European Journal of Pharmacology (v.578, #2-3)

Chronic morphine treatment decreases the Cav1.3 subunit of the L-type calcium channel by Victoria L. Haller; Marissa A. Bernstein; Sandra P. Welch (101-107).
Voltage-gated L- and N-type calcium channels (VOCs) are implicated in the activity of morphine, but their contribution to the expression of opioid tolerance remains uncertain. L- and N-type VOCs are heteropentamers of α1, α2δ, β, and γ subunits. The α1 subunit forms both the ion pore and the binding site for ligands. The Cav1.2 and Cav1.3 are the neuronal dihydropyridine (DHP)-sensitive L-type channel subunit types. The Cav2.2 subunit is found in omega conotoxin GVIA-sensitive N-type calcium channels. Cav1.2 VOC gating properties are phosphorylation-dependent with many kinases implicated. We hypothesized that changes in channel subunit structure or phosphorylation state, induced by chronic opioid exposure, may in part explain changes in calcium regulation observed both in vivo and in vitro. Antibodies, specific for the Cav1.2, Cav1.3, and Cav2.2 subunits of VOCs were employed with Western immunoassays to access whether chronic morphine treatment had an effect on receptor protein levels. The L-type channel Cav1.3 protein, but not the Cav1.2 protein or phosphorylation state, significantly decreased upon chronic morphine treatment. The Cav2.2 subunit protein of the N-type channel of VOCs remained unchanged. The Cav1.3 subunit modification may represent one of many potential adaptive changes in tolerance to morphine-induced changes in intracellular calcium.
Keywords: Morphine; Opioid tolerance; Calcium channel; Protein kinase A; Dihydropyridin;

The regulation of mitochondrial respiration by opening of mKCa channels is age-dependent by André Heinen; Adrian Winning; Wolfgang Schlack; Markus W. Hollmann; Benedikt Preckel; Jan Fräβdorf; Nina C. Weber (108-113).
The protective potency of ischemic preconditioning decreases with increasing age. A key step in ischemic preconditioning is the opening of mitochondrial Ca2+ sensitive K+ (mKCa) channels, which causes mild uncoupling of mitochondrial respiration. We hypothesized that aging reduces the effects of mKCa channel opening on mitochondrial respiration.We measured the effects of mKCa channel opener NS1619 (30 μM) on mitochondrial respiration in isolated heart mitochondria from young (2–3 months) and old (22–26 months) Wistar rats. Oxygen consumption was monitored online after addition of 250 μM ADP (state 3 respiration), and after complete phosphorylation of ADP to ATP (state 4 respiration) in the presence or absence of the mKCa channel blocker paxilline (5 μM). The respiratory control index (RCI) was calculated as state 3/state 4.In mitochondria from young rats, NS1619 increased state 4 respiration by 11.9 ± 4.1% (mean ± S.E.M.), decreased state 3 respiration by 7.6 ± 2.5%, and reduced the RCI from 2.6 ± 0.03 (control) to 2.1 ± 0.06 (all P  < 0.05, n  = 12 for all groups). Paxilline blocked the effect of NS1619 on state 4 respiration (0.7 ± 2.8%), but did not affect the decrease in state 3 respiration; paxilline blunted the decrease of RCI. In mitochondria from old rats, NS1619 had neither effect on state 4 (0.4 ± 1.6%), and state 3 respiration (− 7.4 ± 1.5%), nor on RCI (3.0 ± 0.13 vs. 3.2 ± 0.11, n  = 12).Increasing age reduced the effects of mKCa opening on mitochondrial respiration. This might be one underlying reason of the decreased protective potency of ischemic preconditioning in the aged myocardium.
Keywords: Mitochondria; Respiration; mKCa channel; Aging;

Strong evidence suggests that antidepressants work by induction of neuroplastic changes mediated through regulation of brain-derived neurotrophic factor (BDNF). This study was undertaken to investigate the time-course of the effect of three antidepressants; fluoxetine, imipramine and venlafaxine, which differentially affect monoamine reuptake, on BDNF mRNA expression in the hippocampus. The consequences of increased BDNF in the hippocampus are still indefinite. Here, we also determined the effects on the expression of two other genes (synaptophysin and growth-associated protein-43 (GAP-43)) known to be involved in synapse formation and axonal growth and likely regulated by BDNF. The effects were determined in rats after sub-chronic (7 days) and chronic (14 and 21 days) treatment using semi-quantitative in situ hybridisation. BDNF mRNA levels in the dentate gyrus (DG) were increased after treatment with venlafaxine (7, 14 and 21 days) and imipramine (14 and 21 days), but not after treatment with fluoxetine, indicating that stimulation of BDNF mRNA expression is dependent on the pharmacological profile and on the time-course of drug treatment. A transient increase in synaptophysin mRNA was observed after treatment with venlafaxine and fluoxetine whereas imipramine had no effect. In the CA3 region a reduction of GAP-43 mRNA was observed after treatment with imipramine (21 days) and fluoxetine (7 and 14 days). These results suggest that venlafaxine and imipramine, but not fluoxetine, induce neuroplastic effects in the hippocampus through stimulation of BDNF mRNA expression, and that the effect on BDNF is not directly translated into regulation of synaptophysin and GAP-43 mRNA.
Keywords: Antidepressant; Brain-derived neurotrophic factor; Growth-associated protein-43; Synaptophysin; Hippocampus; Neuroplasticity;

The ability of the sigma1 receptor to interact with a huge range of drug structural classes coupled with its wide distribution in the body has contributed to it being implicated as a possible therapeutic target for a broad array of disorders ranging from substance abuse to depression to Alzheimer's disease. Surprisingly, the reported affinity values for some sigma1 receptor ligands vary more than 50-fold. The potential of the sigma1 receptor as a pharmacotherapeutic target prompted us to develop an unambiguous assay system for measuring the affinity of ligands to the cloned human sigma1 receptor. In the course of characterizing this system and determining the true affinity values for almost three dozen compounds, it was discovered that some dopamine D4 receptor selective compounds bind sigma1 receptors with high affinity. A systematic analysis of haloperidol-like compounds revealed a clear structure–affinity relationship amongst clinically relevant butyrophenones. The antidepressant fluvoxamine, the drug of abuse methamphetamine, and the neurosteroid progesterone were amongst the many ligands whose interactions with the sigma1 receptor were confirmed with our screening assay.
Keywords: Antipsychotics; Psychotomimetics; Psychostimulants; N-methyl-d-aspartate receptor; Opioid receptor; Neuroprotection;

The endocannabinoid system is involved in memory, cognition, and pain perception by the presynaptic cannabinoid CB1 receptor, which is expressed at high levels in many brain regions. Functional studies have shown that activation of cannabinoid CB1 receptors inhibits the synaptic release of many neurotransmitters such as γ-aminobutyric acid, glutamate, acetylcholine and monoamines. Monoamines, however, are known not only to be released from and taken back up at nerve terminals but also at extrasynaptic axonal and somatodendritic sites. Here we present immunocytochemical data documenting cannabinoid CB1 receptor expression on neurite extensions and over cell bodies of serotonergic and dopaminergic neurons.
Keywords: Cannabinoid CB1 receptor; Serotonin; Dopamine; Neuron;

