European Journal of Pharmacology (v.497, #2)

Carbamoylcholine homologs: synthesis and pharmacology at nicotinic acetylcholine receptors by Anders A. Jensen; Ivan Mikkelsen; Bente Frølund; Karla Frydenvang; Lotte Brehm; Jerzy W. Jaroszewski; Hans Bräuner-Osborne; Erik Falch; Povl Krogsgaard-Larsen (125-137).
In a recent study, racemic 3-(N,N-dimethylamino)butyl-N,N-dimethylcarbamate () was shown to be a potent agonist at neuronal nicotinic acetylcholine receptors with a high selectivity for nicotinic over muscarinic acetylcholine receptors [Mol. Pharmacol. 64 (2003) 865–875]. Here we present the synthesis and pharmacological characterization of a series of analogs of , where the methyl group at C-3 has been replaced by different alkyl substituents. Ring systems have been incorporated into the carbon backbone of some of the molecules, or the amino group has been build into ring systems. Furthermore, the (+)- and (−)-enantiomers of have been separated, and X-ray crystallography has revealed that (−)- possesses (S)-configuration. The compounds have been characterized pharmacologically at recombinant nicotinic receptor subtypes. The structure–activity relationship study has provided valuable insight into the mode of interactions of and its analogs with neuronal nicotinic acetylcholine receptors.
Keywords: Nicotinic acetylcholine receptor; Acetylcholine; Agonist; Carbamoylcholine homolog; Muscarinic;

Ionotropic GABA receptors with mixed pharmacological properties of GABAA and GABAC receptors by Kristin Hartmann; Frank Stief; Andreas Draguhn; Christiane Frahm (139-146).
Ionotropic γ-aminobutyric acid (GABA) receptors form a large family of molecular isoforms with distinct properties. We have characterized a distinct new type of GABA receptors in CA1 pyramidal cells in rat hippocampal slices. Somatic application of GABA induced currents which were partially suppressed by (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), a specific antagonist of GABAC receptors. This sensitivity was enhanced when we evoked the currents by the GABAC receptor agonist cis-4-aminocrotonic acid (CACA). However, both GABA- and CACA-evoked currents were sensitive towards bicuculline and thus lack the defining feature of GABAC receptors, which are insensitive towards this antagonist. Spontaneous miniature post-synaptic currents (mIPSCs) revealed a similar pharmacological behaviour. We conclude that juvenile CA1 pyramidal cells express a fraction of ionotropic GABA receptors with mixed pharmacological properties of both, GABAA and GABAC receptors.
Keywords: Hippocampus; mIPSC; GABA -receptor; Rho subunit; TPMPA; CACA;

[3 H] A-369508 ([2-[4-(2-cyanophenyl)-1-piperazinyl]-N-(3-methylphenyl) acetamide): an agonist radioligand selective for the dopamine D4 receptor by Robert B. Moreland; Marc A. Terranova; Renjie Chang; Marie E. Uchic; Mark A. Matulenko; Bruce W. Surber; Andrew O. Stewart; Jorge D. Brioni (147-154).
Tritiation of the dopamine D4 receptor selective agonist A-369508 ([2-[4-(2-cyanophenyl)-1-piperazinyl]-N-(3-methylphenyl) acetamide) has provided a radioligand for the characterization of dopamine D4 receptors. [3 H] A-369508 binds with high affinity to the major human dopamine D4 receptor variants D4.2, D4.4 and D4.7 (K d=1.7, 4, and 1.2 nM, respectively). It also binds to the rat dopamine D4 receptor, (K d=4.4 nM), implying similar binding affinity across human and rat receptors. A-369508 shows >400-fold selectivity over D2L, >350-fold selectivity over 5-HT1A and >700–1000-fold selectivity over all other receptors tested. Agonist activity determined by inhibition of forskolin-induced cAMP in Chinese hamster ovary cells transfected with the human dopamine D4.4 receptor (EC50=7.5 nM, intrinsic activity=0.71) indicates that A-369508 is a potent agonist at the human dopamine D4 receptor. Similar data was observed in other functional assays. [3 H] A-369508 binds to a single, high affinity site on membranes containing the human dopamine D4.4 receptor. When compared to the D2-like antagonist [3 H] spiperone, competition binding for agonists like dopamine and apomorphine were 2–10-fold more potent with [3 H] A-369508, while the antagonists clozapine, haloperidol and L-745870 bind with similar affinity to both ligands. Binding to rat brain regions demonstrated that the most abundant area was cerebral cortex (51.2 fmol/mg protein) followed by hypothalamus, hippocampus, striatum and cerebellum. [3 H] A-369508 is a useful tool to define the localization and physiological role of dopamine D4 receptors in central nervous system and can facilitate measuring accurate affinities (K i) for structure/activity relationship studies designed to identify dopamine D4 receptor selective agonists.
Keywords: Dopamine; D4 receptor; Spiperone; A-369508;

