European Journal of Pharmacology (v.598, #1-3)

Approximately one-third of patients with epilepsy display an inherent resistance to pharmacological therapy, manifest as continuing seizures despite maximal tolerated doses of anti-epileptic drugs. One hypothesis for the underlying mechanism of anti-epileptic drug pharmacoresistance is lower drug entry to the epileptic neurones due to the activity of multidrug efflux pumps from the ATP Binding Cassette (ABC) superfamily at the blood-brain barrier. There has been a steady accumulation of animal and human data supporting this theory, particularly for ABCB1 (P-glycoprotein). However, much of this evidence is indirect. In the present study, several anti-epileptic drugs (carbamazepine, valproic acid, phenytoin, lamotrigine and primidone) were examined for their ability to interact with three ABC transporters that have been implicated pharmacoresistance of anti-epileptic drugs — ABCB1, ABCC1 and ABCG2. Interaction of anti-epileptic drugs with the transporters was assessed by determining whether they could reverse the ability of multidrug ABC transporters to confer a drug resistance phenotype on cancer cell lines. None of these compounds was able to affect the phenotype, suggesting an absence of any interaction with the multidrug transporters. This finding was further investigated by examination of transporter activity; namely the ability to reduce steady-state intracellular [3H]-radiolabelled drug accumulation. None of the anti-epileptic drugs affected labelled drug accumulation by any of the triumvirate of multidrug transporters examined, indicating that they are unlikely to be substrates. The lack of direct modulation by anti-epileptic drugs of ABC transporter function suggests that these proteins do not contribute significantly to resistance in epilepsy.
Keywords: Epilepsy; Anti-epileptic drugs; Drug resistance; ABC transporter; Multidrug efflux; ABCB1; ABCC1; ABCG2;

Activation of high conductance Ca2+-activated K+ channels by sodium tanshinoneII-A sulfonate (DS-201) in porcine coronary artery smooth muscle cells by Yan Yang; Fang Cai; Peng-Yun Li; Miao-Ling Li; Jun Chen; Gui-Lan Chen; Zhi-Fei Liu; Xiao-Rong Zeng (9-15).
High conductance Ca2+ activated K+ channels (BKCa) in vascular smooth muscles play important roles in controlling the vascular tone by determining the level of membrane potential and Ca2+ influx through voltage gated Ca2+ channels. Agents that alter the activity of Ca2+ channels or BKCa thus affect the vascular tone in both physiological and pathological conditions. Danshen, the dried root of Salvia miltiorrhiza, is a commonly used traditional Chinese medicine and is widely used as an effective remedy for cardiovascular and cerebral vascular diseases partly by its vasodilatation. Sodium tanshinoneII-A sulfonate (DS-201) is a water-soluble derivative of Tanshinone IIA, the main active component of Danshen. The purpose of this study was to explore possible mechanisms of vasodilative effects of DS-201 using porcine coronary artery smooth muscle. DS-201 induced relaxation of the coronary smooth muscle which had been contracted with 30 mM KCl, and the relaxation was inhibited by 100 nM iberiotoxin (IbTX), a specific BKCa channel blocker. Using perforated whole-cell recordings and single channel recordings, effects of DS-201 on BKCa were examined. The results showed that DS-201 activated BKCa. Extracellular application of DS-201 at 40, 80 µM under the whole-cell configuration induced increases of the BKCa macroscopic currents by 43.6%, 42.1% respectively, and the spontaneous transient outward K+ currents (STOCs) by 48.7%, 47.4% respectively. In inside-out patches, bath application of 20–150 μM of DS-201 activated BKCa by 5.4–173.2 fold. These results indicate that the vasodilatation by DS-201 is related to activation of BKCa.
Keywords: High conductance Ca2+ activated K+ channel (BKCa); Sodium tanshinoneII-A sulfonate; Porcine coronary artery smooth muscle; Vasodilatation;

