European Journal of Pharmacology (v.487, #1-3)

Regulation of urokinase plasminogen activator by epigallocatechin-3-gallate in human fibrosarcoma cells by Mi H. Kim; Min A. Jung; Young S. Hwang; Min Jeong; Seok M. Kim; Sung J. Ahn; Boo A. Shin; Bong W. Ahn; Young D. Jung (1-6).
(−)-Epigallocatechin-3-gallate (EGCG), a main flavanol of green tea, potently suppressed the urokinase-type plasminogen activator (uPA) expression in human fibrosarcoma HT 1080 cells. EGCG induced not only the suppression of the uPA promoter activity but also the destabilization of uPA mRNA. EGCG inhibited the phosphorylation of extracellular signal-regulated kinases 1 and 2 (Erk-1/2) and P38 mitogen-activated protein kinase (MAPK), but not the phosphorylation of c-jun N-terminal kinase (JNK) and Akt. Specific inhibitors of Erk-1/2 (2′-amino-3′-methoxyflavone, PD98059) and P38 MAPK (pyridinylimidazole, SB203580) were found to suppress the uPA expression and the uPA promoter activity. However, the specific inhibitors did not affect the uPA mRNA stability. These results suggest that EGCG could regulate the uPA expression by at least two different mechanisms: EGCG may inhibit the Erk-1/2 and P38 MAPK, leading to suppression of the uPA promoter activity, and EGCG may destabilize the uPA mRNA in an Erk-1/2- and p38 MAPK-independent way.
Keywords: EGCG ((-)-epigallocatechin-3-gallate); uPA (urokinase-type plasminogen activator); Erk-1/2; P38 MAPK; Transcription; mRNA stability;

Effects of carbocisteine on altered activities of glycosidase and glycosyltransferase and expression of Muc5ac in SO2-exposed rats by Yuji Ishibashi; Fumiyoshi Kobayashi; Akira Idesawa; Akiyoshi Taniguchi; Shigeki Matsuzawa (7-15).
Carbocisteine is a mucoregulatory drug regulating fucose and sialic acid contents in mucus glycoprotein. To investigate the mechanism of carbocisteine action, we evaluated the effects of carbocisteine on the activity of fucosidase, sialidase, fucosyltransferase and sialyltransferase, and on the expression of Muc5ac mRNA in the airway epithelium of SO2-exposed rats. Wistar rats were repeatedly exposed to a 300-ppm SO2 gas for 44 days. Carbocisteine (125 and 250 mg/kg ×2/day) was administered for 25 days after 20 days of SO2 gas exposure. These enzyme activities were measured by fluorogenic substrate or glycoproteinic exogenous acceptor method. The expression levels of Muc5ac mRNA and protein were determined with real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Carbocisteine (250 mg/kg ×2/day) inhibited all the changes in these enzyme activities and the expressions of Muc5ac mRNA and protein in the lung after repeated SO2 exposure. These findings suggest that carbocisteine may normalize fucose and sialic acid contents in mucin glycoprotein through regulation of these enzyme activities, and inhibition of both Muc5ac mRNA and protein expressions in SO2-exposed rats.
Keywords: Carbocisteine; Fucosidase; Sialidase; Fucosyltransferase; Sialyltransferase; Muc5ac;

Pharmacological differentiation of the P2X7 receptor and the maitotoxin-activated cationic channel by Paul M. Lundy; Peggy Nelson; Lei Mi; Robert Frew; Sean Minaker; Cory Vair; Thomas W. Sawyer (17-28).
The ATP-P2X7 receptor subtype and a maitotoxin-activated ion channel were studied to determine factors which identify them as separate entities in the control of a cytotolytic pore. Activation of ATP-P2X7 receptors with 2′-3′-O-(benzylbenzyl) ATP (BzATP) or maitotoxin ion channels resulted in influx of ethidium bromide and cell death. Maitotoxin (25–250 pM)-induced ethidium bromide uptake and cell death was sensitive to extracellular Ca2+, the ionic composition of the buffer, reduced by the calmodulin inhibitor W7, (N-(s-aminohexyl)-5-chloro-1-naphthalenesulfonamide), (10–100 μM) but unaffected by the ATP-P2X7 receptor antagonist oxidized ATP, (adenosine 5′-triphosphate periodate oxidized sodium salt) (oATP). BzATP (10–200 μM)-induced ethidium bromide uptake and cell death were inhibited by oATP, unaffected by W7, inhibited by high ionic concentrations but only slightly dependant on external Ca2+. These results are consistent with the existence of a pharmacological mechanism for controlling cell death consisting of an ATP-P2X7 receptor, a maitotoxin-activated ion channel and a cytolytic pore.
Keywords: ATP-P2X7 receptor; Maitotoxin; Cytolytic pore; Cell death; Ca2+;

Effects of terpenoid phenol derivatives on calcium current in canine and human ventricular cardiomyocytes by János Magyar; Norbert Szentandrássy; Tamás Bányász; László Fülöp; András Varró; Péter P. Nánási (29-36).
Concentration-dependent (10–1000 μM) effects of terpenoid phenol derivatives were studied on L-type Ca2+ current in isolated canine and human ventricular cardiomyocytes using the whole-cell configuration of patch clamp technique. Carvacrol, thymol and eugenol suppressed peak Ca2+ current at +5 mV, having EC50 values and Hill coefficients of 98±11, 158±7 and 187±15 μM and 1.42±0.05, 2.96±0.43 and 1.6±0.1, respectively, in canine myocytes. Zingerone displayed a weak effect (estimated EC50: 2±0.37 mM, Hill coefficient: 0.73±0.07), while vanillin and guaiacol failed to substantially modify Ca2+ current up to the concentration of 1 mM. In addition to tonic block, thymol and carvacrol, but not eugenol, evoked marked rate-dependent block at 2 Hz. Carvacrol and eugenol accelerated inactivation of Ca2+ current and caused leftward shift in the voltage dependence of steady-state inactivation without altering activation kinetics. Carvacrol, but not eugenol, increased the time constant of recovery from inactivation. These effects of carvacrol and eugenol developed rapidly and were largely reversible. In myocytes isolated from undiseased human hearts, the effect of carvacrol was similar to that observed in canine cells. It is concluded that suppression of cardiac Ca2+ currents by phenol derivatives is influenced by the substituent in the benzene ring, and the blocking effect of these drugs may involve interactions with the inactivation machinery of the channel.
Keywords: Cardiac cell; Ca2+ current; Phenol derivative; Electrophysiology; Myocyte;

