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Several toxins are known which account for the ability of some bacteria to initiate or promote carcinogenesis. These ideas are summarised and evidence is discussed for more specific mechanisms involving chymotrypsin and the bacterial chymotryptic enzyme subtilisin. Subtilisin and Bacillus subtilis are present in the gut and environment and both are used commercially in agriculture, livestock rearing and meat processing. The enzymes deplete cells of tumour suppressors such as deleted in colorectal cancer (DCC) and neogenin, so their potential presence in the food chain might represent an important link between diet and cancer. Over-eating increases secretion of chymotrypsin which is absorbed from the gut and could contribute to several forms of cancer linked to obesity. Inhibition of these serine proteases by Bowman-Birk inhibitors in fruit and vegetables could account for some of the protective effects of a plant-rich diet. These interactions represent previously unknown non-genetic mechanisms for the modification of tumour suppressor proteins and provide a plausible explanation contributing to both the pro-oncogenic effects of meat products and the protective activity of a plant-rich diet. The data suggest that changes to farming husbandry and food processing methods to remove these sources of extrinsic proteases might significantly reduce the incidence of several cancers.

Research on the neurobiology of the kynurenine pathway has suffered years of relative obscurity because tryptophan degradation, and its involvement in both physiology and major brain diseases, was viewed almost exclusively through the lens of the well-established metabolite serotonin. With increasing recognition that kynurenine and its metabolites can affect and even control a variety of classic neurotransmitter systems directly and indirectly, interest is expanding rapidly. Moreover, kynurenine pathway metabolism itself is modulated in conditions such as infection and stress, which are known to induce major changes in well-being and behaviour, so that kynurenines may be instrumental in the etiology of psychiatric and neurological disorders. It is therefore likely that the near future will not only witness the discovery of additional physiological and pathological roles for brain kynurenines, but also ever-increasing interest in drug development based on these roles. In particular, targeting the kynurenine pathway with new specific agents may make it possible to prevent disease by appropriate pharmacological or genetic manipulations. The following overview focuses on areas of kynurenine research which are either controversial, of major potential therapeutic interest, or just beginning to receive the degree of attention which will clarify their relevance to neurobiology and medicine. It also highlights technical issues so that investigators entering the field, and new research initiatives, are not misdirected by inappropriate experimental approaches or incorrect interpretations at this time of skyrocketing interest in the subject matter. This article is part of the Special Issue entitled 'The Kynurenine Pathway in Health and Disease'., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Gastro-intestinal (GI) microbiota and the 'gut-brain axis' are proving to be increasingly relevant to early brain development and the emergence of psychiatric disorders. This review focuses on the influence of the GI tract on Brain-Derived Neurotrophic Factor (BDNF) and its relationship with receptors for N-methyl-D-aspartate (NMDAR), as these are believed to be involved in synaptic plasticity and cognitive function. NMDAR may be associated with the development of schizophrenia and a range of other psychopathologies including neurodegenerative disorders, depression and dementias. An analysis of the routes and mechanisms by which the GI microbiota contribute to the pathophysiology of BDNF-induced NMDAR dysfunction could yield new insights relevant to developing novel therapeutics for schizophrenia and related disorders. In the absence of GI microbes, central BDNF levels are reduced and this inhibits the maintenance of NMDAR production. A reduction of NMDAR input onto GABA inhibitory interneurons causes disinhibition of glutamatergic output which disrupts the central signal-to-noise ratio and leads to aberrant synaptic behaviour and cognitive deficits. Gut microbiota can modulate BDNF function in the CNS, via changes in neurotransmitter function by affecting modulatory mechanisms such as the kynurenine pathway, or by changes in the availability and actions of short chain fatty acids (SCFAs) in the brain. Interrupting these cycles by inducing changes in the gut microbiota using probiotics, prebiotics or antimicrobial drugs has been found promising as a preventative or therapeutic measure to counteract behavioural deficits and these may be useful to supplement the actions of drugs in the treatment of CNS disorders.

