European Journal of Pharmacology (v.505, #1-3)

Species comparison and pharmacological characterization of rat and human CB2 cannabinoid receptors by Sutapa Mukherjee; Monique Adams; Kristi Whiteaker; Anthony Daza; Karen Kage; Steven Cassar; Michael Meyer; Betty Bei Yao (1-9).
Pharmacological effects of cannabinoid ligands are thought to be mediated through cannabinoid CB1 and CB2 receptor subtypes. Sequence analysis revealed that rat and human cannabinoid CB2 receptors are divergent and share 81% amino acid homology. Pharmacological analysis of the possible species differences between rat and human cannabinoid CB2 receptors was performed using radioligand binding and functional assays. Pronounced species selectivity at the rat cannabinoid CB2 receptor (50- to 140-fold) was observed with AM-1710 (3-(1,1-Dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo[c]chromen-6-one) and AM-1714 (3-(1,1-Dimethyl-heptyl)-1-9-dihydroxy-benzo[c]chromen-6-one). In contrast, JWH-015 ((2-Methyl-1-propyl-1H-indol-3-yl)-napthalen-1-yl-methanone) was 3- to 10-fold selective at the human cannabinoid CB2 receptor. Endocannabinoid ligands were more human receptor selective. Cannabinoid CB2 receptor antagonist, AM-630 ((6-Iodo-2-methyl-1-(2-morpholin-4-yl-ethyl)-1H-indol-3-yl)-(4-methoxy-phenyl)-methanone) was more potent at the rat receptor in radioligand binding and functional assays than that of the human receptor. The findings of the pharmacological differences between the human and rat cannabinoid CB2 receptors in this study provide critical information for characterizing cannabinoid ligands in in vivo rodent models for drug discovery purpose.
Keywords: GPCR; Cannabinoid; Cannabinoid CB2 receptor; Species selectivity; Pharmacology;

Echinacoside rescues the SHSY5Y neuronal cells from TNFα-induced apoptosis by Min Deng; Jin Yuan Zhao; Peng Fei Tu; Yong Jiang; Zheng Bin Li; Yao Hong Wang (11-18).
We investigated the neuroprotective effect of echinacoside, one of the phenylethanoids isolated from the stems of Cistanches salsa, a Chinese herbal medicine, on tumor necrosis factor-alpha (TNFα)-induced apoptosis in human neuroblastoma (SHSY5Y) cells. Treatment of cultured SHSY5Y cells with TNFα 100 ng ml−1 for 36 h stimulated apoptosis, as demonstrated by typical morphological changes, cell viability, DNA laddering, annexin-V binding, intracellular reactive oxygen species, mitochondrial membrane potential and caspase-3 activity. However, simultaneous treatment with echinacoside (1, 10 or 100 μg ml−1) attenuated the TNFα-mediated apoptosis. The antiapoptotic action of echinacoside was partially dependent on antioxidative stress effects, maintenance of mitochondria function, inhibition of caspase-3 activity and was also associated with increasing the expression of the antiapoptotic protein Bcl2. Thus, echinacoside has the neuroprotective capacity to antagonize TNFα-induced apoptosis in SHSY5Y cells and may be useful in treating some neurodegenerative diseases.
Keywords: Echinacoside; Apoptosis; TNFα; Mitochondrial membrane potential; Reactive oxygen species; Caspase-3;

Basal calcium entry in vascular smooth muscle by Damon Poburko; Philippe Lhote; Tania Szado; Tasneim Behra; Roshanak Rahimian; Bruce McManus; Cornelis van Breemen; Urs T. Ruegg (19-29).
Basal calcium leak into smooth muscle was identified 30 years ago yet remains poorly understood. We characterized this leak measuring 45Ca2+ uptake into cultured rat aortic smooth muscle cells. Wash solution (0 °C) containing lanthanum (3 mM) removed extracellular tracer and increased cellular 45Ca2+ retention more effectively than EGTA (0.2 mM). Basal Ca2+ entry was 1.45×109 Ca2+·cell−1·min−1. This translated to ∼250 μmol−1·min−1 given cell volumes of 4–15 pl as determined by 3-D image reconstruction. Gadolinium (100 μM) blocked 80% of the leak and exhibited a biphasic concentration–response relation (IC50s=1 μM and 2 mM). Organic ion channel blockers also inhibited ∼80% of the leak; 45% by nifedipine (10 μM), 7% was exclusively blocked by SKF 96365 (1-[b-[3-(4-Methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole) (50 μM) and 23% was exclusively sensitive to 2-aminoethoxy-diphenylborate (2-APB, 75 μM). Reverse transcriptase polymerase chain reaction revealed TrpC1, 4 and 6 mRNA, and we propose that 2-APB may selectively block TrpC4-containing channels. We conclude that basal Ca2+ entry is mainly due to a basal open probability of excitable Ca2+-channels.
Keywords: Basal entry; Calcium; Vascular smooth muscle;

This study characterized the functional effects of a novel gastroprokinetic agent, N-[2-(diisopropylamino)ethyl]-2-[(2-hydroxy-4,5-dimethoxybenzoyl)amino]-1, 3-thiazole-4-carboxyamide monohydrochloride trihydrate (Z338), on the muscarinic M1, M2, and M3 receptors expressed in Xenopus oocytes using the two-electrode voltage clamp method. Z-338 did not produce by itself any currents in oocytes expressing muscarinic M1, M3 receptors or muscarinic M2 receptors/G protein-gated inward rectifying K+ channels (Kir3.1 channels). In oocytes expressing muscarinic M1 receptors, Z-338 inhibited the acetylcholine-induced Ca2+-activated Cl current with an IC50 of 1.8 μM. In oocytes expressing muscarinic M2 receptors/Kir3.1 channels, Z-338 inhibited the acetylcholine-induced K+ currents with an IC50 of 10.1 μM, whereas in oocytes expressing muscarinic M3 receptors, Z-338 did not inhibit the acetylcholine-induced Ca2+-activated Cl current in a concentration-dependent manner. These results indicate that Z-338 is a potent antagonist not for muscarinic M3 receptor but for both muscarinic M1 and M2 receptors. Thus, Z-338 is a gastrokinetic agent with a unique profile.
Keywords: Gastrokinetic agent; Muscarinic receptor; Z-338;

