GPER involvement in inflammatory pain.

Chronic pain is a worldwide refractory health disease that causes major financial and emotional burdens and that is devastating for individuals and society. One primary source of pain is inflammation. Current treatments for inflammatory pain are weakly effective, although they usually replace analgesics, such as opioids and non-steroidal anti-inflammatory drugs, which display serious side effects. Emerging evidence indicates that the membrane G protein-coupled estrogen receptor (GPER) may play an important role in the regulation of inflammation and pain. Herein, we focus on the consequences of pharmacological and genetic GPER modulation in different animal models of inflammatory pain. We also provide a brief overview of the putative mechanisms including the direct action of GPER on pain transmission and inflammation.

Role of T CD4+ cells, macrophages, C-low threshold mechanoreceptors and spinal Cav 3.2 channels in inflammation and related pain-like symptoms in murine inflammatory models.

BACKGROUND AND PURPOSE: T-type calcium channels, mainly the Cav 3.2 subtype, are important contributors to the nociceptive signalling pathway. We investigated their involvement in inflammation and related pain-like symptoms. EXPERIMENTAL APPROACH: The involvement of Cav 3.2 and T-type channels was investigated using genetic and pharmacological inhibition to assess mechanical allodynia/hyperalgesia and oedema development in two murine inflammatory pain models. The location of Cav 3.2 channels involved in pain-like symptoms was studied in mice with Cav 3.2 knocked out in C-low threshold mechanoreceptors (C-LTMR) and the use of ABT-639, a peripherally restricted T-type channel inhibitor. The anti-oedema effect of Cav 3.2 channel inhibition was investigated in chimeric mice with immune cells deleted for Cav 3.2. Lymphocytes and macrophages from either green fluorescent protein-targeted Cav 3.2 or KO mice were used to determine the expression of Cav 3.2 protein and the functional status of the cells. KEY RESULTS: Cav 3.2 channels contributed to the development of pain-like symptoms and oedema in the two murine inflammatory pain models. Our results provided evidence of the involvement of Cav 3.2 channels located on C-LTMRs and spinal cord in inflammatory pain. Cav 3.2 channels located in T cells and macrophages contribute to the inflammatory process. CONCLUSION AND IMPLICATIONS: Cav 3.2 channels play crucial roles in inflammation and related pain, implying that targeting of Cav 3.2 channels with pharmacological agents could be an attractive and readily evaluable strategy in clinical trials, to relieve chronic inflammatory pain in patients.

In-Depth Characterization of Somatic and Orofacial Sensitive Dysfunctions and Interfering-Symptoms in a Relapsing-Remitting Experimental Autoimmune Encephalomyelitis Mouse Model.

Among the many symptoms (motor, sensory, and cognitive) associated with multiple sclerosis (MS), chronic pain is a common disabling condition. In particular, neuropathic pain symptoms are very prevalent and debilitating, even in early stages of the disease. Unfortunately, chronic pain still lacks efficient therapeutic agents. Progress is needed (i) clinically by better characterizing pain symptoms in MS and understanding the underlying mechanisms, and (ii) preclinically by developing a more closely dedicated model to identify new therapeutic targets and evaluate new drugs. In this setting, new variants of experimental autoimmune encephalomyelitis (EAE) are currently developed in mice to exhibit less severe motor impairments, thereby avoiding confounding factors in assessing pain behaviors over the disease course. Among these, the optimized relapsing-remitting EAE (QuilA-EAE) mouse model, induced using myelin oligodendrocyte glycoprotein peptide fragment (35-55), pertussis toxin, and quillaja bark saponin, seems very promising. Our study sought (i) to better define sensitive dysfunctions and (ii) to extend behavioral characterization to interfering symptoms often associated with pain during MS, such as mood disturbances, fatigue, and cognitive impairment, in this optimized QuilA-EAE model. We made an in-depth characterization of this optimized QuilA-EAE model, describing for the first time somatic thermal hyperalgesia associated with mechanical and cold allodynia. Evaluation of orofacial pain sensitivity showed no mechanical or thermal allodynia. Detailed evaluation of motor behaviors highlighted slight defects in fine motor coordination in the QuilA-EAE mice but without impact on pain evaluation. Finally, no anxiety-related or cognitive impairment was observed during the peak of sensitive symptoms. Pharmacologically, as previously described, we found that pregabalin, a treatment commonly used in neuropathic pain patients, induced an analgesic effect on mechanical allodynia. In addition, we showed an anti-hyperalgesic thermal effect on this model. Our results demonstrate that this QuilA-EAE model is clearly of interest for studying pain symptom development and so could be used to identify and evaluate new therapeutic targets. The presence of interfering symptoms still needs to be further characterized.