Understanding the neurochemical basis for cognitive function is one of the major goals of neuroscience, with a potential impact on the diagnosis, prevention and treatment of a range of psychiatric and neurological disorders. In this review, the focus will be on a biochemical pathway that remains under-recognized in its implications for brain function, even though it can be responsible for moderating the activity of two neurotransmitters fundamentally involved in cognition - glutamate and acetylcholine. Since this pathway - the kynurenine pathway of tryptophan metabolism - is induced by immunological activation and stress, it also stands in a unique position to mediate the effects of environmental factors on cognition and behaviour. Targeting the pathway for new drug development could, therefore, be of value not only for the treatment of existing psychiatric conditions, but also for preventing the development of cognitive disorders in response to environmental pressures., (© 2013 The British Pharmacological Society.)

Adenosine A(1) receptor antagonists are of potential value in the treatment of cognitive dysfunction. We have developed compound AJ23 (7-methyl-1-phenyl-1,8-dihydro-pyrazolo-(3,4d)(1,2,4)-triazolo(1,5a)-pyrimidin-4-one) as a novel, non-xanthine based antagonist at A(1) receptors. It has micromolar affinity at human A(1) receptors with a 45-fold selectivity for A(1) over A(2A) receptors and little affinity for many other receptors and transporters tested in a screening panel. AJ23 blocks A(1) receptors in the rat hippocampus, increasing the baseline size of excitatory post-synaptic potentials and blocking the inhibitory effects of adenosine. When administered directly into the rodent hippocampus this compound improves consolidation in a step-down avoidance learning task. The results suggest that AJ23 or derivatives may represent possible leads for further chemical development towards a chemically novel group of antagonists at A(1) receptors with potential value as cognitive enhancers., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

The oxidative pathway for the metabolism of tryptophan along the kynurenine pathway generates quinolinic acid, an agonist at N-methyl-D-aspartate receptors, as well as kynurenic acid which is an antagonist at glutamate and nicotinic receptors. The pathway has become recognized as a key player in the mechanisms of neuronal damage and neurodegenerative disorders. As a result, manipulation of the pathway, so that the balance between the levels of components of the pathway can be modified, has become an attractive target for the development of pharmacological agents with the potential to treat those disorders. This review summarizes some of the relevant background information on the pathway itself before identifying some of the chemical strategies for its modification, with examples of their successful application in animal models of infection, stroke, traumatic brain damage, cerebral malaria and cerebral trypanosomiasis., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Several components of the kynurenine pathway of tryptophan metabolism are now recognised to have actions of profound biological importance. These include the ability to modulate the activation of glutamate and nicotinic receptors, to modify the responsiveness of the immune system to inflammation and infection, and to modify the generation and removal of reactive oxygen species. As each of these factors is being recognised increasingly as contributing to major disorders of the central nervous system (CNS), so the potentially fundamental role of the kynurenine pathway in those disorders is presenting a valuable target both for understanding the progress of those disorders and for developing potential drug treatments. This review will summarise some of the evidence for an important contribution of the kynurenines to Huntington's disease and to stroke damage in the CNS. Together with preliminary evidence from a study of kynurenine metabolites after major surgery, an important conclusion is that kynurenine pathway activation closely reflects cognitive function, and may play a significant role in cognitive ability.