Methoctramine analogues inhibit responses to capsaicin and protons in rat dorsal root ganglion neurons by Ian R. Mellor; Jane Ogilvie; Florentina Pluteanu; Richard H. Clothier; Terence L. Parker; Michela Rosini; Anna Minarini; Vincenzo Tumiatti; Carlo Melchiorre (37-50).
We have investigated the possibility that vanilloid receptors have a binding site for polyamines and determined the consequences of binding to such a site. Whole-cell and single-channel patch-clamp recordings were used to investigate the effect of the tetraamine, methoctramine, and 16 of its analogues on capsaicin and proton induced responses of foetal rat dorsal root ganglion neurons. All but two methoctramine analogues inhibited responses to 10 μM capsaicin with IC50 values in the range of 2–70 μM at a holding potential of −100 mV. Inhibition was generally non-competitive and voltage-dependent. Methoctramine at 10 μM reduced the single channel mean open time (>3-fold), but also increased the mean closed time (1.7-fold). Sustained responses to pH 5.4 were antagonized by methoctramine with similar potency to capsaicin responses. Similar data were obtained with adult rat dorsal root ganglion neurons. These data indicate that methoctramine analogues bind to vanilloid receptors to inhibit their function.
Keywords: Vanilloid receptor; Methoctramine; Channel block; Dorsal root ganglion; Patch-clamp; Polyamine;

Nitric oxide mediates cyclosporine-induced impairment of the blood–brain barrier in cocultures of mouse brain endothelial cells and rat astrocytes by Shinya Dohgu; Atsushi Yamauchi; Shinsuke Nakagawa; Fuyuko Takata; Mamiko Kai; Takashi Egawa; Mikihiko Naito; Takashi Tsuruo; Yasufumi Sawada; Masami Niwa; Yasufumi Kataoka (51-59).
The present study was designed to clarify the involvement of nitric oxide (NO) signaling in the adverse effect of cyclosporine on the blood–brain barrier. Cyclosporine increased the permeability of sodium-fluorescein and the cellular accumulation of rhodamine 123, a substrate of P-glycoprotein, in mouse brain endothelial (MBEC4) cells. This effect was markedly enhanced two- to threefold when MBEC4 cells were cocultured with rat astrocytes or C6 glioma cells. Direct and continuous electrochemical measurement of NO demonstrated that cyclosporine dose-dependently increased histamine- and phenylephrine-evoked NO production in MBEC4 cells and astrocytes, respectively. A NO synthase inhibitor (N G-monomethyl-l-arginine) blocked slightly and markedly cyclosporine-induced impairment of the endothelial barrier in the monolayer and coculture system, respectively. These findings suggest that cyclosporine impairs the brain endothelial barrier function by accelerating NO production in the brain endothelial and astroglial cells. This event may be interpreted as triggering the occurrence of cyclosporine neurotoxicity.
Keywords: Cyclosporine; Neurotoxicity; NO (nitric oxide); Blood–brain barrier; Permeability; P-glycoprotein;

We previously reported that rebamipide (2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid) generated oscillations of intracellular Ca2+ concentration ([Ca2+]i) probably through the activation of cholecystokinin type 1 (CCK1) receptors in rat pancreatic acinar cells. Therefore, in the present study, we aimed to establish the pharmacological characteristics of rebamipide in rat pancreatic acinar cells. CCK-8S and rebamipide inhibited [125I]BH-CCK-8S binding to rat pancreatic acinar cell membranes with IC50 values of 3.13 nM and 37.7 μM, respectively. CCK-8S usually evoked [Ca2+]i oscillations at concentrations lower than 50 pM, and it induced biphasic [Ca2+]i increases at higher concentrations. In contrast to CCK-8S, rebamipide only induced [Ca2+]i oscillations at all the concentrations we used in this study. In addition, rebamipide was shown to inhibit high concentrations of CCK-8S-induced biphasic increases in [Ca2+]i, suggesting that rebamipide might be a partial agonist at cholecystokinin CCK1 receptors. Although rebamipide induced [Ca2+]i oscillations by activating the cholecystokinin CCK1 receptors, rebamipide did not cause amylase release and only inhibited CCK-stimulated amylase release reversibly and dose-dependently. However, rebamipide did not inhibit carbachol-, vasoactive intestinal polypeptide (VIP)-, and forskolin-induced amylase releases. These data indicate that rebamipide functions as a partial agonist for Ca2+-mobilizing action, and it is also an antagonist for the amylase-releasing action of CCK.
Keywords: Rebamipide; Cholecystokinin CCK1 receptor; [Ca2+]i oscillation; Amylase;

