European Journal of Pharmacology (v.444, #1-2)

Reactive oxygen and nitrogen species such as superoxide (O2 ) and nitric oxide (NO) are produced under diverse conditions and provoke distinct signaling reactions. The formation of NO has been shown to induce apoptosis and/or necrosis in mesangial cells and to protect other cells such as hepatocytes. Often, NO and O2 are simultaneously generated, which results in their diffusion-controlled interaction and, thus, redirects the signaling properties of either NO or O2 . This has been proven for mesangial cells, where O2 formation attenuates NO-initiated apoptosis. As the mechanisms involved remained unclear, we studied the potential impact of the glutathione redox system and compared the results obtained with mesangial cells with those obtained with Hep G2 hepatocytes. In contrast to mesangial cells, Hep G2 cells appeared resistant to NO donors but displayed massive cell destruction following NO/O2 cogeneration. As a result, we noticed a greater increase in GSSG levels in Hep G2 cells than in mesangial cells. GSH depletion reversed the cell protection in mesangial cells and enhanced the cell damage in Hep G2 cells. NO/O2 -mediated mesangial protection is associated with an increased glutathione reductase activity and an increase in GSH. In conclusion, NO/O2 sensitivity is cell type specific and is determined by the glutathione redox system.
Keywords: Mesangial cell; Hep G2 cell; Cell death; Nitric Oxide (NO); Superoxide; Glutathione;

Effects of clomipramine on neuronal nicotinic acetylcholine receptors by Héctor E. López-Valdés; Jesús Garcı́a-Colunga; Ricardo Miledi (13-19).
The action of the tricyclic antidepressant clomipramine on membrane currents elicited by acetylcholine was studied in Xenopus oocytes expressing neuronal α2β4 nicotinic acetylcholine receptors. Clomipramine inhibited the acetylcholine responses rapidly and reversibly, with a similar IC50 when the oocytes were preincubated with clomipramine (1.3±0.2 μM) or when they were exposed simultaneously with acetylcholine and clomipramine (1.5±0.3 μM). The EC50 was 39.9±2.1 μM for acetylcholine alone and 65.7±3.6 μM for acetylcholine in the presence of 2 μM clomipramine. The inhibitory effect of clomipramine was weakly voltage-dependent, with an electric distance of ∼0.14. Moreover, clomipramine increased the rate of decay of currents elicited by acetylcholine. From all of these, we conclude that clomipramine reversibly and noncompetitively regulates neuronal α2β4 nicotinic acetylcholine receptors by blocking the open receptor–channel complex at a site close to the extracellular vestibule of the channel. The actions of clomipramine on neuronal nicotinic acetylcholine receptors may play an important role in the treatment of mental depression and other mood disorders.
Keywords: Clomipramine; Cholinergic–serotonergic interaction; Nicotinic receptor modulation; Xenopus oocyte; Mental depression;

The lipophilic nitric oxide-liberating drug, 1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162), concentration-dependently induced a Cl secretion in rat colon. At a low concentration (5×10−5 M), the action was Ca2+-dependent, whereas at a high concentration (5×10−4 M), the response was independent from extracellular Ca2+. Fura-2 experiments at isolated colonic crypts revealed that GEA 3162 induced an increase of the cytoplasmic Ca2+ concentration due to an influx of extracellular Ca2+, probably mediated by an activation of a nonselective cation conductance as demonstrated by whole-cell patch-clamp studies. After depolarization of the basolateral membrane, GEA 3162 (5×10−4 M) stimulated a current, which was suppressed by glibenclamide but was resistant against blockade of protein kinases by staurosporine, suggesting an activation of apical Cl channels directly by the nitric oxide (NO) donor. After permeabilizing the apical membrane with the ionophore, nystatin, GEA 3162 (5×10−4 M) activated basolateral K+ conductances and the Na+–K+-ATPase. Thus, the lipophilic NO donor GEA 3162 stimulates a Cl secretion in a Ca2+-dependent and -independent manner.
Keywords: Cl channel; Ca2+; K+ channel; Na+–K+-ATPase; NO; Colon;