The simple diamine diaminoethane (ethylenediamine, EDA) has been shown to activate GABA receptors in the central and peripheral nervous systems, partly by a direct action and partly by releasing endogenous GABA. These effects have been shown to be produced by the complexation of EDA with bicarbonate to form a carbamate. The present work has compared EDA, GABA and β-alanine responses in rat CA1 neurons using extracellular and intracellular recordings, as well as neocortical evoked potentials in vivo. Superfusion of GABA onto hippocampal slices produced depolarisation and a decrease of field epsps, both effects fading rapidly, but showing sensitivity to blockade by bicuculline. EDA produced an initial hyperpolarisation and increase of extracellular field epsp size with no fade and only partial sensitivity to bicuculline, with subsequent depolarisation, while β-alanine produces a much larger underlying hyperpolarisation and increase in fepsps, followed by depolarisation and inhibition of fepsps. The responses to β-alanine, but not GABA or EDA, were blocked by strychnine. In vivo experiments, recording somatosensory evoked potentials, confirmed that EDA produced an initial increase followed by depression, and that this effect was not fully blocked by bicuculline. Overall the results indicate that EDA has actions in addition to the activation of GABA receptors. These actions are not attributable to activation of β-alanine-sensitive glycine receptors, but may involve the activation of sites sensitive to adipic acid, which is structurally equivalent to the dicarbamate of EDA. The results emphasise the complex pharmacology of simple amines in bicarbonate-containing solutions., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The presence of high concentrations of glutamate in the extracellular fluid following brain trauma or ischaemia may contribute substantially to subsequent impairments of neuronal function. In this study, glutamate was applied to hippocampal slices for several minutes, producing over-depolarization, which was reflected in an initial loss of evoked population potential size in the CA1 region. Orthodromic population spikes recovered only partially over the following 60 min, whereas antidromic spikes and excitatory postsynaptic potentials (EPSPs) showed greater recovery, implying a change in EPSP-spike coupling (E-S coupling), which was confirmed by intracellular recording from CA1 pyramidal cells. The recovery of EPSPs was enhanced further by dizocilpine, suggesting that the long-lasting glutamate-induced change in E-S coupling involves NMDA receptors. This was supported by experiments showing that when isolated NMDA-receptor-mediated EPSPs were studied in isolation, there was only partial recovery following glutamate, unlike the composite EPSPs. The recovery of orthodromic population spikes and NMDA-receptor-mediated EPSPs following glutamate was enhanced by the adenosine A1 receptor blocker DPCPX, the A2A receptor antagonist SCH58261 or adenosine deaminase, associated with a loss of restoration to normal of the glutamate-induced E-S depression. The results indicate that the long-lasting depression of neuronal excitability following recovery from glutamate is associated with a depression of E-S coupling. This effect is partly dependent on activation of NMDA receptors, which modify adenosine release or the sensitivity of adenosine receptors. The results may have implications for the use of A1 and A2A receptor ligands as cognitive enhancers or neuroprotectants.

Human African trypanosomiasis, or sleeping sickness, is caused by the protozoan parasites Trypanosoma brucei rhodesiense or Trypanosoma brucei gambiense, and is a major cause of systemic and neurological disability throughout sub-Saharan Africa. Following early-stage disease, the trypanosomes cross the blood-brain barrier to invade the central nervous system leading to the encephalitic, or late stage, infection. Treatment of human African trypanosomiasis currently relies on a limited number of highly toxic drugs, but untreated, is invariably fatal. Melarsoprol, a trivalent arsenical, is the only drug that can be used to cure both forms of the infection once the central nervous system has become involved, but unfortunately, this drug induces an extremely severe post-treatment reactive encephalopathy (PTRE) in up to 10% of treated patients, half of whom die from this complication. Since it is unlikely that any new and less toxic drug will be developed for treatment of human African trypanosomiasis in the near future, increasing attention is now being focussed on the potential use of existing compounds, either alone or in combination chemotherapy, for improved efficacy and safety. The kynurenine pathway is the major pathway in the metabolism of tryptophan. A number of the catabolites produced along this pathway show neurotoxic or neuroprotective activities, and their role in the generation of central nervous system inflammation is well documented. In the current study, Ro-61-8048, a high affinity kynurenine-3-monooxygenase inhibitor, was used to determine the effect of manipulating the kynurenine pathway in a highly reproducible mouse model of human African trypanosomiasis. It was found that Ro-61-8048 treatment had no significant effect (P = 0.4445) on the severity of the neuroinflammatory pathology in mice during the early central nervous system stage of the disease when only a low level of inflammation was present. However, a significant (P = 0.0284) reduction in the severity of the neuroinflammatory response was detected when the inhibitor was administered in animals exhibiting the more severe, late central nervous system stage, of the infection. In vitro assays showed that Ro-61-8048 had no direct effect on trypanosome proliferation suggesting that the anti-inflammatory action is due to a direct effect of the inhibitor on the host cells and not a secondary response to parasite destruction. These findings demonstrate that kynurenine pathway catabolites are involved in the generation of the more severe inflammatory reaction associated with the late central nervous system stages of the disease and suggest that Ro-61-8048 or a similar drug may prove to be beneficial in preventing or ameliorating the PTRE when administered as an adjunct to conventional trypanocidal chemotherapy.