Ursodeoxycholic acid inhibits endothelin-1 production in human vascular endothelial cells by Ji Ma; Haruko Iida; Taisuke Jo; Haruhito Takano; Hitoshi Oonuma; Toshihiro Morita; Teruhiko Toyo-oka; Masao Omata; Ryozo Nagai; Yukichi Okuda; Nobuhiro Yamada; Toshiaki Nakajima (67-74).
Endothelin-1 is known to be implicated in the pathogenesis of hepatobiliary diseases such as cirrhosis, especially in portal hypertension. This study aimed to investigate the effects of ursodeoxycholic acid on endothelin-1 production in human endothelial cells. The effects of ursodeoxycholic acid and its conjugates (tauroursodeoxycholic and glycoursodeoxycholic acids) on endothelin-1 production as well as nitric oxide (NO) in human umbilical vein endothelial cells (HUVECs) were examined. The production of endothelin-1 and nitric oxide in culture medium was measured using enzyme-linked immunosorbent assay (ELISA) and the Griess method, respectively. Endothelin-1 and endothelial nitric oxide synthase (eNOS) mRNA expression were investigated by real-time quantitative reverse transcriptase/polymerase chain reaction (RT-PCR). Ursodeoxycholic acid (30–1000 μM) inhibited endothelin-1 production in a concentration-dependent manner, and ursodeoxycholic acid at concentrations higher than 300 μM increased nitric oxide production in culture medium. The conjugates of ursodeoxycholic acid also increased nitric oxide production and decreased endothelin-1 production, which was less effective than ursodeoxycholic acid. N-nitro-l-arginine-mythel-ester (l-NAME), a nitric oxide synthase (NOS) inhibitor, suppressed the ursodeoxycholic acid-induced nitric oxide production, but it did not antagonize the inhibitory effects of ursodeoxycholic acid on endothelin-1 production. Ursodeoxycholic acid also induced a concentration-dependent decrease in endothelin-1 mRNA expression without significant changes in eNOS mRNA expression. These results provide novel evidence that ursodeoxycholic acid inhibits endothelin-1 production in human endothelial cells, but nitric oxide is not responsible for the inhibitory effect of ursodeoxycholic acid on endothelin-1. Thus, ursodeoxycholic acid therapy may prevent the development of several pathogenesis such as portal hypertension observed in patients with cirrhosis due to the improvement of endothelial function.
Keywords: Ursodeoxycholic acid; Human endothelial cell; Endothelin-1; Nitric oxide; Real-time RT-PCR;

Intrathecal clonidine inhibits mechanical allodynia via activation of the spinal muscarinic M1 receptor in streptozotocin-induced diabetic mice by Kohei Koga; Kenji Honda; Suguru Ando; Ichiro Harasawa; Hiro-o Kamiya; Yukio Takano (75-82).
We examined the involvement of the spinal muscarinic receptors in the clonidine-induced antiallodynic effects. Mechanical sensitivity was assessed by stimulating the hind paw with von Frey filaments. In streptozotocin-treated (200 mg/kg, i.v.) diabetic mice, hypersensitivity to mechanical stimulation appeared 3 days after streptozotocin administration, and persisted for 11 days. This mechanical hypersensitivity (allodynia) was inhibited by the intrathecal (i.t.) injection of clonidine. The muscarinic receptor antagonist atropine (i.t.) and α2-adrenoreceptor antagonist yohimbine (i.t. or subcutaneous injection) abolished the antiallodynic effect of clonidine. The effect was mimicked by the muscarinic M1 receptor antagonist pirenzepine, but not by the muscarinic M2 receptor antagonist methoctoramine or the muscarinic M3 receptor antagonist 4-DAMP (4-diphenyl-acetoxy-N-methylpiperidine methiodide). In addition, the mechanical hypersensitivity in diabetic mice was reduced by the selective muscarinic M1 receptor agonist McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium chloride) (i.t.). These results suggest that spinal muscarinic M1 receptors participate in the antiallodynic effect of clonidine in diabetic mice.
Keywords: Allodynia; Muscarinic receptor; α2-Adrenoreceptor; Spinal cord; Diabetes;

Protection by sustained release of physostigmine and procyclidine of soman poisoning in rats by Ehn-Kyoung Choi; Dongsun Park; Jung-Min Yon; Gyeung-Haeng Hur; Yeon-Cheol Ha; Jeong-Hwan Che; Jongsun Kim; Sunhee Shin; Ja Young Jang; Seock-Yeon Hwang; Yeon-Hee Seong; Dae Joong Kim; Jong-Choon Kim; Yun-Bae Kim (83-91).
The efficacy of a combinational prophylactic regimen on the lethality, convulsions, and loss of morphological and functional integrities of the brain induced by an organophosphate soman was investigated in rats. The rats were implanted subcutaneously with osmotic minipumps containing the combinational prophylactic regimen composed of physostigmine, a reversible cholinesterase inhibitor, and procyclidine, an N-methyl-d-aspartate antagonist possessing anticholinergic action, for 3 days, and intoxicated subcutaneously with soman (160 μg/kg, 1.3 LD50). The doses of combinational regimen in minipumps were optimized to achieve 30–35% inhibition of blood cholinesterase activity by physostigmine and 50–100 ng/ml of blood concentrations of procyclidine as clinically available doses, respectively. In comparison, 1-[([4-(aminocarbonyl)pyridinio]methoxy)methyl]-2-[(hydroxyimino)methyl]pyridinium (HI-6, 125 mg/kg) was administered intraperitoneally 30 min prior to the soman challenge in control groups to reduce mortality of rats without affecting convulsions. Soman induced profound limbic convulsions and 30% mortality, leading to increased blood–brain barrier permeability, neural injuries, learning and memory impairments, and physical incapacitation of survived rats pretreated with HI-6. The combinational regimen, at optimal doses without adverse effects on passive avoidance performances (72 μg/kg/h of physostigmine plus 432 μg/kg/h of procyclidine), exerted full protective effects against lethality, convulsions, blood–brain barrier opening, brain injuries, learning and memory impairments, and physical incapacitation induced by soman. Taken together, it is suggested that the combination of physostigmine and procyclidine, at adequate doses, could be a choice to provide the victims of organophosphate poisoning with chance of intensive care for survival and neuroprotection.
Keywords: Soman; Brain injury; Memory impairment; Prophylactic; Physostigmine; Procyclidine;

