European Journal of Pharmacology (v.465, #3)

Antitumor activity induced by regulatory RNA: possible role of RNA-dependent protein kinase and nuclear factor-κB by Maria Angelica Ehara Watanabe; Liliana Rodrigues Souza; Joana Maria Murad; Fernando Luiz De Lucca (205-210).
Regulatory RNAs are noncoding RNAs that can regulate gene expression. Our previous results showed that regulatory RNAs can induce the production of interleukin-1, interleukin-2, interleukin-8, tumor necrosis factor-α (TNF-α), interferon-γ, and Fas ligand (FasL). These cytokines and FasL are involved in host defense mechanisms against tumors. B16-F10 melanoma cells are highly metastatic to the lungs and we showed that lymphocytes treated with the regulatory B16-RNA reduce significantly the number of metastatic nodules. We also found that B16-RNA activates RNA-dependent protein kinase (PKR) and the active B16-RNA fraction is polyadenylated with a sedimentation coefficient of 18S. Our findings suggest that the antitumor activity of B16-RNA is mediated by PKR through activation of the transcription factor NF-κB. Thus, B16-RNA may act as a regulatory RNA and may regulate gene expression at transcriptional level. This study provides the rationale for the use of B16-RNA as an immunomodulator in melanoma.
Keywords: RNA; RNA; PKR (RNA-dependent protein kinase); NF-κB (nuclear factor-κB); B16-F10 melanoma; TNF-α (tumor necrosis factor-α);

Agonist properties of putative small-molecule somatostatin sst2 receptor-selective antagonists by Caroline Nunn; Daniel Langenegger; Konstanze Hurth; Kerstin Schmidt; Dominique Fehlmann; Daniel Hoyer (211-218).
The availability of antagonist ligands for somatostatin receptors is very limited, with those that are available often displaying agonist properties or limited receptor subtype selectivity. Hay et al. [Bioorg. Med. Chem. Lett. 11 (2001) 2731] recently described the development of small-molecule somatostatin receptor subtype 2 (sst2) selective compounds. This study investigates the binding affinity and functional characteristics of two of those antagonists (2 and 3) and the agonist compound, from which they were derived (1). In radioligand binding studies using the agonist radioligands [125I][Tyr11]SRIF-14 (Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-(125I-Tyr)-Thr-Ser-Cys]-OH), [125I]LTT-SRIF-28 ([Leu8,dTrp22,125I-Tyr25]SRIF-28; Ser-Ala-Asn-Ser-Asn-Pro-Ala-Leu-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-dTrp-Lys-Thr-(125I-Tyr)-Thr-Ser-Cys]-OH), [125I]CGP 23996 (c[Lys-Asu-Phe-Phe-Trp-Lys-Thr-(125I-Tyr)-Thr-Ser]), [125I][Tyr3]octreotide (dPhe-c[Cys-(125I-Tyr)-dTrp-Lys-Thr-Cys]-Thr-OH) and [125I][Tyr10]cortistatin-14 (Pro-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-(125I-Tyr)-Ser-Ser-Cys]-Lys) at human recombinant somatostatin receptors expressed in Chinese hamster lung fibroblast (CCL39) cells and native rat cortex, the compounds bound with high affinity (pK d 6.8–9.7) and selectivity to human sst2 receptors. Some affinity was also observed for sst5 labelled by [125I][Tyr3]octreotide and [125I]CGP 23996. In functional studies at human sst2 receptors expressed in Chinese hamster ovary (CHO) cells, both the agonist 1 and the two putative antagonists 2 and 3 concentration dependently inhibited forskolin-stimulated adenylate cyclase and stimulated luciferase reporter gene expression, with similar efficacy to the natural ligand somatotropin release inhibiting factor (SRIF)-14. Compound 1 had similar potency to SRIF-14, which was in the nanomolar range, whereas 2 and 3 were 10–100-fold less potent. The intrinsic activity of 2 and 3 was too high to allow antagonist studies to be carried out. In conclusion, in contrast to previous findings, all three compounds are potent agonists at recombinant human sst2 receptors.
Keywords: Somatostatin; Somatostatin sst2 receptor 2; Non-peptide antagonist; Luciferase; SRE (serum response element); cAMP;