Affinity of cyamemazine metabolites for serotonin, histamine and dopamine receptor subtypes by Amine Benyamina; Christophe Arbus; Philippe Nuss; Ricardo P. Garay; Gervais Neliat; Ahcène Hameg (142-147).
Animal and human pharmacological studies indicate that the antipsychotic action of cyamemazine results from blockade of dopamine D2 receptors, its anxiolytic properties from serotonin 5-HT2C receptor antagonism and the low incidence of extrapyramidal side effects from a potent 5-HT2A receptor antagonistic action. Cyamemazine is metabolized in monodesmethyl cyamemazine and cyamemazine sulfoxide, which are not known for their affinities for serotonin, dopamine and other brain receptor types considered to mediate central nervous systems effects of drugs. Hence, metabolite affinities were determined in human recombinant receptors expressed in CHO cells (hD2 and hD4.4 receptors, h5-HT1A, h5-HT2A, h5-HT2C and h5-HT7 receptors and hM1, hM2 and hM3 receptors) and HEK-293 cells (h5-HT3 receptors) or natively present in rat cerebral cortex (non-specific α1- and α2-adrenoceptors, GABAA and GABAB receptors) and guinea pig cerebellum (H1 central histamine receptors) membranes. Monodesmethyl cyamemazine showed a neurotransmitter receptor profile similar to that of its parent compound cyamemazine, i.e.: high affinity for h5-HT2A receptors (K i  = 1.5 nM), h5-HT2C receptors (K i  = 12 nM) and hD2 receptors (K i  = 12 nM). Cyamemazine sulfoxide showed high affinity for h5-HT2A receptors (K i  = 39 nM) and histamine H1 receptors (K i  = 15 nM) and a reduced affinity for D2 and 5-HT2C receptors. Therefore, monodesmethyl cyamemazine can contribute to enhance and prolong the therapeutic actions of cyamemazine. Further investigation is required to see if the high affinities of cyamemazine sulfoxide for H1 and 5-HT2A receptors are of therapeutic benefit against sleep onset insomnia and/or sleep maintenance insomnia respectively.
Keywords: Cyamemazine; Affinity; Radioligand binding; Recombinant human receptor; Guinea pig cerebellum; Rat cerebral receptors;

Interactions of attention-deficit/hyperactivity disorder therapeutic agents with the efflux transporter P-glycoprotein by Hao-Jie Zhu; Jun-Sheng Wang; Jennifer L. Donovan; Yan Jiang; Bryan B. Gibson; C. Lindsay DeVane; John S. Markowitz (148-158).
The objective of this study was to assess the potential interactions of the drug transporter P-glycoprotein with attention-deficit/hyperactivity disorder (ADHD) therapeutic agents atomoxetine — and the individual isomers of methylphenidate, amphetamine, and modafinil utilizing established in vitro assay. An initial ATPase assay indicated that both d- and l-methylphenidate have weak affinity for P-glycoprotein. The intracellular accumulation of P-glycoprotein substrates doxorubicin and rhodamine123 in the P-glycoprotein overexpressing cell line LLC-PK1/MDR1 was determined to evaluate potential inhibitory effects on P-glycoprotein. The results demonstrated that all compounds, except both modafinil isomers, significantly increased doxorubicin and rhodamine123 accumulation in LLC-PK1/MDR1 cells at higher concentrations. To investigate the P-glycoprotein substrate properties, the intracellular concentrations of the tested compounds in LLC-PK1/MDR1 and P-glycoprotein negative LLC-PK1 cells were measured in the presence and absence of the P-glycoprotein inhibitor PSC833. The results indicate that the accumulation of d-methylphenidate in LLC-PK1 cells was 32.0% higher than in LLC-PK1/MDR1 cells. Additionally, coadministration of PSC833 leads to 52.9% and 45.6% increases in d-modafinil and l-modafinil accumulation, respectively, in LLC-PK1/MDR1 cells. Further studies demonstrated that l-modafinil transport across LLC-PK1/MDR1 cell monolayers in the basolateral-to-apical (B–A) direction was significantly higher than in the apical-to-basolateral (A–B) direction. PSC833 treatment significantly decreased the transport of l-modafinil in B–A direction. In conclusion, our results suggest that all tested agents with the exception of modafinil isomers are relatively weak P-glycoprotein inhibitors. Furthermore, P-glycoprotein may play a minor role in the transport of d-methylphenidate, d-modafinil, and l-modafinil.
Keywords: P-glycoprotein; Methylphenidate; Amphetamine; Atomoxetine; Modafinil;

Antioxidative and hypocholesterolemic activities of water-soluble puerarin glycosides in HepG2 cells and in C57 BL/6J mice by Mi Ja Chung; Nak-Ju Sung; Cheon-Seok Park; Dong-Keon Kweon; Alberto Mantovani; Tae-Wha Moon; Sung-Joon Lee; Kwan-Hwa Park (159-170).
Puerarin is an isoflavone derived from Kudzu roots and has antioxidant and hypocholesterolemic effects; however, its insolubility often limits its biological availability in vivo. Using a novel transglycosylation process, the solubility of puerarin glycosides was increased > 100-fold, but it was not known whether these modified puerarin glycosides maintained biological activities. We found that water-soluble puerarin glycosides fully maintained antioxidant activities compared with puerarin assessed by radical scavenging activity, reducing power assay, superoxide dismutase activity, and non-site-specific hydroxyl radical scavenging activity. Both puerarin and its glycosides also significantly reduced low-density lipoprotein (LDL) oxidation. Mice fed with puerarin glycosides (0.1% w/w) showed significantly reduced plasma total cholesterol levels, thus, we further investigated their hypocholesterolemic mechanisms by assessing several key gene expressions both in vitro and in vivo. Puerarin and its glycosides induced multiple changes in hepatic cholesterol metabolism. The LDL receptor promoter activity was increased dose-dependently in puerarin glycosides-treated HepG2 cells. Accordingly, the expression of LDL receptor mRNA and protein were also significantly increased in HepG2 cells and mouse livers. The transcription and translation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase were down-regulated both in vitro and in vivo. The cholesterol 7α-hydroxylase (CYP7A1) mRNA levels were not affected in vitro but significantly up-regulated in the mouse livers. Collectively, our results show that puerarin and its glycosides are biologically fully active isoflavone and have antioxidant and hypocholesterolemic effects in HepG2 cells and in C57BL/6J mice. In the livers, hypocholesterolemic effects of puerarin glycoside may be achieved by multiple mechanisms including increasing LDL uptake, reducing cholesterol biosynthesis, and possibly enhancing cholesterol degradation.
Keywords: Puerarin glycoside; Low-density lipoprotein receptor; HMG-CoA reductase; Antioxidant;

Riluzole enhances the activity of glutamate transporters GLAST, GLT1 and EAAC1 by Elena Fumagalli; Marcella Funicello; Thomas Rauen; Marco Gobbi; Tiziana Mennini (171-176).
Riluzole exerts a neuroprotective effect through different mechanisms, including action on glutamatergic transmission. We investigated whether this drug affects glutamate transporter-mediated uptake, using clonal cell lines stably expressing the rat glutamate transporters GLAST, GLT1 or EAAC1. We found that riluzole significantly increased glutamate uptake in a dose-dependent manner; kinetic analysis indicated that the apparent affinity of glutamate for the transporters was significantly increased, with similar effects in the three cell lines. This may facilitate the buffering of excessive extracellular glutamate under pathological conditions suggesting that riluzole's neuroprotective action might be partly mediated by its activating effect on glutamate uptake.
Keywords: Riluzole; Glutamate transporter; Uptake; Cell lines; Synaptosomes;

