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NEUROSCIENCE |
1 Wolfson Institute for Biomedical Research, University College London, Cruciform Building, Gower Street, London WC1E 6BT, UK
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Abstract |
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(Received 31 July 2006;
accepted after revision 8 September 2006;
first published online 14 September 2006)
Corresponding author C. N. Hall: Wolfson Institute for Biomedical Research, University College London, Cruciform Building, Gower Street, London WC1E 6BT, UK. Email: catherine.hall{at}ucl.ac.uk
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Introduction |
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Protection against NO-induced toxicity and control over the spatial and temporal spread of NO under physiological conditions necessitates regulation of the NO concentration, which is dictated by the rates of synthesis and loss. The synthesis pathway, through nitric oxide synthase (NOS) is relatively well characterized at the enzyme level (Alderton et al. 2001), but how the NO signal is terminated remains unclear. Reaction with oxyhaemoglobin in erythrocytes in nearby blood vessels is likely to play a role (Lancaster, 1994; Liu et al. 1998a) and diffusion away from the site of synthesis has also been considered to be important (Wood & Garthwaite, 1994; Lancaster, 1994). The reaction of NO with oxygen in aqueous solution, termed autoxidation, is too slow to be of physiological importance but partitioning of the reactants into lipid membranes may accelerate the process sufficiently to contribute to tissue NO breakdown (Liu et al. 1998b). The reaction of NO with superoxide (O2–) is almost diffusion limited (Kissner et al. 1997) but is limited physiologically by high concentrations of superoxide dismutase (SOD) which scavenges O2– (Beckman & Koppenol, 1996; Wink & Mitchell, 1998), such that this reaction is likely to be of greater significance in pathophysiological situations when O2– production is enhanced, for example during leakage of electrons from the respiratory chain during reperfusion following ischaemia, or from NADPH oxidase in activated microglia (Sankarapandi et al. 1998). NO also reacts at an almost diffusion-limited rate with lipid peroxyl radicals (O'Donnell et al. 1997), which are generated at increased rates during pathological conditions (Moosmann & Behl, 2002) and which account for a component of NO consumption by acutely prepared brain cell suspensions and brain homogenates (Keynes et al. 2005a). Enzymatic NO consumption by lipoxygenases and prostaglandin H synthase (PGHS) in reticulocytes and platelets (O'Donnell et al. 1999, 2000) and unidentified flavohaemoprotein(s) in mammalian cell lines (Gardner et al. 2001; Hallstrom et al. 2004), endothelial cells (Schmidt & Mayer, 2004) and cultured cerebellar glia (Keynes et al. 2005b) have also been reported.
All the above studies of NO inactivation have relied on dispersed tissue preparations and the relevance of any of the processes to intact brain has not been examined. Brain slices, which retain the balance of cell types and connectivity of the intact brain, provide a better model of intact brain than do isolated cells. Indirect evidence supports the existence of NO consumption processes in intact brain tissue, as organotypic hippocampal slice cultures are resistant to levels of NO that are toxic to dispersed cells (Keynes et al. 2004), but no direct evidence for slice NO consumption has yet been reported. Here, we report that cerebellar slices rapidly inactivate NO by a mechanism that is independent of lipid peroxidation and other known mechanisms of NO consumption. The apparent kinetics of NO inactivation predict that this inactivation process will be influential in shaping physiological NO signals when several sources are active.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Cerebellar slice preparation
Experiments used brain tissue from 8-day-old Sprague-Dawley rats. The animals were killed by decapitation as approved by the British Home Office and the local ethics committee. Sagittal slices of cerebellum (400 µm thick) were prepared using a McIlwain tissue chopper. Slices were incubated in shaking, gassed (95% CO2–5% O2) artificial cerebrospinal fluid (aCSF) at 37°C containing (mM): NaCl 120, KCl 2, NaHCO3 26, MgSO4.7H2O 1.19, KH2PO4 1.18, glucose 11, CaCl2 2, L-nitroarginine 0.1 and kynurenic acid 1. After 1 h recovery, slices were transferred to kynurenic acid-free aCSF. All slice experiments were carried out in gassed aCSF at pH 7.45 at 37°C. In the relevant experiments, slices were preincubated for 30 min with the lipid peroxidation inhibitors Trolox and diethylenetiaminepentaacetic acid (DTPA) or for 15 min with sodium cyanide (NaCN) and diphenyleneiodium chloride (DPI).