Paracetamol is a centrally acting analgesic using mechanisms located in the periaqueductal grey.

BACKGROUND AND PURPOSE: We previously demonstrated that paracetamol has to be metabolised in the brain by fatty acid amide hydrolase enzyme into AM404 (N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide) to activate CB1 receptors and TRPV1 channels, which mediate its analgesic effect. However, the brain mechanisms supporting paracetamol-induced analgesia remain unknown. EXPERIMENTAL APPROACH: The effects of paracetamol on brain function in Sprague-Dawley rats were determined by functional MRI. Levels of neurotransmitters in the periaqueductal grey (PAG) were measured using in vivo 1 H-NMR and microdialysis. Analgesic effects of paracetamol were assessed by behavioural tests and challenged with different inhibitors, administered systemically or microinjected in the PAG. KEY RESULTS: Paracetamol decreased the connectivity of major brain structures involved in pain processing (insula, somatosensory cortex, amygdala, hypothalamus, and the PAG). This effect was particularly prominent in the PAG, where paracetamol, after conversion to AM404, (a) modulated neuronal activity and functional connectivity, (b) promoted GABA and glutamate release, and (c) activated a TRPV1 channel-mGlu5 receptor-PLC-DAGL-CB1 receptor signalling cascade to exert its analgesic effects. CONCLUSIONS AND IMPLICATIONS: The elucidation of the mechanism of action of paracetamol as an analgesic paves the way for pharmacological innovations to improve the pharmacopoeia of analgesic agents.

Ethosuximide improves chronic pain-induced anxiety- and depression-like behaviors.

Chronic pain is a heavy burden disease. Current treatments are generally weakly effective or associated with adverse effects. New therapeutic approaches are therefore needed. Recent studies have suggested T-type calcium channels as an attractive target for the treatment of chronic pain. In this perspective, it was decided to perform a preclinical evaluation of the efficacy of ethosuximide, a T-type channel blocker used clinically as an antiepileptic, as a novel pharmacological treatment for chronic pain. Assessment of the effect of ethosuximide was thus made in both nociception and pain-related comorbidities as anxiety and depression are frequently encountered in chronic pain patients. Our results show that such symptoms occurred in three animal models of chronic pain designed to reflect traumatic neuropathic, chemotherapy-induced neuropathic and inflammatory pain conditions. Administration of ethosuximide reduced both chronic pain and comorbidities with a marked intensity ranging from partial reduction to a complete suppression of symptoms. These results make ethosuximide, and more broadly the inhibition of T-type calcium channels, a new strategy for the management of uncontrolled chronic pain, likely to improve not only pain but also the accompanying anxiety and depression.

Inhibition of Cav 3.2 calcium channels: A new target for colonic hypersensitivity associated with low-grade inflammation.

BACKGROUND AND PURPOSE: Abdominal pain associated with low-grade inflammation is frequently encountered in irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) during remission. Current treatments are not very effective and new therapeutic approaches are needed. The role of CaV 3.2 channels, which are important in other chronic pain contexts, was investigated in a murine model of colonic hypersensitivity (CHS) associated with low-grade inflammation. EXPERIMENTAL APPROACH: Low doses of dextran sulfate sodium (DSS; 0.5%) were chronically administered to C57BL/6j mice in drinking water. Their inflammatory state was assessed by systemic and local measures of IL-6, myeloperoxidase, and lipocalin-2 using elisa. Colonic sensitivity was evaluated by the visceromotor responses to colorectal distension. Functional involvement of CaV 3.2 channels was assessed with different pharmacological (TTA-A2, ABT-639, and ethosuximide) and genetic tools. KEY RESULTS: DSS induced low-grade inflammation associated with CHS in mice. Genetic or pharmacological inhibition of CaV 3.2 channels reduced CHS. Cav3.2 channel deletion in primary nociceptive neurons in dorsal root ganglia (CaV 3.2Nav1.8 KO mice) suppressed CHS. Spinal, but not systemic, administration of ABT-639, a peripherally acting T-type channel blocker, reduced CHS. ABT-639 given intrathecally to CaV 3.2Nav1.8 KO mice had no effect, demonstrating involvement of CaV 3.2 channels located presynaptically in afferent fibre terminals. Finally, ethosuximide, which is a T-type channel blocker used clinically, reduced CHS. CONCLUSIONS AND IMPLICATIONS: These results suggest that ethosuximide represents a promising drug reposition strategy and that inhibition of CaV 3.2 channels is an attractive therapeutic approach for relieving CHS in IBS or IBD.