The role of gonadal hormones on opioid receptor protein density in arthritic rats by Matthew C. Kren; Victoria L. Haller; Sandra P. Welch (177-184).
The purpose of this study was to evaluate the effects of the gonadal hormones on the opioid receptor protein levels of Freund's adjuvant-treated (arthritic) male and female Lewis rats. Following a paw pressure nociception assay, the midbrain and spinal cord tissues were collected for comparison of mu, delta, and kappa opioid receptor protein levels. The effects of Freund's adjuvant-induced hyperalgesia resulted in significantly decreased nociception thresholds in both males and females, compared to vehicle treated animals in the paw pressure test. It was hypothesized that the presence or lack thereof of gonadal hormones would alter nociception, an effect temporally correlated with a change in opioid receptor protein expression. Nociceptive thresholds were altered by arthritis in both sexes, but not further altered by gonadal changes in males. A small, but significant increase in threshold was shown in ovariectomized females. In spite of the small gonadal-induced changes in the nociceptive threshold sensitivity to pressure, significant changes in the plasticity of the opioid system were observed. There was a significant increase in kappa opioid receptor protein levels in the spinal cord of arthritic ovariectomized females. Mu opioid receptor and kappa opioid receptor protein levels in the spinal cord tissue of non-arthritic male rats were significantly higher than in arthritic rats, a difference eliminated by gonadectomy. Gonadectomy produced similar results in the mu opioid receptor protein level in the male midbrain tissue as well. Sex differences were observed in both the mu and kappa opioid receptor protein levels. The spinal cord tissue of male rats, regardless of the presence of gonads or arthritis displayed significantly greater levels of mu opioid receptor protein levels than females. The removal of gonadal hormones appears to have opposite effects in males and females in terms of opioid receptor proteins, but not nociception as quantified by the paw pressure test. The role of changes in the plasticity of the opioid systems in response to arthritis or gonadal hormones remains to be elucidated.
Keywords: Gonadal hormones; Arthritis; Antinociception; Opioids;

Corticotropin-releasing factor (CRF) is a neurohormone that mediates stress, anxiety, and affects serotonergic activity. Studies have shown that CRF has dose-dependent opposing effects on serotonergic activity. This effect has been hypothesized to be differentially mediated by CRF1 and CRF2 receptors in the dorsal raphé nucleus. We directly tested this hypothesis by using in vivo microdialysis to determine the effects of CRF and CRF receptor antagonists in the dorsal raphé nucleus on serotonin (5-HT) release in the nucleus accumbens, a brain region implicated in the neuropathology of stress-related psychiatric disorders. Male urethane-anesthetized rats were implanted with a microdialysis probe into the nucleus accumbens, and CRF (0, 100 or 500 ng) was infused into the dorsal raphé. Infusion of CRF into the dorsal raphé nucleus had dose-dependent opposite effects, with 100 ng of CRF significantly decreasing 5-HT levels in the nucleus accumbens and 500 ng CRF significantly increasing accumbal 5-HT levels. In subsequent experiments, the raphé was pre-treated with the CRF1 receptor antagonist antalarmin (0.25 μg) or the CRF2 receptor antagonist antisauvagine-30 (ASV-30; 2 μg) prior to CRF infusion. Antagonism of CRF1 receptors in the dorsal raphé nucleus abolished the decrease in accumbal 5-HT levels elicited by 100 ng CRF, and CRF2 receptor antagonism in the raphé blocked the increase in accumbal 5-HT levels elicited by 500 ng CRF. These results suggest that the opposing effects of dorsal raphé CRF on 5-HT release in the nucleus accumbens are dependent on differential activation of CRF1 and CRF2 receptors in the dorsal raphé nucleus.
Keywords: Microdialysis; Antalarmin; Antisauvigine-30; CRF receptor; Stress;

Nobiletin, a citrus flavonoid with neurotrophic action, augments protein kinase A-mediated phosphorylation of the AMPA receptor subunit, GluR1, and the postsynaptic receptor response to glutamate in murine hippocampus by Kentaro Matsuzaki; Kenichi Miyazaki; Seiichiro Sakai; Hiromu Yawo; Norihito Nakata; Shigeki Moriguchi; Kohji Fukunaga; Akihito Yokosuka; Yutaka Sashida; Yoshihiro Mimaki; Tohru Yamakuni; Yasushi Ohizumi (194-200).
Nobiletin isolated from citrus peels prevents bulbectomy- and amyloid-β protein-induced memory impairment in rodents. In the present study, using combined methods of biochemistry and electrophysiology, we examined the effects of nobiletin on phosphorylation of GluR1 receptor, the subunit of α-amino-3-hydroxy-5-methyl-d-aspartate (AMPA) receptors, and the receptor-mediated synaptic transmission in the hippocampus, a region implicated in memory formation, in culture and/or in slices. Western blot analysis showed that nobiletin-stimulated phosphorylation of multiple protein kinase A (PKA) substrates at 10 min following the treatment in cultured hippocampal neurons. In the cultured neurons, this natural compound also increased not only PKA activity, but also phosphorylation of GluR1 receptor at a PKA phosphorylation site, Ser 845, which has been demonstrated to be critical for synaptic plasticity, including enhancement of postsynaptic glutamate response, and important for spatial memory in vivo. The increased phosphorylation of GluR1 receptor at Ser 845 was abolished by H89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride), the PKA inhibitor, but not U0126 (1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene), the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, in the cultured neurons. An increment of the phosphorylation of GluR1 receptor at Ser 845 was induced by nobiletin in the hippocampal slices as well. Furthermore, our electrophysiological analysis showed that nobiletin potentiated the AMPA receptor-mediated synaptic transmission at Schaffer collateral-CA1 pyramidal cell synapses in the hippocampal slices. This potentiation induced by the natural compound was not accompanied by the changes in paired-pulse ratio, and partially occluded the long-term potentiation, indicating the possible involvement of the postsynaptic mechanism. These findings suggest that nobiletin probably up-regulates synaptic transmission via the postsynaptic AMPA receptors at least partially by stimulation of PKA-mediated phosphorylation of GluR1 receptor in the hippocampus.
Keywords: Nobiletin; Postsynaptic glutamate sensitivity; AMPA receptor; PKA; Hippocampal neurons;

Icilin induces a hyperthermia in rats that is dependent on nitric oxide production and NMDA receptor activation by Zhe Ding; Teresa Gomez; Jennifer L. Werkheiser; Alan Cowan; Scott M. Rawls (201-208).
Icilin (AG-3-5) is a cold-inducing agent that activates the transient receptor potential channels TRPM8 and TRPA1. Both channels are members of the transient receptor potential (TRP) superfamily of ion channels and are activated by cold. Despite the key role of cold-activated TRPM8 and TRPA1 channels in temperature sensation and other physiological processes, the significance of these channels in thermoregulation in conscious animals is poorly understood. Therefore, in the present study we investigated the effects of icilin on body temperature in rats and tested the hypothesis that cold-activated TRP channel activation by icilin causes a hyperthermia which requires nitric oxide (NO) production and NMDA receptor stimulation. Our experiments revealed that icilin (2.5, 5, 7.5 and 10 mg/kg, i.m.) elicits a dose-related hyperthermia that is rapid in onset and of long duration. Pretreating rats with N G-nitro-l-arginine methyl ester hydrochloride (l-NAME) (10, 25 and 50 mg/kg, i.p.), a non-selective NO synthase inhibitor, attenuated the hyperthermia associated with icilin (7.5 mg/kg, i.m.). Pretreatment with (−)-6-[phosphonomethyl-1,2,3,4,4a,5,6,7,8,8a-decahydro-isoquinoline-2-carboxylate] (LY 235959) (0.25, 0.5 and 1 mg/kg, i.p.), a selective NMDA receptor antagonist, also attenuated the icilin-evoked hyperthermia. The administration of icilin (5 and 100 μg) into the lateral cerebroventricle of rats did not affect body temperature, thus indicating a peripheral site of action. These results indicate that icilin, a TRPM8/TRPA1 agonist, produces a dose-related hyperthermia in rats which requires both NO production and NMDA receptor activation.
Keywords: Icilin; TRPM8; Nitric oxide; l-NAME; NMDA; LY 235959; Hyperthermia;