Activation of IP and EP3 receptors alters cAMP-dependent cell migration by Rüdiger Blindt; Anja-K. Bosserhoff; Jürgen vom Dahl; Peter Hanrath; Karsten Schrör; Thomas Hohlfeld; Jutta Meyer-Kirchrath (31-37).
Migration of vascular smooth cells from the media to the intima essentially contributes to neointima formation after percutaneous transluminal angioplasty and stent implantation. The stable prostacyclin mimetic iloprost has been shown to inhibit neointima formation in experimental restenosis, but it is currently unknown whether this may be caused by an antimigratory effect. Hence, the present study analyses (i) the influence of Gs-coupled prostacyclin (IP) receptors on cell migration and (ii) verifies whether EP3 receptors with opposite (i.e., Gi) coupling may conversely stimulate cell migration. In a modified Boyden chamber model, it was shown that iloprost dose-dependently inhibits the migration of primary human arterial smooth muscle cells, which constitutively express the IP receptor. On the other hand, human arterial smooth muscle cell migration was stimulated by the EP3 receptor agonist M&B 28.767. To independently study the effects of these receptors, IP or EP3 receptors were stably overexpressed in chinese hamster ovary cells (CHO-IP and CHO-EP3). Chemotaxis of CHO cells transfected with Gs-coupled IP receptors was concentration-dependently inhibited by iloprost (2–100 nM), while there was no effect of iloprost on mock-transfected CHO. By contrast, CHO-cells that overexpressed EP3 receptors showed a significant, concentration dependent (1–100 nM) increase of cell migration in presence of the selective EP3 agonist M&B 28.767. It is concluded that the prostacyclin mimetic iloprost inhibits vascular cell migration, which probably depends on a Gs-mediated increase of intracellular cAMP. EP3 receptors conversely stimulate CHO migration.
Keywords: IP receptor; EP3 receptor; cAMP; Cell migration;

Pteropodine and isopteropodine positively modulate the function of rat muscarinic M1 and 5-HT2 receptors expressed in Xenopus oocyte by Tai-Hyun Kang; Kinzo Matsumoto; Michihisa Tohda; Yukihisa Murakami; Hiromitsu Takayama; Mariko Kitajima; Norio Aimi; Hiroshi Watanabe (39-45).
Pteropodine and isopteropodine are heteroyohimbine-type oxindole alkaloid components of Uncaria tomentosa (Willd.) DC, a Peruvian medicinal plant known as cat's claw. In this study, the effects of these alkaloids on the function of Ca2+-activated Cl currents evoked by stimulation of G protein-coupled muscarinic M1 acetylcholine and 5-HT2 receptors were studied in Xenopus oocytes in which rat cortex total RNA was translated. Pteropodine and isopteropodine (1–30 μM) failed to induce membrane current by themselves. However, these alkaloids markedly enhanced the current responses evoked by both acetylcholine and 5-hydroxyhyptamine (5-HT) in a concentration-dependent and reversible manner with the maximal effects at 30 μM. Pteropodine and isopteropodine produced 2.7- and 3.3-fold increases in the acetylcholine response with EC50 values of 9.52 and 9.92 μM, respectively, and 2.4- and 2.5-fold increases in the 5-HT response with EC50 values of 13.5 and 14.5 μM, respectively. In contrast, in oocytes injected with total RNA from the rat cerebellum or spinal cord, neither alkaloid had an effect on the metabotropic current responses mediated by glutamate receptor1 and 5 (mGlu1/5) receptors or ionotropic responses mediated by N-methyl-d-aspartate, kainic acid or glycine. Pteropodine and isopteropodine (10 μM) significantly reduced the EC50 values of acetylcholine and 5-HT that elicited current responses, but had no effect on the maximal current responses elicited by acetylcholine and 5-HT. On the other hand, mitraphylline, a stereoisomer of pteropodine, failed to modulate acetylcholine- and 5-HT-induced responses. These results suggest that pteropodine and isopteropodine act as positive modulators of muscarinic M1 and 5-HT2 receptors.
Keywords: Pteropodine; Isopteropodine; Heteroyohimbine-type oxindole alkaloid; Muscarinic M1 receptor; 5-HT2 receptor; Xenopus oocyte;

