TNFR2 signalling in inflammatory diseases.

TNF signals via two receptors, TNFR1 and TNFR2, which play contrasting roles in immunity. Most of the pro-inflammatory effects of TNF are mediated by TNFR1, whereas TNFR2 is mainly involved in immune homeostasis and tissue healing, but also contributes to tumour progression. However, all currently available anti-TNF biologics inhibit signalling via both receptors and there is increasing interest in the development of selective inhibitors; TNFR1 inhibitors for autoimmune disease and TNFR2 inhibitors for cancer. It is hypothesised that selective inhibition of TNFR1 in autoimmune disease would alleviate inflammation and promote homeostasis by allowing TNFR2 signalling to proceed unimpeded. Validation of this concept would pave the way for the development and testing of TNF specific antagonists. Another therapeutic approach being explored is the use of TNFR2 specific agonists, which could be administered alone or in combination with a TNFR1 antagonist.

Interactions of IDO and the Kynurenine Pathway with Cell Transduction Systems and Metabolism at the Inflammation-Cancer Interface.

The mechanisms underlying a relationship between inflammation and cancer are unclear, but much emphasis has been placed on the role of tryptophan metabolism to kynurenine and downstream metabolites, as these make a substantial contribution to the regulation of immune tolerance and susceptibility to cancer. The proposed link is supported by the induction of tryptophan metabolism by indoleamine-2,3-dioxygenase (IDO) or tryptophan-2,3-dioxygenase (TDO), in response to injury, infection or stress. This review will summarize the kynurenine pathway and will then focus on the bi-directional interactions with other transduction pathways and cancer-related factors. The kynurenine pathway can interact with and modify activity in many other transduction systems, potentially generating an extended web of effects other than the direct effects of kynurenine and its metabolites. Conversely, the pharmacological targeting of those other systems could greatly enhance the efficacy of changes in the kynurenine pathway. Indeed, manipulating those interacting pathways could affect inflammatory status and tumor development indirectly via the kynurenine pathway, while pharmacological modulation of the kynurenine pathway could indirectly influence anti-cancer protection. While current efforts are progressing to account for the failure of selective IDO1 inhibitors to inhibit tumor growth and to devise means of circumventing the issue, it is clear that there are wider factors involving the relationship between kynurenines and cancer that merit detailed consideration as alternative drug targets.

Modulation of T cells by tryptophan metabolites in the kynurenine pathway.

Lymphocytes maturing in the thymus (T cells) are key factors in adaptive immunity and the regulation of inflammation. The kynurenine pathway of tryptophan metabolism includes several enzymes and compounds that can modulate T cell function, but manipulating these pharmacologically has not achieved the expected therapeutic activity for the treatment of autoimmune disorders and cancer. With increasing knowledge of other pathways interacting with kynurenines, the expansion of screening methods, and the application of virtual techniques to understanding enzyme structures and mechanisms, details of interactions between kynurenines and other pathways are being revealed. This review surveys some of these alternative approaches to influence T cell function indirectly via the kynurenine pathway and summarizes the most recent work on the development of compounds acting directly on the kynurenine pathway.

Induction of IDO1 and Kynurenine by Serine Proteases Subtilisin, Prostate Specific Antigen, CD26 and HtrA: A New Form of Immunosuppression?