Single-channel activity of L-type Ca2+ channels reconstituted with the β2c subunit cloned from the rat heart by Yasuhiro Kamada; Yoichi Yamada; Michiaki Yamakage; Masato Nagashima; Masaaki Tsutsuura; Takeshi Kobayashi; Sumihiko Seki; Akiyoshi Namiki; Noritsugu Tohse (37-45).
We recently cloned the β2c subunit of the L-type Ca2+ channel as a functional type of β subunit from the rat heart. In order to clarify the contribution of the β2c subunit to native Ca2+ channel function, we investigated the single-channel properties of Ca2+ channels reconstituted with β2a or β2c subunits and compared them with the properties of native channels. In contrast to the Ca2+ channel with β2a subunit, long-lasting closings were dominant in the Ca2+ channel with β2c subunit and the native channel. The ensemble-averaged current of the cells with β2c subunits was comparable to that of the native cardiomyocytes. Many high P o sweeps (mode 2) were observed in the cells with β2a subunits, while only a few high P o sweeps were observed in the cells with β2c subunits and the native cells. These findings suggest that the β2c subunit is one of the functional β subunits in the rat heart.
Keywords: L-type Ca2+ channel; β2c Subunit; Single-channel analysis; Modal gating behavior;

Natural killer cell activity and anti-tumour effects of dehydrocrotonin and its synthetic derivatives by Patricia S. Melo; Giselle Z. Justo; Nelson Durán; Marcela Haun (47-54).
In this work, the anti-tumour properties of dehydrocrotonin and its derivatives were investigated in vitro and in vivo using the Ehrlich ascites tumour model. Treatment of Ehrlich ascites tumour-bearing mice with 20 mg/kg dehydrocrotonin for 4 days significantly increased survival, whereas administration of dehydrocrotonin derivatives was ineffective in affording protection. Compound IV exhibited little activity against Ehrlich tumour cells in vitro. Investigation of the effects of dehydrocrotonin treatment on total natural killer (NK) cell activity of tumour-bearing mice as a possible mechanism of dehydrocrotonin action in vivo revealed that this sesquiterpene lactone significantly improved NK cytotoxicity against YAC-1, a Moloney virus-induced mouse T-cell lymphoma of A/SN origin. As expected, tumour growth in non-treated mice markedly suppressed NK cell cytolysis. No effects on NK functional activity were observed in normal mice receiving dehydrocrotonin. In summary, only the natural compound exhibits anti-tumour efficacy and immunomodulatory actions in vivo, which may be related to its chemical structure.
Keywords: Dehydrocrotonin; Tumour; Cytotoxicity; Natural killer cell; Anti-tumour;

Mutational analysis of the histamine H1-receptor binding pocket of histaprodifens by Martijn Bruysters; Heinz H Pertz; Aloys Teunissen; Remko A Bakker; Michel Gillard; Pierre Chatelain; Walter Schunack; Henk Timmerman; Rob Leurs (55-63).
Histaprodifens constitute a new class of histamine H1-receptor agonists. These ligands can be regarded as hybrid molecules, consisting of a histamine moiety linked at the two-position of the imidazole ring by a propyl chain to two phenyl rings, one of the characteristic features of several H1-receptor antagonists. To delineate the binding site of various histaprodifen-like ligands, we generated mutant histamine H1 receptors, in which various amino acids, involved in the binding of either histamine or H1-receptor antagonists, were replaced by alanine. Wild-type and mutant H1 receptors were transiently expressed in African green monkey kidney cells (COS-7) and evaluated for their interaction with histamine and various histaprodifens by [3H]mepyramine radioligand-binding studies and by nuclear factor κB (NF-κB) reporter-gene assays. Our data show that, within the histamine H1-receptor binding pocket, histaprodifens interact with both agonist and antagonist binding sites, resulting in high affinity histamine H1-receptor agonists.
Keywords: Histaprodifen; Mutational analysis; Histamine H1 receptor; Agonist binding site; Antagonist binding site;

Action of the muscarinic toxin MT7 on agonist-bound muscarinic M1 receptors. by Maria C. Olianas; Abdu Adem; Evert Karlsson; Pierluigi Onali (65-72).
The muscarinic toxin MT7 is the most selective ligand for the muscarinic M1 receptors. Previous studies have shown that the toxin interacts with the antagonist–receptor complex and slows the antagonist dissociation rate, possibly by binding to an allosteric site and impeding the access to and egress from the orthosteric binding pocket. In the present study, we investigated the action of MT7 on agonist-occupied receptors in functional and radioligand binding assays of the cloned human muscarinic M1 receptor expressed in Chinese hamster ovary cells. In time-course experiments, the addition of MT7 rapidly blocked the acetylcholine-stimulated guanosine-5′-O-(3-[35S]thio)triphosphate binding to membrane G proteins. Similarly, in acetylcholine-treated cells MT7 completely stopped the agonist-stimulated [3H]inositol phosphate accumulation. In dissociation experiments using membranes pre-equilibrated with [3H]acetylcholine, the addition of MT7 increased the rate of radioligand dissociation. The data indicate that MT7, while partially stabilizing the antagonist–receptor complex, effectively destabilizes the agonist-occupied muscarinic M1 receptors.
Keywords: Dendroaspis angusticeps toxins; Muscarinic M1 receptor; [35S]GTPγS binding; Phosphoinositide hydrolysis; [3H]Acetylcholine binding; [3H]N-Methyl-scopolamine binding;

