European Journal of Pharmacology (v.413, #1)

The impact of pharmacogenetics for migraine by Roel A Ophoff; Arn M.J.M van den Maagdenberg; Krista I Roon; Michel D Ferrari; Rune R Frants (1-10).
Migraine is a paroxysmal neurological disorder affecting up to 12% of males and 24% of females in the general population. As migraine has been demonstrated to have a strong, but complex, genetic component, pharmacogenetics bears great promise in providing new targets for drug development and optimization of individual specific therapy. Better, preferably prophylactic, treatment of migraine patients is desired because the drugs now used are not effective in all patients, allow recurrence of the headache in a high percentage of patients and sometimes have severe adverse side-effects. With the recent identification of the brain-specific P/Q-type Ca2+channel gene CACNA1A as a pivotal player in the pathogenesis of migraine, the first step has been taken to identify primary biochemical pathways leading to migraine. The work on migraine can also have implications for the increasing number of additional neurological episodic disorders having the common denominator of channelopathy.
Keywords: Migraine; Ca2+ channel; CACNA1A; Triptan; Pharmacogenetics;

Pharmacogenomics of neurodegenerative diseases by Davide Maimone; Roberto Dominici; Luigi M.E. Grimaldi (11-29).
Current knowledge of sporadic degenerative disorders suggests that, despite their multifactorial etiopathogenesis, genetics plays a primary role in orchestrating the pathological events, and even dramatically changes the disease phenotype from patient to patient. Genes may act as susceptibility factors, increasing the risk of disease development, or may operate as regulatory factors, modulating the magnitude and severity of pathogenic processes or the response to drug treatment. The goal of pharmacogenomics is the application of this knowledge to elaborate more specific and effective treatments and to tailor therapies to individual patients according to their genetic profile. Here, we outline the leading theories on the etiopathogenesis of neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer disease, and we review the potential role of genetic variations, such as gene mutations and polymorphisms, in each context. We also suggest potential targets for new therapeutic approaches and variability factors for current treatments based on genotype features. Finally, we propose a few options of preventive therapeutic interventions in patients with a high genetic risk of disease.
Keywords: Amyotrophic lateral sclerosis; Parkinson disease; Alzheimer disease; Gene polymorphism; Gene mutation; Pharmacogenomics;

Open channel and competitive block of nicotinic receptors by pancuronium and atracurium by Cristopher V. Löwenick; Klaus Krampfl; Hajo Schneck; Eberhard Kochs; Johannes Bufler (31-35).
Mouse myotubes were used to investigate effects of the nondepolarizing neuromuscular blocking drugs pancuronium and atracurium on embryonic-type nicotinic acetylcholine receptor channels. Experiments were performed using patch-clamp techniques in combination with devices for ultra-fast solution exchange at outside–out patches. Application of 0.1 mM acetylcholine resulted in a fast current transient. When the peak amplitude was achieved, the current decayed monoexponentially due to desensitization. After application of drugs (pancuronium or atracurium), two different mechanisms of block were observed: (1) open channel block of embryonic-type nicotinic acetylcholine receptor channels after coapplication of blocker and acetylcholine, characterized by decrease of the time constant of current decay; (2) competitive block of embryonic-type nicotinic acetylcholine receptor channels by pancuronium or atracurium after preincubation of outside–out patches with the respective blocker. Different affinities of pancuronium (K B≈0.01 μM) and atracurium (K B≈1 μM) to embryonic-type nicotinic acetylcholine receptor channels were observed.
Keywords: Nicotinic receptor channel; Atracurium; Pancuronium; Open channel block; Competitive block;