NO measurement
For NO measurements, samples (1 ml) were incubated in a stirred vessel (at 37°C) equipped with an NO electrode (ISO-NOP, World Precision Instruments, Stevenage, UK). The chamber was open and samples were continuously gassed over their surface with 95% CO2–5% O2. NO was delivered using the NONOate donors spermine NONOate (Sper/NO), 2-(N,N-diethylamino)-diazenolate-2-oxide, diethylammonium salt (DEA/NO) and (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO; all Alexis Biochemicals, Nottingham, UK). Stock solutions at 100 times the desired final concentration were prepared in 10 mM NaOH and were kept on ice prior to use. Experiments on tissue blocks and homogenates were carried out in the presence of 1000 U ml–1 superoxide dismutase, 100 µM DTPA and 100 µM Trolox.
Haemoglobin–agarose preparation
Haemoglobin (Hb)-coated beaded agarose (Hb–agarose) was used to scavenge bath NO. A 50% v/v suspension was prepared by washing by fltration (0.4 µm pore) with 20 volumes of Tris-HCl (pH 7.4) and resuspension in this same buffer. The Hb was reduced using 10 mM sodium dithionite, which was removed by filtration and washing with more than 50 volumes of aCSF. Finally, the Hb–agarose was resuspended in aCSF to make a 50% v/v suspension and kept on ice until use. Hb reduction was confirmed spectrophotometrically, by observation of the shift in the Soret peak at 412–415 nm (Feelisch et al. 1996). Immediately before the beads were used, the aCSF was aspirated and replaced with freshly gassed aCSF at 37°C.
cGMP measurement and visualization
Slices were inactivated by immersion in boiling buffer (50 mM Tris-HCl and 4 mM EDTA, pH 7.4) and then sonicated. A sample was taken for protein determination and the remainder was centrifuged at 10 000 g for 5 min at 4°C. cGMP in the supernatant was measured by radioimmunoassay. Protein concentrations were measured by the bicinchoninic acid method (BCA Protein Assay Kit, Pierce, IL, USA). cGMP levels were normalized to the response to 100 µM DEA/NO, which generates a maximum cGMP response in a cerebellar slice (data not shown).
Visualization of cGMP was accomplished by immunocytochemistry (de Vente et al. 1998). Slices were fixed for 2 h in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature (21-24°C). Cryoprotection was achieved by overnight incubation in 20% sucrose solution in the phosphate buffer at 4°C. The tissue was then frozen in Tissue-Tek OCT (Raymond Lamb, Eastbourne, UK). Frozen sections (10 µm thick) were cut perpendicular to the plane of the slice and collected on chrome alum/gelatin-coated microscope slides. Slides were rinsed in Tris-buffered saline (TBS) with 0.1% Triton X-100 (pH 7.6) and incubated first with normal donkey serum for 30 min, then with the primary sheep anti-cGMP antibody overnight at 4°C (1: 8000, a gift from J. de Vente, University of Maastricht, the Netherlands). After rinsing, the sections were incubated with donkey anti-sheep biotinylated secondary antibody for 1 h at room temperature (1: 200). Slides were washed and incubated with Vectastain elite ABC complex for 45 min, stained for 4 min with 0.05% 3,3'-diaminobenzidine then counterstained with Mayers haemalum for 15 s. Finally, slides were air-dried and mounted in DPX mounting medium.
Lipid peroxidation assay
Each cerebellar slice was inactivated by addition to 400 µl ice-cold trichloroacetic acid (10% w/v). After homogenization by sonication and then centrifugation at 2000 g for 10 min, thiobarbituric acid reactive species (TBARS) were detected according to a published protocol (Esterbauer & Cheeseman, 1990). Briefly, 300 µl supernatant, 10 µl butylated hydroxytoluene (BHT, 10% w/v) and 300 µl thiobarbituric acid were mixed and heated to 90°C for 20 min, cooled to room temperature and 200 µl transferred to a 96-well plate. Absorbance at 532 and 600 nm was measured spectrophotometrically. The ratio of the difference of the absorbances at 532 and 600 nm to that at 600 nm was compared to that generated from malonaldehyde standards.