The mouse brain adenosine A1 receptor: functional expression and pharmacology by Maria C. Wittendorp; Jacobien von Frijtag Drabbe Künzel; Adriaan P. Ijzerman; Hendrikus W.G.M. Boddeke; Knut Biber (73-79).
The adenosinergic system is involved in many important physiological functions. Adenosine exerts its extracellular effects through four types of G-protein-coupled receptors: A1, A2A, A2B and A3. Adenosine acts as an important regulator of metabolic processes. In the brain adenosine mediates prominent neuroprotective functions via the adenosine A1 receptor. Whereas the pharmacological characteristics of the rat and human adenosine A1 receptor have been intensively studied, the mouse adenosine A1 receptor has not yet been characterised. Accordingly, we have cloned the mouse brain adenosine A1 receptor and present here a pharmacological characterisation of the mouse adenosine A1 receptor using functional studies and radioligand binding assays. The results show that the binding affinities of several ligands for the mouse adenosine A1 receptor are similar to the affinities for the rat and human adenosine A1 receptor with some exceptions.
Keywords: Adenosine receptor; Pharmacology; Brain; Mouse; cAMP assay;

KR-31378 protects neurons from ischemia–reperfusion brain injury by attenuating lipid peroxidation and glutathione loss by Sun-Ok Kim; In Sun Cho; Hee Kyoung Gu; Dong Ha Lee; Hong Lim; Sung-Eun Yoo (81-91).
Neuronal hyperexcitability and oxidative stress play critical roles in neuronal cell death in stroke. Therefore, we studied the effects of (2S,3S,4R)-Nʺ-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N′-benzylguanidine (KR-31378), possessing both antioxidant and K+ channel-modulating activities, on brain ischemia–reperfusion injury models. Treatment with KR-31378 (30 mg/kg, i.v.) significantly reduced infarct area and edema by 24% and 36%, respectively, in rats subjected to 2 h of middle cerebral artery occlusion and 22 h of reperfusion with significant attenuation of elevated lipid peroxidation (99% of normal) and glutathione loss (60% of normal) in ischemic hemisphere. We further studied its neuroprotective mechanism in fetal rat primary mixed cortical culture. Incubation of cortical neurons with KR-31378 protected FeSO4-induced cell death in a concentration-dependent manner (IC50=12 μM). Its neuroprotective effect was neither mimicked by other K+ channel openers nor abolished in the presence of ATP-dependent K+ channel (KATP) blockers, indicating that its effect was not related to K+ channel opening activity. The mechanism of protection is rather attributable to the antioxidant property of KR-31378 since it suppressed the intracellular accumulation of reactive oxygen species and ensured lipid peroxidation by 120% and 80%, respectively, caused by FeSO4. We further studied its effect on antioxidant defense, enzymatic and nonenzymatic systems. Treatment of neurons with FeSO4 resulted in decrease of catalase (8% of control) and glutathione peroxidase (14% of control) activities, which were restored by KR-31378 treatment (70% and 57% of control, respectively). In addition, it attenuated the depletion of glutathione contents (60% of control) caused by FeSO4. These results suggest that KR-31378 exerts a beneficial effect in focal ischemia, which may be attributed to its antioxidant property.
Keywords: Brain ischemia; Oxidative neuronal injury; KR-31378; Neuroprotection; Antioxidant; K+ channel opener;

Pharmacological characterisation of acid-induced muscle allodynia in rats by Alexander Norup Nielsen; Claus Mathiesen; Gordon Blackburn-Munro (93-103).
Previous studies have shown that repeated injections of acidic saline, given into the lateral gastrocnemius muscle of rats, results in a bilateral reduction in withdrawal threshold to tactile stimulation of the hindpaws. We have now characterised this model of muscoskeletal pain pharmacologically, by evaluating the antinociceptive effects of various analgesics after systemic administration. The μ-opioid receptor agonist morphine (3 and 6 mg/kg) produced a particularly prolonged antiallodynic effect. The glutamate receptor antagonists ([8-methyl-5-(4-(N,N-dimethylsulfamoyl)phenyl)-6,7,8,9,-tetrahydro-1H-pyrrolo[3,2-h]-iso-quinoline-2,3-dione-3-O-(4-hydroxybutyric acid-2-yl)oxime] NS1209 and ketamine (6 and 15 mg/kg, respectively), the KCNQ K+ channel openers retigabine and flupirtine (10 and 20 mg/kg, respectively) and the Na+ channel blocker mexiletine (37.5 mg/kg) also significantly increased paw withdrawal threshold, although to a lesser degree than morphine. In contrast, the anticonvulsant lamotrigine (30 mg/kg), the cyclooxygenase-2 inhibitor carprofen (15 mg/kg) and the benzodiazepine diazepam (3 mg/kg) were ineffective. All antinociceptive effects were observed at nonataxic doses as determined by the rotarod test. These results suggest that in this model, muscle-mediated pain can be alleviated by various analgesics with differing mechanisms of action, and that once established ongoing inflammation does not appear to contribute to this process.
Keywords: Central sensitisation; Fibromyalgia; GABA (gamma aminobutyric acid); Glutamate receptor antagonist; Muscle pain; Na+ channel blockers;

