European Journal of Pharmacology (v.725, #C)

Rheumatoid arthritis is most prominently characterized by synoviocyte hyperplasia which therefore serves as an important target for clinical therapy. In the present study, it was observed that menthol, the specific agonist of transient receptor potential melastatin subtype 8 (TRPM8), could induce sustained increases of cytosolic calcium concentration ([Ca2+] c ) in synoviocytes isolated from collagen-induced arthritis rats in dose-dependent manner, which was evidently blocked by applying an extracellular Ca2+-free buffer. Menthol-induced [Ca2+] c increase was also significantly inhibited by potent TRPM8 antagonist capsazepine (CZP), indicating that this [Ca2+] c elevation was mostly attributed to TRPM8-mediated Ca2+ entry. Besides, RT-PCR indeed demonstrated presence of TRPM8 in the synoviocytes. Meanwhile, it was found that menthol evoked production of intracellular reactive oxygen species, which could be abolished by Ca2+ free solutions or CZP. Further experiments showed that menthol reduced the cell numbers and survival of synoviocytes. This reduction was associated with apoptosis as suggested by mitochondrial membrane depolarization, nuclear condensation and a caspase 3/7 apoptotic assay. Menthol-induced death and apoptosis of synoviocytes both were obviously inhibited by CZP, intracellular calcium chelator BAPTA-AM, and reactive oxygen species inhibitor diphenylene iodonium, respectively. Taken together, our data indicated that menthol resulted in synoviocyte death associated with apoptosis via calcium entry and reactive oxygen species production depending on TRPM8 activation.
Keywords: Menthol; TRPM8; Calcium; Synoviocytes; Apoptosis; Reactive oxygen species;

Apolipoprotein B (ApoB) of low-density lipoprotein (LDL) causes endothelial dysfunction in the initial stage of atherogenesis. The present study was designed to explore the underlying molecular mechanisms involved. Rat mesenteric arteries were organ cultured in the presence of different concentrations of ApoB or LDL. Vasodilation induced by acetylcholine was monitored by a sensitive myograph. Nitric oxide (NO), endothelium-dependent hyperpolarizing factor (EDHF) and prostacyclin (PGI2) pathways were characterized by using specific pathway inhibitors. Real-time PCR and immunohistochemistry with confocal microscopy were used to examine alteration of mRNA and protein expressions for NO synthases (eNOS and iNOS) and cycloxygenase (COX), respectively. Lipid peroxidation was measured by thiobarbituric acid reactive substances. In the presence of either LDL or ApoB for 24 h concentration-dependently attenuated the endothelium-dependent vasodilation. Immunohistochemistry staining of endothelial cell marker CD31 was weaker in the presence of LDL, indicating that LDL induced damage to the endothelium. Using the pathway specific inhibitors demonstrated that LDL-induced impairing vasodilation was mainly due to attenuation of NO pathway. This was supported by decreasing mRNA (real-time PCR) and protein expression (immunohistochemistry) for eNOS and iNOS, but not COX, in the presence of LDL. In addition, the levels of lipid peroxidation significantly increased in the presence of LDL for 24 h. In conclusion, ApoB of LDL impairs vasodilation with damaging the endothelium and attenuating the NO-mediated endothelium-dependent relaxation, which might associate with lipid peroxidation and contribute to the development of cardiovascular disease.
Keywords: Apolipoprotein B; Low-density lipoprotein; Endothelial dysfunction; Vasodilation; Lipid peroxidation; Cardiovascular disease;