Adenosine receptors modulate neuronal and synaptic function in a range of ways that may make them relevant to the occurrence, development and treatment of brain ischemic damage and degenerative disorders. A(1) adenosine receptors tend to suppress neural activity by a predominantly presynaptic action, while A(2A) adenosine receptors are more likely to promote transmitter release and postsynaptic depolarization. A variety of interactions have also been described in which adenosine A(1) or A(2) adenosine receptors can modify cellular responses to conventional neurotransmitters or receptor agonists such as glutamate, NMDA, nitric oxide and P2 purine receptors. Part of the role of adenosine receptors seems to be in the regulation of inflammatory processes that often occur in the aftermath of a major insult or disease process. All of the adenosine receptors can modulate the release of cytokines such as interleukins and tumor necrosis factor-alpha from immune-competent leukocytes and glia. When examined directly as modifiers of brain damage, A(1) adenosine receptor (AR) agonists, A(2A)AR agonists and antagonists, as well as A(3)AR antagonists, can protect against a range of insults, both in vitro and in vivo. Intriguingly, acute and chronic treatments with these ligands can often produce diametrically opposite effects on damage outcome, probably resulting from adaptational changes in receptor number or properties. In some cases molecular approaches have identified the involvement of ERK and GSK-3beta pathways in the protection from damage. Much evidence argues for a role of adenosine receptors in neurological disease. Receptor densities are altered in patients with Alzheimer's disease, while many studies have demonstrated effects of adenosine and its antagonists on synaptic plasticity in vitro, or on learning adequacy in vivo. The combined effects of adenosine on neuronal viability and inflammatory processes have also led to considerations of their roles in Lesch-Nyhan syndrome, Creutzfeldt-Jakob disease, Huntington's disease and multiple sclerosis, as well as the brain damage associated with stroke. In addition to the potential pathological relevance of adenosine receptors, there are earnest attempts in progress to generate ligands that will target adenosine receptors as therapeutic agents to treat some of these disorders.

Hypoxic and ischaemic brain damage are believed to involve excessive release of glutamate, and recent work shows that glutamate-induced damage in brain slices can be reduced by preconditioning with hypoxia or glutamate itself. Because adenosine is a powerful preconditioning agent, we have investigated whether adenosine could precondition against glutamate in vitro. In rat hippocampal slices, glutamate depolarization reduced the amplitudes of antidromic- and orthodromic-evoked potentials, with only partial recovery. Applying adenosine before these insults failed to increase that recovery. Ouabain also produced depolarization with partial reversibility, but adenosine pretreatment increased the extent of recovery. The preconditioning effect of adenosine on ouabain responses was prevented by blocking receptors for N-methyl-D-aspartate (NMDA), but not receptors for kainate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and was blocked by inhibiting nitric oxide synthase. Preconditioning was also abolished by the ATP-dependent potassium channel blockers, glibenclamide (cytoplasmic) or 5-hydroxydecanoate (mitochondrial). We conclude that adenosine does not precondition against glutamate in hippocampal slices, but that it does precondition against ouabain with a pharmacology similar to studies in vivo. Ischaemic neuronal damage is a complex of many factors, and because adenosine can precondition against ischaemic neuronal damage, its failure to protect against glutamate highlights limitations of using glutamate alone as a model for ischaemia. Because damage following ischaemia, trauma or excitotoxicity also involves reduced Na(+),K(+)-ATPase activity, and adenosine can precondition against ouabain, we propose that ouabain-induced damage represents an additional or alternative model for the contribution to cell damage of Na(+),K(+)-ATPase loss, this being more relevant to the mechanisms of preconditioning.

