European Journal of Pharmacology (v.409, #1)

Cilostazol prevents impairment of slow axonal transport in streptozotocin-diabetic rats by Yasuchika Yamamoto; Yoshinobu Yasuda; Yoshiaki Komiya (1-7).
We studied the effects of cilostazol, an antiplatelet and vasodilating agent, on axonal transport patterns of cytoskeletal proteins in the motor fibers of sciatic nerve of streptozotocin-induced diabetic rats. Proteins labeled with l-[35S]methionine in 6-mm consecutive segments of the nerve were analyzed electrophoretically following fractionation into Triton-soluble and-insoluble subpopulations. Transport rates of proteins (particularly neurofilaments) in slow component a were reduced by 50% 2 weeks after labeling (4 weeks after streptozotocin). An apparent reduction of tubulin and actin was observed at later intervals after induction of diabetes. Actin transported in slow component b was also impaired, though to a lesser extent than in component a. Cilostazol prevented transport impairment of both slow components a and b without affecting hyperglycemia or reduction in body weight gain. These results suggest that in sciatic motor fibers early defects in slowly transported proteins are more marked in slow component a, and that impairment may be caused primarily by hemodynamic abnormalities.
Keywords: Streptozotocin-diabetic, rat; Cilostazol; Antiplatelet vasodilating agent; Phosphodiesterase inhibitor;

Cytotoxicity and cell cycle effects of the plant alkaloids cryptolepine and neocryptolepine: relation to drug-induced apoptosis by Laurent Dassonneville; Amélie Lansiaux; Aurélie Wattelet; Nicole Wattez; Christine Mahieu; Sabine Van Miert; Luc Pieters; Christian Bailly (9-18).
Cryptolepine and neocryptolepine are two indoloquinoline derivatives isolated from the roots of the african plant Cryptolepis sanguinolenta. These two alkaloids, which only differ by the respective orientation of their indole and quinoline rings, display potent cytotoxic activities against tumour cells and present antibacterial and antiparasitic properties. Our previous molecular studies indicated that these two natural products intercalate into DNA and interfere with the catalytic activity of human topoisomerase II. Here we have extended the study of their mechanism of action at the cellular level. Murine and human leukemia cells were used to evaluate the cytotoxicity of the drugs and their effects on the cell cycle were measured by flow cytometry. Cryptolepine, and to a lesser extent neocryptolepine, provoke a massive accumulation of P388 murine leukemia cells in the G2/M phase. With HL-60 human leukemia cells, the treatment with cryptolepine leads to the appearance of a hypo-diploid DNA content peak (sub-G1) characteristic of the apoptotic cell population. With both P388 and HL-60 cells, cryptolepine proved about four times more toxic than its isomer. But the use of the HL-60/MX2 cell line resistant to the anticancer drug mitoxantrone suggests that topoisomerase II may not represent the essential cellular target for the alkaloids, which are both only two times less toxic to the resistant HL-60/MX2 cells compared to the parental cells. The capacity of the drugs to induce apoptosis of HL-60 human leukemia cells was examined by complementary biochemical techniques. Western blotting analysis revealed that cryptolepine, but not neocryptolepine, induces cleavage of poly(ADP-ribose) polymerase but both alkaloids induce the release of cytochrome c from the mitochondria. The cleavage of poly(ADP-ribose) polymerase observed with cryptolepine correlates with the appearance of a marked sub-G1 peak in the cell cycle experiments. The proteolytic activity of Asp-Glu-Val-Asp- or Ile-Glu-Thr-Asp-caspases was found to be enhanced much more strongly with cryptolepine than with its isomer, as expected from their different cytotoxic potential. Despite the activation of the caspase cascade, we did not detect internucleosomal cleavage of DNA in the HL-60 cells treated with the alkaloids. Altogether, the results shed light on the mechanism of action of these two plant alkaloids.
Keywords: Cryptolepine; Neocryptolepine; Plant alkaloid; Topoisomerase II; Cytotoxicity; Apoptosis;

