European Journal of Pharmacology (v.515, #1-3)

Resveratrol inhibits angiotensin II-induced endothelin-1 gene expression and subsequent proliferation in rat aortic smooth muscle cells by Hung-Hsing Chao; Shu-Hui Juan; Ju-Chi Liu; Hung-Yu Yang; Elaine Yang; Tzu-Hurng Cheng; Kou-Gi Shyu (1-9).
Resveratrol is a phytoestrogen naturally found in grapes and is the major constituent of wine thought to have a cardioprotective effect. The aims of this study were to examine whether resveratrol alters angiotenisn II-induced cell proliferation and endothelin-1 gene expression and to identify the putative underlying signaling pathways in rat aortic smooth muscle cells. Cultured rat aortic smooth muscle cells were preincubated with resveratrol then stimulated with angiotensin II, after which [3H]thymidine incorporation and endothelin-1 gene expression were examined. The intracellular mechanism of resveratrol in cellular proliferation and endothelin-1 gene expression was elucidated by examining the phosphorylation level of angiotensin II-induced extracellular signal-regulated kinase (ERK). The inhibitory effects of resveratrol (1–100 μM) on angiotensin II-induced DNA synthesis and endothelin-1 gene expression were demonstrated with Northern blot and promoter activity assays. Measurements of 2′7′-dichlorofluorescin diacetate, a redox-senstive fluorescent dye, showed a resveratrol-mediated inhibition of intracellular reactive oxygen species generated by the effects of angiotensin II. The inductive properties of angiotensin II and H2O2 on ERK phosphorylation and activator protein-1-mediated reporter activity were found reversed with resveratrol and antioxidants such as N-acetyl-cysteine. In summary, we speculate that resveratrol inhibits angiotensin II-induced cell proliferation and endothelin-1 gene expression, and does so in a manner which involves the disruption of the ERK pathway via attenuation of reactive oxygen species generation. Thus, this study provides important insight into the molecular pathways that may contribute to the proposed beneficial effects of resveratrol on the cardiovascular system.
Keywords: Endothelin-1; Resveratrol; Angiotensin II; Smooth muscle cell; Reactive oxygen species; ERK (extracellular signal-regulated kinase);

Aripiprazole's low intrinsic activities at human dopamine D2L and D2S receptors render it a unique antipsychotic by Yoshihiro Tadori; Takashi Miwa; Katsura Tottori; Kevin D. Burris; Arlene Stark; Toyoki Mori; Tetsuro Kikuchi (10-19).
Aripiprazole is the first clinically approved atypical antipsychotic agent having dopamine D2 receptor partial agonist activities. To evaluate aripiprazole's agonist and antagonist properties, we established a Chinese hamster ovary cell line expressing high and low densities of the long and short isoforms of human dopamine D2 receptors, then compared its properties with 7-{3-[4-(2,3-dimethylphenyl)piperazinyl]propoxy}-2(1H)-quinolinone (OPC-4392), S(−)-3-(3-hydroxyphenyl)-N-n-propylpiperidine ((−)-3-PPP), and terguride (other partial agonists) using forskolin-stimulated cAMP accumulation as an index. In cells expressing high receptor densities, all partial agonists predominantly behaved as agonists. However, in cells expressing low receptor densities, the partial agonists showed significantly lower maximal effects than dopamine. Aripiprazole showed the lowest intrinsic activities. In addition, all compounds blocked the action of dopamine with a maximum effect equal to that of each compound alone. Aripiprazole's low intrinsic activities may account for the clinical finding that, unlike the other partial agonists, it is substantially active against both positive and negative symptoms of schizophrenia.
Keywords: Aripiprazole; Antipsychotic; Partial agonist; Dopamine D2 receptor; Receptor density; Schizophrenia;

