It has been postulated that the G protein-coupled receptor, GPR55, is a third cannabinoid receptor. Given that the ligands at the CB(1) and CB(2) receptors are effective analgesic and anti-inflammatory agents, the role of GPR55 in hyperalgesia associated with inflammatory and neuropathic pain has been investigated. As there are no well-validated GPR55 tool compounds, a GPR55 knockout (GPR55(-/-)) mouse line was generated and fully backcrossed onto the C57BL/6 strain. General phenotypic analysis of GPR55(-/-) mice revealed no obvious primary differences, compared with wild-type (GPR55(+/+)) littermates. GPR55(-/-) mice were then tested in the models of adjuvant-induced inflammation and partial nerve ligation. Following intraplantar administration of Freund's complete adjuvant (FCA), inflammatory mechanical hyperalgesia was completely absent in GPR55(-/-) mice up to 14 days post-injection. Cytokine profiling experiments showed that at 14 days post-FCA injection there were increased levels of IL-4, IL-10, IFN gamma and GM-CSF in paws from the FCA-injected GPR55(-/-) mice when compared with the FCA-injected GPR55(+/+) mice. This suggests that GPR55 signalling can influence the regulation of certain cytokines and this may contribute to the lack of inflammatory mechanical hyperalgesia in the GPR55(-/-) mice. In the model of neuropathic hypersensitivity, GPR55(-/-) mice also failed to develop mechanical hyperalgesia up to 28 days post-ligation. These data clearly suggest that the manipulation of GPR55 may have therapeutic potential in the treatment of both inflammatory and neuropathic pain.

GPR55 is a G protein-coupled receptor activated by L-␣-lysophosphatidylinositol and suggested to have roles in pain signaling, bone morphogenesis, and possibly in vascular endothelial cells. It has affinity for certain cannabinoids (molecules that interact with the cannabinoid CB 1 and CB 2 receptors), but investigation of its functional role in cell-based systems and in tissue has been limited by a lack of selective pharmacological tools. Here, we present our characterization of GPR55 in the yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK293) cells. We describe GSK494581A (1-{2-fluoro-4-[1-(methyloxy)ethyl]phenyl}-4-{[4Ј-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}piperazine), a selective small-molecule ligand of GPR55 identified through diversity screening. GSK494581A is one of a series of benzoylpiperazines originally identified and patented as inhibitors of the glycine transporter subtype 1 (GlyT1). The structure-activity relationship between GPR55 and GlyT1 is divergent across this series. The most GPR55-selective example is GSK575594A (3-fluoro-4-(4-{[4Ј-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}-1-piperazinyl) aniline), which is approximately 60-fold selective for GPR55 (pEC 50 ϭ 6.8) over GlyT1 (pIC 50 ϭ 5.0). Several exemplars with activity at GPR55 and GlyT1 have been profiled at a broad range of other molecular targets and are inactive at cannabinoid receptors and all other targets tested. The benzoylpiperazine agonists activate human GPR55 but not rodent GPR55, suggesting that the relatively low level of sequence identity between these orthologs (75%) translates to important functional differences in the ligand-binding site.

Neuropeptide-expressing small diameter sensory neurones are thought to be vital in generating inflammatory hyperalgesic responses. Within the dorsal root ganglion (DRG), both the levels of the neuropeptide calcitonin gene-related peptide (CGRP) and the numbers of CGRP-immunoreactive (CGRP-IR) DRG neurones have been shown to increase in a number of acute adjuvant-induced inflammatory pain models. The aim of this study was to look specifically at changes in numbers of CGRP-IR DRG neurones in a chronic model of inflammatory joint pain following complete Freund's adjuvant (CFA) injection into the rat knee. In this model, there were significant increases in the number of ipsilateral CGRP-IR small DRG neurones at days 1, 16 and 35 following intra-articular CFA, compared to saline-injected sham animals. This correlated with the behavioural readouts of hypersensitivity and knee joint inflammation at the same time points. There was also a significant increase in the number of ipsilateral CGRP-IR medium DRG neurones and contralateral CGRP-IR small DRG neurones at day 1. Following dosing of CFA-injected rats with rofecoxib (Vioxx) or paracetamol, there was a significant decrease in the number of ipsilateral CGRP-IR small and medium DRG neurones in rofecoxib- but not paracetamol-treated rats. These data also correlated with behavioural readouts where hypersensitivity and knee joint inflammation were significantly reduced by rofecoxib but not paracetamol treatment. In conclusion, these data show that changes in ipsilateral CGRP expression within small DRG neurones are consistent with behavioural readouts in both time course, rofecoxib and paracetamol studies in this model of chronic inflammatory pain.