Modafinil is a novel wake-promoting drug used for the treatment of narcolepsy, the mechanism of action of which remains unclear. Previous studies have shown that modafinil produces a different pattern of c-Fos activation in the brain to the classical stimulants amphetamine and methylphenidate. Modafinil, given i.p. to urethane-anesthetized rats, is associated with an increase in histamine release from the anterior hypothalamus, indicating that its behavioral actions may involve histaminergic systems. In the present study, the effects of modafinil on histamine release using in vivo microdialysis and locomotor activity in freely moving rats were examined, and compared with those of the classical psychostimulant methylphenidate. Modafinil (75 and 150 mg/kg, i.p.) increased both histamine release and locomotor activity, significantly. Methylphenidate (3 mg/kg, i.p.) also increased locomotor activity to the same extent as modafinil (150 mg/kg, i.p.) without stimulating histamine release. Depletion of neuronal histamine using α-fluoromethylhistidine abolished the effect of modafinil on locomotor activity in mice but had no effect on methylphenidate-induced locomotion. Examination of the effects of modafinil and methylphenidate on locomotor activity in the dark phase at doses that produced comparable effects in the light phase showed that the effect of modafinil in the dark phase was less than that of methylphenidate, a possible indication that modafinil-induced locomotor activity may be partly related to its wake-promoting actions. These findings suggest that the locomotor effects of modafinil but not of methylphenidate, involve the central histaminergic systems.
Keywords: Modafinil; Methylphenidate; The histaminergic system; Locomotor activity; Microdialysis;

Schizophrenia is a serious psychiatric disorder that is most frequently treated with the administration of antipsychotics. Although onset of schizophrenia typically occurs in late adolescence, the majority of preclinical research on the behavioral effects of antipsychotics and their mechanism(s) of action has been conducted on adult male animals. In this study, the acute effects of haloperidol (0.03–0.3 mg/kg, i.p.) and clozapine (1–10 mg/kg, i.p.) on locomotor activity were examined in juvenile [postnatal day 22 (PN22)], adolescent (PN40), and adult (> PN70) rats of both sexes. Subsequently, in order to determine whether tolerance to the activity suppressive effects of these drugs would occur in adolescents, PN40 rats were dosed and assessed for an additional nine days. While all groups exhibited some degree of suppression following acute administration of both drugs, juvenile rats were considerably more sensitive to this effect. With sub-chronic administration during late adolescent development (PN40–PN49), tolerance failed to develop. These results emphasize the importance of age in pharmacological characterization of antipsychotics and suggest that pre-adolescents may have enhanced sensitivity to the motor effects of these drugs. Further, they suggest that, similar to adults, older adolescents may not develop tolerance to the activity suppression induced by these two antipsychotics.
Keywords: Adolescence; Clozapine; Haloperidol; Locomotion; Sex differences; Tolerance;

Aripiprazole is an atypical antipsychotic that acts as a partial agonist at the dopamine D2 receptor. It has been mainly investigated in dopamine-based models of schizophrenia, while its effects on glutamate-based paradigms have remained to be further characterized. Due to its unique mechanism of action, aripiprazole has also been considered as a replacement medication for psychostimulant abuse. Thus, in the present study we tested the hypothesis that aripiprazole would prevent the motor hyperactivity induced by psychostimulant and psychotomimetic drugs that act either by dopaminergic or glutamatergic mechanisms. Male Swiss mice received injections of aripiprazole (0.1–1 mg/kg) followed by drugs that enhance the dopamine-mediated neurotransmission, amphetamine (3 mg/kg) or cocaine (5 mg/kg), or by glutamate NMDA-receptor antagonists, ketamine (60 mg/kg) or MK-801 (0.4 mg/kg). Independent groups also received aripiprazole (0.1–1 mg/kg) or haloperidol (0.5 mg/kg) and were tested for catalepsy. All doses of aripiprazole were effective in preventing the motor stimulant effects of amphetamine and cocaine. Moreover, the higher dose also prevented the effects of ketamine and MK-801. The present study reports the effects of aripiprazole in dopaminergic and glutamatergic models predictive of antipsychotic activity, suggesting that both may be useful for screening novel partial agonists with antipsychotic activity. It also shows that aripiprazole may prevent the acute effects of psychostimulant drugs without significant motor impairment.
Keywords: Aripiprazole; Antipsychotics; Psychostimulants; Schizophrenia; Dopamine; Glutamate;

Taurine and ethanol interactions: Behavioral effects in mice by Brett C. Ginsburg; Richard J. Lamb (228-237).
Taurine is an abundant amino acid in the brain that shares pharmacological effects and similar potency with ethanol. Recently, taurine-containing beverages have been reported to enhance the euphoric effects of ethanol, though the extent of this effect and the role of taurine remain speculative. The present study was designed to explore interactions between taurine and ethanol on several behaviors including locomotion, ataxia, and loss of righting. Two strains of mice, C57BL/6J and DBA/2J mice, were used to examine potential strain differences. In the first experiment, effects of various doses of taurine (0.3–3.0 g/kg), ethanol (1.0–4.2 g/kg), or taurine in combination with ethanol were assessed in a within-subjects design. Although taurine did not appear to alter effects of ethanol on any measure in either strain, the development of tolerance to locomotor effects and sensitization to ataxic effects of ethanol in DBA/2J mice complicated interpretation of these results. In a second experiment, drug-naïve mice were assigned to one of four treatment groups: saline + saline, saline + ethanol (1.78 g/kg), taurine (1.78 g/kg) + saline, or ethanol + taurine. In this experiment, taurine pretreatment significantly attenuated the locomotor-stimulating effect of ethanol in both strains (but to a greater extent in C57BL/6J mice) and appeared to reduce the ataxic effects of ethanol in C57BL/6J mice. In conclusion, the interaction between taurine and ethanol is subtle. Further, results are inconsistent with the notion that taurine plays a major role in the locomotor, ataxic, or loss of righting effects of ethanol.
Keywords: Sleep-time; Alcohol; Locomotor; (Mouse);

The effect of taurolidine on experimental thrombus formation by Levent Kaptanoglu; Hasan Fehmi Kucuk; Elif Colak; Necmi Kurt; Sadık Mehmet Bingul; Huseyin Akyol; Oguzhan Aziz Torlak; Fatma Yazici (238-241).
Venous thrombosis can be the source of emboli, a significant health risk encountered throughout surgical and medical clinics. Taurolidine is an antimicrobial agent used to prevent intraabdominal adhesion formation and sepsis in experimental and clinical trials. The aim of this study is to evaluate effect of taurolidine on experimental thrombus formation and make a comparison with low-molecular weight heparin. Four groups of ten Wistar-Albino rats (300–350 g) were used; with the first and second groups each being administered 10 and 20 mg of taurolidine, the third group low-molecular weight heparin and the fourth group saline solution (control group) respectively. Experimental thrombus formation was performed in rats in the area of the abdominal inferior vena cava by using a combination of stasis and hypercoagulability described by Wessler et al. [Wessler, S., Reimer, S.M., Sheps, M.C., 1959. Biologic assay of a thrombosis inducing activity in human serum. J. Appl. Physiol. 14:943–946.]. Thrombocyte count, the weight of thrombus, prothrombin time and activated partial thromboplastin time and activities of coagulation factors were measured and compared across groups. Thrombus weights in the taurolidine treated groups were lower than the control group and greater than the low-molecular weight heparin treated group. Taurolidine was found to decrease activities of coagulation factors V, VIII, IX, XI and XII. Taurolidine showed no effect on activated partial thromboplastin time and prothrombin time values; however, it decreased thrombus weight, but not as much as low-molecular weight heparin. The cause of these findings in our study may be related to the minimized effect of taurolidine on factor II, VII, and X activities. These effects likely render the agent ineffective in the prevention of venous thrombosis. Taurolidine was found to be less effective than low-molecular weight heparin in prevention of thrombus formation.
Keywords: Taurolidine; Low-molecular weight heparin; Thrombus formation;