Mirtazapine-induced corelease of dopamine and noradrenaline from noradrenergic neurons in the medial prefrontal and occipital cortex by Paola Devoto; Giovanna Flore; Luigi Pira; Giorgio Longu; Gian Luigi Gessa (105-111).
The novel antidepressant mirtazapine has been shown to increase extracellular noradrenaline and dopamine in the medial prefrontal cortex. Our previous studies indicate that extracellular dopamine in the cerebral cortex originates largely from noradrenergic terminals, such release being controlled by α2-adrenoceptors. Because mirtazapine inhibits α2-adrenoceptors, the possibility that it might corelease dopamine and noradrenaline was investigated. By means of microdialysis, the effect of mirtazapine on extracellular dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and noradrenaline in the medial prefrontal cortex, densely innervated by dopaminergic and noradrenergic neurons, and in the occipital cortex, receiving equal noradrenergic but scarce dopaminergic projections, was compared. Basal extracellular concentration of noradrenaline was similar in both cortices, while dopamine in the occipital cortex was only about 50% lower than in the medial prefrontal cortex, reflecting noradrenergic rather than dopaminergic projections. The intraperitoneal (i.p.) administration of mirtazapine (5 and 10 mg/kg) increased extracellular dopamine, DOPAC and noradrenaline to approximately the same extent in both cortices, an effect totally suppressed by the α2-adrenoceptors agonist clonidine (0.15 mg/kg, i.p.). To exclude the possibility that mirtazapine-induced increase in dopamine might result from reduced dopamine removal from extracellular space, noradrenaline and dopamine uptake mechanisms were blocked by perfusing 100 μM desipramine into either cortex. The combined i.p. administration of mirtazapine (5 mg/kg) and the local perfusion of desipramine produced an additional increase in extracellular dopamine, DOPAC and noradrenaline in the medial prefrontal cortex and occipital cortex compared with the increase produced by either drug given alone. The results suggest that mirtazapine by inhibiting α2-adrenoceptors produces a corelease of noradrenaline and dopamine from noradrenergic terminals in the cerebral cortex.
Keywords: α2-Adrenoceptor antagonist; Desipramine; DOPAC; HPLC; Microdialysis; (Rat);

The clinical free radical scavenger, edaravone, protects cochlear hair cells from acoustic trauma by Tsuyoshi Takemoto; Kazuma Sugahara; Takeshi Okuda; Hiroaki Shimogori; Hiroshi Yamashita (113-116).
It is known that reactive oxygen species have toxicity to the cochlea. We investigated the effect of edaravone, a free radical scavenger for clinical use, on the cochleae of guinea pigs subjected to acoustic trauma. We assessed auditory brainstem response (ABR) thresholds to evaluate cochlear function and observed the sensory epithelium. After noise exposure (130 dB SPL, 3 h), we observed that the auditory brainstem response threshold shift in edaravone-treated ears was significantly less than that in untreated ears. This result suggests that edaravone protected the cochleae from acoustic trauma.
Keywords: Edaravone; Reactive oxygen species; Cochlear protection; Noise trauma;

Morphine withdrawal-induced c-fos expression in the heart: a peripheral mechanism by Ana González-Cuello; M.Victoria Milanés; M.Teresa Castells; M.Luisa Laorden (117-124).
We previously demonstrated that hyperactivity of cardiac noradrenergic pathways observed during morphine withdrawal is mediated by peripheral mechanisms. In the present study, naloxone methiodide (quaternary derivative of naloxone that does not cross the blood–brain barrier) and naloxone were administered to morphine-dependent rats and Fos immunostaining was used as a reflection of neuronal activity. Dependence on morphine was induced by 7-day chronic subcutaneous (s.c.) implantation of six morphine pellets (75 mg). Morphine withdrawal was precipitated by administration of naloxone methiodide (5 mg/kg, s.c.) or naloxone (5 mg/kg, s.c.) on day 8. Using immunohistochemical staining of Fos, present results indicate that the administration of naloxone methiodide or naloxone to morphine-dependent rats induced marked Fos immunoreactivity within the cardiomyocyte nuclei. Moreover, Western blot analysis revealed a peak expression of c-fos in the right and left ventricles after naloxone methiodide- or naloxone-precipitated withdrawal. In addition, in the hypothalamic paraventricular nucleus (PVN), Fos expression was increased after naloxone—but not after naloxone methiodide—administration to morphine-dependent rats. These results suggest that the activation of c-fos expression observed during morphine withdrawal in the heart is due to intrinsic mechanisms outside the central nervous system (CNS).
Keywords: Morphine withdrawal; Fos; Heart; Hypothalamic paraventricular nucleus; Naloxone; Naloxone methiodide;