Pharmacodynamic effects of atorvastatin versus rosuvastatin in coronary artery disease patients with normal platelet reactivity while on dual antiplatelet therapy—The PEARL randomized cross-over study by Francesco Pelliccia; Giuseppe Rosano; Giuseppe Marazzi; Cristiana Vitale; Ilaria Spoletini; Ferdinando Franzoni; Giuseppe Speziale; Marina Polacco; Cesare Greco; Carlo Gaudio (18-22).
High platelet reactivity during co-administration of clopidogrel and a CYP3A4-metabolized statin (i.e. atorvastatin) can be lowered by switching to a non-CYP3A4-metabolized statin (i.e rosuvastatin). Aim of this study was to verify if atorvastatin and rosuvastatin have different pharmacodynamic effects also when platelet reactivity while on dual antiplatelet therapy (DAPT) is normal at baseline. A total of 122 stable coronary artery disease patients receiving DAPT (clopidogrel 75 mg plus aspirin 100 mg) who had evidence of normal platelet reactivity after a 1-week statin wash-out entered the trial. Patients were randomly assigned to atorvastatin (40 mg day, n=61) or rosuvastatin (20 mg day, n=61) for 30 days. After another 1-week wash-out to avoid any carryover effect, cross-over was performed, and patients were switched to the other drug which was continued for 30 days. Platelet reactivity (expressed as P2Y(12) reaction units (PRU) by the VerifyNow assay [Accumetrics, San Diego, California]) was measured after 1-week statin wash-out and at the end of each treatment period. High platelet reactivity was defined as a PRU value >235. After 30-day atorvastatin, platelet reactivity did not significantly change as compared with pre-treatment evaluation (119±66 vs. 136±59 PRU, NS), with 2 patients only showing a PRU>235. Similarly, after 30-day rosuvastatin, platelet reactivity was unchanged vs. baseline (135±46 vs. 128±62 PRU, NS), with PRU>235 occurring in 3 patients. Atorvastatin does not negatively affect DAPT as compared with rosuvastatin when is given to stable coronary artery disease patients with normal platelet reactivity while in statin wash-out (ClinicalTrials.gov Identifier: NCT01567774).
Keywords: Clopidogrel; Coronary artery disease; Percutaneous coronary intervention; Platelet reactivity;

A role for diallyl trisulfide in mitochondrial antioxidative stress contributes to its protective effects against vascular endothelial impairment by Li-Li Liu; Li Yan; Yuan-Hong Chen; Guo-Hua Zeng; Ying Zhou; He-Ping Chen; Wei-Jie Peng; Ming He; Qi-Ren Huang (23-31).
Persistent hyperglycemia increases a systemic oxidative stress, causing the onset of vascular endothelial dysfunction and atherosclerosis. Diallyl trisulfide (DAT), a natural organosulfur compound in garlic, has been reported to have actions of dilating blood vessels and antibacteria, etc. In this study, models of obese diabetic rat in vivo and high glucose concentration (HG)-induced endothelial cell injury in vitro were used to investigate the protective effects of DAT on vascular endothelial injury and its underlying mechanisms. In the in vivo model, the obese diabetic rats were injected venously with DAT (5.0 mg kg−1  d−1) and Vitamin E (1.0 mg kg−1  d−1) respectively, once daily for 7 consecutive days. In the in vitro model, HG-injured HUVEC were treated with or without DAT (25 µmol L−1, 50 µmol L−1, 100 µmol L−1) or Vitamin E (25 µmol L−1) respectively for 24 h. The extents of vascular endothelial injury and protective effects of DAT were evaluated. The results both in vivo and in vitro displayed that DAT-treatment significantly attenuated the endothelial cell impairments. Besides, DAT-treatment markedly decreased the levels of malondialdehyde (MDA) and reactive oxygen species, whereas elevated the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in mitochondrium. Moreover, DAT-treatment considerably improved mitochondrial respiration function. Taken together, our results suggest that DAT protects vascular endothelium from HG or hyperglycemia induced-injury by reducing mitochondrial oxidative stress. The findings provide a novel insight for DAT to potentially treat the oxidative stress diseases, i.e., atherosclerosis, diabetes, and neurodegenerative diseases.
Keywords: Diallyl trisulfide; Endothelial cells; Diabetes mellitus; Oxidative stress;