Several serine proteases have been linked to autoimmune disorders and tumour initiation although the mechanisms are not fully understood. Activation of the kynurenine pathway enzyme indoleamine-2,3-dioxygenase (IDO1) modulates cellular activity in the brain, tolerogenesis in the immune system and is a major checkpoint in cancer development. We now report that IDO1 mRNA and IDO1 protein expression (generating kynurenine) are induced in human monocyte-derived macrophages by several chymotryptic serine proteases with direct links to tumorigenesis, including Prostate Specific Antigen (PSA), CD26 (Dipeptidyl-peptidase-4, CD26/DPP-4), High Temperature Requirement protein-A (HtrA), and the bacterial virulence factor subtilisin. These proteases also induce expression of the pro-inflammatory cytokine genes IL1B and IL6. Other serine proteases tested: bacterial glu-C endopeptidase and mammalian Pro-protein Convertase Subtilase-Kexin-3 (PCSK3, furin), urokinase plasminogen activator (uPA), cathepsin G or neutrophil elastase, did not induce IDO1, indicating that the reported effects are not a general property of all serine proteases. The results represent a novel mechanism of activating immunosuppressive IDO1 and inducing kynurenine generation which, together with the production of inflammatory cytokines, would contribute to tumour initiation and progression, providing a new target for drug development. In addition, the proteasomal S20 serine protease inhibitor carfilzomib, used in the treatment of myeloma, prevented the induction of IDO1 and cytokine gene expression, potentially contributing to its clinical anti-cancer activity.

Dependence and Guidance Receptors-DCC and Neogenin-In Partial EMT and the Actions of Serine Proteases.

The Epithelial-Mesenchymal Transition (EMT) is an important concept in understanding the processes of oncogenesis, especially with respect to the relationship between cell proliferation and metastatic properties such as spontaneous cell motility, chemotaxic migration and tissue invasion. EMT is now recognized as a more complex phenomenon than an all-or-nothing event, in which different components of the EMT may have distinct roles in the physio-pathological regulation of cell function and which may in turn depend on differential interactions with cell constituents and metabolic products. This mini-review summarizes recent work on the induction of cancer properties in parallel with the presence of EMT activities in the presence of serine proteases, with the focus on those tumor suppressors known as "dependence" receptors such as neogenin and Deleted in Colorectal Cancer (DCC). It is concluded that various forms of partial EMT should be given more detailed investigation and consideration as the results could have valuable implications for the development of disease-specific and patient-specific therapies.

Serine protease modulation of Dependence Receptors and EMT protein expression.

Expression of the tumour suppressor Deleted in Colorectal Cancer (DCC) and the related protein neogenin is reduced by the mammalian serine protease chymotrypsin or the bacterial serine protease subtilisin, with increased cell migration. The present work examines whether these actions are associated with changes in the expression of cadherins, ß-catenin and vimentin, established markers of the Epithelial-Mesenchymal Transition (EMT) which has been linked with cell migration and tumour metastasis. The results confirm the depletion of DCC and neogenin and show that chymotrypsin and subtilisin also reduce expression of ß-catenin in acutely prepared tissue sections but not in human mammary adenocarcinoma MCF-7 or MDA-MB-231 cells cultured in normal media, or primary normal human breast cells. A loss of ß-catenin was also seen in low serum media but transfecting cells with a dcc-containing plasmid induced resistance. E-cadherin was not consistently affected but vimentin was induced by low serum-containing media and was increased by serine proteases in MCF-7 and MDA-MB-231 cells in parallel with increased wound closure. Vimentin might contribute to the promotion of cell migration. The results suggest that changes in EMT proteins depend on the cells or tissues concerned and do not parallel the expression of DCC and neogenin. The increased cell migration induced by serine proteases is not consistently associated with the expression of the EMT proteins implying either that the increased migration may be independent of EMT or supporting the view that EMT is not itself consistently related to migration. (241).

Obesity and Cancer: Existing and New Hypotheses for a Causal Connection.

Existing explanations of obesity-associated cancer emphasise direct mutagenic effects of dietary components or hormonal imbalance. Some of these hypotheses are reviewed briefly, but recent evidence suggests a major role for chronic inflammation in cancer risk, possibly involving dietary content. These ideas include the inflammation-induced activation of the kynurenine pathway and its role in feeding and metabolism by activation of the aryl hydrocarbon receptor (AHR) and by modulating synaptic transmission in the brain. Evidence for a role of the kynurenine pathway in carcinogenesis then provides a potentially major link between obesity and cancer. A second new hypothesis is based on evidence that serine proteases can deplete cells of the tumour suppressors Deleted in Colorectal Cancer (DCC) and neogenin. These enzymes include mammalian chymotryptic proteases released by pro-inflammatory neutrophils and macrophages. Blood levels of chymotrypsin itself increase in parallel with food intake. The mechanistically similar bacterial enzyme subtilisin is widespread in the environment, animal probiotics, meat processing and cleaning products. Simple public health schemes in these areas, with selective serine protease inhibitors and AHR antagonists and could prevent a range of intestinal and other cancers.