Memantine before Mastectomy Prevents Post-Surgery Pain: A Randomized, Blinded Clinical Trial in Surgical Patients.

BACKGROUND: Neuropathic pain following surgical treatment for breast cancer with or without chemotherapy is a clinical burden and patients frequently report cognitive, emotional and quality of life impairment. A preclinical study recently showed that memantine administered before surgery may prevent neuropathic pain development and cognitive dysfunction. With a translational approach, a clinical trial has been carried out to evaluate whether memantine administered before and after mastectomy could prevent the development of neuropathic pain, the impairment of cognition and quality of life. METHOD: A randomized, pilot clinical trial included 40 women undergoing mastectomy in the Oncology Department, University Hospital, Clermont-Ferrand, France. Memantine (5 to 20 mg/day; n = 20) or placebo (n = 20) was administered for four weeks starting two weeks before surgery. The primary endpoint was pain intensity measured on a (0-10) numerical rating scale at three months post-mastectomy. RESULTS: Data analyses were performed using mixed models and the tests were two-sided, with a type I error set at α = 0.05. Compared with placebo, patients receiving memantine showed at three months a significant difference in post-mastectomy pain intensity, less rescue analgesia and a better emotional state. An improvement of pain symptoms induced by cancer chemotherapy was also reported. CONCLUSIONS: This study shows for the first time the beneficial effect of memantine to prevent post-mastectomy pain development and to diminish chemotherapy-induced pain symptoms. The lesser analgesic consumption and better well-being of patients for at least six months after treatment suggests that memantine could be an interesting therapeutic option to diminish the burden of breast cancer therapy. TRIAL REGISTRATION: Clinicaltrials.gov NCT01536314.

Cholinergic Neurotransmission in the Posterior Insular Cortex Is Altered in Preclinical Models of Neuropathic Pain: Key Role of Muscarinic M2 Receptors in Donepezil-Induced Antinociception.

Neuropathic pain is one of the most debilitating pain conditions, yet no therapeutic strategy has been really effective for its treatment. Hence, a better understanding of its pathophysiological mechanisms is necessary to identify new pharmacological targets. Here, we report important metabolic variations in brain areas involved in pain processing in a rat model of oxaliplatin-induced neuropathy using HRMAS (1)H-NMR spectroscopy. An increased concentration of choline has been evidenced in the posterior insular cortex (pIC) of neuropathic animal, which was significantly correlated with animals' pain thresholds. The screening of 34 genes mRNA involved in the pIC cholinergic system showed an increased expression of the high-affinity choline transporter and especially the muscarinic M2 receptors, which was confirmed by Western blot analysis in oxaliplatin-treated rats and the spared nerve injury model (SNI). Furthermore, pharmacological activation of M2 receptors in the pIC using oxotremorine completely reversed oxaliplatin-induced mechanical allodynia. Consistently, systemic treatment with donepezil, a centrally active acetylcholinesterase inhibitor, prevented and reversed oxaliplatin-induced cold and mechanical allodynia as well as social interaction impairment. Intracerebral microdialysis revealed a lower level of acetylcholine in the pIC of oxaliplatin-treated rats, which was significantly increased by donepezil. Finally, the analgesic effect of donepezil was markedly reduced by a microinjection of the M2 antagonist, methoctramine, within the pIC, in both oxaliplatin-treated rats and spared nerve injury rats. These findings highlight the crucial role of cortical cholinergic neurotransmission as a critical mechanism of neuropathic pain, and suggest that targeting insular M2 receptors using central cholinomimetics could be used for neuropathic pain treatment. SIGNIFICANCE STATEMENT: Our study describes a decrease in cholinergic neurotransmission in the posterior insular cortex in neuropathic pain condition and the involvement of M2 receptors. Targeting these cortical muscarinic M2 receptors using central cholinomimetics could be an effective therapy for neuropathic pain treatment.

