register interest

John Davis

Research Area: Drug Discovery
Technology Exchange: Drug discovery
Scientific Themes: Physiology, Cellular & Molecular Biology and Protein Science & Structural Biology

The Alzheimer’s Research UK Oxford Drug Discovery Institute (ODDI) couples the deep disease knowledge and biology expertise of the academic community with high quality, innovative drug discovery technologies. This initiative is based on juxtaposing high quality drug discovery expertise alongside a deep scientific and academic understanding of patients, disease mechanisms, and model systems. The intent is to translate cutting edge academic science into drug discovery, and to prosecute projects from target to lead status, and beyond. 

Housed within The Target Discovery Institute at the University of Oxford, the ODDI is uniting collaborative efforts for target identification with sophisticated target development capabilities. Led by the CSO, Dr John Davis, the ODDI focuses on novel targets in the dementia therapeutic area, bringing together researchers from Biology, Chemistry, Psychiatry, and Neuroscience. The institute is part of a newly formed, world-class, network of three drug discovery institutes, sponsored by Alzheimer’s Research UK. 

The Oxford Drug Discovery Institute is one of three Institutes within the Alzheimer’s Research UK Drug Discovery Alliance, working alongside Institutes at the University of Cambridge and University College London. The Alliance will accelerate the discovery of novel, effective therapeutics for Alzheimer’s disease and other neurodegenerative diseases.

Alzheimer’s Research UK unites more than 1000 dementia researchers from across the UK to support and streamline dementia research. The charity is dedicated to funding the best minds and forging the most effective partnerships to nurture discovery and ideas, as well as translating findings from this pioneering research into benefits for people with dementia. For more information, visit ARUK’s website.

Name Department Institution Country
Paul Brennan Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Professor Simon Lovestone University of Oxford United Kingdom
Chas Bountra Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Dr Caleb Webber Department of Physiology, Anatomy and Genetics University of Oxford United Kingdom
Professor Susan Lea Sir William Dunn School of Pathology University of Oxford United Kingdom
Daniel Ebner Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Benedikt Kessler Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Oleg Fedorov Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Priestley RS, Cheung J, Murphy EJ, Ehebauer MT, Davis JB, Di Daniel E. 2019. A novel high-content imaging-based technique for measuring binding of Dickkopf-1 to low-density lipoprotein receptor-related protein 6. J Pharmacol Toxicol Methods, 95 pp. 47-55. | Show Abstract | Read more

INTRODUCTION: Dickkopf-related protein 1 (Dkk1) is a secreted protein ligand of low-density lipoprotein receptor-related protein 6 (LRP6), which antagonises canonical Wnt signalling. Elevated Dkk1 levels have been linked to Alzheimer's disease (AD), with protein blockade protective in pre-clinical AD models, suggesting inhibitors of Dkk1-LRP6 binding may have therapeutic utility against AD. Cell-based Dkk1-LRP6 assays reported in the literature use either modified Dkk1 protein and/or do not possess suitable throughput for drug screening. Here we report a novel immunocytochemical-based assay utilising high-content imaging (HCI) and automated data analysis suitable for the screening of protein and small-molecule inhibitors of Dkk1-LRP6 binding. METHODS: We developed an immunocytochemical (ICC) protocol to detect specific binding of exogenous human Dkk1 protein to human LRP6 transiently expressed in HEK293 cells. Images were generated using the PerkinElmer Operetta HCI System, after which quantitative data was generated using the PerkinElmer Columbus™ System. RESULTS: Our ICC technique and analysis pipeline allowed measurement of cell membrane-localised, LRP6-specific Dkk1 binding, normalised at individual cellular events. Saturation binding demonstrated concentration-dependent Dkk1 binding to LRP6, with a KD in keeping with reported values. Association kinetic experiments demonstrated the utility of the technique to investigate Dkk1 binding kinetics. Human Dkk members Dkk2 and Dkk4 fully displaced Dkk1 binding in a competition assay, while Dkk3 and Soggy-1/DkkL1 exhibited non-complete displacement of Dkk1. Finally gallocyanine, a previously reported inhibitor of Dkk1-LRP6 binding, fully displaced Dkk1 near the expected IC50. DISCUSSION: In conclusion, we provide a validated cell-based assay, suitable for the screening of inhibitors of Dkk1-LRP6 binding, and provide the basis for additional assay development, investigating Dkk1-LRP6 pharmacology.

Horsburgh K, Wardlaw JM, van Agtmael T, Allan SM, Ashford MLJ, Bath PM, Brown R, Berwick J, Cader MZ, Carare RO et al. 2018. Small vessels, dementia and chronic diseases - molecular mechanisms and pathophysiology. Clin Sci (Lond), 132 (8), pp. 851-868. | Show Abstract | Read more

Cerebral small vessel disease (SVD) is a major contributor to stroke, cognitive impairment and dementia with limited therapeutic interventions. There is a critical need to provide mechanistic insight and improve translation between pre-clinical research and the clinic. A 2-day workshop was held which brought together experts from several disciplines in cerebrovascular disease, dementia and cardiovascular biology, to highlight current advances in these fields, explore synergies and scope for development. These proceedings provide a summary of key talks at the workshop with a particular focus on animal models of cerebral vascular disease and dementia, mechanisms and approaches to improve translation. The outcomes of discussion groups on related themes to identify the gaps in knowledge and requirements to advance knowledge are summarized.

Scholz J, Rathmell JP, David WS, Chad DA, Broderick AC, Perros SG, Shin NS, Wells JL, Davis JB, DiMaggio CJ et al. 2016. A standardized clinical evaluation of phenotypic diversity in diabetic polyneuropathy. Pain, 157 (10), pp. 2297-2308. | Show Abstract | Read more

Diabetic polyneuropathy (DPN) is a major cause of neuropathic pain and a frequent target condition in analgesic treatment trials. Differences in the clinical symptoms and signs associated with DPN suggest distinct pathophysiological mechanisms underlying nerve damage and dysfunction that are likely to have therapeutic relevance. The aim of this study was to develop a tool for the bedside assessment of painful neuropathies such as DPN that captures the diversity of phenotypes. Sixty-one patients with type 2 diabetes and painful neuropathy, 19 patients with painless DPN, 25 patients with type 2 diabetes but no clinical evidence of neuropathy, and 20 healthy control subjects completed a structured interview (47 items) and a standardized physical examination (39 items). After analyzing critical features of pain and painless symptoms and examining the outcome of physical tests of sensory function, we determined principal components of the phenotypic variance among patients. Increased sensitivity to mechanical or thermal stimuli and, to a lesser extent, the sensory quality of pain or paresthesia were the most discriminating elements of DPN phenotypes. Correlation patterns of symptoms and signs indicated the involvement of functionally distinct nerve fiber populations. We combined interview questions and physical tests identifying these differences in a shortened assessment protocol that we named Standardized Evaluation of Pain and Somatosensory Function (StEPS). The protocol StEPS generates a phenotypic profile of patients with neuropathy. Separate intensity ratings for spontaneous painful symptoms and pain evoked by standard stimuli support a detailed documentation of neuropathic pain and its response to analgesic treatment.

Sadeghian M, Marinova-Mutafchieva L, Broom L, Davis JB, Virley D, Medhurst AD, Dexter DT. 2012. Full and partial peroxisome proliferation-activated receptor-γ agonists, but not δ agonist, rescue of dopaminergic neurons in the 6-OHDA parkinsonian model is associated with inhibition of microglial activation and MMP expression. J Neuroimmunol, 246 (1-2), pp. 69-77. | Show Abstract | Read more

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor that has been shown to have anti-inflammatory and matrix metalloproteinase (MMP) inhibitor properties. PPARγ agonists have been shown to have neuroprotective effects in various neurodegeneration models where inflammation is implicated, including models of Parkinson's disease. However, no studies have looked at the effects of partial PPARγ agonists. EXPERIMENTAL APPROACH: The neuroprotective effects of the PPARγ full agonist, pioglitazone (20 mg/kg), partial PPARγ agonist GW855266X (15 mg/kg) and PPAR-δ full agonist GW610742X (10 mg/kg) were investigated in the 6-hydroxydopamine (6-OHDA) model of Parkinson's disease when administered prior to or post 6-OHDA lesioning. The integrity of the nigrostriatal system was assessed by assessing the numbers dopaminergic neurons in the substantia nigra (SN) and by assessing striatal dopamine content. The degree of microglia activation in the SN was also immunohistochemistry assessed utilizing the marker OX-6 for activated microglia and CD-68 a marker for phagocytic microglia. Additionally we performed immunocytochemistry for MMP3 in the SN. Finally, we investigated whether a period of drug withdrawal for a further 7 days affected the neuroprotection produced by the PPARγ agonists. KEY RESULTS: Both pioglitazone and GW855266X protected against 6-OHDA induced loss of dopaminergic neurons in the substantia nigra and depletion of striatal dopamine when administered orally twice daily for either 1) 7 day prior to and 7 days post lesioning or 2) for 7 days starting 2 days post lesioning when neurons will be severely traumatized. 6-OHDA lesioning was associated with an increase in microglia activation and in numbers of MMP-3 immunoreactive cells which was attenuated by pioglitazone and GW855266X. Neuroprotective effects were not replicated using the PPARδ agonist GW610742X. Subsequent withdrawal of both pioglitazone and GW855266X, for a further 7 days negated any neuroprotective effect suggesting that long-term administration may be required to attenuate the inflammatory response. CONCLUSIONS AND IMPLICATIONS: For the first time a partial PPAR-γ agonist has been shown to be neuroprotectory when administered post lesioning in a parkinsonian model. Effects may be via the inhibition of microglial and MMP activation and support further research.

Michaki V, Guix FX, Vennekens K, Munck S, Dingwall C, Davis JB, Townsend DM, Tew KD, Feiguin F, De Strooper B et al. 2012. Down-regulation of the ATP-binding cassette transporter 2 (Abca2) reduces amyloid-β production by altering Nicastrin maturation and intracellular localization. J Biol Chem, 287 (2), pp. 1100-1111. | Show Abstract | Read more

Clinical, pharmacological, biochemical, and genetic evidence support the notion that alteration of cholesterol homeostasis strongly predisposes to Alzheimer disease (AD). The ATP-binding cassette transporter-2 (Abca2), which plays a role in intracellular sterol trafficking, has been genetically linked to AD. It is unclear how these two processes are related. Here we demonstrate that down-regulation of Abca2 in mammalian cells leads to decreased amyloid-β (Aβ) generation. In vitro studies revealed altered γ-secretase complex formation in Abca2 knock-out cells due to the altered levels, post-translational modification, and subcellular localization of Nicastrin. Reduced Abca2 levels in mammalian cells in vitro, in Drosophila melanogaster and in mice resulted in altered γ-secretase processing of APP, and thus Aβ generation, without affecting Notch cleavage.