Expression of the transient receptor potential vanilloid 1 (TRPV1) in LNCaP and PC-3 prostate cancer cells and in human prostate tissue by María G. Sánchez; Ana M. Sánchez; Beatriz Collado; Sophie Malagarie-Cazenave; Nuria Olea; María J. Carmena; Juan C. Prieto; Inés Díaz-Laviada (20-27).
Vanilloid receptor subtype-1 (TRPV1), the founding member of the vanilloid receptor-like transient receptor potential channel family, is a non-selective cation channel that responds to noxious stimuli such as low pH, painful heat and irritants.In the present study, we show, as means of reverse transcriptase-polymerase chain reaction and Western blot analysis, that the vanilloid TRPV1 receptor is expressed in the prostate epithelial cell lines PC-3 and LNCaP as well as in human prostate tissue. The kinetic parameters inferred from [125I]-resiniferatoxin binding were in concordance with data of TRPV1 receptors expressed in other tissues. The contribution of the endogenously expressed TRPV1 channel to intracellular calcium concentration increase in the prostate cells was studied by measuring changes in Fura-2 fluorescence by fluorescence microscopy. Addition of capsaicin, (R)-methanandamide and resiniferatoxin to prostate cells induced a dose-dependent increase in the intracellular calcium concentration that was reversed by the vanilloid TRPV1 receptor antagonist capsazepine. These results indicate that the vanilloid TRPV1 receptor is expressed and functionally active in human prostate cells.
Keywords: Vanilloids; Capsaicin; TRPV1; Prostate;

Trapidil suppresses platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell (VSMC) proliferation by inhibiting Raf-1/extracellular signal-regulated kinase (ERK) via cAMP/protein kinase A (PKA). We examined whether trapidil inhibits PDGF-induced VSMC migration and investigated its mechanisms of action. VSMC migration was inhibited to a similar extent by trapidil and forskolin. A PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H89) blocked the inhibition by forskolin to a greater degree than that by trapidil. Trapidil but not forskolin suppressed PDGF-stimulated RhoA activation. In the presence of both H89 and (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate, an inhibitor of Rho-associated kinase (ROCK), trapidil and forskolin inhibited migration to a similar extent. Thus, in addition to cAMP/PKA activation, trapidil inhibits RhoA/ROCK activation, which may be important in trapidil's inhibitory effect on migration.
Keywords: Rho GTP-binding protein; Angioplasty; Trapidil; Protein–serine–threonine kinase; Forskolin;

Kir6.1 subunit is one of the pore-forming components of KATP channel complex. The endogenous modulation of Kir6.1 subunit function has been largely unknown. Whether acetylcholine modulated the function of Kir6.1 subunit stably expressed in human embryonic kidney (HEK-293) cells was examined in the present study using the whole-cell patch-clamp technique. Acetylcholine from 1–100 μM concentration-dependently stimulated the heteologously expressed and PNU-37883A sensitive Kir6.1 channels (p  < 0.05). Co-expression of sulphonylurea receptor 1 subunit with Kir6.1 significantly inhibited the stimulatory effect of acetylcholine on KATP currents. Pretreatment of the transfected HEK-293 cells with atropine, α-bungarotoxin, mecamylamine, prazocine, propranolol, or dihydro-β-erythroidine hydrobromide did not alter the stimulatory effect of acetylcholine on Kir6.1 currents. When intracellular ATP was increased from 0.3 mM to 5 mM, acetylcholine at 10 μM still exhibited its stimulatory effect (−16.4 ± 2.3 to −25.5 ± 3.8 pA/pF, n  = 8, p  < 0.05). In conclusion, we have demonstrated an excitatory effect of acetylcholine on Kir6.1 channels, which is mediated neither by an acetylcholine receptor-dependent mechanism, nor by alteration in ATP metabolism.
Keywords: Acetylcholine; Neurotransmitter; KATP channel; Patch clamp; Smooth muscle cell; Stable transfection;

Induction of human cholesterol 7α-hydroxylase in HepG2 cells by 2,4,6-trihydroxyacetophenone by Juree Charoenteeraboon; Kasem Nithipatikom; William B. Campbell; Pawinee Piyachaturawat; Prapon Wilairat; Pornpimol Rongnoparut (43-46).
In animal the plasma cholesterol-lowering activity of 2,4,6-trihydroxyacetophenone (THA) is due to enhanced cholesterol 7α-hydroxylase (CYP7A1) activity. We have examined the effect of THA on CYP7A1 activity and mRNA level in HepG2 cells. THA stimulated CYP7A1 activity in a concentration- and time-dependent manner. After exposure for 24 h, 1 μM THA induced CYP7A1 activity 160 ± 8% and mRNA level 166 ± 21% (mean ± S.E.M.) of control. Moreover THA antagonized the inhibitory regulation of chenodeoxycholic acid on CYP7A1 mRNA expression. These results indicated that THA increases CYP7A1 activity in human HepG2 cells by stimulating mRNA transcription.
Keywords: Chenodeoxycholic acid; Cholesterol 7α-hydroxylase; CYP7A1; HepG2 cell; Induction; 2,4,6-Trihydroxyacetophenone;