MC3T3-E1 osteoblast-like cells represent a suitable model for studying osteogenic development in vitro. The current investigation extends our previous work on the response of these cells to hydrogen peroxide by considering the effects of reactive oxygen species from other sources, and by determining whether differentiation alters sensitivity to oxidative damage. Aspects of hydrogen peroxide-mediated apoptotic and necrotic death were also examined. Cell viability was determined using the Alamar Blue assay; and accompanying morphological changes monitored by phase-contrast microscopy. Sensitivity to hydrogen peroxide increased significantly in cultures which had been induced to differentiate. Hydrogen peroxide and copper (II) ions, when combined, produced greater damage than hydrogen peroxide alone, whilst the hydroxyl radical scavengers mannitol or dimethylsulphoxide had no effect. Cyclosporin A and nicotinamide afforded partial protection. The tryptophan metabolite, 3-hydroxykynurenine significantly reduced viability, although 3-hydroxyanthranilic acid did not. The xanthine/xanthine oxidase system also reduced cell viability, an effect prevented by catalase but potentiated by superoxide dismutase. S-nitroso-N-acetylpenicillamine did not impair viability at the concentrations tested. Cultures were resistant to mitochondrial poisoning by potassium cyanide, but succumbed to 24-h exposures to 3-nitropropionic acid (1 mM). The results reveal a differential sensitivity of MC3T3-E1 cells to hydrogen peroxide-induced oxidative stress, an enhancement of sensitivity by cellular differentiation, and a potential preference for the glycolytic pathway by MC3T3-E1 cells. This study gives new insight into how bone cells may succumb to the toxic effects of oxidative stress generated by different stimuli and has relevance to conditions such as osteoporosis.

Agonists at A(1) receptors and antagonists at A(2A) receptors are known to be neuroprotective against excitotoxicity. We set out to clarify the mechanisms involved by studying interactions between adenosine receptor ligands and endogenous glutamate in cultures of rat cerebellar granule neurons (CGNs). Glutamate and the selective agonist N-methyl-D: -aspartate (NMDA), applied to CGNs at 9 div (days in vitro), both induced cell death in a concentration-dependent manner, which was attenuated by treatment with the NMDA receptor antagonists dizocilpine, D: -2-amino-5-phosphono-pentanoic acid (D: -AP5) or kynurenic acid (KYA), but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Glutamate toxicity was reduced in the presence of all of the following: cyclosporin A (CsA), a blocker of the membrane permeability transition pore, the caspase-3 inhibitor, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (Z-DEVD-fmk), the poly (ADP-ribose) polymerase (PARP-1) inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ), and nicotinamide. This is indicative of involvement of both apoptotic and necrotic processes. The A(1) receptor agonist, N (6)-cyclopentyladenosine (CPA), and the A(2A) receptor antagonist 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazo-5-yl-amino]ethyl)phenol (ZM241385) afforded significant protection, while the A(1) receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and the A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxyamidoadenosine (CGS21680) had no effect. These results confirm that glutamate-induced neurotoxicity in CGNs is mainly via the NMDA receptor, but show that a form of cell death which exhibits aspects of both apoptosis and necrosis is involved. The protective activity of A(1) receptor activation or A(2A) receptor blockade occurs against this mixed profile of cell death, and appears not to involve the selective inhibition of classical apoptotic or necrotic cascades.