Sadeghian M, Marinova-Mutafchieva L, Broom L, Davis JB, Virley D, Medhurst AD, Dexter DT. 2012. Full and partial peroxisome proliferation-activated receptor-gamma agonists, but not delta agonist, rescue of dopaminergic neurons in the 6-OHDA Parkinsonian model is associated with inhibition of microglial activation and MMP expression Journal of Neuroimmunology,

Broom L, Marinova-Mutafchieva L, Sadeghian M, Davis JB, Medhurst AD, Dexter DT. 2011. Neuroprotection by the selective iNOS inhibitor GW274150 in a model of Parkinson disease. Free Radic Biol Med, 50 (5), pp. 633-640. | Show Abstract | Read more

Neuroinflammation and the activation of inducible nitric oxide synthase (iNOS) have been proposed to play a role in the pathogenesis of Parkinson disease (PD). In this study we investigated the effects of the selective iNOS inhibitor GW274150 in the 6-OHDA model of PD. 6-OHDA administration was associated with increased numbers of cells expressing iNOS. Administration of the iNOS inhibitor twice daily for 7 days, beginning 2 days after the 6-OHDA lesioning, led to a significant neuroprotection as shown by assessment of the integrity of the nigrostriatal system by tyrosine hydroxylase immunocytochemistry and HPLC assessment of striatal dopamine content. However, GW274150 displayed a bell-shaped neuroprotective profile, being ineffective at high doses. 6-OHDA lesioning was associated with an increase in microglial activation as assessed by the MHC II antigen OX-6 and the number of matrix metalloproteinase 9 (MMP-9)-immunopositive cells. NO is a known modulator of MMP-9, and iNOS inhibition was associated with decreased numbers of MMP-9-immunopositive cells, culminating in a reduction in the numbers of reactive microglia. Withdrawal of GW274150 for a further 7 days negated any neuroprotective effects of iNOS inhibition, suggesting that the damaging effects of inflammation last beyond 7 days in this model and the continued administration of the drug may be required.

White JPM, Calcott G, Jenes A, Hossein M, Paule CC, Santha P, Davis JB, Ma D, Rice ASC, Nagy I. 2011. Xenon reduces activation of transient receptor potential vanilloid type 1 (TRPV1) in rat dorsal root ganglion cells and in human TRPV1-expressing HEK293 cells Life Sciences, 88 (3-4), pp. 141-149. | Read more

Andratsch M, Mair N, Constantin CE, Scherbakov N, Benetti C, Quarta S, Vogl C, Sailer CA, Uceyler N, Brockhaus J et al. 2009. A Key Role for gp130 Expressed on Peripheral Sensory Nerves in Pathological Pain Journal of Neuroscience, 29 (43), pp. 13473-13483. | Read more

Lambert GA, Davis JB, Appleby JM, Chizh BA, Hoskin KL, Zagami AS. 2009. The effects of the TRPV1 receptor antagonist SB-705498 on trigeminovascular sensitisation and neurotransmission Naunyn-Schmiedeberg's Archives of Pharmacology, 380 (4), pp. 311-325. | Read more

Marinova-Mutafchieva L, Sadeghian M, Broom L, Davis JB, Medhurst AD, Dexter DT. 2009. Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6-hydroxydopamine model of Parkinson's disease. J Neurochem, 110 (3), pp. 966-975. | Show Abstract | Read more

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson's disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6-OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen-cluster of differentiation 11b - anti-complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage-myeloid associated antigen immunoreactivity was observed at day 3 post-lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage-myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.

Roberts JC, Friel SL, Roman S, Perren M, Harper A, Davis JB, Richardson JC, Virley D, Medhurst AD. 2009. Autoradiographical imaging of PPARgamma agonist effects on PBR/TSPO binding in TASTPM mice. Exp Neurol, 216 (2), pp. 459-470. | Show Abstract | Read more

Chronic inflammation is known to occur in the brains of Alzheimer's Disease (AD) patients, including the presence of activated microglia close to amyloid plaques. We utilised real time autoradiography and immunohistochemistry to investigate microglial activation and the potential anti-inflammatory effects of PPARgamma agonists in the Thy-1 APP695swe/Thy-1 PS-1.M146V (TASTPM) overexpressing transgenic mouse model of AD. An age dependent increase in specific [3H](R)-PK11195 binding to peripheral benzodiazepine receptors (PBR)/translocator protein (18 kDa) (TSPO) was observed in the cortex of TASTPM mice compared to wild type mice, indicative of microglial activation. This was consistent with immunohistochemical data showing age-dependent increases in CD68 immunoreactivity co-localised with amyloid beta (Abeta) deposits. In 10 month old TASTPM mice, pioglitazone (20 mg/kg) and ciglitazone (50 mg/kg) significantly reduced [3H](R)-PK11195 and [3H]DPA-713 binding in cortex and hippocampus, indicative of reduced microglial activation. In AD brain, significant [3H](R)-PK11195 and [3H]DPA-713 binding was observed across all stages of the disease. These results support the use of PBR/TSPO autoradiography in TASTPM mice as a functional readout of microglial activation to assess anti-inflammatory drugs prior to evaluation in AD patients.

Constantin CE, Mair N, Sailer CA, Andratsch M, Xu Z-Z, Blumer MJF, Scherbakov N, Davis JB, Bluethmann H, Ji R-R, Kress M. 2008. Endogenous Tumor Necrosis Factor   (TNF ) Requires TNF Receptor Type 2 to Generate Heat Hyperalgesia in a Mouse Cancer Model Journal of Neuroscience, 28 (19), pp. 5072-5081. | Read more

Cutler P, Akuffo EL, Bodnar WM, Briggs DM, Davis JB, Debouck CM, Fox SM, Gibson RA, Gormley DA, Holbrook JD et al. 2008. Proteomic identification and early validation of complement 1 inhibitor and pigment epithelium-derived factor: Two novel biomarkers of Alzheimer's disease in human plasma PROTEOMICS – CLINICAL APPLICATIONS, 2 (4), pp. 467-477. | Read more

Davis JB, Bountra C, Richardson J. 2008. Perspectives of Alzheimer's disease treatments. Handb Clin Neurol, 89 pp. 273-290. | Read more

Akuffo EL, Davis JB, Fox SM, Gloger IS, Hosford D, Kinsey EE, Jones NA, Nock CM, Roses AD, Saunders AM et al. 2008. The discovery and early validation of novel plasma biomarkers in mild-to-moderate Alzheimer's disease patients responding to treatment with rosiglitazone Biomarkers, 13 (6), pp. 618-636. | Read more

Gunthorpe MJ, Hannan SL, Smart D, Jerman JC, Arpino S, Smith GD, Brough S, Wright J, Egerton J, Lappin SC et al. Characterization of SB-705498, a Potent and Selective Vanilloid Receptor-1 (VR1/TRPV1) Antagonist That Inhibits the Capsaicin-, Acid-, and Heat-Mediated Activation of the Receptor Journal of Pharmacology and Experimental Therapeutics, 321 (3), pp. 1183-1192. | Read more

Hussain I, Hawkins J, Harrison D, Hille C, Wayne G, Cutler L, Buck T, Walter D, Demont E, Howes C et al. 2007. Oral administration of a potent and selective non-peptidic BACE-1 inhibitor decreases ?-cleavage of amyloid precursor protein and amyloid-? production in vivo Journal of Neurochemistry, 100 (3), pp. 802-809. | Read more

Medhurst AD, Atkins AR, Beresford IJ, Brackenborough K, Briggs MA, Calver AR, Cilia J, Cluderay JE, Crook B, Davis JB et al. 2007. GSK189254, a novel H3 receptor antagonist that binds to histamine H3 receptors in Alzheimer's disease brain and improves cognitive performance in preclinical models. J Pharmacol Exp Ther, 321 (3), pp. 1032-1045. | Show Abstract | Read more

6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) is a novel histamine H(3) receptor antagonist with high affinity for human (pK(i) = 9.59 -9.90) and rat (pK(i) = 8.51-9.17) H(3) receptors. GSK189254 is >10,000-fold selective for human H(3) receptors versus other targets tested, and it exhibited potent functional antagonism (pA(2) = 9.06 versus agonist-induced changes in cAMP) and inverse agonism [pIC(50) = 8.20 versus basal guanosine 5'-O-(3-[(35)S]thio)triphosphate binding] at the human recombinant H(3) receptor. In vitro autoradiography demonstrated specific [(3)H]GSK189254 binding in rat and human brain areas, including cortex and hippocampus. In addition, dense H(3) binding was detected in medial temporal cortex samples from severe cases of Alzheimer's disease, suggesting for the first time that H(3) receptors are preserved in late-stage disease. After oral administration, GSK189254 inhibited cortical ex vivo R-(-)-alpha-methyl[imidazole-2,5(n)-(3)H]histamine dihydrochloride ([(3)H]R-alpha-methylhistamine) binding (ED(50) = 0.17 mg/kg) and increased c-Fos immunoreactivity in prefrontal and somatosensory cortex (3 mg/kg). Microdialysis studies demonstrated that GSK189254 (0.3-3 mg/kg p.o.) increased the release of acetylcholine, noradrenaline, and dopamine in the anterior cingulate cortex and acetylcholine in the dorsal hippocampus. Functional antagonism of central H(3) receptors was demonstrated by blockade of R-alpha-methylhistamine-induced dipsogenia in rats (ID(50) = 0.03 mg/kg p.o.). GSK189254 significantly improved performance of rats in diverse cognition paradigms, including passive avoidance (1 and 3 mg/kg p.o.), water maze (1 and 3 mg/kg p.o.), object recognition (0.3 and 1 mg/kg p.o.), and attentional set shift (1 mg/kg p.o.). These data suggest that GSK189254 may have therapeutic potential for the symptomatic treatment of dementia in Alzheimer's disease and other cognitive disorders.

Medhurst AD, Briggs MA, Bruton G, Calver AR, Chessell I, Crook B, Davis JB, Davis RP, Foley AG, Heslop T et al. 2007. Structurally novel histamine H3 receptor antagonists GSK207040 and GSK334429 improve scopolamine-induced memory impairment and capsaicin-induced secondary allodynia in rats. Biochem Pharmacol, 73 (8), pp. 1182-1194. | Show Abstract | Read more

GSK207040 (5-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-2-pyrazinecarboxamide) and GSK334429 (1-(1-methylethyl)-4-({1-[6-(trifluoromethyl)-3-pyridinyl]-4-piperidinyl}carbonyl)hexahydro-1H-1,4-diazepine) are novel and selective non-imidazole histamine H(3) receptor antagonists from distinct chemical series with high affinity for human (pK(i)=9.67+/-0.06 and 9.49+/-0.09, respectively) and rat (pK(i)=9.08+/-0.16 and 9.12+/-0.14, respectively) H(3) receptors expressed in cerebral cortex. At the human recombinant H(3) receptor, GSK207040 and GSK334429 were potent functional antagonists (pA(2)=9.26+/-0.04 and 8.84+/-0.04, respectively versus H(3) agonist-induced changes in cAMP) and exhibited inverse agonist properties (pIC(50)=9.20+/-0.36 and 8.59+/-0.04 versus basal GTPgammaS binding). Following oral administration, GSK207040 and GSK334429 potently inhibited cortical ex vivo [(3)H]-R-alpha-methylhistamine binding (ED(50)=0.03 and 0.35 mg/kg, respectively). Functional antagonism of central H(3) receptors was demonstrated by blockade of R-alpha-methylhistamine-induced dipsogenia in rats (ID(50)=0.02 and 0.11 mg/kg p.o. for GSK207040 and GSK334429, respectively). In more pathophysiologically relevant pharmacodynamic models, GSK207040 (0.1, 0.3, 1 and 3mg/kg p.o.) and GSK334429 (0.3, 1 and 3mg/kg p.o.) significantly reversed amnesia induced by the cholinergic antagonist scopolamine in a passive avoidance paradigm. In addition, GSK207040 (0.1, 0.3 and 1mg/kg p.o.) and GSK334429 (3 and 10mg/kg p.o.) significantly reversed capsaicin-induced reductions in paw withdrawal threshold, suggesting for the first time that blockade of H(3) receptors may be able to reduce tactile allodynia. Novel H(3) receptor antagonists such as GSK207040 and GSK334429 may therefore have therapeutic potential not only in dementia but also in neuropathic pain.