[Nphe1,Arg14,Lys15]N/OFQ-NH2 is a competitive antagonist of NOP receptors in the periaqueductal gray by Lih-Chu Chiou; Yan-Yu Liao; Remo Guerrini; Girolamo Calo' (47-53).
Nociceptin/orphanin FQ (N/OFQ) and N/OFQ peptide (NOP) receptors are implicated in many physiological functions including pain regulation. This study quantitatively investigated the interaction of a novel NOP receptor antagonist, UFP-101 ([Nphe1,Arg14,Lys15]N/OFQ-NH2), with N/OFQ in the ventrolateral periaqueductal gray, a crucial midbrain area for pain regulation. N/OFQ concentration-dependently activated G-protein coupled inwardly rectifying K+ (GIRK) channels in ventrolateral neurons of periaqueductal gray slices. UFP-101 antagonized N/OFQ-induced GIRK channel activation in a concentration-dependent manner and produced a parallel shift of the concentration–response curve of N/OFQ. The pA 2 value estimated from Schild plot is 6.92 ± 0.06. At concentrations up to 1 μM, UFP-101 had no effect on membrane current per se and did not affect the GIRK current activated by [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin, a μ-opioid receptor agonist. It is concluded that UFP-101 is a potent and competitive peptide antagonist of NOP receptors that mediate GIRK channel activation in ventrolateral periaqueductal gray neurons.
Keywords: [Nphe1,Arg14,Lys15]N/OFQ-NH2; UFP-101; Nociceptin; Orphanin FQ; NOP receptor; Potassium channel; Periaqueductal gray;

The anticonvulsant effects produced by mixtures of oxcarbazepine and gabapentin (two second-generation antiepileptic drugs) in numerous fixed-ratio combinations of 1:1, 1:2, 1:5, 1:10, 1:15, and 1:20 were examined isobolographically in the mouse maximal electroshock seizure model. Results displayed that mixtures of both drugs at the fixed-ratios of 1:2, 1:5, 1:10, 1:15, and 1:20 exerted supra-additive (synergistic) interactions against electroconvulsions. Only a fixed-ratio of 1:1 was indifferent with isobolography, although the combination displayed the trend towards supra-additivity. Furthermore, the combinations of oxcarbazepine with gabapentin, administered at their median effective doses (ED50 mixs), did not alter motor performance of animals challenged with the chimney test. Additionally, neither gabapentin nor oxcarbazepine affected total brain concentrations of co-administered drug, indicating a pharmacodynamic nature of interaction between these antiepileptics. Finally, based on preclinical data presented here the combination of oxcarbazepine and gabapentin is of particular importance for further therapy in patients with refractory partial seizures.
Keywords: Oxcarbazepine; Gabapentin; Electroshock maximal; Isobolographic analysis; Pharmacodynamic interaction; Seizure;

Estradiol protects against alteration of protein kinase Cɛ in a binge model of ethanol dependence and withdrawal by Marianna Eunsun Jung; Stephanie Jacobs; Mridula Rewal; Andrew Wilson; James William Simpkins (62-72).
This study tested the hypothesis that a binge type of ethanol intake and ethanol withdrawal disturbs protein kinase C (PKC) homeostasis in a manner protected by 17β-estradiol. Ovariectomized rats implanted with 17β-estradiol or oil pellets received ethanol (7.5% weight/volume, 7 days) or control solution by a gavage method. The cerebelli were collected during ethanol exposure or ethanol withdrawal to assess the activity, protein levels, and cellular distribution of PKCɛ and total PKC, using an ATP phosphorylation and immunoblot assays. While both ethanol exposure and ethanol withdrawal increased membrane protein levels and membrane translocation, only ethanol withdrawal enhanced activity of PKCɛ. Ethanol withdrawal not ethanol exposure increased the three parameters of total PKC. 17β-Estradiol treatment prevented these changes in PKC profiles. These data suggest that an excessive episodic intake of ethanol followed by ethanol withdrawal disturbs PKC homeostasis and cellular distribution of PKC, in particular PKCɛ, in a manner that is protected by estrogen. PKCɛ appears more vulnerable during ethanol withdrawal than during ethanol exposure.
Keywords: Ethanol withdrawal; 17β-estradiol; Protein kinase Cɛ; Membrane translocation;