The tryptophan metabolite kynurenic acid can block glutamate at ionotropic receptors, but recent evidence suggests a more potent antagonistic action at alpha7 nicotinic receptors for acetylcholine on cultured neurons. The present study examines activity of kynurenic acid at those nicotinic receptors, which mediate cholinergic neurotransmission onto interneurons in the rat hippocampus. Intracellular recordings were made from pyramidal cells and interneurons in the presence of atropine, bicuculline methobromide, (3-aminopropyl)(diethoxymethyl)-phosphinic acid [CGP35348, to block gamma-aminobutyric acid (GABA)(B) receptors] and 3-tropanyl-3,5-dichlorobenzoate (MDL 72222, to block 5-HT3 receptors). In the added presence of glutamate antagonists 2-amino-5-phosphono-pentanoic acid and 6-cyano-7-nitroquinoxaline-2,3-dione, interneurons exhibited a residual excitatory postsynaptic potential (EPSP) that could be blocked by the nicotinic alpha7 receptor blocker methyl-lycaconitine, but not by dihydro-beta-erythroidine which blocks alpha4beta2 receptors. Kynurenic acid reduced the amplitude of these EPSPs with an EC50 of 136 microM. The amplitudes of nicotinic spontaneous miniature EPSPs were also reduced by methyl-lycaconitine and kynurenic acid. The results show that kynurenic acid is more potent in blocking nicotinic EPSPs compared with the full, glutamate-mediated EPSPs, but it was substantially less potent than has been reported in cultures, possibly because of differences in the accessibility of synaptic and extrasynaptic receptors. It is suggested that blockade of nicotinic synaptic transmission may be relevant to the actions of kynurenic acid in the hippocampus, but that in the intact brain this activity is likely to be comparable in importance to the blockade of glutamate-mediated transmission.

Quinolinic acid is an agonist at glutamate receptors sensitive to N-methyl-D-aspartate (NMDA). It has been implicated in neural dysfunction associated with infections, trauma, and ischemia, although its neurotoxic potency is relatively low. This study was designed to examine the effects of a combination of quinolinic acid and the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). Compounds were administered to the hippocampus of anesthetized male rats, animals being allowed to recover for 7 days before histological analysis of the hippocampus for neuronal damage estimated by counting of intact, healthy neurons. A low dose of quinolinic acid or IL-1beta produced no damage by itself, but the two together induced a significant loss of pyramidal neurons in the hippocampus. Higher doses produced almost total loss of pyramidal cells. Intrahippocampal TNF-alpha produced no effect alone but significantly reduced the neuronal loss produced by quinolinic acid. The adenosine A(2A) receptor antagonist ZM241385 reduced neuronal loss produced by the combinations of quinolinic acid and IL-1beta. The results suggest that simultaneous quinolinic acid and IL-1beta, both being induced by cerebral infection or injury, are synergistic in the production of neuronal damage and could together contribute substantially to traumatic, infective, or ischemic cerebral damage. Antagonism of adenosine A(2A) receptors protects neurons against the combination of quinolinic acid and IL-1beta., ((c) 2007 Wiley-Liss, Inc.)

