European Journal of Pharmacology (v.452, #2)

PSAB-OFP, a selective α7 nicotinic receptor agonist, is also a potent agonist of the 5-HT3 receptor by Lisa M Broad; Catherine Felthouse; Ruud Zwart; Gordon I McPhie; Kathy H Pearson; Peter J Craig; Louise Wallace; Richard J Broadmore; John R Boot; Martine Keenan; S.Richard Baker; Emanuele Sher (137-144).
5-Hydroxytryptamine 3 (5-HT3) and α7 nicotinic receptors share high sequence homology and pharmacological cross-reactivity. An assessment of the potential role of α7 receptors in many neurophysiological processes, and hence their therapeutic value, requires the development of selective α7 receptor agonists. We used a recently reported selective α7 receptor agonist, (R)-(−)-5′Phenylspiro[1-azabicyclo[2.2.2] octane-3,2′-(3′H)furo[2,3-b]pyridine (PSAB-OFP) and confirmed its activity on human recombinant α7 receptors. However, PSAB-OFP also displayed high affinity binding to 5-HT3 receptors. To assess the functional activity of PSAB-OFP on 5-HT3 receptors we studied recombinant human 5-HT3 receptors expressed in Xenopus oocytes, as well as native mouse 5-HT3 receptors expressed in N1E-115 neuroblastoma cells, using whole-cell patch clamp and Ca2+ imaging. Our results show that PSAB-OFP is an equipotent, partial agonist of both α7 and 5-HT3 receptors. We conclude that it will be necessary to identify the determinant of this overlapping pharmacology in order to develop more selective α7 receptor ligands.
Keywords: α7 Nicotinic receptor; 5-HT3 receptor; Cross-reactivity; Spiroazabicyclic;

Assessment of the cytoprotective role of adenosine in an in vitro cellular model of myocardial ischemia by Sandrine Bès; Blandine Ponsard; Mounia El Asri; Cindy Tissier; David Vandroux; Luc Rochette; Pierre Athias (145-154).
This work aimed to detect functional adenosine receptors in isolated rat cardiomyocytes and to study the influence of stimulation of these receptors in an in vitro model of ischemia. Cultures of cardiomyocytes were prepared from newborn rat ventricles. The contractions were photometrically monitored. In this preparation, adenosine induced a positive chronotropic response. This effect was reproduced by CGS 21680 (2-(4-[2-carboxyethyl]-phen-ethyl-amino) adenosine-5′N-ethylunosamide), a specific adenosine A2 receptor agonist, and antagonized by DMPX (3,7-dimethyl-1-propargylxanthine), an adenosine A2 receptor antagonist. However, R-PIA (R-N 6-(2-phenylisopropyl)-adenosine; a specific adenosine A1 receptor agonist) induced a negative chronotropic effect that was abolished by its corresponding adenosine A1 antagonist DPCPX (1,3-dipropyl-8-cyclo-pentyl-adenosine). Substrate-free hypoxia, as simulation of ischemia, induced a progressive decrease and then arrest of spontaneous cell contractions. The spontaneous rhythmic contractile activity was restored during reoxygenation following simulated ischemia. Adenosine A1 receptor stimulation with R-PIA induced a decrease of hypoxia-induced damage. This effect was antagonized by DPCPX, an adenosine A1 receptor antagonist. Conversely, the cells treated with CGS 21680 did not display complete recovery after reoxygenation. In addition, this effect was abolished by DMPX, since the cells recovered normal function after reoxygenation. To conclude, it appeared that cardiomyocytes possess both functional adenosine A1 and A2 receptors and that only the activation of adenosine A1 receptor had a cytoprotective effect against simulated ischemia-induced cardiac cell injury.
Keywords: Cardiomyocyte; Cell culture; Electrophysiology; Cell contraction; Adenosine receptor; Simulated ischemia-reperfusion; (Rat);