Modelling
All partial differential equations were solved using the pdepe function in MATLAB 6.5 (The Mathworks Inc., Natick, MA, USA). All other modelling used Mathcad 2001i (Mathsoft, Bagshot, UK).
NO profile across the slice
Diffusion of NO into the slice from the bathing medium approximates to one-dimensional diffusion from two infinite planar sources of NO, as the vast majority of NO entering the slice will be from the slice surfaces rather than the edges. As such, it can be described by Fick's second law of diffusion. The overall change in concentration over time at a certain position in the slice (x) is given by the net of diffusion and inactivation by the slice. Were the inactivation process first order with respect to NO, this would be described by eqn (1), if it were Michaelis-Menten in nature, eqn (2) would apply.
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| (1) |
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| (2) |
cGMP profile across the slice and predicted NO concetration–cGMP response curves
The cGMP concentration at each position in the slice can be calculated from the NO concentration from the Hill equation for NOGC (eqn (3)). Normalizing cGMP levels to the maximum that can be produced (cGMPmax
= 1), the concentration at which it is half-maximum (KGC) is 1.6 nM and the slope (n) is 2 (parameters were found by fitting the NO–cGMP data in Fig. 1C with eqn (3)). Normalization means that cGMP becomes a dimensionless variable, representing the fraction of the maximum cGMP concentration that is produced.
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| (3) |
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A complication of the method using steady-state NO delivery is that Sper/NO may enter the slice and release NO within the tissue, increasing cGMP production above that predicted here. The measured cGMP (cGMPtotal), expressed as a fraction of the maximum, is therefore given by eqn (4), where cGMPbathNO is that predicted from eqns (2 and 3) to be produced by diffusion of NO into the slice from the bathing solution and cGMPdonorNO is that produced from NO released from the donor within the slice.
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| (4) |
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| (5) |
If the simplifying assumptions are made, that (i) a given part of the slice is either empty or full of cGMP and (ii) the donor is equally distributed throughout the slice, then the fraction of the tissue that has no cGMP is given by 1 (the normalized maximum cGMP produced in the slice) minus the cGMP produced by diffusion of NO from the bath (cGMPbathNO). The cGMP produced by release of NO from the donor is therefore given by eqn (6).
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| (6) |
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| (7) |
Quantifying NO consumption by tissue blocks
When DETA/NO is added to a suspension of tissue blocks, a number of processes are at play: NO is being released into the suspension at a given constant rate, which is given by eqn (8), where x is the stoichiometry of NO release from DETA/NO (1.6; Griffiths & Garthwaite, 2001) and kDETA/NO is the rate constant of release from DETA/NO, and is given by eqn (9). The half-life (t1/2) for DETA/NO at 37°C is 20.5 h.
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| (8) |
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| (9) |
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| (10) |
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| (11) |
).
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| (12) |
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| (13) |
; eqn (15)), is calculated from the Km and Vmax for NO consumption, the diffusion coefficient, the ‘radius’ of a block (R; 200 µm) and the external NO concentration ([NO]o).
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| (14) |
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| (15) |
can be used to find by reading from published graphs. V'max can be calculated from eqn (12) and the protein concentration which would give this level of inactivation can be calculated from eqn (13). Plots can therefore be constructed of NO plateau versus protein concentration, as for the experimental data. Modelling autoxidation
If inactivation of NO by the slice is due to autoxidation, its concentration throughout the slice will be given by eqn (16).
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| (16) |
Modelling physiological NO profiles
Step-wise activation of neuronal NOS (100 ms).
The following derivations of the Michaelis-Menten equation describe the generation of the NO signal at various rates of NO synthesis (eqn. 17) and the decline once NO production is halted (eqn. 18).
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| (17) |
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| (18) |
Dynamic activation of NOS.
Sabatini et al. (2002) used measurements of Ca2+ concentrations to simulate Ca2+–calmodulin binding in dendritic spines following synaptic stimulation of NMDA receptors. Assuming that neuronal NOS activation matches that of Ca2+–calmodulin binding during such a Ca2+ transient, neuronal NOS activity was modelled using eqn (19).
