European Journal of Pharmacology (v.592, #1-3)

Previous studies have shown that tachykinin peptide ligands of the tachykinin NK1 receptor exhibit functional selectivity with respect to signal activation and desensitization. The differences are most dramatic between the naturally occurring peptides substance P (RPKPQQFFGLM-NH2) and ranatachykinin C (HNPASFIGLM-NH2). To understand the structural features of the peptides that underlie these differences, four peptide analogs have been designed and tested. The analogs were designed to assess the major structural differences between substance P and ranatachykinin C, including the role of the N-terminal Arg and the substitution of the mid-region Glns with Ala and Ser (Q5 replaced with A and/or Q6 replaced with S). Receptor binding, receptor activation of intracellular calcium fluxes, and receptor desensitization of the rat tachykinin NK1 receptor were quantified for each ligand. All of the peptides bound to the rat tachykinin NK1 receptor with high affinity, produced robust calcium signal activation, and led to agonist-induced receptor desensitization. It was found that deletion of the N-terminal Arg of substance P or replacement of either or both Q5 and Q6 altered the functional selectivity of substance P based on the relationship of receptor binding to receptor activation and activation to desensitization. When considered in light of our previously published nuclear magnetic resonance structure data, the data presented herein suggest that the one, five and six positions of the substance P backbone are key structural residues that govern the relative degree of tachykinin peptide-mediated receptor signaling and desensitization.
Keywords: Substance P; Tachykinin; NK1 receptor; G protein coupled receptor; Nuclear magnetic resonance; Receptor binding; Calcium responses; Desensitization;

The purpose of this study was to investigate possible efflux mechanisms involved in amphetamine derivative transport such as for 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), para-methoxyamphetamine (p-MA), dexamphetamine and pseudoephedrine, especially across pH gradients that exist in intestinal or kidney transport. This was determined using our Caco-2 subclone, CLEFF9. Transport of the amphetamine derivatives was evaluated at pH 7.4 and pH 6/7.4 ± efflux inhibitors. Na+–H+ transporter inhibition via carbonyl cyanide-4-trifluoromethoxy phenylhydrazone (FCCP), and metabolic inhibition using Na-azide and Na-orthovanadate were also conducted, as well as using noradrenalin, adrenalin and other inhibitors of a range of carrier mediated transport systems such as histamine, organic cation transporters and dopamine carrier systems. At pH 7.4, the rate of transport for dexamphetamine, pseudoephedrine and MDMA in both apical to basolateral and reverse directions was all very rapid, confirming extensive passive diffusion at systemic pH. However, creating a pH 6.0/7.4 gradient showed marked increase in basolateral to apical transport of all amphetamines tested, with dexamphetamine, MDEA, MDMA and p-MA having a net efflux ratio of around 16, 14, 13 and 11 respectively and this was not reversed with P-glycoprotein inhibitors. Azide, FCCP, adrenalin, noradrenalin and reserpine were able to reduce the efflux by 2 to 3 fold, although tetraethylammonium could not. This suggested that extraneuronal monoamine transporters (hEMT) could be involved. This data suggests that elevated endogenous adrenalin levels may reduce amphetamine removal from the body based on these in vitro studies. Also, the use of stomach acid lowering drugs could result in more rapid systemic uptake of these amphetamine derivatives.
Keywords: MDMA; MDEA; p-MA; Epinephrine; Noradrenalin; Reserpine;

Effects of tamoxifen and raloxifene on normal human endometrial cells in an organotypic in vitro model by Merja Bläuer; Pentti K. Heinonen; Päivi Rovio; Timo Ylikomi (13-18).
The selective estrogen receptor modulator tamoxifen is widely used in breast cancer therapy though its use is associated with an elevated risk of endometrial carcinoma. An organotypic culture model was employed here to examine the effects of tamoxifen and raloxifene, a related compound with no known adverse uterine effects, on epithelial cells of the premenopausal human endometrium. Changes in the expression levels of the proliferation marker Ki67, and estrogen and progesterone receptors were evaluated. No change in the Ki67 index compared to untreated controls was detected in cultures exposed to tamoxifen or tamoxifen + estradiol. In response to tamoxifen, the level of progesterone receptor-expressing organoids was shown to vary markedly between individual samples, whereas no change in estrogen receptor expression could be demonstrated. A significant decrease in Ki67 expression was observed in raloxifene-exposed cultures. Raloxifene or raloxifene + estradiol had no effect on progesterone receptor expression. The expression of estrogen receptor was markedly inhibited in response to raloxifene or raloxifene + estradiol in all but two samples displaying an intense estrogen receptor labelling. The present observations add to current clinical data on the respective estrogen receptor agonist and antagonist activities of tamoxifen and raloxifene on the human uterus by providing novel insights into the interindividual variation in cellular responses. Our organotypic model may have uses as an alternative to animal experimentation in preclinical screening of the endometrial effects of selective estrogen receptor modulators and may serve as a tool in personalized medicine by identifying patients with an increased risk of developing endometrial pathologies.
Keywords: Tamoxifen; Raloxifene; Selective estrogen receptor modulator; Endometrium; Organotypic;