Mitogen-activated protein kinases (MAPKs), a family of signal transduction mediators important in a host of cellular activities, include the extracellular signal-regulated kinases Erk1 and Erk2. We determined whether 5-HT1A receptors activate Erk1/2 in rat brain in vivo, as they do in recombinant cell lines. In contrast to the effect in cells, the 5-HT1A receptor agonist 8-hydroxy-N,N-diproylaminotetralin (8-OH-DPAT) dose- and time-dependently decreased basal levels of phosphorylated Erk1/2 (phospho-Erk1/2) in rat hippocampus (ED50 ∼0.1 mg/kg, maximum ∼90%) without altering total Erk1/2. The effects were kinase-specific, as 8-OH-DPAT did not modify phosphorylated or total levels of the MAPKs c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAPK. Moreover, 8-OH-DPAT did not modify phospho-Erk1/2 in striatum or frontal cortex. The effect of 8-OH-DPAT was blocked by pretreatment with the selective 5-HT1A receptor antagonists N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY 100,635), 1-(2-methoxyphenyl)-4-(4-[2-phthalimido]butyl)piperazine (NAN-190) and 4-fluoro-N-(2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl)-N-(2-pyridinyl)benzamide dihydrochloride (p-MPPF), but not by the weak partial agonist/antagonist 8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro(4.5)decane-7,9-dione dihydrochloride (BMY 7378). Other 5-HT1A receptor agonists (buspirone, gepirone and ipsapirone) also reduced phospho-Erk1/2 levels in hippocampus. 8-OH-DPAT also reduced the levels of the upstream activator of Erk1/2, phosphorylated extracellular signal-regulated kinase kinase (phospho-MEK1/2), and at least one potential downstream target, the nuclear transcription factor phospho-Elk-1. The region- and kinase-specific effects suggest that the Erk1/2 signal transduction cascade is likely an important differential mediator of 5-HT1A receptor-regulated events in the central nervous system.
Keywords: 5-HT (5-hydroxytryptamine, serotonin); Hippocampus; 5-HT1A receptor; Phosphorylation; Erk1/2; MAP (mitogen-activated protein) kinase; Immunoblotting;

Previous studies indicate that in antinociceptive procedures employing thermal, chemical and electrical stimuli, opioids are generally more potent in male than female rodents. The purpose of the present study was to examine nociception and opioid antinociception in male and female rats using a mechanical nociceptive stimulus. Results indicated that males had a higher threshold for nociception, and in tests in which a constant pressure was applied to the hindpaw, the paw withdrawal latencies were consistently longer in males. Opioids with activity at the μ receptor, including levorphanol, morphine, dezocine, buprenorphine, butorphanol and nalbuphine, were generally more potent and/or effective in males. In contrast, sex differences were not consistently observed with the κ-opioid receptor agonists spiradoline, (5,7,8b)-N-methyl-N[2-1(1-pyrrolidinyl),1-oxaspiro[4,5]dec-8-yl benzeneacetamide (U69,593), trans-(±)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488), enadoline, ethylketocyclazocine, and nalorphine. These findings suggest that males and females differ in their responsiveness to mechanical nociception and that sex differences in sensitivity to κ-, but not μ-, opioid receptor agonists are specific to certain nociceptive stimulus modalities.
Keywords: Sex difference; μ-Opioid receptor agonist; κ-Opioids receptor agonist; Antinociception; Mechanical; (Rat);

