, University of Bath, Claverton Down, Bath, BA2 7AY, UK
`bDepartment of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK
`cClinical Pharmacology, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, Charterhouse Square, London, EC1M 6BQ, UK
`
`Received 2 April 1998
`
`Abstract Xanthine oxidoreductase (XOR) catalyses the reduc-
`tion of the therapeutic organic nitrate, nitroglycerin (glyceryl
`trinitrate, GTN), as well as inorganic nitrate and nitrite, to nitric
`oxide (NO) under hypoxic conditions in the presence of NADH.
`Generation of nitric oxide is not detectable under normoxic
`conditions and is inhibited by the molybdenum site-specific
`inhibitors, oxypurinol and (3)BOF 4272. These enzymic
`reactions provide a mechanism for generation of NO under
`hypoxic conditions where nitric oxide synthase does not function,
`suggesting a vasodilatory role in ischaemia.
`z 1998 Federation of European Biochemical Societies.
`
`Key words: Xanthine oxidoreductase; Nitric oxide; Nitrate;
`Nitrite
`
`1. Introduction
`
`Nitric oxide (NO) is widely recognised as mediating the
`relaxation of smooth muscle in vasodilation and as initiating
`many other important biological functions, including inhibi-
`tion of platelet aggregation and adhesion [1,2]. Its generally
`accepted physiological source is NO synthase [3], a complex
`enzyme which is totally dependent on oxygen for its activity
`and consequently ine¡ective in a hypoxic environment, where
`the vasodilatory properties of NO might be seen to be advan-
`tageous.
`Organic nitrates have been used therapeutically for over 100
`years in the treatment and prophylaxis of angina pectoris [4].
`These drugs, which include glyceryl trinitrate (GTN) and iso-
`sorbide dinitrate (ISDN), have a potent vasodilator activity,
`the most likely mechanism for which is reduction to nitric
`oxide in the endothelium and/or vascular smooth muscle [5].
`The mechanisms by which organic nitrates are reduced in vivo
`to NO have never been fully explained. Endogenous sulphy-
`dryl groups have been implicated in the generation of S-nitro-
`sothiols, which subsequently break down to produce NO [6,7].
`Enzymic metabolism has also been proposed, involving gluta-
`thione-S-transferases [8,9] or members of the cytochrome
`P-450 family of enzymes [10,11]. On the other hand, evidence
`has been presented showing that glutathione-S-transferases
`are capable of reduction of nitrate to nitrite but not to NO
`[12].
`Xanthine oxidoreductase (XOR) is a complex molybdo£a-
`
`*Corresponding author. Fax: (44) (1225) 323847.
`E-mail: c.r.stevens@bath.ac.uk
`
`Abbreviations: XOR, xanthine oxidoreductase; GTN, glyceryl trini-
`trate; NO, nitric oxide
`
`voprotein, which has been studied as the essentially pure en-
`zyme for almost 60 years [13]. In addition to molybdenum
`and FAD,
`it contains two iron-sulphur redox centres and
`has a wide substrate speci¢city, typically hydroxylating pu-
`rines and concomitantly reducing either NAD(cid:135) (dehydrogen-
`ase form, EC 1.1.1.204) or molecular oxygen (primarily the
`oxidase form EC 1.1.3.22) [14]. The latter reaction generates
`the reactive oxygen species, superoxide anion and hydrogen
`peroxide, and it is this ability, with its implications for ischae-
`mia-reperfusion injury, that has led to the enzyme’s becoming
`a focus of research activity over the last two decades [15,16].
`Much less well known is XOR’s capacity to reduce inorganic
`nitrate to nitrite under conditions of low oxygen tension [17^
`19]; a property that is perhaps not surprising in view of the
`enzyme’s many similarities with the assimilatory nitrate reduc-
`tases of fungi, algae and higher plants. Like XOR, the nitrate
`reductases contain both molybdenum and FAD redox centres
`and utilise NAD(P)H as reducing substrate [20].
`We now report that, under hypoxic conditions and in the
`presence of NADH, XOR is capable of catalysing the reduc-
`tion of GTN, as well as inorganic nitrate and nitrite, to NO.