Microbial carcinogenic toxins and dietary anti-cancer protectants.

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.

Kynurenine Pathway Activation in Human African Trypanosomiasis.

Background: The kynurenine pathway of tryptophan oxidation is associated with central nervous system (CNS) inflammatory pathways. Inhibition of this pathway ameliorates CNS inflammation in rodent models of the late (meningoencephalitic) stage of human African trypanosomiasis (HAT). In this study, we evaluate whether the kynurenine pathway is activated in clinical HAT and associated with CNS inflammatory responses. Methods: We measured cerebrospinal fluid (CSF) tryptophan and kynurenine metabolite concentrations in patients infected with Trypanosoma brucei rhodesiense, using liquid chromatography-mass spectrometry. Results: Kynurenine concentration in CSF was increased in both the early and late stages of disease, with a progressive increase in tryptophan oxidation associated with stage progression. Kynurenine pathway activation was associated with increases in neuroinflammatory markers, but there was no clear relationship to neurological symptoms. Conclusions: CNS kynurenine pathway activation occurs during HAT, including cases prior to the current diagnostic cutoff for late-stage infection, providing evidence for early CNS involvement in HAT. Metabolite data demonstrate that the kynurenine-3-monooxygenase and kynurenine aminotransferase branches of the kynurenine pathway are active. The association between tryptophan oxidation and CNS inflammatory responses as measured by CSF interleukin 6 (IL-6) concentration supports a role of kynurenine metabolites in the inflammatory pathogenesis of late-stage HAT.

Quinolinic acid induces neuritogenesis in SH-SY5Y neuroblastoma cells independently of NMDA receptor activation.

Glutamate and nicotinamide adenine dinucleotide (NAD+ ) have been implicated in neuronal development and several types of cancer. The kynurenine pathway of tryptophan metabolism includes quinolinic acid (QA) which is both a selective agonist at N-methyl-D-aspartate (NMDA) receptors and also a precursor for the formation of NAD+ . The effect of QA on cell survival and differentiation has therefore been examined on SH-SY5Y human neuroblastoma cells. Retinoic acid (RA, 10 µm) induced differentiation of SH-SY5Y cells into a neuronal phenotype showing neurite growth. QA (50-150 nm) also caused a concentration-dependent increase in the neurite/soma ratio, indicating differentiation. Both RA and QA increased expression of the neuronal marker ß3-tubulin in whole-cell homogenates and in the neuritic fraction assessed using a neurite outgrowth assay. Expression of the neuronal proliferation marker doublecortin revealed that, unlike RA, QA did not decrease the number of mitotic cells. QA-induced neuritogenesis coincided with an increase in the generation of reactive oxygen species. Neuritogenesis was prevented by diphenylene-iodonium (an inhibitor of NADPH oxidase) and superoxide dismutase, supporting the involvement of reactive oxygen species. NMDA itself did not promote neuritogenesis and the NMDA antagonist dizocilpine (MK-801) did not prevent quinolinate-induced neuritogenesis, indicating that the effects of QA were independent of NMDA receptors. Nicotinamide caused a significant increase in the neurite/soma ratio and the expression of ß3-tubulin in the neuritic fraction. Taken together, these results suggest that QA induces neuritogenesis by promoting oxidizing conditions and affecting the availability of NAD+ , independently of NMDA receptors.

Selective depletion of tumour suppressors Deleted in Colorectal Cancer (DCC) and neogenin by environmental and endogenous serine proteases: linking diet and cancer.

