European Journal of Pharmacology (v.541, #3)

The new orally active iron chelator ICL670A exhibits a higher antiproliferative effect in human hepatocyte cultures than O-trensox by Karine Chantrel-Groussard; François Gaboriau; Nicole Pasdeloup; René Havouis; Hanspeter Nick; Jean-Louis Pierre; Pierre Brissot; Gérard Lescoat (129-137).
By comparing the antiproliferative effect of the iron chelators ICL670A and O-trensox in the human hepatoma cell line HUH7 and human hepatocyte cultures, we have shown that ICL670A decreased cell viability, inhibited DNA replication and induced DNA fragmentation more efficiently than O-trensox. O-trensox and ICL670A induced a cell cycle blockade in G0–G1 and S phases respectively. In parallel, ICL670A inhibited polyamine biosynthesis by decreasing ornithine decarboxylase and spermidine/spermine N1-acetyltransferase activities. O-trensox increased polyamine biosynthesis and particularly putrescine level by stimulating spermidine–spermine N1-acetyltransferase activity which could activate the polyamine retro-conversion pathway. Moreover, the two chelators exhibit some cytotoxic effect in the two culture models; ICL670A was more cytotoxic than O-trensox and higher concentrations of the two chelators were necessary to induce a cytotoxicity in primary cultures versus hepatoma cells. These results suggested that ICL670A has the most efficient antitumoral effect, blocks cell proliferation by a pathway different of O-trensox and may constitute a potential drug for anticancer therapy.
Keywords: Iron chelation; Human hepatocyte; Cell proliferation; Polyamine metabolism;

Dipyridamole is a nucleoside transport inhibitor and a non-selective phosphodiesterase inhibitor. However, the mechanisms by which dipyridamole exerts its anti-inflammatory effects are not completely understood. In the present study, we investigated the role of mitogen-activated kinase phosphatase-1 (MKP-1) in dipyridamole's anti-inflammatory effects. We show that dipyridamole inhibited interleukin-6 and monocyte chemoattractant protein-1 secretion, inducible nitric oxide synthase protein expression, nitrite accumulation, and cyclooxygenase-2 (COX-2) induction in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Dipyridamole inhibited the nuclear factor kappa B (NF-κB) signaling pathway as demonstrated by inhibition of the inhibitor of NF-κB (IκB) phosphorylation, IκB degradation, p65 translocation from the cytosol to the nucleus, and transcription of the reporter gene. Dipyridamole also inhibited LPS-stimulated p38 mitogen-activated protein kinase (p38 MAPK) and IκB kinase-beta (IKK-β) activities in RAW 264.7 cells. A p38 MAPK inhibitor, SB 203580, inhibited LPS-stimulated COX-2 expression and IKK-β activation suggesting that LPS may activate the NF-κB signaling pathway via upstream p38 MAPK activation. Furthermore, dipyridamole stimulated transient activation of MKP-1, a potent inhibitor of p38 MAPK function. Knockdown of MKP-1 by transfecting MKP-1 siRNA or inhibition of MKP-1 by the specific inhibitor, triptolide, significantly reduced the inhibitory effects of dipyridamole on COX-2 expression induced by LPS. Taken together, these data suggest that dipyridamole exerts its anti-inflammatory effect via activation of MKP-1, which dephosphorylates and inactivates p38 MAPK. Inactivation of p38 MAPK in turn inhibits IKK-β activation and subsequently the NF-κB signaling pathway that mediates LPS-induced cyclooxygenase-2 expression in RAW 264.7 cells.
Keywords: Lipopolysaccharide; Nitric oxide; Mitogen-activated kinase phosphatase-1; Cyclooxygenase-2; Signal transduction; RAW 264.7 macrophage; Anti-inflammatory effect;