`We believe that these ¢ndings, coupled with the vascular lo-
`cation of the enzyme, suggest a role for XOR not only in the
`metabolism of GTN but also as a source of NO derived from
`endogenous nitrate and nitrite, under ischaemic conditions
`ranging from sub-normoxia to anoxia when NO synthase
`does not function.
`
`2. Methods and materials
`
`2.1. Materials
`Bovine xanthine oxidase (1.4 U/mg) was obtained from Biozyme,
`Blaenavon, UK. GTN (David Bull Laboratories, Warwick, UK), in
`injectable form, was diluted in 100 mM potassium phosphate bu¡er,
`pH 7.2, containing 0.9% NaCl (PPB) to give a stock concentration of
`10 mM. Inorganic nitrate (KNO3) and nitrite (NaNO2) (Sigma, Poole,
`UK) were treated in the same manner. NADH (Sigma) was diluted in
`PPB to a stock concentration of 1 mM. Oxypurinol (Sigma) and
`(3)BOF-4272 (Otsuka Pharmaceutical Factory, Japan) were made
`up in PPB also at stock concentrations of 1 mM. All solutions were
`freshly prepared on the day of use and maintained on ice until re-
`quired.
`
`2.2. Determination of nitric oxide
`Nitric oxide determinations were made by using an ozone chemilu-
`minescence assay in a continuous £ow apparatus (Sievers NOA 280)
`that allows the real time analysis of NO production. The apparatus
`was modi¢ed to allow a constant stream of nitrogen to £ow into the
`reaction chamber. Chemiluminescence data were collected by a data
`acquisition system; the mean NO produced in parts per billion (ppb)
`was calculated from readings taken every second and shown as NO
`ppb/s.
`Progress curves, of molar production of NO against time, were
`
`0014-5793/98/$19.00 (cid:223) 1998 Federation of European Biochemical Societies. All rights reserved.
`PII S 0 0 1 4 - 5 7 9 3 ( 9 8 ) 0 0 4 3 0 - X
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`226
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`T.M. Millar et al./FEBS Letters 427 (1998) 225^228
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`calculated by taking into account the gas £ow and successively inte-
`grating the ppb/s curves. Reactions were carried out in a ¢nal volume
`of 1 ml at 37‡C in an atmosphere of 6 1% oxygen (Stathkelvin oxy-
`gen electrode).
`
`2.3. Determination of inhibition constants
`In inhibition experiments, Ki values were determined by ¢tting to
`the inhibition function i = I/I+C where I is the concentration of in-
`hibitor, C is a constant and i = 1-vi/vo, in which vo = uninhibited rate,
`vi = inhibited rate. Assuming competitive inhibition, C = Ki(1+S/Km).
`Here S is the concentration of the competing substrate (GTN) and Km
`is its Michaelis constant [21].
`
`3. Results
`
`3.1. XOR-catalysed reduction of GTN to NO
`At low oxygen tension, NO is generated when XOR is in-
`cubated in the presence of GTN and NADH. A typical rate
`vs. time pro¢le is shown in Fig. 1, in which it can be seen that,
`after a lag phase, a steady state rate (represented by the pla-
`teau) is achieved. Fig. 1 also illustrates that introduction of air
`abolished NO generation. Steady-state rates were shown to be
`directly proportional to enzyme concentration. Generation of
`NO was found to be dependent on each component of the
`reaction mixture, without any one of which NO production
`was not detectable. The reaction followed Michaelis-Menten
`kinetics (Fig. 2) with apparent Km and Vmax values for GTN
`of 0.33 (cid:254) 0.05 mM and 1.83 (cid:254) 0.08U1037 mol/min/mg, respec-
`tively.
`Substitution of xanthine for NADH as reducing substrate
`under the above conditions gave no detectable NO produc-
`tion. The molybdenum site-directed XOR inhibitors, oxypuri-
`nol [22] and (3)BOF-4272 [23] inhibited, in a dose-dependent
`manner, NO production catalysed by XOR in the presence of
`GTN and NADH. Xanthine and, to a lesser extent, hypoxan-
`thine similarly inhibited NO generation. Ki values determined
`as described in the Section 2 are oxypurinol, 2.8U1037 M;
`(3)BOF-4272, 3.2U1038 M; xanthine, 2.5U1037 M; hypo-
`xanthine, 2.5U1036 M. Representative semilogarithmic plots
`
`Fig. 2. Hanes-Woolf plot of NO production catalysed by XOR in
`the presence of GTN and 300 WM NADH. Experimental conditions
`are described in the legend to Fig. 1 for a given concentration of
`GTN; progress curves were derived as outlined in Section 2. Inset
`shows the Michaelis-Menten curve ¢tted to the data.