Glutamate has toxic effects on a number of tissues, partly by inducing toxic (e.g., oxidative) stress, whereas adenosine can be protective. Since there is evidence that glutamate and adenosine receptors are present in bone, we set out to study whether oxidative stress, induced by hydrogen peroxide (H2O2), affected viability in the MC3T3-E1 osteoblast-like cell line and whether treatment with adenosine receptor ligands attenuated this. Hydrogen peroxide (100 microM to 5 mM) reduced the viability of the MC3T3-E1 cells, while catalase reversed this cell loss and itself had some mitogenic effect. Superoxide dismutase (SOD) increased the number of viable cells alone but failed to modify significantly the effect of H2O2 treatments. Glutamate (100 microM, 1 mM) and NMDA (10 microM), applied alone for up to 1 h, had a mitogenic effect (P < 0.05). Adenosine A1 and A2A receptor agonists and antagonists at low and high concentrations showed some mitogenic effects when added singly, but only high concentrations of the agonists showed significant protection against cell death resulting from H2O2 treatments. Contributions from both apoptotic and necrotic pathways were implicated in the H2O2-induced cell loss as was demonstrated by the use of the caspase-3 inhibitor (Z-DEVD-fmk) and the PARP-1 inhibitor (DPQ). The results demonstrate that hydrogen peroxide was toxic to MC3T3-E1 cells, whereas glutamate was not and may even have a trophic influence. Adenosine and its receptors afforded some protection to osteoblasts against cellular death mediated partly by apoptosis and partly by necrosis.

Activation of adenosine A1 receptors raised spike thresholds and induced a dissociation of excitatory postsynaptic potential (EPSP) spike coupling in hippocampal pyramidal neurones. This effect could be prevented by activation of A2A adenosine receptors. The A1 receptor agonist N6-cyclopentyladenosine caused a dissociation of the EPSP spike coupling recorded extracellularly and increased the threshold for spike generation measured intracellularly. These effects were prevented by the A2A receptor agonist CGS21680. A series of agents interfering with adenylate cyclase activity, protein kinase A or C, or nitric oxide synthase had no effect on these responses to N6-cyclopentyladenosine. Superfusion with barium or glibenclamide prevented both the dissociation of EPSP spike coupling and the increase of spike threshold. It is concluded that a barium- and glibenclamide-sensitive potassium current may be involved in the postsynaptic effects of A1 receptors on spike threshold, and it is suggested that a similar suppression of a potassium current by A2A receptors could underlie the inhibition of A1 receptor responses., (Copyright (c) 2004 S. Karger AG, Basel.)

We have recently observed that the free radical-generating mixture of xanthine and xanthine oxidase (X/XO) can suppress the inhibitory effects of adenosine on synaptic transmission in the hippocampus, but that this action can be mimicked by xanthine alone. We have now clarified the mechanism of these interactions by using the new, potent and highly selective inhibitor of xanthine oxidase, 1-(3-cyano-4-neopentyloxyphenyl)pyrazole-4-carboxylic acid (Y-700). Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region of rat hippocampal slices. X/XO induced a long-lasting increase of fEPSP slope and significantly reduced the presynaptic inhibitory effect of adenosine. Both these actions were prevented by Y-700 at a concentration of only 200nM. Similarly the superfusion of xanthine alone increased fEPSP slope and reduced sensitivity to adenosine but these effects were also prevented by Y-700. The results indicate that the antagonism of adenosine responses by X/XO or by xanthine alone are entirely attributable to the activity of the added or endogenous XO activity, probably generating free radicals, and are not likely to be caused by a direct antagonistic action at the xanthine-sensitive site on the adenosine receptor.

The kynurenine pathway from tryptophan generates compounds which can act on glutamate receptors in peripheral tissues or modulate free radical activity. We have measured the concentrations of several of these compounds in the plasma of patients with rheumatoid arthritis (RA) and osteoporosis (OP) before treatment with drugs and then at monthly intervals for 6 months during treatment. Kynurenine analysis was performed by HPLC. Compared with healthy controls, RA patients showed significantly decreased baseline levels of tryptophan, 3-hydroxykynurenine and 3-hydroxyanthranilic acid and increased levels of kynurenine and xanthurenic acid, while kynurenic acid concentrations were normal. Different results were recorded from patients with OP with only a significant reduction in tryptophan and 3-hydroxyanthranilic acid when compared with healthy controls. During 6 months of treating the RA patients with prednisolone or methotrexate, and the OP patients with raloxifene or etidronate and calcium there were significant therapeutic responses and a significant trend towards a reduction in levels of neopterin in RA patients receiving methotrexate but no changes in the profiles of tryptophan metabolites. The results are consistent with the induction of indoleamine-2,3-dioxygenase (IDO) in both RA and OP but with far greater activation of the pathway in the much more inflammatory condition, i.e. RA. It is concluded that there are changes in the kynurenine pathway, which may modify the activation of tissue glutamate receptors, in RA and OP, but that these are not affected by the drug treatments studied.