8-OH-DPAT acts on both 5-HT1A and 5-HT7 receptors to induce hypothermia in rodents by Peter B Hedlund; Lisa Kelly; Curt Mazur; Timothy Lovenberg; J.Gregor Sutcliffe; Pascal Bonaventure (125-132).
Studies using selective drugs and knockout mice have demonstrated that the 5-HT7 receptor plays an instrumental role in serotonin-induced hypothermia. There is also evidence supporting an involvement of the 5-HT1A receptor, although mainly from studies using 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A/7 receptor agonist. Here we studied the effects of 8-OH-DPAT and selective antagonists for the 5-HT1A and 5-HT7 receptors on body temperature in rats, wild-type (5-HT7 +/+) mice and knockout (5-HT7 −/−) mice. At lower doses (0.3–0.6 mg/kg, i.p.), 8-OH-DPAT decreased body temperature in 5-HT7 +/+ mice but not in 5-HT7 −/− mice. At a higher dose (1 mg/kg, i.p.) 8-OH-DPAT induced hypothermia in both 5-HT7 −/− and 5-HT7 +/+ mice. The 5-HT1A receptor antagonist (S)-N-tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide (WAY-100135) (10 mg/kg, i.p.) inhibited the effect of 8-OH-DPAT at all doses in rats and mice. In 5-HT7 +/+ mice the selective 5-HT7 receptor antagonist (R)-3-(2-(2-(4-methylpiperidin-1-yl)-ethyl)pyrrolidine-1-sulfonyl)phenol (SB-269970) (10 mg/kg, i.p.) fully inhibited the hypothermia induced by 0.3 mg/kg 8-OH-DPAT, but not that of higher doses. In rats, SB-269970 caused a 60% inhibition of the hypothermia induced by 0.3 mg/kg 8-OH-DPAT. Thus, both 5-HT7 and 5-HT1A receptors are involved in a complex manner in thermoregulation, with the 5-HT7 receptor being more important at lower, possibly more physiological, concentrations.
Keywords: Thermoregulation; Hypothermia; Serotonin; In vivo; Knockout;

Effect of combined administration of 5-HT1A or 5-HT1B/1D receptor antagonists and antidepressants in the forced swimming test by Ewa Tatarczyńska; Aleksandra Kłodzińska; Katarzyna Stachowicz; Ewa Chojnacka-Wójcik (133-142).
In the present study, we examined effects of the selective serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitor citalopram, the 5-HT/noradrenaline reuptake inhibitor imipramine, the selective noradrenaline reuptake inhibitor desipramine or the monoamine oxidase-A inhibitor moclobemide, administered in combination with the 5-HT1A receptor antagonist N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridynyl)cyclohexanecarboxamide (WAY 100635) or the 5-HT1B/1D 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) and the 5-HT1B receptor antagonist N-[3-(2-dimethylamino) ethoxy-4-methoxyphenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-(1,1′-biphenyl)-4-carboxamide (SB 216641) in the forced swimming test in rats. When given alone, citalopram (20 and 30 mg/kg), imipramine (20 mg/kg), desipramine (20 mg/kg), moclobemide (20 mg/kg), WAY 100635 (0.1 and 1 mg/kg), GR 127935 (10 and 20 mg/kg) or SB 216641 (2 mg/kg) did not shorten the immobility time of rats. Co-administration of WAY 100635 (0.1 and 1 mg/kg) and citalopram (20 mg/kg), or imipramine (20 mg/kg), or moclobemide (20 mg/kg) did not affect the immobility time of rats, whereas WAY 100635 given jointly with desipramine (20 mg/kg) induced a weak anti-immobility effect. GR 127935 (10 and 20 mg/kg) or SB 216641 (2 mg/kg) co-administered with imipramine, desipramine or moclobemide, but not citalopram, produced a significant anti-immobility action in the forced swimming test in rats. These results indicate that the blockade of 5-HT1B rather than 5-HT1A receptors may facilitate the anti-immobility effect of imipramine, desipramine or moclobemide in the forced swimming test. No interaction was observed between 5-HT1A or 5-HT1B/1D receptor antagonists and citalopram.
Keywords: Citalopram; Imipramine; Desipramine; Moclobemide; 5-HT1A/5-HT1B receptor antagonist; Forced swimming test in rats;

Influence of central administration ATP-dependent K+ channel on morphine state-dependent memory of passive avoidance by Mohammad R Zarrindast; Mohammad R Jafari; Shamseddin Ahmadi; Bijan Djahanguiri (143-148).
Pre-training injection of a moderate dose of morphine (5–10 mg/kg) in a step-down passive avoidance task induced state-dependent learning with impaired memory retrieval on the test day. The impairment of memory was restored after the pre-test administration of the same dose of the drug. We have studied the effect of intracerebroventricular administration of naloxone and KATP channel modulators (glibenclamide and diazoxide) on the test day on restoration of memory by morphine in mice. The effect of scopolamine on restoration of memory on the test-day by glibenclamide was studied as well. Naloxone pretreatment (0.006, 0.025 and 0.1 μg/mouse) reversed the effect of pre-test morphine administration. The KATP channel blocker, glibenclamide (0.1, 0.5 and 1 μg/mouse), showed effects similar to those of pre-test administration of morphine. Glibenclamide tended to potentiate the morphine response. Scopolamine (0.15 and 0.30 μg/mouse) prevented the effect of glibenclamide on the restoration of memory. The pre-test administration of different doses of diazoxide (1.7, 5 and 15 μg/mouse), a KATP channel opener, showed no effect on restoration of memory when used alone but decreased morphine state-dependence. Diazoxide blocked the effects of glibenclamide on memory restoration. It is concluded that KATP channel modulators may be involved, at least in part, in morphine state dependence through a cholinergic system mechanism.
Keywords: Morphine; Memory; Passive avoidance; Intracerebroventricular; KATP channel modulators; Scopolamine; (Mouse);