We evaluated the effect of several K+ channel blockers on the peripheral antinociception induced by dipyrone using the rat paw pressure test, in which sensitivity is increased by intraplantar injection (2 μg) of prostaglandin E2. Dipyrone administered locally into the right hindpaw (50, 100 and 200 μg) elicited a dose-dependent antinociceptive effect which was demonstrated to be local, since only higher doses produced an effect when injected in the contralateral paw. The specific blockers of ATP-sensitive K+ channels glibenclamide (40, 80 and 160 μg/paw) and tolbutamide (80, 160 and 320 μg/paw) antagonized the peripheral antinociception induced by dipyrone (200 μg/paw). Charybdotoxin (2 μg/ paw), a blocker of large conductance Ca2+-activated K+ channels, and dequalinium (50 μg/paw), a selective blocker of small conductance Ca2+-activated K+ channels, did not modify the effect of dipyrone. This effect was also unaffected neither by intraplantar administration of non-specific voltage-dependent K+ channel blockers tetraethylammonium (1700 μg) and 4-aminopyridine (100 μg) nor cesium (500 μg), a non-specific K+ channel blocker. These results suggest that the peripheral antinociceptive effect of dipyrone may result from activation of ATP-sensitive K+ channels, while other K+ channels appear not to be involved in the process.
Keywords: K+ channel blocker; Antinociception; Dipyrone; K+ channel;

Role of spin trapping and P2Y receptor antagonism in the neuroprotective effects of 2,2′-pyridylisatogen tosylate and related compounds by Kevin Menton; Michael Spedding; Pierre Gressens; Pascal Villa; Toni Williamson; Anthony Markham (53-60).
2,2′-Pyridylisatogen tosylate (PIT) is both an allosteric modulator of P2Y receptors, and an immine oxide, acting as a spin trap for free radicals. PIT (10 mg kg−1, i.p.) was found to be a powerful neuroprotective agent in protecting against the lesions induced by 15 μg S-bromo-willardiine injected into the cortex or white matter of 5-day-old mice pups. As the multiple effects of PIT may induce both beneficial and deleterious effects, a reanalysis of the structure–activity relationship was undertaken. PIT (50 μM) and 2,3′-pyridylisatogen were potent antagonists of responses to ATP in the taenia preparation of the guinea-pig caecum, but 2,3′-nitrophenylisatogen was not. The reactive immine oxide group could be substituted by a keto moiety (N-(2′-pyridyl)phthalide) while maintaining antagonism of responses to ATP, equivalent to PIT. Thus, antagonism of P2Y receptors was not restricted to the isatogen nucleus. Other spin traps did not antagonise P2Y receptors, although dimethyl-pyrroline-N-oxide (DMPO) increased the sensitivity of responses to ATP. Both N-(2′-pyridyl)phthalide and 2,3′-nitrophenylisatogen was less neuroprotective than PIT (10 mg kg−1, i.p.) in protecting against the S-bromo-willardiine-induced lesions in mice, implying that both antagonism of P2Y receptors and the immine oxide moiety may be important for the neuroprotective effects of PIT. However, the usefulness of the neuroprotection was limited because, in motoneurones obtained from rat embryos, PIT (10–100 μM) exacerbated cell death.
Keywords: 2,2′-Pyridylisatogen; Immine oxide; Spin trap; Motoneuron; ATP antagonist; P2Y receptor; Neuroprotection; AMPA receptor;