We have previously shown the involvement of Na+ channel as well as N-type and P/Q-type Ca2+ channels in the oxygen and glucose deprivation-induced injury in rat cerebrocortical slices. In the present study, we investigated the influence of halothane on the cerebroprotective effects of a variety of Na+ and Ca2+ channel blockers in rat cerebrocortical slices. The hypoxic injury was attenuated by Na+ channel blockers including tetrodotoxin, lidocaine and dibucaine, and Ca2+ channel blockers, such as verapamil, ω-agatoxin IVA and ω-conotoxin GVIA. Halothane abolished the protective effects of lidocaine, dibucaine and verapamil, all of which block the respective cation channels in a voltage-dependent manner, without affecting the actions of tetrodotoxin, ω-agatoxin IVA and ω-conotoxin GVIA, which reveal voltage-independent blockade. On the other hand, the nitric oxide synthesis estimated from the extracellular cyclic GMP formation was elevated during exposure to hypoxia. All channel blockers tested here attenuated hypoxia-evoked nitric oxide synthesis. Halothane blocked almost completely these actions of lidocaine and verapamil. Moreover, the Na+ and Ca2+ channel blockade by these compounds, as determined by veratridine- and KCl-stimulated nitric oxide synthesis, respectively, was also reversed by halothane. These findings suggest that an anesthetic agent halothane reversed the Na+ and Ca2+ channel blockade of several voltage-dependent ion channel blockers, leading to the attenuation of their cerebroprotective actions. Therefore, the influence of halothane anesthesia should be taken into consideration for the evaluation of neuroprotective action of Na+ and Ca2+ channel blockers.
Keywords: Halothane; Ca2+ channel blocker; Na+ channel blocker; Hypoxia; Nitric oxide (NO) synthesis; Cerebral cortex;

In the present study, we investigated three drug-induced long-QT syndromes in isolated rabbit Purkinje fibers in order to identify the relationship of action potential duration (APD), triangulation of action potentials (APD90–APD40) and early afterdepolarizations. Isolated rabbit Purkinje fibers were superperfused in Tyrode solution with solvent, indapamide (1×10−4 M, an I ks blocker mimicking long QT1), dofetilide (1×10−9, 1×10−8 or 1×10−7 M, an I kr blocker mimicking long QT2) or anthopleurin (1×10−8 M, an inhibitor of the inactivation of the I Na+ current mimicking long QT3) (n=8 per group) for 25 min, and stimulated at 1 Hz for 20 min and at 0.2 Hz for another 5 min. Indapamide did not change APD and triangulation or elicit early afterdepolarizations even in the presence of β-adrenergic stimulation with isoproterenol. Dofetilide concentration-dependently prolonged APD90, increased triangulation and elicited early afterdepolarizations. Anthopleurin markedly increased APD90 as well as triangulation and elicited early afterdepolarizations. The induction of early afterdepolarizations by dofetilide and anthopleurin was associated with a prolongation of APD90 or an increase in triangulation, but not with a change in APD40. Moreover, the degree of the increase in the triangulation was larger than that of APD90 in long QT2 (dofetilide-induced) and long QT3 (anthopleurin-induced) models in isolated rabbit Purkinje fibers. Our present study indicates that rabbit Purkinje fibers can be used as long QT2 (dofetilide-mimicking) and LQT3 (anthopleurin-mimicking) syndrome models, and confirms that drug-induced long QT1 (indapamide-mimicking) is absent. Our present study also shows the relationship between a prolongation of APD90 or increase in triangulation and the induction of early afterdepolarizations with dofetilide (I kr blocker) and anthopleurin (I Na modulator) in isolated rabbit Purkinje fibers.
Keywords: APD (action potential duration); Triangulation; Afterdepolarization; Purkinje fiber; Long QT syndrome;

Effects of histamine and opioid systems on memory retention of passive avoidance learning in rats by Mohammad-Reza Zarrindast; Maryam Eidi; Akram Eidi; Shahrbano Oryan (193-197).
The present study investigated the effect of interactions between histamine receptor agents and the opioid peptidergic system on memory retention of passive avoidance learning in rats. Post-training intracerebroventricular (i.c.v.) injections were carried out in all the experiments. Administration of histamine (20 μg/rat) reduced, but the histamine H1 receptor antagonist, pyrilamine (20 and 50 μg/rat), and the histamine H2 receptor antagonist, cimetidine (10 and 50 μg/rat), increased memory retention in rats. The histamine receptor antagonists decreased the response induced by histamine. Morphine (1–10 μg/rat) reduced, while pentazocine (5 and 10 μg/rat) or the opioid receptor antagonist, naloxone (5 and 15 μg/rat), increased memory retention. The combination of histamine with morphine showed potentiation. Effects of pyrilamine and cimetidine were attenuated by morphine. The responses to pentazocine and naloxone also were decreased by histamine. It is concluded that the histaminergic system has an interaction with opioidergic system that is involved in the memory retention process.
Keywords: Retention; Passive avoidance; Histaminergic agent; Opioid; (Rat);