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| (19) |
80 ms). Values for maximum synthesis from 0.5 to 2 µM s–1 were used to generate NO profiles which were calculated by finding the net of synthesis and inactivation (eqn (20)).
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| (20) |
Modelling NO signals in three dimensions.
Spatially discrete NO signalling was investigated by modelling diffusion, synthesis and inactivation in three dimensions and constraining NO synthesis to 0.5 µm diameter ‘terminal boutons’ (dimensions in Palay & Chan-Palay, 1974). The density of parallel fibre synapses in rat cerebellum is just under 109 synapses µm–3, or approximately 1 synapse µm–3 (Napper & Harvey, 1988). Assuming that neuronal NOS is distributed uniformly within but constrained to boutons, then half of the total tissue volume is capable of producing NO, such that the maximum rate of synthesis within a bouton will be 1.6 µM s–1, rather than 0.8 µM s–1. The relevant equation (eqn (21)) describes radial diffusion away from a central point, where NO synthesis (v1) is positive within boutons and zero outside them.
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| (21) |
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Results |
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Cerebellar slices inactivate NO
Constant concentrations of NO were generated by adding the NO donor, Sper/NO to aCSF. The half-life of the donor (39 min at 37°C) is long compared with the duration of the experiment, so that NO was effectively released at a constant rate. Within 10 min, the NO concentration in the gassed aCSF reached a plateau (Fig. 1A) when the rate of NO breakdown by reaction with oxygen equals the rate of release. By adding slices to different concentrations of Sper/NO (after 10 min pre-equilibration), different constant concentrations of NO could be administered.
When slices were exposed to varying NO concentrations in this way, cGMP rose to form steady state levels after 1 min (Fig. 1B). The steady states, graded to the NO concentration, arise because of the combination of NOGC receptor desensitization and a slow hydrolysis of cGMP by phosphodiesterases (Bellamy et al. 2000; Mo et al. 2004). Most subsequent experiments were carried out with cGMP at steady state (2 min incubations).
Immunohistochemical staining of the cross-section of a cerebellar slice for cGMP revealed that at intermediate bath NO concentrations (100 nM) the edges of the slice stained but the centre did not, signifying that NO failed to access this region in active concentrations (Fig. 2B). A concentration gradient therefore existed across the slice, as predicted for tissue NO inactivation. The thickness of the band of cGMP staining was roughly uniform, indicating that the mechanism by which NO is inactivated has a grossly similar distribution throughout the cerebellar layers. Incubation with higher concentrations allowed NO to penetrate the whole slice in concentrations sufficient to activate NOGC receptors maximally, as indicated by robust and uniform cGMP staining throughout the slice thickness (Fig. 2C). No cGMP staining was apparent in the absence of NO application (Fig. 2A). Longer incubations (up to 8 min) had no effect on the patterns of staining at different NO concentrations (data not shown), consistent with cGMP being at steady state (Fig. 1B).
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1 µM compared to 1.6 nM), as expected for access of NO to the slice interior being substantially hindered by NO inactivation. Role of lipid peroxidation
In cerebellar cell suspensions, NO consumption is predominantly the result of ascorbate and iron together initiating the formation of lipid peroxyl radicals, which avidly react with NO (O'Donnell et al. 1997; Keynes et al. 2005a). To determine whether this process accounts for NO consumption in cerebellar slices, we tested the transition metal chelator, DTPA and the vitamin E analogue, Trolox (Britt et al. 1992), both of which inhibit lipid peroxidation-dependent NO consumption in the cell suspensions (Keynes et al. 2005a). Slices were stimulated with both a sub-EC50 concentration of NO (240 ± 30 nM NO, n = 10; generated from 10 µM Sper/NO), and a maximal concentration (100 µM DEA/NO). With a 30 min preincubation period, neither inhibitor significantly affected the resultant cGMP levels (Fig. 3A). Equally, there was no significant difference between control slices and those incubated with Trolox when endogenous NO synthesis was stimulated by exposure to 100 µM NMDA (Fig. 3B). Some slices were incubated with Trolox from the time of slice preparation, which caused a reduction in cGMP accumulation in response to the submaximal NO concentration (Fig. 3C). This is indicative of an increase, rather than a decrease in inactivation, suggesting oxidative stress reactions such as lipid peroxidation impair rather than cause slice NO consumption.