Passage of erythropoietic agents across the blood–brain barrier: a comparison of human and murine erythropoietin and the analog darbepoetin alfa by William A. Banks; Nelson L. Jumbe; Catherine L. Farrell; Michael L. Niehoff; Anne C. Heatherington (93-101).
Studies have suggested that erythropoietin (EPO) may be used to treat stroke in both animals and humans. It is thought to exert its effects directly on the brain and studies with therapeutic doses have shown that it can cross the blood–brain barrier. Here, we compared in a blinded fashion the ability of three erythropoietic agents (murine erythropoietin, human erythropoietin, and darbepoetin alfa, an analog of human erythropoietin in clinical use) to cross the blood–brain barrier of the mouse. High-performance liquid chromatography (HPLC) results showed that all three erythropoietic agents were enzymatically resistant in brain and blood. The unidirectional blood-to-brain influx rates (K i) as measured by multiple-time regression analysis showed that all the erythropoietic agents crossed the blood–brain barrier at about the same rate as albumin, suggesting that they cross the blood–brain barrier by way of the extracellular pathways. No saturable component to influx was found, but indirect evidence suggested a brain-to-blood efflux system. The percent of the intravenously injected dose taken up per gram of brain (%Inj/g) ranged from 0.05 to 0.1 %Inj/g among the three erythropoietic agents and peaked about 3 h after IV injection. For other substances, this range of %Inj/g is known to produce direct effects on brain function. We conclude that erythropoietic agents cross the blood–brain barrier by way of the extracellular pathways in amounts that are likely sufficient to explain their neuroprotective effects.
Keywords: Brain; Pharmacokinetics; Drug delivery; Blood–brain barrier; Erythropoietin; Stroke; Neuroprotection;

Effects on rat thalamic proteome by acute and subchronic MK-801-treatment by Linda Paulson; Peter Martin; Elisabeth Ljung; Kaj Blennow; Pia Davidsson (103-109).
Since the symptoms of intoxication with non-competitive N-methyl-d-aspartate (NMDA) receptor antagonists closely mimic symptoms in patients with schizophrenia, [+]-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cycloheptene-5,10-iminehydrogenmaleate (MK-801)-treated rodents are often used as a model for schizophrenia. In most studies, acute injections of MK-801 to rats have been used, but in some studies, longer periods of treatment have been performed. In our previous work, alterations in mRNA/protein expression were screened in the cerebral cortex of MK-801 treated rats. Different proteins were altered in different treatment courses of MK-801. The main objective of the present study was to evaluate different treatment periods of treatment with MK-801 in rats as a model for schizophrenia. Thalamus proteins from treated (acute, six and 12 days) and control rats were analyzed with two-dimensional gel electrophoresis and mass spectrometry. Our results show that different treatment times of MK-801 to rats give different biochemical results. Therefore, it is important to use the same treatment time in studies that will be compared.
Keywords: MK-801; Schizophrenia; Thalamus; Proteomic; Acute; Subchronic;

Lack of evidence of direct mitochondrial involvement in the neuroprotective effect of minocycline by Sylvie Cornet; Brigitte Spinnewyn; Sylvie Delaflotte; Christelle Charnet; Véronique Roubert; Christine Favre; Hamida Hider; P. Etienne Chabrier; Michel Auguet (111-119).
Minocycline has been reported to exert neuroprotection through inhibition of inflammatory processes and of mitochondrial cell death pathway. To further characterize the neuroprotective effect of minocycline, we determined its efficacy in different neuronal damage paradigms involving inflammation or mitochondrial dysfunction. In transient global ischaemia in gerbils, minocycline reduced hippocampal neuronal damage measured by peripheral type benzodiazepine binding sites density, a marker of microglial activation. The antiinflammatory properties of minocycline were confirmed on the model of carrageenan-induced paw oedema in rats. The use of two experimental animal models involving administration of mitochondrial toxins inhibiting a different complex of the mitochondrial respiratory chain permitted the exploration of the mitochondrial impact of minocycline. Although minocycline exhibited a marked efficacy in 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP; complex I inhibitor)-induced neurotoxicity in mice, it was ineffective in malonate (complex II inhibitor)-induced striatal lesion in rats. In vitro investigations on energized mitochondria isolated from rat liver showed that minocycline (1 μM) did not inhibit the swelling induced by MPP+(1-methyl-4-phenylpyridinium). Moreover, higher concentrations of minocycline induced swelling. From these experiments, the neuroprotective activity of minocycline appears more related to its antiinflammatory activity than to a direct beneficial action on mitochondria.
Keywords: Minocycline; Neuroprotection; Mitochondria; Inflammation;

Nicergoline reverts haloperidol-induced loss of detoxifying-enzyme activity by Mariapia Vairetti; Andrea Ferrigno; Pier Luigi Canonico; Angelo Battaglia; Francantonio Bertè; Plinio Richelmi (121-125).
We evaluated the effects of nicergoline on antioxidant defense enzymes (detoxifying enzymes), during chronic treatment with haloperidol in rats. Chronic use of haloperidol (10 weeks, 1.5 mg/kg/day) induces a significant decrease in glutathione reductase, glutathione peroxidase and superoxide dismutase activity, in selected areas of the brain. Co-administration of nicergoline (20 days, 10 mg/kg/day) significantly restored the activity of these enzymes to levels comparable to those observed in control rats. These observations suggest beneficial effects of nicergoline in the prevention and in the treatment of haloperidol-induced side effects.
Keywords: Haloperidol; Nicergoline; Glutathione reductase; Glutathione peroxidase; Superoxide dismutase;

BAY 59-3074 {3-[2-cyano-3-(trifluoromethyl)phenoxy]phenyl-4,4,4-trifluoro-1-butane-sulfonate} is a structurally novel cannabinoid CB1/CB2 receptor partial agonist with analgesic properties. The present study was performed to confirm its receptor binding profile in a highly sensitive in vivo assay. Rats (n=10) learned to discriminate BAY 59-3074 (0.5 mg/kg, p.o., t−1 h) from vehicle in a fixed-ratio: 10, food-reinforced two-lever procedure after a median number of 28 training sessions. BAY 59-3074 generalized dose-dependently (ED50: 0.081 mg/kg, p.o.) and the cue was detectable between 0.25 and 4 h after administration. The selective cannabinoid CB1 receptor antagonist SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] blocked the discriminative effects of BAY 59-3074 (ID50: 1.79 mg/kg, i.p.). Complete generalization was also obtained after i.p. administration of BAY 59-3074 (ED50 value: 0.41 mg/kg), and the reference cannabinoids BAY 38-7271 [(−)-(R)-3-(2-hydroxymethylindanyl-4-oxy)phenyl-4,4,4-trifluoro-1-butanesulfonate, 0.011 mg/kg], CP 55,940 {(−)-cis-3-[2-hydroxy-4(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol, 0.013 mg/kg}, HU-210 [(−)-11-OH-Δ8-tetrahydrocannabinol dimethylheptyl, 0.022 mg/kg], WIN 55,212-2 [(R)-4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo [3,2,1-ij] quinolin-6-one, 0.41 mg/kg] and (−)-Δ9-tetrahydrocannabinol (0.41 mg/kg). Non-cannabinoids with analgesic properties, such as morphine, amitriptyline, carbamazepine, gabapentin and baclofen, did not generalize to the cue. It is concluded that the discriminative stimulus effects of BAY 59-3074 are specifically mediated by cannabinoid CB1 receptor activation.
Keywords: Analgesic; Animal model; Cannabinoid receptor; Drug discrimination;