Effects of betahistine on the spatiotemporal response properties of vestibulospinal neurons to labyrinthine volleys by Massimo Barresi; Luca Bruschini; Guido Li Volsi; Diego Manzoni (73-82).
Betahistine, a drug used in the treatment of vestibular disorders, speeds-up the recovery from hemilabyrinthectomy in experimental animals, likely through the activation of histamine receptors. In order to better understand the mechanism of action of this drug we investigated, in adult, urethane anesthetized rats, whether betahistine modifies the spatial (directional) and temporal response properties of vestibular nuclear neurons to the labyrinthine input, as well as the convergence of different labyrinthine signals on single units. Extracellular single-unit activity was recorded from the caudal, spinal-projecting region of the vestibular nuclei during tilt of the animal, before and after i.p. injection of betahistine. The two orthogonal directions of maximal and minimal response to tilt, as well as the corresponding gains were determined for each neuron. Betahistine reduced the maximal response gain of units showing larger basal values of this parameter and increased it in neurons with smaller basal values, while the minimal response gain was on the average raised. These changes led to a significant decrease in the spatial specificity of the neurons, suggesting that betahistine affects the process of spatiotemporal convergence on vestibular units, likely through a rearrangement of the various inputs. This could be related to the effect of the drug on vestibular compensation.
Keywords: Betahistidine; Vestibular disorder; Electrophysiology;

We studied the effects of chronic oral nicotine on brain low affinity nicotine binding sites. Mice received nicotine in the drinking water for 4 or 7 weeks. Receptor binding was measured at 24 or 48 h after cessation of nicotine administration with [3H]methyllycaconitine, an antagonist in α7 and α3/α6β2β3* nicotinic receptors in striatum, midbrain, hippocampus and cortex. Chronic nicotine for 4 weeks resulted in a significant increase in the [3H]methyllycaconitine binding in the striatum and cortex, whereas after 7 weeks the increase in binding could be found in the hippocampus but not in the other brain areas studied. For comparison, high affinity nicotine binding sites (mostly α4β2) were measured with [3H]epibatidine after 7-week chronic nicotine treatment. [3H]Epibatidine binding sites were increased in the hippocampus, midbrain and cortex, but not in the striatum. The up-regulation of [3H]methyllycaconitine binding was significant at 24 h but that of [3H]epibatidine binding sites was not observed until at 48 h after cessation of chronic nicotine. These results suggest that up-regulation of low affinity nicotine binding sites does occur during chronic nicotine administration. Furthermore, the low affinity and high affinity binding differ clearly as regards regions and duration suggesting that different nicotinic receptors respond differently to nicotine administration.
Keywords: α7 nicotinic receptor; Neuronal nicotinic receptor; Nicotine; Receptor binding; Methyllycaconitine; Epibatidine;

Nicotine physical dependence in the mouse: Involvement of the α7 nicotinic receptor subtype by Sheri D. Grabus; Billy R. Martin; M. Imad Damaj (90-93).
Although chronic nicotine produces dependence in mice, the role of specific nicotinic receptors has not been examined in knockout animals. The present study utilized α7 nicotinic receptor knockout mice to explore the role of this receptor subunit in nicotine dependence. Mice were chronically exposed to nicotine (0 or 200 μg/ml) in their drinking water and assayed for somatic withdrawal signs, hyperalgesia (tail-flick and hot-plate) and spontaneous activity following nicotine cessation. Nicotine withdrawal produced increased somatic signs in both strains and hyperalgesia in wild-type, but not in knockout animals. These results indicate that the α7 nicotinic receptor subunit may mediate some aspects of nicotine dependence.
Keywords: Nicotine; Withdrawal; Hyperalgesia; Knockout;

Up-regulation of GABAB receptors by chronic administration of the GABAB receptor antagonist SCH 50,911 by Fabio Pibiri; Giovanni Carboni; Mauro A.M. Carai; Gian Luigi Gessa; M. Paola Castelli (94-98).
Chronic treatment of mice with the specific γ-aminobutyric acidB (GABAB) receptor antagonist (2S)(+)-5,5-dimethyl-2-morpholineacetic acid (SCH 50,911) increased both the number of GABAB receptors in the whole brain (measured as [3H]CGP 54626 [S-(R,R)]-3-[[1-(3,4-dichlorophenyl)amino]-2-hydroxypropyl](cyclohexylmethyl)phosphinic acid hydrochloride binding) and the ability of baclofen to activate GABAB receptor coupled G-protein (measured as % reduction of the EC50 of baclofen to activate [35S]GTPγS binding).The results indicate that persistent blockade of GABAB receptors leads to their compensatory up-regulation and suggest that GABAB receptors are tonically activated by endogenous GABA.
Keywords: GABAB receptor; GTPγS binding; SCH 50,911; Chronic treatment; (Mice);