PKC412 induces apoptosis through a caspase-dependent mechanism in human keloid-derived fibroblasts by Ayako Nakazono-Kusaba; Fumi Takahashi-Yanaga; Yoshikazu Miwa; Sachio Morimoto; Masutaka Furue; Toshiyuki Sasaguri (155-160).
There is no established pharmacological therapy for skin keloids, a wound healing disorder. In this study, we investigated the effect of N-benzoyl staurosporine (PKC412), a protein kinase C inhibitor, on human keloid-derived fibroblasts to examine whether this agent is applicable for the treatment of keloid formation. Although PKC412 induced apoptosis in keloid fibroblasts in a time- and dose-dependent manner, the effective concentration of this agent was much higher than that of staurosporine. Western blotting showed that both PKC412 (10 μM) and staurosporine (100 nM) cleaved pro-caspase-3 to active forms. An in vitro caspase assay also showed that PKC412 and staurosporine elevated caspase-3 activities. Carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), a caspase inhibitor with a broad spectrum, inhibited caspase-3 activities stimulated by PKC412 and staurosporine; however, only PKC412-induced apoptosis, but not staurosporine-induced apoptosis, was prevented by Z-VAD-FMK. These results suggested that PKC412-induced apoptosis, but not staurosporine-induced apoptosis, is mainly mediated by the caspase-dependent mechanism.
Keywords: Keloid; Apoptosis; PKC412; Staurosporine; Caspase;

The involvement of α2A-adrenoceptors in morphine analgesia, tolerance and withdrawal in mice by Ümit Kazim Özdoǧan; Janne Lähdesmäki; Kristo Hakala; Mika Scheinin (161-171).
α2-Adrenoceptor agonists potentiate opioid analgesia and alleviate opioid withdrawal. The effects of two α2-adrenoceptor agonists, clonidine (2 mg/kg) and dexmedetomidine (20 and 100 μg/kg), and the α1-adrenoceptor antagonist prazosin (0.5 mg/kg) were tested on morphine analgesia, tolerance, and withdrawal in wild-type and α2A-adrenoceptor knock-out (KO) mice. Analgesia and tolerance were assessed with the tail-flick test. Withdrawal was precipitated with naloxone. Prazosin potentiated morphine analgesia equally in both genotypes. Clonidine and dexmedetomidine had no analgesic effects in α2A-adrenoceptor KO mice, but morphine analgesia and tolerance were similar in both genotypes. α2A-Adrenoceptor KO mice exhibited 70% fewer naloxone-precipitated jumps than wild-type mice; weight loss was similar in both genotypes. The α2-adrenoceptor agonists reduced opioid withdrawal signs only in wild-type mice. We conclude that α2A-adrenoceptors are not directly involved in morphine analgesia and tolerance, and not critical for potentiation of morphine analgesia by prazosin, but that α2A-adrenoceptors modulate the expression of opioid withdrawal signs in mice.
Keywords: Morphine withdrawal; Morphine tolerance; Analgesia; Prazosin; α2-Adrenoceptor agonist; α2A-Adrenoceptor;

