European Journal of Pharmacology (v.516, #3)

Mitochondrial dysfunction is related to necrosis-like programmed cell death induced by A23187 in CEM cells by Keigo Hamahata; Souichi Adachi; Hiroshi Matsubara; Masayuki Okada; Tsuyoshi Imai; Ken-ichiro Watanabe; Shin-ya Toyokuni; Masaki Ueno; Shigeo Wakabayashi; Yuki Katanosaka; Satoshi Akiba; Masaru Kubota; Tatsutoshi Nakahata (187-196).
We have previously reported that calcium ionophore A23187 differentially induces necrosis in CEM cells, a T-lymphoblastic leukemia cell line, and apoptosis in HL60 cells, a promyelocytic leukemia cell line. Stimulation with VP16, however, induces typical apoptosis in both cell lines. Necrosis in CEM cells, characterized by cell shrinkage and clustering, began within 5 min of treatment. Swelling of the mitochondria, lumpy chromatin condensation and intact plasma membranes were evident by electron microscopy. These A23187-mediated changes in CEM cells were suppressed by clonazepam or CGP37157, inhibitors of the mitochondrial Na+/Ca2+ exchanger. The changes, however, were not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. In both CEM and HL60 cells, intra-cellular calcium increased with similar amplitude within 1 min of treatment with 2 μM A23187. Intra-mitochondrial calcium increased with clonazepam pre-treatment alone in both CEM and HL60 cells. However, intra-mitochondrial calcium did not change drastically in response to A23187 in CEM or HL60 cells, either untreated or pre-treated with clonazepam. A23187 induces necrosis in CEM cells concurrent with mitochondrial dysfunction, which is independent of the mitochondrial permeability transition, but affected by intra-mitochondrial calcium, while HL60 cells lack these early changes. Differences in the responses to A23187 between these two cell lines might derive from differences in the susceptibility of the mitochondrial membrane to rapid increases in intra-cellular calcium.
Keywords: Apoptosis; Necrosis; Mitochondria; Calcium ionophore; Clonazepam; Bcl-2;

Effect of a cyclohexenonic long-chain fatty alcohol on calcium mobilization by Emmanuel Jover; Jose-Luis Gonzalez de Aguilar; Bang Luu; Bernadette Lutz-Bucher (197-203).
Cyclohexenonic long-chain fatty alcohols constitute a family of synthetic compounds with trophic, secretagogue and antioxidant properties. Despite their multiple biological actions in neuronal and non-neuronal tissues, the intracellular mechanisms underlying CFA activity remain unknown. In the present study, we show that 3-(15-hydroxypentadecyl)-2,4,4-trimethyl-2-cyclohexen-1-one (tCFA15) directly mobilizes Ca2+ in the pituitary neural lobe synaptosomes and in primary sensory neurons from dorsal root ganglia. This effect is dependent on the presence of extracellular Ca2+, but does not involve transmembrane voltage-operated calcium channels. Using a combination of pharmacological agents that block or deplete intracellular Ca2+ stores, our results suggest the implication of a calcium induced–calcium release mechanism evoked by tCFA15-induced Ca2+ influx. To our knowledge, these findings constitute the first attempt towards the comprehension of the biological actions of cyclohexenonic long-chain fatty alcohols at a molecular level.
Keywords: Calcium; Fura-2; Long-chain fatty alcohol; Pituitary neural lobe; Primary sensory neuron; Trimethyl-cyclohexenonic alcohol;

Ethyl 2-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-4-imidazolecarboxylate is a novel positive modulator of GABAA receptors by Maria Paola Mascia; Battistina Asproni; Fabio Busonero; Giuseppe Talani; Elisabetta Maciocco; Amedeo Pau; Riccardo Cerri; Enrico Sanna; Giovanni Biggio (204-211).
Ethyl 2-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-4-imidazolecarboxylate (TG41) enhanced the binding both of γ-aminobutyric acid (GABA) and of flunitrazepam to rat cerebral cortical membranes. Electrophysiological recordings from Xenopus oocytes expressing various recombinant GABAA receptor subtypes revealed that TG41 enhanced the function of all receptor subunit combinations tested. The potency of TG41 at receptors containing α1, β2, and γ2L subunits was greater than that of alphaxalone, etomidate, propofol, or pentobarbital. The potency of TG41 was also greater at receptors containing α1 or α2 subunits than at those containing α4 and it was markedly higher at receptors containing β2 or β3 subunits than at those containing β1. This drug induced a reversible loss of the righting reflex in Xenopus tadpoles and it elicited hypnosis (5 mg/kg) after intravenous administration in rats. These results indicate that the pharmacological profile of TG41 is similar to that of general anesthetics which potentiate the activity of GABAA receptors containing the β2 or β3 subunit.
Keywords: γ-Aminobutyric acid; Transmembrane domain; Ethyl 2-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-4-imidazolecarboxylate;