Inhibition by agmatine on morphine-induced conditioned place preference in rats by Xiao-Li Wei; Rui-Bin Su; Xin-Qiang Lu; Yin Liu; Shou-Zhong Yu; Ben-Li Yuan; Jin Li (99-106).
Our previous studies demonstrated the ability of exogenous agmatine to inhibit tolerance to and physical dependence on morphine in mice, rats and monkeys. The present study further evaluated the effect of agmatine on the psychological dependence induced by morphine in conditioned place preference assay. Agmatine (0.75–20 mg/kg, s.c.) co-administered with morphine during the conditioning sessions completely abolished the acquisition of morphine-induced conditioned place preference in rats, which was associated with activation of imidazoline receptors. Agmatine (0.75–10 mg/kg, s.c.) administered on the test day inhibited the expression of the place preference. After 30 days of extinction of conditioned place preference, agmatine 2.5 and 40 mg/kg inhibited the priming effect of morphine 0.5 mg/kg on the place preference. Furthermore, agmatine inhibited the increased expression of FosB in the nucleus accumbens caused by chronic morphine. All these results suggest that agmatine could inhibit morphine-induced psychological dependence and relapses by affecting the expression of transcription factor FosB.
Keywords: Agmatine; Morphine; Conditioned place preference; Imidazoline receptor;

Effects of the 5-hydroxytryptamine (5-HT)1A/1B/2C receptor agonist N-[3-(trifluoromethyl)phenyl] piperazine (TFMPP, 0–3.0 mg/kg s.c.) and the 5-HT2C receptor agonist 8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one (WAY 161503, 0–3.0 mg/kg s.c.) in place conditioning were measured in male Sprague–Dawley rats. Effects of TFMPP, alone and with the 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl]-N-2-pyridinyl-cyclohexanecarboxamine (WAY 100635), the 5-HT1B receptor antagonist N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-1,1′-biphenyl-4-carboxamide (GR 127935) or the 5-HT2C receptor antagonist 6-chloro-5-methyl-1-[[2-(2-methylpyrid-3-yloxy)pyrid-5-yl]carbamoyl]indoline (SB 242084) and of WAY 161503 alone and with SB 242084 on locomotor activity were also assessed. Neither TFMPP nor WAY 161503 induced place conditioning. WAY 161503 (1.0 and 3.0 mg/kg s.c.) decreased locomotor activity; SB 242084 (1.0 mg/kg i.p.) blocked this effect. Reduced locomotor activity following TFMPP was blocked by SB 242084 but not WAY 100635 (0.1 mg/kg s.c.) or GR 127935 (3.0 mg/kg s.c.). Behaviourally relevant levels of 5-HT2C receptor stimulation may not exert reinforcing effects, although other studies indicate that such manipulations alter reinforcing effects of drugs of abuse.
Keywords: 5-HT2C receptor; Place conditioning; Locomotor activity; (Rat);

Dose response effects of lithium chloride on conditioned place aversions and locomotor activity in rats by Christine M. Tenk; Martin Kavaliers; Klaus-Peter Ossenkopp (117-127).
The present study examined the multi-variable locomotor activity effects of lithium chloride (LiCl) treatment in male rats. Of interest was a determination of which variables might show a dose–response relationship in LiCl-induced conditioned place aversions. Automated open-fields were partitioned into two chambers distinct in tactile and visual cues. A control group [n  = 8] received saline (NaCl; 0.15 M) paired with both chambers while three LiCl groups (0.15 M; 32 mg/kg [n  = 7], 95 mg/kg [n  = 7], 127 mg/kg [n  = 7]) received LiCl paired with the normally preferred chamber and saline paired with the non-preferred chamber. During extinction trials, rats were allowed to choose between the two chambers to provide an index of conditioned place aversions. Locomotor activity and its distribution within the chambers were also assessed during both conditioning and extinction trials. Dose-dependent decreases occurred in all measures of locomotor activity following LiCl administration during conditioning. During extinction trials, place aversions developed in animals conditioned with LiCl. LiCl-treated rats spent significantly less time in the LiCl-paired chamber relative to controls but not in a dose-dependent manner. Animals that had been conditioned with 95 or 127 but not 32 mg/kg LiCl, displayed significantly more vertical activity in the LiCl-paired chamber than controls during extinction trials. These findings indicate that, in addition to producing dose-dependent unconditioned effects on locomotor activity, LiCl also produces dose-dependent conditioned effects on vertical activity. These conditioned rearing response effects provide a valid measure of the conditioned avoidance response that provides evidence for dose-dependent LiCl-induced conditioned place aversions.
Keywords: Lithium chloride; Place conditioning; Biased conditioning apparatus; Nausea; Toxin; Long-Evans rat; Open-field;