Clomipramine block of the hERG K+ channel: Accessibility to F656 and Y652 by Su-Hyun Jo; Hee-Kyung Hong; Seon Ha Chong; Kwang Hee Won; Sung Jun Jung; Han Choe (19-25).
Clomipramine is a tricyclic antidepressant for psychiatric disorders that can induce QT prolongation, which may lead to torsades de pointes. Since blockade of cardiac human ether-a-go-go-related gene (hERG) channels is an important cause of acquired long QT syndrome, we investigated the acute effects of clomipramine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. We examined the effects of clomipramine on the hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. Clomipramine induced a concentration-dependent decrease in the current amplitude at the end of the voltage steps and hERG tail currents. The IC50 for clomipramine needed to block the hERG current in Xenopus oocytes decreased progressively relative to the degree of depolarization. The fractional electrical distance was estimated to be δ  = 0.83. The IC50 for the clomipramine-induced blockade of the hERG currents in HEK293 cells at 36 °C was 0.13 μM at + 20 mV. Clomipramine affected the channels in the activated and inactivated states but not in the closed states. The clomipramine-induced blockade of hERG was found to be use-dependent, exhibiting a more rapid onset and a greater steady-state block at the higher frequencies of activation. The S6 domain mutations, Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG-current blockade. These results suggest that clomipramine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of clomipramine.
Keywords: Arrhythmia; Clomipramine; hERG channel; LQT; Rapidly activating delayed rectifier K+ current; Torsades de pointes; Tricyclic antidepressant;

Cloning and pharmacological characterization of the dog histamine H4 receptor by Wen Jiang; Herman D. Lim; Mai Zhang; Pragnya Desai; Heng Dai; Patricia M. Colling; Rob Leurs; Robin L. Thurmond (26-32).
The histamine H4 receptor has been shown to have a role in chemotaxis and mediator release in various types of immune cells and has been implicated in mediating inflammation in vivo. Previous work has shown that there were differences in the histamine H4 receptor sequence of different species and these translated into changes in the pharmacology of the receptors. To help further understand the nature of these differences, we have cloned and expressed the histamine H4 receptor of dog (Canis familiaris). The dog histamine H4 receptor has a 61–71% homology with the receptors from other species, with a 71% homology to the human receptor. The affinity for histamine at the dog histamine H4 receptor is 18 nM and is 3-fold lower than the human ortholog. A number of previously described histamine H4 receptor ligands were tested for affinity at the dog histamine H4 receptor and histamine showed the highest affinity of the ligands tested. In addition, the histamine H4 receptor selective antagonist, JNJ 7777120, had a K i value of 50 nM and acts as an antagonist at the dog receptor. In general, agonists of the human histamine H4 receptor were also agonists of the dog receptor albeit with different efficacy levels. The cloning and in vitro pharmacological characterization of the dog histamine H4 receptor provide useful information for future studies using dog models as well as in understanding the ligand–receptor interactions of the receptor.
Keywords: Histamine receptor; Ligand binding; G protein-coupled receptor; Antihistamine;

Guanabenz, an α2-selective adrenergic agonist, activates Ca2+-dependent chloride currents in cystic fibrosis human airway epithelial cells by Caroline Norez; Clarisse Vandebrouck; Fabrice Antigny; Luc Dannhoffer; Marc Blondel; Frédéric Becq (33-40).
In cystic fibrosis respiratory epithelial cells, the absence or dysfunction of the chloride channel CFTR (Cystic Fibrosis Transmembrane conductance Regulator) results in reduced chloride ion transport. In contrast, Ca2+-stimulated Cl secretion is intact in cystic fibrosis airway epithelia. One possible target for drug discovery aiming at treating cystic fibrosis is to correct the ionic imbalance through stimulation of alternative ionic pathways that may compensate the failure of epithelial Cl conductance. Here, using a simple high-throughput screening assay to search for Cl channels modulators in the cystic fibrosis nasal epithelial cell line JME-CF15, the compound guanabenz (Wytensin®), an α2-selective adrenergic agonist was found positive. Using iodide effluxes and electrophysiological recordings, we showed that guanabenz-activated (EC50  = 831 nM) a DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid) sensitive and Ca2+ dependent Cl channel (CaCC). Guanabenz activated a linear Cl channel with unitary single-channel conductance of 8 pS. Recording calcium signals in CF15 cells showed that guanabenz increased the intracellular Ca2+ concentration stimulating an influx of Ca2+. In the absence of extracellular Ca2+, the guanabenz effects on Ca2+ influx and activation of CaCC were both abolished. These data demonstrate that guanabenz activates Ca2+-dependent Cl channels via a Ca2+ influx in human cystic fibrosis airway epithelial cells.
Keywords: CaCC; Ca2+ influx; Chloride channels; Cystic fibrosis; Human airway epithelial cells; Guanabenz; Pharmacology;

Hypoxia-inducible factor-1 (HIF-1) is the central mediator of cellular responses to low oxygen and vital to many aspects of cancer biology. In a search for HIF-1 inhibitors, we identified a quassinoid 6α-tigloyloxychaparrinone (TCN) as an inhibitor of HIF-1 activation from Ailantus altissima. We here demonstrated the effect of TCN on HIF-1 activation induced by hypoxia or CoCl2. TCN showed the potent inhibitory activity against HIF-1 activation induced by hypoxia in various human cancer cell lines. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently, whereas it did not affect the expressions of HIF-1β and topoisomerase-I. Furthermore, TCN prevented hypoxia-induced expression of HIF-1 target genes for vascular endothelial growth factor (VEGF) and erythropoietin. Further analysis revealed that TCN strongly inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. Moreover, the levels of phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2), mitogen-activated protein (MAP) kinase-interacting protein kinase-1 (MNK1) and eukaryotic initiation factor 4E (eIF4E) were significantly suppressed by the treatment of TCN, without changing the total levels of these proteins. Our data suggested that TCN may exhibit anticancer activity by inhibiting HIF-1α translation through the inhibition of eIF4E phosphorylation pathway and thus provide a novel mechanism for the anticancer activity of quassinoids. TCN could be a new HIF-1-targeted anticancer agent and be effective on mammalian target of rapamycin (mTOR)-targeted cancer therapy, in which mTOR inhibition increases eIF4E phosphorylation.
Keywords: 6α-tigloyloxychaparrinone; Quassinoid; HIF-1α translation; eIF4E; Antitumor activity;