Westaway SM, et al. EA. 2006. N-Tetrahydroquinolinyl, N-Quinolinyl and N-Isoquinolinyl Biaryl Carboxamides as Antagonists of TRPV1. ChemInform, 37 (51), | Read more

Westaway SM, Chung Y-K, Davis JB, Holland V, Jerman JC, Medhurst SJ, Rami HK, Stemp G, Stevens AJ, Thompson M et al. 2006. N-Tetrahydroquinolinyl, N-quinolinyl and N-isoquinolinyl biaryl carboxamides as antagonists of TRPV1 Bioorganic & Medicinal Chemistry Letters, 16 (17), pp. 4533-4536. | Read more

Skaper SD, Facci L, Culbert AA, Evans NA, Chessell I, Davis JB, Richardson JC. 2006. P2X7 receptors on microglial cells mediate injury to cortical neurons in vitro Glia, 54 (3), pp. 234-242. | Read more

Rami HK, Thompson M, Stemp G, Fell S, Jerman JC, Stevens AJ, Smart D, Sargent B, Sanderson D, Randall AD et al. 2006. Discovery of SB-705498: A potent, selective and orally bioavailable TRPV1 antagonist suitable for clinical development Bioorganic & Medicinal Chemistry Letters, 16 (12), pp. 3287-3291. | Read more

Anand U, Otto WR, Casula MA, Day NC, Davis JB, Bountra C, Birch R, Anand P. 2006. The effect of neurotrophic factors on morphology, TRPV1 expression and capsaicin responses of cultured human DRG sensory neurons. Neurosci Lett, 399 (1-2), pp. 51-56. | Show Abstract | Read more

We have studied the effect of key neurotrophic factors (NTFs) on morphology, levels of the vanilloid receptor-1 (TRPV1) and responses to capsaicin in adult human sensory neurons in vitro. Avulsed dorsal root ganglia (DRG, n = 5) were cultured with or without a combination of nerve growth factor (NGF), glial cell (line)-derived growth factor (GDNF) and neurotrophin3 (NT3) for 5 days. In the absence of NTFs, the diameter of neurons ranged from 20 to 100 microm (mean 42 +/- 4 microm). Adding NTFs caused a significant increase in neuronal sizes, up to 120 microm (mean diameter 62 +/- 5 microm, P < 0.01, t-test), an overall 35% increase of TRPV1-positive neurons (P < 0.003), and notably of large TRPV1-positive neurons > 80 microm (P < 0.05). Responses to capsaicin were significantly enhanced with calcium ratiometry (P < 0.0001). Short duration (1h) exposure of dissociated sensory neurons to NTFs increased numbers of TRPV1-positive neurons, but not of TRPV3, Nav 1.8 and IK1 and the morphological size-distribution remained similar to intact post-mortem DRG neurons. NTFs thus increase size, elevate TRPV1 levels and enhance capsaicin responses in cultured human DRG neurons; these changes may relate to pathophysiology in disease states, and provide an in vitro model to study novel analgesics.

APOSTOLIDIS A, POPAT R, YIANGOU Y, COCKAYNE D, FORD APDW, DAVIS JB, DASGUPTA P, FOWLER CJ, ANAND P. 2005. DECREASED SENSORY RECEPTORS P2X 3 AND TRPV1 IN SUBUROTHELIAL NERVE FIBERS FOLLOWING INTRADETRUSOR INJECTIONS OF BOTULINUM TOXIN FOR HUMAN DETRUSOR OVERACTIVITY Journal of Urology, 174 (3), pp. 977-983. | Read more

Gopinath P, Wan E, Holdcroft A, Facer P, Davis JB, Smith GD, Bountra C, Anand P. 2005. Increased capsaicin receptor TRPV1 in skin nerve fibres and related vanilloid receptors TRPV3 and TRPV4 in keratinocytes in human breast pain. BMC Womens Health, 5 (1), pp. 2. | Show Abstract | Read more

BACKGROUND: Breast pain and tenderness affects 70% of women at some time. These symptoms have been attributed to stretching of the nerves with increase in breast size, but tissue mechanisms are poorly understood. METHODS: Eighteen patients (n = 12 breast reduction and n = 6 breast reconstruction) were recruited and assessed for breast pain by clinical questionnaire. Breast skin biopsies from each patient were examined using immunohistological methods with specific antibodies to the capsaicin receptor TRPV1, related vanilloid thermoreceptors TRPV3 and TRPV4, and nerve growth factor (NGF). RESULTS: TRPV1-positive intra-epidermal nerve fibres were significantly increased in patients with breast pain and tenderness (TRPV1 fibres / mm epidermis, median [range] - no pain group, n = 8, 0.69 [0-1.27]; pain group, n = 10, 2.15 [0.77-4.38]; p = 0.0009). Nerve Growth Factor, which up-regulates TRPV1 and induces nerve sprouting, was present basal keratinocytes: some breast pain specimens also showed NGF staining in supra-basal keratinocytes. TRPV4-immunoreactive fibres were present in sub-epidermis but not significantly changed in painful breast tissue. Both TRPV3 and TRPV4 were significantly increased in keratinocytes in breast pain tissues; TRPV3, median [range] - no pain group, n = 6, 0.75 [0-2]; pain group, n = 11, 2 123, p = 0.008; TRPV4, median [range] - no pain group, n = 6, [0-1]; pain group, n = 11, 1 [0.5-2], p = 0.014). CONCLUSION: Increased TRPV1 intra-epidermal nerve fibres could represent collateral sprouts, or re-innervation following nerve stretch and damage by polymodal nociceptors. Selective TRPV1-blockers may provide new therapy in breast pain. The role of TRPV3 and TRPV4 changes in keratinocytes deserve further study.

Bulmer DCE, Jiang W, Hicks GA, Davis JB, Winchester WJ, Grundy D. 2005. Vagal selective effects of ruthenium red on the jejunal afferent fibre response to ischaemia in the rat Neurogastroenterology and Motility, 17 (1), pp. 102-111. | Read more

Matthews PJ, Aziz Q, Facer P, Davis JB, Thompson DG, Anand P. 2004. Increased capsaicin receptor TRPV1 nerve fibres in the inflamed human oesophagus. Eur J Gastroenterol Hepatol, 16 (9), pp. 897-902. | Show Abstract | Read more

BACKGROUND: Gastro-oesophageal reflux disease (GORD) patients commonly describe symptoms of heartburn and chest pain. The capsaicin receptor vanilloid receptor 1 (TRPV1) (VR1) is a cation channel expressed by sensory neurones and activated by heat, acid pH and ethanol, which may trigger burning pain. AIM: To study the distribution of TRPV1-expressing nerve fibres in oesophageal mucosal biopsies from patients with symptomatic oesophagitis and in control subjects. METHODS: Biopsies were taken at gastroscopy from the distal oesophagus of seven symptomatic oesophagitis patients and seven asymptomatic patients undergoing investigation for iron-deficiency anaemia. These biopsies were studied by immunohistochemistry using affinity-purified antibodies to TRPV1 and to the neuronal marker peripherin. The density of oesophageal epithelial TRPV1 innervation was assessed by calculating the proportion of papillae in each oesophageal epithelium biopsy specimen containing TRPV1-immunoreactive fibres. RESULTS: TRPV1-immunoreactive nerves were distributed within the lamina propria in healthy subjects and in oesophagitis patients. The percentage of papillae positive for TRPV1 was elevated in oesophagitis patients compared with controls. Peripherin fibre density was not significantly different between the groups. CONCLUSIONS: TRPV1-immunoreactive sensory nerve fibres are expressed in human oesophageal mucosa both in health and in disease. Increased TRPV1 expression in the inflamed oesophagus may mediate the heartburn in oesophagitis, and TRPV1 blockers may provide novel treatment.

Rami HK, Thompson M, Wyman P, Jerman JC, Egerton J, Brough S, Stevens AJ, Randall AD, Smart D, Gunthorpe MJ, Davis JB. 2004. Discovery of small molecule antagonists of TRPV1 Bioorganic & Medicinal Chemistry Letters, 14 (14), pp. 3631-3634. | Read more

Amadesi S. 2004. Protease-Activated Receptor 2 Sensitizes the Capsaicin Receptor Transient Receptor Potential Vanilloid Receptor 1 to Induce Hyperalgesia Journal of Neuroscience, 24 (18), pp. 4300-4312. | Read more

Szelenyi Z, Hummel Z, Szolcsanyi J, Davis JB. 2004. Daily body temperature rhythm and heat tolerance in TRPV1 knockout and capsaicin pretreated mice European Journal of Neuroscience, 19 (5), pp. 1421-1424. | Read more

Gunthorpe MJ, Rami HK, Jerman JC, Smart D, Gill CH, Soffin EM, Luis Hannan S, Lappin SC, Egerton J, Smith GD et al. 2004. Identification and characterisation of SB-366791, a potent and selective vanilloid receptor (VR1/TRPV1) antagonist Neuropharmacology, 46 (1), pp. 133-149. | Read more

Roberts JC, Davis JB, Benham CD. 2004. [3H]Resiniferatoxin autoradiography in the CNS of wild-type and TRPV1 null mice defines TRPV1 (VR-1) protein distribution Brain Research, 995 (2), pp. 176-183. | Read more

Benham CD, Gunthorpe MJ, Davis JB. 2003. TRPV channels as temperature sensors Cell Calcium, 33 (5-6), pp. 479-487. | Read more

Rigoni M, Trevisani M, Gazzieri D, Nadaletto R, Tognetto M, Creminon C, Davis JB, Campi B, Amadesi S, Geppetti P, Harrison S. 2003. Neurogenic responses mediated by vanilloid receptor-1 (TRPV1) are blocked by the high affinity antagonist, iodo-resiniferatoxin British Journal of Pharmacology, 138 (5), pp. 977-985. | Read more

Chan CLH, Facer P, Davis JB, Smith GD, Egerton J, Bountra C, Williams NS, Anand P. 2003. Sensory fibres expressing capsaicin receptor TRPV1 in patients with rectal hypersensitivity and faecal urgency. Lancet, 361 (9355), pp. 385-391. | Show Abstract | Read more

BACKGROUND: Faecal urgency and incontinence with rectal hypersensitivity is a distressing, unexplained disorder that is inadequately treated. We aimed to determine whether expression of the heat and capsaicin receptor vanilloid receptor 1 (TRPV1 or VR1) was changed in rectal sensory fibres, and to correlate nerve fibre density with sensory abnormalities. METHODS: We compared full-thickness rectal biopsy samples from nine patients with physiologically characterised rectal hypersensitivity with tissue samples from 12 controls. Sensory thresholds to rectal balloon distension and heating the rectal mucosa were measured before biopsy. We assessed specimens with immunohistochemistry and image analysis using specific antibodies to TRPV1; nerve growth factor (NGF) receptor tyrosine kinase A; glial cell line-derived neurotrophic factor (GDNF); neuropeptides calcitonin gene-related peptide (CGRP) and substance P; the related vanilloid receptor-like protein (VRL) 2; glial markers S-100 and glial fibrillary acid protein (GFAP); and the nerve structural marker peripherin. FINDINGS: In rectal hypersensitivity, nerve fibres immunoreactive to TRPV1 were increased in muscle, submucosal, and mucosal layers: in the mucosal layer, the median% area positive was 0.44 (range 0.30-0.59) in patients who were hypersensitive and 0.11 (0.00-0.21) in controls (p=0.0005). The numbers of peripherin-positive fibres also increased in the mucosal layer (hypersensitive 3.00 [1.80-6.50], controls 1.20 [0.39-2.10]: (p=0.0002). The increase in TRVP1 correlated significantly with the decrease in rectal heat (p=0.03) and the distension (p=0.02) sensory thresholds. The thresholds for heat and distension were also significantly correlated (p=0.0028). Expression of nerve fibres positive for GDNF (p=0.001) and tyrosine kinase A (p=0.002) was also increased, as were cell bodies of the submucosal ganglia immunoreactive to CGRP (p=0.0009). INTERPRETATION: Faecal urgency and rectal hypersensitivity could result from increased numbers of polymodal sensory nerve fibres expressing TRPV1. The triggering factor or factors remain uncertain, but drugs that target nerve terminals that express this receptor, such as topical resiniferatoxin, deserve consideration.