Nicotine produces antidepressant-like actions: Behavioral and neurochemical evidence by Piotr Popik; Martyna Krawczyk; Tomasz Kos; Irena Nalepa; Marta Kowalska; Tadeusz Witarski; Lucyna Antkiewicz-Michaluk; Jerzy Vetulani (128-133).
Converging lines of evidence indicate the involvement of nicotinic acetylcholine receptors in depressive illness and antidepressant drug action. We investigated the effects of sub-chronic and chronic treatment with imipramine, nicotine and their combination on: (a) the ability of a dopamine-mimetic challenge to produce locomotor stimulation and (b) cortical density of β-adrenoceptors. One week of treatment with imipramine (10 mg/kg, twice daily) did not result in an altered response to the apomorphine (0.15 mg/kg) challenge, but after 2 weeks, the imipramine-treated rats demonstrated hyperactivity. Conversely, such increased locomotor response was observed in rats treated with nicotine (0.4 mg/kg, twice daily) for 1 but not for 2 weeks. Groups treated with nicotine + imipramine for 1 and 2 weeks demonstrated equally high hyperactivity in response to the apomorphine challenge. This effect was not different from the effects of 1-week treatment with nicotine or 2-week treatment with imipramine. The density of β-adrenoceptors was equally decreased by 2 (but not 1) weeks of the treatment with imipramine, nicotine and their combination. The present behavioral and neurochemical data suggest the antidepressant-like effect of the chronic treatment with nicotine. It appears that the potentiation of the dopamine-mimetic-induced hyperactivity cannot be explained by β-adrenoceptor down-regulation.
Keywords: Depression; Acetylcholine nicotinic receptor; Antidepressant; Imipramine; Dopamine-mimetic-induced locomotor stimulation; β-adrenoceptor down-regulation;

Dexamethasone prevents impairment of endothelium-dependent relaxation in arteries cultured with fetal bovine serum by Takahisa Murata; Natsuko Suzuki; Hideyuki Yamawaki; Koichi Sato; Masatoshi Hori; Hideaki Karaki; Hiroshi Ozaki (134-141).
In the present study, we assessed the effects of dexamethasone on fetal bovine serum-induced dysfunction of mesenteric endothelial cells using an organ culture procedure. In rabbit mesenteric arteries cultured in the presence of 10% fetal bovine serum for 7 days, the endothelium-dependent, nitric oxide (NO)-mediated relaxations caused by substance P and ionomycin were decreased as compared to those in non-treated arteries. Dexamethasone (3 μM) inhibited the proliferative stimuli-induced endothelial dysfunction without affecting the contractility or NO susceptibility of smooth muscle cells. Cross-sectioned hematoxylin–eosin staining and whole-mount CD31 staining indicated that chronic proliferative stimulation induced detachment of endothelial cells from the tunica intima in some regions, and also caused thickening of the arterial wall and shortening of the internal diameter. Endothelial NO synthesis (eNOS) mRNA expression was also decreased by the treatment with fetal bovine serum. The dexamethasone treatment did not inhibit the smooth muscle hypertrophy, but it inhibited the peeling of endothelial cells and recovered the eNOS mRNA expression. These results suggest that DEX ameliorate the impairments of arterial relaxation induced by proliferative stimuli and that these beneficial effects may be mediated by maintaining the adhesion of endothelial cells to the vascular wall and/or by recovering eNOS mRNA expression.
Keywords: Organ culture; Endothelium-dependent relaxation; Dexamethasone;