Blockade of anaesthetic-induced preconditioning in the hyperglycaemic myocardium by Nina C. Weber; Christine Goletz; Ragnar Huhn; Yvonne Grueber; Benedikt Preckel; Wolfgang Schlack; Dirk Ebel (48-54).
Preconditioning by volatile anaesthetics is blocked by hyperglycaemia. The regulation of mitogen-activated protein kinases during this effect has yet not been investigated.For infarct size measurements, anaesthetized rats were subjected to 25 min coronary artery occlusion followed by 120 min reperfusion. Control animals were not further treated. One group was preconditioned by two 5-min periods of desflurane inhalation (desflurane preconditioning, Des-preconditioning, 1MAC), each followed by 10-min washout. Four groups received glucose 50% in order to achieve blood glucose concentrations between 22.2 and 33.3 mM/l. Glucose infusion started 40 min before ischaemia (early hyperglycaemia, EH) and stopped with the onset of artery occlusion with (EH+Des-preconditioning) or without (EH) preconditioning. The other two groups received glucose during ischaemia (late hyperglycaemia, LH), again with (LH+Des-preconditioning) or without (LH) preconditioning. Additional hearts were excised for Western blot of mitogen-activated protein kinases.Infarct size was reduced from 51.7 ± 9.0% in controls to 28.8 ± 11.8% after Des-preconditioning (P< 0.01 vs Con). Hyperglycaemia alone did not affect infarct size (EH, 51.5 ± 9.0%, LH, 44.3 ± 16.9%), but EH as well as LH blocked Des-preconditioning (49.1 ± 12.3%, P< 0.01, 48.1 ± 17.6%, P< 0.05 vs Des-preconditioning). All three mitogen-activated protein kinases showed a similar time course pattern of phosphorylation in the Des-preconditioning, EH and EH+Des-preconditioning group.Despite the lack of cardioprotection, mitogen-activated protein kinases are activated in hyperglycaemic myocardium. Therefore, it can be assumed that the hyperglycaemic induced blockade of Des-preconditioning is situated downstream or in parallel of these mitogen-activated protein kinases or involves different signal transduction pathways.
Keywords: Hyperglycaemia; Preconditioning; Desflurane; Mitogen-activated protein kinase;

We investigated the effect of the p38 mitogen-activated protein kinase inhibitor SB239063 on inflammation and neurogenesis after ischemia in organotypic hippocampal slice cultures. Our study shows that after oxygen–glucose deprivation, the p38 mitogen-activated protein kinase (MAPK) and the extracellular-signal-regulated kinase 1/2 (ERK1/2) are strongly activated. The p38 MAPK phosphorylation returned to basal levels within 1 h after oxygen–glucose deprivation, whereas the ERK1/2 phosphorylation reached the basal level only after 24 h. Treatment with 20 μM and 100 μM SB239063 strikingly reduced cell death after oxygen–glucose deprivation and significantly diminished microglia activation in the cornu ammonis (CA-region), but not in the area dentata. Levels of the pro-inflammatory cytokine IL-1β were reduced by 84% after treatment with SB239063 whereas the cytokines IL-6 and TNF-α were not affected. After 6 days, neurogenesis was significantly increased in the posterior periventricle. Based on these findings, our study shows that anti-inflammatory treatment with SB239063 reduces cell death, inflammation and microglia activation and, at high concentrations, enhances the oxygen–glucose deprivation-induced neurogenesis in the posterior periventricle.
Keywords: SB239063; Inflammation; Neuroprotection; Neurogenesis; Microglia; Ischemia;

Role of α2-adrenoceptors in enhancement of antinociceptive effect in diabetic mice by Yuji Omiya; Mitsutoshi Yuzurihara; Yasuyuki Suzuki; Yoshio Kase; Toru Kono (62-66).
The present studies investigated behavioral and neurochemical aspects of the noradrenergic and serotonergic nervous systems in streptozotocin-induced diabetic mice. We previously reported that intrathecal (i.t.) injection of norepinephrine significantly potentiated antinociception in diabetic mice compared to that in non-diabetic mice, and that antinociception due to norepinephrine injection was completely abolished by pretreatment with yohimbine, an α2-adrenoceptor antagonist. The present studies demonstrated that i.t. injection of clonidine also showed more-potent antinociceptive activity in diabetic mice than in non-diabetic mice, but that i.t. methoxamine injection did not affect diabetic or non-diabetic mice. The antinociceptive potency due to i.t. injection of 5-HT was significantly lower in diabetic than in non-diabetic mice. In a neurochemical study, we found that the density of [3H]-rauwolscine binding sites in spinal α2-adrenoceptors was significantly higher in diabetic than in non-diabetic mice, but that the binding affinity was unchanged. Spinal norepinephrine turnover was determined by measuring the decline in tissue norepinephrine concentration at 3 h after injection of the tyrosine hydroxylase inhibitor α-methyl-p-tyrosine. The spinal norepinephrine concentration decreased to 43.7% from the baseline in non-diabetic mice, while it was 21.0% in diabetic mice. These results suggest that, based on the decrease of norepinephrine release in the spinal cord, up-regulation of spinal α2-adrenoceptors caused the increase of antinociception due to i.t. injection of an α2-adrenoceptor agonist in streptozotocin-induced diabetic mice, and it seemed that the stimulation of α2-adrenoceptors potentiated the antinociceptive effect. Thus, the spinal noradrenergic systems play an important moderating role in diabetes-induced neuropathic pain.
Keywords: Antinociception; Diabetes; Descending pain inhibitory system; Spinal cord; α2-adrenoceptor;