Halenaquinone, a novel phosphatidylinositol 3-kinase inhibitor from a marine sponge, induces apoptosis in PC12 cells by Hironori Fujiwara; Kimihiro Matsunaga; Mika Saito; Shinji Hagiya; Ken-Ichi Furukawa; Hideshi Nakamura; Yasushi Ohizumi (37-45).
In nerve growth factor-treated PC12 cells, 12b-methyl-(S)-1H-benzo[6,7]phenanthro[10,1-bc]furan-3,6,8,11(2H,12bH)-tetrone (halenaquinone) caused cytotoxicity in a concentration-dependent manner (EC50 value; 10 μM). Gel electrophoretic DNA analysis of PC12 cells treated with halenaquinone (10 μM) and 11-(acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-[1S-(1α,6bα,9aβ,11α,11bβ)]-3H-furo[4,3,2-de]indeno[4,5-h]-2-benzopyran-3,6,9-trione (wortmannin) (3 μM) showed a typical apoptotic DNA ladder. In the flow cytometric analysis, halenaquinone caused apoptosis in a concentration- and time-dependent manner (EC50 value; 10 μM), whereas 2,3-dihydro-12b-methyl-(S)-1H-benzo[6,7]phenanthro[10,1-bc]furan-6,8,11(12bH)-trione (xestoquinone) with the methylene group at the C-3 position failed to cause apoptosis, suggesting that the carbonyl group at the C-3 position in halenaquinone is important for exerting apoptotic effects in PC12 cells. Phosphatidylinositol 3-kinase was inhibited by halenaquinone (IC50 value; 3 μM) as well as wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. Halenaquinone inhibited phosphatidylinositol 3-kinase activity at lower concentrations than those at which it induced apoptosis in PC12 cells. These results suggest that halenaquinone causes the death of PC12 cells through an apoptotic process and that the mechanism of halenaquinone-induced apoptosis may be partially explained by the inhibition of phosphatidylinositol 3-kinase activity.
Keywords: Halenaquinone; Apoptosis; Phosphatidylinositol 3-kinase inhibitor; PC12 cell; Pharmacological tool;

Voltage-dependent inhibition of brain Na+ channels by American ginseng by Dong Liu; Bei Li; Yi Liu; Anoja S. Attele; John W. Kyle; Chun-Su Yuan (47-54).
American ginseng (Panax quinquefolius) is a major species of ginseng that has many pharmacological effects. Studies have demonstrated that constituents of ginseng have neuroprotective effects during ischemia. Neuronal damage during ischemic episodes has been associated with abnormal Na+ fluxes. Drugs that block voltage-dependent Na+ channels provide cytoprotection during cerebral ischemia. We thus hypothesized that American ginseng may block Na+ channels. In this study, effects of an American ginseng aqueous extract was evaluated in tsA201 cells transfected with cDNA expressing α subunits of the Brain2a Na+ channel using the whole-cell patch clamp technique. We found that American ginseng extract tonically and reversibly blocked the channel in a concentration- and voltage-dependent manner. It shifted the voltage-dependence of inactivation by 14 mV (3 mg/ml) in the hyperpolarizing direction and delayed recovery from inactivation, whereas activation of the channel was unaffected. Ginsenoside Rb1, a major constituent of the American ginseng extract, produced similar effects. The data were compared with the actions of lidocaine, a Na+ channel blocker. Our results suggest that Na+ channel block by American ginseng extract and Rb1 was primarily due to interaction with the inactive state of the channel. Inhibition of the Na+ channel activity by American ginseng extract may contribute to its neuroprotective effect during ischemia.
Keywords: American ginseng; Ginsenoside Rb1; Lidocaine; tsA201 cell; Brain2a α subunits; Patch clamp; Na+ current;

Heterotrimeric GTP-binding (G) proteins, termed Ge, have a role in the late stages of the adrenocorticotrophin (ACTH) secretory pathway in the mouse AtT-20/D16-16 anterior pituitary tumour cell line. The wortmannin sensitivity of Ge-controlled mechanisms in AtT-20 cells was investigated to provide information on the possible mechanisms linking Ge with secretion. Permeabilised cells exposed to calcium ions (10−9 to 10−3 M), guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S) (10−8 to 10−4 M) and mastoparan (10−8 to 10−5 M) demonstrated a significant and concentration-dependent stimulation of ACTH secretion from non-stimulated levels for all three agents. Coincubation with wortmannin (10−5 M) significantly inhibited both calcium-independent and -stimulated secretion. The effect of wortmannin was concentration-dependent being maximal at 10−6 M. The study shows that wortmannin inhibits both calcium-independent and -stimulated secretion from permeabilised AtT-20 cells indicating a role for phosphatidylinositol-3 kinase in determining the size of the readily releasable pool of ACTH and/or in mediating calcium/Ge-evoked secretion from this pool.
Keywords: G-protein; Wortmannin; Phosphatidylinositol 3-kinase; Exocytosis; ACTH (adrenocorticotrophin); Pituitary;