Long-lasting change in 5-HT2A receptor-mediated behavior in rats after a single footshock by Takeshi Izumi; Katsuji Suzuki; Takeshi Inoue; Xiao Bai Li; Yuji Maki; Ihoko Muraki; Yuji Kitaichi; Syogo Hashimoto; Tsukasa Koyama (199-204).
To investigate the long-term functional change in the 5-HT2A receptor after acute stress, we examined the effect of single footshock on head shake behavior induced by the 5-HT2A receptor agent (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) in rats. Head shakes were evoked in a dose-dependent manner by 0.1–10 mg/kg of DOI, and the maximal response was attenuated by a single footshock given 24 h before. This suggests that there is a decrease in the number of functionally effective 5-HT2A receptors. The single footshock-induced reduction in head shakes evoked by DOI was observed immediately and 24 h after footshock, and lasted until 1 and 2 weeks after footshock. Because there were no changes in the [3H]ketanserin binding of the frontal cortex 1 week after footshock, decreases in head shakes were not due to the down-regulation of 5-HT2A receptors evoked by footshock.
Keywords: DOI; 5-HT2A receptor; Head shake; 5-HT (5-hydroxytryptamine, serotonin); Stress;

Role of endothelial adenosine receptor-mediated vasorelaxation in ethanol-induced hypotension in hypertensive rats by Moez Rekik; Mahmoud M El-Mas; Jamal S Mustafa; Abdel A Abdel-Rahman (205-214).
Our previous findings showed that chronic ethanol feeding lowers blood pressure in spontaneously hypertensive rats. The present study investigated the role of the adenosine receptor–endothelial nitric oxide (NO) pathway in the hypotensive response to ethanol. Changes in blood pressure were evaluated in radiotelemetered pair-fed rats receiving liquid diet with or without ethanol (2.5% or 5%, w/v) for 12 weeks. The vasorelaxant activity of the adenosine analogue 5′-N-ethylcarboxamidoadenosine (NECA) in isolated aortic rings obtained from ethanol and control rats were evaluated. Ethanol (2.5% and 5%) lowered blood pressure in a dose-dependent manner. The hypotension started at week 1, reached its maximum at week 4 and remained so thereafter. In aortas with intact endothelium, NECA (10−10 to 10−4 M) produced a concentration-dependent relaxation of the phenylephrine-precontracted aortas. Compared with control rats, ethanol (2.5% and 5%) caused significant and concentration-related increases in NECA responses. This effect of ethanol was attenuated by the adenosine receptor antagonist 8-sulfophenyltheophylline and the nitric oxide synthase inhibitor N G-monomethyl-l-arginine (l-NMMA). Further, endothelium denudation abolished the ethanol-evoked enhancement of NECA responses. The vasorelaxant responses to acetylcholine or sodium nitroprusside in aortic rings were not influenced by ethanol. In conclusion, the present findings suggest that chronic ethanol enhances the NO-dependent vasorelaxant responses to adenosine receptor activation and this may explain, at least partly, the mechanism of the hypotensive effect of ethanol in spontaneously hypertensive rats.
Keywords: Ethanol; NECA (5′-N-ethylcarboxamidoadenosine); Nitric oxide (NO); Blood pressure; Radiotelemetry; Aorta;