Antagonists of steroid receptors may interfere with seizure phenomena. The present study deals with effects of aminoglutethimide and spironolactone on the action of carbamazepine and diphenylhydantoin in amygdala-kindled rats of both genders. Co-administration of the antimineralocorticoid with carbamazepine at their ineffective doses (50 and 15 mg/kg, respectively) led to significant reduction of the seizure and afterdischarge durations. No anticonvulsant effect was observed when spironolactone was combined with diphenylhydantoin.The concomitant treatment of aminoglutethimide and carbamazepine (both drugs at their subprotective doses of 5 and 15 mg/kg, respectively) resulted in antiseizure activity in respect of all measured parameters, including the afterdischarge threshold, seizure severity, seizure duration and afterdischarge duration. The similar combination of aminoglutethimide with diphenylhydantoin (2.5 mg/kg) significantly shortened the seizure and afterdischarge durations.The antiseizure effect of tested combinations was not sex-dependent and not reversed by hydrocortisone pretreatment. Pharmacokinetic events may be involved only in the interaction between spironolactone and carbamazepine. Among various chemoconvulsants, bicuculline reversed the action of aminoglutethimide on carbamazepine and diphenylhydantoin. The effect of aminoglutethimide on diphenylhydantoin was also abolished by N-methyl-d-aspartic acid and aminophylline. In conclusion, our results suggest that doses of carbamazepine and diphenylhydantoin should be modified in epileptic patients concomitantly treated with aminoglutethimide or spironolactone.
Keywords: Aminoglutethimide; Spironolactone; Carbamazepine; Diphenylhydantoin; Amygdala-kindled seizures;

Effect of milnacipran on extracellular monoamine concentrations in the medial prefrontal cortex of rats pre-treated with lithium by Yuji Kitaichi; Takeshi Inoue; Shin Nakagawa; Takeshi Izumi; Tsukasa Koyama (219-226).
Antidepressants are effective in most patients with depression, but sometimes have sub-optimal effects. Thus, there is a need to use more powerful antidepressants when dealing with treatment-resistant cases. Lithium carbonate is widely used for this purpose. We investigated the acute effects of milnacipran, a serotonin-noradrenaline reuptake inhibitor, on extracellular serotonin, dopamine and noradrenaline concentrations, in the rat medial prefrontal cortex. The effects of milnacipran were examined in rats following 7 days of treatment with lithium, and in untreated controls. The lithium group had significantly greater basal levels of extracellular serotonin than the control group. Milnacipran (3 mg/kg) combined with lithium treatment caused a greater increase in extracellular noradrenaline and dopamine levels than milnacipran alone. Milnacipran (3 and 30 mg/kg) combined with lithium treatment also caused a greater increase in extracellular serotonin levels than milnacipran alone. Thus, lithium might augment the antidepressant effects of serotonin-noradrenaline reuptake inhibitors by augmenting serotonin release.
Keywords: Lithium carbonate; Milnacipran; In vivo microdialysis; Noradrenaline; Dopamine; Serotonin;

In this study, the non-reactivating effects of oximes in the hippocampus of the rat are investigated. The potassium (51 mM) evoked release of [3H]-acetylcholine and the liberation of [3H]-choline were determined in hippocampal slices following in vitro exposure to soman and five oximes (toxogonin, HI-6, HLö-7, P2S and 2-PAM) in separate experiments by superfusion. In the absence of soman, toxogonin and HLö-7 in particular induced a concentration dependent significant increase in the evoked release of [3H]-acetylcholine. There was also a significant effect of HI-6, but the effect was much smaller. Two pralidoxime salts, P2S (methanesulfonate salt) and 2-PAM (methiodide salt), had similar but lower effects that were only observed at relatively high concentrations. Experiments performed following complete inhibition of the acetylcholinesterase activity by soman (1.0 μM) showed that HI-6 and HLö-7 induced a significant decrease in the potassium-evoked release of [3H]-acetylcholine, while the liberation of [3H]-choline increased. Toxogonin, P2S and 2-PAM did not reduce significantly the evoked release of [3H]-acetylcholine. Only limited reactivation of the acetylcholinesterase activity was observed in superfusion experiments with toxogonin, HI-6, P2S and 2-PAM following exposure of hippocampal slices to soman. However, HLö-7 was proved to be relatively more effective in reactivating the acetylcholinesterase activity at high concentrations (50 and 200 μM). The acetylcholinesterase activity was reactivated to approximately 12% and 40% of control, respectively. It is concluded that HI-6 and HLö-7 have important non-acetylcholinesterase reactivating properties following soman poisoning, as may be seen by the significant reduction in the evoked release of [3H]-acetylcholine effected by these oximes. HLö-7 is of particular interest in view of its ability to additionally improve reactivation of the acetylcholinesterase activity.
Keywords: Acetylcholine; HI-6; HLö-7; Oxime; Pralidoxime; Toxogonin; Soman;