The effect of gliclazide and glibenclamide on preconditioning of the human myocardium by Mahmoud Loubani; Alan Fowler; Nicholas B. Standen; Manuel Galiñanes (142-149).
The cardioprotection of ischaemic preconditioning may be abolished in diabetic patients especially when some oral hypoglycaemics are used. The dose–response effect of gliclazide and glibenclamide on ischaemic preconditioning and the action of glibenclamide on signal transduction in human myocardium were investigated using right atrial appendages from cardiac surgery patients. Glibenclamide (0.1, 1, 3 and 10 μM) and gliclazide (1, 10, 30 and 100 μM) were added for 10 min prior to ischaemic preconditioning. The cardioprotection was abolished by glibenclamide at all concentrations and by gliclazide at supratherapeutic concentrations of 30 and 100 μM. Glibenclamide abolished the protective effect of mitoKATP channel opening but not that of protein kinase C (PKC) or p38 mitogen activated protein kinase (p38MAPK) activation. In conclusion, glibenclamide and gliclazide differential effects may be a result of differential sensitivities. Glibenclamide does not block protection conferred by either PKC or p38MAPK activation. These findings may have clinical implications in ischaemic heart disease.
Keywords: Gliclazide; Glibenclamide; Human myocardium; Preconditioning;

Endothelial mechanisms underlying responses to acetylcholine in the horse deep dorsal penile vein by Ana Cristina Martínez; Dolores Prieto; Medardo Hernández; Luis Rivera; Paz Recio; Albino García-Sacristán; Sara Benedito (150-159).
This study evaluates the mechanisms underlying endothelium-dependent responses to acetylcholine in horse deep dorsal penile veins. Acetylcholine-induced relaxation was abolished by endothelium removal, the soluble guanylyl cyclase-inhibitor, and the nitric oxide (NO) synthase inhibitors. Acetylcholine-induced relaxation was inhibited by high K+ concentrations and blockade of large-conductance Ca2+-activated potassium (BKCa) channels, and voltage-dependent potassium (Kv) channels. Relaxations were unaffected by a small-conductance KCa (SKCa) channel blocker, or an ATP-sensitive potassium (KATP) channel blocker. Relaxation in response to a NO donor was unaffected by KCa channel blockers, but inhibited by high K+ concentrations and a Kv channel blocker. In the presence of a NO synthase inhibitor, acetylcholine-induced contractions were inhibited by a cyclooxygenase blocker and abolished by endothelial removal. The contractile response was competitively inhibited by muscarinic receptor antagonists, high affinity M1 and M3 antagonists, while the M2 antagonist had no effect. The pharmacological profile suggests that acetylcholine contraction is mediated by muscarinic M1 receptors. Our findings indicate that acetylcholine-induced relaxation in the horse deep dorsal penile vein is essentially mediated by NO, acting via the cGMP-dependent pathway and opening of K+ channels. The contraction elicited by acetylcholine is prostanoid-mediated and induced by endothelial muscarinic M1 receptor activation.
Keywords: Acetylcholine; Nitric oxide; Cyclic GMP; Potassium channel; Prostanoid; Endothelial muscarinic receptor; Horse dorsal penile vein;

Comparison of the gastroprokinetic effects of ghrelin, GHRP-6 and motilin in rats in vivo and in vitro by Inge Depoortere; Benedicte De Winter; Theo Thijs; Joris De Man; Paul Pelckmans; Theo Peeters (160-168).
Ghrelin and motilin form a new family of structurally related peptides. We compared the gastroprokinetic effects of ghrelin, the ghrelin receptor agonist, growth hormone releasing peptide 6 (GHRP-6), and motilin in rats in vivo and in vitro.Ghrelin, GHRP-6 or motilin (10–150 μg/kg) were injected i.p. and the effects on gastric emptying and transit were measured after intragastric application of Evans blue. In antral and fundic strips the effect of motilin, ghrelin or GHRP-6 was studied during electrical field stimulation (EFS) in the absence and presence of NG-nitro-l-arginine methyl ester hydrochloride (l-NAME) (300 μM).Ghrelin and GHRP-6 but not motilin accelerated gastric emptying and transit in rats. Ghrelin was more potent than GHRP-6 and the dose–response relationship for ghrelin but not for GHRP-6 was bell-shaped. In fundic or antral strips, neural responses to EFS consisted of an on-relaxation that was reversed into a cholinergically mediated contraction by addition of the nitric oxide (NO)-synthase blocker, l-NAME. The post-stimulus off-contraction was cholinergically mediated. Under normal conditions, the ghrelin agonists reduced the on-relaxations in fundic strips and increased the cholinergic off-contractions in antral and fundic strips. The concentration response curves in muscle strips of the fundus were bell-shaped with maximal effects for ghrelin at 1.2 μM (on-responses) and 0.66 μM (off-responses) and for GHRP-6 at 0.50 μM (on-responses) and 0.26 μM (off-responses). No effects were observed with motilin between 1 nM and 0.1 μM. Studies in the presence of l-NAME confirmed the effect of the ghrelin agonists on cholinergic excitatory motor responses. No effects were observed with motilin under the different experimental conditions. The presence of growth hormone secretagogue receptor 1a transcripts in the strip preparations was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR).Ghrelin and GHRP-6 but not motilin accelerate gastric emptying and transit by activating cholinergic excitatory pathways in the enteric nervous system in addition to the known vagal pathways.
Keywords: Peptide hormone; Gastric emptying; Intestinal transit; Enteric nervous system;