Impaired working memory by repeated neonatal MK-801 treatment is ameliorated by galantamine in adult rats by Yun-Ai Su; Run-Hu Huang; Xiao-Dong Wang; Ji-Tao Li; Tian-Mei Si (32-39).
Early life blockade of the NMDA receptor by using MK-801, a non-competitive NMDA receptor antagonist, induces behavioral changes that mimic several features of schizophrenia. In the current study, we first examined the effects of neonatal MK-801 treatment in male Sprague-Dawley rats on locomotor activity, prepulse inhibition and spatial working memory in adolescence (postnatal day 35, PND35) and adulthood (PND63). Next, we investigated the effects of an acetylcholinesterase inhibitor, galantamine, on working memory deficits induced by MK-801 treatment. Rats were treated with either saline or MK-801 (0.25 mg/kg twice daily) at PND 5–14, and the long-term behavioral effects were investigated. MK-801 treated rats showed moderate working memory impairments in adolescence but a pronounced deficit in adulthood. However, locomotion and prepulse inhibition at two life stages were not affected by this treatment. Systemic administration of galantamine (1 mg/kg) 30 min before each training session significantly improved neonatal MK-801-induced working memory deficits in adulthood. In conclusion, these results suggest that the neonatal MK-801 treatment-induced selective working memory deficit is related to a change in brain cholinergic systems.
Keywords: Schizophrenia; Working memory; MK-801; Galantamine;

Solasodine protects rat brain against ischemia/reperfusion injury through its antioxidant activity by Tejas Sharma; Vishal Airao; Nimesh Panara; Devendra Vaishnav; Vishavas Ranpariya; Navin Sheth; Sachin Parmar (40-46).
Ischemic stroke is the second leading cause of death worldwide. The major limitation of stroke management is the lack of clinically effective therapy. Antioxidants have been demonstrated as potent neuroprotective agents by enhancing the defense mechanism(s), whereas reducing the oxidative stress in the ischemic stroke models. In the present study, we evaluated neuroprotective potential of solasodine, an antioxidant glycoalkaloid of Solanum species, against global model of ischemia in rats. Ischemia/reperfusion (I/R)-injury produced marked elevation in lipid peroxidation (LPO) and nitric oxide (NO), whereas superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) levels were decreased in experimental animals. Prior administration of solasodine (100 and 200 mg/kg, p.o.) significantly heightened SOD, CAT, GSH and total thiols, whereas reduced LPO and NO levels in the brain. Interestingly, brain coronal sectioning and histopathology studies revealed a marked reversal of I/R-provoked neuronal damage in the solasodine treatment groups. Taken together, our study, for the first time, demonstrates neuroprotective potential of solasodine against global ischemia-induced cerebral injury in experimental rats. We propose that the neuroprotection offered by solasodine could be attributed, at least in part, to its anti-oxidant property.Display Omitted
Keywords: Antioxidants; Global ischemia; Neuroprotection; Oxidative stress; Solasodine; Stroke;

Neuroprotective effects of propofol on ER stress-mediated apoptosis in neuroblastoma SH-SY5Y cells by Ai Nakajima; Mayumi Tsuji; Manami Inagaki; Yurie Tamura; Masumi Kato; Akifumi Niiya; Yuki Usui; Katsuji Oguchi (47-54).
Anesthetic treatment has been associated with widespread apoptotic neurodegeneration in the neonatal rodent brain. It has recently been suggested that propofol, a short-acting intravenous anesthetic agent, may have a potential as a neuroprotective agent. An apoptotic pathway mediated through endoplasmic reticulum (ER) stress has been attracting attention. ER stress is associated with accumulation of unfolded or misfolded proteins in ER, and ER stress-induced apoptosis is implicated in a wide range of diseases, including ischemia/reperfusion injury, neurodegeneration, and diabetes. We investigated whether thapsigargin-induced ER stress is prevented by propofol in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were pretreated with various concentrations of propofol (1–10 μM) for 3 h before co-treatment with 0.5 μM thapsigargin and propofol for 20 h. Levels of ssDNA, specific evidence of apoptosis, and biomarkers of ER stress (mRNA expression of Chop and sXbp-1) were determined. We also assayed calpain and caspase-4 activities and intracellular Ca2+ ([Ca2+] i ) levels. Thapsigargin-induced increases in ssDNA levels, expressions of ER stress biomarkers, activities of caspase-4 and calpain, and level of [Ca2+] i were suppressed by co-incubation with propofol. Our data indicate the possibility that propofol inhibits the Ca2+ release from ER at clinically employed dose levels. These results demonstrate that propofol suppresses the ER stress-induced apoptosis in this cell system, and may have the neuroprotective potency. It may also be a promising agent for preventing damage from cerebral ischemia or edema.
Keywords: Neuroprotective potency of propofol; Anesthetic treatment; ER stress-mediated apoptosis; Ischemia; Neurodegeneration;

