European Journal of Pharmacology (v.399, #1)

T-588 inhibits astrocyte apoptosis via mitogen-activated protein kinase signal pathway by Kazuhiro Takuma; Takashi Fujita; Yuji Kimura; Masato Tanabe; Akiko Yamamuro; Eibai Lee; Koichi Mori; Yutaka Koyama; Akemichi Baba; Toshio Matsuda (1-8).
The effect of (1R)-1-benzo[b]thiophen-5-yl-2-[2-(diethylamino)ethoxy]ethan-1-ol hydrochloride (T-588), a cognition enhancer, on reperfusion injury was studied in cultured rat astrocytes. T-588 at 1–10 μM partially protected astrocytes against reperfusion injury after exposure to Ca2+-free medium or hydrogen peroxide. Nerve growth factor (NGF) had a similar protective effect. Addition of both T-588 and NGF resulted in complete protection against Ca2+ reperfusion injury. T-588 did not stimulate NGF production in astrocytes. The effect of T-588 on Ca2+ reperfusion injury including apoptosis was inhibited by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase inhibitor 2′-amino-3′-methoxyflavone (PD98059), but not by the phosphoinositide 3-kinase inhibitor wortmannin. The effect of NGF was inhibited by PD98059 and wortmannin. T-588 stimulated rapidly the phosphorylation of ERK, but did not affect that of Akt in astrocytes. These findings suggest that the ERK MAP kinase pathway has a role in the protective effects of T-588 and NGF.
Keywords: Ca2+ reperfusion; T-588; NGF (nerve growth factor); PD98059; MAP (mitogen-activated protein) kinase; Astrocyte;

3-Morpholinosydnonimine (SIN-1) and K+ channels in smooth muscle cells of the rabbit and guinea pig carotid arteries by Jean-François Quignard; Michel Félétou; Catherine Corriu; Thierry Chataigneau; Gillian Edwards; Arthur H Weston; Paul M Vanhoutte (9-16).
Experiments were designed to determine the subtype of K+ channels activated by the nitrovasodilator 3-morpholinosydnonimine (SIN-1) in smooth muscle cells of the rabbit and guinea pig carotid arteries. Membrane potential was recorded in isolated segments with intracellular microelectrode and K+ currents in freshly dissociated smooth muscle cells, with the patch–clamp technique. In the guinea pig carotid artery, SIN-1 caused a glibenclamide-sensitive hyperpolarization. The nitrovasodilator did not affect the whole-cell K+ current, but activated a glibenclamide-sensitive K+ current. In the rabbit carotid artery, SIN-1 induced only an iberiotoxin-sensitive repolarization in phenylephrine-depolarized tissue and in isolated cells, enhanced the activity of an iberiotoxin-sensitive K+ current. These findings demonstrate that the population of K+ channels activated by nitric oxide (NO) is species-dependent and support the conclusion that, in the guinea pig carotid artery, in contrast to the rabbit carotid artery, the release of NO cannot account for the responses attributed to endothelium-derived hyperpolarizing factor (EDHF).
Keywords: 3-morpholinosydnonimine (SIN-1); Endothelium-derived hyperpolarizing factor (EDHF); K+ channel; K+ channel; Membrane potential;