Evaluation of native GABAA receptors containing an α5 subunit by Ming Li; Andras Szabo; Howard C Rosenberg (63-72).
The type A receptor for γ-aminobutyric acid (GABA), or GABAA receptor, is a pentamer of highly variable quaternary structure. It includes two α subunits, drawn from a pool of six genes, which largely determine benzodiazepine pharmacology of the receptor. In brain sections, both [3H]RY-80 (ethyl-8-acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylate) and [3H]L-655,708 (ethyl (S)-11,12,13,13a-tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate), which are selective for the benzodiazepine site of α5 subunit-containing receptors, showed high-affinity, specific binding, but to fewer regions than did the nonselective benzodiazepine, [3H]flunitrazepam. The pattern mirrored α5 mRNA distribution, and was similar to that previously reported for [3H]L-655,708 binding. Displacement of [3H]RY-80 bound to hippocampal homogenates, and of [3H]flunitrazepam bound to cerebellar and hippocampal homogenates showed comparable displacement by flumazenil (K i's 5–7 nM). However, the K i's for diazepam and for clobazam to displace [3H]RY-80 binding in hippocampus were about fourfold higher than for [3H]flunitrazepam, and the K i for clonazepam was sixfold larger, suggesting that these benzodiazepine receptor agonists bind with relatively lower affinity at hippocampal α5-containing receptors.
Keywords: GABAA receptor; Autoradiography; [3H]RY-80; [3H]L-655,708; Clonazepam;

Modulation of a 40-kDa catecholamine regulated protein by dopamine receptor antagonists by Niki Sharan; Venugopalan D. Nair; Ram K. Mishra (73-79).
Previous reports have shown that catecholamine regulated proteins (CRP) are central nervous system specific and covalently bind to catecholamines. In the present study, we report the subcellular localization and differential modulation of a 40-kDa catecholamine regulated protein (CRP40) by dopamine D1 and D2 receptor antagonists. CRP40 was found to be localized with nuclear and synaptosomal/mitochondrial and fractions. Chronic treatment with dopamine D2 receptor antagonist haloperidol in rats significantly increased the levels of CRP40 in the striatum, whereas, chronic R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390) dopamine D1 receptor antagonist administration significantly decreased striatal CRP40 levels. Moreover, acute haloperidol treatment did not alter the levels of CRP40 in any of the brain regions. Despite a sequence homology with the heat shock protein 70 (HSP70), levels of HSP70 remained unchanged after either drug treatment, suggesting a distinct function of CRP40 than HSP70. These results further suggest that CRP40 play an important role in dopaminergic neuronal function and the dopamine D1 receptor-mediated signaling pathway may be involved in the regulation of CRP40.
Keywords: Catecholamine regulated protein; Dopamine; Heat shock protein; Neurodegenerative disease; Haloperidol; SCH 23390;

Effects of a novel uncompetitive NMDA receptor antagonist, MRZ 2/579 on ethanol self-administration and ethanol withdrawal seizures in the rat by Przemyslaw Bienkowski; Pawel Krzascik; Eliza Koros; Wojciech Kostowski; Anna Scinska; Wojciech Danysz (81-89).
It has been repeatedly reported that NMDA receptors may contribute to ethanol-induced discriminative stimulus effects and withdrawal syndrome. However, the role of NMDA receptors in the reinforcing properties of ethanol remains unclear. The aim of the present study was to evaluate effects of the novel low-affinity, uncompetitive NMDA receptor antagonist, 1-amino-1,3,3,5,5-pentamethyl-cyclohexane hydrochloride (MRZ 2/579), on ethanol self-administration and ethanol withdrawal-associated seizures in rats. Both an operant (lever pressing for ethanol) and non-operant two-bottle choice setups were employed to initiate ethanol self-administration. In another procedure, forced treatment with high doses (9–15 g/kg/day) was used to induce physical dependence on ethanol. MRZ 2/579 delivered chronically by osmotic minipumps (9.6 mg/day, s.c.) did not alter either operant or non-operant ethanol drinking behaviour in a maintenance phase of ethanol self-administration. In contrast, repeated daily injections of the drug (5 mg/kg, i.p.) led to a progressive decrease in operant responding for ethanol. MRZ 2/579 (0.5–7.5 mg/kg, i.p.) and another low-affinity NMDA receptor antagonist, memantine (1–10 mg/kg, i.p.) dose-dependently suppressed ethanol withdrawal seizures with efficacies comparable with that of a standard benzodiazepine derivative, diazepam. The results of the present study indicate that: (i) intermittent administration of MRZ 2/579 may lead to a gradual decrease of operant responding for ethanol; and (ii) the group of low-affinity uncompetitive NMDA receptor antagonists may be an interesting alternative to benzodiazepines in the treatment of alcohol withdrawal.
Keywords: Ethanol self-administration; Ethanol withdrawal; NMDA receptor antagonist; MRZ 2/579; (Rat);