Delta-9-tetrahydrocannabinol differently affects striatal c-Fos expression following haloperidol or clozapine administration by Giorgio Marchese; Angela Sanna; Gianluca Casu; Paola Casti; Gabriele Pinna Spada; Stefania Ruiu; Luca Pani (16-20).
It was previously shown that haloperidol, but not clozapine, induced intense rat catalepsy when co-administered with delta-9-tetrahydrocannabinol. The present study investigated whether similar alterations could be observed on striatal c-Fos immunoreactivity after administration of the same drug combinations. Western Blot and immunocytochemistry stereological analyses indicated that delta-9-tetrahydrocannabinol (0.5 mg/kg) increased striatal c-Fos immunoreactivity induced by haloperidol (0.1 mg/kg). Conversely, no significant alterations of striatal c-Fos immunoreactivity were observed after injections of clozapine (10 mg/kg) + vehicle, clozapine + delta-9-tetrahydrocannabinol or vehicle + delta-9-tetrahydrocannabinol. The present results indicate that the behavioral effects induced by delta-9-tetrahydrocannabinol in haloperidol- and clozapine-treated rats are associated with different striatal c-Fos expressions.
Keywords: Cannabinoid; Antipsychotic; Signal transduction; Basal ganglia;

Multiplicative interactions to enhance gabapentin to treat neuropathic pain by Ken-ichiro Hayashida; James C. Eisenach (21-26).
We previously reported that gabapentin activates the bulbospinal-spinal noradrenergic–cholinergic pathway to produce analgesia in rats after nerve injury. Also, gabapentin interacts synergistically with a cholinesterase inhibitor donepezil to produce analgesia. Duloxetine, a serotonin/noradrenaline re-uptake inhibitor, has been used for the treatment of neuropathic pain and should amplify the noradrenergic mechanisms recruited by gabapentin. In the present study, we determined the interaction between duloxetine and gabapentin with and without donepezil when administered by the clinically preferred oral route in rats after spinal nerve ligation. The ED50 value of gabapentin, donepezil, and duloxetine to reduce mechanical hypersensitivity after nerve injury was 45, 3.7, and 32 mg/kg, respectively. In the examination of two drug combinations, oral duloxetine with either gabapentin or donepezil were additive to reduce hypersensitivity. The combination of all three drugs yielded a synergistic interaction with an observed ED50 at 1/4th the predicted dose of additivity, likely due to the gabapentin–donepezil interaction. This three drug combination did not affect motor coordination or show signs of sedation in the rotarod test. Analgesia by the combination of these three drugs was reversed by intrathecal injection either of the α2-adrenoceptor antagonist idazoxan or by the muscarinic receptor antagonist atropine. These results suggest that the combination of these drugs, which stimulate and augment the bulbospinal-spinal noradrenergic–cholinergic pathway, lowers the dose requirement for each drug to reduce hypersensitivity after nerve injury without sedative effects. The current study provides the rationale for clinical study of the combination of gabapentin, donepezil and duloxetine to treat neuropathic pain.
Keywords: Neuropathic pain; Descending inhibition; Gabapentin; Donepezil; Duloxetine;

Bromocriptine, a dopamine D2 receptor agonist with the structure of the amino acid ergot alkaloids, induces neurite outgrowth in PC12 cells by Toru Oda; Toshiaki Kume; Yasuhiko Izumi; Yuki Takada-Takatori; Tetsuhiro Niidome; Akinori Akaike (27-31).
To investigate whether dopamine agonists induce neurite outgrowth, we examined the effects of dopamine D2 receptor agonists such as bromocriptine, talipexole, and pramipexole in PC12 cells, a well-studied model of neurite outgrowth. Bromocriptine significantly induced neurite outgrowth in a concentration-dependent manner. Neither talipexole nor pramipexole induced neurite outgrowth. Domperidone and sulpiride, dopamine D2 receptor antagonists, did not have any effect on the bromocriptine-induced neurite outgrowth. These results suggest that the stimulation of dopamine D2 receptors would not affect neurite outgrowth in nerve regeneration. Next, we investigated how bromocriptine-induced neurite outgrowth. Bromocriptine is not only a dopamine D2 receptor agonist but also an ergot alkaloid. We examined the involvement of the structure of ergot alkaloids in the effect of bromocriptine. Ergot alkaloids have been divided into two groups: amino acid ergot alkaloids, including bromocriptine, and amine ergot alkaloids. Amino acid ergot alkaloids, such as ergocornine and ergotamine, induced neurite outgrowth, but amine ergot alkaloids, including ergometrine and methylergometrine, did not. These results indicate that the structure of amino acid ergot alkaloids is important for the effect of bromocriptine. Moreover, the effect of bromocriptine was inhibited by a src inhibitor and a mitogen-activated protein kinase kinase (MEK) inhibitor. Taken together, bromocriptine-induced neurite outgrowth via src and the MEK/extracellular signal-regulated kinase 1/2 signaling pathway in PC12 cells.
Keywords: Neurite outgrowth; Bromocriptine; Dopamine D2 receptor agonist; Ergot alkaloid;

