European Journal of Pharmacology (v.418, #3)

We have isolated from a human genomic library the human 5-hydroxytryptamine 5-HT5A and 5-HT5B genes. The human 5-HT5A gene encodes a protein with similar characteristics to its mouse homologue. When expressed in monkey COS-7 cells, the human 5-HT5A receptor displayed a high affinity for tritiated 5-carbamidotryptamine ([3H]5-CT; K D=2.8 nM) and iodinated lysergic acid diethylamide ([125I]LSD; K D=187 pM). These binding sites displayed the following displacement profile: Ergotamine>Methiothepin>5-CT, Ritanserin>5-HT. Reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed the presence of human 5-HT5A mRNA in the central nervous system but not in peripheral organs. When expressed in Xenopus oocytes, the 5-HT5A receptor was able to couple to the inwardly rectifying K+ channel, GIRK1. In contrast to the human 5-HT5A gene and the mouse 5-HT5B gene, the human 5-HT5B gene does not encode a functional protein because its coding sequence is interrupted by stop codons. Our results suggest, therefore, that the 5-HT5B receptor has been lost during evolution after the divergence between rodents and primates. The 5-HT5B receptor is the first example of a brain-specific protein that is absent in human.
Keywords: 5-HT5A receptor; G protein-coupled receptor; Evolution; Null; (Human);

The effects of ethanol and higher alcohols on 45Ca2+ fluxes, mediated by voltage-dependent Ca2+ channels (VDCCs), were investigated in inside-out transverse (T)-tubule membrane vesicles from rabbit skeletal muscle. 45Ca2+ effluxes were induced by membrane potentials generated via establishing K+ gradients across the vesicles, and were significantly inhibited by the inorganic Ca2+ channel blocker La3+ (1 mM) and the Ca2+ channel antagonist nifedipine (1–10 μM). Ethanol, in the concentration range of 100–400 mM, caused a significant suppression of depolarization-induced 45Ca2+ fluxes. Ethanol also functionally modulated the effect of nifedipine (1–10 μM) and the Ca2+ channel agonist Bay K 8644 (1 μM) on Ca2+ effluxes. Pretreatment with pertussis toxin (5 μg/ml) or phorbol 12-myrstate 13-acetate (PMA, 50 nM) did not affect the ethanol inhibition of 45Ca2+ fluxes. Further experiments with alcohols revealed that butanol, hexanol, octanol and decanol also significantly inhibited 45Ca2+ effluxes. However, undecanol and dodecanol did not cause any significant change on 45Ca2+ fluxes, indicating that the effects of alcohols on 45Ca2+ effluxes exhibit a cut-off phenomenon. In radioligand binding studies, it was found that at the concentrations used in flux studies, alcohols did not alter the characteristics of the specific binding of [3H]PN 200-110 to T-tubule membranes. Results indicate that ethanol directly inhibits the function of voltage-dependent Ca2+ channels without modulating the specific binding of Ca2+ channel ligands of the dihydropyridine class, and that this inhibition is independent of intracellular Ca2+ levels.
Keywords: Ca2+ channel; Ethanol; Alcohol; Skeletal muscle;