Supra-spinal FAAH is required for the analgesic action of paracetamol in an inflammatory context.

Paracetamol (acetaminophen) is the most commonly used analgesic in the world. Recently, a new view of its action has emerged: that paracetamol would be a pro-drug that should be metabolized by the FAAH enzyme into AM404, its active metabolite. However, this hypothesis has been demonstrated only in naive animals, a far cry from the clinical pathologic context of paracetamol use. Moreover, FAAH is a ubiquitous enzyme expressed both in the central nervous system and in the periphery. Thus, we explored: (i) the involvement of FAAH in the analgesic action of paracetamol in a mouse model of inflammatory pain; and (ii) the contributions of central versus peripheral FAAH in this action. The analgesic effect of paracetamol was evaluated in thermal hyperalgesia, mechanical allodynia and hyperalgesia induced by an intra-plantar injection of carrageenan (3%) in FAAH knock-out mice or their littermates. Moreover, the contribution of the central and peripheral enzymes was explored by comparing the effect of a global FAAH inhibitor (URB597) to that of a peripherally restricted FAAH inhibitor (URB937) on paracetamol action. Here, we show that in a model of inflammatory pain submitted to different stimuli, the analgesic action of paracetamol was abolished when FAAH was genetically or pharmacologically inhibited. Whereas a global FAAH inhibitor, URB597 (0.3 mg/kg), reduced the anti-hyperalgesic action of paracetamol, a brain-impermeant FAAH inhibitor, URB937 (0.3 mg/kg), had no influence. However, administered intracerebroventricularly, URB937 (5 µg/mouse) reduced the action of paracetamol. These results demonstrate that the supra-spinally-located FAAH enzyme is necessary for the analgesic action of paracetamol.

Low doses of dextromethorphan have a beneficial effect in the treatment of neuropathic pain.

N-methyl-D-aspartate receptor (NMDAR) antagonists may be given in persistent neuropathic pain, but adverse events especially with ketamine may limit their clinical use. Less central and cognitive adverse events are described with dextromethorphan and memantine. These molecules have been explored in many preclinical and clinical studies, but data are conflicting as regards neuropathic pain alleviation. Dextromethorphan and memantine have been administered to animals after spinal nerve ligation (SNL) to evaluate their antinociceptive/cognitive effects and associated molecular events, including the phosphorylation of several tyrosine (pTyr(1336), pTyr(1472)) residues in the NR2B NMDAR subunit. Spinal nerve ligation and sham animals received dextromethorphan (10 mg/kg, i.p.), memantine (20 mg/kg, i.p.) or saline (1 mL/kg, i.p.). These drugs were administered once symptoms of allodynia and hyperalgesia had developed. Tests were carried out before and after surgery. Tactile allodynia, mechanical hyperalgesia and spatial memory were, respectively, evaluated by von Frey, Randall & Selitto and Y-maze tests and molecular events by Western blot analysis. Spinal nerve-ligated animals displayed nociception and impaired spatial memory. Dextromethorphan, but not memantine, reversed neuropathic pain (NP) symptoms, restored spatial memory integrity and decreased the expression of pTyr(1336)NR2B. Following postoperative administration of dextromethorphan, this study has demonstrated for the first time a concordance between behaviour, cognitive function and molecular events via pTyr(1336)NR2B for neuropathic pain alleviation. Confirmation of these findings in patients would constitute a major step forward in the treatment of neuropathic pain and in the improvement of cognitive function and quality of life.

A polyamine-deficient diet prevents oxaliplatin-induced acute cold and mechanical hypersensitivity in rats.