Effects of cytidine 5′-diphosphocholine (CDP-choline) on the thermal nociceptive threshold in streptozotocin-induced diabetic mice by Junzo Kamei; Masahiro Ohsawa; Shigeo Miyata; Kazuki Endo; Hiroyuki Hayakawa (32-36).
Neuropathy accompanied by abnormal sensory perception is the most common complication in insulin-dependent and -independent diabetes mellitus. Since there are very few effective therapeutic regimens for sensory abnormalities in diabetes, we examined the effect of cytidine 5′-diphosphocholine (CDP)-choline on the thermal nociceptive threshold in streptozotocin-induced diabetic mice using the tail-flick test. Diabetic mice showed a shorter tail-flick latency at 1–4 weeks after streptozotocin treatment and a longer tail-flick latency after 8–12 weeks. This hyper- and hypoalgesia in diabetic mice was almost completely inhibited by daily treatment with CDP-choline (100mg/kg/day, p.o.) beginning on the day of streptozotocin treatment. Daily treatment with CDP-choline beginning 5 weeks after streptozotocin treatment attenuated the development of hypoalgesia. Diabetic mice showed a significant increase in Na+-K+-ATPase activity at 3 weeks after streptozotocin treatment, whereas Na+-K+-ATPase activity was decreased at 12 weeks after treatment. These alterations were normalized by daily treatment with CDP-choline (100mg/kg/day, p.o.) beginning the day of streptozotocin treatment. These results provide evidence to support the therapeutic potency of CDP-choline on the development of thermal hyper- and hypoalgesia and the progression of thermal hypoalgesia in diabetic mice. Moreover, these effects of CDP-choline may result from the normalization of Na+-K+-ATPase activity.
Keywords: Diabetes; Cytidine 5′-diphosphocholine; Na+-K+-ATPase; Neuropathy; Tail-flick test; Hyperalgesia; Hypoalgesia;

Antidepressant-like effect of folic acid: Involvement of NMDA receptors and l-arginine-nitric oxide-cyclic guanosine monophosphate pathway by Patrícia de Souza Brocardo; Josiane Budni; Kelly Ribas Lobato; Manuella Pinto Kaster; Ana Lúcia S. Rodrigues (37-42).
Antidepressant-like activity of folic acid in forced swimming test and in the tail suspension test was demonstrated previously by our group. In this study we investigated the involvement of N-methyl-d-aspartate (NMDA) receptors and l-arginine-nitric oxide (NO)-cyclic guanosine monophosphate pathway in its antidepressant-like effect in the forced swimming test in mice. The antidepressant-like effect of folic acid (10 nmol/site, i.c.v.) was prevented by the pretreatment of mice with NMDA (0.1 pmol/site, i.c.v.), l-arginine (750 mg/kg, i.p., substrate for nitric oxide synthase), S-nitroso-N-acetyl-penicillamine (SNAP, 25 µg/site, i.c.v, a NO donor) or sildenafil (5 mg/kg, i.p., phosphodiesterase 5 inhibitor). The administration of 7-nitroindazole (25 and 50 mg/kg, i.p., a specific neuronal nitric oxide synthase (nNOS) inhibitor) or methylene blue (20 mg/kg, i.p., direct inhibitor of both nitric oxide synthase and soluble guanylate cyclase) in combination with a sub-effective dose of folic acid (1 nmol/site, i.c.v.) reduced the immobility time in the FST as compared with either drug alone. Together the results suggest that the antidepressant-like effect of folic acid in the forced swimming test is dependent on an inhibition of either NMDA receptors or NO and cGMP synthesis.
Keywords: Antidepressant; Folic acid; Forced swimming test; l-arginine-nitric oxide-cGMP pathway;

SONU20176289, a compound combining partial dopamine D2 receptor agonism with specific serotonin reuptake inhibitor activity, affects neuroplasticity in an animal model for depression by Adina T. Michael-Titus; Monika Albert; Gregory J. Michael; Thomas Michaelis; Takashi Watanabe; Jens Frahm; Olga Pudovkina; Marieke G.C. van der Hart; Mayke B. Hesselink; Eberhard Fuchs; Boldizsár Czéh (43-50).
We investigated the efficacy of SONU20176289, a member of a group of novel phenylpiperazine derivatives with a mixed dopamine D2 receptor partial agonist and specific serotonin reuptake inhibitor (SSRI) activity, in a chronic stress model of depression in male tree shrews. Animals were subjected to a 7-day period of psychosocial stress before treatment for 28 days with SONU20176289 (6 mg/kg/day, p.o.), during which stress was maintained. Stress reduced the in vivo brain concentrations of N-acetyl-aspartate, total creatine, and choline-containing compounds, as measured by localized proton magnetic resonance spectroscopy. Post mortem analyses revealed a reduced adult dentate cell proliferation and a decreased GluR2 expression in the prefrontal cortex. All these alterations were prevented by concomitant administration of SONU20176289. The results provide further support to the concept that antidepressant treatments may act by normalizing disturbed neuroplasticity, and indicate that combining dopamine D2 receptor agonism with SSRI activity may serve as an effective tool in the treatment of depressive/anxiety syndromes.
Keywords: GluR2; AMPA; Prefrontal cortex; Adult neurogenesis; Proton magnetic resonance spectroscopy; Antidepressant;