Marshall ICB, Owen DE, Cripps TV, Davis JB, McNulty S, Smart D. 2003. Activation of vanilloid receptor 1 by resiniferatoxin mobilizes calcium from inositol 1,4,5-trisphosphate-sensitive stores British Journal of Pharmacology, 138 (1), pp. 172-176. | Read more

Ralevic V, Jerman JC, Brough SJ, Davis JB, Egerton J, Smart D. 2003. Pharmacology of vanilloids at recombinant and endogenous rat vanilloid receptors Biochemical Pharmacology, 65 (1), pp. 143-151. | Read more

Jerman JC, Gray J, Brough SJ, Ooi L, Owen D, Davis JB, Smart D. 2002. Comparison of effects of anandamide at recombinant and endogenous rat vanilloid receptors British Journal of Anaesthesia, 89 (6), pp. 882-887. | Read more

Smith GD, Gunthorpe MJ, Kelsell RE, Hayes PD, Reilly P, Facer P, Wright JE, Jerman JC, Walhin J-P, Ooi L et al. 2002. TRPV3 is a temperature-sensitive vanilloid receptor-like protein Nature, 418 (6894), pp. 186-190. | Read more

Trevisani M, Smart D, Gunthorpe MJ, Tognetto M, Barbieri M, Campi B, Amadesi S, Gray J, Jerman JC, Brough SJ et al. 2002. Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1 Nature Neuroscience, 5 (6), pp. 546-551. | Read more

Benham CD, Davis JB, Randall AD. 2002. Vanilloid and TRP channels: a family of lipid-gated cation channels Neuropharmacology, 42 (7), pp. 873-888. | Read more

Watanabe H, Davis JB, Smart D, Jerman JC, Smith GD, Hayes P, Vriens J, Cairns W, Wissenbach U, Prenen J et al. 2002. Activation of TRPV4 Channels (hVRL-2/mTRP12) by Phorbol Derivatives Journal of Biological Chemistry, 277 (16), pp. 13569-13577. | Read more

Gunthorpe MJ, Benham CD, Randall A, Davis JB. 2002. The diversity in the vanilloid (TRPV) receptor family of ion channels Trends in Pharmacological Sciences, 23 (4), pp. 183-191. | Read more

Riccio A, Mattei C, Kelsell RE, Medhurst AD, Calver AR, Randall AD, Davis JB, Benham CD, Pangalos MN. 2002. Cloning and functional expression of human short TRP7, a candidate protein for store-operated Ca2+ influx. J Biol Chem, 277 (14), pp. 12302-12309. | Show Abstract | Read more

The regulation and control of plasma membrane Ca(2+) fluxes is critical for the initiation and maintenance of a variety of signal transduction cascades. Recently, the study of transient receptor potential channels (TRPs) has suggested that these proteins have an important role to play in mediating capacitative calcium entry. In this study, we have isolated a cDNA from human brain that encodes a novel transient receptor potential channel termed human TRP7 (hTRP7). hTRP7 is a member of the short TRP channel family and is 98% homologous to mouse TRP7 (mTRP7). At the mRNA level hTRP7 was widely expressed in tissues of the central nervous system, as well as some peripheral tissues such as pituitary gland and kidney. However, in contrast to mTRP7, which is highly expressed in heart and lung, hTRP7 was undetectable in these tissues. For functional analysis, we heterologously expressed hTRP7 cDNA in an human embryonic kidney cell line. In comparison with untransfected cells depletion of intracellular calcium stores in hTRP7-expressing cells, using either carbachol or thapsigargin, produced a marked increase in the subsequent level of Ca(2+) influx. This increased Ca(2+) entry was blocked by inhibitors of capacitative calcium entry such as La(3+) and Gd(3+). Furthermore, transient transfection of an hTRP7 antisense expression construct into cells expressing hTRP7 eliminated the augmented store-operated Ca(2+) entry. Our findings suggest that hTRP7 is a store-operated calcium channel, a finding in stark contrast to the mouse orthologue, mTRP7, which is reported to enhance Ca(2+) influx independently of store depletion, and suggests that human and mouse TRP7 channels may fulfil different physiological roles.

De Petrocellis L, Davis JB, Di Marzo V. 2001. Palmitoylethanolamide enhances anandamide stimulation of human vanilloid VR1 receptors FEBS Letters, 506 (3), pp. 253-256. | Read more

Bisogno T, Hanuš L, De Petrocellis L, Tchilibon S, Ponde DE, Brandi I, Moriello AS, Davis JB, Mechoulam R, Di Marzo V. 2001. Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide British Journal of Pharmacology, 134 (4), pp. 845-852. | Read more

Gray J, Haran MM, Schneider K, Vesce S, Ray AM, Owen D, White IR, Cutler P, Davis JB. 2001. Evidence That Inhibition of Cathepsin-B Contributes to the Neuroprotective Properties of Caspase Inhibitor Tyr-Val-Ala-Asp-Chloromethyl Ketone Journal of Biological Chemistry, 276 (35), pp. 32750-32755. | Read more

Vellani V, Mapplebeck S, Moriondo A, Davis JB, McNaughton PA. 2001. Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide. J Physiol, 534 (Pt 3), pp. 813-825. | Show Abstract | Read more

1. The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. 2. Maximal activation of PKC by a brief application of phorbol 12-myristate 13-acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65-fold in DRG neurones and 2.18-fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31-8220 prevented the enhancement caused by PMA. 3. Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. 4. Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. 5. Removing external Ca(2+) enhanced the response to a low concentration of capsaicin 2.40-fold in DRG neurones and 3.42-fold in HEK293 cells. Activation of PKC in zero Ca(2+) produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. 6. The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin-gated current. 7. The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium also potentiates activation, and PKC activation then has no further effect. The results are consistent with a model in which phosphorylation of VR1 by PKC increases the probability of channel gating by agonists, and in which dephosphorylation occurs by a calcium-dependent process.

M. G, G. S, J. D, A. R. 2001. Characterisation of a human acid-sensing ion channel (hASIC1a) endogenously expressed in HEK293 cells Pfl�gers Archiv European Journal of Physiology, 442 (5), pp. 668-674. | Read more

De Petrocellis L, Harrison S, Bisogno T, Tognetto M, Brandi I, Smith GD, Creminon C, Davis JB, Geppetti P, Di Marzo V. The vanilloid receptor (VR1)-mediated effects of anandamide are potently enhanced by the cAMP-dependent protein kinase Journal of Neurochemistry, 77 (6), pp. 1660-1663. | Read more

Dass N, Davis JB, Sanger GJ. 2001. Responses to E-capsaicin in detrusor tissue from both wildtype and vanilloid receptor-1 null mice BRITISH JOURNAL OF PHARMACOLOGY, 133 pp. U10-U10.

Di Marzo V, Lastres-Becker I, Bisogno T, De Petrocellis L, Milone A, Davis JB, Fernandez-Ruiz JJ. 2001. Hypolocomotor effects in rats of capsaicin and two long chain capsaicin homologues European Journal of Pharmacology, 420 (2-3), pp. 123-131. | Read more

De Petrocellis L, Bisogno T, Maccarrone M, Davis JB, Finazzi-Agrò A, Di Marzo V. 2001. The Activity of Anandamide at Vanilloid VR1 Receptors Requires Facilitated Transport across the Cell Membrane and Is Limited by Intracellular Metabolism Journal of Biological Chemistry, 276 (16), pp. 12856-12863. | Read more

Ralevic V, Kendall DA, Jerman JC, Davis JB, Middlemiss DN, Smart D. 2001. Low pH modulation of recombinant vanilloid receptors and perivascular capsaicin-sensitive sensory neurotransmission. Auton Neurosci, 88 (1-2), pp. 36-44. | Show Abstract | Read more

The effect of low pH on capsaicin-sensitive sensory neurotransmission in the rat isolated mesenteric arterial bed and at recombinant (rVR1) vanilloid receptors was investigated. Mesenteric sensory neurogenic vasorelaxation elicited by electrical field stimulation was reversibly inhibited by lowering pH from 7.4 to 6.9 and 6.3. Capsaicin-induced vasorelaxation was not different at pH 6.9, but was attenuated at pH 6.3. Vasorelaxation to calcitonin gene-related peptide, the principal sensory motor neurotransmitter in rat mesenteric arteries, was not different at pH 6.9 or pH 6.3. In rVR1-transfected HEK293 cells, acidic conditions enhanced the affinities of capsaicin and capsazepine at rVR1, but did not affect the potency of carbachol at endogenous muscarinic receptors. Following inactivation of endogenous acid-sensitive ion channels, lowering pH (6.0-4.5) directly increased [Ca2+]i in rVR1-HEK293 cells (EC50 5.5). This response was abolished by 1 microM capsazepine. In conclusion, a decrease in pH (to 6.9 and 6.3) enhances the affinity of capsaicin at rVR1, but inhibits sensory neurotransmission in the rat mesenteric arterial bed. This likely explains why there is no evidence of an enhancement of sensitivity to capsaicin at endogenous vanilloid receptors, as observed with rVR1. When pH is reduced still further (6.0-5.5) there is direct activation of rVR1.

Smart D, Jerman JC, Gunthorpe MJ, Brough SJ, Ranson J, Cairns W, Hayes PD, Randall AD, Davis JB. 2001. Characterisation using FLIPR of human vanilloid VR1 receptor pharmacology. Eur J Pharmacol, 417 (1-2), pp. 51-58. | Show Abstract | Read more

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.

Di Marzo V, Bisogno T, De Petrocellis L, Brandi I, Jefferson RG, Winckler RL, Davis JB, Dasse O, Mahadevan A, Razdan RK, Martin BR. 2001. Highly selective CB(1) cannabinoid receptor ligands and novel CB(1)/VR(1) vanilloid receptor "hybrid" ligands. Biochem Biophys Res Commun, 281 (2), pp. 444-451. | Show Abstract | Read more

Anandamide and the metabolically stabler analogs, (R)-1'-methyl-2'-hydroxy-ethyl-arachidonamide (Met-AEA) and N-(3-methoxy-4-hydroxy-benzyl)-arachidonamide (arvanil), are CB(1) cannabinoid and VR(1) vanilloid receptors agonists. We synthesized 1',1'-dimethylheptyl-arvanil (O-1839) and six other AEA analogs obtained by addition of either a hydroxy, cyano, or bromo group on the C-20 atom of 1,1'-dimethylpentyl-Met-AEA (O-1811, O-1812 and O-1860, respectively) or 1,1'-dimethylpentyl-arvanil (O-1856, O-1895 and O-1861, respectively). The compounds were tested for their (i) affinity for CB(1) and CB(2) receptors, (ii) capability to activate VR1 receptors, (iii) inhibitory effect on the anandamide hydrolysis and on the anandamide membrane transporter, and (iv) cannabimimetic activity in the mouse 'tetrad' of in vivo assays. O-1812 is the first ligand ever proven to be highly (500- to 1000-fold) selective for CB(1) vs both VR(1) and CB(2) receptors, while O-1861 is the first true "hybrid" agonist of CB(1)/VR(1) receptors and a compound with potential therapeutic importance. The activities of the seven compounds in vivo did not correlate with their activities at either CB(1) or VR(1) receptors, thus suggesting the existence of other brain sites of action mediating some of their neurobehavioral actions in mice.