Selective inhibition of amine oxidases differently potentiate the hypophagic effect of benzylamine in mice by Grazia Banchelli; Carla Ghelardini; Laura Raimondi; Nicoletta Galeotti; Renato Pirisino (91-99).
In mice deprived of food for 12 h, the i.c.v. or i.p. administration of benzylamine, a substrate common to both monoamine oxidase B and semicarbazide-sensitive benzylamine oxidases, dose-dependently inhibited feeding. This effect was significantly potentiated by selective monoamine oxidase A and B inhibition, suggesting that central monoamines, known to be substrates of these enzymes may be released. The i.p. administration of semicarbazide-sensitive benzylamine oxidase inhibitors, B24 (3,5-ethoxy-4-aminomethylpyridine) and MDL 72274 ((E)-2-phenyl-3-chloroallylamine) strongly potentiated the effect of i.p. but not i.c.v.-administered benzylamine. The hypophagic effect of benzylamine was evaluated following i.c.v. administration, in comparison with the effect of the sympathomimetic compound amphetamine or the K+ channel blocker tetraethylammonium, as reference compounds. Our results make it possible to define benzylamine as a centrally acting hypophagic compound devoid of amphetamine-like motor stimulatory effects and point to a role of B24 and MDL 72274 as specific peripheral enhancers of the pharmacological effects of benzylamine.
Keywords: Food consumption; Anorectic activity; Monoamine oxidase A; Monoamine oxidase B; Semicarbazide-sensitive benzylamine oxidases; 5-HT1A receptor; MDL 72274; B24; WAY 100635;

Cardiovascular action of a cardioselective Ca2+channel blocker AH-1058 in conscious dogs assessed by telemetry by Akira Takahara; Hideki Dohmoto; Ryota Yoshimoto; Atsushi Sugiyama; Keitaro Hashimoto (101-108).
AH-1058, 4-(5H-Dibenzo[a,d]cyclohepten-5-ylidene)-1-[(E)-3-(3-methoxy-2-nitro)phenyl-2-propenyl]piperidine hydrochloride, is a novel Ca2+channel blocker exerting cardioselective action in isolated or anesthetized canine heart preparations. To clarify the cardiac and hemodynamic action of AH-1058 in conscious dogs, we assessed the effects of the drug on the hemodynamic parameters continuously recorded by telemetry in conscious unrestrained beagle dogs, and its cardiovascular effects were compared with those of verapamil, disopyramide and atenolol. Oral administration of AH-1058 (0.15, 0.3 and 0.6 mg/kg) reduced the systolic blood pressure and maximal upstroke velocity of the left ventricular pressure (LVdP/dt max), increased heart rate and prolonged the QA interval in a dose-dependent manner whereas the drug did not affect diastolic blood pressure. Verapamil at 10 mg/kg reduced systolic and diastolic blood pressure with little effect on heart rate, LVdP/dt max and QA interval. Disopyramide at 20 mg/kg increased systolic and diastolic blood pressure, decreased LVdP/dt max and prolonged the QA interval with little changes in heart rate. Atenolol at 10 mg/kg decreased LVdP/dt max and prolonged the QA interval with little changes in systolic blood pressure, diastolic blood pressure and heart rate. The time course of the cardiohemodynamic action of AH-1058 was longer than those of the other drugs. These results suggest that AH-1058 is a long-acting cardiodepressive drug, and its hemodynamic profile is obviously different from that of disopyramide and atenolol. This unique cardiovascular profile may be beneficial for the treatment of certain pathological processes in which selective inhibition of the ventricular Ca2+channels would be the target of drug therapy.
Keywords: AH-1058; Cardioselective Ca2+channel blocker; Hemodynamics; Telemetry;