The combination of selective serotonin reuptake inhibitors with atypical antipsychotic drugs exhibits beneficial effects in treatment-resistant depression. We investigated the effects of a 2-week treatment with a low fluoxetine dose (3 mg/kg per day) plus a single injection of olanzapine (3 mg/kg) on the dialysate concentration of noradrenaline, dopamine and serotonin (5-HT) in the medial prefrontal cortex of the rat. Chronic fluoxetine increased only 5-HT levels whereas single olanzapine administration increased the concentration of catecholamines and decreased that of 5-HT to a comparable extent in vehicle- and fluoxetine-treated rats. Therefore, it is possible that the therapeutic benefit of this pharmacological combination may not be associated to changes in the cortical concentration of monoamines, but to postsynaptic blockade of monoaminergic receptors.
Keywords: Medial prefrontal cortex; Fluoxetine; Olanzapine; Microdialysis; Osmotic minipump;

Panicolytic-like effect induced by the stimulation of GABAA and GABAB receptors in the dorsal periaqueductal grey of rats by Cíntia H. Bueno; Hélio Zangrossi; Regina L. Nogueira; Vanessa P. Soares; Milena B. Viana (239-246).
Activation of GABAA and benzodiazepine receptors within the dorsal periaqueductal grey inhibits the escape behaviour evoked by the electrical stimulation of this midbrain area, a defensive reaction that has been related to panic. Nevertheless, there is no evidence indicating whether the same antiaversive effect is also observed in escape responses evoked by species-specific threatening stimuli. In the present study, male Wistar rats were injected intra-dorsal periaqueductal grey with the benzodiazepine receptor agonist midazolam (10, 20 and 40 nmol), the GABAA receptor agonist muscimol (2, 4 and 8 nmol), the GABAB receptor agonist baclofen (2, 4 and 8 nmol), or with the benzodiazepine inverse agonist FG 7142 (20, 40 and 80 pmol) and tested in an ethologically-based animal model of anxiety, the elevated T-maze. Besides escape, this test also allows the measurement of inhibitory avoidance which has been related to generalised anxiety disorder. Midazolam, muscimol and baclofen impaired escape, a panicolytic-like effect, without altering inhibitory avoidance. FG 7142, on the other hand, facilitated both avoidance and escape reactions, suggesting an anxiogenic and panicogenic-like effect, respectively. The data suggest that GABAA/benzodiazepine and GABAB receptors within the dorsal periaqueductal grey are involved in the control of escape behaviour and that a failure in this regulatory mechanism may be of importance in panic disorder.
Keywords: Dorsal periaqueductal grey; GABA; Benzodiazepine; Elevated T-maze; Generalised anxiety; Panic disorder;

Differential action of domperidone to modify emesis and behaviour induced by apomorphine in the ferret by Angie H.Y. Lau; Man-P. Ngan; John A. Rudd; David T.W. Yew (247-252).
The action of domperidone (1 mg/kg, i.p.) on spontaneous behaviour and the emesis and behavioural change induced by apomorphine (0.25 mg/kg, s.c.) were studied in the ferret. Domperidone was inactive to modify spontaneous behaviour but apomorphine-induced emesis and increased locomotor activity (distance travelled and velocity of movement; P  < 0.05); the emesis, but not the modification of locomotor activity was antagonized significantly (P  < 0.01) by domperidone. However, apomorphine did not modify significantly other behavioural measures (i.e. lip licking, rearing, burrowing, backward walking, curling-up activity, or defecatory frequency; P  > 0.05). The action of apomorphine to modify behaviour and its interaction with domperidone in this species is discussed in relation to animal models of nausea.
Keywords: Apomorphine; Domperidone; Nausea; Emesis; Dopamine;