De Petrocellis L, Bisogno T, Davis JB, Pertwee RG, Di Marzo V. 2000. Overlap between the ligand recognition properties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsaicin-like activity. FEBS Lett, 483 (1), pp. 52-56. | Show Abstract | Read more

Some synthetic agonists of the VR1 vanilloid (capsaicin) receptor also inhibit the facilitated transport into cells of the endogenous cannabinoid anandamide (arachidonoylethanolamide, AEA). Here we tested several AEA derivatives containing various derivatized phenyl groups or different alkyl chains as either inhibitors of the AEA membrane transporter (AMT) in intact cells or functional agonists of the VR1 vanilloid receptor in HEK cells transfected with the human VR1. We found that four known AMT inhibitors, AM404, arvanil, olvanil and linvanil, activate VR1 receptors at concentrations 400-10000-fold lower than those necessary to inhibit the AMT. However, we also found three novel AEA derivatives, named VDM11, VDM12 and VDM13, which inhibit the AMT as potently as AM404 but exhibit little or no agonist activity at hVR1. These compounds are weak inhibitors of AEA enzymatic hydrolysis and poor CB(1)/CB(2) receptor ligands. We show for the first time that, despite the overlap between the chemical moieties of AMT inhibitors and VR1 agonists, selective inhibitors of AEA uptake that do not activate VR1 (e.g. VDM11) can be developed.

Gray CW, Ward RV, Karran E, Turconi S, Rowles A, Viglienghi D, Southan C, Barton A, Fantom KG, West A et al. 2000. Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response. Eur J Biochem, 267 (18), pp. 5699-5710. | Show Abstract | Read more

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.

Gunthorpe MJ, Harries MH, Prinjha RK, Davis JB, Randall A. 2000. Voltage- and time-dependent properties of the recombinant rat vanilloid receptor (rVR1). J Physiol, 525 Pt 3 (3), pp. 747-759. | Show Abstract | Read more

Whole-cell voltage-clamp techniques were used to investigate the capsaicin-, voltage- and time-dependent properties of the rat vanilloid receptor (rVR1) stably expressed in human embryonic kidney (HEK) 293 cells. At a holding potential of -70 mV, application of capsaicin (0.03-30 microM) to HEK 293 cells expressing the rVR1 receptor led to the appearance of inward currents (EC50, 497 nM; Hill coefficient, nH, 2.85) which were reversibly antagonized by 10 microM capsazepine. Current-voltage relationships, determined using depolarizing or hyperpolarizing voltage ramps, had reversal potentials close to 0 mV, exhibited substantial outward rectification and possessed a region of negative slope conductance at holding potentials negative to around -70 mV. Further experiments indicated that the outward rectification and the region of negative slope conductance did not result from external block of the channel by either Ba2+, Ca2+ or Mg2+. During our characterization of rVR1, it became apparent that the rectification behaviour of this receptor was not entirely instantaneous as might be expected for a ligand-gated ion channel, but rather displayed clear time-dependent components. We characterized the kinetics of these novel gating properties in a series of additional voltage-step experiments. The time-dependent changes in rVR1-mediated conductance due to membrane depolarization or repolarization occurred with bi-exponential kinetics. On depolarization to +70 mV the time-dependent increase in outward current developed with mean time constants of 6.7 +/- 0.7 and 51.8 +/- 18.4 ms, with the faster time constant playing a dominant role (64.4 +/- 3.8 %). Similar kinetics also described the decay of 'tail currents' observed on repolarization. Furthermore, these time-dependent changes appeared to be unaffected by the removal of extracellular divalent cations and were not significantly voltage dependent. Our data reveal that rVR1 exhibits substantial time- and voltage-dependent gating properties that may have significance for the physiology of sensory transduction of nociceptive signals.

Ray AM, Owen DE, Evans ML, Davis JB, Benham CD. 2000. Caspase inhibitors are functionally neuroprotective against oxygen glucose deprivation induced CA1 death in rat organotypic hippocampal slices. Brain Res, 867 (1-2), pp. 62-69. | Show Abstract | Read more

We have explored the neuroprotective efficacy of the cell penetrant caspase inhibitor, Ac-YVAD-cmk, in a hippocampal slice model of neuronal cell death induced by oxygen and glucose deprivation. Organotypic hippocampal slice cultures were prepared from 8 to 10-day-old rats and maintained for 10 to 12 days in vitro. Pre-treatment with Ac-YVAD-cmk prior to 45 min oxygen and glucose deprivation was neuroprotective as measured by propidium iodide uptake, with an EC(50) between 1 and 10 micromol/l. Ac-YVAD-cmk was also able to preserve synaptic function in the organotypic hippocampal slice cultures 24 h after oxygen and glucose deprivation. Ac-YVAD-cmk prevented the increase in histone-associated DNA fragmentation induced by oxygen and glucose deprivation. Interleukin-1beta did not reverse the protective effect of Ac-YVAD-cmk, and interleukin-1 receptor antagonist alone was not protective. These results show that caspase inhibitors are neuroprotective in a hippocampal slice culture system, using structural, biochemical and electrophysiological endpoints, and that this effect is not a result of inhibition of interleukin-1beta production.

Jerman JC, Brough SJ, Prinjha R, Harries MH, Davis JB, Smart D. 2000. Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology. Br J Pharmacol, 130 (4), pp. 916-922. | Show Abstract | Read more

The vanilloid receptor (VR1) is a ligand-gated ion channel, which plays an important role in nociceptive processing. Therefore, a pharmacological characterization of the recently cloned rat VR1 (rVR1) was undertaken. HEK293 cells stable expressing rVR1 (rVR1-HEK293) were loaded with Fluo-3AM and then incubated at 25 degrees C for 30 min with or without various antagonists or signal transduction modifying agents. Then intracellular calcium concentrations ([Ca(2+)](i)) were monitored using FLIPR, before and after the addition of various agonists. The rank order of potency of agonists (resiniferatoxin (RTX)>capsaicin>olvanil>PPAHV) was as expected, and all were full agonists. The potencies of capsaicin and olvanil, but not RTX or PPAHV, were enhanced at pH 6.4 (pEC(50) values of 7.47+/-0.06, 7.16+/-0.06, 8.19+/-0.06 and 6.02+/-0.03 respectively at pH 7.4 vs 7.71+/-0.05, 7.58+/-0.14, 8.10+/-0.05 and 6.04+/-0.08 at pH 6.4). Capsazepine, isovelleral and ruthenium red all inhibited the capsaicin (100 nM)-induced Ca(2+) response in rVR1-HEK293 cells, with pK(B) values of 7.52+/-0.08, 6.92+/-0.11 and 8.09+/-0.12 respectively (n=6 each). The response to RTX and olvanil were also inhibited by these compounds. None displayed any agonist-like activity. The removal of extracellular Ca(2+) abolished, whilst inhibition of protein kinase C with chelerythrine chloride (10 microM) partially (approximately 20%) inhibited, the capsaicin (10 microM)-induced Ca(2+) response. However, tetrodotoxin (3 microM), nimodipine (10 microM), omega-GVIA conotoxin (1 microM), thapsigargin (1 microM), U73122 (3 microM) or H-89 (3 microM) had no effect on the capsaicin (100 nM)-induced response. In conclusion, the recombinant rVR1 stably expressed in HEK293 cells acts as a ligand-gated Ca(2+) channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor.

Ward RV, Jennings KH, Jepras R, Neville W, Owen DE, Hawkins J, Christie G, Davis JB, George A, Karran EH, Howlett DR. 2000. Fractionation and characterization of oligomeric, protofibrillar and fibrillar forms of beta-amyloid peptide. Biochem J, 348 Pt 1 (1), pp. 137-144. | Show Abstract | Read more

The beta-amyloid (Abeta) peptide, a major component of senile plaques in Alzheimer's disease brain, has been shown previously to undergo a process of polymerization to produce neurotoxic forms of amyloid. Recent literature has attempted to define precisely the form of Abeta responsible for its neurodegenerative properties. In the present study we describe a novel density-gradient centrifugation method for the isolation and characterization of structurally distinct polymerized forms of Abeta peptide. Fractions containing protofibrils, fibrils, sheet structures and low molecular mass oligomers were prepared. The fractionated forms of Abeta were characterized structurally by transmission electron microscopy. The effects on cell viability of these fractions was determined in the B12 neuronal cell line and hippocampal neurons. Marked effects on cell viability in the cells were found to correspond to the presence of protofibrillar and fibrillar structures, but not to monomeric peptide or sheet-like structures of polymerized Abeta. Biological activity correlated with a positive reaction in an immunoassay that specifically detects protofibrillar and fibrillar Abeta; those fractions that were immunoassay negative had no effect on cell viability. These data suggest that the effect of Abeta on cell viability is not confined to a single conformational form but that both fibrillar and protofibrillar species have the potential to be active in this assay.

Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, Harries MH, Latcham J, Clapham C, Atkinson K et al. 2000. Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature, 405 (6783), pp. 183-187. | Show Abstract | Read more

The vanilloid receptor-1 (VR1) is a ligand-gated, non-selective cation channel expressed predominantly by sensory neurons. VR1 responds to noxious stimuli including capsaicin, the pungent component of chilli peppers, heat and extracellular acidification, and it is able to integrate simultaneous exposure to these stimuli. These findings and research linking capsaicin with nociceptive behaviours (that is, responses to painful stimuli in animals have led to VR1 being considered as important for pain sensation. Here we have disrupted the mouse VR1 gene using standard gene targeting techniques. Small diameter dorsal root ganglion neurons isolated from VR1-null mice lacked many of the capsaicin-, acid- and heat-gated responses that have been previously well characterized in small diameter dorsal root ganglion neurons from various species. Furthermore, although the VR1-null mice appeared normal in a wide range of behavioural tests, including responses to acute noxious thermal stimuli, their ability to develop carrageenan-induced thermal hyperalgesia was completely absent. We conclude that VR1 is required for inflammatory sensitization to noxious thermal stimuli but also that alternative mechanisms are sufficient for normal sensation of noxious heat.

Hayes P, Meadows HJ, Gunthorpe MJ, Harries MH, Duckworth DM, Cairns W, Harrison DC, Clarke CE, Ellington K, Prinjha RK et al. 2000. Cloning and functional expression of a human orthologue of rat vanilloid receptor-1. Pain, 88 (2), pp. 205-215. | Show Abstract | Read more

Capsaicin, resiniferatoxin, protons or heat have been shown to activate an ion channel, termed the rat vanilloid receptor-1 (rVR1), originally isolated by expression cloning for a capsaicin sensitive phenotype. Here we describe the cloning of a human vanilloid receptor-1 (hVR1) cDNA containing a 2517 bp open reading frame that encodes a protein with 92% homology to the rat vanilloid receptor-1. Oocytes or mammalian cells expressing this cDNA respond to capsaicin, pH and temperature by generating inward membrane currents. Mammalian cells transfected with human VR1 respond to capsaicin with an increase in intracellular calcium. The human VR1 has a chromosomal location of 17p13 and is expressed in human dorsal root ganglia and also at low levels throughout a wide range of CNS and peripheral tissues. Together the sequence homology, similar expression profile and functional properties confirm that the cloned cDNA represents the human orthologue of rat VR1.