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Role of superoxide ions or red blood cells
As slices do not inactivate NO by the same mechanism that predominates in dispersed cells, we investigated other known mechanisms for NO breakdown. A contribution of the reaction of NO with superoxide ions (forming peroxynitrite) was tested by incubating slices with 1000 U ml–1 SOD or 200 µM of the cell-permeable SOD analogue, Mn(III)tetrakis(4–benzoic acid)porphyrin (MnTBAP).
Only MnTBAP significantly increased the accumulation of cGMP (Fig. 4A). The NO concentration generated (by 10 µM Sper/NO) in the presence of 200 µM MnTBAP, however, was increased more than 2-fold compared to control (Fig. 4B). This increase occurred even in the presence of SOD, and SOD alone did not affect the NO profile generated by 10 µM Sper/NO (not shown). This indicated that the increased NO production in the presence of MnTBAP was not due to scavenging of superoxide, but instead suggested that MnTBAP increased the rate of release of NO from the donor. In support of this hypothesis, the initial rate of release of NO was greater in the presence of MnTBAP (0.57 µM min–1) than in aCSF alone (0.10 µM min–1). The cGMP generated in the presence of MnTBAP was entirely as predicted from the resulting increased NO concentration (Fig. 4C). This, together with the lack of effect of SOD, suggested that superoxide does not contribute significantly to NO inactivation.
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Role of known NO-consuming enzymes
The involvement of PGHS was addressed by preincubating slices with 20 µM indomethacin, which inhibits PGHS-mediated NO consumption by activated platelets (O'Donnell et al. 2000; Fig. 4D). There was no significant effect on NO-evoked cGMP accumulation in the slices. Lipoxgenases have also been implicated in NO breakdown (O'Donnell et al. 1999; Coffey et al. 2001) but the effect of inhibition of these enzymes with the usual inhibitor, 5,8,11,14-eicosatetraynoic acid (ETYA; O'Donnell et al. 1999; Coffey et al. 2001), could not be tested as ETYA quenched NO release from 10 µM Sper/NO. This quench was relieved by addition of 1000 U ml–1 SOD, indicating that ETYA caused generation of superoxide ions, which reacted with NO (data not shown). Generation of superoxide in the slices would cause artefactually high NO consumption, confounding interpretation of any inhibitory effect on biological NO consumption. In any case, lipoxygenases are also inhibited by Trolox (Panganamala et al. 1977), presumably by reaction with the enzyme-bound lipid peroxyl radical that consumes NO (O'Donnell et al. 1999). As Trolox does not effect NO inactivation (Fig. 3A), lipoxygenases are unlikely to underlie slice NO consumption.
Effect of cyanide
NO consumption by intact and homogenized colorectal cancer (CaCo-2) cells, endothelial cells, cerebellar glia and forebrain synaptosomes has been found to be at least partially inhibited by the haem poison cyanide (NaCN) and the flavoprotein inhibitor DPI (Gardner et al. 2001; Schmidt & Mayer, 2004; Keynes et al. 2005b). Incubation with these compounds at concentrations that have maximal effects in dispersed preparations (100 µM NaCN and 50 µM DPI) had no effect on cGMP accumulation in response to 10 µM Sper/NO or 100 µM DEA/NO (Fig. 5A). It has previously been shown that 100 µM NaCN reduces brain slice ATP levels to 69% of control levels (Banay-Schwartz et al. 1974). A concentration of 50 µM DPI is sufficient to inhibit NO and cGMP production in response to 100 µM NMDA (vehicle-treated slices, 243 ± 19 pmol cGMP (mg protein)–1; 50 µM DPI-treated slices, 12 ± 12 pmol cGMP (mg protein)–1; n = 8). The lack of an inhibitory effect is therefore not due to a lack of slice permeability of these compounds. The lack of cyanide sensitivity was not due to the indirect method of gauging NO levels. Chopping sagittal cerebellar slices additionally in the coronal plane produces tissue blocks that retain good histological preservation (Garthwaite et al. 1980) and an intact NMDA receptor–cGMP pathway (Garthwaite, 1985) but their reduced dimensions mean that they can be held in suspension by fast stirring, allowing direct measurement of NO levels using an electrochemical probe. Different numbers of blocks were added to the probe chamber and NO accumulation in response to the slow NO releaser, DETA/NO (100 µM) and then NaCN (100 µM) recorded (Fig. 5B). As the concentration of blocks was increased, the plateau NO concentrations generated in the presence of DETA/NO became progressively lower (Fig. 5B) because of tissue NO consumption. Addition of 100 µM NaCN once the plateau had been achieved had no effect. When the blocks were homogenized, however, NO consumption was increased by addition of NADPH and this increase was inhibited by NaCN (Fig. 5C), as previously observed in homogenates of whole brain, synaptosomes and cultured glial cells (Keynes et al. 2005b). Intact brain preparations therefore consume NO in a qualitatively different manner from homogenates of the same tissue.