Multi-probe microdialysis was used to investigate the interaction between the release of noradrenaline and dopamine in the medial prefrontal cortex. Retrograde microdialysis was used to stimulate or inhibit the activity of the locus coeruleus for a restricted period of time, and the response of extracellular noradrenaline and dopamine in the ipsilateral and contralateral medial prefrontal cortex was recorded with microdialysis probes. Infusion of clonidine into the locus coeruleus (100 μM for 45 min) suppressed noradrenaline release and slightly inhibited dopamine release in the ipsilateral medial prefrontal cortex. Application of carbachol to the locus coeruleus (100 μM for 45 min) stimulated both the noradrenaline and dopamine release in the ipsilateral medial prefrontal cortex. No changes were seen in the contralateral medial prefrontal cortex. In the ipsilateral nucleus accumbens, extracellular noradrenaline levels increased, but dopamine levels remained unchanged. Application to the locus coeruleus (during 10 min) of the glutamate receptor agonists N-methyl-d-aspartate (NMDA) (300 μM) or kainate (100 μM) strongly increased extracellular noradrenaline and dopamine levels in the ipsilateral medial prefrontal cortex. However, in the contralateral probe the release of dopamine (but not of noradrenaline) was also stimulated. Application of carbachol to the locus coeruleus was used as a model to further investigate the presumed noradrenaline–dopamine interaction. In a series of dual-probe experiments, α1-, α2-, and β-adrenoceptor antagonists (prazosin, idazoxan, propranolol) or a reuptake-inhibitor (nomifensine) was administered during carbachol stimulation of the locus coeruleus. Prazosin and propranolol were administered systemically in a dose of 3 mg/kg, whereas idazoxan (10 μM) and nomifensine (100 μM) were infused into the medial prefrontal cortex. However, none of these pretreatments modified the effects of the control carbachol-infusions. The results did not identify a receptor-interaction or a common reuptake site that explained the presumed interaction between dopamine and noradrenaline in the medial prefrontal cortex. Therefore, the noradrenaline–dopamine interaction hypothesis could not be confirmed or refuted.
Keywords: Noradrenaline; Dopamine; Cortex, prefrontal; Microdialysis;

Stimulatory effects of centrally injected κ-opioid receptor agonists on gastric acid secretion in urethane-anesthetized rats by Satomi Ishihara; Shizuko Tsuchiya; Syunji Horie; Toshihiko Murayama; Kazuo Watanabe (187-194).
Gastric acid secretion has been proposed to be regulated by opioid receptors in the central nervous system (CNS). However, whether the effect of morphine is stimulatory or inhibitory, and the role of type specificity of opioid receptors have not been established. We investigated the effects of centrally injected opioid receptor agonists on gastric acid secretion in the perfused stomach of urethane-anesthetized rats. Injection of morphine (1–30 μg/rat, μ-opioid receptor agonist) into the fourth cerebroventricle inhibited the secretion stimulated by i.v. injection of 2-deoxy-d-glucose. Morphine itself did not show an inhibitory effect. In contrast, injection of κ1-opioid receptor agonists such as (5α,7α,8β)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4.5]dec-8-yl)benzeneacetamide (U59593, 0.3–3 μg) and (trans)-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide hydrochloride (U50488H, 10 μg) and the κ2-opioid receptor agonist, bremazocine (3 μg), into the lateral cerebroventricle markedly stimulated secretion. The effect of U59593 was inhibited by naloxone and norbinaltorphimine (an antagonist of κ-opioid receptors) and in vagotomized rats. [d-Pen2-d-Pen5]enkephalin (10 μg, δ-opioid receptor agonist) had no effect on secretion. The dual roles of the opioid system in the CNS in gastric acid secretion are discussed.
Keywords: Gastric acid secretion; Opioid receptor type; Central injection; (Rat);

Sildenafil increases diclofenac antinociception in the formalin test by Raquel Asomoza-Espinosa; Rosario Alonso-López; Teresa Mixcoatl-Zecuatl; Patricia Aguirre-Bañuelos; Jorge E Torres-López; Vinicio Granados-Soto (195-200).
The antinociceptive activity of an inhibitor of phosphodiesterase 5, alone or combined with diclofenac, was assessed in the formalin test. Local administration of diclofenac produced a significant antinociception in both phases of the formalin test in female Wistar rats. In contrast, 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [3,4-d]pyrimidin-5-yl)phenylsulfonyl]-4-methyl piperazine (sildenafil, an inhibitor of phosphodiesterase 5) produced significant antinociception, only during the second phase of the formalin test. Non-effective doses of sildenafil (25–100 μg/paw) significantly increased the antinociceptive effect of an inactive dose of diclofenac (25 μg) in both phases of the test. The antinociception produced by the drugs alone or the combination was due to a local action, as its administration in the contralateral paw was ineffective. Since sildenafil is a potent and selective inhibitor of phosphodiesterase 5, our results suggest that this drug produced its antinociceptive activity, and increased that of diclofenac, probably through the inhibition of cyclic GMP degradation.
Keywords: Diclofenac; Sildenafil; cGMP; Synergism; Antinociception;