α2-Adrenoceptor-stimulated GTPγS binding in rat brain: an autoradiographic study by H.Kevin Happe; David B Bylund; L.Charles Murrin (17-27).
Agonist-stimulated [35S]GTPγS binding by α2-adrenoceptors was examined in rat brain by autoradiography. Epinephrine, norepinephrine, dexmedetomidine and brimonidine stimulated [35S]GTPγS binding in a dose-dependent manner. Agonist-stimulated binding was blocked by the specific α2-adrenoceptor antagonist (1,4-benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride (RX821002). Each α2-adrenoceptor agonist stimulated [35S]GTPγS binding in the same brain regions, corresponding to α2-adrenoceptor distribution determined by [125I]para-iodoclonidine autoradiography. The order of antagonist potency (RX821002>idazoxan>rauwolscine>phentolamine>prazosin), and weak inhibition by propranolol and selective serotonin antagonists, indicate that epinephrine-stimulated [35S]GTPγS binding is mediated primarily by α2-adrenoceptors. Several antagonists increased [35S]GTPγS binding at very high concentrations, and this effect had anatomic and pharmacologic characteristics of binding mediated by 5-HT1A receptors. These studies demonstrate functional linkage of α2-adrenoceptors to G proteins in tissue sections, thus providing data on neuroanatomic localization and a means to examine drug specificity at α2-adrenoceptors in different brain regions.
Keywords: Signal transduction; G protein; α2-Adrenoceptor; G protein coupled receptor; Second messenger; 5-HT receptor; 5-HT1A receptor;

Clomethiazole is neuroprotective in a variety of animal models of ischaemic stroke, but the mechanism is unclear. This study examined whether clomethiazole is able to modify spreading depression elicited in rat hippocampal slices. When spreading depression was induced by superfusion with high K+ medium (50 mM), clomethiazole at 100 μM reduced its duration. Both the amplitude and duration of spreading depression were reduced at 200 μM. Clomethiazole at 200 μM tended to reduce the amplitude of the K+-induced shift in direct current (DC) potential but this was not statistically significant. When a pair of K+ pulses were presented, 30 min apart, the second produced a smaller DC potential than the first. Clomethiazole at 200 μM increased the size of the ratio of these responses. Superfusion with a hypoxic solution induced spreading depression observed as a shift in the DC field potential. The amplitude of this was decreased significantly by clomethiazole at 200 μM. With intracellular recordings, the effects of clomethiazole were quantified by measuring the time from the peak K+-induced depolarisation to the recovery of membrane potential following the period of hyperpolarisation. Clomethiazole did not reduce this period significantly. It is concluded that clomethiazole can reduce some forms of spreading depression, but only at the higher concentrations tested. It is unlikely that this effect contributes to its neuroprotective properties.
Keywords: Clomethiazole; Chlormethiazole; Spreading depression; Hippocampus; Hypoxia; Neuroprotection;

Interaction of topiramate with conventional antiepileptic drugs in mice by Mariusz Świąder; Jacek Kotowski; Maciej Gąsior; Zdzisław Kleinrok; Stanisław J. Czuczwar (35-41).
Topiramate [2,3:4,5-bis-O-(1-methyl-ethylidene-)-β-d-fructopyranose sulfamate], administered intraperitoneally (i.p.) up to 5 mg/kg, did not influence the threshold for electroconvulsions. In doses of 10–30 mg/kg, topiramate significantly raised the threshold. This novel antiepileptic drug, in subprotective doses, enhanced the protective activity of i.p. given valproate, carbamazepine, dihenylhydantoin and phenobarbital against maximal electroshock-induced convulsions in mice. The potentiation induced by topiramate (2.5–5 mg/kg) was most profound for carbamazepine and phenobarbital. The anticonvulsive activity of valproate and diphenylhydantoin was potentiated by topiramate only at 5 mg/kg. Topiramate (5 mg/kg) combined with valproate, phenobarbital and diphenylhydantoin did not alter their free plasma levels but its combination with carbamazepine resulted in an increased free plasma level of this antiepileptic drug. Treatment with topiramate (5 mg/kg) alone or in combination with the studied antiepileptics (providing 50% protection against maximal electroshock) resulted in no adverse effects, as measured in the chimney test (motor coordination) or passive avoidance task (long-term memory). In contrast, valproate administered alone at its ED50 against maximal electroshock impaired motor coordination. It is noteworthy that valproate and carbamazepine at their respective ED50 values of 248 and 11.2 mg/kg disturbed long-term memory. The results provide an experimental basis for rational polytherapy.
Keywords: Topiramate; Antiepileptic drug; Electroshock, maximal; Drug interaction; Seizure;