Drug interaction between methamphetamine and antihistamines: behavioral changes and tissue concentrations of methamphetamine in rats by Tomohiro Okuda; Yukiko Ito; Naoto Nakagawa; Takanori Hishinuma; Hiroki Tsukamoto; Kentaro Iwabuchi; Takehiko Watanabe; Kiyoyuki Kitaichi; Junichi Goto; Kazuhiko Yanai (135-144).
Methamphetamine is a psychomotor stimulant, whereas first generation antihistamines cause sedation. Several studies have demonstrated that first generation antihistamines potentiate methamphetamine-induced psychomotor activation and two possible mechanisms have been postulated. One is blockage of the central histaminergic neuron system and the other is inhibition of dopamine reuptake. However, the exact mechanism is still controversial. In this study, we examined in behavioral tests the effects of selected antihistamines on methamphetamine-induced psychomotor activation in rats, and measured plasma and brain tissue concentrations of methamphetamine. We found that some antihistamines significantly potentiate methamphetamine-induced psychomotor activation in rats and that plasma and brain tissue concentrations of methamphetamine in rats treated with methamphetamine in combination with d-chlorpheniramine were markedly higher than those in rats treated with methamphetamine alone. These results suggest that the potentiating effects of antihistamines are due to not only central effects but also the alteration of the pharmacokinetics of methamphetamine.
Keywords: Methamphetamine; Antihistamine; Prepulse inhibition; Locomotor activity; Methamphetamine concentration;

Effect of MS-153 on the acquisition and expression of conditioned fear in rats by XiaoBai Li; Takeshi Inouei; Tomohiro Abekawai; Fang YiRui; Tsukasa Koyama (145-149).
Pavlovian fear conditioning is one of the most extensively studied and reliable behavioral paradigms used to investigate the mechanisms involved in fear and anxiety. Increased glutamatergic neurotransmission may play an important role in mediating fear conditioning. The present study assessed whether (R)-(−)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), a novel cerebroprotective agent that inhibits the release of glutamate and enhances glutamate uptake, affects the acquisition and expression of conditioned fear. The rats received administration of MS-153 (i.p.) at 3, 10, and 30 mg/kg, 30 min before footshock and 24 h after footshock. Freezing behavior was measured in the chamber where they had previously received footshock for the acquisition experiments. For the expression experiments, the rats received MS-153 (i.p.) at the same doses 23.5 h after footshock and 30 min before expression testing. MS-153 significantly attenuated the acquisition and expression of freezing behavior. In addition, MS-153 administration did not affect locomotor activity. The present results suggest that extracellular glutamate is involved in fear conditioning, and that MS-153 has an anxiolytic effect by decreasing endogenous glutamate neurotransmission.
Keywords: MS-153; Contextual fear conditioning; Glutamate uptake and release; NMDA receptor antagonist; Fear and anxiety;

Anandamide vehicles: a comparative study by Visitación López-Miranda; Esperanza Herradón; María Teresa Dannert; Ángela Alsasua; María Isabel Martín (151-161).
Among the studies that investigate the vasorelaxation induced by anandamide, one of the most frequent differences is the use of distinct solvents that could modify vascular function and explain the controversial results described. The aims of this study were: to evaluate the influence of different cannabinoid vehicles in vascular function of rat aorta, and to compare the vasorelaxation induced by anandamide dissolved in different vehicles. Vehicles were: ethanol (70%), Tween 80/ethanol (2:1 and 1:1), 1:1:18 (Tween 80/ethanol/saline) and dimethylsulphoxide (DMSO) 0.5%. All the vehicles tested, except DMSO 0.5%, modified the vascular and/or the endothelial function in rat aorta rings. Anandamide caused a time- and concentration-dependent vasorelaxation in all the experimental groups except in ethanol group, but the mechanisms involved in its vasorelaxation appear to be different depending on the vehicle used. The results obtained with vehicles containing Tween 80 suggest a non-endothelial component in the vasorelaxation caused by anandamide, while those obtained with DMSO at 0.5% suggest an endothelial component in this vasorelaxation.
Keywords: Anandamide; Vehicle; Vasorelaxation; aorta;

Effects of SEA0400, a novel inhibitor of the Na+/Ca2+ exchanger, on myocardial stunning in anesthetized dogs by Teisuke Takahashi; Kenzo Takahashi; Michihito Onishi; Taizo Suzuki; Yu Tanaka; Tomomi Ota; Shigeru Yoshida; Shiro Nakaike; Toshio Matsuda; Akemichi Baba (163-168).
Activation of the Na+/Ca2+ exchanger may contribute to Ca2+ overload during reperfusion after transient ischemia. We examined the effects of 2-[4-[(2,5-difluorophenyl) methoxy]phenoxy]-5-ethoxyaniline (SEA0400), a selective inhibitor of Na+/Ca2+ exchange, on a canine model of ischemia/reperfusion injury (myocardial stunning). Myocardial stunning was induced by a 15-min occlusion of the left anterior descending coronary artery followed by a 4-h reperfusion in anesthetized open-chest dogs. Reperfusion gradually restored myocardial percent segment shortening but remained depressed during a 4-h reperfusion period. A bolus intravenous injection of SEA0400 (0.3 or 1.0 mg/kg), given 1 min before reperfusion, improved significantly the recovery of percent segment shortening in the ischemic/reperfused myocardium. SEA0400 did not affect the hemodynamics and electrocardiogram parameters. In addition, SEA0400 did not affect reperfusion-induced change in coronary blood flow. These results suggest that the Na+/Ca2+ exchanger is involved in the stunned myocardium of dogs after reperfusion, and that SEA0400 has a protective effect against myocardial stunning in dogs.
Keywords: Na+/Ca2+ exchanger; SEA0400; Myocardial stunning;