The present study was designed to investigate the effect of montelukast sodium, a leukotriene D4 receptor antagonist, and 1,2,3,4,tetrahydroisoquinoline, a leukotriene D4 synthetic pathway inhibitor, on the development of morphine dependence in a mouse model of naloxone-induced opioid withdrawal syndrome. Morphine (5 mg/kg, i.p.) was administered twice daily for a period of 5 days following which a single injection of naloxone (8 mg/kg, i.p.) precipitated the opioid withdrawal syndrome in mice. Behavioral observations were made for a period of 30 min immediately after naloxone treatment. The withdrawal syndrome was quantitatively assessed in terms of withdrawal severity score and the frequency of jumping, rearing, fore paw licking and circling. Montelukast sodium as well as 1,2,3,4,tetrahydroisoquinoline, markedly and dose dependently (p  < 0.01) attenuated the morphine-naloxone-induced opioid withdrawal syndrome in mice. However, administration of montelukast sodium or 1,2,3,4,tetrahydroisoquinoline did not alter the activity of the central nervous system, assessed in terms of locomotor activity count thus ruling out any per se sedative action of montelukast sodium. Further, pretreatment with montelukast sodium or 1,2,3,4,tetrahydroisoquinoline did not alter the acute analgesic effect of morphine. Thus, leukotriene D4 may be involved in the development of opioid dependence and the precipitation of its withdrawal syndrome and thus may serve as a viable pharmacological target to tackle the problem of opioid addiction.
Keywords: Morphine dependence; Withdrawal syndrome; Leukotriene D4 receptors; Montelukast sodium; 1,2,3,4,tetrahydroisoquinoline; Gamma glutamyl transpeptidase;

The aim of the present study was to determine whether various glutamate receptor antagonists could affect ethanol withdrawal-induced anxiety-like behavior measured in the elevated plus-maze test in rats. In our study, memantine (8 and 12 mg/kg), a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, did not show any effect on ethanol withdrawal anxiety. Acamprosate (NMDA and metabotropic glutamate5 (mGlu5) receptor antagonist), at a dose of 400 mg/kg showed anxiolytic-like effect, thus increasing the percent of time spent in open arms and open arms entries. Antagonists of group I mGlu receptors, such as MTEP ([(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine, mGlu5 receptor) or EMQMCM (3-ethyl-2-methyl-quinolin-6-yl-(4-methoxy-cyclohexyl)-methanone methanesulfonate, mGlu1 receptor), caused similar effects to acamprosate. In contrast to acamprosate and MTEP, EMQMCM (5 mg/kg) elevated the ethanol withdrawal-induced decrease in locomotion. When given alone to the saline-treated group, EMQMCM indicated anxiolytic-like effect. Our results imply a crucial role of mGlu5 receptor in an anxiety-like effect of ethanol withdrawal because MTEP (a selective mGlu5 receptor antagonist) and acamprosate (which also indirectly inhibits mGlu5 receptor) attenuated ethanol withdrawal anxiety-like behavior without influence on ethanol withdrawal hypolocomotion and did not show any effect in the saline-treated groups. However, difference in anxiolytic-like potency between both these group I mGlu receptors antagonists may be due to the recent experimental design. Therefore, taking into account a positive correlation between ethanol withdrawal-induced anxiety and relapse to ethanol drinking, our results suggest that mGlu receptor antagonists of group I (similarly to acamprosate) could prevent relapse to drinking and, therefore they might be useful in therapy of alcoholism.
Keywords: Ethanol withdrawal; Anxiety; Elevated plus-maze; Memantine; Acamprosate; MTEP; EMQMCM; NMDA; mGlu receptor; Behavior;