BACKGROUND: Oxaliplatin is an anticancer drug used for the treatment of advanced colorectal cancer, but it can also cause painful peripheral neuropathies. The pathophysiology of these neuropathies has not been yet fully elucidated, but may involve spinal N-methyl-D-aspartate (NMDA) receptors, particularly the NR2B subunit. As polyamines are positive modulators of NMDA-NR2B receptors and mainly originate from dietary intake, the modulation of polyamines intake could represent an interesting way to prevent/modulate neuropathic pain symptoms by opposing glutamate neurotransmission. METHODS: The effect of a polyamine deficient diet was investigated in an animal model of oxaliplatin-induced acute pain hypersensitivity using behavioral tests (mechanical and cold hypersensitivity). The involvement of spinal glutamate neurotransmission was monitored by using a proton nuclear magnetic resonance spectroscopy based metabolomic approach and by assessing the expression and phosphorylation of the NR2B subunit of the NMDA receptor. RESULTS: A 7-day polyamine deficient diet totally prevented oxaliplatin-induced acute cold hypersensitivity and mechanical allodynia. Oxaliplatin-induced pain hypersensitivity was not associated with an increase in NR2B subunit expression or phosphorylation, but with an increase of glutamate level in the spinal dorsal horn which was completely prevented by a polyamine deficient diet. As a validation that the oxaliplatin-induced hypersensitivity could be due to an increased activity of the spinal glutamate system, an intrathecal administration of the specific NR2B antagonist, ifenprodil, totally reversed oxaliplatin-induced mechanical and cold hypersensitivity. CONCLUSION: A polyamine deficient diet could represent a promising and valuable nutritional therapy to prevent oxaliplatin-induced acute pain hypersensitivity.

Fatty acid amide hydrolase-dependent generation of antinociceptive drug metabolites acting on TRPV1 in the brain.

The discovery that paracetamol is metabolized to the potent TRPV1 activator N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404) and that this metabolite contributes to paracetamol's antinociceptive effect in rodents via activation of TRPV1 in the central nervous system (CNS) has provided a potential strategy for developing novel analgesics. Here we validated this strategy by examining the metabolism and antinociceptive activity of the de-acetylated paracetamol metabolite 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA), both of which may undergo a fatty acid amide hydrolase (FAAH)-dependent biotransformation to potent TRPV1 activators in the brain. Systemic administration of 4-aminophenol and HMBA led to a dose-dependent formation of AM404 plus N-(4-hydroxyphenyl)-9Z-octadecenamide (HPODA) and arvanil plus olvanil in the mouse brain, respectively. The order of potency of these lipid metabolites as TRPV1 activators was arvanil = olvanil>>AM404> HPODA. Both 4-aminophenol and HMBA displayed antinociceptive activity in various rodent pain tests. The formation of AM404, arvanil and olvanil, but not HPODA, and the antinociceptive effects of 4-aminophenol and HMBA were substantially reduced or disappeared in FAAH null mice. The activity of 4-aminophenol in the mouse formalin, von Frey and tail immersion tests was also lost in TRPV1 null mice. Intracerebroventricular injection of the TRPV1 blocker capsazepine eliminated the antinociceptive effects of 4-aminophenol and HMBA in the mouse formalin test. In the rat, pharmacological inhibition of FAAH, TRPV1, cannabinoid CB1 receptors and spinal 5-HT3 or 5-HT1A receptors, and chemical deletion of bulbospinal serotonergic pathways prevented the antinociceptive action of 4-aminophenol. Thus, the pharmacological profile of 4-aminophenol was identical to that previously reported for paracetamol, supporting our suggestion that this drug metabolite contributes to paracetamol's analgesic activity via activation of bulbospinal pathways. Our findings demonstrate that it is possible to construct novel antinociceptive drugs based on fatty acid conjugation as a metabolic pathway for the generation of TRPV1 modulators in the CNS.

Memantine, a promising drug for the prevention of neuropathic pain in rat.