Relative roles of endothelial relaxing factors in cyclosporine-induced impairment of cholinergic and β-adrenergic renal vasodilations by Mahmoud M El-Mas; Mahmoud M Mohy El-Din; Sahar M El-gowilly; Fouad M Sharabi (149-158).
Vascular toxicity is a major adverse effect for the immunosuppressant drug cyclosporine A. The present study sought to characterize the relative roles of the endothelium-derived relaxing factors (nitric oxide, endothelium-derived hyperpolarizing factor [EDHF], and prostaglandins) in the cyclosporine-induced impairment of renovascular responsiveness to acetylcholine receptor or β-adrenoceptor activation. Changes evoked by cyclosporine in the responses to either vasorelaxant were evaluated in phenylephrine-preconstricted isolated perfused rat kidneys in the absence and presence of N G-nitro-l-arginine methyl ester (l-NAME, nitric oxide synthase inhibitor), tetraethylammonium (K+ channel blocker), or diclophenac (cyclooxygenase inhibitor). Acetylcholine (0.03–2 nmol) vasodilations were significantly inhibited by prior treatment with l-NAME, tetraethylammonium, or diclophenac, suggesting a role for nitric oxide, EDHF, and prostaglandins in acetylcholine vasodilations. Isoprenaline (0.125–4 μmol) vasodilations were inhibited by l-NAME and tetraethylammonium versus no effect for diclophenac. Cyclosporine (1–4 μM) produced a concentration-related inhibition of vasodilations relaxations produced by either vasodilator. Cyclosporine-induced inhibition of acetylcholine vasodilations was attenuated in tissues pretreated with l-NAME or tetraethylammonium but not diclophenac, implicating nitric oxide and EDHF in cyclosporine–acetylcholine interaction. On the other hand, the inhibition of isoprenaline vasodilations by cyclosporine was virtually abolished by l-NAME. In cyclosporine-treated kidneys, exposure to l-arginine, the substrate of nitric oxide synthesis, fully restored isoprenaline vasodilations to control levels and significantly increased acetylcholine vasodilations. It is concluded that the identity and relative contributions of endothelial factors to renal vasodilatory responses as well as to the inhibition of these responses by cyclosporine largely depend on the vasodilator stimulus.
Keywords: Cyclosporine; Perfused kidney; Endothelium-derived relaxing factor; Acetylcholine; Isoprenaline;

Mechanisms of antiplatelet and antithrombotic activity of midazolam in in vitro and in vivo studies by George Hsiao; Ming-Yi Shen; Duen-Suey Chou; Yi Chang; Lin-Wen Lee; Chien-Huang Lin; Joen-Rong Sheu (159-166).
Midazolam is widely used as a sedative and anesthetic induction agent. The aim of this study was to systematically examine the inhibitory mechanisms of midazolam in platelet aggregation. In this study, midazolam concentration-dependently (15 and 30 μM) inhibited platelet aggregation in washed human platelets stimulated by thrombin (0.05 U/ml). Midazolam (15 and 30 μM) also inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in platelets stimulated by thrombin (0.05 U/ml). In addition, midazolam (15 and 30 μM) increased the formation of cyclic AMP but not cyclic GMP or nitric oxide. The thrombin-evoked increase in pHi was markedly inhibited in the presence of midazolam (15 and 30 μM). Rapid phosphorylation of a platelet protein of molecular weight (Mr.) 47,000 (P47), a marker of protein kinase C activation, was triggered by thrombin (0.05 U/ml). This phosphorylation was markedly inhibited by midazolam (15 and 30 μM). Midazolam (30 μM) did not significantly reduce the electron spin resonance signal intensity of hydroxyl radicals in activated platelets. In the vivo study, intravenous injection of midazolam (10 μg/g) significantly prolonged the latent period of inducing platelet plug formation in mesenteric venules. These results indicate that midazolam can significantly prevent thrombus formation in vivo. Its antiplatelet activity may be involved in the inhibition of the activation of phospholipase C and the Na+/H+ exchanger and increased cyclic AMP formation. These lead to lower intracellular Ca+2 mobilization and phosphorylation of P47.
Keywords: Midazolam; Platelet aggregation; Phospholipase C; Cyclic AMP; Na+/H+ exchanger; Thrombosis;

Protein kinase A increases electrical stimulation-induced neuronal nitric oxide release in rat mesenteric artery by Mercedes Ferrer; Margarita Sánchez; Maria del Carmen Martı́n; Iván Márquez-Rodas; Maria Jesús Alonso; Mercedes Salaices; Gloria Balfagón (167-173).
The aim of this study was to analyse the possible influence of cyclic AMP–protein kinase A (cAMP–PKA) activation on neuronal nitric oxide (NO) release induced by electrical field stimulation in mesenteric arteries from Wistar Kyoto (WKY) rats. Western blot experiments demonstrated the expression of neuronal NO synthase (nNOS) in mesenteric artery from WKY rats; however, electrical field stimulation alone did not induce detectable NO release. Preincubation with forskolin allowed NO release induced by electrical field stimulation, which was abolished by: the neuronal toxine tetrodotoxin, the nNOS inhibitors 7-nitroindazole or N ω-propil-l-arginine (NPLA), and the PKA inhibitors N-(2-(p-Bromocinnamylamino) ethyl 5-isoquinolinesulfonamide hydrochloride (H-89) or (9R,10S,12S)-2,3,9,10,11, 12-Hexahydro-10-9-methyl-1-oxo-9,12-epoxy-1H-diindolo(1,2,3-fg:3,2,1k)pyrrolo(3,4-l)(1,6) benzodiazocine-10-carboxylic acid hexyl ester (KT-5720). Preincubation with prostacyclin also allowed the NO release induced by electrical field stimulation which was significantly decreased by: the neuronal toxine tetrodotoxin, the nNOS inhibitors 7-nitroindazole or NPLA, and the PKA inhibitors H-89 or KT-5720. The NOS inhibitor N ω-nitro-l-arginine methyl ester (l-NAME) did not modify the vasoconstrictor response induced by electrical field stimulation. However, in the presence of forskolin or prostacyclin, l-NAME increased the vasoconstrictor response to electrical field stimulation. These results indicate that forskolin and prostacyclin allow neuronal NO release induced by electrical field stimulation through a mechanism involving cAMP–PKA activation in rat mesenteric arteries.
Keywords: Nitrergic innervation; Mesenteric artery; cAMP; PKA (protein kinase A); Forskolin;