KR-31372 inhibits KDR/Flk-1 tyrosine phosphorylation via K+ ATP channel opening in its antiangiogenic effect by Ki Young Kim; Sun-Ok Kim; Hong Lim; Sung-Eun Yoo; Ki Whan Hong (219-228).
The aim of this study was to identify the signaling pathway of the antiangiogenesis by (2R,3R,4S)-Nʺ-cyano-N-(6-nitro-3,4-dihydro-hydroxy-2-methyl-2-dimethoxymethyl 2H-1-benzopyran-4yl)-N′-benzylguanidine (KR-31372). KR-31372 inhibited the in vitro basal tube formation using Matrigel-coated plate and in vivo neovascularizations in mice induced by Matrigel containing vascular endothelial growth factor (VEGF165, 5 ng/ml). VEGF165 markedly increased cell proliferation using 5-bromo-2′-deoxyuridine incorporation and chemotactic migration using transwell chamber in human umbilical vein endothelial cells, those of which were significantly suppressed by pretreatment with KR-31372 and levcromakalim concentration dependently. The suppression of all these variables were strongly antagonized by glibenclamide, ATP-sensitive K+ channel blocker. KR-31372 (10−6–10−4 M) and levcromakalim (10−5 M) concentration-dependently suppressed the VEGF165-induced increases in KDR/Flk-1 tyrosine phosphorylation as well as the extracellular signal-related kinase 1/2 (ERK1/2), p38 MAK and p125FAK tyrosine phosphorylation. These variables were significantly antagonized by glibenclamide. In conclusion, KR-31372 significantly inhibited the KDR/Flk-1 tyrosine phosphorylation-linked ERK1/2, p38 MAPK and p125FAK tyrosine phosphorylation via mediation of K+ ATP channel opening, thereby resulting in antiangiogenesis.
Keywords: KR-31372; K+ channel opener, ATP-sensitive; VEGF (vascular endothelial growth factor); p125FAK; KDR/Flk-1; Endothelial cell; umbilical vein, human;

Organoammonium hydroselenites: antitumor action through radical balance regulation by Pavel Arsenyan; Irina Shestakova; Kira Rubina; Ilona Domracheva; Alena Nesterova; Kristina Vosele; Olga Pudova; Edmunds Lukevics (229-235).
Organoammonium hydroselenites were synthesized and investigated as potential selective, anticancer prodrugs. These compounds were studied in vitro on human fibrosarcoma (HT-1080), hamster kidney endothelial (BHK 21) and normal mouse embryonic fibroblasts (NIH 3T3). Most of them were very active against HT-1080 (0.6–5.3 g/ml). Amino acid hydroselenites readily increased the nitric oxide (NO) concentration in the culture medium of HT-1080 cells (up to TG100=1500%); however, 4-amidohydroximinomethylpyridinium hydroselenite (TG100=24%) and o-phenanthrolinium hydroselenite (TG100=50%) were free radical inhibitors. All compounds were glutathione peroxidase inhibitors; some of them could also prevent hydrogen peroxide degradation by inhibition of catalase. The influence of the investigated ammonium hydroselenites on tumor cell (HT-1080) morphology was examined. The substances studied were also active in vivo against sarcoma S-180. The role of organoammonium hydroselenites as free radical regulators and their therapeutic antitumor are discussed.
Keywords: Antitumor activity; Catalase; Hydroselenite; Glutathione peroxidase; Morphology; Nitric oxide (NO); Selenium;

Characterization of muscarinic receptor subtypes in the rostral ventrolateral medulla and effects on morphine-induced antinociception in rats by Kenji Abe; Kyoji Taguchi; Masatoshi Kato; Iku Utsunomiya; Toshiyuki Chikuma; Hiroshi Hojyo; Tadashi Miyatake (237-249).
The present study investigated the role of muscarinic receptor subtypes in the nucleus reticularis gigantocellularis/nucleus reticularis gigantocellularis alpha of the rat rostral ventrolateral medulla in morphine-induced antinociception. The antinociceptive effects of morphine were evoked by systemic administration or microinjection into the nucleus reticularis gigantocellularis/nucleus reticularis gigantocellularis alpha. Administration of morphine produced antinociception for hot plate and tail immersion responses to noxious heat stimuli. These effects were antagonized by prior exposure to naloxone and inhibited by mecamylamine pretreatment. Morphine-induced antinociception was significantly inhibited by atropine in a dose-dependent manner. Muscarinic toxin-1 and pirenzepine inhibited morphine-induced antinociception for both the hot plate and tail immersion tests. At a dose of 5 nmol/site, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) also inhibited morphine-induced antinociception, although low doses of this drug did not significantly affect hot plate test response latency when morphine was systemically administered. These results suggest that the antinociceptive effects induced by morphine in part involve the muscarinic M1 and M3 receptors of the rat nucleus reticularis gigantocellularis/nucleus reticularis gigantocellularis alpha.
Keywords: Morphine; Antinociception; Nucleus reticularis gigantocellularis/nucleus reticularis gigantocellularis alpha; Muscarinic antagonist; (Rat);