Neuromuscular blocking agents block carotid body neuronal nicotinic acetylcholine receptors by Malin Jonsson; Nicholas Wyon; Sten G.E. Lindahl; Bertil B. Fredholm; Lars I. Eriksson (173-180).
Neuromuscular blocking agents predominantly block muscle type nicotinic acetylcholine receptors as opposed to the neuronal type. However, there is growing evidence that neuromuscular blocking agents have affinity to some neuronal nicotinic acetylcholine receptors. The carotid body chemoreceptor as the essential oxygen-sensing cell, relies on cholinergic signalling. Atracurium and vecuronium impair carotid body chemoreceptor activity during hypoxia. Here, we characterize atracurium and vecuronium as antagonists at nicotinic receptors of the carotid body chemoreceptor. Isolated rabbit carotid body preparations with carotid sinus nerve were used, and chemoreceptor activities were recorded. There was a concentration-dependent reduction in the chemoreceptor responses to nicotine, with an IC50 to 50 μg nicotine of 3.64 and 1.64 μM and to 500 μg nicotine of 27.00 μM and 7.29 μM for atracurium and vecuronium, respectively. It is concluded that atracurium and vecuronium depress nicotine-induced chemoreceptor responses of the carotid body in a dose-dependent fashion.
Keywords: Carotid body; Nicotine; Atracurium; Vecuronium; Acetylcholine receptor; Anesthetic;

Effect of sumatriptan in different models of pain in rats by Alessandra Ottani; Eleonora Ferraris; Daniela Giuliani; Chiara Mioni; Alfio Bertolini; Emilio Sternieri; Anna Ferrari (181-186).
The effect of sumatriptan in two standard algesimetric tests and in a model of cephalalgia was evaluated in rats. The pain threshold was measured by the hot-plate and the writhing tests; cephalalgia was produced by injecting bradykinin (10 μg in a volume of 10 μl) into a common carotid artery. Sumatriptan was subcutaneously (s.c.) injected at the doses of 4, 8, 24 or 42 mg/kg; morphine (5 or 10 mg/kg s.c.) and indomethacin (5 or 10 mg/kg s.c) were used as standard analgesic drugs. Sumatriptan had no analgesic activity either in the hot-plate test or in the writhing test. On the other hand, at 24 and 42 mg/kg it dose-dependently reduced the response to the intracarotid injection of bradykinin (vocalization and tachypnea), this effect being prevented by the 5-HT1B receptor antagonist, isamoltane. The 5-HT1D receptor antagonist BRL15572 prevented the effect of sumatriptan on bradykinin-induced tachypnea, but not the effect of sumatriptan on bradykinin-induced vocalization. These data demonstrate that sumatriptan is significantly effective in a reliable animal model of cephalalgia, while having no systemic analgesic activity.
Keywords: Hot-plate; Writhing test; Migraine-like model; Bradykinin; Rat; Sumatriptan;

The pathophysiology of Parkinson's disease and l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia are characterised by an imbalance between activity of the direct and indirect pathways regulated by dopamine D1 and D2 receptors, respectively. In this study, we investigated the effects of treatments combining adenosine A1 and metabotropic glutamate 5 (mGlu5) receptors modulators on locomotion induced by dopamine D1 receptor activation in the reserpine-treated rats. Administration of the adenosine A1 receptor agonist and mGlu5 receptor antagonist resulted in the significant reduction of dopamine D1 receptor agonist-induced locomotion. The combination of adenosine A1 receptor agonist with mGlu5 receptor antagonist had no greater effect than these compounds alone. However, the adenosine A1 receptor antagonist attenuated the inhibitory effect of mGlu5 receptor antagonist. The data suggest that the effect of mGlu5 receptor blockade on locomotion elicited by dopamine D1 receptor stimulation involves activation of adenosine A1 receptors. This interaction can improve our understanding of pathophysiology of l-DOPA-induced dyskinesia.
Keywords: mGlu5 receptor; Adenosine receptor; Dopamine receptor; Parkinson's disease; l-DOPA-induced dyskinesia; Locomotion;