Repeated treatment with the dopamine D2/D3 receptor agonist quinpirole produces a sensitized behavioral response in rats manifested as an increase in locomotor activity. Pre-treatment with certain monoamine oxidase inhibitors, such as Ro 41-1049 [N-(2-aminomethyl)-5-(3-fluorophenyl)-4-thiazolecarboxamide HCl], changes the sensitized response from locomotion to stationary, self-directed mouthing. In this study, the effects of quinpirole sensitization, with and without pre-treatment with Ro 41-1049, were determined on dopamine D2-like receptors in the nucleus accumbens and the striatum. Long-Evans rats were pre-treated with Ro 41-1049 (1 mg/kg) 90 min prior to administration of quinpirole (0.5 mg/kg, 8 injections, every 3–4 days). Dopamine D2-like receptor binding was determined 3 days after the last injection by ex vivo radioligand assays using [3H]spiperone and [3H]quinpirole. Densities of [3H]spiperone- and [3H]quinpirole-labeled sites were both increased 32% in the nucleus accumbens of rats with demonstrated locomotor sensitization to quinpirole. In contrast, the density of dopamine D2-like receptors in quinpirole-sensitized rats pre-treated with Ro 41-1049 was not different from saline controls. These findings support the involvement of alterations in dopamine D2-like receptors in the development of locomotor sensitization to quinpirole and suggest that modification of these alterations in dopamine D2-like receptors contributes to the change from sensitized locomotion to mouthing observed when rats are pre-treated with Ro 41-1049.
Keywords: Dopamine D2 receptor; Sensitization; Quinpirole; Striatum; Nucleus accumbens;

Persistence of tolerance to the anaesthetic effect of allopregnanolone in male rats by Şahruh Türkmen; Göran Wahlström; Torbjörn Bäckström; Inga-Maj Johansson (73-80).
Both acute and chronic tolerance can develop to allopregnanolone—a gamma-aminobutyric acid (GABA)-modulatory progesterone metabolite. Here we investigated if acute tolerance to allopregnanolone persisted for 1 or 2 days after the induction and thus could be the initial part of chronic tolerance. Male rats were anaesthetised with allopregnanolone (i.v) to the deep anaesthesia level of the silent second (SS), which is an EEG burst suppression of 1 s or more. They were divided into four groups: SS1—anaesthesia to the first silent second; LAn (long anaesthesia)—90 min anaesthesia at the SS level; SS2;D1—90 min anaesthesia and SS induction 1 day later; SS2;D2—90 min anaesthesia and SS induction 2 days later. Allopregnanolone concentrations in tissue and serum were analysed. Levels of the GABAA receptor α2, α4, γ2(S+L) and δ subunits mRNAs were analysed by in situ hybridisation. Acute tolerance was induced during the 90 min anaesthesia. Tolerance persisted for 1 day, since the dose of allopregnanolone needed to induce a new SS anaesthesia was increased after 1 day. The level of α4 subunit mRNA expression in the ventral posteriomedial nucleus of thalamus was negatively related to the tolerance parameters, the SS dose of allopregnanolone and ΔSS (SS dose difference between days). Allopregnanolone threshold anaesthesia lasting 90 min induces acute tolerance that persisted for at least 1 day, which could be used as the start of a chronic tolerance. The α4 subunit may be involved in allopregnanolone caused effects in the brain.
Keywords: GABAA receptor (Gamma-aminobutyric acid A); In situ hybridisation; Neurosteroid; Burst suppression threshold; Allopregnanolone tolerance;

Centrally administered neuromedin U elevates plasma adrenaline by brain prostanoid TP receptor-mediated mechanisms in rats by Tsuyoshi Sasaki; Takahiro Shimizu; Hiroshi Wakiguchi; Kunihiko Yokotani (81-86).
Neuromedin U is a hypothalamic peptide involved in energy homeostasis and stress responses. The peptide, when administered intracerebroventricularly (i.c.v.), decreases food intake and body weight while increasing body temperature and heat production. We examined the effect of i.c.v. administered neuromedin U on plasma catecholamines with regard to the brain prostanoid using anesthetized rats. Neuromedin U (0.1, 0.5 and 1 nmol/animal, i.c.v.) effectively elevated plasma adrenaline (a maximal response was obtained at 0.5 nmol/animal), but had little effect on plasma noradrenaline. However, intravenously administered neuromedin U (0.5 nmol/animal) had no effect on plasma catecholamines. Neuromedin U (0.5 nmol/animal, i.c.v.)-induced elevation of plasma adrenaline was effectively reduced by intracerebroventricular pretreatments with indomethacin (an inhibitor of cyclooxygenase) (0.6 and 1.2 µmol/animal), furegrelate (an inhibitor of thromboxane A2 synthase) (0.9 and 1.8 µmol/animal) and (+)-S-145 (a blocker of prostanoid TP receptors) (250 and 625 nmol/animal), respectively. The neuromedin U-induced adrenaline response was also abolished by acute bilateral adrenalectomy. These results suggest that centrally administered neuromedin U evokes the secretion of adrenaline from the adrenal medulla by brain prostanoid TP receptor-mediated mechanisms in rats.
Keywords: Brain; Neuromedin U; Cyclooxygenase; Thromboxane A2; Adrenal medulla;

The critical role of invading peripheral macrophage-derived interleukin-6 in vincristine-induced mechanical allodynia in mice by Norikazu Kiguchi; Takehiko Maeda; Yuka Kobayashi; Toshikazu Kondo; Masanobu Ozaki; Shiroh Kishioka (87-92).
Although the clinical use of vincristine is limited by its adverse effect, neuropathic pain, the mechanism of this effect is poorly understood. Recently, reports demonstrated that inflammatory and immune responses play an important role in the neuropathic pain that follows peripheral nerve injury. In this study, we examined the role of macrophage-derived interleukin (IL)-6 in vincristine-induced mechanical allodynia. Vincristine sulfate (0.01–0.1 mg/kg, i.p.) was administered to male ICR mice and BALB/c mice once per day for 7 or 14 days. Mechanical allodynia was evaluated by withdrawal responses, using von Frey filaments from day 0 to day 28. In both ICR mice and BALB/c mice, significant dose-dependent increases in the percentage of withdrawal responses were observed from day 3 to day 28 following repeated administration of vincristine (0.1 mg/kg). As determined by immunohistochemistry, the number of macrophages in the region of the sciatic nerve and lumbar dorsal root ganglion was significantly increased on day 7 of vincristine administration. The expression of IL-6 was increased by vincristine administration and was co-localized in the invading macrophage. Moreover, a neutralizing antibody of IL-6, which was injected into areas surrounding the sciatic nerve on day 0, 3, and 6, significantly attenuated vincristine-induced mechanical allodynia from day 7 to day 28. In addition, the incidence of vincristine-induced mechanical allodynia in IL-6 knockout mice was lower than that in wild type mice from day 3 to day 28. These results suggest that the invading peripheral macrophage-derived IL-6 plays a critical role in vincristine-induced mechanical allodynia.
Keywords: Vincristine; Allodynia; Interleukin-6; Macrophage; Sciatic nerve; Neuropathic pain;