Lipopolysaccharide increases degradation of central monoamines: An in vivo microdialysis study in the nucleus accumbens and medial prefrontal cortex of mice by Floor van Heesch; Jolanda Prins; Jan Pieter Konsman; Gerdien A.H. Korte-Bouws; Koen G.C. Westphal; Joanna Rybka; Berend Olivier; Aletta D. Kraneveld; S. Mechiel Korte (55-63).
Peripheral administration of lipopolysaccharide (LPS) in rodents induces anhedonia, i.e. the inability to experience pleasure. Recently, we reported that serotonin transporter (SERT) function is required for LPS-induced anhedonia. Less is known about the effect of LPS on the biological activity of dopamine transporters (DAT) and norepinephrine transporters (NET). Therefore, in vivo microdialysis was performed in the nucleus accumbens and medial prefrontal cortex of C57BL6/J mice exposed to saline or LPS (133 µg/kg i.p.). To investigate the possible involvement of different monoamine transporters, the triple reuptake inhibitor DOV 216,303 or saline was i.p. injected 30 min before the saline/LPS injection. The dose of LPS, shown to decrease responding for brain stimulation reward in mice, significantly increased extracellular levels of monoamine metabolites (5-HIAA, DOPAC and HVA) in the nucleus accumbens and medial prefrontal cortex. Remarkably, DOV 216,303 abolished LPS-induced DOPAC and HVA formation in the nucleus accumbens, suggesting that LPS increases DAT activity in this brain area. DOV 216,303 also inhibited LPS-induced DOPAC and HVA formation in the medial prefrontal cortex. Since DAT density is very low in this brain structure, reuptake of DA predominantly takes place via NET, suggesting that LPS increases DAT and NET activity in the medial prefrontal cortex. Furthermore, DOV 216,303 pretreatment prevented LPS-induced 5-HIAA formation only in the medial prefrontal cortex, indicating that LPS increases prefrontal SERT activity. In conclusion, the present findings suggest that peripheral LPS increases DAT activity in the nucleus accumbens and increases NET and SERT activity in the medial prefrontal cortex of mice.
Keywords: Lipopolysaccharide; Serotonin transporter; Dopamine transporter; Norepinephrine transporter; Triple reuptake inhibitor; Microdialysis;

Palmitoylethanolamide inhibits rMCP-5 expression by regulating MITF activation in rat chronic granulomatous inflammation by Daniele De Filippis; Annapina Russo; Daniela De Stefano; Mariateresa Cipriano; Davide Esposito; Gianluca Grassia; Rosa Carnuccio; Giulia Russo; Teresa Iuvone (64-69).
Chronic inflammation, a condition frequently associated with several pathologies, is characterized by angiogenic and fibrogenic responses that may account for the development of granulomatous tissue. We previously demonstrated that the chymase, rat mast cell protease-5 (rMCP-5), exhibits pro-inflammatory and pro-angiogenic properties in a model of chronic inflammation sustained by mast cells (MCs), granuloma induced by the subcutaneous carrageenan-soaked sponge implant in rat. In this study, we investigated the effects of palmitoylethanolamide (PEA), an anti-inflammatory and analgesic endogenous compound, on rMCP-5 mRNA expression and Microphtalmia-associated Transcription Factor (MITF) activation in the same model of chronic inflammation. The levels of rMCP-5 mRNA were detected using semi-quantitative RT-PCR; the protein expression of chymase and extracellular signal-regulated kinases (ERK) were analyzed by western blot; MITF/DNA binding activity and MITF phosphorylation were assessed by electrophoretic mobility shift assay (EMSA) and immunoprecipitation, respectively. The administration of PEA (200, 400 and 800 µg/ml) significantly decreased rMCP-5 mRNA and chymase protein expression induced by λ-carrageenan. These effects were associated with a significant decrease of MITF/DNA binding activity and phosphorylated MITF as well as phosphorylated ERK levels.In conclusion, our results, showing the ability of PEA to inhibit MITF activation and chymase expression in granulomatous tissue, may yield new insights into the understanding of the signaling pathways leading to MITF activation controlled by PEA.
Keywords: Chronic inflammation; Palmitoylethanolamide; Rat mast cell protease; Microphtalmia-associated Transcription Factor;