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The extent to which the NO consumption described here contributes to physiological and pathological effects of NO depends on the kinetics. By using diffusional modelling it is possible to derive values for the inactivation rates that would account for the experimental data. The model was a simple one, in which slices are treated as homogenous slabs into which NO diffuses in a Fickian manner, and which consume NO by a process that is describable either by a first-order decay (corresponding to a fixed half-life) or by saturable (Michaelis-Menten) kinetics (for details of modelling see Methods).
The rate of NO consumption determines the steepness of the NO concentration gradient existing across the slice thickness at steady state (Fig. 6A). At each point, the NO concentration can be translated into a cGMP level (Fig. 6B) by reference to the NO concentration–cGMP response curve obtained at steady state (see eqn (3) in Methods). Then, the total slice cGMP can be calculated as a fraction of the maximum attainable, giving predicted concentration–response curves at different rates of tissue NO inactivation (Fig. 6C and D). When first-order decay was considered the curves were much shallower than the experimental data (Fig. 6C). A better fit was obtained by assuming saturable inactivation kinetics with the Michaelis-Menten parameters being Vmax
2 µM s–1 and Km
100 nM but the predicted NO concentration–cGMP response curve was still shallower than that found experimentally (Fig. 6D). In reality, the slice is exposed not only to NO diffusing from the bath, but also from NO released within the tissue itself, from donor that has diffused into the slice (Fig. 7). Both corrected and uncorrected curves overlay at low NO concentrations, but at higher NO concentrations, the corrected curves are steeper than the uncorrected curves, giving an improved fit to the experimental data (Fig. 6D, dashed lines). NOGC desensitizes and any effect of this was assessed by incorporating an exponential time-dependence (
= 6.9 s) into the maximal NOGC activity, such that it followed the desensitization kinetics previously described (Bellamy et al. 2000). This had no impact on the NO concentration–cGMP response curves, indicating that desensitization does not occur before the steady-state NO levels are established. Varying the Km while fixing the Vmax (2 µM s–1), generated a family of curves that converged at around 1 nM (Fig. 8A), with the experimental data fitting a Km of between 1 and 10 nM. Fixing the Km at 1 nM, the data are again best fitted by an inactivation process with a Vmax of 1–2 µM s–1 (Fig. 8B). In short, the experimental data are consistent with a saturable tissue consumption process having a Vmax of 1–2 µM s–1 and a Km of 1–10 nM.
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Influence of accelerated autoxidation
With the model, the potential significance of reaction of NO with oxygen (autoxidation) within the tissue can be discerned. In aqueous phases, this process is slow but it may be accelerated as much as 13-fold in lipid due to increased solubility of NO in this phase (Liu et al. 1998b). However, incorporating NO breakdown by autoxidation into the diffusional model above (see Methods) showed no observable difference between the curves using kinetics for normal autoxidation and for accelerated autoxidation (Fig. 8D), implying a negligible impact.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The identity of this process remains unclear, but it seems independent of several known pathways for NO breakdown, namely reaction with haemoglobin in red blood cells, superoxide, molecular oxygen and lipid peroxyl radicals or catalytic consumption by PGHS and lipoxygenase. Its insensitivity to NaCN and DPI also suggest it is different from the flavohaemoprotein(s) postulated to consume NO in several dispersed cell preparations (Gardner et al. 2001; Schmidt & Mayer, 2004; Keynes et al. 2005b).