The present data provide the first in vivo evidence that the proinflammatory chemokine, Regulated on Activation Normal T cell Expressed and Secreted (RANTES/CCL5) microinjected directly into the periaqueductal grey in rats, a brain region critical to the processing of pain signals, and a primary site of action of many analgesic compounds, induced hyperalgesia. Pretreatment with antibodies against RANTES/CCL5 prevented the hyperalgesic response, indicating that RANTES/CCL5 is able to interfere with the control of hyperalgesia at the level of the periaqueductal grey and suggesting that chemokine blockers could have analgesic properties.
Keywords: RANTES/CCL5; Periaqueductal grey; Hyperalgesia; Chemokine; Pain;

Behavioral characterization of the mGlu group II/III receptor antagonist, LY-341495, in animal models of anxiety and depression by Anton Y. Bespalov; Marcel M. van Gaalen; Irina A. Sukhotina; Karsten Wicke; Mario Mezler; Hans Schoemaker; Gerhard Gross (96-102).
There is a growing body of evidence indicating that stimulation of metabotropic glutamate type II receptors (mGlu2/3) reduces anxiety in laboratory animals and humans. Surprisingly, it was reported that mGlu2/3 receptor antagonists have antidepressant- and anxiolytic-like activities in laboratory animal studies as well. The present study aimed to resolve this controversy by characterizing behavioral effects of a selective mGlu2/3 receptor antagonist, LY-341495, in a variety of animal models sensitive to clinically used anxiolytic and antidepressant agents. In agreement with previous reports, LY-341495 (0.3–3 mg/kg, i.p.) reduced immobility in the mouse forced swim test. LY-341495 was also effective in the marble burying test in mice, although similar effects were observed after administration of various drugs including methamphetamine. Further, LY-341495 had no effects in the elevated plus maze and stress-induced hyperthermia tests in mice, as well as on punished drinking (Geller–Seifter's test) and differential reinforcement of low rates of responding (DRL) in rats. It is concluded that behavioral profile of mGlu2/3 receptor antagonists as represented by LY-341495 is different from that of conventional anxiolytic and antidepressant drugs.
Keywords: Anxiety; Depression; Animal model; Metabotropic glutamate receptor; LY-341495;

Aripiprazole inhibits marble-burying behavior via 5-hydroxytryptamine (5-HT)1A receptor-independent mechanisms by Nobuaki Egashira; Ryoko Okuno; Michihiko Matsushita; Moe Abe; Kenichi Mishima; Katsunori Iwasaki; Ryoji Nishimura; Ryozo Oishi; Michihiro Fujiwara (103-108).
Aripiprazole is a first next-generation atypical antipsychotic drug with dopamine system stabilizing, serotonin (5-hydroxytryptamine, 5-HT) 5-HT1A receptor partial agonistic, and 5-HT2A receptor antagonistic properties. In the present study, we examined the effect of aripiprazole on marble-burying behavior, which has been considered an animal model of obsessive–compulsive disorder, and compared this with the effects of other atypical antipsychotics such as olanzapine and quetiapine. Aripiprazole (1 mg/kg, i.p.) inhibited marble-burying behavior without affecting the locomotor activity in mice. Conversely, olanzapine (3 mg/kg, i.p.) and quetiapine (100 mg/kg, p.o.) showed significant suppression of locomotor activity and impairment of motor coordination at the dose that inhibited marble-burying behavior. On the other hand, a selective 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane (WAY100635, 3 mg/kg, i.p.) markedly antagonized the inhibition of marble-burying behavior by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 3 mg/kg, i.p.), a selective 5-HT1A/7 receptor agonist. By contrast, WAY100635 at the same dose had no effect on the inhibition of marble-burying behavior by aripiprazole (1 mg/kg, i.p.). Quinpirole, a dopamine D2 receptor agonist, showed significant suppression of locomotor activity at the dose that inhibited marble-burying behavior. Conversely, L-741,626, a selective dopamine D2 receptor antagonist, at a dose of 10 mg/kg inhibited marble-burying behavior without affecting the locomotor activity. On the other hand, ketanserin, a 5-HT2A receptor antagonist, had no effect on the marble-burying behavior. These findings suggest that aripiprazole may be a useful drug for the treatment of obsessive–compulsive disorder, and that aripiprazole inhibits the marble-burying behavior via 5-HT1A receptor-independent mechanisms.
Keywords: Marble-burying behavior; Aripiprazole; Olanzapine; Quetiapine; Antipsychotic; 5-HT1A receptor; Obsessive–compulsive disorder;