Hydrogen sulfide (H2S) has been proposed as a novel gas-transmittter, which plays multiple physiological and pathological functions in various body systems, including gastrointestinal tract. The present study was undertaken to investigate the effects and mechanisms of H2S pharmacological preconditioning on gastric epithelial cells ischemia–reperfusion (I/R) injury. We report here that sodium hydrosulfide (NaHS), an H2S donor, concentration-dependently suppressed I/R-induced cellular injury and apoptotic cell death. This protection effect was also confirmed by endogenous over-producing H2S. Furthermore, NaHS also prevented I/R-induced oxidative stress and inflammatory responses, evidenced by increases in GSH level, decreases in MDA contents, reactive oxygen species generation and secretions of NO, IL-6 and TNF-α. NaHS also prevented I/R-induced p38- and c-Jun NH2-terminal kinase (JNK)-mitogen-activated protein kinase (MAPK) phosphorylation and NF-κB activation. H2S also induced Keap1 s-sulfhydration, and further Keap1/Nrf2 disassociation and Nrf2 activation. H2S exerted its protective effect through reactive oxygen species clearance, inhibition of p38 and JNK dependent cell apoptosis and NF-κB dependent inflammation pathway. Our results provide evidence that H2S may have potential therapeutic value in acute gastric mucosal lesion, which is often caused by ischemia/reperfusion.
Keywords: H2S; Oxidative stress; s-Sulfhydration; Keap1; NF-κB;

Contribution of dopaminergic and adenosinergic systems in the antinociceptive effect of p-chloro-selenosteroid by Marcel Henrique Marcondes Sari; Ana Cristina Guerra Souza; Suzan Gonçalves Rosa; Diego Souza; Oscar Endrigo Dorneles Rodrigues; Cristina Wayne Nogueira (79-86).
This study investigated the antinociceptive action of p-chloro-selenosteroid (PCS), administered by intragastric route (i.g.) to mice against acute models. The contribution of adenosinergic, dopaminergic, serotonergic, nitric oxide and opioid systems was investigated. It was evaluated if the administration of PCS triggers toxic effect. Treatment with PCS (10 mg/kg) reduced writhing induced by acetic acid and its effect lasts up to 48 h after treatment. The compound caused an inhibition in neurogenic and inflammatory phases of nociception and in paw edema induced by formalin. The licking behavior triggered by glutamate was reduced by PCS. In the tail-immersion test, PCS elicited an increase in delta latency response. Pretreatment with caffeine (3 mg/kg, intraperitoneally [i.p.]) and SCH58261 (3 mg/kg, i.p.), antagonist at adenosinergic receptors, SCH23390 (0.05 mg/kg, i.p.) and sulpiride (5 mg/kg, i.p.), antagonist at dopaminergic receptors, caused a reduction in the antinociceptive action of PCS in the glutamate test. By contrast, pretreatment with WAY100635 (0.7 mg/kg, i.p.), ketanserin (0.3 mg/kg, i.p.), ondasentron (0.5 mg/kg, i.p.), l-arginine (600 mg/kg, i.p.) and naloxone (1 mg/kg, subcutaneous [s.c.]) did not abolish the antinociceptive effect caused by PCS (10 mg/kg, i.g.) administration. The animals treated with PCS did not show alterations in locomotor and exploratory activities, in biochemical parameters evaluated, food and water consumption, as well as in the body weight. These results clearly showed the antinociceptive action of PCS in different animal models without causing acute toxic effects in mice. Adenosinergic and dopaminergic systems seem to be related to the mechanisms by which PCS elicits antinociception.
Keywords: Organoselenium; Selenium; Antinociceptive; Adenosinergic system; Dopaminergic system; Mice;

Opioids are among the most frequently used analgesics for treatment of severe pain. However, certain of their side-effects, particularly ventilatory disturbances, often restrict their use. Separation of analgesia from respiratory depression has long been a goal in the basic research and therapeutic use of opioids. This report briefly describes opioid-induced respiratory depression and possible pharmacological strategies to counteract this without affecting analgesia.
Keywords: Opioid; Respiratory depression; Cholinesterase inhibitor; Serotonergic agonist; Phosphodiesterase inhibitor; Methylxanthine;