The factors affecting gut activity in inflammatory bowel disease are unclear, but purines and kynurenines may be involved in the regulation of neuronal activity and therefore gut motility and secretion. We have measured the serum levels of these compounds in patients and in sex- and age-matched controls. Purines and kynurenines were analysed using HPLC. The levels of tryptophan and its metabolites 3-hydroxykynurenine, 3-hydroxyanthranilic acid and xanthurenic acid were unchanged in all patients. However, the levels of kynurenine and kynurenic acid were significantly elevated in patients with inflammatory bowel disease when compared to control subjects. There were no significant differences between patients and controls for any of the purines analysed or for neopterin. In the inflammatory bowel disease patients serum lipid peroxidation products were significantly elevated when compared to control subjects, suggesting the presence of increased oxidative stress consistent with inflammatory activity. The elevated level of kynurenic acid may represent either a compensatory response to elevated activation of enteric neurones, or a primary abnormality, which induces a compensatory increase in gut activity, but may indicate a role for kynurenine modulation of glutamate receptors in the symptoms of inflammatory bowel disease.

Central nervous system tissue of mice infected with Plasmodium berghei ANKA (PbA) exhibits similar histopathological features to those in post-mortem human cerebral malaria (CM) tissue. In this study, the neurochemical characteristics of PbA-infected and control mice were compared. Substance P-containing neurones were almost completely lost from the cortex and striatum of PbA-infected mice seven days after inoculation, whereas the intensity of calbindin immunolabelling was increased compared with controls. Neuropeptide Y- and somatostatin-containing neurones were dramatically reduced in number only in the cortex of day 7 post-inoculation mice compared with controls. This neurochemical pattern in mice with CM is similar to that previously reported to be produced in rats by quinolinic acid. Since the level of quinolinic acid is known to be raised in the brains of PbA-infected mice, the results would be consistent with a role for quinolinic acid in the production of brain damage in fatal murine CM.

The activation of adenosine A1, A2 andA3 receptors can protect neurones against damage generated by mechanical or hypoxic/ischaemic insults as well as excitotoxins. A1 receptors are probably effective by suppressing transmitter release and producing neuronal hyperpolarisation. They are less likely to be of therapeutic importance due to the plethora of side effects resulting from A1 agonism, although the existence of receptor subtypes and recent synthetic chemistry efforts to increase ligand selectivity, may yet yield clinically viable compounds. Activation of A2A receptors can protect neurons, although there is much uncertainty as to whether agonists are acting centrally or via a peripheral mechanism such as altering blood flow or immune cell function. Selective antagonists at the A2A receptor, such as 4-(2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-yl-amino]ethyl)phenol (ZM 241385) and 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261), can also protect against neuronal death produced by ischaemia or excitotoxicity. In addition, A2A receptor antagonists can reduce damage produced by combinations of subthreshold doses of the endogenous excitotoxin quinolinic acid and free radicals. Since the A2A receptors do not seem to be activated by normal endogenous levels of adenosine, their blockade should not generate significant side effects, so that A2A receptor antagonists appear to be promising candidates as new drugs for the prevention of neuronal damage. Adenosine A3 receptors have received less attention to date, but agonists are clearly able to afford protection against damage when administered chronically. Given the disappointing lack of success of NMDA receptor antagonists in human stroke patients, despite their early promise in animal models, it is possible that A2A receptor antagonists could have a far greater clinical utility.