The basic observation that exposure of cerebellar slices to NO results in a marked gradient of NO at steady state (as judged by the profile of cGMP immunostaining) is as predicted from a high rate of NO inactivation, a property confirmed by direct measurement of NO consumption by intact blocks of cerebellum kept in suspension. This being so, the steady-state NO concentration–cGMP response curve for NO in whole slices is dictated much more by the rate of NO consumption than by the kinetics of activation of NOGC receptors. The situation with NO therefore becomes very similar to that applying to the neurotransmitter glutamate whose inward diffusion into brain slices is limited by the activity of transporters to the extent that very high external concentrations are needed to activate NMDA receptors throughout the tissue (Garthwaite, 1985). In both cases (glutamate and NO), the shift in apparent potency is about 1000-fold compared with their potencies measured using isolated cells, signifying that the rates of inactivation relative to their physiological concentrations are similar. This implies that the NO consumption mechanism may be of comparable importance in protecting the brain from high NO concentrations and for shaping physiological NO signals.
Without identifying it or having the means to alter its activity, the physiological significance of the NO inactivation mechanism is hard to address experimentally. The problem is compounded by the difficulties in reliably measuring endogenous NO signals in intact tissues (for discussion see Keynes & Garthwaite, 2004). Nevertheless, a number of predictions can be made from the apparent kinetics of the process derived by modelling, which suggested that NO consumption is saturable, with a Vmax of 1–2 µM s–1 and a Km of 1–10 nM.
Most previous models of physiological NO signals assumed that micromolar concentrations were achieved (Wood & Garthwaite, 1994; Lancaster, 1994; Philippides et al. 2000) and that the biological half-life of NO in tissue was about 5 s, based on measurements of the rate of loss of NO as it was perfused over preparations of aorta (Palmer et al. 1987). One prediction was that NO from a single source could influence a very large number of synapses (2 million, Wood & Garthwaite, 1994) and that tissue inactivation of NO would only minimally affect NO profiles, unless multiple sources were active simultaneously. A recent paper by Garthwaite (2005) predicts physiological NO concentrations to be in the low nanomolar range, in keeping with more recent biochemical and electrophysiological estimates (see discussion by Hall & Garthwaite (2005) and Garthwaite (2005)), but does not address tissue consumption.
Because the basic features of a physiological NO signal (e.g. its amplitude and duration) are not known, we were guided by the experimentally measured maximum rates of NO synthesis in cerebellar tissue and considered three different scenarios: homogenous NO synthesis in a tissue volume activated in a step-wise fashion; transient NO synthesis in a tissue volume mirroring the time course of a synaptic NMDA receptor-mediated rise in cytosolic Ca2+; and, finally, spatially limited NO synthesis in dispersed boutons.
The cerebellum has one of the highest levels of NOS in the brain, the maximal rate of NO formation in homogenates amounting to 50 nmol min–1 (g tissue)–1 (Salter et al. 1995) or, assuming 1 ml (g tissue)–1, 0.8 µM s–1. In the first two scenarios, NOS rates of around this value were assumed to be occurring within a discrete tissue volume wherein multiple sources of NO are simultaneously active, a situation that may be analogous to the event-related activation of discrete brain regions in vivo (Buckner & Koutstaal, 1998; Shoham et al. 1999). It is further assumed that NO inactivation is homogeneous, consistent (at a gross level) with the results in cerebellar slices (Fig. 2B). If global NO synthesis is prolonged and occurs at a rate less than the Vmax for inactivation (2 µM s–1), it is predicted that the NO-consuming mechanism rapidly translates a given synthesis rate into a steady-state NO concentration (Fig. 9A) and thus a corresponding degree of activity of the NOGC receptor. When the concentration on cessation of synthesis is within the presumed physiological range (<10 nM). NO disappears with a half-life of less than 10 ms.