The contribution of α2-adrenoceptor and opioid receptor mechanisms to antinociception differs in Lewis and Fischer 344 rats by Gonzalo Herradón; Lydia Morales; Carmen Pérez-Garcı́a; Luis F. Alguacil (251-256).
Lewis and Fischer 344 (F344) rats differ in their physiological and pharmacological responses to a variety of environmental stimuli, which have been partially attributed to endogenous opioid function. Since opioid and α2-adrenoceptor mechanisms are closely related, we have comparatively examined the contribution of both systems to antinociception in female Lewis and F344 rats by the tail-flick method. Basal responses of F344 and Lewis rats were found to be similar, both showing a slight but significant increase in reaction time along the experimental period which was not completely reversed by naloxone. Morphine exhibited a bell-shaped dose–response curve in Lewis rats, these animals being more sensitive than F344 at 1 and 5 mg/kg but less sensitive at 10 mg/kg. Clonidine up to 0.1 mg/kg was more active in F344 rats. The α2-adrenoceptor antagonist yohimbine provoked a higher hyperalgesic effect in Lewis rats and decreased morphine antinociception in both strains. The existence of a balanced contribution of opioid and α2-adrenoceptor mechanisms to control pain transmission in both strains is discussed.
Keywords: Antinociception; Lewis rat; F344 rat; Morphine; Yohimbine; Clonidine;

Evaluation of the novel antiepileptic drug, AWD 131-138, for benzodiazepine-like discriminative stimulus and reinforcing effects in squirrel monkeys by Sevil Yasar; Jack Bergman; Patrik Munzar; Godfrey Redhi; Christine Tober; Norbert Knebel; Michael Zschiesche; Carol Paronis (257-265).
AWD 131-138 {1-(4-chlorophenyl)-4-morpholino-imidazolin-2-one}, a new low-affinity partial benzodiazepine receptor agonist with potent anticonvulsant and anxiolytic properties in rodent models, was studied in squirrel monkeys trained to discriminate intramuscular (i.m.) injections of midazolam (0.3 mg/kg) from injections of vehicle. Diazepam produced midazolam-like responding at cumulative doses of 1.0 and 3.0 mg/kg i.m. and decreased rates of responding at 3.0 mg/kg (plasma levels of about 400 ng/ml). In contrast, AWD 131-138 did not produce midazolam-like responding or alter response rates at cumulative doses up to 18.0 mg/kg i.m. (plasma levels over 2100 ng/ml). Other monkeys were trained to intravenously (i.v.) self-administer cocaine (56.0 μg/kg/injection). When AWD 131-138 (10–100 μg/kg/injection) was studied by substitution, responding declined to vehicle substitution levels within three sessions. At the dose of 100 μg/kg i.v. AWD 131-138, sufficient drug was self-administered during the first session (about 3.5 mg/kg) to produce plasma levels above 1000 ng/ml, yet responding over the next two sessions dropped to vehicle levels. The failure of AWD 131-138 to produce benzodiazepine-like discriminative effects and the absence of drug self-administration behavior when substituted for cocaine suggest that its abuse liability is low.
Keywords: Anticonvulsant; Benzodiazepine; AWD 131-138; Midazolam; Diazepam; Drug discrimination; Self-administration;

Facilitation of ischemia-induced release of dopamine and neuronal damage by dexamethasone in the rat striatum by Toshihiko Mitsuyo; Naoto Adachi; Toshihiro Yorozuya; Etsuo Tabo; Takumi Nagaro; Tatsuru Arai (267-274).
Glucocorticoids have been reported to aggravate ischemia-induced neuronal damage in both humans and experimental animals. Because an excess release of neurotransmitters is closely related to the outcome of ischemic neuronal damage, we evaluated the effects of dexamethasone on monoaminergic release and histological outcome. Changes in the extracellular concentrations of monoamines and their metabolites in the striatum produced by occlusion of the middle cerebral artery for 20 min were measured using a microdialysis high-performance liquid chromatography procedure, and the effects of intracerebroventricular administration of dexamethasone (10 μg) were evaluated in halothane-anesthesized rats. The histological outcome was evaluated by light microscopy 7 days after ischemia. Additionally, the effects of lesioning of the substantia nigra were estimated. The extracellular concentrations of neither dopamine nor serotonin were affected by the administration of dexamethasone in the nonischemic state. The occlusion of the middle cerebral artery produced a marked increase in the extracellular concentration of dopamine in the striatum, the peak value being 240 times that before ischemia. The preischemic administration of dexamethasone enhanced the increase in dopamine level during ischemia, and the peak value in the dexamethasone group was 640% of that in the vehicle group. After 7 days, ischemic neuronal damage in the dexamethasone group was severe compared with that in the vehicle group. In rats receiving the substantia nigra lesion, the ischemic release of dopamine was abolished, and the aggravation of ischemic neuronal damage by dexamethasone was completely alleviated. Changes in the release of monoamines may be a contributing factor in the development of the ischemic neuronal damage induced by glucocorticoids.
Keywords: Cerebral ischemia; Dexamethasone; Dopamine; Substantia nigra;