N-methyl-D-aspartate (NMDA) receptor antagonists are used for post-surgery neuropathic pain but severe side-effects limit their clinical use. Memantine, when given after surgery, shows conflicting results as regard to neuropathic pain alleviation. Here memantine is administered in animals before or after spinal nerve ligation (SNL) in order to evaluate the induced antinociceptive/cognitive effects and associated molecular events, including the phosphorylation of several tyrosine (pTyr(1336), pTyr(1472)) and serine (pSer(1303)) residues in the NR2B subunit of the NMDA receptor. Spinal nerve ligated and sham animals received memantine (20mg/kg/day) or vehicle (1ml/kg/day) by intraperitoneal route. Pre-emptive protocol started 4 days before surgery and continued for 2 days post-surgery. In the post-operative protocol, the 7 day-treatment began on the day of surgery. Tests were done before and after surgery. Tactile allodynia, mechanical hyperalgesia and spatial memory were evaluated by von Frey, Randall & Selitto and Y-maze-tests respectively, and molecular events by western-blot analysis. Spinal nerve ligated animals displayed nociception, impaired memory and increased expression of the 3 phosphorylated residues. Post-operative memantine had no beneficial effect. Pre-emptive memantine prevented the development of post-surgical nociception, impairment of spatial memory and did not increase the expression of pTyr(1472)NR2B at spinal, insular and hippocampal levels. Memantine administered a few days before surgery is a promising strategy to alleviate neuropathic pain development and impairment of cognitive function in animals. The pivotal role of pTyr(1472)NR2B must be studied further, and these findings will now be challenged in patients for the prevention of postsurgical neuropathic pain.

αv integrin: a new gastrin target in human pancreatic cancer cells.

AIM: To analyse αv integrin expression induced by gastrin in pancreatic cancer models. METHODS: αv integrin mRNA expression in human pancreatic cancer cells was analysed using a "cancer genes" array and confirmed by real-time reverse transcription-polymerase chain reaction (PCR). Western blotting and semi-quantitative immunohistochemistry were used to examine protein levels in human pancreatic cancer cell lines and pancreatic tissues, respectively. The role of αv integrin on gastrin-induced cell adhesion was examined using blocking anti-αv integrin monoclonal antibodies. Adherent cells were quantified by staining with crystal violet. RESULTS: Using a "cancer genes" array we identified αv integrin as a new gastrin target gene in human pancreatic cancer cells. A quantitative real-time PCR approach was used to confirm αv integrin gene expression. We also demonstrate that Src family kinases and the PI 3-kinase, two signalling pathways specifically activated by the CCK-2 receptor (CCK2R), are involved in gastrin-mediated αv integrin expression. In contrast, inhibition of the ERK pathway was without any effect on αv integrin expression induced by gastrin. Our results also show that gastrin modulates cell adhesion via αv integrins. Indeed, in vitro adhesion assays performed on fibronectin show that gastrin significantly increases adhesion of pancreatic cancer cells. The use of blocking anti-αv integrin monoclonal antibodies completely reversed the increase in cell-substrate adhesion induced by gastrin. In addition, we showed in vivo that the targeted CCK2R expression in the pancreas of Elas-CCK2 mice, leads to the overexpression of αv integrin. This process may contribute to pancreatic tumour development observed in these transgenic animals. CONCLUSION: αv integrin is a new gastrin target in pancreatic cancer models and contributes to gastrin effects on cell adhesion.

Phosphorylation of spinal N-methyl-d-aspartate receptor NR1 subunits by extracellular signal-regulated kinase in dorsal horn neurons and microglia contributes to diabetes-induced painful neuropathy.

The N-methyl-d-aspartate receptor (NMDAR) contributes to central sensitization in the spinal cord, a phenomenon which comprises various pathophysiological mechanisms responsible for neuropathic pain-like signs in animal models. NMDAR function is modulated by post-translational modifications including phosphorylation, and this is proposed to underlie its involvement in the production of pain hypersensitivity. As in diabetic patients, streptozotocin-induced diabetic rats exhibit or not somatic mechanical hyperalgesia; these rats were named DH and DNH respectively. At three weeks of diabetes, we present evidence that somatic mechanical hyperalgesia was correlated with an enhanced phosphorylation of the NMDAR NR1 subunit (pNR1) in the rat spinal cord. This increase was not found in normal and DNH rats, suggesting that this regulation was specific to hyperalgesia. Double immunofluorescence studies revealed that the numbers of pNR1-immunoreactive neurons and microglial cells were significantly increased in all laminae (I-II and III-VI) of the dorsal horn from DH animals. Western-blots analysis showed no change in NR1 protein levels, whatever the behavioural and glycemic status of the animals. Chronic intrathecal treatment (5µg/rat/day for 7days) by U0126 and MK801, which blocked MEK (an upstream kinase of extracellular signal-regulated protein kinase: ERK) and the NMDAR respectively, simultaneously suppressed somatic mechanical hyperalgesia developed by diabetic rats and decreased pNR1. These results indicate for the first time that increased expression of pNR1 is regulated by ERK and the NMDAR via a feedforward mechanism in spinal neurons and microglia and represents one mechanism involved in central sensitization and somatic mechanical hyperalgesia after diabetes.