Enhancement of brain kynurenic acid production by anticonvulsants—Novel mechanism of antiepileptic activity? by Tomasz Kocki; Marian Wielosz; Waldemar A. Turski; Ewa M. Urbanska (147-151).
In this study, we describe the effect of antiepileptic drugs on the production of kynurenic acid in rat cortical slices, and on the activity of kynurenic acid biosynthetic enzymes, kynurenine aminotransferases (KATs I and II) in the brain tissue. Phenobarbital, felbamate, phenytoin and lamotrigine (all at 0.5–3.0 mM) enhanced kynurenic acid production in vitro, and stimulated the activity of KAT I. In contrast, vigabatrin, gabapentin and tiagabine inhibited kynurenic acid synthesis in cortical slices with IC50 of 3.9 (2.8–7.9), 3.7 (2.5–5.4) and 7.5 (3.5–14.3) mM, respectively. Vigabatrin, gabapentin and tiagabine reduced also the activity of KAT I with IC50 of 1.6 (1.1–2.4), 0.1 (0.01–0.15), 0.9 (0.7–1.2) mM, and the activity of KAT II with IC50 values of 6.0 (4.8–7.5), 0.2 (0.1–0.3) and 2.0 (1.5–2.6) mM, respectively. In conclusion, the enhancement of kynurenic acid formation displayed by carbamazepine, phenytoin, phenobarbital, felbamate and lamotrigine seems to be a novel mechanism, synergistic with other actions of these drugs, and potentially valuable in terms of better control of epilepsy.
Keywords: Kynurenic acid; Kynurenine aminotransferase; Antiepileptic drug; Epilepsy; In vitro; Brain;

Centrally administered histamine evokes the adrenal secretion of noradrenaline and adrenaline by brain cyclooxygenase-1- and thromboxane A2-mediated mechanisms in rats by Takahiro Shimizu; Shoshiro Okada; Naoko Yamaguchi; Tsuyoshi Sasaki; Lianyi Lu; Kunihiko Yokotani (152-157).
Plasma adrenaline is originated from adrenal medulla, while plasma noradrenaline reflects the release from sympathetic nerves in addition to the secretion from adrenal medulla. The present study was designed to characterize the source of plasma catecholamines induced by centrally administered histamine, with regard to the brain prostanoids. Intracerebroventricularly (i.c.v.) administered histamine (1, 5 and 10 μg/animal) elevated plasma noradrenaline and adrenaline (noradrenaline < adrenaline) in a dose-dependent manner. Ketoprofen (a selective inhibitor of cyclooxygenase-1) (100, 250 and 500 μg/animal, i.c.v.) dose-dependently reduced the histamine (5 μg/animal, i.c.v.)-induced elevation of both catecholamines, while NS-398 (a selective inhibitor of cyclooxygenase-2) (250 and 500 μg/animal, i.c.v.) had no effect. The histamine-induced response was dose-dependently attenuated by furegurelate (an inhibitor of thromboxane A2 synthase) (250 and 500 μg/animal, i.c.v.), and abolished by acute bilateral adrenalectomy. These results suggest that centrally administered histamine evokes plasma noradrenaline and adrenaline from adrenal medulla by brain cyclooxygenase-1- and thromboxane A2-mediated mechanisms in rats.
Keywords: Histamine; Cyclooxygenase-1; Thromboxane A2; Catecholamines; Adrenal medulla;

The pharmacological profile of a γ-aminobutyric acid A (GABAA) receptor depends upon subunit composition. Studies using recombinant expression systems suggest that δ-subunit containing GABAA receptors are particularly sensitive to the actions of the GABAA partial agonist, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP, gaboxadol). Here we investigated the actions of THIP in mutant mice lacking the GABAA receptor δ-subunit gene. Using the chloride flux assay, we determined that the actions of THIP were reduced by 21% in the cortical, but not cerebellar, membranes of knockout mice. Similar results were seen with another GABAA agonist, muscimol. Moreover, δ-subunit knockout mice exhibited a 54% reduction in sensitivity to the hypnotic actions of THIP as assessed by the loss of righting reflex test. These data support the notion that δ-containing GABAA receptors are at least partially responsible for the actions of THIP, and contribute to the growing literature suggesting that the pharmacological specificity of GABAA receptors depends on which subunits are present or absent.