Amphetamine increases extracellular dopamine and induces locomotor and stereotypical behaviors in rats. This study examined the effect of the dopamine D2/D3 receptor antagonist sulpiride (50 mg/kg s.c.) on the dopaminergic response to amphetamine (0.5, 2.0, or 8.0 mg/kg i.p.) in male Sprague-Dawley rats. Extracellular dopamine in the striatum was monitored using in vivo microdialysis and high performance liquid chromatography with electrochemical detection. Dopamine concentration curves were analyzed using non-linear regression and residual F-testing. Amphetamine enhanced extracellular dopamine in a dose-dependent manner. Sulpiride augmented the increase in dopamine induced by 0.5 and 2 mg/kg amphetamine by decreasing the rate of dopamine concentration fall off in the extracellular space (P<0.05). Sulpiride also potentiated the amount of dopamine increased by 8 mg/kg amphetamine, but did so by affecting the maximum concentration achieved (P<0.05), not the onset or offset rates. We conclude that the primary effect of a dopamine D2/D3 receptor antagonist is a potentiation of the effect of amphetamine on extracellular striatal dopamine levels, which may contribute to the enhanced stereotypic effects observed when paired with amphetamine.
Keywords: Amphetamine; Dopamine D2 receptor; Sulpiride; Dopamine receptor antagonist; Microdialysis; Striatum;

Intravenous administration of ecstasy (3,4-methylendioxymethamphetamine) enhances cortical and striatal acetylcholine release in vivo by Elio Acquas; Paola Marrocu; Augusta Pisanu; Cristina Cadoni; Gerald Zernig; Alois Saria; Gaetano Di Chiara (207-211).
The effect of intravenous administration of 3,4-methylendioxymethamphetamine (MDMA), in a range of doses (0.32–3.2 mg/kg) that have been shown to maintain self-administration behaviour in rats, on in vivo acetylcholine release from rat prefrontal cortex and dorsal striatum was studied by means of microdialysis with vertical concentric probes. Intravenous administration of MDMA dose-dependently increased basal acetylcholine release from the prefrontal cortex to 57±21%, 98±20%, 102±7% and 141±14% above baseline, at doses of 0.32, 0.64, 1.0 and 3.2 mg/kg, respectively. MDMA also stimulated striatal acetylcholine release at the dose of 3.2 mg/kg i.v. (the maximal increase being 32±3% above baseline) while at the dose of 1 mg/kg i.v., MDMA failed to affect basal acetylcholine output. Administration of MDMA also dose-dependently stimulated behaviour. The results of the present study show that MDMA affects measures of central cholinergic neurotransmission in vivo and suggest that at least some of the psychomotor stimulant actions of MDMA might be positively coupled with an increase in prefrontal cortical and striatal acetylcholine release.
Keywords: Acetylcholine; (±)-3,4-Methylendioxymethamphetamine (MDMA, ecstasy); Microdialysis; Prefrontal cortex; Striatum;

Repeated haloperidol injections increase extracellular striatal ascorbate. Because ascorbate release depends on glutamate uptake, we assessed this mechanism in the haloperidol effect. Linear staircase voltammetry was combined with intrastriatal infusions of l- or d-glutamate or saline in behaving rats after 7 or 21 days of haloperidol (0.5 mg/kg, s.c.). Control animals, receiving either vehicle or no treatment, responded to l-, but not d-glutamate or saline infusion with a 50% increase in ascorbate. In contrast, glutamate-evoked ascorbate release disappeared after 7 but reappeared after 21 days of haloperidol. Thus, increased striatal ascorbate release following chronic haloperidol cannot be explained by an enhanced response to glutamate.
Keywords: Ascorbate; Glutamate; Haloperidol; Striatum; Voltammetry;