The effect of protein kinase C (PKC) inhibitors on the induction of endothelin ETB receptors during organ culture was examined in isolated segments of the rat mesenteric artery. After 24 h of organ culture, the endothelin ETB receptor agonist sarafotoxin 6c (S6c) induced a strong contraction compared to fresh segments. The contractile response after 24-h organ culture to S6c was studied in presence (30-min preincubation) or absence, after 24-h treatment, of the PKC inhibitors staurosporine, K252a and Ro31-7549. Exposure to staurosporine or K252a in presence and after 24-h treatment reduced the S6c contraction. In contrast, presence of 2-1[1-3(aminopropyl)indol-3-yl]-3(1-methyl-1H-indol-3-yl)maleimide (Ro31-7549), did not affect the S6c-induced contraction, whereas 24-h treatment abolished the increase of contraction. The PKA inhibitor N-(2-[bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H89) did not affect the S6c responses. The mRNA expressions of endothelin ETB receptors (analysed with real-time PCR) were abolished after 24-h treatment with the PKC inhibitors. These results suggest that PKC is involved in the endothelin ETB receptor upregulation following organ culture.
Keywords: PKC (protein kinase C); Endothelin; Sarafotoxin 6c; Endothelin ETB receptor; Ro31-7549; Staurosporine; K252a; H89;

The effect of anti-migraine compounds on nitric oxide-induced dilation of dural meningeal vessels by Simon Akerman; David J Williamson; Holger Kaube; Peter J Goadsby (223-228).
Migraine is characteristically accompanied by a throbbing quality of head pain thought to involve trigeminovascular afferents. Administration of nitric oxide (NO) donors provides the most reliable model of migraine induction in humans. The present studies used intravital microscopy to monitor the effect of local meningeal nerve stimulation and NO on dural blood vessels and any modulation of that effect by anti-migraine compounds. NO caused an immediate and reproducible dilation of meningeal blood vessels that was partially blocked by sumatriptan and indomethacin, while flunarizine and histamine H1 and H2 receptor antagonists were unable to block the dilation. Indomethacin also inhibited the neurogenic dilation while flunarizine did not. The present studies demonstrate that NO is unlikely to interact with histamine to produce its dilatory response. Sumatriptan and indomethacin inhibit the NO response by inhibiting trigeminal activation and calcitonin gene-related peptide (CGRP) release. Flunarizine does not modify either the neurogenic vasodilator response or the NO meningeal dilator response at least acutely.
Keywords: Migraine; Nitric oxide (NO); Meningeal artery; Intravital microscopy; Trigeminal;

We tested if there is a direct relationship between reactive oxygen species and citric acid-induced airway constriction. Guinea pigs were divided into two groups: control and dimethylthiourea (a hydroxyl radical scavenger). The animals in each group were further separated into four subgroups: baseline, recovery 2–3 min, recovery 10 min, and recovery 20 min. Each animal was anesthetized, cannulated, paralyzed, and artificially ventilated. Citric acid aerosol inhalation caused the following significant changes in the control group during the recovery period: airway constriction for at least 20 min, increases in luminol-amplified t-butyl hydroperoxide-initiated chemiluminescence counts in the bronchoalveolar lavage samples at 2–3 and 20 min, an increase in bronchoalveolar lavage fluid substance P level at 2–3 min, and elevations in the bronchoalveolar lavage fluid total cell and neutrophil numbers at 20 min. All citric acid-induced alterations were prevented by dimethylthiourea pretreatment. These results suggest that citric acid inhalation induces the initial release of reactive oxygen species and tachykinins, which causes further cellular infiltration and sustained airway constriction.
Keywords: Airway reactivity; Chemiluminescence count; Inflammatory cell; Tachykinin;