A gastrin precursor, gastrin-gly, upregulates VEGF expression in colonic epithelial cells through an HIF-1-independent mechanism.

One of the major angiogenic factor released by tumor cells is VEGF. Its high expression is correlated with poor prognosis in colorectal tumors. In colon cancer, gastrin gene expression is also upregulated. In these tumors, gastrin precursors are mainly produced and act as growth factors. Recently, a study has also shown that the gastrin precursor, G-gly induced in vitro tubules formation by vascular endothelial cells suggesting a potential proangiogenic role. Here, we demonstrate that stimulation of human colorectal cancer cell lines with G-gly increases the expression of the proangiogenic factor VEGF at the mRNA and protein levels. In addition, blocking the progastrin autocrine loop leads to a downregulation of VEGF. Although HIF-1 is a major transcriptional activator for VEGF our results suggest an alternative mechanism for VEGF regulation in normoxic conditions, independent of HIF-1 that involves the PI3K/AKT pathway. Indeed we show that G-gly does not lead to HIF-1 accumulation in colon cancer cells. Moreover, we found that G-gly activates the PI3K/AKT pathway and inhibition of this pathway reverses the effects of G-gly observed on VEGF mRNA and protein levels. In correlation with these results, we observed in vivo, on colon tissue sections from transgenic mice overexpressing G-gly, an increase in VEGF expression in absence of HIF-1 accumulation. In conclusion, our study demonstrates that gastrin precursors, known to promote colon epithelial cells proliferation and survival can also contribute to the angiogenesis process by stimulating the expression of the proangiogenic factor VEGF via the PI3K pathway and independently of hypoxia conditions.

Endocannabinoid and serotonergic systems are needed for acetaminophen-induced analgesia.

Acetaminophen is the most used analgesic/antipyretic drug. Its unclear mechanism of action could rely on cyclooxygenase inhibition, NO synthesis blockade or reinforcement of the serotonergic system. Here we show that in thermal, mechanical and chemical pain tests, AM-251, a specific CB(1) receptor antagonist, abolished the analgesic action of acetaminophen, which was also lost in CB(1) receptor knockout mice. Moreover, acetaminophen was shown unable to bind to CB(1) receptors demonstrating an indirect involvement of these receptors in the analgesic effect of this compound. Accordingly with these results, we also demonstrated that the inhibition of FAAH, an enzyme involved in the cerebral metabolism of acetaminophen into AM404, known to reinforce the activity of the endocannabinoid system, suppressed the antinociceptive effect of acetaminophen. In addition, similarly to the interaction of acetaminophen with bulbospinal serotonergic pathways and spinal serotonin receptors, we observed that the antinociceptive activity of ACEA, a CB(1) receptor agonist, was inhibited by lesion of bulbospinal serotonergic pathways and antagonists of spinal 5-HT receptors. We therefore propose that acetaminophen-induced analgesia could involve the following sequence: (1) FAAH-dependent metabolism of acetaminophen into AM404; (2) indirect involvement of CB(1) receptors by this metabolite; (3) endocannabinoid-dependent reinforcement of the serotonergic bulbospinal pathways, and (4) involvement of spinal pain-suppressing serotonergic receptors.

Acetaminophen recruits spinal p42/p44 MAPKs and GH/IGF-1 receptors to produce analgesia via the serotonergic system.