Feeding conditions differentially affect the neurochemical and behavioral effects of dopaminergic drugs in male rats by Rajkumar J. Sevak; Wouter Koek; William Anthony Owens; Aurelio Galli; Lynette C. Daws; Charles P. France (109-115).
The high co-morbidity of eating disorders and substance abuse suggests that nutritional status can impact vulnerability to drug abuse. These studies used rats to examine the effects of food restriction on dopamine clearance in striatum and on the behavioral effects of amphetamine (locomotion, conditioned place preference), the dopamine receptor agonist quinpirole (yawning), and the dopamine receptor antagonist raclopride (catalepsy). Amphetamine increased locomotion and produced conditioned place preference. Food restriction reduced dopamine clearance, which was restored by repeated treatment with amphetamine or by free feeding. Food restriction also decreased sensitivity to quinpirole-induced yawning and raclopride-induced catalepsy; normal sensitivity to both drugs was restored by free feeding. The same amphetamine treatment that normalized dopamine clearance, failed to restore normal sensitivity to quinpirole or raclopride, suggesting that in food-restricted rats the activity of dopamine transporters and dopamine receptors is differentially affected by pathways that are stimulated by amphetamine. These studies show that modest changes in nutritional status markedly alter dopamine neurotransmission and the behavioral effects of direct-acting dopamine receptor drugs (agonist and antagonist). These results underscore the potential importance of nutritional status (e.g., glucose and insulin) in modulating dopamine neurotransmission and in so doing they begin to establish a neurochemical link between the high co-morbidity of eating disorders and drug abuse.
Keywords: Dopamine; Receptor; Chronoamperometry; Locomotion; Conditioned place preference; Quinpirole; Raclopride; Amphetamine;

Mouse strain differences in immobility and sensitivity to fluvoxamine and desipramine in the forced swimming test: Analysis of serotonin and noradrenaline transporter binding by Yumi Sugimoto; Yoshinobu Kajiwara; Kazufumi Hirano; Shizuo Yamada; Noriko Tagawa; Yoshiharu Kobayashi; Yoshihiro Hotta; Jun Yamada (116-122).
Strain differences in immobility time in the forced swimming test were investigated in five strains of mice, namely, ICR, ddY, C57BL/6, DBA/2 and BALB/c mice. There were significant strain differences. The immobility times of ICR, ddY and C57BL/6 mice were longer than those of DBA/2 and BALB/c mice. Immobility times were not significantly related to locomotor activity in these strains. There were also differences in sensitivity to the selective serotonin reuptake inhibitor (SSRI) fluvoxamine. In ICR, ddY and C57BL/6 mice, fluvoxamine did not affect immobility time, while it reduced the immobility time of DBA/2 and BALB/c mice dose-dependently. The noradrenaline reuptake inhibitor desipramine decreased immobility time in all strains of mice. Serotonin (5-HT) transporter binding in the brains of all five strains of mice was also investigated. Analysis of 5-HT transporter binding revealed significant strain differences, being lower in DBA/2 and BALB/c mice than in other strains of mice. The amount of 5-HT transporter binding was correlated to baseline immobility time. However, there was no significant relation between noradrenaline transporter binding and immobility time. These results suggest that the duration of baseline immobility depends on the levels of 5-HT transporter binding, leading to apparent strain differences in immobility time in the forced swimming test. Furthermore, differences in 5-HT transporter binding may cause variations in responses to fluvoxamine.
Keywords: Forced swimming test; Strain difference; Depression; SSRI; Serotonin transporter; Noradrenaline transporter; (Mouse);

Clozapine-induced myocarditis: Role of catecholamines in a murine model by Ju-Feng Wang; Jiang-Yong Min; Thomas G. Hampton; Ivo Amende; Xinhua Yan; Sohail Malek; Walter H. Abelmann; Alan I. Green; John Zeind; James P. Morgan (123-127).
Clozapine, an atypical antipsychotic, is very effective in the treatment of resistant schizophrenia. However, cardiotoxicity of clozapine, particularly in young patients, has raised concerns about its safety. Increased catecholamines have been postulated to trigger an inflammatory response resulting in myocarditis, dilated cardiomyopathy, and death, although this has not yet been thoroughly studied. Here, we used the mouse to study whether clozapine administration could cause adverse myocarditis associated with an increase in catecholamines. Male Balb/C mice, age ~ 6 weeks, were administered 5, 10 or 25 mg/kg clozapine daily for 7 and 14 days; one group was administered 25 mg/kg clozapine plus 2 mg/kg propranolol for 14 days. Saline-treated mice served as controls. Heart sections were stained with hematoxylin and eosin for histopathological examination. Plasma catecholamines were measured with HPLC. Myocardial TNF-alpha concentrations were determined by ELISA. Histopathology of clozapine-treated mice showed a significant dose-related increase in myocardial inflammation that correlated with plasma catecholamine levels and release of TNF-alpha. Propranolol significantly attenuated these effects. A hypercatecholaminergic state induced by clozapine could explain the occurrence of myocarditis in some patients. Our data suggest that a beta-adrenergic blocking agent may be effective in reducing the incidence and severity of clozapine-induced myocarditis.
Keywords: Atypical antipsychotic; Norepinephrine; Epinephrine; TNF-alpha; Cardiotoxicity; Balb/C mice;

Primary and secondary capture of platelets onto inflamed femoral artery endothelium is dependent on P-selectin and PSGL-1 by Oscar Ö. Braun; Jan E. Slotta; Michael D. Menger; David Erlinge; Henrik Thorlacius (128-132).
Platelets constitute a key role in vascular injuries, however, the detailed mechanisms behind platelet–endothelial cell and platelet–leukocyte interactions in the femoral artery are not yet fully elucidated. We used intravital fluorescence microscopy of the femoral artery in C57BL/6 mice to study primary and secondary capture of platelets onto endothelial cells as well as onto adherent platelets and leukocytes in vivo. By use of monoclonal antibodies, the role of P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) in these adhesive interactions in mice exposed to endotoxin was determined. Intravenous injection of endotoxin significantly increased gene expression of P-selectin as well as platelet tethering, rolling and adhesion in the femoral artery. Pretreatment with the anti-PSGL-1 antibody decreased platelet tethering by 85%, platelet rolling by 88% and platelet adhesion by 96%. Immunoneutralization of P-selectin reduced platelet tethering by 91%, platelet rolling by 98%, and platelet adhesion by 97%. In addition, inhibition of P-selectin and PSGL-1 completely abolished secondary capture of platelets onto adherent platelets and leukocytes. Our data show that P-selectin and PSGL-1 mediate early interactions between platelets and other cells, including endothelial cells and leukocytes, in inflamed arteries. These novel results suggest that interference with P-selectin and PSGL-1 may be a useful target in strategies aiming to protect the vascular wall during arterial inflammation.
Keywords: Adhesion; Inflammation; Platelet; P-selectin;