Angiotensin-converting enzyme inhibition and angiotensin AT1-receptor antagonism equally improve endothelial vasodilator function in l-NAME-induced hypertensive rats by Vito De Gennaro Colonna; Antonello Rigamonti; Simona Fioretti; Sara Bonomo; Barbara Manfredi; Paolo Ferrario; Mauro Bianchi; Ferruccio Berti; Eugenio E. Muller; Giuseppe Rossoni (253-259).
Male Sprague–Dawley rats given N ω-nitro-l-arginine methyl ester (l-NAME) in drinking water for 8 weeks showed: (1) a clear-cut increase in systolic blood pressure; (2) a consistent decrease of endothelial-cell nitric oxide synthase (eNOS) gene expression in aortic tissue; (3) a marked reduction of plasma nitrite/nitrate concentrations; (4) a reduction of the relaxant activity of acetylcholine (ACh, from 10−10 to 10−4 M) on norepinephrine-precontracted aortic rings (reduction by 48 ± 5%); (5) a marked decrease (−58%) of the basal release of 6-keto-prostaglandin F1α (6-keto-PGF1α) from aortic rings. In l-NAME-treated rats, administration in the last 4 weeks of either the angiotensin-converting enzyme (ACE) inhibitor enalapril (10 mg/kg/day in tap water) or the angiotensin AT1-receptor antagonist losartan (10 mg/kg/day in tap water) decreased systolic blood pressure levels, completely restored eNOS mRNA levels in aortic tissue and plasma nitrite/nitrate levels, and allowed a consistent recovery of both the relaxant activity of acetylcholine and the generation of 6-keto-PGF1α. Coadministration of icatibant, a bradykinin B2-receptor antagonist (200 μg/kg/day), with enalapril blunted the stimulatory effect of the ACE inhibitor on eNOS mRNA expression, circulating levels of nitrite/nitrate, the relaxant activity of ACh and the release of 6-keto-PGF1α in l-NAME-treated rats. The generation of 6-keto-PGF1α from aortic rings was also decreased in rats coadministered icatibant with losartan. These findings indicate that (1) the ACE inhibitor enalapril and the angiotensin AT1-receptor blocker losartan are equally effective to reverse NAME-induced endothelial dysfunction; (2) the beneficial effect of enalapril on the endothelial vasodilator function in l-NAME-treated rats is mediated by bradykinin B2-receptor activation; and (3) the enhanced endothelial generation of prostacyclin induced by losartan in l-NAME rats is also mediated by bradykinin B2-receptor activation.
Keywords: Endothelial dysfunction; NO (nitric oxide); Hypertension; Bradykinin; Angiotensin-converting enzyme inhibitor; AT1-receptor antagonist;

N ω-hydroxy-l-arginine homologues and hydroxylamine as nitric oxide-dependent vasorelaxant agents by Petra Beranova; Karel Chalupsky; Andrei L. Kleschyov; Christa Schott; Jean-Luc Boucher; Daniel Mansuy; Thomas Munzel; Bernard Muller; Jean-Claude Stoclet (260-267).
Endothelium-independent relaxant activities of N ω-hydroxy-l-arginine (l-NOHA) homologues and hydroxylamine, a possible intermediate in nitric oxide (NO) formation, were examined in rat aortic rings. Addition of one –CH2– group to the –(CH2) x – chain between the α-amino acid and the hydroxyguanidine group (x  = 4) almost abolished–while deletion of one or two –CH2– (x  = 1 or 2) enhanced–the relaxant activity of l-NOHA homologues. N ω-hydroxy-nor-l-arginine- (x  = 2) and hydroxylamine-induced relaxations were blunted by a NO scavenger and by inhibitors of the guanylyl cyclase pathway, but not by NO synthase or cytochrome P 450 inhibitors (except 7-ethoxyresorufin). However, aortic NO formation was detected (using electron paramagnetic resonance) in the presence of concentrations of these compounds higher than those producing relaxation. These findings support the view that endothelium-independent vasorelaxations induced by both l-NOHA homologues with a required chain length x  ≤ 3 and hydroxylamine are mediated by NO-dependent activation of guanylyl cyclase, through a 7-ethoxyresorufin-inhibited mechanism.
Keywords: N ω-hydroxy-l-arginine homologue; Hydroxylamine; Nitric oxide release; Vasorelaxation; Electron paramagnetic resonance;