Mapplebeck S, Davis JB, McNaughton PA. 2000. Effect of protein kinase C (PKC) on the heat activation of the capsaicin-gated ion channel (VR1) EUROPEAN JOURNAL OF NEUROSCIENCE, 12 pp. 123-123.

Jerman JC, Brough SJ, Davis JB, Middlemiss DN, Smart D. 2000. The anandamide transport inhibitor AM404 is an agonist at the rat vanilloid receptor (VR1) BRITISH JOURNAL OF PHARMACOLOGY, 129 pp. U44-U44.

Smart D, Jerman JC, Brough SJ, Davis JB. 2000. Characterisation using FLIPR of human vanilloid receptor-i(HVR1) pharmacology. EUROPEAN JOURNAL OF NEUROSCIENCE, 12 pp. 124-124.

Christie G, Hussain I, Smith L, Mansfield F, Bresciani L, Davis JB, Gray CW, Markwell R, Wadsworth H, King R et al. 2000. Peptidyl boronic acids inhibit the degradation of presenilin-l in presenilin-l transfected cells ALZHEIMERS REPORTS, 3 (3), pp. 169-175.

Smart D, Gunthorpe MJ, Jerman JC, Nasir S, Gray J, Muir AI, Chambers JK, Randall AD, Davis JB. 2000. The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br J Pharmacol, 129 (2), pp. 227-230. | Show Abstract | Read more

The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 microM) and capsaicin (1 microM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 microM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC(50) values of 5. 94+/-0.06 (n=5) and 7.13+/-0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK(B) of 7.40+/-0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1.

Jerman JC, Brough SJ, Davis JB, Middlemiss DN, Smart D. 2000. The endogenous lipid anandamide is an agonist AT rat and human vanilloid (VR1) receptors. EUROPEAN JOURNAL OF NEUROSCIENCE, 12 pp. 124-124.

Owen DE, Gray J, Davis JB. 2000. Apomorphine is neuroprotective in a model of oxidative glutamate toxicity EUROPEAN JOURNAL OF NEUROSCIENCE, 12 pp. 32-32.

Gunthorpe MJ, Smith GD, Davis JB, Randall AD. 2000. Characterisation of human vanilloid receptor-1 and an endogenous acid sensing ion channel expressed in HEK293 cells EUROPEAN JOURNAL OF NEUROSCIENCE, 12 pp. 24-24.

Smith GD, Clarke CE, Meadows HJ, Benham CD, Randall AD, Davis JB. 2000. Identification of an amino acid responsible for acid mediated potentiation of vanilloid receptor (VR1) function. EUROPEAN JOURNAL OF NEUROSCIENCE, 12 pp. 384-384.

Beher D, Elle C, Underwood J, Davis JB, Ward R, Karran E, Masters CL, Beyreuther K, Multhaup G. 1999. Proteolytic fragments of Alzheimer's disease-associated presenilin 1 are present in synaptic organelles and growth cone membranes of rat brain. J Neurochem, 72 (4), pp. 1564-1573. | Show Abstract | Read more

Previous studies have demonstrated the molecular linkage of three causative genes for early-onset Alzheimer's disease: the presenilin 1 gene on chromosome 14, the presenilin 2 gene on chromosome 1, and the amyloid precursor protein gene on chromosome 21. In the present study, we have investigated the distributions of the approximately 20-kDa C-terminal and approximately 30-kDa N-terminal fragments of presenilin 1 and the amyloid precursor protein in rat brain and compared them with the distribution of several marker proteins. The fragments of presenilin 1 are present in synaptic plasma membranes, neurite growth cone membranes, and small synaptic vesicles of rat brain. Both proteolytic fragments are coenriched in the corresponding tissue fractions. Based on this observation, it seems likely that N- and C-terminal presenilin 1 fragments form a functional unit while remaining associated. In contrast to a predominant subcellular localization of presenilin 1 to the endoplasmic reticulum and Golgi apparatus in different cell lines, our results indicate that rat brain presenilin 1 fragments exit from these biosynthetic compartments to reach synaptic organelles in neurons.

Karran EH, Allsop D, Christie G, Davis J, Gray C, Mansfield F, Ward RV. 1998. Presenilins--in search of functionality. Biochem Soc Trans, 26 (3), pp. 491-496. | Show Abstract | Read more

The discovery of the PS proteins, the complexities of their biochemistry and their potential involvement in signalling pathways and in apoptosis have galvanized research into AD. To date, the aspect of the functionality of the PSs most relevant to the pathology of AD is the effect of PS FAD mutants to increase the proportion of A beta 42 produced from cells. This, coupled to the observation that gamma-secretase cleavage is considerably reduced in neurons derived from PS-1 knockout mice, argues strongly that PS plays a very direct role in the proteolytic processing of APP.

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Sagara Y, Dargusch R, Chambers D, Davis J, Schubert D, Maher P. 1998. Cellular mechanisms of resistance to chronic oxidative stress. Free Radic Biol Med, 24 (9), pp. 1375-1389. | Show Abstract | Read more

Oxidative stress is implicated in several pathologies such as AIDS, Alzheimer's disease, and Parkinson's disease, as well as in normal aging. As a model system to study the response of cells to oxidative insults, glutamate toxicity on a mouse nerve cell line, HT-22, was examined. Glutamate exposure kills HT-22 via a nonreceptor-mediated oxidative pathway by blocking cystine uptake and causing depletion of intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species and, ultimately, apoptotic cell death. Several HT-22 subclones that are 10-fold resistant to exogenous glutamate were isolated and the mechanisms involved in resistance characterized. The expression levels of neither heat shock proteins nor apoptosis-related proteins are changed in the resistant cells. In contrast, the antioxidant enzyme catalase, but not glutathione peroxidase nor superoxide dismutase, is more highly expressed in the resistant than in the parental cells. In addition, the resistant cells have enhanced rates of GSH regeneration due to higher activities of the GSH metabolic enzymes gamma-glutamylcysteine synthetase and GSH reductase, and GSH S-transferases activities are also elevated. As a consequence of these alterations, the glutamate resistant cells are also more resistant to organic hydroperoxides and anticancer drugs that affect these GSH enzymes. These results indicate that resistance to apoptotic oxidative stress may be acquired by coordinated changes in multiple antioxidant pathways.

Culvenor JG, Maher F, Evin G, Malchiodi-Albedi F, Cappai R, Underwood JR, Davis JB, Karran EH, Roberts GW, Beyreuther K, Masters CL. 1997. Alzheimer's disease-associated presenilin 1 in neuronal cells: evidence for localization to the endoplasmic reticulum-Golgi intermediate compartment. J Neurosci Res, 49 (6), pp. 719-731. | Show Abstract | Read more

The recently identified Alzheimer's disease-associated presenilin 1 and 2 (PS1 and PS2) genes encode two homologous multi membrane-spanning proteins. Rabbit antibodies to the N-terminal domain of PS1 detected PS1 in human neuroblastoma SH-SY5Y wild type and PS1 transfectants (SY5Y-PS1) as well as in mouse P19, in CHO-K1 and CHO-APP770 transfected cells, in rat cerebellar granule and hippocampal neurons, and astrocytes. Immunoblotting detected full-length protein of 50 kDa, and a major presumptive cleavage product of 30 kDa. The immunofluorescence pattern resembled labeling of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) marker protein ERGIC-53. PS1 distribution showed slight condensation after brefeldin A and more marked condensation after incubation of cells at 16 degrees C, characteristic of the ERGIC compartment. Double labeling showed colocalization of ERGIC-53 with PS1 in the SY5Y-PS1 cells. PS1 labeling of SY5Y-PS1 and P19 cells showed overlap of the cis-Golgi marker p210 and colocalization with p210 after brefeldin A which causes redistribution of p210 to the ERGIC. Expression of PS1 did not change in level or cellular distribution during development of neurons in culture. Double labeling for the amyloid precursor protein (APP) and PS1 on SY5Y-PS1 cells and CHO-APP770 cells showed some overlap under control conditions. These results indicate that PS1 is a resident protein of the ERGIC and could be involved in trafficking of proteins, including APP, between the ER and Golgi compartments.

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Scopus

Culvenor JG, Maher F, Evin GV, Malchiodi-Albedi F, Cappai R, Underwood JR, Davis JB, Karran EH, Roberts GW, Beyreuther K, Masters CL. 1997. Alzheimer's disease-associated presenilin 1 in neuronal cells: Evidence for localization to the endoplasmic reticulum-Golgi intermediate compartment Journal of Neuroscience Research, 49 (6), pp. 719-731. | Show Abstract | Read more

The recently identified Alzheimer's disease-associated presenilin 1 and 2 (PS1 and PS2) genes encode two homologous multi membrane-spanning proteins. Rabbit antibodies to the N-terminal domain of PS1 detected PS1 in human neuroblastoma SH-SY5Y wild type and PS1 transfectants (SY5Y-PS1) as well as in mouse P19, in CHO-K1 and CHO-APP770 transfected cells, in rat cerebellar granule and hippocampal neurons, and astrocytes. Immunoblotting detected full-length protein of 50 kDa, and a major presumptive cleavage product of 30 kDa. The immunofluorescence pattern resembled labeling of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) marker protein ERGIC-53. PS1 distribution showed slight condensation after brefeldin A and more marked condensation after incubation of cells at 16°C, characteristic of the ERGIC compartment. Double labeling showed colocalization of ERGIC-53 with PS1 in the SY5Y-PS1 cells. PS1 labeling of SY5Y-PS1 and P19 cells showed overlap of the cis-Golgi marker p210 and colocalization with p210 after brefeldin A which causes redistribution of p210 to the ERGIC. Expression of PS1 did not change in level or cellular distribution during development of neurons in culture. Double labeling for the amyloid precursor protein (APP) and PS1 on SY5Y-PS1 cells and CHO-APP770 cells showed some overlap under control conditions. These results indicate that PS1 is a resident protein of the ERGIC and could be involved in trafficking of proteins, including APP, between the ER and Golgi compartments.

Toye AM, Davis JB, Jones OTG. 1997. Amyloid beta induces markers of oxidative stress in human clonal neural cell line SH-SY5Y. JOURNAL OF NEUROCHEMISTRY, 69 pp. S49-S49.

Ward RV, Davis JB, Gray CW, Barton AJ, Bresciani LG, Caivano M, Murphy VF, Duff K, Hutton M, Hardy J et al. 1996. Presenilin-1 is processed into two major cleavage products in neuronal cell lines. Neurodegeneration, 5 (4), pp. 293-298. | Show Abstract | Read more

Presenilin-1 (PS-1) has been identified as the protein encoded by the chromosome 14 locus that, when mutated, leads to familial Alzheimer's disease (FAD). Using PS-1 transfected SHSY5Y neuroblastoma cells, we have demonstrated by immunodetection, using polyclonal antibodies, that PS-1 is processed to give two fragments: an N-terminal 28 kDa fragment, and a C-terminal 18 kDa fragment. In a number of non-transfected cell types, most PS-1 is detected as the cleaved products. The molecular weights of the PS-1 cleavage products suggest that the cleavage point will most probably be within a region of the hydrophilic loop domain coded for by either exon 8 or 9 of the PS-1 gene. The clustering of FAD mutations within exon 8 strongly suggests that it encodes a key functional domain. It seems likely that the cleavage of PS-1 is crucial to some aspect of its functionality. An understanding of this process will give insights into the pathology of AD, and may offer new opportunities for therapeutic intervention.