The effect of urocortin II administration on the coronary circulation and cardiac function in the anaesthetized pig is nitric-oxide-dependent by Elena Grossini; Claudio Molinari; David A.S.G. Mary; Paolo Marino; Giovanni Vacca (242-248).
We planned to determine the primary effects and mechanisms of urocortin II, a member of the corticotrophin-releasing factor (CRF) family highly expressed in the cardiovascular system, on coronary blood flow and myocardial function in vivo. Urocortin II was infused into the left anterior descending coronary artery in 25 anaesthetized pigs whilst measuring haemodynamic variables, coronary blood flow, ventricular dP/dt max cardiac output and percentage of segmental shortening. This infusion was repeated after blockade of the autonomic nervous system, nitric-oxide synthase (NOS) or subtype 2 of the CRF receptors. In all experiments changes in heart rate and aortic blood pressure were prevented. Intra-coronary urocortin II increased, within 60 s, coronary blood flow (15 ± 3.2%, P  < 0.05), dP/dt max (12.7 ± 2.6%, P  < 0.05), cardiac output (16 ±2.3%, P  < 0.05) and percentage of segmental shortening (19.8 ± 3.8%, P  < 0.05). Blockade of NOS abolished only the coronary effects whereas blockade of subtype 2 of the CRF receptors abolished all cardiac and coronary effects. It was shown for the first time that urocortin II administration primarily increases coronary blood flow and myocardial function through the release of nitric oxide and activation of subtype 2 of the CRF receptors in the anaesthetized pig. This provides a mechanism through which a local increase of urocortin II levels can help improve a compromised cardiovascular function.
Keywords: Coronary blood flow; Corticotrophin releasing factor; Myocardial contractility; Nitric oxide; Urocortin II;

Effect of rosuvastatin treatment on plasma visfatin levels in patients with primary hyperlipidemia by Michael S. Kostapanos; Christos S. Derdemezis; Theodosios D. Filippatos; Haralampos J. Milionis; Dimitrios N. Kiortsis; Alexandros D. Tselepis; Moses S. Elisaf (249-252).
Visfatin is a novel adipokine involved in the process of atherosclerosis. We assessed the effect of rosuvastatin on plasma visfatin levels in patients with primary hyperlipidemia. Eighty hyperlipidemic patients without evidence of cardiovascular disease were randomized to receive either rosuvastatin 10 mg/day or therapeutic lifestyle changes intervention. Plasma visfatin levels were determined at baseline and after 12-weeks post-randomization. Rosuvastatin induced a significant decrease in plasma visfatin levels (17.1 ± 2.1 versus 15.5 ± 2.0 ng/ml, P  = 0.03). This effect correlated with baseline visfatin levels (r  = 0.51, P  < 0.01) and was independent of any lipid-lowering actions of rosuvastatin.
Keywords: Adipokine; Hyperlipidemia; Rosuvastatin; Visfatin;

Mechanisms of the dilator action of cryptotanshinone on rat coronary artery by Francis F.Y. Lam; John H.K. Yeung; Kam M. Chan; Penelope M.Y. Or (253-260).
In this study, we have investigated the actions of cryptotanshinone, an active, lipophilic component of the medicinal herb danshen (Salvia miltiorrhiza), on rat isolated coronary artery rings precontracted with 1 μM 5-hydroxytryptamine (5-HT) and its action compared to the ethanol-extractable fraction of the herb. Extraction of the ethanol-soluble fraction from danshen provided a yield of 1%. The amount of cryptotanshinone determined in this ethanol extract was 3.682%, and it was 6 times more potent than the extract in relaxing 5-HT-precontracted coronary artery rings; IC50 values were 2.65 ± 0.15 μg/ml and 15.82 ± 1.07 μg/ml, respectively. Involvement of endothelium-dependant mechanisms in their dilator effects were investigated by pretreatment of the artery rings with a cyclooxygenase inhibitor flurbiprofen (10 μM), a nitric oxide synthase inhibitor N G-nitro-l-arginine methyl ester (L-NAME, 100 μM), a muscarinic receptor antagonist atropine (100 nM), and by mechanical removal of the endothelium; none of these procedures produced a significant change on their dilator actions. Involvement of endothelium-independent mechanisms was investigated in endothelium-denuded artery rings pretreated with a β-adrenoceptor antagonist propranolol (100 nM), an adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536, 100 μM), a guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM), and a potassium channel inhibitor tetraethylammonium (TEA, 100 mM); these also produced no change on their dilator actions. The possible involvement of Ca2+ channels was investigated in artery rings incubated with Ca2+-free buffer and primed with 1 μM 5-HT for 5 min prior to adding CaCl2 to elicit contraction. The danshen ethanol extract (100 μg/ml) abolished the CaCl2-induced vasoconstriction, whereas, cryptotanshinone (30 μg/ml) produced 59% inhibition. These findings suggest their vasorelaxant effects are independent of pathways mediated by the endothelium, muscarinic receptors, β-adrenoceptors, adenylyl cyclase, and guanylyl cyclase, whereas, inhibition of Ca2+ influx in the vascular smooth muscle cells is important for their vasodilator actions. The high vasodilator potency and the quantity of salvianolic acid B contained in danshen ethanolic extract suggest it is an important constituent in this medicinal herb.
Keywords: Cryptotanshinone; Danshen; Calcium channel; Coronary artery; (Salvia miltiorrhiza);

Negative inotropic effect of selective AT2 receptor stimulation and its modulation by the endocardial endothelium by Paulo Castro-Chaves; Susana Soares; Ricardo Fontes-Carvalho; Adelino F. Leite-Moreira (261-269).
Angiotensin II is an octapeptide whose effects are mediated by two types of receptors. AT1 receptors are responsible for the vasoconstrictor, positive inotropic and growth promoting properties, while AT2 receptors have been linked to vasodilator and anti-mitogenic properties. In this study we investigated the effects of selective AT2 receptor stimulation on myocardial contractility and lusitropy. Effects of selective AT2 receptor activation were evaluated in rabbit right papillary muscles (n  = 96) by adding increasing concentrations of H-9395, an AT2 receptor agonist, alone or in presence of a selective AT1 receptor antagonist (ZD-7155), or alternatively, by adding increasing concentrations of angiotensin II in presence of ZD-7155. In the latter conditions, selective AT2 receptor activation was also performed in presence of NG-nitro-l-Arginine, indomethacin, proadifen, hydroxocobalamin, apamin plus charybdotoxin, Hoe-140 or PD-123,319, as well as, after endocardial endothelium removal. Selective AT2 stimulation induced a negative inotropic and lusitropic effect in the first three protocols. This effect was completely abolished after selective removal of the endocardial endothelium and blunted in presence of Hoe-140, hydroxocobalamin, apamin plus charybdotoxin and PD-123,319, but maintained in presence of NG-nitro-l-Arginine, indomethacin or proadifen. Selective AT2 receptor stimulation induces a negative inotropic and lusitropic effect, which is modulated by endocardial endothelium and mediated by bradykinin B2 receptors through NO release and calcium dependent potassium channels activation. Such findings may help to better understand the therapeutic effects of selective AT1 antagonists, which are increasingly used for treating cardiovascular diseases.
Keywords: Angiotensin II; AT2 receptor; Endocardial endothelium; Inotropism;