Activation of the mitogen-activated protein kinase (MAPK/ ERK) signal transduction pathway may mediate excitotoxic neuronal cell death in vitro and during ischemic brain injury in vivo. However, little is known, of the upstream regulation or downstream consequences of ERK activation under these conditions. Magnesium removal has been described to induce hyperexcitability and degeneration in cultured hippocampal neurones. Here, we show that neurotoxicity evoked by Mg 2+ removal in primary hippocampal neurones stimulates ERK, but not p38, phosphorylation. Removal of Mg 2+ also resulted in induction of the MAPK/ERK substrate mitogen-and stressresponse kinase 1 (MSK1) and induced phosphorylation of the MSK1 substrate, the transcription factor cAMP response element binding protein (CREB). Neuronal death and phosphorylation of components in this cascade were inhibited by the Raf inhibitor SB-386023, by the MEK inhibitor U0126, or by the MSK1 inhibitors H89 and Ro318220. Importantly, this form of cell death was inhibited in hippocampal neurones cultured from MSK1-/-mice and inhibitors of Raf or MEK had no additive neuroprotective effect. Together, these data indicate that MSK1 is a physiological kinase for CREB and that this activity is an essential component of activity-dependent neuronal cell death. Keywords: cAMP-responsive element binding protein, excitotoxicity, hippocampal neurones, mitogen and stressresponse kinase-1, mitogen-activated protein kinase, neuroprotection.

Phosphorylation-dependent intracellular signalling cascades that utilise ERKl/ERK2 MAP kinases play key roles as components of integrated signal transduction networks by which a variety of activated cell surface receptors elicit cellular responses to environmental cues. Excessive glutamate receptor stimulation and calcium influx are characteristic features of ischemic stroke, animal models of which also exhibit induction of ERKIMAPK activity. Moreover, excessive synaptic activity in cultured hippocampal neurons can trigger neuronal degeneration, concommitant with induction of ERK/MAPK activity. To explore further the nature of both upstream regulators and downstream effectors of ERK/MAPK activation in such a model, we have employed direct activity assays of candidate protein kinases in conjunction with a repertoire of potent and selective small molecule protein kinase inhibitors. Biochemical analyses, together with the neuroprotective properties of some of these compounds, define additional necessary components of an ERK/MAPK-mediated pathway by which excessive synaptic activity associated with neuronal injury signals to modulate transcription factor activity. Comparison with models of ERK/MAPK-mediated survival in other neuronal systems, and implications for therapeutic intervention in acute stroke, will be discussed. 569Hyper glucose-effect on PI3K/Akt signaling pathway leads to enhancement of oxidative cell damage in insulinoma BetaTC cells.Supraphysiologic levels of high glucose are known to cause glucotoxic effects to change cellular responsiveness against various cellular stresses such as cytokines, growth factors, and oxidants. Using mouse insulinoma BetaTC cells, we examined whether hyper glucose conditions could affect the cellular response against oxidative stress by hydrogen peroxide (H202). In hyper glucose conditions with 28mM glucose in the medium, the cell viability was drastically decreased under H 2 0 2 treatment, compared to intact viability with normal glucose conditions (5.5mM glucose).To examine the molecular mechanism of the distinctive responses of the cells between normal and hyper glucose conditions, we focused on PI3K/Akt signaling pathway for cell survival. In contrast to highly responsive states of PI3K and Akt phosphorylation during H 2 0 2 treatment in normal glucose conditions, the activation of PI3K/Akt pathway was transient and weak in the hyper glucose conditions. Moreover, we found a 54kDa PI3K-associated protein with phosphotyrosine in the normal culture but not in the hyper glucose one, and it suggests that the 54kDa protein may be involved in the PI3K/Akt signaling pathway working for H 2 0 2 resistant mechanism. Our results suggest that the hyper glucose conditions suppress the tyrosine phosphorylation of specific signaling proteins, such as 54kDa PI3K-associated protein, leading to diminish the cell survival signals of PI3K/Akt against oxidative stress in insulinoma cells.SAP-1 (_stomach cancer-associated protein-tyrosine ehosphatase-1) is a transmembrane-type protein-tyros...