Exploratory behaviour and grooming after repeated restraint and chronic mild stress: effect of desipramine by Paolo S D'Aquila; Alessandra T Peana; Vittorio Carboni; Gino Serra (43-47).
In a previous study, we have recently shown that chronic treatment with desipramine either reduced or potentiated the locomotor response to the dopamine D2-like receptor agonist quinpirole, a behavioural response mediated by the mesolimbic dopamine system, depending on whether the animals were subjected, respectively, to repeated restraint or to chronic mild stress (different stressors randomly presented). In this study, we examined the interaction between prolonged exposure to either repeated restraint stress or chronic mild stress with the chronic administration of the antidepressant desipramine on two spontaneous behaviours, in which an involvement of the mesolimbic dopamine system has been suggested: novelty-induced exploratory activity and grooming. Exploratory activity in the open field was reduced by chronic mild stress regardless of the drug treatment, while it was not influenced by restraint stress. Desipramine reduced exploratory activity in rats subjected to restraint stress. Restraint stress increased grooming and desipramine reversed this effect, while increasing grooming in the chronic mild stress group. These findings suggest that antidepressants exert their effect by opposing the modifications induced by stress. The available experimental evidence is consistent with the hypothesis that an important role in the observed behavioural changes is played by the mesolimbic dopamine system.
Keywords: Antidepressant drug; Desipramine; Grooming; Locomotor activity; Mesolimbic dopamine system; Open field; Restraint stress; Chronic mild stress;

5-HT1B/D receptor agonist, SKF99101H, induces locomotor hyperactivity in the guinea pig by Michael F O'Neill; David R Dobson; Graham J Sanger (49-55).
Previous studies in guinea pigs have shown that while a serotonin 5-HT1B/D receptor agonist, GR46611, does not induce locomotor activation when given alone, it markedly enhances the locomotor response to selective 5-HT1A receptor agonists, 8-OH-DPAT and buspirone. In these studies, we found that another 5-HT1B/D agonist, 3-(2-dimethylaminoethyl)-4-chloro-5-propoxyindole hemifumarate (SKF99101H), significantly elevated locomotor activity in guinea pigs when given alone. We assessed the relative contribution of 5-HT11A and 5-HT1B/D receptors in the mediation of this effect.Activity was measured by photobeam interrupts in opaque Perspex cylinders linked to a computer. SKF99101H (20 mg/kg s.c.) significantly increased the locomotor activity in guinea pigs. The locomotor stimulant effect of SKF99101H (20 mg/kg s.c) was reversed by the selective 5-HT1B/D receptor antagonist N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)[1,1biphenyl]4-carboxamide (GR127935; 0.06–0.25 mg/kg s.c.). The 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride (WAY100635; 0.05–0.25 mg/kg s.c.), slightly but significantly attenuated the hyperactivity induced by SKF99101H. These findings suggest that 5-HT1B/D receptor agonists may require concomitant activation of 5-HT1A receptors to induce locomotor activity in guinea pigs. The 5-HT2A receptor antagonist 6[2-[4-[bis(4-fluorophenyl)methylene]-1-piperidinyl]-ethyl]-7-methyl-5H-thiazol[3,2-a]pyrimidin-5-one (ritanserin) had no effect on SKF99101H-induced hyperactivity, suggesting that these receptors are not involved in the mediation of SKF99101H-induced hyperactivity. SKF99101H-induced hyperactivity was significantly attenuated by the D1 dopamine receptor antagonist SCH 23390 (0.005–025 mg/kg), but not by the D2 dopamine receptor antagonist raclopride (0.25–1.0 mg/kg), possibly suggesting the selective involvement of D1 dopaminergic receptors in the mediation of the stimulant actions of the 5-HT1B/D agonist.
Keywords: 5-HT1B/D receptor; Locomotor activity; (Guinea pig); SKF99101H; GR127935; WAY100635;