BACKGROUND: The related tumour suppressor proteins Deleted in Colorectal Cancer (DCC) and neogenin are absent or weakly expressed in many cancers, whereas their insertion into cells suppresses oncogenic behaviour. Serine proteases influence the initiation and progression of cancers although the mechanisms are unknown. METHODS: The effects of environmental (bacterial subtilisin) and endogenous mammalian (chymotrypsin) serine proteases were examined on protein expression in fresh, normal tissue and human neuroblastoma and mammary adenocarcinoma lines. Cell proliferation and migration assays (chemoattraction and wound closure) were used to examine cell function. Cells lacking DCC were transfected with an ectopic dcc plasmid. RESULTS: Subtilisin and chymotrypsin selectively depleted DCC and neogenin from cells at nanomolar concentrations without affecting related proteins. Cells showed reduced adherence and increased migration, but after washing they re-attached within 24 h, with recovery of protein expression. These effects are induced by chymotryptic activity as they are prevented by chymostatin and the soybean Bowman-Birk inhibitor typical of many plant protease inhibitors. CONCLUSIONS: Bacillus subtilis, which secretes subtilisin is widely present in soil, the environment and the intestinal contents, while subtilisin itself is used in meat processing, animal feed probiotics and many household cleaning agents. With chymotrypsin present in chyme, blood and tissues, these proteases may contribute to cancer development by depleting DCC and neogenin. Blocking their activity by Bowman-Birk inhibitors may explain the protective effects of a plant diet. Our findings identify a potential non-genetic contribution to cancer cell behaviour which may explain both the association of processed meats and other factors with cancer incidence and the protection afforded by plant-rich diets, with significant implications for cancer prevention.

Dependence receptor involvement in subtilisin-induced long-term depression and in long-term potentiation.

The serine protease subtilisin induces a form of long-term depression (LTD) which is accompanied by a reduced expression of the axo-dendritic guidance molecule Unco-ordinated-5C (Unc-5C). One objective of the present work was to determine whether a loss of Unc-5C function contributed to subtilisin-induced LTD by using Unc-5C antibodies in combination with the pore-forming agents Triton X-100 (0.005%) or streptolysin O in rat hippocampal slices. In addition we have assessed the effect of subtilisin on the related dependence receptor Deleted in Colorectal Cancer (DCC) and used antibodies to this protein for functional studies. Field excitatory postsynaptic potentials (fEPSPs) were analyzed in rat hippocampal slices and protein extracts were used for Western blotting. Subtilisin produced a greater loss of DCC than of Unc-5C, but the antibodies had no effect on resting excitability or fEPSPs and did not modify subtilisin-induced LTD. However, antibodies to DCC but not Unc-5C did reduce the amplitude of theta-burst long-term potentiation (LTP). In addition, two inhibitors of endocytosis - dynasore and tat-gluR2(3Y) - were tested and, although the former compound had no effect on neurophysiological responses, tat-gluR2(3Y) did reduce the amplitude of subtilisin-induced LTD without affecting the expression of DCC or Unc-5C but with some loss of PostSynaptic Density Protein-95. The results support the view that the dependence receptor DCC may be involved in LTP and suggest that the endocytotic removal of a membrane protein or proteins may contribute to subtilisin-induced LTD, although it appears that neither Unc-5C nor DCC are involved in this process.

Prenatal activation of maternal TLR3 receptors by viral-mimetic poly(I:C) modifies GluN2B expression in embryos and sonic hedgehog in offspring in the absence of kynurenine pathway activation.