A therapeutic dose of zolpidem has limited abuse-like effects in drug-naïve females: A pilot study by Stephanie C. Licata; David M. Penetar; Steven Dunlap; Scott E. Lukas (64-67).
Zolpidem has abuse potential, particularly among individuals with histories of drug abuse. This double-blind, placebo-controlled, cross over pilot study investigated the subjective effects of zolpidem (10 mg) in drug-naïve females. Over the course of a 5-h period vital signs were monitored and a series of computerized questionnaires was administered. Results indicate that zolpidem engendered subjective effects characteristic of hypnotic drugs, but reduced ratings of drug liking, willing to take again, and willing to pay for, relative to placebo. Thus, a therapeutic dose of zolpidem may have limited potential for misuse among females who have no experience with drugs of abuse.
Keywords: Zolpidem; Benzodiazepine; Subjective effect; Abuse liability; Human volunteer;

Putrescine modulation of acute activation of the β-adrenergic system in the left atrium of rat by Carmen Bordallo; Begoña Cantabrana; Lucía Velasco; Lorena Secades; Clara Meana; Miriam Méndez; Javier Bordallo; Manuel Sánchez (68-74).
Endogenous polyamines mediate acute metabolic effects and cardiac hypertrophy associated to β-adrenoceptor stimulation. The aim of this study is to characterize the role of polyamines on β-adrenoceptor system mediated responses. To this end, the functional interaction of polyamine modifying drugs on isoproterenol-elicited cardiotonic effect, in isolated left atria of male Wistar rats, and their effects on [3H]dihydroalprenolol (DHA) binding on β-adrenoceptors and on adenylyl cyclase activity of membrane heart were studied. Polyamines interact with β-adrenoceptors in rat heart, as shown by the displacement of [3H]DHA binding. Furthermore, putrescine (but not spermidine or spermine) increased adenylyl cyclase activity, elicited a positive inotropism and increased intracellular cAMP. The putrescine effect on adenylyl cyclase was not antagonized by the β-adrenoceptors blockers, alprenolol and ICI-118,551, and facilitated the isoproterenol effect. Neither alprenolol, atenolol nor ICI-118,551 antagonized putrescine-elicited positive inotropism. However, the effect was abolished in preparations with desensitized β-adrenoceptors. α-Difluoromethylornithine, an inhibitor of ornithine decarboxylase, antagonized the effect of isoproterenol on inotropism and cAMP increase. In addition, putrescine might elicit effects by mechanisms independent of β-adrenoceptor system, since in left atria with functional desensitized receptors an interaction with ouabain-elicited cardiotonic effect was observed. These results suggest that putrescine may act as a low affinity agonist on β-adrenoceptors and modulate acute responses mediated by β-adrenoceptors. These findings may be of importance in the physiology and in diseases involving cardiac β-adrenoceptors.
Keywords: β-adrenoceptor; Polyamine; Left atrium; Heart; Inotropism; (Rat);

Quercetin antagonism of Bay K 8644 effects on rat tail artery L-type Ca2+ channels by Simona Saponara; Giampietro Sgaragli; Fabio Fusi (75-80).
The functional interaction between two L-type Ca2+ channel activators, quercetin and (S)-(−)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate (Bay K 8644), has been investigated in vascular smooth muscle cells. L-type Ca2+ currents [I Ca(L)] were recorded in freshly isolated rat tail main artery myocytes using the whole-cell patch-clamp method. Bay K 8644 increased I Ca(L) in a concentration-dependent manner with a pEC50 value of 8.25. Pre-incubation of myocytes with concentrations of quercetin per se ineffective as an L-type Ca2+ channel activator (0.1 and 0.3 μM) inhibited significantly the maximal response evoked by Bay K 8644, but left unaltered its potency. Quercetin (0.1 μM) prevented the hyperpolarizing shift of the steady-state inactivation curve induced by 0.1 μM Bay K 8644 and its stimulation of I Ca(L) tail current intensity without modifying Bay K 8644-induced effects on I Ca(L) activation, inactivation, deactivation kinetics as well as on use-dependence and recovery from inactivation. Quercetin at nutritionally meaningful concentrations, limited the responsiveness of vascular L-type Ca2+ channels to the pharmacological stimulation operated by Bay K 8644. These data contribute to a better understanding of quercetin effects on experimental in vivo cardioprotection.
Keywords: Bay K 8644; Quercetin; Rat tail artery myocyte; Whole-cell patch-clamp;

