Biochem. J. (2009) 419, 411–418 (Printed in Great Britain) doi:10.1042/BJ20082419 411 Cytochrome P450 oxidoreductase participates in nitric oxide consumption by rat brain Catherine N. HALL1,2, Robert G. KEYNES1 and John GARTHWAITE Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, U.K. In low nanomolar concentrations, NO (nitric oxide) functions inhibited NO consumption by brain membranes and the amount as a transmitter in brain and other tissues, whereas near-micro- of CYPOR in several cell types correlated with their rate of NO molar NO concentrations are associated with toxicity and cell consumption. NO was also consumed by purified CYPOR but death. Control of the NO concentration, therefore, is critical this activity was found to depend on the presence of the vitamin for proper brain function, but, although its synthesis pathway E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2- is well-characterized, the major route of breakdown of NO in carboxylic acid), included in the buffer as a precaution against brain is unclear. Previous observations indicate that brain cells inadvertent NO consumption by lipid peroxidation. In contrast, actively consume NO at a high rate. The mechanism of this NO consumption by brain membranes was independent of Trolox. consumption was pursued in the present study. NO consumption Hence, it appears that, during the purification process, CYPOR by a preparation of central glial cells was abolished by cell becomes separated from a partner needed for NO consumption. lysis and recovered by addition of NADPH. NADPH-dependent Cytochrome P450 inhibitors inhibited NO consumption by brain consumption of NO localized to cell membranes and was inhibited membranes, making these proteins likely candidates. by proteinase K, indicating the involvement of a membrane- bound protein. Purification of this activity yielded CYPOR (cyto- Key words: brain, cytochrome P450 oxidoreductase (CYPOR), chrome P450 oxidoreductase). Antibodies against CYPOR NADPH, nitric oxide. INTRODUCTION Control of the amplitude and duration of changes in NO concentration is therefore likely to critically affect both the NO (nitric oxide) is an intercellular signalling molecule with a manner in which NO can act physiologically and also whether it role in several neurophysiological functions, including the acute has any pathological effects. The NO concentration experienced modulation of neuronal excitability, the longer-term synaptic by a cell will be determined by the relative rates of NO changes associated with learning, and the development of the synthesis and breakdown but, although the mechanism of NO syn- nervous system [1]. Its major physiological receptor is the NO GC thesis from L-arginine is relatively well characterized, there is (NO-activated guanylyl cyclase, also known by its homogenate- no known dedicated consumption pathway for NO in the brain, based name, soluble guanylyl cyclase), through which it although a number of enzymes have been proposed to fulfil this stimulates the production of the second messenger cGMP. cGMP function in other tissues [10–14]. One such protein is CYPOR has numerous targets, including cyclic nucleotide gated ion (cytochrome P450 oxidoreductase), which is involved in an channels, protein kinases and phosphodiesterases, mediating the extremely avid NO consumption by a colorectal cancer cell line short- and long-term modulations of neuronal function [2,3]. [15]. A process with similar properties [membrane localization These physiological pathways are engaged by low nanomolar and NAD(P)H dependence] has also been reported in cultured concentrations of NO. The dynamic range of the NOGC receptor, endothelial cells [16]. as measured in intact cells, is between 0.1 and 10 nM NO Previous work has revealed that brain tissue actively consumes [4–6], suggesting that this is the range of NO concentrations NO [17–19]. In dissociated brain cells, part of the NO con- normally experienced by cells. Indeed, electrical stimulation sumption was found to be caused by lipid peroxidation, which is of cerebellar brain slices yielded 4 nM NO, as measured by likely to be of particular relevance to pathology, but inhibition of electrodes positioned at the slice surface [7]. Even lower NO lipid peroxidation unmasked another consumption process [18]. concentrations may also be physiologically relevant, as NO- The present study aimed to identify this mechanism. dependent phosphorylation events have been reported after exposure to sub-nanomolar NO concentrations [6]. At higher concentrations, NO may be linked with pathophy- siology. NO inhibits the respiratory chain enzyme, cytochrome MATERIALS AND METHODS c oxidase, with an IC50 of 60–120 nM at physiological oxygen concentrations [4,8], and micromolar NO levels can produce cell All compounds were purchased from Sigma (Poole, U.K.) unless damage via reaction with superoxide and production of the highly otherwise stated. All tissue culture media components were oxidising species peroxynitrite [9]. purchased from Invitrogen (Paisley, U.K.). Abbreviations used: CYPOR, cytochrome P450 oxidoreductase; DETA/NO, diethylenetriamine NONOate; DHEA, dehydroepiandrosterone; DPI, diphenyleneiodonium chloride; DTPA, diethylenetriaminepentaacetic acid; L-NNA, L-nitroarginine; NO, nitric oxide; NOGC, NO-activated guanylyl cyclase; NOS, NO synthase; SOD, superoxide dismutase; Trolox, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid. 