Exhaled Interleukine-6 and 8-isoprostane in chronic obstructive pulmonary disease: effect of carbocysteine lysine salt monohydrate (SCMC-Lys) by Giovanna E. Carpagnano; O. Resta; Maria P. Foschino-Barbaro; Antonio Spanevello; Antonio Stefano; Giuseppe Di Gioia; Gaetano Serviddio; Enzo Gramiccioni (169-175).
Chronic obstructive pulmonary disease (COPD) is characterized by an airways inflammation and by an enhanced generation of reactive oxygen species. The aim of our study was to assess the inflammation and the oxidative stress in airways of COPD patients with acute exacerbation of disease and in stability. Furthermore, we investigated the anti-inflammatory and antioxidant effects of 6 months treatment with carbocysteine lysine salt monohydrate (SCMC-Lys) in COPD. We studied 30 mild acute COPD, 10 mild stable COPD and 15 healthy subjects. 8-isoprostane and Interleukine-6 were measured in their breath condensate through immunoassay. Significantly higher concentrations of exhaled 8-isoprostane and Interleukine-6 were found in acute COPD patients compared to stable COPD and healthy controls (21.8±5.1 vs. 13.2±2.0 vs. 4.7±1.8 pg/ml and 7.4±0.9 vs. 5.8±0.2 vs. 2.7±0.6 pg/ml, p<0.0001). COPD patients treated with SCMC-Lys showed a marked reduction of exhaled 8-isoprostane and Interleukine-6 (8.9±1.5 and 4.6±0.8 pg/ml, p<0.0001). These findings suggest that there is an increase of 8-isoprostane and Interleukine-6 concentrations in the breath condensate of COPD patients compared to healthy controls especially during acute exacerbations of the disease. Moreover, we showed an anti-inflammatory and antioxidant effect of short-term administration of SCMC-Lys in COPD, suggesting the importance of a further placebo-controlled study that should evaluate the effects of this drug.
Keywords: SCMC-Lys (Carbocysteine lysine salt monohydrate); COPD; Breath condensate; 8-isoprostane; Interleukin-6;

We examined the contribution of nitric oxide (NO) on the contractile impairment in diaphragm muscles of endotoxemic rats. Force–frequency relationship was depressed 24 h after lipopolysaccharide administration. 7-Nitroindazole, aminoguanidine and 1H-[1,2,4]Oxadiazole (4,3-a)quinoxalin-1-one (ODQ) partially restored the contractile impairment, Nω-Nitro-l-Arginine (L-NNA) was ineffective. K+ contractions were reduced by 50% in endotoxemic muscles, 7-nitroindazole partially recovered, while aminoguanidine and L-NNA were ineffective. Verapamil reduced contractility to a greater extent in endotoxemic muscles. Caffeine and ryanodine contractions were augmented during endotoxemia without NOS contribution. L-NNA, 7-nitroindazole, ODQ and hemoglobin did not affect, but aminoguanidine completely restored partially inhibited neurotransmission by d-tubocurarine. Endotoxemia did not change membrane potentials and neurotransmitter release but slightly increased excitability. At this stage of endotoxemia, (1) constitutive NOS appears to be the dominant isoform, (2) NO does not have a major role on contractile dysfunction and (3) impairment could be explained by altered sensitivity of the voltage sensor. (4) NO does not substantially modulate neuromuscular transmission in normal and endotoxemic rats.
Keywords: NO (Nitric oxide); Lipopolysaccharide; Contractility; Excitation–contraction coupling; Neuromuscular junction; Skeletal muscle;

Angiogenesis, cell proliferation and apoptosis in gastric ulcer healing. Effect of a selective cox-2 inhibitor by Susana Sánchez-Fidalgo; Inés Martín-Lacave; Matilde Illanes; Virginia Motilva (187-194).
To elucidate the role of cyclooxygenase-2, we compared the effects of rofecoxib, a selective cyclooxygenase-2 inhibitor, and ibuprofen, a nonselective cyclooxygenase inhibitor, on the evolution of acetic-acid-induced gastric ulcers in rats, evaluating growth factor expression, the angiogenic process, cell proliferation and cell apoptosis. Levels of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), angiogenesis and cell proliferation were analysed by immunohistochemical methods, and apoptosis was evaluated by an enzyme immunoassay. Both growth factors and microvessels appeared to be abundant in the granulation tissue of the ulcer bed. Rofecoxib (2.5 mg/kg/day) and ibuprofen (100 mg/kg/day) delayed ulcer healing, but only rofecoxib treatment provoked a reduction of bFGF expression and inhibition of the development of new microvessels. No changes in VEGF expression were detected. Results also showed that proliferation and apoptosis were increased in control ulcerated animals. Rofecoxib reduced significantly both processes. These findings demonstrate that a reduction of bFGF expression and an antiangiogenic action, as well as proliferation/apoptosis inhibition, are some of the mechanisms possibly implicated in the delay in ulcer healing seen after the administration of the highly selective COX-2 inhibitor rofecoxib.
Keywords: NSAID; Rofecoxib; Ibuprofen; bFGF expression; VEGF expression; Apoptosis; Proliferation;

Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a nuclear hormone receptor super family that has recently been implicated in atherosclerosis, inflammation, cancer, infertility, and demyelination. Oxidative stress, neutrophil infiltration, proinflammatory cytokines, and the exhibition of luminal acid play a role in the pathogenesis of gastric injury induced by ischemia–reperfusion. Rosiglitazone, a specific PPAR-γ ligand, has been shown to have antiinflammatory activity, but its effects on experimental ischemia–reperfusion gastric injury remain unknown. We have investigated the effects of the rosiglitazone on gastric injury caused by ischemia following reperfusion in rats. Tumour necrosis factor-alpha (TNF-α) levels and changes in enzymatic activities of myeloperoxidase, as a marker of neutrophils infiltration, xanthine oxidase, superoxide dismutase, and glutathione peroxidase, were determined. Histological analysis of the lesions was also carried out. Pretreatment with 1 or 4 mg/kg of rosiglitazone ameliorated the gastric damage induced by clamping the celiac artery for 30 min followed by 60 min of reperfusion. It significantly (P<0.05) reduced the index of neutrophil infiltration and the levels of the cytokine. Rosiglitazone did not revert the reduced glutathione peroxidase activity but enhanced significantly (P<0.01) the decreased xanthine oxidase and superoxide dismutase activities in gastric mucosa of ischemic rats. In conclusion, rosiglitazone reduces the damage in ischemia–reperfusion gastric injury and alleviates the inflammatory response and the oxidative events.
Keywords: PPAR-γ; Rosiglitazone; Ischemia–reperfusion; Reactive oxygen specie; Neutrophil; Cyclooxygenase;

Preventive effect of Y-27632, a selective Rho-kinase inhibitor, on ischemia/reperfusion-induced acute renal failure in rats by Kohji Teraishi; Hayato Kurata; Atsushi Nakajima; Masanori Takaoka; Yasuo Matsumura (205-211).
We evaluated the effects of Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate], a selective Rho-kinase inhibitor, on ischemic acute renal failure. Ischemic acute renal failure in rats was induced by clamping the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after the contralateral nephrectomy. Y-27632 administration (1, 10, and 100 μg/kg, i.p.) before ischemia dose-dependently attenuated the ischemia/reperfusion-induced renal dysfunction and histological damage, such as tubular necrosis. The ischemia/reperfusion-induced renal dysfunction was also overcome by postischemic treatment with Y-27632 at 100 μg/kg, i.p. Myeloperoxidase activity in the kidney after ischemia/reperfusion was significantly increased, being the maximal level at 6 h after the reperfusion, and this increase was also suppressed by Y-27632 (100 μg/kg, i.p.). These results indicate that Y-27632 prevents the development of ischemia/reperfusion-induced acute renal failure, and the effect is related to the suppression of the enhanced myeloperoxidase activity in an early phase after reperfusion, thereby suggesting that the Rho/Rho-kinase pathway plays a key role in the pathogenesis of ischemic acute renal failure.
Keywords: Ischemia; Reperfusion; Acute renal failure; Rho-kinase;

The oxidation of low-density lipoprotein (LDL) is an important event in the development of atherosclerosis. In the present study, the antioxidant properties of two novel 2-biphenylmorpholine compounds (EP2306 and EP2302) were studied. Both compounds inhibited dose-dependently the in vitro oxidation of LDL induced by copper ions. EP2306 and EP2302 increased significantly the lag phase of the oxidation reaction at 0.1 and 10 μM, respectively, whereas they reduced the rate of the reaction at 1 and 10 μM, respectively. This inhibitory effect was not due to a free radical scavenging or copper-chelating activity of EP2300 compounds. Moreover, EP2306 and EP2302 inhibited 12-lipoxygenase activity dose-dependently with IC50 values of 454 and 318 μM, respectively, but had no effect on 15-lipoxygenase activity. In hyperlipidaemic rabbits treated with EP2306 for 4 weeks, there was a decrease in thiobarbituric acid-reactive substance (TBARS) levels and a significant increase in total peroxyl radical-trapping potential (TRAP) levels as compared to control animals. The present data suggest that EP2300 compounds are effective inhibitors of copper-mediated LDL oxidation in vitro. Moreover, EP2306 acts as an antioxidant in hyperlipidaemic rabbits, a property which could be beneficial in reducing atherosclerosis.
Keywords: Antioxidant; 2-Biphenylmorpholine compound; Low-density lipoprotein; Atherosclerosis; Copper-mediated oxidation; (Rabbit);

A novel Syk kinase-selective inhibitor blocks antigen presentation of immune complexes in dendritic cells by Kosuke Nakashima; Toshio Kokubo; Michitaka Shichijo; Ying-Fu Li; Takeshi Yura; Noriyuki Yamamoto (223-228).
The initiation of antigen presentation by dendritic cells requires proper internalization of antigens through various mechanisms. Internalization of immune complexes via Fc receptors has been shown to be around 100 times more efficient than the internalization of non-complexed antigens. Spleen tyrosine kinase (Syk) plays an essential role in the signaling cascade initiated by immunoglobulin receptors. We used a selective Syk inhibitor, 7-(3,4-dimethoxyphenyl)-N-1H-indazol-6-ylimidazo[1,2-c]pyrimidin-5-amine dihydrochloride (compound-D), to evaluate the role of Syk in antigen presentation by mouse bone marrow-derived dendritic cells. In line with our expectation, compound-D concentration-dependently inhibited the internalization of immune complexes but not that of antigen itself. Furthermore, when dendritic cells were pretreated with compound-D, the ability of dendritic cells to present immune complex antigens to Th2 cells was attenuated, parallel by a reduced release of interleukin-4 production in Th2 cells. Therefore, Syk kinase activity is a critical component in the process of Fcγ receptor-mediated internalization of immune complex antigens in dendritic cells, and Syk kinase inhibitors may be beneficial in selectively suppressing antibody-mediated antigen presentation in allergic diseases.
Keywords: Syk; Bone marrow-derived dendritic cell; Immune complex; Fcγ receptor; Antigen internalization; Antigen presentation;