Involvement of KATP channels in diethylstilbestrol-induced relaxation in rat aorta by Carmen Martı́nez; Manuel Sánchez; Agustı́n Hidalgo; Marı́a José Garcı́a de Boto (109-116).
The estrogens prevent cardiovascular diseases that among other effects could be related to the modulation of the vascular tone via modifying ionic channel permeability. ATP-sensitive K+ (KATP) channels seem to be involved in diethylstilbestrol-induced relaxation in isolated rat aorta precontracted by noradrenaline (30 nM), since the effect is inhibited by glibenclamide (1–10 μM), and 1 mM tetraethylammonium, but not by 30 mM tetraethylammonium or paxilline. The antiestrogen tamoxifen, the inhibitor of protein kinase A, Rp-cAMPS, and the inhibitor of ornithine decarboxylase, difluoromethylornithine, antagonized diethylstilbestrol-induced relaxation. The association of glibenclamide with these compounds separately did not modify the effect of glibenclamide alone on diethylstilbestrol-induced relaxation. Functional KATP channels are present in rat aorta, since diazoxide induced relaxation sensitive to glibenclamide. Papaverine, dibutyryl cyclic AMP and spermine relaxed isolated rat aorta although this was not sensitive to glibenclamide. The relaxation to forskolin was antagonized by glibenclamide. We conclude that diethylstilbestrol-induced relaxation in rat aorta is related to the modulation of KATP channels. Cyclic AMP-dependent mechanisms and polyamine synthesis may mediate this modulation.
Keywords: Diethylstilbestrol; K+-channel; Vasorelaxation; Aorta; Smooth muscle;

Effects of a dual L/N-type Ca2+ channel blocker cilnidipine on neurally mediated chronotropic response in anesthetized dogs by Tomoyuki Konda; Akira Takahara; Kazutoshi Maeda; Hideki Dohmoto; Ryota Yoshimoto (117-120).
We investigated the effects of an L-type and N-type Ca2+ channel blocker, cilnidipine, on neurally mediated chronotropic responses to clarify the anti-autonomic profile of cilnidipine in anesthetized dogs. Pretreatment with cilnidipine (0.3, 1.0 and 3.0 μg/kg, i.v.), which decreased mean blood pressure by 5 to 31 mm Hg, inhibited the changes in heart rate and plasma norepinephrine concentration induced by bilateral carotid artery occlusion, whereas it had no effect on vagal nerve stimulation-induced bradycardia. These results suggest that antihypertensive and antisympathetic doses of cilnidipine fail to influence chronotropic responses mediated by parasympathetic nerve activation in the in vivo canine heart.
Keywords: N-type Ca2+ channel; Cilnidipine; Bilateral carotid artery occlusion; Vagal nerve stimulation;

Oxygen radicals mediate the final exacerbation of endothelin-1-induced gastric ulcer in rat by Spyridon Lazaratos; Yoko Irukayama-Tomobe; Takashi Miyauchi; Katsutoshi Goto; Akira Nakahara (121-129).
We investigated the role of xanthine oxidase-derived oxygen radicals in the development of endothelin-1-induced gastric ulcer. Mucosal lipid peroxidation showed a peak 24 h after injection, while gastric mucosal haemodynamics were fully restored 26 h after endothelin-1 injection. Allopurinol and oxypurinol, but not superoxide dismutase or catalase, protected the gastric mucosa 24 h after endothelin-1 injection. Oxypurinol antagonized both the vasoconstrictor effect of endothelin-1 and the decrease in gastric ATP. All treatments on the second day after endothelin-1 injection significantly reduced gastric mucosal damage. Xanthine oxidase-derived oxygen radicals contributed largely to the exacerbation but they did not mediate the onset of endothelin-1-induced gastric ulcer. Pretreatment with probucol (500 mg/kg, p.o.) also protected the gastric mucosa from endothelin-1-induced mucosal injury by its antioxidant activity. Oxypurinol was gastroprotective through its vasoactive and energy saving actions. The haemodynamic background of endothelin-1-induced gastric ulcer consists of long lasting ischaemia and subsequent “reperfusion” which may be responsible for the late burst of oxygen radicals.
Keywords: Endothelin-1; Gastric ulcer experimental; Oxygen radical; Mucosal blood flow; ATP;