Acute administration of the Ca2+ channel antagonist amlodipine has been shown to facilitate memory for several types of learning in adult animals and to improve retention in aging mice. This study reports three experiments investigating the effect of chronic amlodipine treatment on retention in mice. In the first experiment, groups of mice were treated with either amlodipine or vehicle once a day for 14 days prior to training on a spatial discrimination task. Immediately after training, animals were given a single dose of amlodipine or the vehicle and tested for retention 24 h later. Both groups showed facilitated retention, thereby demonstrating that chronic amlodipine treatment did not produce desensitization to the facilitating effects of a post training treatment. In the second experiment, chronic treatments were administered once daily for 14 days beginning 24 h after training on one-way active avoidance and retention was tested on day 15. Results showed that chronic amlodipine attenuated spontaneous forgetting, but surprisingly, a similar enhancement could be achieved by a single treatment administered 1 day after training. In the third experiment, amlodipine was given either before or immediately after 10 daily training sessions in the one-way active avoidance task. Results showed that chronic treatment accelerated rate of learning. These findings confirm the memory facilitating properties of amlodipine under conditions of chronic drug administration.
Keywords: Ca2+ channel antagonist; Amlodipine; Memory enhancement; Maze learning; Active avoidance; Chronic drug administration;

Effects of phorbol 12,13-diacetate on human isolated bronchus by Benjamin Sarriá; Consuelo Pedrós; Genaro Galán; Julio Cortijo; Esteban J Morcillo (65-73).
Protein kinase C appears to be involved in the regulation of airway contractility. Phorbol 12,13-diacetate (PDA; 0.01–10 μM), a protein kinase C activator, produced a transient relaxation followed by a sustained contraction of human isolated bronchus. Different protein kinase C inhibitors (calphostin C, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine) (H-7), nifedipine (NIF; 1 μM) or incubation with Ca2+-free medium, inhibited the spasmogenic response to phorbol, while ouabain (10 μM) suppressed only the initial relaxation. These results indicate that the initial relaxation, in response to PDA, is related to the activation of Na+/K+-ATPase, while the ensuing contraction depends on extracellular Ca2+ entry.Incubation with PDA (1–5 μM) depressed the maximal relaxation to theophylline and caffeine obtained at 37°C but augmented the spasmogenic responses to methylxanthines (10 mM) obtained in cooled preparations. These effects do not result apparently from increased extracellular entry of Ca2+, but instead, from facilitation of the release of Ca2+ from intracellular stores.
Keywords: Bronchus, human, isolated; Phorbol 12,13-diacetate; Theophylline; Caffeine;

Role of BKCa channels and cyclic nucleotides in synergistic relaxation of trachea by Kirsi Vaali; Liang Li; Tuula Lähteenmäki; Heikki Vapaatalo (75-84).
β-Adrenoceptor agonists, nitric oxide (NO), and NO donors have been shown to mediate their effects through large conductance Ca2+-activated K+ (BKCa) channels. The mechanism of the synergistic effect of the β2-adrenoceptor agonist, salbutamol, and an NO donor, sodium nitroprusside, was studied in guinea pig tracheal preparations. Salbutamol (0.1 nM) and sodium nitroprusside (0.33 μM) alone relaxed the acetyl-β-methylcholine chloride (methacholine)-contracted preparations only by 0.5% and 28%, respectively, but their combination caused a maximum of 60% relaxation (at 3 min), which stabilized to 40% (at 10 min). Iberiotoxin, a selective inhibitor of the BKCa channels, did not abolish the synergistic effect. 3-isobutyl-1-methylxanthine (IBMX) did not modify relaxation evoked by the drugs. Concentrations of cyclic nucleotides did not correlate with relaxations as a function of time. The mechanism of synergy remains to be clarified. The results show that NO is an important modulator in the relaxation of guinea pig trachea induced by β2-adrenoceptor agonists in vitro.
Keywords: Nitric oxide (NO); β2-Adrenoceptor agonist; Smooth muscle; cGMP; cAMP; Iberiotoxin;