Various GABA-mimetic drugs differently affect cocaine-evoked hyperlocomotion and sensitization by Małgorzata Filip; Małgorzata Frankowska; Anna Gołda; Magdalena Zaniewska; Jerzy Vetulani; Edmund Przegaliński (163-170).
To substantiate the notion that cocaine behavioral effects may be influenced by γ-aminobutyric acid (GABA) neurotransmission male Wistar rats were injected with gabapentin (a cyclic GABA analogue), tiagabine (a GABA reuptake inhibitor), or vigabatrin (a GABA transaminase inhibitor) before acute or repeated treatment with cocaine evoking either locomotor hyperactivation or sensitization. Gabapentin (1–30 mg/kg), tiagabine (2.5–10 mg/kg) or vigabatrin (75–250 mg/kg) attenuated the cocaine (10 mg/kg)-induced hyperactivation and in the highest doses they also decreased basal locomotor activation. Vigabatrin (75–250 mg/kg) dose-dependently reduced the development of cocaine sensitization in rats treated repeatedly (days 1–5) with cocaine (10 mg/kg) and then challenged with cocaine (10 mg/kg) following 5-day withdrawal; the remaining drugs were ineffective. When injected acutely with a cocaine challenge dose, gabapentin (3–10 mg/kg) or vigabatrin (150 mg/kg), but not tiagabine, significantly attenuated the expression of cocaine sensitization. The present results show that enhanced GABA-ergic neurotransmission exerted inhibitory actions on acute responses to cocaine, however, only in a case of vigabatrin the inhibition seems to be unrelated to the inhibitory effect of the drugs on basal locomotor activity. The finding that vigabatrin protected against the development and the expression of cocaine sensitization further supports its therapeutic potential in the treatment of cocaine dependence.
Keywords: Cocaine; GABA neurotransmission; Locomotor activation; Behavioral sensitization; (Rat);

Ranolazine, a novel anti-anginal agent, does not alter isosorbide dinitrate- or sildenafil-induced changes in blood pressure in conscious dogs by Gong Zhao; Eric Messina; Xiaobin Xu; Manuel Ochoa; Sobrina Serpillon; John Shryock; Luiz Belardinelli; Thomas H. Hintze (171-176).
Effects of ranolazine on isosorbide dinitrate- and on sildenafil-induced changes in mean arterial pressure and heart rate were assessed in conscious dogs. Dogs (n  = 7) were chronically instrumented for measurements of mean arterial pressure and heart rate. Bolus intravenous injections of either isosorbide dinitrate (0.2 mg/kg) or sildenafil (0.5 mg/kg) caused biphasic changes in mean arterial pressure and heart rate: a transient (∼ 20 s) decrease in mean arterial pressure and an increase in heart rate, followed by prolonged (10–15 min) decreases in mean arterial pressure by 11 ± 1.6 and 11 ± 2.2 mm Hg, respectively. Infusion of ranolazine alone (plasma concentrations = 4 or 8 μM) for 10 min did not significantly affect mean arterial pressure and heart rate. The transient hypotension and tachycardia caused by isosorbide dinitrate were not altered by ranolazine. The sildenafil-induced transient tachycardia (Δ change: 114 ± 10 beats/min) was significantly (P  < 0.05) blunted by either 4 (Δ change: 71 ± 8 beats/min) or 8 (Δ change: 66 ± 9 beats/min) μM ranolazine. However, the sildenafil-induced transient decrease in mean arterial pressure was not altered by ranolazine. During ranolazine infusion (4–5 or 8–10 μM), isosorbide dinitrate and sildenafil caused prolonged decreases in mean arterial pressure. These results indicate that except for a blunting of the transient tachycardia caused by sildenafil, ranolazine at concentrations up to 10 μM does not alter changes in mean arterial pressure and heart rate induced by either isosorbide dinitrate or sildenafil in conscious dogs.
Keywords: Ranolazine; Long-acting nitrate; Sildenafil; Blood pressure; Heart rate; Conscious dog;