`
`of relative rates vs. inhibitor concentration for (3)BOF-4272,
`xanthine and oxypurinol are displayed in Fig. 3.
`
`3.2. XOR-catalysed reduction of inorganic nitrate and nitrite
`to NO
`XOR catalysed the reduction of both inorganic nitrate and
`nitrite to NO in the presence of NADH. Reduction of nitrate
`followed Michaelis-Menten kinetics (Fig. 4), giving apparent
`Km and Vmax values of 0.29 (cid:254) 0.6 mM and 9.7 (cid:254) 0.3U1038
`mol/min/mg, respectively. In the case of nitrite reduction,
`the data, ¢tted to a Michaelis-Menten hyperbola (Fig. 5, in-
`set), gave operational apparent Km and Vmax values of
`22.9 (cid:254) 8.1 mM and 3.73 (cid:254) 0.72U1036 mol/min/mg, respec-
`tively. However, the Hanes-Woolf linear plot (Fig. 5) shows
`systematic deviation from Michaelis-Menten kinetics.
`
`Fig. 1. Chemiluminescence data showing the time dependence of
`rates of NO production (ppb/s) catalysed by XOR in the presence
`of 3 mM GTN and 300 WM NADH in an atmosphere containing
`6 1% oxygen. Solutions of GTN and NADH, in 100 mM potassium
`phosphate bu¡er, pH 7.4, were mixed and purged with a continuous
`stream of nitrogen over 5 min before addition (arrow) of a similarly
`purged solution of XOR (1 U, 0.71 mg) in the same bu¡er. At the
`time point indicated (arrow) the £ow of nitrogen was replaced by
`air.
`
`Fig. 3. Inhibition, by oxypurinol, (3)BOF-4272, and xanthine of
`NO production catalysed by XOR in the presence of 3 mM GTN
`and 300 WM NADH. Experimental conditions are described in the
`legend to Fig. 1 except that inhibitor, at the appropriate concentra-
`tion, was added immediately prior to addition of enzyme. Inhibi-
`tions are displayed as semilogarithmic plots of percentage inhibition
`vs. inhibitor concentration; vi is the inhibited rate and vo is the rate
`in the absence of inhibitor. The solid lines are drawn using parame-
`ters obtained from the ¢ts of inhibition data as described in Section
`2. Error bars on experimental points represent (cid:254) S.E.M. of dupli-
`cate determinations from each of two experiments.
`
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`T.M. Millar et al./FEBS Letters 427 (1998) 225^228
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`227
`
`4. Discussion
`
`We show that, under conditions of low oxygen tension,
`XOR catalyses the reduction of the organic nitrate, GTN,
`to NO in the presence of NADH.
`The ability of NADH to act as a reducing substrate for
`XOR has long been known [24] but has been little studied
`and is not generally recognised. In air-saturated medium,
`both dehydrogenase and oxidase forms of XOR show
`NADH oxidase activity [25], generating superoxide anion
`and hydrogen peroxide at maximal rates of NADH oxidation
`of approximately 3U1037 mol/min/mg. This compares with a
`maximal rate of 1.8U1037 mol NO/min/mg XOR determined
`at 1% oxygen in the present studies. Considering a XOR con-
`centration of 1034 mg/mg endothelial cell protein (Harrison,
`unpublished data), this rate appoximates to 20 pmol NO/min/
`mg cell XOR protein; a rate comparable with that determined
`by Feelisch et al. [26] in endothelial cells exposed to GTN. In
`view of the likely competition between GTN and molecular
`oxygen for NADH-generated reducing equivalents on XOR, it
`is to be expected that NO production will increase as oxygen
`tension falls further. Conversely, it appears from our results
`that, as normoxic conditions are approached, oxygen com-
`petes increasingly for available electrons.