The mechanisms underlying the capsaicin-induced relaxation of the acetylcholine- as well as KCl-contraction were studied by measuring isometric force and phosphorylation of 20-kDa regulatory light chain subunit of myosin (MLC20) in ileal longitudinal smooth muscles of rats. Capsaicin relaxed acetylcholine- and KCl-stimulated preparations in a concentration-dependent manner; the former was less sensitive to capsaicin than the latter and maximum responses to capsaicin (a percentage of papaverine-induced relaxation) were 70.6±7.5%, n=10 and 97.1±0.9%, n=13, P<0.05, respectively. The response showed no desensitization. Like nifedipine, capsaicin relaxed the tissue precontracted with an agonist of L-type Ca2+ channels as well. The relaxant effect of capsaicin was not inhibited by capsazepine (a selective antagonist of vanilloid VR1 receptors), nitro-l-arginine, indomethacin, guanethidine, nor by inhibitors of soluble guanylate cyclase. Capsaicin inhibited acetylcholine-induced transient contraction in a Ca2+-free, EGTA solution. Phosphorylation of MLC20 (a percentage of phosphorylated to total MLC20) was increased 1 min after application of 10 μM acetylcholine (7.8±2.0%, n=6 vs. 22.6±3.2%, n=6) and of 65.9 mM KCl (2.2±0.3%, n=8 vs. 10.7±1.7%, n=12). Capsaicin reduced the KCl-induced increase more markedly than acetylcholine-induced increase in MLC20 phosphorylation. When the tissue was contracted for 20 min with acetylcholine, MLC20 phosphorylation was increased, and capsaicin reduced markedly the contraction and abolished MLC20 phosphorylation both elicited by acetylcholine. It is suggested that capsaicin relaxes the rat ileum via its direct action on smooth muscle, and that capsaicin inhibits contractile mechanisms involving extracellular Ca2+ influx via non-L-type Ca2+ channels, possibly via store-operated Ca2+ channels and Ca2+ release from intracellular storage sites. The effects of capsaicin on acetylcholine- and KCl-induced contraction could be explained by a decrease in MLC20 phosphorylation.
Keywords: Capsaicin; Capsazepine; Ca2+ influx; Myosin light chain phosphorylation;

Antiobesity effects of A-331440, a novel non-imidazole histamine H3 receptor antagonist by Arthur A. Hancock; Youssef L. Bennani; Eugene N. Bush; Timothy A. Esbenshade; Ramin Faghih; Gerard B. Fox; Peer Jacobson; Victoria Knourek-Segel; Kathleen M. Krueger; Merrill E. Nuss; Jia Bao Pan; Robin Shapiro; David G. Witte; Betty B. Yao (183-197).
Histamine affects homeostatic mechanisms, including food and water consumption, by acting on central nervous system (CNS) receptors. Presynaptic histamine H3 receptors regulate release of histamine and other neurotransmitters, and histamine H3 receptor antagonists enhance neurotransmitter release. A-331440 {4′-[3-(3(R)-(dimethylamino)-pyrrolidin-1-yl)-propoxy]-biphenyl-4-carbonitrile} is a histamine H3 receptor antagonist which binds potently and selectively to both human and rat histamine H3 receptors (K i≤25 nM). Mice were stabilized on a high-fat diet (45 kcal % lard) prior to 28-day oral b.i.d. dosing for measurement of obesity-related parameters. A-331440 administered at 0.5 mg/kg had no significant effect on weight, whereas 5 mg/kg decreased weight comparably to dexfenfluramine (10 mg/kg). A-331440 administered at 15 mg/kg reduced weight to a level comparable to mice on the low-fat diet. The two higher doses reduced body fat and the highest dose also normalized an insulin tolerance test. These data show that the histamine H3 receptor antagonist, A-331440, has potential as an antiobesity agent.
Keywords: Antiobesity; Histamine H3 receptor antagonist; Magnetic resonance imaging;

Expression of heat shock protein 70 and its mRNAs during ischemia–reperfusion in the rat prostate by Motoaki Saito; Lika Tominaga; Eiji Nanba; Ikuo Miyagawa (199-203).
We investigated the expression of heat shock protein (HSP) 70 and its mRNAs during ischemia–reperfusion in the rat prostate. Eight-week-old rats were divided into six groups: a control group, a 30-min ischemia group, and 30-min ischemia+30-min, 60-min, 1-day, and 1-week reperfusion groups (groups A, B, C, D, E, and F, respectively). In vivo real-time blood flow and HSP 70-1 and 70-2 mRNAs and proteins in the prostate were measured using laser Doppler flow meter, real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) methods, respectively. Clamping of the aorta decreased blood flow to 10% of the basal level. The expressions of HSP 70-1/2 mRNAs increased in groups B, C, and D, and decreased in groups E and F. The expression of HSP 70 proteins was increased after a short interval of increase in their mRNAs. Our data indicated that the expressions of HSP 70 proteins and their mRNAs are dramatically changed during ischemia–reperfusion in the rat prostate.
Keywords: Prostate; Ischemia–reperfusion injury; Heat shock protein 70; Real-time PCR; (Rat);