K+-depolarization (KCl)-activated Ca2+ entry permitting sustained force-maintenance in tonic vascular smooth muscle has long been attributed solely to activation of L-type voltage-operated Ca2+ channels (VOCs). We used the transient receptor potential channel (TRP) blocker, 2-aminoethoxydiphenyl borate (2-APB), to test the hypothesis that KCl activates additional Ca2+ entry pathways. 2-APB alone caused a transient weak increase in force, a sustained weak increase in basal [Ca2+]i and myosin light chain phosphorylation, and inhibition of KCl-induced force, [Ca2+]i and myosin light chain phosphorylation. 2-APB did not appear to block VOCs, because 2-APB did not inhibit 30 nM Bay k 8644-induced increases in [Ca2+]i. Moreover, although 1 μM nifedipine abolished the increase in [Ca2+]i produced by α-adrenergic receptor activation, 2-APB produced an additional reduction in [Ca2+]i below the basal level. These data support the conclusion that membrane depolarization activates 2-APB-sensitive TRPs in addition to VOCs to permit strong force-maintenance in tonic vascular smooth muscle.
Keywords: 2-APB; Signal transduction; Cell signaling; Calcium channel blocker; Fura-2; Myosin light chain phosphorylation; Contraction;

Sildenafil, tadalafil, vardenafil and verapamil induced concentration-dependent relaxation of the rabbit corpus cavernosum muscle precontracted with noradrenaline. The maximal relaxation (%) at 20 μM was 61.4 ± 6.9, 32.4 ± 5.4, 100.0 ± 5.5 and 86.6 ± 5.1 (n  = 5 each) respectively. Pre-incubation of cavernosal muscle strips with N ω-nitro-l-arginine or guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) but not adenylate cyclase inhibitor, cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine] (MDL12330A) culminated in only a 20–30% reduction in muscle relaxant action of the 3 phosphodiesterase inhibitors. This suggests that another mechanism of relaxation independent of nitric oxide–cGMP or cAMP pathway was involved. Higher concentrations of sildenafil (100 μM) and vardenafil (10 and 100 μM) produced non-competitive antagonism of noradrenaline-induced contraction characterized by reduced maximal effect. In contrast, tadalafil was devoid of significant effect on noradrenaline. On K+-depolarized tissues, sildenafil was as potent as vardenafil whereas tadalafil was the least effective in relaxing K+-induced tone. The maximal relaxation (% of K+-induced tone) at 20 μM sildenafil, tadalafil and vardenafil was respectively 84.1 ± 6.5, 9.0 ± 19.9, and 88.9 ± 6.2 (n  = 5 each). In addition, verapamil, sildenafil and vardenafil were more efficacious than tadalafil in reversing tonic contractions by Ca2+ channel activator, 1,4,dihydro-2,6-dimethyl-5-nitro-4-[2(triflouromethyl)phenyl]pyridine-3-carboxylic acid methyl ester (BAY K-8644). These results indicate that vardenafil and sildenafil possess direct muscle relaxant potential possibly via inhibiting Ca2+ influx through both receptor-operated and voltage-dependent Ca2+ channels whereas tadalafil appears capable of inhibiting receptor-operated transmembrane Ca2+ entry only.
Keywords: Sildenafil; Tadalafil; Vardenafil; Corpus cavernosum; Signal transduction;

The protective effect of nitroglycerin on gastrointestinal and renal side effects of lornoxicam in rats by Selda Sen; Firuzan Kacan Doger; Serdar Sen; Osman N Aydın; Aslıhan Karul; Turhan Dost (191-197).
The aim of this study is firstly, to determine the preventive effect of chronic usage of combination of nitroglycerin and lornoxicam on gastrointestinal and renal side effects and secondly, to investigate the oxidative and antioxidative effects of this combination in rats.Thirty-seven Wistar male rats were divided into five groups for 15 days; isotonic group (n  = 8, sodium chloride 0.09, Group ISO), lornoxicam group (n  = 8, lornoxicam 1.3 mg/kg, Group L), nitroglycerin group (n  = 6, nitroglycerin 1 mg/kg, Group NTG), lornoxicam–nitroglycerin combination group (n  = 8, 1.3 mg/kg lornoxicam + 1 mg/kg nitroglycerin, Group L–NTG), and control group (n  = 7, no drug was administered, Group C). Nitric oxide, malondialdehyde, reduced glutathione (GSH), catalase, interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha concentrations were measured before drug injection and on fifteenth day in all blood samples. Gastrointestinal and renal biopsies were performed on fifteenth day.Two rats died on tenth and twelfth days in Group L. There were significant differences in Group L compared to the other groups for the lesions of stomach and kidney (p  = 0.01, p  = 0.028 respectively). Gastric ulceration was occurred in a rat in Group L. Malondialdehyde, TNF-α, and IL-6 levels decreased in NTG and L–NTG groups, whereas catalase and glutathion levels increased in NTG, L and L–NTG groups compared to control group (p  < 0.05).Lornoxicam may cause gastrointestinal and renal side effects without oxidative stress. Adding nitroglycerin to lornoxicam for chronic treatment may prevent these side effects and enhance antioxidative effect compared to the use of lornoxicam alone in rats.
Keywords: Lornoxicam; Nitroglycerin; Oxidative stress; Antioxidative effects; Side effects;