The mechanism of action of acetaminophen is currently widely discussed. Direct inhibition of cyclooxygenase isoforms remains the commonly advanced hypothesis. We combined behavioral studies with molecular techniques to investigate the mechanism of action of acetaminophen in a model of tonic pain in rats. We show that acetaminophen indirectly stimulates spinal 5-hydroxytryptamine (5-HT)1A receptors in the formalin test, thereby increasing transcript and protein levels of low-affinity neurotrophin receptor, insulin-like growth factor-1 (IGF-1) receptor alpha subunit, and growth hormone receptor and reducing the amount of somatostatin 3 receptor (sst3R) mRNA. Those cellular events seem to be important for the antinociceptive activity of acetaminophen. Indeed, down-regulation of sst3R mRNA depends on acetaminophen-elicited, 5-HT1A receptor-dependent increase in neuronal extracellular signal-regulated kinase 1/2 (ERK1/2) activities that mediate antinociception. In addition, spinal growth hormone (GH) and IGF-1 receptors would also be involved in the antinociceptive activity of the analgesic at different degrees. Our results show the involvement of specific 5-HT1A receptor-dependent cellular events in acetaminophen-produced antinociception and consequently indicate that inhibition of cyclooxygenase activities is not the exclusive mechanism involved. Furthermore, we propose that the mechanisms of 5-HT1A receptor-elicited antinociception and the role of the spinal ERK1/2 pathway in nociception are more intricate than suspected so far and that the GH/IGF-1 axis is an interesting new player in the regulation of spinal nociception.

Diabetes-induced mechanical hyperalgesia involves spinal mitogen-activated protein kinase activation in neurons and microglia via N-methyl-D-aspartate-dependent mechanisms.

Molecular mechanisms underlying diabetes-induced painful neuropathy are poorly understood. We have demonstrated, in rats with streptozotocin-induced diabetes, that mechanical hyperalgesia, a common symptom of diabetic neuropathy, was correlated with an early increase in extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) phosphorylation in the spinal cord and dorsal root ganglion at 3 weeks after induction of diabetes. This change was specific to hyperalgesia because nonhyperalgesic rats failed to have such an increase. Immunoblot analysis showed no variation of protein levels, suggesting a post-translational regulation of the corresponding kinases. In diabetic hyperalgesic rats, immunocytochemistry revealed that all phosphorylated mitogen-activated protein kinases (MAPKs) colocalized with both the neuronal (NeuN) and microglial (OX42) cell-specific markers but not with the astrocyte marker [glial fibrillary acidic protein (GFAP)] in the superficial dorsal horn-laminae of the spinal cord. In these same rats, a 7-day administration [5 microg/rat/day, intrathecal (i.t.)] of 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), and anthra(1,9-cd)pyrazol-6(2H)-one (SP600125), which inhibited MAPK kinase, p38, and JNK, respectively, suppressed mechanical hyperalgesia, and decreased phosphorylation of the kinases. To characterize the cellular events upstream of MAPKs, we have examined the role of the NMDA receptor known to be implicated in pain hypersensitivity. The prolonged blockade of this receptor during 7 days by (5R, 10S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]-cyclohepten-5-10-imine hydrogen maleate (MK801; 5 microg/rat/day, i.t.), a noncompetitive NMDA receptor antagonist, reversed hyperalgesia developed by diabetic rats and blocked phosphorylation of all MAPKs. These results demonstrate for the first time that NMDA receptor-dependent phosphorylation of MAPKs in spinal cord neurons and microglia contribute to the establishment and longterm maintenance of painful diabetic hyperalgesia and that these kinases represent potential targets for pain therapy.

c-Jun NH(2)-terminal kinase pathway in growth-promoting effect of the G protein-coupled receptor cholecystokinin B receptor: a protein kinase C/Src-dependent-mechanism.

The proliferative effects of gastrin on normal and malignant gastrointestinal tissues have been shown to be mediated by a G protein-coupled receptor (GPCR), the cholecystokinin B receptor. The c-Jun NH(2)-terminal kinase (JNK) pathway has been implicated in the regulation of mitogenesis by growth factors or cytokines. However, the contribution of this signaling cascade to the proliferative effects of GPCR remains largely unknown. Here, we show that cholecystokinin B receptor occupancy by gastrin leads to the activation of the JNK pathway. The mechanism involves certain protein kinase C isoforms and Src family kinases other than p60Src. The complex p130Cas/CrkII, known to be involved in JNK activation, is also activated in response to gastrin by a protein kinase C- and Src-dependent mechanism. However, gastrin-induced CrkII and JNK pathways are independent. Using a dominant negative mutant of c-Jun, we blocked the ability of gastrin to induce DNA synthesis, demonstrating a major role of the JNK pathway in the growth-promoting effect of a GPCR agonist.