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2009 Biochemical Society 412 C. N. Hall, R. G. Keynes and J. Garthwaite NO measurement discarded. The synaptosome pellet was resuspended in incubation For NO measurements, samples (1 ml) were incubated in an buffer using 8 strokes of the Potter homogenizer. openstirredvesselat37◦C equipped with an NO electrode (ISO-NOP, World Precision Instruments, Stevenage, U.K.). NO T47D cells was delivered using DETA/NO [diethylenetriamine NONOate Control T47D cells and those that had previously been (diazeniumdiolate); Alexis Biochemicals, Nottingham, U.K.]. transfected to overexpress CYPOR [20] were a generous gift Stock solutions of DETA/NO were prepared in 10 mM NaOH, from Kaye Williams (School of Pharmacy and Pharmaceutical kept on ice, and diluted 100-fold or more into the experimental Sciences, University of Manchester, Manchester, U.K.). They solution. were cultured in RPMI medium containing 10% foetal calf serum, supplemented with 2 mM glutamine. When confluent, cells were Tissue preparation harvested in the same manner as cultured glia. Animals were killed by decapitation and associated exsanguina- tion, before removal of the brains, except when blood was taken. Lysates and cell fractionation In this case, rats were anaethetised with 5% isoflurane in oxygen Lysates were prepared from the intact suspensions by freezing to at 2 litres/min, and were bled by cardiac puncture before being −20 ◦C, thawing and brief sonication. Lysis was verified visually killed by cervical dislocation. All procedures were in accordance under a light microscope. with the U.K. Home Office guidelines and approved by the local Membrane and cytosolic fractions were prepared by centri- ethics committee. fugation at 53000 rev./min using a TLA-100.2 rotor (Beckman Instruments) for 1 h. The supernatant was retained and membranes Glia were resuspended in incubation buffer by sonication. Protein concentrations of membranes refer to the concentration of protein Glial cultures were prepared as described in [18]. Cultures were in the pre-spun sample. used after 6–10 days in vitro at which stage they were fully confluent. Immunohistochemical staining indicated that 77% Whole brain homogenates and membranes of the cells were astrocytic, 7% neuronal and 16% microglial (results not shown). Membranes from forebrains from 8–12-day-old Sprague–Dawley To prepare the suspension of mixed glia for studies of rat pups were homogenized in 25 mM Tris/HCl, pH 7.45 using NO consumption, dishes were washed with ∼100 ml of cell an Ultraturrax homogenizer. Crude membranes were prepared incubation buffer (20 mM Tris/HCl, 130 mM NaCl, 5 mM KCl, by centrifugation of homogenate at 5 mg protein/ml at 1.2 mM Na2HPO4 and 11 mM glucose, adjusted to pH 7.45 at 53000 rev./min using a TLA-100.2 rotor for 1 h. To prepare 37 ◦C) and incubated with 30 ml of 0.05% (w/v) trypsin, 0.53 mM a purer preparation, homogenates were centrifuged for 1 h EDTA in HBSS (Hanks balanced salt solution) for 15 min at 37 ◦C at 50000 g, the supernatant discarded and the membranes to dissociate the cells, which were washed and resuspended at resuspended and centrifuged for a further 1 h at 50000 g,before 3 × 106 cells/ml in incubation buffer. Cell viability was verified final resuspension in 25 mM Tris/HCl, pH 7.45, at ∼10 mg/ml at more than 95% based on Trypan Blue staining. protein. This dual spin was found to increase the recovered activity more than the single 53000 rev./min (using a TLA-100.2 rotor) − ◦ Platelets and white blood cells (used in Figure 2A) spin (results not shown). Membranes were then stored at 80 C for later use. Platelets and white blood cells were prepared from adult Sprague–Dawley rat blood. Whole blood was collected into acid Solubilization and chromatography citrate dextrose solution (12.5%) and centrifuged at 300 g for 10 min at 20 ◦C. The platelet-rich plasma was removed and the Forebrain membranes (0.8 mg/ml protein) were solubilized by ◦ centrifugation repeated to eliminate residual red and white blood gentle agitation for 1 h at 4 C in buffer containing 25 mM cells from the supernatant. White cells were aspirated from the Tris/HCl, pH 7.45, 100 mM KCl, 10% (v/v) glycerol and top of the pellet and suspended in platelet buffer containing 3 mM dodecyl maltoside (Calbiochem, Nottingham, U.K.). Any 137 mM NaCl, 0.5 mM MgCl2, 0.55 mM NaH2PO4,2.7mMKCl, remaining particulate material was removed by centrifugation at 25 mM Hepes and 5.6 mM glucose, pH 7.45 at 37 ◦C.