In vitro neuronal and vascular responses to 5-hydroxytryptamine: modulation by 4-methylthioamphetamine, 4-methylthiomethamphetamine and 3,4-methylenedioxymethamphetamine by James E.J. Murphy; James J. Flynn; Dara M. Cannon; Patrick J. Guiry; Peter McCormack; Alan W. Baird; Gethin J. McBean; Alan K. Keenan (61-67).
4-Methylthioamphetamine and 4-methylthiomethamphetamine are thioarylethylamines structurally related to 3,4-methylenedioxymethamphetamine (MDMA, ‘Ecstasy’). This study compared effects of these agents and MDMA on 5-hydroxytryptamine (5-HT) signalling systems in the brain and vasculature in vitro. Both 4-methylthioamphetamine and 4-methylthiomethamphetamine (100 μM) reduced the rate of specific high affinity [3H]5-HT reuptake in isolated rat brain synaptosomes to 14% and 10% of control, respectively. The concentration required for half-maximal inhibition (IC50) of [3H]5-HT reuptake by 4-methylthioamphetamine (0.27 μM) was significantly lower (P<0.005) than that for inhibition by MDMA (1.28 μM) and that for inhibition by 4-methylthiomethamphetamine (0.89 μM). Both 4-MTA and 4-MTMA caused a significant release of preloaded [3H]5-HT from synaptosomes, but were significantly less effective than MDMA at the concentrations tested (1–100 μM). In isolated rat aorta, a 15-min preincubation with 4-methylthioamphetamine or 4-methylthiomethamphetamine significantly reduced the maximal contraction (E max) induced by 5-HT to 71% or 91% of control (P<0.05 in each case), respectively. In addition, 4-methylthiomethamphetamine (100 μM), but not 4-methylthioamphetamine, significantly increased the concentration of 5-HT required for half-maximal contraction (EC50) from 4.13 to 20.08 μM (P<0.0001). In contrast, MDMA did not significantly alter the E max or the EC50 of 5-HT-induced aortic contraction. It is concluded that both 4-methylthioamphetamine and 4-methylthiomethamphetamine are potent inhibitors of [3H]5-HT reuptake in the brain. Furthermore, unlike MDMA, they both directly inhibit 5-HT-mediated vascular contraction. These results suggest that these compounds may be potentially more harmful than MDMA in the context of human misuse.
Keywords: 4-MTA (4-methylthioamphetamine); 4-MTMA (4-methylthiomethamphetamine); MDMA (3,4-methylenedioxymethamphetamine); 5-HT (5-hydroxyhyptamine, serotonin); Reuptake; Synaptosome; Aortic contraction;

Effect of the amisulpride isomers on rat catalepsy by Giorgio Marchese; Francesco Bartholini; Stefania Ruiu; Paola Casti; Pierluigi Saba; Gian Luigi Gessa; Luca Pani (69-74).
The substituted benzamide amisulpride is currently administered in its racemic form. In the present study, the biochemical and cataleptogenic profiles of the two enantiomers (R+ and S−) were compared with those of the racemic mixture. Displacement binding studies showed that the (S−)-isomer possesses an higher affinity for dopamine D2-like receptor (K i 5.2±0.4 nM) compared to (R+)-amisulpride (K i 244±12 nM) and to (RS)-amisulpride (K i 9.8±0.8 nM). In contrast, (S−)-amisulpride binds the α2-receptor with an affinity (K i 1528±45 nM) lower than that of the (R+)-isomer (K i 375±34 nM) and of (RS)-amisulpride (K i 783±27 nM). The bar test was used to evaluate the catalepsy induced by each drug. (RS)-amisulpride induced catalepsy only at very high doses (>100 mg/kg, s.c.) whereas, (S−)-amisulpride produced a catalepsy at a lower dose (30 mg/kg, s.c.) and (R+)-amisulpride did not produce any catalepsy up to the dose of 75 mg/kg. Interestingly, (R+)-amisulpride reduced the catalepsy induced by (S−)-amisulpride (50 mg/kg, s.c.) or haloperidol (0.3 mg/kg, s.c.), at the doses of 50 or 30 mg/kg, respectively. These results indicate that the weak cataleptic properties of (RS)-amisulpride might partially rely on its (R+)-isomer and provide a further explanation for the atypical properties of amisulpride as an antipsychotic.
Keywords: Antipsychotic; Benzamide; Extrapyramidal symptom;