Previous studies from our laboratory as well as from others have suggested that the thiazolidinediones have the capacity to act as insulinomimetic agents, especially in the liver. In order to further characterize this insulinomimetic action, we evaluated the effect of troglitazone, a representative thiazolidinedione, on lactate- and glucagon-stimulated gluconeogenesis, in the presence or absence of insulin (10 nM) in isolated rat hepatocytes. The antigluconeogenic effect of troglitazone under basal (lactate-stimulated) conditions was found to be due to an elevation in the fructose 2,6-bisphosphate content, which was, however, not mediated by an activation of 6-phosphofructo 2-kinase. Troglitazone (125 and 250 μM) in the absence of insulin, produced a dose-dependent reduction in glucagon-stimulated gluconeogenesis, thereby suggesting an insulinomimetic effect. In addition, troglitazone (125 and 250 μM), in combination with insulin, produced an additive inhibition of gluconeogenesis during glucagon-stimulated conditions. However, unlike insulin, the metabolic mechanism responsible for these effects (in the presence or absence of insulin) does not involve fructose 2,6-bisphosphate.
Keywords: Troglitazone; Fructose 2,6-bisphosphate; Hepatocyte; Insulin; Thiazolidinedione;

The antifolate drug methotrexate is mainly eliminated from the body by renal tubular secretion via organic anion transporters. In clinical situations, severe methotrexate toxicity, due to an increase in serum concentrations, was observed after coadministration with nonsteroidal anti-inflammatory drugs (NSAIDs) or probenecid. In this study, we examined the effects of NSAIDs and probenecid on methotrexate transport via the rat renal organic anion transporter rOAT1, using Xenopus laevis oocytes. [3H]Methotrexate uptake was markedly stimulated in the rOAT1 cRNA-injected oocytes, and this uptake was inhibited by probenecid and various NSAIDs, whereas the influence of salicylate was less. The Dixon plots showed that probenecid, indomethacin and salicylate competitively inhibited rOAT1 with apparent K i values of 15.8 μM, 4.2 μM and 1.0 mM, respectively. These findings demonstrate that rOAT1 is the major site of the transporter-mediated interaction between methotrexate and NSAIDs and/or probenecid, leading to a decrease in renal excretion of methotrexate.
Keywords: OAT1 (organic anion transporter 1); p-Aminohippurate; Methotrexate; Probenecid; NSAID (nonsteroidal anti-inflammatory drug); Kidney; (Rat);

Effect of intrathecal administration of serotonin in chronic pain models in rats by Laurent Bardin; Jeannot Schmidt; Abdelkrim Alloui; Alain Eschalier (37-43).
The present study examined the effects of intrathecal (i.t.) administration of 5-hydroxytryptamine (5-HT; 0.1–100 μg) on mechanical hyperalgesia associated with neuropathic pain (chronic constriction of the sciatic nerve model and diabetic model) and inflammatory pain (carrageenan and polyarthritic models) in rats. Results demonstrated that the hyperalgesia observed in the mononeuropathic and diabetic rats was attenuated by 5-HT; the active dose, however, was 100- to 1000-fold higher than that required in normal rats, and was moderately effective. In the two experimental models of inflammatory pain, 5-HT was not markedly or similarly active. In the carrageenan model, 5-HT at the highest dose was only weakly effective whereas in the polyarthritic model it was inactive.Together, these results show that 5-HT has antinociceptive effects in several rat pain models, except in the model of diffuse pain (polyarthritic rats). Its antinociceptive effects in these models, however, are slight and differ from those observed in normal rats.
Keywords: 5-HT (5-hydroxytryptamine, serotonin); Chronic pain; Spinal cord; Paw pressure test; (Rat);