`By analogy with the nitrate reductases [20] it might be an-
`ticipated that GTN acts at the molybdenum site of XOR. This
`is consistent with our demonstration of inhibition of NO pro-
`duction by oxypurinol [22], (3)BOF-4272 [23]. It is well es-
`tablished that NADH donates electrons and molecular oxygen
`accepts electrons at the FAD site of XOR [14].
`Regarding the pathway of NO production from GTN, the
`present data are consistent with initial reduction to inorganic
`nitrite, although determination of the latter by the commonly
`used Griess reaction [27] was not feasible in the presence of
`NADH as electron donor. Certainly, XOR was found readily
`to catalyse the reduction of nitrite to NO. Rates were 30^50-
`fold faster than those determined with GTN or inorganic
`nitrate as reducing substrates, suggesting that reduction of
`nitrate is the rate-limiting step in NO production in these
`latter cases.
`On the basis of these considerations, a plausible case can be
`made for a role for XOR in metabolism of GTN and of other
`
`Fig. 4. Hanes-Woolf plot of NO production catalysed by XOR in
`the presence of inorganic nitrate and 300 WM NADH. Experiments
`were carried out and analysed as described in the legend to Fig. 2.
`Inset shows the Michaelis-Menten curve ¢tted to the data.
`
`Fig. 5. Hanes-Woolf plot of NO production catalysed by XOR in
`the presence of inorganic nitrite and 300 WM NADH. Experiments
`were carried out and analysed as described in the legend to Fig. 2.
`Inset shows the Michaelis-Menten curve ¢tted to the data.
`
`organic nitrates in vivo (we also observed conversion of ISDN
`to NO by XOR; data not shown). Endothelial cells have been
`identi¢ed as a site of bioactivation of organic nitrates to NO
`[27], a process deemed to be enzymically catalysed [27]. XOR
`is known to be present at relatively high levels in endothelial
`cells [28], in which its enzymic activity has been shown to be
`increased in hypoxia [29,30]. Under conditions of ischaemia,
`NADH levels will rise, and as oxygen tension falls NO gen-
`eration will be increasingly favoured. It is relevant that organ-
`ic nitrates are generally more e¡ective in the venous circula-
`tion than in large coronary arteries or (still less e¡ective) in
`arterioles [1,31], a comparison that re£ects the distribution of
`XOR activity [32]. It is worth noting that hypoxanthine in-
`hibits XOR-catalysed generation of NO, albeit less e¡ectively
`than does xanthine. Concentrations of hypoxanthine are gen-
`erally assumed to be elevated in ischaemia [33,34] and, de-
`pending on the actual levels attained, rates of NO production
`via this route could be a¡ected. However, hypoxanthine lev-
`els, most commonly determined in complete ischaemia, are
`uncertain under conditions of reduced blood £ow [34]. More-
`over, rates of NO generation clearly depend upon oxygen
`tension, itself subject to wide variation in the pathological
`state. It is, accordingly, di⁄cult to predict the magnitude of
`these e¡ects in vivo.
`XOR-catalysed generation of NO under hypoxic conditions
`can be seen as complementary to the activity of NO synthase,
`which requires oxygen [3]. Thus, in ischaemic blood vessels,
`XOR catalyses the metabolism of GTN to NO, which medi-
`ates vasodilation and relief from angina. Under these hypoxic
`conditions meanwhile, NO synthase is induced [35^37] and,
`being dependent on molecular oxygen for its function, is set to
`take over NO production as the oxygen tension rises and
`XOR-catalysed NO production shuts down. This complemen-
`tarity is also relevant in the absence of ingested organic ni-
`trates, when circulating inorganic nitrates and/or nitrites could
`act as oxidising substrates for XOR-catalysed generation of
`NO under ischaemic conditions. Such a role for XOR would
`be consistent with our ¢ndings and with those of Zweier et al.
`who recently reported formation of NO in biological tissues
`that was independent of NO synthase [38].
`
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`Acknowledgements: The Bone and Joint Research Group receives pro-
`gramme grant support from the Arthritis and Rheumatism Council
`for the United Kingdom.
`
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