Reduction of cerebral injury in stroke-prone spontaneously hypertensive rats by amlodipine by Erwin L.A Blezer; Klaas Nicolay; Roel Goldschmeding; Hein A Koomans; Jaap A Joles (75-81).
Dihydropyridine Ca2+ channel antagonists, initiated together with high salt intake, prevent the development of hypertension and subsequent cerebral damage in stroke-prone spontaneously hypertensive rats (SHRSP). We hypothesized that the dihydropyridine Ca2+ channel antagonist amlodipine (approximately 15 mg/kg/day) could also reverse established hypertension and cerebral damage. SHRSP drank 1% NaCl from 8 weeks of age. Cerebral damage (cerebral edema and blood–brain barrier integrity) was investigated with magnetic resonance imaging twice a week. Systolic blood pressure was measured weekly. All rats developed severe hypertension and subsequent cerebral damage (defined as day 0). Untreated controls (n=7) died at day 12 (range: 7–28). Oral treatment with amlodipine (n=7), initiated at day 0, reduced systolic blood pressure, reversed cerebral edema and restored blood–brain barrier integrity. Systolic blood pressure remained low and eventually rats died after 450 days (range: 350–580) showing nephrosis but no recurrence of cerebral damage. In conclusion, established hypertension and cerebral damage are reversed by amlodipine in SHRSP.
Keywords: Cerebral edema; Dihydropyridine Ca2+ channel antagonist; Magnetic resonance imaging; Hypertension; Hypertensive encephalopathy; (Rat);

Effects of acamprosate and some polyamine site ligands of NMDA receptor on short-term memory in rats by Przemyslaw Mikolajczak; Irena Okulicz-Kozaryn; Ewa Kaminska; Lidia Niedopad; Anna Polanska; Joanna Gebka (83-96).
The aim of this study was to evaluate the effect of multiple acamprosate (500.0 mg/kg, p.o.) administration on short-term memory, using the social recognition test in rats. Ifenprodil (1.0 mg/kg, i.p.), arcaine (5.0 mg/kg, i.p.) and spermidine (20.0 mg/kg, i.p.) were chosen as polyamine ligands and their action or interaction with acamprosate was also studied. The doses used did not show any sedative activity, which was assessed by measuring locomotor activity and the hypnotic effect of ethanol. The findings suggest that acute acamprosate treatment did not impair short-term memory. Multiple acamprosate and a single spermidine or arcaine administration led to better performance in the memory test, whereas no significant difference was observed in ifenprodil-treated rats. Co-administration of a single arcaine or spermidine dose with multiple acamprosate produced worse results. This means that the effect of repeated acamprosate administration can be changed by the co-administration of other polyamine ligands, so that care should be taken in interpreting.
Keywords: Acamprosate; Memory; Polyamines; Ifenprodil;

Role of endothelium-derived relaxing factors in adrenomedullin-induced vasodilation in the rat kidney by Rosemary Wangensteen; Andrés Quesada; Juan Sainz; Juan Duarte; Félix Vargas; Antonio Osuna (97-102).
The present study aimed to evaluate the contributions of endothelium-derived hyperpolarizing factor (EDHF), the nitric oxide (NO)-cGMP pathway, and prostaglandins to adrenomedullin-induced vasodilation in isolated rat kidney. Inhibition of the NO-cGMP pathway with N ω-nitro-l-arginine methyl ester (l-NAME) or 1H-[1,2,4]oxadiazolo-[4,3a]quinoxalin-1-one (ODQ) reduced the maximal vasodilator response to adrenomedullin by approximately 50%. Pretreatment of the vessels with the potassium channel inhibitor, tetraethylammonium or increased extracellular K+, also decreased the maximal response to adrenomedullin by approximately 50%. The simultaneous administration of blockers of both endothelium-derived relaxing factors had a combined effect that almost suppressed adrenomedullin-induced vasodilation. The administration of indomethacin did not modify the renal response to adrenomedullin. Our results suggest that the vasodilator response to adrenomedullin in the isolated perfused kidney of rats is mediated by EDHF and NO to a similar extent. Our data also provide evidence that prostaglandins play no role in the vasodilator response to adrenomedullin in the renal vasculature.
Keywords: Kidney; Adrenomedullin; Vasodilation; Nitric oxide (NO); EDHF (endothelium-derived hyperpolarizing factor);