Long-term amiodarone treatment causes cardioselective hypothyroid-like alteration in gene expression profile by Rong-qian Shi; Jong-Kook Lee; Yoshitaka Hayashi; Yoko Takeuchi; Fukushi Kambe; Sugiko Futaki; Hisao Seo; Yoshiharu Murata; Itsuo Kodama (270-278).
The long-term cardiac effects of amiodarone resemble many aspects of hypothyroidism. The anti-arrhythmic potential of amiodarone may therefore be the result of a drug-induced, local hypothyroid-like condition. To investigate this controversial issue, we compared gene expression profiles in the hearts of rats treated with amiodarone with those of rats with hypothyroidism. Wistar male rats were assigned to 3 groups (n  = 6–8): Control, systemic hypothyroidism (Hypothyroidism) and amiodarone treatment (Amiodarone, 150 mg/kg/day, p.o., 4 weeks). Electrocardiogram (ECG) recordings, gene profiling by DNA microarray and Northern blotting were carried out. Amiodarone, like Hypothyroidism, caused significant prolongation of RR and QT intervals in ECGs. Microarray analysis of 8435 genes in the left ventricular myocardium revealed a significant similarity in expression profiles between Hypothyroidism and Amiodarone (R  = 0.63, p  < 0.00001). The gene expression profiles of Hypothyroidism and Amiodarone showed closer correlation when top 100 up-regulated and 100 down-regulated genes in Hypothyroidism (total 200 genes) were analyzed (R  = 0.78, p  < 0.00001). Northern blots of left ventricular myocardium showed a parallel decrease in mRNAs for myosin heavy chain (MHC)-alpha and a parallel increase for myosin heavy chain (MHC)-beta in Hypothyroidism and Amiodarone. In the liver and pituitary, in contrast, Northern blots showed quite different changes in the transcripts of the representative T3-responsive genes in the Hypothyroidism and Amiodarone. In conclusion, long-term treatment with amiodarone causes cardioselective hypothyroid-like alterations in gene expression profiles. The potent anti-arrhythmic activity of amiodarone may be attributable, in part at least, to this unique transcriptional remodeling.
Keywords: Amiodarone; Thyroid hormone; ECG; DNA microarray analysis; Gene expression profile;

Glucagon like peptide-2 induces intestinal restitution through VEGF release from subepithelial myofibroblasts by Kerem Bulut; Christian Pennartz; Peter Felderbauer; Juris J. Meier; Matthias Banasch; Daniel Bulut; Frank Schmitz; Wolfgang E. Schmidt; Peter Hoffmann (279-285).
Glucagon like peptide-2 (GLP-2) exerts intestinotrophic actions, but the underlying mechanisms are still a matter of debate. Recent studies demonstrated the expression of the GLP-2 receptor on fibroblasts located in the subepithelial tissue, where it might induce the release of growth factors such as keratinocyte growth factor (KGF) or vascular endothelial growth factor (VEGF). Therefore, in the present studies we sought to elucidate the downstream mechanisms involved in improved intestinal adaptation by GLP-2. Human colonic fibroblasts (CCD-18Co), human colonic cancer cells (Caco-2 cells) and rat ileum IEC-18 cells were used. GLP-2 receptor mRNA expression was determined using real time RT-PCR. Conditioned media from CCD-18Co cells were obtained following incubation with GLP-2 (50–250 nM) for 24 h. Cell viability was assessed by a 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT)-assay, and wound healing was determined with an established migration-assay. Transforming Growth Factor beta (TGF-β), VEGF and KGF mRNA levels were determined by RT-PCR. Protein levels of VEGF and TGF-β in CCD-18Co cells following GLP-2 stimulation were determined using ELISA. Neutralizing TGF-β and VEGF-A antibodies were utilized to assess the role of TGF-β and VEGF-A in the process of wound healing. GLP-2 receptor expression was detected in CCD-18Co cells. Conditioned media from CCD-18Co cells dose-dependently induced proliferation in Caco-2 cells, but not in IEC-18 cells. Conditioned media also enhanced cell migration in IEC-18 cells (P  < 0.01), while migration was even inhibited in Caco-2 cells (P  < 0.0012). GLP-2 significantly stimulated mRNA expression of VEGF and TGF-β, but not of KGF in CCD-18Co. The migratory effects of GLP-2 were completely abolished in the presence of TGF-β and VEGF-A antibodies. GLP-2 exerts differential effects on the epithelium of the small intestine and the colon. Thus, in small intestinal cells GLP-2 stimulates wound repair, whereas no such effects were observed in colonic cells. The mechanisms underlying GLP-2 induced intestinal wound repair seem to involve the secretion of VEGF and, subsequently, TGF-β from subepithelial fibroblasts, whereas KGF appeared to be less important.
Keywords: Glucagon like peptide-2; Mucosal injury; VEGF; Subepithelial myofibroblast; Intestinal wound healing; Intestinal cell proliferation;

Contribution of prostaglandin D2 via prostanoid DP receptor to nasal hyperresponsiveness in guinea pigs repeatedly exposed to antigen by Kiyoshi Yasui; Fujio Asanuma; Yosuke Hirano; Michitaka Shichijo; Masashi Deguchi; Akinori Arimura (286-291).
We examined the role of prostanoid DP receptor in nasal blockage in an experimental allergic rhinitis model in guinea pigs. Local inhalation of prostaglandin D2 (PGD2) to the nasal cavity resulted in an increase in intranasal pressure in guinea pigs actively sensitized by repeated antigen exposure but not in non-sensitized guinea pigs. Nasal hyperresponsiveness was observed when the guinea pigs were exposed to histamine and U-46619 (11α, 9α-epoxymethano-PGH2; a thromboxane (TX) A2 mimetic) after repeated antigen exposure. S-5751 ((Z)-7-[(1R,2R,3S,5S)-2-(5-hydroxybenzo[b]thiophen-3-ylcarbonylamino)-10-norpinan-3-yl]hept-5-enoic acid), a prostanoid DP receptor antagonist, inhibited not only PGD2-induced nasal blockage but also nasal hyperresponsiveness to histamine and U-46619 in sensitized guinea pigs. Combined exposure of the nasal cavity of guinea pigs to an aerosol of PGD2 with histamine or U-46619 at sub-threshold concentrations synergistically caused a marked increase in intranasal pressure. These responses were significantly suppressed by S-5751. These results suggest that PGD2 plays a critical role in the increase in intranasal pressure via prostanoid DP receptor, probably through synergistically enhancing the nasal response with other chemical mediators released from mast cells and other inflammatory cells activated by allergens.
Keywords: PGD2; Prostanoid DP receptor; Allergic rhinitis; Nasal blockage; Nasal hyperresponsiveness; S-5751; Histamine; TXA2; U46619;

Protection of cellular and mitochondrial functions against liver ischemia by N-benzyl-N′-(2-hydroxy-3,4-dimethoxybenzyl)-piperazine (BHDP), a sigma1 ligand by Anis Klouz; Dorra Ben Saïd; Henda Ferchichi; Nadia Kourda; Lobna Ouanes; Mohamed Lakhal; Jean-Paul Tillement; Didier Morin (292-299).
We investigated the antiischemic properties of a new compound N-benzyl-N′-(2-hydroxy-3,4-dimethoxybenzyl)-piperazine (BHDP), having high affinity and selectivity for the sigma1 receptor, in two different models of ischemia. The first was an experimental model of rat liver normothermic ischemia-reperfusion. Rats were pretreated with different doses of BHDP (0.5, 2.5 or 10 mg/kg/day, or solvent alone) and subjected to 90 min normothermic ischemia followed by either 30 or 120 min reperfusion. The second model was a hypothermic model of ischemia in which livers were incubated for 24 h at 4 °C in a preservation solution in the absence or presence of increasing BHDP concentrations (0.5, 2.5 or 10 μg/ml). These different ischemic conditions induced huge alterations in hepatocyte functions (membrane leakage of alanine aminotransferase and aspartate aminotransferase, decreased metabolic capacities evaluated by the ability of the liver to transform lidocaine, alterations of mitochondrial functions characterized by a decrease in ATP synthesis and the appearance of histological damages). Pretreatment of rats with BHDP alleviated these deleterious ischemia-reperfusion effects in a dose-dependent manner at both the cellular and mitochondrial levels. The protection of mitochondrial functions was almost complete at a dosage of 10 mg/kg/day during normothermic ischemia and 10 μg/ml in the preservation liquid during hypothermic ischemia. In addition, BHDP significantly reduced the histological damage. These data demonstrate that BHDP protects liver against the deleterious effects of ischemia-reperfusion and suggest that sigma1 receptors play an important role in the protective effect.
Keywords: BHDP; Mitochondria; Normothermic ischemia-reperfusion; Hypothermic preservation; Liver; Monoethylglycinexylidide;