Activation of the immune system during pregnancy is believed to lead to psychiatric and neurological disorders in the offspring, but the molecular changes responsible are unknown. Polyinosinic:polycytidylic acid (poly(I:C)) is a viral-mimetic double-stranded RNA complex which activates Toll-Like-Receptor-3 and can activate the metabolism of tryptophan through the oxidative kynurenine pathway to compounds that modulate activity of glutamate receptors. The aim was to determine whether prenatal administration of poly(I:C) affects the expression of neurodevelopmental proteins in the offspring and whether such effects were mediated via the kynurenine pathway. Pregnant rats were treated with poly(I:C) during late gestation and the offspring were allowed to develop to postnatal day 21 (P21). Immunoblotting of the brains at P21 showed decreased expression of sonic hedgehog, a key protein in dopaminergic neuronal maturation. Expression of α-synuclein was decreased, while tyrosine hydroxylase was increased. Disrupted in Schizophrenia-1 (DISC-1) and 5-HT2C receptor levels were unaffected, as were the dependence receptors Unc5H1, Unc5H3 and Deleted in Colorectal Cancer (DCC), the inflammation-related transcription factor NFkB and the inducible oxidative enzyme cyclo-oxygenase-2 (COX-2). An examination of embryo brains 5 h after maternal poly(I:C) showed increased expression of GluN2B, with reduced doublecortin and DCC but no change in NFkB. Despite altered protein expression, there were no changes in the kynurenine pathway. The results show that maternal exposure to poly(I:C) alters the expression of proteins in the embryos and offspring which may affect the development of dopaminergic function. The oxidation of tryptophan along the kynurenine pathway is not involved in these effects.

An expanding range of targets for kynurenine metabolites of tryptophan.

The kynurenine pathway of tryptophan metabolism accounts for most of the tryptophan that is not committed to protein synthesis and includes compounds active in the nervous and immune systems. Kynurenine acts on the aryl hydrocarbon receptor, affecting the metabolism of xenobiotics and promoting carcinogenesis. Quinolinic acid is an agonist at N-methyl-D-aspartate receptors (NMDARs), but is also pro-oxidant, has immunomodulatory actions, and promotes the formation of hyperphosphorylated tau proteins. Kynurenic acid blocks NMDARs and α7-homomeric nicotinic cholinoceptors and is also an agonist at the orphan G-protein-coupled receptor GPR35. 3-Hydroxykynurenine and 3-hydroxyanthranilic acid have pronounced redox activity and regulate T cell function. Cinnabarinic acid can activate metabotropic glutamate receptors. This review highlights the increasing range of molecular targets for components of the kynurenine pathway in both the nervous and immune systems in relation to their relevance to disease and drug development.

A novel dihydro-pyrazolo(3,4d)(1,2,4)triazolo(1,5a)pyrimidin-4-one (AJ23) is an antagonist at adenosine A(1) receptors and enhances consolidation of step-down avoidance.

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.

Involvement of kynurenines in Huntington's disease and stroke-induced brain damage.

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.

Kynurenine metabolism predicts cognitive function in patients following cardiac bypass and thoracic surgery.

Cardiac surgery involving extra-corporeal circulation can lead to cognitive dysfunction. As such surgery is associated with signs of inflammation and pro-inflammatory mediators activate tryptophan oxidation to neuroactive kynurenines which modulate NMDA receptor function and oxidative stress, we have measured blood concentrations of kynurenines and inflammatory markers in 28 patients undergoing coronary arterial graft surgery and, for comparison, 28 patients undergoing non-bypass thoracic surgery. A battery of cognitive tests was completed before and after the operations. The results show increased levels of tryptophan with decreased levels of kynurenine, anthranilic acid and 3-hydroxyanthranilic acid associated with bypass, and a later increase in kynurenic acid. Levels of neopterin and lipid peroxidation products rose after surgery in non-bypass patients whereas tumour necrosis factor-α and S100B levels increased after bypass. Changes of neopterin levels were greater after non-bypass surgery. Cognitive testing showed that the levels of tryptophan, kynurenine, kynurenic acid and the kynurenine/tryptophan ratio, correlated with aspects of post-surgery cognitive function, and were significant predictors of cognitive performance in tasks sensitive to frontal executive function and memory. Thus, anaesthesia and major surgery are associated with inflammatory changes and alterations in tryptophan oxidative metabolism which predict, and may play a role in, post-surgical cognitive function.

Glutamate-induced depression of EPSP-spike coupling in rat hippocampal CA1 neurons and modulation by adenosine receptors.

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.

Adenosine receptors and neurological disease: neuroprotection and neurodegeneration.

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.

Adenosine preconditions against ouabain but not against glutamate on CA1-evoked potentials in rat hippocampal slices.

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.