Prostanoid DP1 receptor agonist inhibits the pruritic activity in NC/Nga mice with atopic dermatitis by Iwao Arai; Norikazu Takano; Yuki Hashimoto; Nobuko Futaki; Masanori Sugimoto; Nobutaka Takahashi; Tomoyuki Inoue; Shiro Nakaike (229-235).
NC/Nga mice have similar pathological and behavioral features of human atopic dermatitis and are used as a model of the disease. Under conventional circumstances, spontaneous and persistent scratching is frequent and can lead to the onset of skin inflammation. We examined the effects of several prostanoids and their related compounds on the scratching behavior of NC/Nga mice. Among them, topically applied prostaglandin D2, prostaglandin E1, prostaglandin E2 and prostaglandin I2 significantly suppressed the scratching, the order of inhibitory activities being prostaglandin D2≫prostaglandin I2>prostaglandin E1=prostaglandin E2. Prostaglandin D2 metabolite, prostaglandin J2 also significantly suppressed the scratching but not so 13,14-dihydro-15-keto-prostaglandin D2, and 15-deoxy-Δ12,14-prostaglandin J2. The order of the inhibitory activities of these prostaglandin D2 metabolites depended on affinity of the prostanoid DP1 receptor but not on the DP2 receptor (chemoattractant receptor-homologous molecule expressed on T helper2 cells, CRTH2) and PPAR-γ receptors. Likewise, topically applied arachidonic acid significantly suppressed the scratching while indomethacin enhanced it. Pretreatment of arachidonic acid increased the skin prostaglandins (prostaglandin D2, prostaglandin E2, prostaglandin F and 6-keto-prostaglandin F) contents, but indomethacin decreased the prostaglandin D2 and prostaglandin E2 contents. On the other hand, prostaglandin D2 and indomethacin had no apparent effects on histamine-induced scratching of ICR mice. These results suggested that prostaglandin D2 plays a physiological role in inhibiting pruritis of NC/Nga mice via their specific prostanoid DP1 receptors, and that prostaglandin D2 and/or a prostanoid DP1 receptor agonist may have therapeutic effects for cases of consecutive skin inflammation.
Keywords: Prostaglandin D2; Scratch; Atopic dermatitis; NC/Nga, mouse; Pruritus;

Involvement of the active metabolites in the inhibitory activity of K579 on rat plasma dipeptidyl peptidase IV by Kotaro Takasaki; Hidenori Takada; Takao Nakajima; Kimihisa Ueno; Junko Ushiki; Katsuya Higo (237-241).
K579 ((S)-1-[4-methyl-1-(2-pyrimidinyl)-4-piperidylamino]acetyl-2-pyrrolidinecarbonitrile), which is a long-acting and a slow binding dipeptidyl peptidase IV inhibitor, preserved the endogenously secreted active forms of glucagon-like peptide-1, augmented the insulin response and ameliorated the glucose excursion during oral glucose tolerance test in rats. In this study, we measured plasma concentrations of K579 after oral administration to rats. However, K579 was eliminated rapidly from plasma after oral administration to rats. Therefore, we postulated that there are active metabolites of K579 in rat plasma. We investigated the effect of K579 on plasma dipeptidyl peptidase IV activity using bile duct-cannulated rats. The duration of inhibitory action of plasma dipeptidyl peptidase IV after the administration of K579 in bile duct-cannulated rats was shorter than that in sham-operated rats. Moreover, we investigated the effect of bile obtained from K579-treated rat on plasma dipeptidyl peptidase IV activity in normal rats. The bile collected from K579-treated rats exhibited tardive and potent inhibitory activity of normal rat plasma. These results suggest that K579 sustained the duration of inhibitory action of plasma dipeptidyl peptidase IV by the character as a slow-binding inhibitor and, as well, by the presence of metabolites of K579, which exhibit the inhibitory activity of dipeptidyl peptidase IV.
Keywords: Dipeptidyl peptidase IV; Active metabolite; Bile; (Rat);

Altered glucose homeostasis in α2A-adrenoceptor knockout mice by Veronica Fagerholm; Tove Grönroos; Päivi Marjamäki; Tapio Viljanen; Mika Scheinin; Merja Haaparanta (243-252).
To elucidate the functions of α2-adrenoceptor subtypes in metabolic regulation, we determined plasma glucose and insulin levels and tissue uptake of the glucose analogue 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG) in C57Bl/6J wild-type (WT) and α2A-adrenoceptor knockout (α2A-KO) mice at baseline and following α2-adrenoceptor agonist ((+)-4-(S)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole (dexmedetomidine)) and antagonist (4-[2-ethyl-2,3-dihydro-1H-inden-2-yl]-1H-imidazole (atipamezole)) administration. Basal glucose levels were 30% lower in α2A-KO mice than in WT mice. In WT mice, dexmedetomidine lowered insulin and elevated glucose levels, and atipamezole reduced glucose levels. In α2A-KO mice, neither drug affected the glucose or insulin levels. [18F]FDG uptake was investigated in plasma, heart, liver, kidney, pancreas, lung, fat, and skeletal muscle. Cardiac [18F]FDG uptake was a sensitive indicator of sympathetic function. Liver [18F]FDG uptake conformed to the plasma glucose levels. In α2A-KO mice, drug effects on [18F]FDG tissue uptake were absent. Thus, the α2A-adrenoceptor is the α2-adrenoceptor subtype primarily involved in the regulation of blood glucose homeostasis in vivo.
Keywords: α2-Adrenoceptor knockout; Atipamezole; Dexmedetomidine; 18FDG; Fluorodeoxyglucose;

Ziprasidone suppresses olanzapine-induced increases in ingestive behaviour in the rat by Shona L. Kirk; Joanna C. Neill; Declan N.C. Jones; Gavin P. Reynolds (253-254).
Many atypical antipsychotic drugs, such as olanzapine, induce significant weight gain. However, ziprasidone produces minimal weight gain, the mechanism of which remains unclear. The aim of the present study was to investigate whether ziprasidone would reduce the acute effect of olanzapine on feeding behaviour. The results suggest that ziprasidone suppresses the significant increases in food intake produced by olanzapine, indicating that it has an intrinsic protective mechanism against drug-induced increases in food intake.
Keywords: Antipsychotic; Weight gain; (Rat);

Author index (255-257).

Keyword index (259-263).