TC-2559: A novel orally active ligand selective at neuronal acetylcholine receptors by Merouane Bencherif; Andrew J Bane; Craig H Miller; Gary M Dull; Gregory J Gatto (45-55).
TC-2559 [(E)-N-Methyl-4-[3-(5-ethoxypyridin)yl]-3-buten-1-amine] is a novel nicotinic agonist markedly more selective than recently reported novel nicotinic receptor ligands (selectivity ratio for central nervous system (CNS) to peripheral nervous system (PNS)>4000). TC-2559 competes effectively with [3H]-nicotine binding (K i=5 nM) but not with [125I]-bungarotoxin (>50,000 nM). Dopamine release from striatal synaptosomes and ion flux from thalamic synaptosomes indicate that TC-2559 is potent and efficacious in the activation of CNS receptors and significantly reduced glutamate-induced neurotoxicity in vitro. TC-2559 has no detectable effects on muscle and ganglion-type nicotinic acetylcholine receptors at concentrations up to 1 mM. TC-2559 significantly attenuates scopolamine-induced cognitive deficits in a step-through passive avoidance task. Acute and repeated oral dosing of TC-2559 enhances performance in a radial arm maze task. In contrast to the effects of equimolar concentrations of (−) nicotine, TC-2559 does not induce hypothermia and locomotor activity is not enhanced following repeated daily administration of 14 days. TC-2559 has a markedly enhanced CNS-PNS selectivity ratio and an intra-CNS selectivity as evidenced by the improved cognition without increased locomotor activity. The in vitro and in vivo studies in the present study suggest that TC-2559 has the desired profile to be further evaluated as a potential therapeutic agent for neurodegenerative diseases.
Keywords: TC-2559; Nicotinic receptor; Cognition; Central nervous system;

Phencyclidine (PCP) is widely used as an animal model of schizophrenia. In rats, acute PCP treatment increased locomotor activity and induced stereotyped behaviours consisting of head weaving, turning and backpedalling. PCP had differential regional effects on c-fos expression in rat brain, suggesting different patterns of neuronal activity. The most prominent immunostaining was observed in the cortical regions. To elucidate the role of nitric oxide, an important intracellular messenger, in the mechanism of action of PCP the effects of nitric oxide synthase inhibitor N G-nitro-l-arginine methyl ester (l-NAME) were studied in PCP-treated animals. l-NAME potentiated PCP-induced behaviours and c-fos expression in many brain regions. The greatest increases were observed in the frontal, retrosplenial granular cortex, cerebellum, thalamic and subthalamic nuclei. While PCP alone induced low c-fos expression in the entorhinal cortex, with almost no expression in the rostral part of caudate putamen, animals pretreated with l-NAME showed marked activation in these brain areas. These results strongly indicate the involvement of the nitric oxide system in the mechanism of action of PCP.
Keywords: Phencyclidine; Nitric oxide (NO); N G-nitro-l-arginine methyl ester; Behaviour; c-fos; (Rat);

Increased plasma corticosterone, aggressiveness and brain monoamine changes induced by central injection of pertussis toxin by Do-Hoon Kim; Jun-Sub Jung; Ji-Jing Yan; Hong-Won Suh; Bong-Ki Son; Yung-Hi Kim; Dong-Keun Song (67-72).
The effects of intracerebroventricular (i.c.v.) injection of pertussis toxin, a specific inhibitor of Gi/Go proteins, on plasma corticosterone levels, aggressiveness, and hypothalamic and hippocampal monoamines and their metabolites levels were examined in mice. Plasma corticosterone level was markedly increased at 3 h after pertussis toxin injection (0.03 and 0.2 μg/mouse), peaked at 6 h and was still increased for up to 6 days after injection. Mice injected with pertussis toxin (0.2 μg/mouse) did not show weight gain between day 0 and day 6 after injection. In addition, pertussis toxin (0.2 μg/mouse) induced a progressive increase in aggressiveness, i.e. a decrease in attack latency and an increase in number of attacks, on day 1 and 6 after injection. Brain monoamines and their metabolites levels were changed on day 1 and 6 after pertussis toxin injection (0.2 μg/mouse): in the hypothalamus, levels of dopamine and 3,4-dihydroxyphenylacetic acid were increased, norepinephrine level decreased, and 5-hydroxyindole acetic acid (5-HIAA) level was markedly increased, with no changes in 5-hydroxytryptamine (5-HT) level, whereas in the hippocampus, 5-HT level was significantly decreased, with no changes in 5-HIAA and catecholamines. These results suggest that signal transduction through Gi/Go proteins in the brain is involved in the modulation of hypothalamo-pituitary-adrenal axis, aggressiveness, and monoamine levels in vivo.
Keywords: Hypothalamo-pituitary-adrenal axis; Aggressive behavior; Monoamine; Pertussis toxin; (Mouse);