Characterization of contractile 5-hydroxytryptamine receptor subtypes in the in situ autoperfused kidney in the anaesthetized rat by Asunción Morán; Ana-Vega Ortiz de Urbina; Maria-Luisa Martín; Mónica García; Alicia Rodriguez-Barbero; Fernando Dorado; Luis San Román (133-137).
Using several 5-hydroxytryptamine (5-HT) agonists and antagonists, we attempted to characterize the receptor subtypes involved in the contractile response to 5-HT in the in situ autoperfused rat kidney.An intra-arterial (i.a.) bolus injection of 5-HT (0.00000125 to 0.1 µg/kg) increased renal perfusion pressure in a dose-dependent way but did not affect the systemic blood pressure. The selective 5-HT2 receptor agonist α-methyl-5-HT (α-methyl-5-hydroxytryptamine) and the non-selective 5-HT2C receptor agonist (1-(3-chlorophenyl)piperazine), m-CPP, caused a local vasoconstrictor effect in the autoperfused rat kidney, whereas BW723C86, a selective 5-HT2B receptor agonist, the 5-HT1 receptor agonist 5-carboxamidotryptamine, 5-CT, and the selective 5-HT3 receptor agonist m-CPBG (1-(m-chlorophenyl)-biguanide) did not modify the renal perfusion pressure. The vasoconstrictor effect elicited by α-methyl-5-HT and m-CPP was significantly decreased by ritanserin (a 5-HT2 receptor antagonist), SB 206553 (3,5-Dihydro-5-methyl-N-3pyridinylbenzo[1,2.b:4,5-b']dipyrrole(1H)-carboxamide hydrochloride), a selective 5-HT2B/2C receptor antagonist and enalapril, but was not modified by pretreatment with spiperone (a 5-HT2A receptor antagonist). The results of protein expression analyses allow us to postulate that 5HT-SRC (a 5-HT2C receptor protein) is expressed in renal tissue and differentially expressed in renal artery. Our data suggest also that the serotonergic vasoconstrictor response induced in the in situ autoperfused rat kidney would be mediated by local 5-HT2C receptor activation.
Keywords: 5-hydroxytryptamine; 5-HT2C receptor; Autoperfused rat kidney;

A protective role of unfolded protein response in mouse ischemic acute kidney injury by Worapat Prachasilchai; Hiroko Sonoda; Naoko Yokota-Ikeda; Sayaka Oshikawa; Chie Aikawa; Kazuyuki Uchida; Katsuaki Ito; Takashi Kudo; Kazunori Imaizumi; Masahiro Ikeda (138-145).
Although renal ischemia-reperfusion is known to activate the unfolded protein response, the renal site and role of activation of this response following the insult in vivo remains largely unknown. Here we studied the renal spatio-temporal expression pattern of glucose-regulated protein (GRP) 78, a central regulator of the unfolded protein response network, following renal ischemia-reperfusion and the effects of the specific chemical unfolded protein response inducers, tunicamycin and thapsigargin, on renal ischemia-reperfusion injury in mice. Renal ischemia-reperfusion resulted in expression of the spliced form of the X-box binding protein-1 (XBP-1s) transcript, an unfolded protein response target, at 1 and 2 h after the insult. This response was followed by an increase in the GRP78 transcript and protein. The increased amount of GRP78 protein after ischemia-reperfusion was largely localized in proximal tubule cells. Pretreatment with tunicamycin or thapsigargin significantly ameliorated renal dysfunction and injury after ischemia-reperfusion. Taken together with these results, the unfolded protein response was activated following renal ischemia-reperfusion at sites that are susceptible to ischemia-reperfusion injury, and this activation had a protective effect against renal ischemia-reperfusion injury in vivo. Molecules involved in the unfolded protein response may offer new opportunities for pharmacological intervention against renal ischemia-reperfusion injury, which is an important cause of acute kidney injury.
Keywords: Ischemia-reperfusion injury; Endoplasmic reticulum stress; Unfolded protein response; Glucose-regulated protein 78; X-box binding protein; Tunicamycin; Thapsigargin;

Thalidomide (α-naphtylimidoglutarimide), a psychoactive drug that readily crosses blood-brain barrier, has been shown to exhibit anti-inflammatory, anti-angiogenic, immunomodulatory properties through a mechanism that is not fully established. Keeping these properties in mind, we tried to find out the anti-inflammatory properties of thalidomide in mouse model of acute inflammation by introducing K. pneumoniae B5055 in BALB/c mice via intranasal route. The intranasal instillation of bacteria in this mouse model of acute pneumonia induced inflammation accompanied with significant increase in neutrophil infiltration in the lungs and also increased production of mediators of inflammation (i.e. malondialdehyde, myeloperoxidase and nitric oxide) in the lung tissue. The animals, which received thalidomide alone orally or in combination with augmentin, 30 min prior to bacterial instillation into the lungs via intranasal route, showed significant (P  < 0.05) decrease in neutrophil influx into the lungs and there was significant (P  < 0.05) decrease in the production of malondialdehyde, nitric oxide and myeloperoxidase activity. But the augmentin treatment alone did not decrease the malondialdehyde, myeloperoxidase and nitric oxide significantly (P  > 0.05) as compared to the control group. We therefore conclude that thalidomide ameliorates lung inflammation induced by K. pneumoniae B5055 without significantly (P  < 0.05) decreasing the bacterial load in the lung tissue whereas augmentin takes care of bacterial proliferation. Hence, it can be used as an adjunct therapy along with antibiotics as an anti-inflammatory or an immunomodulatory agent in case of acute lung infection.
Keywords: Thalidomide; Inflammation; Malondialdehyde; Myeloperoxidase; Nitric oxide; Augmentin; Klebsiella pneumoniae B5055;