Age-dependent effect of secretagogues during colonic maturation by Libor Mrnka; Sergej Beskid; Oksana Sevastyanova; Jana Bryndová; Jiří Pácha (268-275).
Intestinal secretory response is altered during colonic development. The aim of this report was to study the developmental changes of the Ca2+- and cAMP-induced regulatory pathways with special attention to the direct and indirect effect of secretagogues on the colonic epithelium. We investigated the effect of bethanechol, 5-hydroxytryptamine (5-HT), and histamine on Cl secretion and stimulation of intracellular Ca2+ ([Ca2+]i) and cAMP in the distal colon of suckling, weanling and adult rats. In the presence of tetrodotoxin, immature colon of suckling and weanling rats displayed higher potency (EC50) of 5-HT to stimulate Cl secretion, whereas the potency of histamine was not changed during development. The potency of bethanechol was reduced during weaning and partially restored in adulthood. 5-HT increased cAMP level similarly in both neonatal and adult colonic crypts, but the adults had higher basal level of cAMP than suckling rats. Also the effect of bethanechol on [Ca2+]i was independent of colonic maturation. The results suggest that colonic Cl secretion displays developmental changes of regulation depending on the non-neural secretagogue-signalling pathway and that these developmental changes seem to be localized somewhere outside colonocytes.
Keywords: Colonic secretion; Ontogeny; Ion transport development; Bethanechol; Histamine; 5-Hydroxytryptamine;

Inhibitory effects of putative peptidic urotensin-II receptor antagonists on urotensin-II-induced contraction of cat isolated respiratory smooth muscle by David J. Behm; Zhaohui Ao; Valeria Camarda; Nambi V. Aiyar; Douglas G. Johns; Stephen A. Douglas (276-281).
Urotensin-II is purported to influence pulmonary function by modulating smooth muscle tone/growth. In the present study, Northern blot and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated the presence of UT receptor mRNA in cat trachea, bronchi and lung parenchyma. Urotensin-II contracted cat isolated trachea and bronchi with similar potencies (pEC50s 8.61 ± 0.07–8.81 ± 0.10). Contractile efficacies ranged from 19 ± 9% to 63 ± 11% KCl in the primary and secondary bronchi. The peptidic UT receptor antagonists BIM-23127, SB-710411 and GSK248451 (7.18 ± 0.12, 7.52 ± 0.08 and 9.05 ± 0.16 cat recombinant UT pKis) inhibited urotensin-II-induced contraction of cat isolated trachea with pKbs 6.36 ± 0.11, 6.74 ± 0.07 and 9.27 ± 0.12, respectively. As such, feline lung contains significant amounts of UT mRNA and this receptor appears to be functionally coupled to bronchoconstriction (the peptidic tool compound GSK248451 representing a sub-nanomolar inhibitor of such effects). These findings suggest that the cat represents a suitable species for future studies designed to assess the effects of the urotensin-II receptor on pulmonary (patho)physiology.
Keywords: Urotensin-II; GPR14; UT receptor; BIM-23127; SB-710411; GSK248451; Trachea; Bronchus;

Anti-inflammatory and analgesic effects of atorvastatin in a rat model of adjuvant-induced arthritis by Michele M. Barsante; Ester Roffê; Celina M. Yokoro; Wagner L. Tafuri; Danielle G. Souza; Vanessa Pinho; Maria Salete De A. Castro; Mauro M. Teixeira (282-289).
Statins exert favorable effects on lipoprotein metabolism but may also possess anti-inflammatory effects. Here, we explored the effects of atorvastatin in a model of adjuvant-induced arthritis in rat. Oral treatment with atorvastatin (1–10 mg/kg) from days 10 to 15 after arthritis induction caused inhibition of the increase in paw volume. Maximal inhibition occurred at a dose of 10 mg/kg. At this dose, atorvastatin markedly ameliorated the histopathological findings of joints obtained from day 16 of arthritic animals. This was mirrored by an effective blockade of neutrophil influx, as assessed by the tissue myeloperoxidase levels. The concentrations of the cytokines interleukin-1β, interleukin-6 and tumor necrosis factor-α and the chemokines CCL5 and CCL2 were significantly decreased in arthritic rats treated with atorvastatin. In contrast, the levels of interleukin-10 were enhanced by the drug treatment. The drug also prevented the hypernociception observed in the inflamed joints. These data clearly illustrate the therapeutic potential of a statin-sensitive pathway in inflammatory arthritis.
Keywords: Arthritis; Statin; Inflammation; Nociception; Cytokine;

Author index (290-292).

Keyword index (293-299).