Tacrolimus (FK506) has protective actions against murine bleomycin-induced acute lung injuries by Tadatsura Koshika; Yoshitaka Hirayama; Yoshitaka Ohkubo; Seitaro Mutoh; Akitoshi Ishizaka (169-178).
The effects of tacrolimus on murine acute lung injury were tested, especially in comparison to dexamethasone. Acute lung injury was induced by intratracheal instillation of bleomycin. Oral tacrolimus significantly improved survival rates of bleomycin-exposed mice, while cyclosporin A or dexamethasone did not. After instillation of bleomycin (day 0), a migration of neutrophils into alveolar spaces peaked on day 3, with concomitant increases of chemokines. On day 6, marked morphological changes in the lungs were observed. All these changes were significantly inhibited by tacrolimus. Furthermore, DNA ladder and immunohistochemical analyses of lungs showed that apoptosis of lung cells appeared on day 6 and was abolished only by the treatment of tacrolimus. These results suggest that both anti-inflammatory and anti-apoptotic action of tacrolimus contribute to improvement of bleomycin-induced acute lung injury.
Keywords: Neutrophil; MCP-1 (Monocyte chemoattractant protein-1); Dexamethasone; Acute lung injury; Cyclosporin A; Tacrolimus;

CEP-11004, an inhibitor of the SAPK/JNK pathway, reduces TNF-α release from lipopolysaccharide-treated cells and mice by John R. Ciallella; Michael Saporito; Soren Lund; Marcel Leist; Henrik Hasseldam; Natalie McGann; Charles S. Smith; Donna Bozyczko-Coyne; Dorothy G. Flood (179-187).
CEP-11004, a mixed lineage kinase (MLK) inhibitor, was examined for its effects on tumor necrosis factor-alpha (TNF-α) production in human THP-1 monocytes, mouse BV-2 microglia, and C57Bl/6 mice. CEP-11004 inhibited TNF-α secretion up to 90% in THP-1 cells incubated with 3 μg/ml lipopolysaccharide, with an IC50 of 137 ± 14 nM. CEP-11004 also inhibited TNF-α production in lipopolysaccharide-stimulated microglial cells, but did not inhibit the initial increase in TNF-α mRNA expression as measured by real-time polymerase chain reaction (PCR). The mitogen-activated protein kinases (MAPKs) phospho-c-jun N-terminal kinase (JNK), phospho-p38, and phospho-MAPK kinase 4 (MKK4) levels were increased in THP-1 cells following lipopolysaccharide treatment, and were reduced by CEP-11004 treatment. For in vivo studies, CEP-11004 was injected 2 h prior to lipopolysaccharide (20 mg/kg) administration. CEP-11004 significantly inhibited TNF-α production at doses of 1–10 mg/kg as measured by enzyme-linked immunosorbent assay (ELISA). These results suggest that MLK blockade may be useful in inhibiting pro-inflammatory cytokine production in a wide range of diseases.
Keywords: Mixed lineage kinase; Tumor necrosis factor-alpha; MAP kinase inhibition;

Erratum to “Prevention of progressive joint destruction in adjuvant induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR217840” [Eur. J. of Pharmacol. 508 (2005) 239–247] by Takeshi Ishikawa; Fusako Nishigaki; Susumu Miyata; Yoshitaka Hirayama; Kyoko Minoura; Junko Imanishi; Masahiro Neya; Tsuyoshi Mizutani; Yoshimasu Imamura; Yoshitaka Ohkubo; Seitaro Mutoh (188).

Author index (189-190).

Keyword index (191-194).