Targeted disruption of the A2A adenosine receptor reduces in-vitro prostate contractility in mature mice by Katherine T. Gray; Jennifer L. Short; Catherine Ledent; Sab Ventura (151-157).
Prostatic A2A adenosine receptors mediate varied effects. This study aimed to test whether genetic disruption of this receptor affects prostate contractility. Prostates taken from mice which were homozygous (A2AR−/−) and heterozygous (A2AR+/−) for the disrupted A2A adenosine receptor gene and wild-type littermates (A2AR +/+) were mounted in organ baths. Contractile responses to nerve stimulation and noradrenaline were measured in the presence of various pharmacological tools. Electrical field stimulation (0.5 ms pulse duration, 60 V, 0.1–20 Hz) yielded frequency-dependent contractions while exogenous administration of noradrenaline (10 nM–1 mM) or tyramine (1 μM–1 mM) produced concentration-dependent responses. Contractile responses to electrical field stimulation from A2AR−/− and A2AR+/− prostates were reduced when compared to A2A+/+ prostates (P  = 0.013, n  = 33–36). Prazosin (0.3 μM) inhibited electrical field stimulation-induced responses in prostates from A2AR+/+ and A2AR+/− mice (P  ≤ 0.016, n  = 5–7) but not A2AR−/− mice (P  = 0.400, n  = 6). Tetrodotoxin abolished electrical field stimulation-induced responses in all prostates (P  < 0.001, n  = 5–7). NF 449 and ZM 241385 were without effect (P  ≤ 0.421, n  = 4–6). There were no genotype differences in noradrenaline or tyramine concentration–response curves (P  ≥ 0.180, n  = 10–13). Prazosin (0.3 μM) and cocaine (10 μM) attenuated the responses induced by noradrenaline (P  < 0.001, n  = 6–7) and tyramine (P  < 0.001, n  = 5–6), respectively, in all genotypes. Disruption of the A2A adenosine receptor leads to reduced nerve mediated contractile responses of the prostate in mature mice.
Keywords: Smooth muscle; Neuromuscular transmission; α1-adrenoceptor;

Differential modulation of inflammatory pain by a selective estrogen receptor beta agonist by Luis R. Gardell; Lene Hyldtoft; Andria L. Del Tredici; Carsten B. Andersen; Luke C. Fairbairn; Birgitte W. Lund; Magnus Gustafsson; Mark R. Brann; Roger Olsson; Fabrice Piu (158-159).
To understand the contribution of the estrogen receptor beta, the potent and selective agonist ERb-131 was evaluated in animal models of inflammatory pain. In paradigms of acute and persistent inflammatory pain, ERb-131 did not alleviate the nociception induced by either carrageenan or formalin. However, in the chronic complete Freund's adjuvant model, ERb-131 resolved both inflammatory and hyperalgesic components. Thus, ERb-131 is sufficient to alleviate chronic but not acute inflammatory pain states.
Keywords: Inflammation; Pain; Estrogen receptor beta;

The antipsychotics clozapine and olanzapine increase plasma glucose and corticosterone levels in rats: Comparison with aripiprazole, ziprasidone, bifeprunox and F15063 by Marie-Bernadette Assié; Elisabeth Carilla-Durand; Laurent Bardin; Mireille Maraval; Monique Aliaga; Nathalie Malfètes; Michèle Barbara; Adrian Newman-Tancredi (160-166).
Several novel antipsychotics activate serotonin 5-HT1A receptors as well as antagonising dopamine D2/3 receptors. Such a pharmacological profile is associated with a lowered liability to produce extrapyramidal side effects and enhanced efficacy in treating negative and cognitive symptoms of schizophrenia. However, 5-HT1A receptor agonists increase plasma corticosterone and many antipsychotics disturb the regulation of glucose. Here, we compared the influence on plasma glucose and corticosterone of acute treatments with ‘new generation’ antipsychotics which target dopamine D2/3 receptors and 5-HT1A receptors, with that of atypical antipsychotics, and with haloperidol. Olanzapine and clozapine, antipsychotics that are known to produce weight gain and diabetes in humans, both at 10 mg/kg p.o., substantially increased plasma glucose (from 0.8 to 1.7 g/l) at 1 h after administration, an effect that returned to control levels after 4 h. In comparison, F15063 (40 mg/kg p.o.) was without effect at any time point. Olanzapine and clozapine dose-dependently increased plasma glucose concentrations as did SLV313 and SSR181507. Haloperidol and risperidone had modest effects whereas aripiprazole, ziprasidone and bifeprunox, antipsychotics that are not associated with metabolic dysfunction in humans, and F15063 had little or no influence on plasma glucose. The same general pattern of response was found for plasma corticosterone levels. The present data provide the first comparative study of conventional, atypical and ‘new generation’ antipsychotics on glucose and corticosterone levels in rats. A variety of mechanisms likely underlie the hyperglycemia and corticosterone release observed with clozapine and olanzapine, whilst the balance of dopamine D2/3/5-HT1A interaction may contribute to the less favourable impact of SLV313 and SSR181507 compared with that of bifeprunox and F15063.
Keywords: Antipsychotic; Plasma corticosterone; Plasma glucose; F15063; 5-HT1A receptor; Dopamine D2 receptor; (rat);