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Finally, we modelled NO synthesis from discrete boutons (0.5 µm diameter), chosen to resemble parallel fibre terminals, a major source of NO in the cerebellum (Shibuki & Kimura, 1997). This approach also allows assessment of the relative contributions of diffusion and NO consumption to spatial NO profiles.
In all cases, the derived peak NO concentration scaled with the rate of NO synthesis (not shown), as has been previously deduced (Philippides et al. 2005). With a single source active, inactivation was predicted to have a minimal influence on the NO profile, which was solely determined by diffusion (Fig. 10A). This seemed to be because with such a small source, diffusion was sufficiently fast for NO to reach a steady state before inactivation could impact upon the concentration. Slowing the diffusion rate constant increased the impact of inactivation (not shown). A single bouton produced only low picomolar levels of NO, even at presumed maximal rates of NO synthesis, suggesting that release from a single site would be insufficient to produce a biological effect. When a larger source was simulated (a whole granule cell; diameter, 10 µm), the kinetics of inactivation influenced the concentration and spatial extent of the NO profile (Fig. 10B), constraining the NO signal closer to the cell body than when inactivation was absent. However, the NO concentrations achieved were still in the low picomolar range, suggesting that more than one NO-producing cell would have to be active at a given time for a biologically relevant NO signal to be produced.
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These conclusions are not consistent, however, with reports of NO-dependent synaptic plasticity at a single parallel fibre–Purkinje cell synapse (Casado et al. 2002). Two possible explanations for this discrepancy are that, firstly, NO synthesis from adjacent boutons, or within the parallel fibre axon, can summate to generate physiologically significant NO concentrations, such that the parallel fibre represents a much bigger source than a single bouton. Secondly, it is possible that the NO synthesis rate used in the simulations is too low. The rates used here were based on experimental data from cerebellar homogenates (Salter et al. 1995), but variations in tissue NOS distribution, cofactor availability and oxygenation could result in endogenous synthesis rates that are higher or lower than those used here. In this respect it is notable that the maximum measured rate of purified neuronal NOS is only 3–4 molecules s–1 at 10°C (Stuehr et al. 2004), a rate that, assuming it to be 4- to 8-fold higher at 37°C, corresponds to a maximum synthesis rate of about 12–32 molecules (NOS molecule) s–1. A recent model (Garthwaite, 2005) used a maximal source strength of 20 000 molecule s–1 as a point source (equivalent to 1000 synaptic NOS molecules each synthesizing 20 (molecules NO) s–1 at a steady rate). The same steady-state source strength distributed evenly in a bouton equates to a synthesis rate of 500 µM s–1 and generates a scaled-up profile of those in Fig. 10A (not shown). This will produce 3 nM NO at the surface of the bouton and 150 pM at 5 µm from the bouton centre, a profile that is again purely determined by diffusion, not inactivation. It may therefore be possible, depending on physiological conditions and the in vivo kinetics of NOS, to generate sufficient NO at a single site to produce a physiological effect. At more distant sites, however (e.g. neighbouring synapses), NO would be too low to be physiologically relevant, even with this high rate of synthesis, and summation of NO signals may be required. In these circumstances, the kinetics of inactivation will critically determine the sphere of influence of NO, constraining it closer to the active tissue.
In summary, cerebellar slices consume NO by an unidentified mechanism, with kinetics that predict that it will significantly impinge on endogenous NO signals that derive from multiple sites. Modulation of this process and its subcellular distribution may well affect the spatial and temporal patterns of NO signals and therefore influence the properties of physiological and pathophysiological NO profiles. Further characterization and identification of this mechanism is clearly of paramount importance to fully understand its impact. There is also a practical implication of the results in that avid NO inactivation by slices generates a sharp concentration gradient across the slice thickness when exogenous NO is administered. Even at applied NO concentrations that are clearly submaximal for cGMP synthesis therefore the responses generated by the slice will be very heterogeneous, being in the pathophysiological concentration range (0.1–1 µM) at the edge of the slice (e.g. producing inhibition of mitochondrial respiration and associated events), and physiological (or subphysiological) towards the centre of the tissue. This heterogeneity needs to be considered when designing and interpreting experiments using exogenous NO.
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