Healing properties of malabaricone B and malabaricone C, against indomethacin-induced gastric ulceration and mechanism of action by Debashish Banerjee; Ajay K. Bauri; Ranjit K. Guha; Sandip K. Bandyopadhyay; Subrata Chattopadhyay (300-312).
The healing activity of malabaricone B and malabaricone C, the major antioxidant constituents of the spice Myristica malabarica against the indomethacin-induced gastric ulceration in mice has been studied. The histological indices revealed maximum ulceration on the 3rd day after indomethacin administration, which was effectively healed by malabaricone B, malabaricone C (each 10 mg/kg body weight/day) and omeprazole (3 mg/kg body weight/day) for 3 days. Compared to the untreated ulcerated mice, treatment with malabaricone B, malabaricone C and omeprazole reduced the ulcer indices by 60.3% (P  < 0.01), 88.4% and 86.1% respectively (P  < 0.001). All the test samples accelerated ulcer healing than observed in natural recovery even after 7 days. Stomach ulceration reduced the total antioxidant status of plasma by 41% (P  < 0.05), which was significantly increased by malabaricone B (36%, P  < 0.01), malabaricone C (61%, P  < 0.001) and omeprazole (53%, P  < 0.001). Compared to the ulcerated untreated mice, those treated with malabaricone B reduced the levels of thiobarbituric acid reactive substances and protein carbonyls by 17% and ∼ 34% respectively (P  < 0.05), while malabaricone C and omeprazole reduced the parameters almost equally (∼ 30%, P  < 0.01, and ∼ 40%, P  < 0.01 respectively). Likewise, all the test samples reduced the oxidation of protein and non-protein thiols significantly (P  < 0.05). The antioxidant activity of the test samples could partly account their healing capacities. However, the differential potency of them was explainable by considering their relative abilities to modulate mucin secretion, PGE2 synthesis and expression of EGF receptor and COX isoforms, malabaricone C being most effective in controlling all these factors.
Keywords: Antioxidant; EGF receptor; Histopathology; Mucin; Prostaglandin;

Inhibitory effects of synthetic somatostatin receptor subtype 4 agonists on acute and chronic airway inflammation and hyperreactivity in the mouse by Krisztián Elekes; Zsuzsanna Helyes; László Kereskai; Katalin Sándor; Erika Pintér; Gábor Pozsgai; Valéria Tékus; Ágnes Bánvölgyi; József Németh; Tamás Szűts; György Kéri; János Szolcsányi (313-322).
Somatostatin released from activated capsaicin-sensitive afferents of the lung inhibits inflammation and related bronchial hyperreactivity presumably via somatostatin 4 receptors (sst4). The aim of this study was to examine the effects of TT-232, a heptapeptide sst4/sst1 receptor agonist and J-2156, a high affinity sst4 receptor-selective peptidomimetic agonist in airway inflammation models. Acute pneumonitis was evoked by intranasal lipopolysaccharide 24 h before measurement. Chronic inflammation was induced by ovalbumin inhalation on days 28, 29 and 30 after i.p. sensitization on days 1 and 14. Semiquantitative histopathological scoring was based on perivascular/peribronchial oedema, neutrophil/macrophage infiltration, goblet cell hyperplasia in the acute model and eosinophil infiltration, mucosal oedema, mucus production and epithelial cell damage in chronic inflammation. Myeloperoxidase activity of the lung was measured spectrophotometrically to quantify granulocyte accumulation and the broncoalveolar lavage fluid was analysed by flow cytometry. Carbachol-induced bronchoconstriction was assessed by unrestrained whole body plethysmography and its calculated indicator, enhanced pause (Penh) was determined. TT-232 and J-2156 induced similar inhibition on granulocyte recruitment and histopathological changes in both models, although macrophage infiltration in LPS-induced inflammation was unaltered by either compounds. Both agonists diminished inflammatory airway hyperresponsiveness. Since their single administration after the development of the inflammatory reactions also inhibited carbachol-induced bronchoconstriction, somatostatin sst4 receptor activation on bronchial smooth muscle cells is likely to be involved in their anti-hyperreactivity effect. These results suggest that stable, somatostatin sst4 receptor-selective agonists could be potential candidates for the development of a completely novel group of anti-inflammatory drugs for the treatment of airway inflammation and hyperresponsiveness.
Keywords: Somatostatin sst4 receptor; Airway inflammation; Bronchial hyperreactivity; Lipopolysaccharide; Ovalbumin; Whole body plethysmography; Interleukin-1β; Myeloperoxidase activity;

Inducible nitric oxide synthase and cyclooxygenase-2 participate in anti-inflammatory and analgesic effects of the natural marine compound lemnalol from Formosan soft coral Lemnalia cervicorni by Yen-Hsuan Jean; Wu-Fu Chen; Chang-Yi Duh; Shi-Ying Huang; Chi-Hsin Hsu; Chan-Shing Lin; Chun-Sung Sung; I-Ming Chen; Zhi-Hong Wen (323-331).
Lemnalol (8-isopropyl-5-methyl-4-methylene-decahydro-1,5-cyclo-naphthalen-3-ol) is a natural compound isolated from the marine soft coral Lemnalia cervicorni. In the present study, the anti-inflammatory and anti-nociceptive properties of lemnalol were investigated in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and carrageenan-injected rats, respectively. Our results demonstrate that lemnalol significantly inhibited the expression of the pro-inflammatory proteins, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in LPS-stimulated RAW 264.7 cells. An in vivo inflammation model was induced by intraplantar injection of carrageenan into rat hind paws. An intramuscular injection of lemnalol (15 mg/kg) 10 min before carrageenan injection resulted in significant inhibition of carrageenan-induced rat paw edema and thermal hyperalgesia behavior. Western blot experiments revealed that the carrageenan-induced expression of iNOS and COX-2 in paw tissue was significantly down-regulated by lemnalol. Moreover, post-intrathecal injection of lemnalol produced a dose-dependent anti-nociceptive effect in carrageenan-injected rats (1 and 5 μg). The present results indicate that the marine-derived compound lemnalol had anti-inflammatory and analgesic effects in LPS-stimulated RAW 264.7 cells and carrageenan-injected rats, respectively. In addition, inhibition of elevated iNOS and COX-2 protein expression as well as neurophil infiltration of carrageenan-injected paws may be involved in the beneficial effects of lemnalol.
Keywords: Lemnalol; iNOS; COX-2; Intrathecal; Carrageenan; Inflammation; Analgesia;