A low nicotine concentration augments vesicle motion and exocytosis triggered by K+ depolarisation of chromaffin cells by Antonio M.G. de Diego; Laura Tapia; Rocío M. Álvarez; Marta Mosquera; Lorena Cortés; Inmaculada López; Luis M. Gutiérrez; Luis Gandía; Antonio G. García (81-86).
Tobacco smokers have an increased risk of cardiovascular disease; this is likely associated to an enhanced catecholamine release by circulating nicotine. Here, we have explored how low concentrations of nicotine in the range of those found in the blood of tobacco smokers, might affect the release of catecholamines in bovine chromaffin cells. We have combined patch-clamp and Ca2+ imaging techniques to study cell excitability, cytosolic Ca2+ transients, vesicle movement, and secretory responses. We found that low concentrations of nicotine (1.5–3 μM) did not enhance catecholamine release by themselves. However, they drastically augmented the catecholamine release response triggered by a supramaximal K+ depolarising pulse. Furthermore, low nicotine concentrations caused slight depolarisation with superimposed action potentials, a transient elevation of [Ca2+] c and augmented Ca2+-dependent vesicle motion underneath the plasmalemma. We suggest that low nicotine concentrations overload the secretory machinery with secretory vesicles, which cause chromaffin cells to respond with an exaggerated adrenaline release into the circulation during stress. This might contribute to the higher cardiovascular risk of tobacco smokers.
Keywords: Exocytosis; Nicotine; Nicotinic receptors; Chromaffin cell;

Vasodilator responses induced by okadaic acid were investigated in canine basilar artery precontracted with 80 mM KCl. Okadaic acid (1 µM) relaxed the artery and this relaxant effect was partially inhibited by Gö6976, a conventional protein kinase C inhibitor, and calphostin C, an inhibitor of conventional and novel PKCs. Rottlerin, a specific inhibitor of PKCδ, did not influence okadaic acid's effect. KCl increased phosphorylation of 20,000-Dalton myosin light chain (MLC20) at Ser-19. Okadaic acid additionally increased MLC20 phosphorylation at Thr-18 and Thr-9, resulting in triphosphorylation of MLC20. This phosphorylation was inhibited by Gö6976. Okadaic acid stimulated phosphorylation of PKCα and 17,000-Dalton PKC-potentiated inhibitory phosphoprotein (CPI-17), and Gö6976 inhibited these phosphorylations. These results suggest that okadaic acid's relaxant effect involves MLC20 triphosphorylation through a direct phosphorylation by PKCα and an indirect phosphorylation by inhibition of myosin light chain phosphatase through PKCα-mediated CPI-17 phosphorylation.
Keywords: Okadaic acid; Myosin light chain; Phosphorylation; Protein kinase Cα; CPI-17; Canine basilar artery;

Effects of three metabolites of propiverine on voltage-dependent L-type calcium currents in human atrial myocytes by Melinda Wuest; Torsten Christ; Nicole Hiller; Manfred Braeter; Ursula Ravens (94-97).
The non-selective muscarinic receptor antagonist propiverine impairs L-type Ca2+ currents (I Ca,L) in human detrusor smooth muscle cells and atrial cardiomyocytes. Here, we have investigated the effects of three metabolites of propiverine on human cardiac I Ca,L. Propiverine reduced I Ca ,L with a − logIC50 [M] value of 4.1, M-5 only showed minor effect on I Ca ,L at high concentrations, M-6 did not influence I Ca ,L at all. Like the parent compound M-14 also reduced I Ca ,L (− logIC50 [M] = 4.6). We conclude, that propiverine and M-14 reduce cardiac I Ca ,L at higher concentrations than in detrusor cells and therefore preferentially reduce I Ca ,L in the urinary bladder than in the heart.
Keywords: Propiverine and metabolite; L-type Ca2+ current; Human atrial myocyte;

The isolated, perfused trachea preparation has been used to compare reactivity of the intact airway in response to differential exposure of the mucosal (intraluminal) and serosal (extraluminal) surfaces to contractile and relaxant agonists and other agents, and to gain insight into the modulatory role of the epithelium and the pathways involved. The apparatus has also been configured for simultaneous measurement of transepithelial potential difference and changes in tracheal diameter, thereby providing parallel observations of epithelial and smooth muscle function and reactivity to drugs. The transepithelial potential difference is a product of transepithelial resistance and short circuit current, and the present study describes a novel isolated, perfused tracheal apparatus which allows simultaneous measurement of transepithelial potential difference, transepithelial resistance and mechanical responses of the smooth muscle. The apparatus was validated using well-known ion transport inhibitors [intraluminal amiloride and 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB), extraluminal ouabain and bumetanide], bronchoactive agonists (extraluminal methacholine, histamine and terbutaline), and osmolytes (intraluminal d-mannitol and NaCl) to induce epithelium-derived relaxing factor-mediated relaxations. This apparatus will facilitate investigation of interactions between the epithelium and smooth muscle in airways that retain their in situ structure, and signaling mechanisms potentially involved in the regulation of airway smooth muscle tone.
Keywords: Perfused trachea; Transepithelial potential difference; Transepithelial resistance; Airway smooth muscle; Epithelium; Guinea pig;