Deterioration of the protein kinase C–KATP channel pathway in regulation of coronary flow in hypercholesterolaemic rabbits by Eva Pongo; Zsolt Balla; Kanigula Mubagwa; Willem Flameng; Istvan Edes; Zoltan Szilvassy; Peter Ferdinandy (217-223).
We studied the effect of experimental hypercholesterolaemia/atherosclerosis on changes in coronary flow and cardiac function, induced by protein kinase C and ATP-sensitive K+ (KATP) channel modulators in isolated Langendorff-perfused rabbit hearts. Both phorbol 12-myristate-13-acetate (PMA) and phorbol 12,13-dibutyrate (PDB, 0.1 μM each), activators of protein kinase C, decreased, whereas staurosporine, (0.1 μM), a protein kinase C inhibitor, increased coronary flow and left ventricular dP/dt, an index of ventricular contractility. Glyburide (5–50 μM), a KATP channel inhibitor, blocked the effect of staurosporine. The phorbol esters were without effect in the presence of pinacidil (5 μM), a KATP channel activator. Neither the protein kinase C modulators nor glyburide produced any effect on coronary flow and left ventricular contractility, when the hearts were prepared from animals either maintained on a cholesterol (1.5%)-enriched diet or treated with lovastatin (5 mg/kg/day per os). Treatment with farnesol (1 mg/kg twice a day for 7 days intravenously) restored the reactivity of hearts from either hypercholesterolaemic or lovastatin-treated animals to protein kinase C modulators. We conclude that non-cholesterol mevalonate products are necessary for the functional integrity of the protein kinase C–KATP channel pathway in the rabbit heart.
Keywords: Protein kinase C; KATP channel; Hypercholesterolaemia; Farnesol; Heart;

AG-041R, a novel indoline-2-one derivative, induces systemic cartilage hyperplasia in rats by Hidetomo Kitamura; Atsuhiko Kato; Toru Esaki (225-230).
AG-041R (3R-1-(2,2-diethoxyethyl)-3-((4 methylphenyl)aminocarbonylmethyl)-3-((4-methylphenyl) ureido)-indoline-2-one) is a novel small compound synthesized as a cholecystokinin-2 (CCK2)/gastrin receptor antagonist. In the course of the development of this compound, we discovered unexpectedly that oral administration of a high dose for 4 weeks markedly induced systemic cartilage hyperplasia. This change was histologically observed in the auricles, the trachea, the marginal region of the femoral condyle, the xiphoid process and intervertebral disks in rats. Daily intraarticular injections of AG-041R into rat knee joints for 3 weeks also caused cartilage hyperplasia in the marginal region of the femoral condyle, but no hyperplasia was observed in any other cartilage. We have confirmed that chondrogenic activity of AG-041R is an intrinsic property of the compound, and is not due to its CCK2/gastrin receptor antagonistic actions. These results indicate that AG-041R is a novel stimulator of chondrogenesis, and can be expected to be a potent therapeutic agent for cartilage disorders.
Keywords: Systemic cartilage hyperplasia; Indoline-2-one derivative;

Cloricromene, a semi-synthetic coumarin derivative, inhibits tumor necrosis factor-α production at a pre-transcriptional level by Emanuela Corsini; Laura Lucchi; Marco Binaglia; Barbara Viviani; Carla Bevilacqua; Giovanni Monastra; Marina Marinovich; Corrado L. Galli (231-237).
Cloricromene decreases myocardial infarct size after ischemic-reperfusion injury in vivo, and it has been suggested that this is due to inhibition of tumor necrosis factor-α (TNF-α). The purpose of this work was to characterize the mechanism of cloricromene-induced inhibition of TNF-α in rat macrophages. Cloricromene inhibited lipopolysaccharide-induced TNF-α release in a dose-dependent manner (IC50=5.9±0.8 μM). This was not due to cytotoxicity, as cloricromene was well tolerated up to 500 μM. Cloricromene inhibited lipopolysaccharide-induced expression of TNF-α mRNA, which suggests a pre-transcriptional effect. We then investigated the early signal transduction pathway triggered by lipopolysaccharide. The binding of lipopolysaccharide to its receptor CD14 activates protein kinase C and nuclear factor-κB (NF-κB). Cloricromene inhibited NF-κB activation in a dose-dependent manner, but affected protein kinase C translocation only slightly. We then established that cloricromene inhibited lipopolysaccharide-induced cellular oxidative activity, which is important for NF-κB activation. Our results show that cloricromene interferes with the early signal transduction pathway triggered by lipopolysaccharide.
Keywords: Macrophage; Anti-inflammatory; NF-κB (nuclear factor-κB); Lipopolysaccharide;

Author index (239-240).

Keyword index (241-245).