The role of dopamine in the effects of midazolam on ingestive behaviour was investigated using microstructural analysis of licking behaviour in the rat. Midazolam (1.8 mg/kg i.p.) was administered alone or in combination with the dopamine D2 receptor antagonist raclopride (0.1 and 0.3 mg/kg i.p.). The effect on licking patterns during 60 s exposure to a range of concentrations of sucrose solution was recorded using an automated lickometer. Midazolam increased the total number of licks via an increase in mean bout duration, an effect consistent with the proposal that these drugs enhance palatability. Midazolam also decreased the intrabout lick rate, probably because of muscle relaxant effects. Pre-treatment with raclopride blocked midazolam-induced increases in mean bout duration, at doses that by themselves were ineffective, but did not reverse the decrease in intrabout lick rate. These data point to the interdependence of benzodiazepine and dopamine substrates in the mediation of palatability.
Keywords: Benzodiazepine; Dopamine; Licking behaviour; Bout structure; Palatability;

The effects of the selective serotonin (5-hydroxytryptamine (5-HT)) reuptake inhibitor fluvoxamine, given alone or in combination with the benzodiazepine anxiolytic diazepam on the defensive freezing behavior of mice in the conditioned fear stress paradigm were examined. Fluvoxamine (5–20 mg/kg, i.p.) induced a dose-dependent reduction in freezing behavior. In contrast, while low doses of diazepam (0.125 and 0.25 mg/kg, i.p.) reduced the freezing behavior, such effects were not observed with high doses of diazepam (0.5 and 1 mg/kg, i.p.). In the combination study, fluvoxamine (20 mg/kg, i.p.) did not reduce the freezing behavior in mice that had been pretreated with diazepam (0.125–1 mg/kg, i.p.). None of the doses of fluvoxamine and diazepam used in the present study had any effects on motor activity under non-stressed conditions. These results suggest that benzodiazepines may negatively influence the clinical efficacy of selective 5-HT reuptake inhibitors in the treatment of anxiety disorders.
Keywords: Fluvoxamine; Diazepam; Anxiety; Conditioned fear stress; Defensive freezing behavior; (Mouse);

Nitric oxide synthase-independent release of nitric oxide induced by KCl in the perfused mesenteric bed of the rat by V.E Mendizabal; I Poblete; A Lomniczi; V Rettori; J.P Huidobro-Toro; E Adler-Graschinsky (85-91).
The aim of the present study was to test whether the contractile responses elicited by KCl in the rat mesenteric bed are coupled to the release of nitric oxide (NO). Contractions induced by 70 mM KCl were coincident with the release of NO to the perfusate. The in vitro exposure to the nitric oxide synthase (NOS) inhibitor l-N ω-nitro-l-arginine methyl ester, l-NAME (1–100 μM) potentiated the vascular responses to 70 mM KCl and, unexpectedly, increased the KCl-stimulated release of NO. Moreover, even after the chronic treatment with l-NAME (70 mg/kg/day during 4 weeks), the KCl-induced release of NO was not reduced, whereas the potentiation of contractile responses was indeed achieved. The possibility that NOS had not been completely inhibited under our experimental conditions can be precluded because NOS activity was significantly inhibited after both l-NAME treatments. After the in vitro treatment with 1 to 100 μM l-NAME, the inhibition of NOS was concentration-dependent (from 50% to 90%). With regard to the basal release of NO, the inhibition caused by l-NAME was not concentration-dependent and reached a maximum of 40%, suggesting that basal NO outflow is only partially dependent on NOS activity. An eventual enhancement of NOS activity caused by KCl was disregarded because the activity of this enzyme measured in homogenates from mesenteric beds perfused with 70 mM KCl was significantly reduced. On the other hand, endothelium removal, employed as a negative control, almost abolished NOS activity, whereas the incubation with the Ca2+ ionophore A23187, employed as a positive control, induced an increase in NOS activity. It is concluded that in the mesenteric arterial bed of the rat, the contractile responses elicited by depolarization through KCl are coincident with a NOS-independent release of NO. This observation, which differs from the results obtained with noradrenaline, do not support the use of KCl as an alternative contractile agent whenever the participation of NO is under study.
Keywords: Nitric oxide (NO); Mesenteric bed; N ω-nitro-l-arginine methyl ester; Depolarization; KCl; Nitric oxide (NO) synthase;