An inhibitory effect of A20 on NF-κB activation in airway epithelium upon influenza virus infection by Akira Onose; Shu Hashimoto; Shinichi Hayashi; Shuichiro Maruoka; Fumio Kumasawa; Kenji Mizumura; Itsuro Jibiki; Ken Matsumoto; Yasuhiro Gon; Tomoko Kobayashi; Noriaki Takahashi; Yasuko Shibata; Yoshimitsu Abiko; Toshikatsu Shibata; Kazufumi Shimizu; Takashi Horie (198-204).
Influenza is a major disease in humans. The reemergence of avian influenza A viruses has indicated that hyperinflammatory responses are closely related to the severity of disease. Influenza virus infection induces nuclear transcription factor kappaB (NF-κB) activation. NF-κB and NF-κB-dependent gene products promote lung inflammation and injury. Therefore, it is important to investigate the means to attenuate NF-κB activation. A20 is a cytoplasmic zinc finger protein that inhibits NF-κB activity, However, little is known about the role of A20 in influenza virus infection. Here, we have examined the role of A20 in influenza virus infection-induced NF-κB promoter activation in human bronchial epithelial cells. The results showed that (1) A20 protein and mRNA are inducible and expressed in the lung from mice and human bronchial epithelial cells upon influenza virus infection; (2) NF-κB promoter activation was induced in bronchial epithelial cells upon influenza virus infection; and (3) overexpression by transient transfection of A20 attenuated NF-κB promoter activation in bronchial epithelial cells. These results indicate that A20 may function as a negative regulator of NF-κB-mediated lung inflammation and injury upon influenza virus infection, thereby protecting the host against inflammatory response to influenza virus infection.
Keywords: A20; Influenza virus; NF-κB; Bronchial epithelium; Inflammation;

Protective effects of α1-acid glycoprotein and serum amyloid A on concanavalin A-induced liver failure via interleukin-6 induction by ME3738 by Hiroyuki Kuzuhara; Yoshihisa Nakano; Nobuyuki Yamashita; Masako Imai; Yuji Kawamura; Tohru Kurosawa; Shoji Nishiyama (205-210).
We examined whether the 22β-methoxyolean-12-ene-3β,24(4β)-diol (ME3738)-mediated selective induction of interleukin-6 increased α1-acid glycoprotein and serum amyloid A expression, and whether these proteins protected against liver injury in vitro and in vivo. ME3738 treatment in male mice increased gene expression of α1-acid glycoprotein subtypes and serum amyloid A 2 genes, and plasma concentration of serum amyloid A. Treatment with α1-acid glycoprotein at 5 mg/animal or serum amyloid A at 0.03 and 0.1 mg/animal prior to concanavalin A administration reduced multifocal necrosis in the liver. Treatment with α1-acid glycoprotein and serum amyloid A, but not α1-antitrypsin, protected Hep G2 cells against cell injury. These results suggest that α1-acid glycoprotein and serum amyloid A, increased by ME3738-induced interleukin-6, might protect against concanavalin A-induced liver injury.
Keywords: Protective effect; Acute phase protein; Liver injury; Interleukin-6; ME3738;

Author index (211-213).

Keyword index (214-218).