Davis JB. 1996. Oxidative mechanisms in beta-amyloid cytotoxicity. Neurodegeneration, 5 (4), pp. 441-444. | Show Abstract | Read more

Amyloid beta-peptide has been demonstrated to be toxic for primary and clonal neuronal cell lines in vitro. Oxidative mechanisms have been implicated in this pathway at several points, including the aggregation of beta-amyloid necessary for cytotoxic activity, generation of radicals by the peptide itself, and intracellularly in response to toxic beta-amyloid peptides. Supporting an oxidative hypothesis are the observations that cells mount a stress response to beta-amyloid similar to that seen in response to oxidative stress and that they may be rescued from cytotoxicity by antioxidants, inhibitors of oxidative enzyme metabolism, and overexpression of antioxidant enzymes. Although the source(s) of the oxygen radicals has not yet been identified, altered antioxidant enzyme levels and oxidative by-products in Alzheimer's disease brain samples relate the in vitro studies to the human disease.

Maher P, Davis JB. 1996. The role of monoamine metabolism in oxidative glutamate toxicity. J Neurosci, 16 (20), pp. 6394-6401. | Show Abstract | Read more

Glutamate kills neuronal cells by either a receptor-mediated pathway or the inhibition of cystine uptake, the "oxidative pathway." Antioxidants can block cell death initiated by either pathway, suggesting that toxicity is dependent on the production of free radicals. We provide evidence that in a neuronal cell line, glutamate toxicity via the oxidative pathway requires monoamine metabolism as a source of free radicals. Glutamate toxicity is inhibited by monoamine oxidase (MAO) type-A-specific inhibitors, but only at concentrations much higher than those required to inhibit classical type-A MAO. Toxicity is not inhibited by MAO type-B-specific inhibitors at any concentration. Furthermore, treatment of cells with agents that block monoamine uptake inhibits glutamate toxicity. These results suggest that an enzyme distinct from MAO is involved in monoamine metabolism and demonstrate a relationship between glutamate toxicity and monoamine metabolism. These data also have implications for the understanding and treatment of neurodegenerative disorders in which glutamate toxicity is thought to be involved.

Davis JB, Maher P. 1994. Protein kinase C activation inhibits glutamate-induced cytotoxicity in a neuronal cell line. Brain Res, 652 (1), pp. 169-173. | Show Abstract | Read more

A neuronal cell line, HT-22, is sensitive to glutamate cytotoxicity via a non-receptor mediated oxidative pathway. 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, blocks this glutamate-induced cell death. Down-regulation of protein kinase C eliminates the protection against glutamate cytotoxicity afforded by TPA. The data suggest that protein kinase C activation blocks an early step in the cytotoxic pathway.

Behl C, Davis JB, Lesley R, Schubert D. 1994. Hydrogen peroxide mediates amyloid beta protein toxicity. Cell, 77 (6), pp. 817-827. | Show Abstract | Read more

Amyloid beta protein (A beta) is a 40-43 amino acid peptide that is associated with plaques in the brains of Alzheimer's patients and is cytotoxic to cultured neurons. Using both primary central nervous system cultures and clonal cell lines, it is shown that a number of anti-oxidants protect cells from A beta toxicity, suggesting that at least one pathway to A beta cytotoxicity results in free radical damage. A beta causes increased levels of H2O2 and lipid peroxides to accumulate in cells. The H2O2-degrading enzyme catalase protects cells from A beta toxicity. Clonal cell lines selected for their resistance to A beta toxicity also become resistant to the cytolytic action of H2O2. In addition, A beta induces the activity of NF-kappa B, a transcription factor thought to be regulated by oxidative stress. Finally, A beta-induced H2O2 production and A beta toxicity are blocked by reagents that inhibit flavin oxidases, suggesting that A beta activates a member of this class of enzymes. These results show that the cytotoxic action of A beta on neurons results from free radical damage to susceptible cells.

Behl C, Davis JB, Klier FG, Schubert D. 1994. Amyloid beta peptide induces necrosis rather than apoptosis. Brain Res, 645 (1-2), pp. 253-264. | Show Abstract | Read more

Amyloid beta peptide (A beta P), a major component of Alzheimer's disease plaques, is toxic to rat pheochromocytoma PC12 cells and to rat cortical neurons. A reduction in cell survival could be detected after 24 h incubation with 0.01 to 20 microM of the 25-35 peptide fragment (beta 25-35) of A beta P. To study the mechanism of cell death induced by A beta P, the morphological as well as the biochemical features of neuronal cell death were analyzed. To distinguish between necrosis and apoptosis, PC12 cell death caused by beta 25-35 was compared to that induced by serum deprivation, a process known to be apoptotic in these cells. The DNA-degradation pattern of A beta P treated cells appeared random rather than at distinct internucleosomal sites as with apoptosis. Electron microscopic studies of NGF-treated PC12 cells and cortical primary cultures exposed to 20 microM beta 25-35 revealed immediate cellular damage such as vacuolization of the cytoplasm, breakdown of Golgi-apparatus and other membrane systems, and neurite disintegration. This was followed by total collapse of the cytoplasm and cell lysis. These data show that A beta P toxicity occurs via a necrotic rather than an apoptotic pathway.

HOWLETT DR, PARVATHY S, FINN HJ, DAVIS JB, CLARK MSG, ROBERTS GW. 1994. RESISTANCE TO BETA-A4 NEUROTOXICITY IN PC12 CELLS NEUROBIOLOGY OF AGING, 15 pp. S141-S141.

WETZEL R, WOOD SJ, DAVIS JB, HURLE MR. 1994. PROLINE SUBSTITUTIONS IN BETA-PEPTIDE FRAGMENTS ELIMINATE BOTH FIBRIL FORMATION AND CELLULAR TOXICITY NEUROBIOLOGY OF AGING, 15 pp. S50-S50.

Goodearl AD, Davis JB, Mistry K, Minghetti L, Otsu M, Waterfield MD, Stroobant P. 1993. Purification of multiple forms of glial growth factor. J Biol Chem, 268 (24), pp. 18095-18102. | Show Abstract

Glial growth factors (GGFs) were purified from bovine pituitaries using an in vitro rat Schwann cell mitogenesis assay. In addition to an approximately 34-kDa species termed GGF-I, similar in molecular mass to a previously identified molecule (Lemke, G. E., and Brockes, J. P. (1984) J. Neuroscience 4, 75-83), two species named GGF-II and GGF-III were characterized with apparent molecular masses of approximately 59 and approximately 45 kDa, respectively. Highly purified preparations of all species share a similar dose-dependent stimulation of Schwann cell DNA synthesis at nanomolar concentrations. Forskolin synergizes with all three GGFs, shifting their dose dependence 3-8-fold into the sub-nanomolar range. The GGFs, which contain N-linked carbohydrate groups not essential for their in vitro mitogenic effects, are three distinct members of a novel family of glial cell mitogens.

Marchionni MA, Goodearl AD, Chen MS, Bermingham-McDonogh O, Kirk C, Hendricks M, Danehy F, Misumi D, Sudhalter J, Kobayashi K. 1993. Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system. Nature, 362 (6418), pp. 312-318. | Show Abstract | Read more

Glial growth factors, proteins that are mitogenic for Schwann cells, and several ligands for the p185erbB2 receptor, are products of the same gene. Alternative splicing of the messenger RNA generates an array of putative membrane-attached, intracellular and secreted signalling proteins, at least some of which are expressed in the developing spinal cord and brain. These factors are probably important in the development and regeneration of the nervous system.

Davis JB, McMurray HF, Schubert D. 1992. The amyloid beta-protein of Alzheimer's disease is chemotactic for mononuclear phagocytes. Biochem Biophys Res Commun, 189 (2), pp. 1096-1100. | Show Abstract | Read more

The cellular pathology of Alzheimer's disease includes an accumulation of microglia surrounding the amyloid plaques. We report that human amyloid beta-protein is chemotactic for murine resident peritoneal macrophages and rat microglia, which may account for the increased density of microglia in plaques. A maximal chemotactic response was observed at 1-10nM, with a 2.5 fold increase in activity over controls for both classes of mononuclear phagocytes. The neurotoxic peptide fragment (25-35) of amyloid beta-protein is similarly chemotactic, while a control scrambled version and the precursor protein are not chemotactic. These results indicate that beta-protein may influence plaque formation via the recruitment of phagocytes, with consequent implications for the future development of treatments for Alzheimer's disease.

Davis JB, Goodearl AD. 1991. The axon may control Schwann cell responses to growth factors. Ann N Y Acad Sci, 633 (1 Glial-Neurona), pp. 535-536. | Read more

Davis JB, Stroobant P. 1990. Platelet-derived growth factors and fibroblast growth factors are mitogens for rat Schwann cells. J Cell Biol, 110 (4), pp. 1353-1360. | Show Abstract | Read more

Rat sciatic nerve Schwann cells in culture respond to a limited range of mitogens, including glial growth factor, transforming growth factors beta-1 and beta-2 (TGF-beta 1, TGF-beta 2), some cell membrane-associated factors, and to agents such as cholera toxin and forskolin which raise intracellular levels of cAMP. These responses require the presence of FCS, which exhibits little or no mitogenic activity in the absence of other factors. However, we recently found that forskolin greatly potentiates the mitogenic signal from TGFs-beta 1 and beta 2, raising the possibility that cAMP might couple other factors to mitogenesis. We have therefore screened a range of candidate mitogens using DNA synthesis assays. Other than TGFs-beta and glial growth factor, none of the factors tested were mitogenic in the presence of 10% serum alone. With the addition of forskolin, however, porcine PDGF, human PDGF, acidic and basic FGF were potent mitogens for rat Schwann cells, stimulating DNA synthesis and increasing cell number. Cholera toxin and dibutyrylcyclicAMP, but not 1,9-dideoxyforskolin, can substitute for forskolin indicating that the mitogenic effect is mediated via adenylyl cyclase activation. Porcine PDGF gave half-maximal stimulation at 15 pM, and human PGDF an equivalent response at 1 nM. Basic FGF was half maximal at 5 pM, acidic FGF at 1 nM. The recognition of PDGFs and FGFs as mitogens for Schwann cells has many implications for the study of Schwann cell proliferation in the development and regeneration of nerves, and in Schwann cell tumorigenesis.

Ridley AJ. 1989. Transforming growth factors-beta 1 and beta 2 are mitogens for rat Schwann cells The Journal of Cell Biology, 109 (6), pp. 3419-3424. | Read more

Davis JB, Bowyer DE. 1989. Macrophages modify β-VLDL by proteolysis and enhance subsequent lipid accumulation in arterial smooth muscle cells Atherosclerosis, 77 (2-3), pp. 203-208. | Read more

Mauricio R, Benn C, Davis J, Dawson G, Dawson LA, Evans A, Fox N, Gallacher J, Hutton M, Isaac J et al. 2019. Tackling gaps in developing life-changing treatments for dementia. Alzheimers Dement (N Y), 5 pp. 241-253. | Show Abstract | Read more

Since the G8 dementia summit in 2013, a number of initiatives have been established with the aim of facilitating the discovery of a disease-modifying treatment for dementia by 2025. This report is a summary of the findings and recommendations of a meeting titled "Tackling gaps in developing life-changing treatments for dementia", hosted by Alzheimer's Research UK in May 2018. The aim of the meeting was to identify, review, and highlight the areas in dementia research that are not currently being addressed by existing initiatives. It reflects the views of leading experts in the field of neurodegeneration research challenged with developing a strategic action plan to address these gaps and make recommendations on how to achieve the G8 dementia summit goals. The plan calls for significant advances in (1) translating newly identified genetic risk factors into a better understanding of the impacted biological processes; (2) enhanced understanding of selective neuronal resilience to inform novel drug targets; (3) facilitating robust and reproducible drug-target validation; (4) appropriate and evidence-based selection of appropriate subjects for proof-of-concept clinical trials; (5) improving approaches to assess drug-target engagement in humans; and (6) innovative approaches in conducting clinical trials if we are able to detect disease 10-15 years earlier than we currently do today.