Muscarinic receptors mediating contraction of female mouse urinary bladder: effects of oestrogen by Sherie Ma; Margot E Story; Jocelyn N Pennefather (205-211).
Muscarinic receptors mediating contraction of bladder detrusor muscle from female mice were examined. Mice were untreated (A) or treated with oestradiol cypionate (200 μg/kg) 24 h (B) or 96 h (C) before experimentation, or were pregnant (day 17) (D). Saturation radioligand binding experiments using [3H]quinuclidinyl benzilate ([3H] QNB) indicated similar muscarinic receptor densities and affinities in bladders from groups A and B. Neither oestrogen treatment nor pregnancy altered pD 2 estimates for methacholine. Maximum responses to methacholine and high-K+ physiological salt solution (KPSS) were significantly greater (P<0.05) in tissues from groups C and D than in A and B. Potencies of other muscarinic receptor agonists were similar in groups A and B with an order of acetylcholine plus physostigmine (10 μM)≈methacholine plus physostigmine (10 μM)>methacholine≈acetylcholine>bethanechol. Antagonist pK B estimates were similar in bladders from groups A and B with a rank order of: atropine≥4-diphenyl acetoxy-N-methyl piperidine methiodide>parafluorohexahydrosiladifenidol≈pirenzepine>himbacine, implicating muscarinic M1 and/or M5 as well as muscarinic M3 receptors in mediating methacholine-induced bladder contraction.
Keywords: Bladder; Female mouse; Oestrogen; Muscarinic M1, M3 and M5 receptors; Pregnancy;

6ʺ-Azidohex-2ʺ-yne-cannabidiol: a potential neutral, competitive cannabinoid CB1 receptor antagonist by Adèle Thomas; Ruth A Ross; Bijali Saha; Anu Mahadevan; Raj K Razdan; Roger G Pertwee (213-221).
Previous experiments with the mouse vas deferens have shown that cannabidiol produces surmountable antagonism of cannabinoid CB1 receptor agonists at concentrations well below those at which it binds to cannabinoid CB1 receptors and antagonizes α1-adrenoceptor agonists insurmountably. It also enhances electrically evoked contractions of this tissue. We have now found that subtle changes in the structure of cannabidiol markedly influence its ability to produce each of these effects, suggesting the presence of specific pharmacological targets for this non-psychoactive cannabinoid. Our experiments were performed with cannabidiol, 6ʺ-azidohex-2ʺ-yne-cannabidiol, abnormal-cannabidiol and 2′-monomethoxy- and 2′,6′-dimethoxy-cannabidiol. Of these, 6ʺ-azidohex-2ʺ-yne-cannabidiol was as potent as cannabidiol in producing surmountable antagonism of (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (R-(+)-WIN55212) in vasa deferentia. However, it produced this antagonism with a potency that matched its cannabinoid CB1 receptor affinity, suggesting that, unlike cannabidiol, it is a competitive cannabinoid CB1 receptor antagonist. Moreover, since it did not enhance the amplitude of electrically evoked contractions, it may be a neutral cannabinoid CB1 receptor antagonist.
Keywords: Cannabidiol; Abnormal-cannabidiol; WIN55212; Cannabinoid CB1 receptor; Novel cannabinoid receptor antagonist; Mouse vas deferens;

Preventive effect of teprenone on acute gastric mucosal lesion progression in compound 48/80-treated rats by Yoshiji Ohta; Takashi Kobayashi; Kazuo Inui; Junji Yoshino; Akira Kitagawa; Saburo Nakazawa (223-232).
The preventive effect of teprenone (6,10,14,18-teramethyl-5,9,13,17-nonadecatetaene-2-one), an anti-ulcer drug, on acute gastric mucosal lesion progression was examined in rats with a single intraperitoneal (i.p.) injection of compound 48/80 (0.75 mg/kg). Teprenone (20, 100 or 200 mg/kg), which was orally administered 0.5 h after compound 48/80 treatment at which time gastric mucosal lesions appeared, prevented gastric mucosal lesion development at 3 h after the treatment dose-dependently. Gastric mucosal tissues of compound 48/80-treated rats showed increases in myeloperoxidase (an index of neutrophil infiltration) and xanthine oxidase activities and thiobarbituric acid reactive substances (an index of lipid peroxidation) content and decreases in Se-glutathione peroxidase activity and hexosamine and vitamin E contents at 3 h after the treatment. Post-administered teprenone attenuated all these changes dose-dependently. These results indicate that teprenone prevents acute gastric mucosal lesion progression in compound 48/80-treated rats possibly by suppressing gastric mucus depletion, neutrophil infiltration and oxidative stress in the gastric mucosal tissue.
Keywords: Compound 48/80; Gastric mucosal lesion; Teprenone; Gastric mucus; Neutrophil infiltration; Oxidative stress;

Effects of lipopolysaccharide on epithelium-dependent relaxation in coaxial bioassay by U.Burcin Ismailoglu; Inci Sahin-Erdemli; Arzu Sungur; Mustafa Ilhan (233-239).
This study investigated the effects of airway inflammation elicited by intraperitoneal and intratracheal lipopolysaccharide administration to guinea pigs on the activity of tracheal epithelium-derived relaxant factor (EpDRF). Acetylcholine induced epithelium-dependent relaxation in precontracted rat anococygeus muscle placed in guinea pig trachea (coaxial bioassay). Indomethacin, N-nitro-l-arginine methyl ester, aminoguanidine and l-canavanine did not alter this relaxation excluding the role of prostaglandins and nitric oxide (NO). Intraperitoneal lipopolysaccharide potentiated the acetylcholine response, which was reversed by aminoguanidine and l-canavanine while intratracheal lipopolysaccharide inhibited acetylcholine-induced relaxation. Lipopolysaccharide pretreatments did not cause epithelial damage but induced inflammatory cell infiltration. These results suggested that systemic lipopolysaccharide administration did not alter the EpDRF response but resulted in NO synthase induction, thus NO participated in relaxation to acetylcholine in coaxial bioassay system. On the other hand, airway inflammation induced by intratracheal lipopolysaccharide attenuated the synthesis/release of EpDRF without altering the epithelium morphology.
Keywords: Trachea; Anococcygeus muscle;

Author index (241-243).

Keyword index (245-249).