Anti-inflammatory effect of 1-methylnicotinamide in contact hypersensitivity to oxazolone in mice; involvement of prostacyclin by Krzysztof Bryniarski; Rafal Biedron; Andrzej Jakubowski; Stefan Chlopicki; Janusz Marcinkiewicz (332-338).
1-methylnicotinamide (MNA) displays anti-inflammatory effects in patients with contact dermatitis, though the mechanisms involved remain unknown. Herein, we examined the anti-inflammatory effects of MNA and its parent molecule, nicotinamide, in the contact hypersensitivity reaction to oxazolone in CBA/J inbred mice. Feeding mice with MNA or nicotinamide (100 mg/kg, 10 days) resulted in the inhibition of the development of contact hypersensitivity reaction by 37% and 35%, respectively, as assessed by the magnitude of ear swelling. This effect was not associated with changes in the expression of adhesion molecules (CD49d+ and CD54+) on CD4+ and CD8+ oxazolone-specific T lymphocytes, the major cell component of an inflammatory infiltrate in contact hypersensitivity reaction. Furthermore, in the adoptive transfer model of contact hypersensitivity reaction, pretreatment of mice (recipients of oxazolone-specific T cells), with MNA, resulted in a remarkable anti-inflammatory effect (inhibition of contact hypersensitivity reaction by 66%). Interestingly, in the presence of prostanoid IP receptor antagonist R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO-3244794) (10 mg/kg) the MNA was inactive. In summary, pretreatment with MNA profoundly attenuated contact hypersensitivity reaction in vivo. In particular, the vessel dependent phase of contact hypersensitivity reaction was affected, in spite of the fact that MNA did not alter the expression of adhesive molecules on oxazolone-specific T lymphocytes. However, the anti-inflammatory action of MNA was completely reversed by the antagonist of prostanoid IP receptor. Accordingly, our results demonstrate for the first time that anti-inflammatory properties of MNA are linked to endothelial, PGI2-mediated mechanisms.
Keywords: 1-Methylnicotinamide; Nicotinamide; Contact hypersensitivity reaction; Oxazolone; Lymphocytes T; Adhesions; Prostacyclin; Endothelium;

Reactive oxygen metabolites (ROMs) and inducible nitric oxide synthase (iNOS) are involved in pathogenesis of inflammatory bowel disease. In this study, we examined the effects of 2,3,5,4′-tetrahydroxystilbene-2-O-beta-d-glucoside (THSG), an active component extracted from Polygonum multiflorum Thunb, on acetic acid-induced acute colitis and mitomycin C-induced chronic colitis. The inflammatory degree was assessed by histology and myeloperoxidase (MPO) activity. Nitric oxide (NO), malondialdehyde (MDA) and superoxide dismutase (SOD) levels were determined with biochemical methods. In addition, inducible nitric oxide synthase (iNOS) expression was innumohistochemically studied. In acetic acid-induced acute model, THSG (60 and 120 mg/kg) significantly ameliorated colon damage, inhibited the increase of acetic acid-induced MPO activity, depressed MDA and NO level, and enhanced SOD activity. Moreover, the effects of 120 mg/kg THSG were better than that of positive control drug, 5-aminosaliylic acid (5-ASA). In mitomycin C-induced model, THSG (60 mg/kg) administered for 7 days and 24 days, significantly improved colon damage and inhibited MPO activity and MDA content while increased SOD activity only on the 7th day and debased NO level on the 24th day. Furthermore, on the 24th day, the effects of THSG were prior to that of 5-ASA. Additionally, THSG (60 mg/kg) could inhibit iNOS expression in both models. In conclusion, THSG exerts protective effects on experimental colitis through alleviating oxygen and nitrogen free radicals level and down-regulating iNOS expression.
Keywords: THSG (2,3,5,4′-tetrahydroxystilbene-2-O-beta-d-glucoside); Colitis; Acetic acid; Mitomycin C; Myeloperoxidase; Superoxide dismutase; Malondialdehyde; Nitric oxide; Inducible nitric oxide synthase;

α1-Adrenoceptors and extracellular signal-regulated kinases 1 and 2 (ERK1/2) regulate salivary secretion. However, whether α1-adrenoceptors couple to ERK1/2 activation and the specific α1-adrenoceptor subtypes involved in salivary glands is unknown. Western blotting of ERK1/2 phosphorylation showed phenylephrine activated ERK1/2 by 2–3-fold in submandibular gland slices and 3–4-fold in submandibular acinar (SMG-C10) cells with an EC50 of 2.7 ± 2 μM. ERK1/2 activation was blocked by either prazosin or HEAT, indicating α1-adrenoceptors stimulate ERK1/2 in native glands and SMG-C10 cells. Inhibition of [125I]HEAT binding by 5-methylurapidil (selective for α1A over α1B/α1D), but not BMY 7378 (selective for α1D over α1A/α1B), was biphasic and best-fit by a two-site binding model with Ki H and Ki L values for 5-methylurapidil of 0.64 ± 0.3 and 91 ± 7 nM, respectively, in SMG-C10 membranes. From these binding data, we obtained subtype-selective concentrations of 5-methylurapidil to determine the α1-adrenoceptor subtype/s activating ERK1/2 in SMG-C10 cells. 5-methylurapidil (20 nM) did not affect phenylephrine- or A-61603- (α1A-selective agonist) induced ERK1/2 activation; whereas, 30 μM chloroethylclonidine (α1B-selective antagonist) inhibited ERK1/2 activation by phenylephrine, indicating α1B-adrenoceptors, but not α1A-adrenoceptors, activate ERK1/2 in submandibular cells. We also examined α1-adrenoceptor location and dependence on cholesterol-rich microdomains for activating ERK1/2. Sucrose density gradient centrifugation showed 71 ± 3% of α1-adrenoceptor binding sites were in plasma membranes. Cholesterol-disrupting agents filipin and methyl-β-cyclodextrin inhibited phenylephrine-stimulated ERK1/2. These results show only α1B-adrenoceptors activate ERK1/2 and suggest subtype-specific ERK1/2 signaling by α1B-adrenoceptors may be determined by localization to cholesterol-rich microdomains in submandibular cells.
Keywords: α1-Adrenoceptor subtype; Extracellular signal-regulated kinase; Submandibular gland acinar cell;

Reduced blood glucose levels, increased insulin levels and improved glucose tolerance in α2A-adrenoceptor knockout mice by Eriika Savontaus; Veronica Fagerholm; Olli Rahkonen; Mika Scheinin (359-364).
α2-Adrenoceptors regulate insulin secretion and sympathetic output. In the present study, α2A-adrenoceptor knockout (α2A-KO) mice and their C57BL/6J wild-type (WT) controls were used to assess the glucoregulatory role of the α2A-adrenoceptor subtype in vivo. Fasting and glucose-stimulated blood glucose and plasma insulin levels were determined with or without (± )-propranolol (5 mg/kg) or atropine (10 mg/kg) pre-treatment. Intraperitoneal glucose (1 g/kg) and insulin (0.5 and 1.0 IU/kg) tolerance tests were performed. Fasting plasma glucagon and corticosterone levels were measured. Blood glucose levels (mean ± S.E.M.) were lower in α2A-KO males (7.2 ± 0.6 mM) and females (7.2 ± 0.2 mM) than in WT males (9.8 ± 0.3 mM) and females (9.1 ± 0.3 mM). Plasma insulin levels were higher in α2A-KO males (2.2 ± 0.5 μg/l) and females (1.7 ± 0.3 μg/l) than in WT males (0.7 ± 0.1 μg/l) and females (0.8 ± 0.2 μg/l). These differences remained after pharmacological β-adrenoceptor and muscarinic acetylcholine receptor inhibition. In spite of a tendency for slightly decreased insulin sensitivity in α2A-KO mice, glucose tolerance in α2A-KO mice was significantly better than in WT mice. However, glucose-stimulated insulin secretion was not increased in α2A-KO mice compared to WT controls. Plasma glucagon levels, but not corticosterone levels, were elevated in α2A-KO mice. These results suggest that lack of inhibitory pancreatic β-cell α2A-adrenoceptor function results in hyperinsulinaemia, reduced blood glucose levels and improved glucose tolerance in α2A-KO mice, and demonstrate a key role for the α2A-adrenoceptor in adrenergic regulation of blood glucose and insulin homeostasis.
Keywords: α2A-adrenoceptor; Diabetes; Insulin; Glucagon; Glucose; (Mouse);