Rami HK, Thompson M, Stemp G, Fell S, Jerman JC, Stevens AJ, Smart D, Sargent B, Sanderson D, Randall AD et al. 2006. Discovery of SB-705498: A potent, selective and orally bioavailable TRPV1 antagonist suitable for clinical development Bioorganic & Medicinal Chemistry Letters, 16 (12), pp. 3287-3291. | Read more

Rami HK, Thompson M, Wyman P, Jerman JC, Egerton J, Brough S, Stevens AJ, Randall AD, Smart D, Gunthorpe MJ, Davis JB. 2004. Discovery of small molecule antagonists of TRPV1 Bioorganic & Medicinal Chemistry Letters, 14 (14), pp. 3631-3634. | Read more

Chan CLH, Facer P, Davis JB, Smith GD, Egerton J, Bountra C, Williams NS, Anand P. 2003. Sensory fibres expressing capsaicin receptor TRPV1 in patients with rectal hypersensitivity and faecal urgency. Lancet, 361 (9355), pp. 385-391. | Show Abstract | Read more

BACKGROUND: Faecal urgency and incontinence with rectal hypersensitivity is a distressing, unexplained disorder that is inadequately treated. We aimed to determine whether expression of the heat and capsaicin receptor vanilloid receptor 1 (TRPV1 or VR1) was changed in rectal sensory fibres, and to correlate nerve fibre density with sensory abnormalities. METHODS: We compared full-thickness rectal biopsy samples from nine patients with physiologically characterised rectal hypersensitivity with tissue samples from 12 controls. Sensory thresholds to rectal balloon distension and heating the rectal mucosa were measured before biopsy. We assessed specimens with immunohistochemistry and image analysis using specific antibodies to TRPV1; nerve growth factor (NGF) receptor tyrosine kinase A; glial cell line-derived neurotrophic factor (GDNF); neuropeptides calcitonin gene-related peptide (CGRP) and substance P; the related vanilloid receptor-like protein (VRL) 2; glial markers S-100 and glial fibrillary acid protein (GFAP); and the nerve structural marker peripherin. FINDINGS: In rectal hypersensitivity, nerve fibres immunoreactive to TRPV1 were increased in muscle, submucosal, and mucosal layers: in the mucosal layer, the median% area positive was 0.44 (range 0.30-0.59) in patients who were hypersensitive and 0.11 (0.00-0.21) in controls (p=0.0005). The numbers of peripherin-positive fibres also increased in the mucosal layer (hypersensitive 3.00 [1.80-6.50], controls 1.20 [0.39-2.10]: (p=0.0002). The increase in TRVP1 correlated significantly with the decrease in rectal heat (p=0.03) and the distension (p=0.02) sensory thresholds. The thresholds for heat and distension were also significantly correlated (p=0.0028). Expression of nerve fibres positive for GDNF (p=0.001) and tyrosine kinase A (p=0.002) was also increased, as were cell bodies of the submucosal ganglia immunoreactive to CGRP (p=0.0009). INTERPRETATION: Faecal urgency and rectal hypersensitivity could result from increased numbers of polymodal sensory nerve fibres expressing TRPV1. The triggering factor or factors remain uncertain, but drugs that target nerve terminals that express this receptor, such as topical resiniferatoxin, deserve consideration.

Smith GD, Gunthorpe MJ, Kelsell RE, Hayes PD, Reilly P, Facer P, Wright JE, Jerman JC, Walhin J-P, Ooi L et al. 2002. TRPV3 is a temperature-sensitive vanilloid receptor-like protein Nature, 418 (6894), pp. 186-190. | Read more

Trevisani M, Smart D, Gunthorpe MJ, Tognetto M, Barbieri M, Campi B, Amadesi S, Gray J, Jerman JC, Brough SJ et al. 2002. Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1 Nature Neuroscience, 5 (6), pp. 546-551. | Read more

Vellani V, Mapplebeck S, Moriondo A, Davis JB, McNaughton PA. 2001. Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide. J Physiol, 534 (Pt 3), pp. 813-825. | Show Abstract | Read more

1. The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. 2. Maximal activation of PKC by a brief application of phorbol 12-myristate 13-acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65-fold in DRG neurones and 2.18-fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31-8220 prevented the enhancement caused by PMA. 3. Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. 4. Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. 5. Removing external Ca(2+) enhanced the response to a low concentration of capsaicin 2.40-fold in DRG neurones and 3.42-fold in HEK293 cells. Activation of PKC in zero Ca(2+) produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. 6. The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin-gated current. 7. The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium also potentiates activation, and PKC activation then has no further effect. The results are consistent with a model in which phosphorylation of VR1 by PKC increases the probability of channel gating by agonists, and in which dephosphorylation occurs by a calcium-dependent process.

De Petrocellis L, Bisogno T, Davis JB, Pertwee RG, Di Marzo V. 2000. Overlap between the ligand recognition properties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsaicin-like activity. FEBS Lett, 483 (1), pp. 52-56. | Show Abstract | Read more

Some synthetic agonists of the VR1 vanilloid (capsaicin) receptor also inhibit the facilitated transport into cells of the endogenous cannabinoid anandamide (arachidonoylethanolamide, AEA). Here we tested several AEA derivatives containing various derivatized phenyl groups or different alkyl chains as either inhibitors of the AEA membrane transporter (AMT) in intact cells or functional agonists of the VR1 vanilloid receptor in HEK cells transfected with the human VR1. We found that four known AMT inhibitors, AM404, arvanil, olvanil and linvanil, activate VR1 receptors at concentrations 400-10000-fold lower than those necessary to inhibit the AMT. However, we also found three novel AEA derivatives, named VDM11, VDM12 and VDM13, which inhibit the AMT as potently as AM404 but exhibit little or no agonist activity at hVR1. These compounds are weak inhibitors of AEA enzymatic hydrolysis and poor CB(1)/CB(2) receptor ligands. We show for the first time that, despite the overlap between the chemical moieties of AMT inhibitors and VR1 agonists, selective inhibitors of AEA uptake that do not activate VR1 (e.g. VDM11) can be developed.

Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, Harries MH, Latcham J, Clapham C, Atkinson K et al. 2000. Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature, 405 (6783), pp. 183-187. | Show Abstract | Read more

The vanilloid receptor-1 (VR1) is a ligand-gated, non-selective cation channel expressed predominantly by sensory neurons. VR1 responds to noxious stimuli including capsaicin, the pungent component of chilli peppers, heat and extracellular acidification, and it is able to integrate simultaneous exposure to these stimuli. These findings and research linking capsaicin with nociceptive behaviours (that is, responses to painful stimuli in animals have led to VR1 being considered as important for pain sensation. Here we have disrupted the mouse VR1 gene using standard gene targeting techniques. Small diameter dorsal root ganglion neurons isolated from VR1-null mice lacked many of the capsaicin-, acid- and heat-gated responses that have been previously well characterized in small diameter dorsal root ganglion neurons from various species. Furthermore, although the VR1-null mice appeared normal in a wide range of behavioural tests, including responses to acute noxious thermal stimuli, their ability to develop carrageenan-induced thermal hyperalgesia was completely absent. We conclude that VR1 is required for inflammatory sensitization to noxious thermal stimuli but also that alternative mechanisms are sufficient for normal sensation of noxious heat.

Hayes P, Meadows HJ, Gunthorpe MJ, Harries MH, Duckworth DM, Cairns W, Harrison DC, Clarke CE, Ellington K, Prinjha RK et al. 2000. Cloning and functional expression of a human orthologue of rat vanilloid receptor-1. Pain, 88 (2), pp. 205-215. | Show Abstract | Read more

Capsaicin, resiniferatoxin, protons or heat have been shown to activate an ion channel, termed the rat vanilloid receptor-1 (rVR1), originally isolated by expression cloning for a capsaicin sensitive phenotype. Here we describe the cloning of a human vanilloid receptor-1 (hVR1) cDNA containing a 2517 bp open reading frame that encodes a protein with 92% homology to the rat vanilloid receptor-1. Oocytes or mammalian cells expressing this cDNA respond to capsaicin, pH and temperature by generating inward membrane currents. Mammalian cells transfected with human VR1 respond to capsaicin with an increase in intracellular calcium. The human VR1 has a chromosomal location of 17p13 and is expressed in human dorsal root ganglia and also at low levels throughout a wide range of CNS and peripheral tissues. Together the sequence homology, similar expression profile and functional properties confirm that the cloned cDNA represents the human orthologue of rat VR1.

Smart D, Gunthorpe MJ, Jerman JC, Nasir S, Gray J, Muir AI, Chambers JK, Randall AD, Davis JB. 2000. The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br J Pharmacol, 129 (2), pp. 227-230. | Show Abstract | Read more

The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 microM) and capsaicin (1 microM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 microM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC(50) values of 5. 94+/-0.06 (n=5) and 7.13+/-0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK(B) of 7.40+/-0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1.

Maher P, Davis JB. 1996. The role of monoamine metabolism in oxidative glutamate toxicity. J Neurosci, 16 (20), pp. 6394-6401. | Show Abstract | Read more

Glutamate kills neuronal cells by either a receptor-mediated pathway or the inhibition of cystine uptake, the "oxidative pathway." Antioxidants can block cell death initiated by either pathway, suggesting that toxicity is dependent on the production of free radicals. We provide evidence that in a neuronal cell line, glutamate toxicity via the oxidative pathway requires monoamine metabolism as a source of free radicals. Glutamate toxicity is inhibited by monoamine oxidase (MAO) type-A-specific inhibitors, but only at concentrations much higher than those required to inhibit classical type-A MAO. Toxicity is not inhibited by MAO type-B-specific inhibitors at any concentration. Furthermore, treatment of cells with agents that block monoamine uptake inhibits glutamate toxicity. These results suggest that an enzyme distinct from MAO is involved in monoamine metabolism and demonstrate a relationship between glutamate toxicity and monoamine metabolism. These data also have implications for the understanding and treatment of neurodegenerative disorders in which glutamate toxicity is thought to be involved.

Behl C, Davis JB, Lesley R, Schubert D. 1994. Hydrogen peroxide mediates amyloid beta protein toxicity. Cell, 77 (6), pp. 817-827. | Show Abstract | Read more

Amyloid beta protein (A beta) is a 40-43 amino acid peptide that is associated with plaques in the brains of Alzheimer's patients and is cytotoxic to cultured neurons. Using both primary central nervous system cultures and clonal cell lines, it is shown that a number of anti-oxidants protect cells from A beta toxicity, suggesting that at least one pathway to A beta cytotoxicity results in free radical damage. A beta causes increased levels of H2O2 and lipid peroxides to accumulate in cells. The H2O2-degrading enzyme catalase protects cells from A beta toxicity. Clonal cell lines selected for their resistance to A beta toxicity also become resistant to the cytolytic action of H2O2. In addition, A beta induces the activity of NF-kappa B, a transcription factor thought to be regulated by oxidative stress. Finally, A beta-induced H2O2 production and A beta toxicity are blocked by reagents that inhibit flavin oxidases, suggesting that A beta activates a member of this class of enzymes. These results show that the cytotoxic action of A beta on neurons results from free radical damage to susceptible cells.

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