Antisecretory effect of somatostatin on gastric acid via inhibition of histamine release in isolated mouse stomach by Midori Komasaka; Syunji Horie; Kazuo Watanabe; Toshihiko Murayama (235-243).
Somatostatin is known to inhibit gastric acid secretion via both inhibition of histamine release from gastric enterochromaffin-like cells and direct inhibition of parietal cell function. We tried to clarify which of these two mechanisms plays a more important role in the inhibition of gastric acid section by somatostatin using isolated mouse stomach preparations. The gastric acid secretion stimulated by histamine was not inhibited by pretreatment with somatostatin (1 μM), but somatostatin abolished acid secretion induced by 4-[[[(3-chlorophenyl)amino]carbonyl]oxy]-N,N,N,-trimethyl-2-butynyl-1-aminium chloride (McN-A-343), a muscarinic M1 receptor agonist. In addition, the histamine-H2 receptor antagonist famotidine also completely inhibited the secretion stimulated by McN-A-343. Similarly, pretreatment with both somatostatin and famotidine completely abolished pentagastrin-induced acid secretion. Somatostatin partially inhibited the acid secretion stimulated by bethanechol. The late sustained acid secretion induced by bethanechol was reduced more strongly by somatostatin than the initial peak secretion. In addition, somatostatin had no effect on the transient increase in bethanechol-induced acid secretion in famotidine-pretreated preparations. Somatostatin had no effect on basal histamine secretion in isolated mouse stomach preparations, but markedly reduced histamine release induced by McN-A-343 and bethanechol. The present study showed that the acid secretory response via the endogenous histamine-mediated pathway was inhibited by somatostatin, but the response to a direct activation of gastric parietal cells was not. These results suggest that the inhibition of histamine release from enterochromaffin-like cells plays a more important role in the inhibition of gastric acid secretion by somatostatin than the direct inhibition of parietal cells. In addition, somatostatin inhibited the sustained acid secretion more strongly than the initial peak secretion after the cholinergic stimulation.
Keywords: Somatostatin; Gastric acid secretion; Histamine release; Gastric parietal cell; Enterochromaffin-like cell; Muscarinic acetylcholine receptor agonist; Stomach;

The subtypes of muscarinic receptors for neurogenic bladder contraction in rats by Hiroyasu Hirose; Ikuo Aoki; Toshifumi Kimura; Toru Fujikawa; Tomoshige Numazawa; Kaori Sasaki; Masaru Nishikibe; Kazuhito Noguchi (245-253).
We evaluated in vivo functional selectivity profiles for muscarinic M2 and M3 subtypes of four muscarinic antagonists: Compound A (a novel muscarinic receptor antagonist with M2-sparing antagonistic activity), darifenacin, (a muscarinic M3 receptor antagonist); methoctramine (a muscarinic M2 receptor antagonist) and tolterodine (a nonselective muscarinic receptor antagonist), and compared the inhibition potency on distention-induced bladder contraction in rats. In an in vivo functional study, Compound A (0.03–10 mg/kg, i.v.) showed antimuscarinic activity with high selectivity for M3 (salivation) over M2 (bradycardia) (>100-fold). Darifenacin (0.01–0.3 mg/kg, i.v.) showed only slight selectivity for M3 over M2 (3.7-fold). Methoctramine (0.003–1 mg/kg, i.v.) showed the reverse selectivity profile (0.077-fold). Tolterodine (0.003–0.3 mg/kg, i.v.) showed less selectivity (1.2-fold). Compound A at M3 inhibitory doses (0.1 and 0.3 mg/kg, i.v.) showed inhibition in a distention-induced neurogenic bladder contraction model, and its maximal inhibitory effects were about 60% at an even higher dose (3 mg/kg). Methoctramine at M2 inhibitory doses (0.03 and 0.1 mg/kg, i.v.) did not significantly affect distention-induced bladder contraction. When tolterodine and darifenacin caused inhibition of distention-induced bladder contraction, its maximal inhibitory effects were similar to that of Compound A. Therefore, these findings suggest that Compound A would be an excellent pharmacological tool to give a better understanding of which subtypes of muscarinic receptors act in bladder function so far, and muscarinic M3, but not M2, receptors mainly mediate the cholinergic component of distention-induced bladder contraction.
Keywords: Muscarinic receptor antagonist; Urinary bladder; Micturition reflex; Bradycardia; Salivation;