Monocyte migration: A novel effect and signaling pathways of catestatin by Margot Egger; Arno G.E. Beer; Markus Theurl; Wilfried Schgoer; Benjamin Hotter; Tobias Tatarczyk; Danijela Vasiljevic; Silke Frauscher; Josef Marksteiner; Josef R. Patsch; Peter Schratzberger; Angela M. Djanani; Sushil K. Mahata; Rudolf Kirchmair (104-111).
Several members of the neuropeptide family exert chemotactic actions on blood monocytes consistent with neurogenic inflammation. Furthermore, chromogranin A (CgA) containing Alzheimer plaques are characterized by extensive microglia activation and such activation induces neuronal damage. We therefore hypothesized that the catecholamine release inhibitory peptide catestatin (hCgA352–372) would induce directed monocyte migration. We demonstrate that catestatin dose-dependently stimulates chemotaxis of human peripheral blood monocytes, exhibiting its maximal effect at a concentration of 1 nM comparable to the established chemoattractant formylated peptide Met-Leu-Phe (fMLP). The naturally occurring catestatin variants differed in their chemotactic property insofar as that the Pro370Leu variant was even more potent than wild type, whereas the Gly364Ser variant was less effective. Specificity of this effect was shown by inhibition of catestatin-induced chemotaxis by a specific neutralizing antibody. In addition, catestatin mediated effect was blocked by dimethylsphingosine and treatment with endothelial differentiation gene (Edg)-1 and Edg-3 antisense RNA as well as by incubation with pertussis toxin and genistein indicating involvement of tyrosine kinase receptor-, G-protein- and sphingosine-1-phosphate signaling. Catestatin also stimulated Akt- and extracellular signal related kinase (ERK)-phosphorylation and catestatin-induced chemotaxis was blocked by blockers of phosphoinositide-3 (PI-3) kinase and nitric oxide as well as by inhibition of the mitogen-activated protein kinases (MAPK) system indicating involvement of these signal transduction pathways. In summary, our data indicate that catestatin induces monocyte chemotaxis by activation of a variety of signal transduction pathways suggesting a role of this peptide as an inflammatory cytokine.
Keywords: Monocyte/Macrophage; Neuropeptide; Chemokine; Chemotaxis; Signal transduction;

Rebamipide affects the efficiency of colchicine for the herpes simplex virus-induced inflammation in a Behcet's disease mouse model by Dongsik Bang; Bunsoon Choi; Hyuk Jae Kwon; Eun-So Lee; Sungnack Lee; Seonghyang Sohn (112-117).
Rebamipide inhibits free radicals derived from activated neutrophils and decreases the inhibiting inflammatory cytokine. Behcet's disease (BD) is a chronic, multi-systemic inflammatory disorder with arthritic, gastrointestinal, mucocutaneous, ocular, vascular, and central nervous system involvement. This disease has a chronic course with periodic exacerbations and progressive deterioration. To study the effect of rebamipide treatment to BD-like mice, combination treatment with rebamipide and colchicine was compared to colchicine treatment. Colchicine is one of the most frequently prescribed medicine to the patients with BD. For each BD mouse, 200 μl gastric fluid or 2 μg colchicine or 150 μg rebamipide or 2 μg colchicine plus 150 μg rebamipide was treated orally once per day. Treatment was done for 5 consecutive days. Two hour or 20 days after last administration, spleens were isolated for RT-PCR and real time PCR, and serum was collected for ELISA. In the combination treated group, TNF alpha, MIP-1 alpha, p22 phox, p47 phox, and gp91 phox mRNA expressions were lower than rebamipide treated or colchicine treated groups by reverse transcriptase PCR. NADPH oxidase subunits mRNA were markedly downregulated compared to the colchicine treated group by real time PCR. At 20 days after administration, combination treatment decreased 23.5% of the severity score compared to before administration. In contrast, colchicine treatment decreased 14.3% of the severity score compared to before administration. Rebamipide helped the function of colchicine to improve the HSV induced BD-like symptoms by inhibiting the expression of NADPH oxidase in vivo mouse model.
Keywords: Rebamipide; Behcet's disease; Herpes simplex virus; Mouse model; NADPH oxidase;