In the present study, the effects of morphine sensitization on impairment of memory formation and the state-dependent learning by morphine have been investigated in mice. Pretraining administration of morphine (0.5, 2.5 and 5 mg/kg) dose dependently decreased the learning of a one-trial passive avoidance task. Pretest administration of morphine (0.5, 2.5 and 5 mg/kg) induced state-dependent retrieval of the memory acquired under pretraining morphine influence. Pretraining or pretest administration of naloxone (0.25, 0.5 and 1 mg/kg) reversed both responses to morphine (5 mg/kg). Amnesia induced by pretraining morphine was significantly reversed in morphine-sensitized mice which had previously received once daily injections of morphine [20 and 30 mg/kg, subcutaneously (s.c.)] for 3 days. Morphine sensitization tended to reverse but did not significantly affect morphine state-dependent memory. The inhibition of morphine-induced amnesia in morphine-sensitized mice was decreased by once daily administration of naloxone (0.5, 1 and 2 mg/kg) 30 min prior to injection of morphine (20 mg/kg/day×3 days). Three-days administration of 1-phenyl-7,8-dihydroxy-2,3,4,5-tetrahydro-1H-3-benzazepine HCL (SKF 38393; 8, 16 and 32 mg/kg) or SCH 23390; R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine HCL (0.01, 0.05 and 0.1 mg/kg) before morphine (for 3 days) and during morphine-sensitization, decreased and increased, the amnesia induced by pretraining morphine, respectively. Similar administration of quinpirole (0.5, 1 and 2 mg/kg) or sulpiride (25, 50 and 100 mg/kg) before morphine also decreased and increased the amnesia induced by pretraining morphine, respectively. The results suggest that morphine sensitization affects the impairment of memory formation, but not the facilitation of retrieval induced by morphine and thus it is postulated that dopamine receptors may play an important role in this effect.
Keywords: Morphine; Naloxone; Dopamine receptor agonists and antagonists; Sensitization; (Mouse);

The cannabis gateway hypothesis purports that early exposure to cannabis is a risk factor for subsequent use of other addictive drugs, e.g., psychostimulants. Neurobiological sensitization, consistent with a gateway hypothesis, was currently studied in regard to amphetamine response. Rats were exposed to the cannabinoid receptor agonist WIN 55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone] 1.25 mg/kg, intraperitoneally; i.p. for 5 days during early adolescence. Amphetamine (0.5 mg/kg, i.p.) or WIN 55,212-2 (1.25 mg/kg, i.p.) was administered in late adolescence and in vivo dopamine levels were simultaneously measured in the nucleus accumbens. Locomotor and stereotyped behaviors were also monitored in rats pretreated with WIN 55,212-2 (0.625, 1.25 or 2.5 mg/kg) or Δ-9-tetrahydrocannabinol (0.75, 1.5 or 3.0 mg/kg, i.p.) for 5 days during early adolescence and challenged with amphetamine (0.5 or 2.0 mg/kg) in late adolescence or as adults. Pretreatment with WIN 55,212-2 or Δ-9-tetrahydrocannabinol during early adolescence did not alter the dopaminergic or behavioral responses to amphetamine in adolescence or adulthood. In conclusion, these findings do not support the cannabis gateway hypothesis in regard to subsequent amphetamine exposure.
Keywords: THC; Psychostimulants; Cross-sensitization; Locomotion; Microdialysis; (Rat);

Risperidone reduces K+ currents in human atrial myocytes and prolongs repolarization in human myocardium by Pascale Gluais; Michèle Bastide; Daniel Grandmougin; Georges Fayad; Monique Adamantidis (215-222).
The antipsychotic agent risperidone has been shown to cause QT prolongation. In rabbit heart preparations, we have demonstrated that risperidone markedly lengthened action potential duration and blocked the delayed rectifier current, I Kr. The current study was designed to investigate the risperidone effects: (i) on the main K+ repolarizing currents on human atrial myocytes, using whole-cell patch clamp recordings; (ii) on action potentials recorded from human atrial and ventricular myocardium using conventional microelectrodes. We found that: (1) risperidone (3–30 μM) reduced significantly the sustained current, I sus, and 30 μM decreased significantly the transient outward current I to but was without effect on the inward rectifier current I K1; (2) risperidone (0.3–10 μM) lengthened significantly the final repolarization of the atrial action potential and risperidone (10 μM) markedly lengthened the final repolarization in ventricular myocardium. This study showed that risperidone exerts direct electrophysiological effects on human preparations but only at relatively high concentration.
Keywords: Risperidone; Heart; Outward potassium current; Action potential;