Relaxation induced by milrinone and rolipram in human penile arteries and veins by Gloria Segarra; Pascual Medina; José M. Vila; Juan B. Martinez-León; Cristina Domenech; Fernando Prieto; Salvador Lluch (103-106).
We studied the relaxant effects of milrinone, an inhibitor of phosphodiesterase 3, and rolipram, an inhibitor of phosphodiesterase 4, on contracted human penile dorsal artery and deep dorsal vein. Vascular rings from 12 multi-organ donors were suspended in organ baths for isometric recording of tension. Both milrinone and rolipram inhibited (100%) the contraction induced by noradrenaline and shifted the relaxation–response curves to the cAMP forming agents prostaglandin E1 and forskolin to the left. The findings indicate that the cAMP pathway appears to be a main determinant of relaxation in human penile vessels.
Keywords: Penile artery; Penile vein; Milrinone; Rolipram; cAMP pathway;

Triglyceride-lowering effect of pitvastatin in a rat model of postprandial lipemia by Taro Aoki; Yasunobu Yoshinaka; Hiroyuki Yamazaki; Hideo Suzuki; Taro Tamaki; Fumiyasu Sato; Masaki Kitahara; Yasushi Saito (107-113).
The triglyceride-lowering effect of pitavastatin, a potent 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, was investigated in a rat model of postprandial lipemia. Plasma triglyceride levels started to increase 4 h after the fat load, reached the maximum at 6 h and then gradually decreased. A single dose of pitavastatin (1 mg/kg) significantly suppressed chylomicron-triglyceride secretion into the lymph by 40% and delayed the elevation of plasma triglyceride. Pitavastatin at 1 mg/kg decreased the 6-h plasma triglyceride levels by 53% and at 0.5 mg/kg decreased the 0–12 h area under the curve (AUC) of triglyceride levels by 56%. Atorvastatin also caused decreases, but to a lesser extent. Pitavastatin, and atorvastatin to a lesser extent, reduced the activity of the intestinal microsomal triglyceride transfer protein (MTP) at 6 h. These results suggested that a single dose of pitavastatin lowered postprandial triglyceride levels in rats by decreasing chylomicron-triglyceride secretion, probably through a reduction of intestinal MTP activity and triglyceride droplet formation in the endoplasmic reticulum.
Keywords: Pitavastatin; 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitor; Model of postprandial lipemia; Lymph chylomicron; Microsomal triglyceride transfer protein;

Nicotine suppresses gastric wound repair via the inhibition of polyamine and K+ channel expression by Vivian Y. Shin; Edgar S.L. Liu; Marcel W.L. Koo; Jiing C. Luo; Wallace H.L. So; Chi H. Cho (115-121).
Nicotine is one of the most representative components in cigarette smoke leading to gastric ulceration. Both ornithine decarboxylase and potassium ion (K+) channels are essential for cell growth and wound repair. The aim of the present study is to elucidate the causative relationship of these two factors during wound healing and the influence of nicotine on this healing process in rat gastric mucosal epithelial cells (RGM-1). Nicotine markedly inhibited cell migration and proliferation in RGM-1 cells. The latter effect was significantly antagonized by a nicotinic receptor blocker, mecamylamine. Nicotine also suppressed ornithine decarboxylase activity significantly. Our data showed that inhibition of cell proliferation and ornithine decarboxylase activity by nicotine was accompanied with a reduction in K+ channel protein expression, all of which were significantly alleviated by spermidine pretreatment. These results suggested that there was a cause/effect link between ornithine decarboxylase and K+ channel on wound repair. Nicotine in cigarette smoke inhibited this healing process and delayed wound repair in gastric epithelial cells.
Keywords: Nicotine; Cell migration; Cell proliferation; Ornithine decarboxylase; K+ channel; Wound healing; Nicotine receptor;