Differential involvement of cyclooxygenase isoforms in neutrophil migration in vivo and in vitro by Gustavo Batista Menezes; Rafael Machado Rezende; Pedro Elias Marques Pereira-Silva; André Klein; Denise Carmona Cara; Janetti Nogueira Francischi (118-122).
Pretreatment using celecoxib, a cyclooxygenase (COX) 2 inhibitor, or indomethacin, a nonselective COX inhibitor, reduced lypopolyssaccharide (LPS)-induced leukocyte migration to the rat peritoneal cavity. The effect of celecoxib (12 mg/kg) or indomethacin (2 mg/kg) on neutrophil chemotaxis induced by formyl-methionyl-leucyl-phenylalanine (FMLP) in an in vitro chemotactic assay (Boyden chamber) was investigated. Celecoxib and indomethacin inhibited chemotaxis induced by FMLP (Control = 26.6 ± 1.45, Celecoxib = 12.8 ± 3.04, Indomethacin = 6.26 ± 2.19 cells/field). When observed under intravital microscopy, a mouse cremaster preparation was used to assess the microvasculature to further investigate which step of cell recruitment was affected by these drugs. Celecoxib and indomethacin inhibited leukocyte migration induced by 0.05 μg/kg LPS injected into the cremaster muscle. However, the effect of celecoxib was associated with reduced cell rolling and adhesion, whereas indomethacin was only effective at inhibiting cell adhesion. Furthermore, SC560 pretreatment (a COX-1 selective inhibitor) of normal or LPS-challenged tissues did not alter leukocyte migration or cell adhesion, but it did enhance leukocyte rolling activity in both cases. Taken together, these results indicate that: 1) COX-1 activity is mainly related to leukocyte traffic under physiological conditions, and 2) COX-2 activity is mainly related to cell traffic under inflammatory conditions in vascular beds, suggesting a possible effect of selective COX-2 inhibitors on the expression of adhesion molecules.
Keywords: Leukocyte recruitment; Cyclooxygenase inhibitors; Boyden chamber; Intravital microscopy;

Blockade of cytosolic phospholipase A2 and 5-lipoxygenase activation in neutrophils by a natural isoflavanquinone abruquinone A by Mei-Feng Hsu; Ling-Chu Chang; Sheng-Chih Chen; Sheng-Chu Kuo; Hsiao-Yun Lee; Min-Chi Lu; Jih-Pyang Wang (123-131).
Abruquinone A, a natural isoflavanquinone, suppressed A23187- and formyl-Met-Leu-Phe (fMLP)-induced production of thromboxane B2 and leukotriene B4 from rat neutrophils. This compound failed to inhibit the enzymatic activity of ram seminal vesicles cyclooxygenase (COX) and human recombinant 5-lipoxygenase (5-LO) in cell-free systems. Abruquinone A diminished the arachidonic acid release from [3H]arachidonic acid-loaded neutrophils stimulated with either fMLP or A23187, whereas it had no inhibitory effect on the cytosolic phospholipase A2 (cPLA2) activity of neutrophil cytosolic fraction. Based on the Western blot analysis, the nuclear membrane recruitment of cPLA2 and 5-LO was inhibited by abruquinone A in A23187- as well as in fMLP-stimulated cells. Moreover, the phosphorylation of both cPLA2 and extracellular signal regulated kinases (ERKs) induced by fMLP and A23187 was attenuated by abruquinone A in a parallel concentration-dependent manner. Abruquinone A attenuated both fMLP- and ionomycin-mediated [Ca2+]i elevation in a concentration range that inhibited the recruitment of cPLA2 to nuclear membrane. These results indicate that the blockade of leukotriene B4 production by abruquinone A implicates the attenuation of 5-LO membrane translocation. Inhibition of thromboxane B2 production by abruquinone A is due to the attenuation of cPLA2 membrane recruitment and/or cPLA2 phosphorylation through the blockade of [Ca2+]i elevation and ERK activation, respectively.
Keywords: Neutrophil; Abruquinone A; Cytosolic phospholipase A2; Arachidonic acid release; Extracellular signal regulated kinase; Intracellular free-Ca2+; 5-Lipoxygenase;