Drug–drug interactions of Z-338, a novel gastroprokinetic agent, with terfenadine, comparison with cisapride, and involvement of UGT1A9 and 1A8 in the human metabolism of Z-338 by Shigeru Furuta; Emiko Kamada; Takeshi Omata; Tohru Sugimoto; Yoshihiro Kawabata; Keiko Yonezawa; Xiaochun Cheryl Wu; Tadashi Kurimoto (223-231).
In the present study, the inhibitory properties of N-[2-(diisopropylamino)ethyl]-2-[(2-hydroxy-4,5-dimethoxybenzoyl)amino]-1,3-thiazole-4-carboxamide monohydrochloride trihydrate (Z-338), a novel gastroprokinetic agent, were investigated and compared with those of cisapride to establish its potential for drug–drug interactions. There was no notable inhibition of terfenadine metabolism or of any of the isoforms of cytochrome P450 (CYP1A1/2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4) by Z-338 in in vitro studies using human liver microsomes. Z-338 was mainly metabolized to its glucuronide by UGT1A9 (UDP glucoronosyltransferase 1 family, polypeptide A9) and UGT1A8, and did not show marked inhibition of P-glycoprotein activity. On the other hand, cisapride strongly inhibited CYP3A4 and markedly inhibited CYP2C9.Furthermore, we used the whole-cell patch-clamp technique to investigate the effects of Z-338 and cisapride on potassium currents in human embryonic kidney (HEK) 293 cells transfected with the human ether-a-go-go-related gene (hERG). Z-338 had no significant effect on hERG-related current at the relatively high concentration of 10 μM. In contrast, the inhibition by Z-338 was very small compared with that of cisapride at 10 nM, which was a thousand-fold lower concentration.In the prediction method for the drug interaction between terfenadine and cisapride based on the K i and PK parameters, we suggest the possibility that terfenadine mainly affect the QT interval, since its plasma concentration would be markedly increased, but cisapride may not be changed. Thus, in contrast with cisapride, Z-338 did not inhibit CYP and the hERG channel, and is predominantly metabolized by glucuronide conjugation, Z-338 is considered unlikely to cause significant drug–drug interactions when coadministered with CYP substrates at clinically effective doses.
Keywords: Z-338; Terfenadine; Cisapride; hERG; CYP inhibition;

Antitussive effect of NS-398, a selective cyclooxygenase-2 inhibitor, in guinea pigs by Junzo Kamei; Yasuhiro Matsunawa; Akiyoshi Saitoh (233-239).
Several reports have demonstrated that the number of capsaicin-induced coughs is increased in the presence of prostaglandins in the airway. Moreover, it has been reported that the expression of cyclooxygenase-2, which converts arachidonic acid to prostaglandins, was found in cultured human airway epithelial cells in the absence of inflammatory cytokine stimulation. Thus, it is possible that cyclooxygenase-2 inhibitor may produce an antitussive effect. To test this hypothesis, we investigated the effects of N-[2-(cyclohexyloxy)-4-nitrofenyl]-methane sulfonamide (NS-398), a selective cyclooxygenase-2 inhibitor, and 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl-pyrazole (SC-560), a selective cyclooxygenase-1 inhibitor, on capsaicin-induced coughs in guinea pigs. NS-398 (1–10 mg/kg, p.o.) dose-dependently and significantly reduced the number of capsaicin-induced coughs. In contrast, SC-560 (10 mg/kg, p.o.) did not reduce the number of capsaicin-induced coughs. The antitussive effect of NS-398 (10 mg/kg, p.o.) was not antagonized by pretreatment with methysergide (3 mg/kg, i.p.), a non-selective serotonin (5-HT) receptor antagonist, or glibenclamide (10 mg/kg, i.p.), an ATP-sensitive K+ channel blocker. Furthermore, although NS-398 did not significantly affect the cough reflex induced by substance P (10-16 M), it significantly reduced the capsaicin-induced release of substance P in bronchoalveolar lavage fluid (BALF). The present findings clearly show that cyclooxygenase-2 inhibitor, but not cyclooxygenasez-1 inhibitor, has a potent antitussive effect. Furthermore, it is possible that the antitussive action of NS-398 does not depend on centrally acting mechanisms, since 5-HT receptors play an important role in the cough-depressant activities of centrally acting antitussive drugs. NS-398 may exert peripheral antitussive effects by inhibiting the release of substance P from capsaicin-sensitive afferent C-fibers in the airways. These results suggest that cyclooxygenase-2 inhibitors may have a therapeutic benefit in reducing coughs.
Keywords: Cyclooxygenase inhibitor; Cough reflex; Antitussive effect; NS-398; SC-560;