Protective effect of a novel antioxidant non-steroidal anti-inflammatory agent (compound IA) on intestinal viability after acute mesenteric ischemia and reperfusion by Dimitrios Poussios; Ioanna Andreadou; Apostolos Papalois; Eleni Rekka; Nikolaos Gavalakis; Kyriaki Aroni; Panos N Kourounakis; Constantinos Fotiadis; Michael N Sechas (275-280).
Reactive oxygen species play an important role in the basic pathophysiology of ischemia–reperfusion injury. We investigated whether the administration of a novel non-steroidal anti-inflammatory compound with antioxidant properties, the compound [5-(2-amino-ethylamino)-1-phenyl-2-pentanone] (compound IA), has a beneficial effect on the repair process of the intestinal mucosa after transient mesenteric ischemia in a randomized blind trial. Six groups of rats were subjected to a model of 60 min of intestinal ischemia that was produced by occluding the superior mesenteric artery. At the end of ischemia, compound IA was administered intravenously and the clamp was removed allowing reperfusion. At 60 min after reperfusion, animals were sacrificed and a 10 cm section of terminal ileum was resected. The outcome was evaluated by histopathologic assessment, measurement of polymorphonuclear leukocytes and the extent of lipid peroxidation measuring the small intestine tissue malondialdehyde. After 1 h of reperfusion, the mucosal damage was less in IA-treated rats compared with the control group. Moreover, the number of polymorphonuclear leukocytes in intestinal mucosa was significantly lower in IA group. Compound IA resulted in a statistically significant reduction of the concentration of small intestine tissue malondialdehyde, compared to those of controls. Administration of compound IA decreased the mucosal damage in rats that were subjected to 60 min of ischemia followed by 60 min of reperfusion. The mechanism of compound IA action is considered to be mediated via its potent antioxidant, free radical scavenging activities and inhibition of polymorphonuclear leukocytes infiltration.
Keywords: Antioxidant; Non-steroidal anti-inflammatory agent; Acute mesenteric ischemia; Intestinal viability; Ischemia–reperfusion;

Inhibitory activity of kinetin on free radical formation of activated platelets in vitro and on thrombus formation in vivo by George Hsiao; Ming-Yi Shen; Kuan-Hung Lin; Chin-Yi Chou; Nien-Hsuan Tzu; Chien-Huang Lin; Duen-Suey Chou; Tzeng-Fu Chen; Joen-Rong Sheu (281-287).
Kinetin has been shown to have anti-aging effects on several different systems, including plants and human cells. Recently, we demonstrated that kinetin markedly inhibited platelet aggregation in washed human platelets. In the present study, an electron spin resonance (ESR) method was used to further evaluate the scavenging activity of kinetin on the free radicals formed. Kinetin (70 and 150 μM) concentration dependently reduced the ESR signal intensity of hydroxyl radicals in collagen (1 μg/ml)-activated platelets. Furthermore, kinetin was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at doses of 4 and 6 mg/kg. In addition, intravenous injection of kinetin (4 and 6 mg/kg) significantly prolonged the bleeding time by approximately 1.9- and 2.1-fold as compared with normal saline in severed mesenteric arteries of rats. A continuous infusion of kinetin (0.6 mg/kg/min) for 10 min also significantly increased the bleeding time by about 2.3-fold, and the bleeding time returned to baseline within 120 min after cessation of kinetin infusion. Platelet thrombi formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. When kinetin was administered at 13 and 14 mg/kg in mice pretreated with fluorescein sodium (5 mg/kg), the occlusion time was significantly prolonged. In conclusion, these results suggest that kinetin has effective free radical-scavenging activity in vitro and antithrombotic activity in vivo. Treatment with kinetin may lower the risk of thromboembolic-related disorders. Therefore, kinetin may be a potential therapeutic agent for arterial thrombosis, but its toxicity must be further assessed.
Keywords: Kinetin; Hydroxyl radical; Bleeding time; Occlusion time; Arterial thrombosis;

Carbon monoxide modulates the response of human basophils to FcεRI stimulation through the heme oxygenase pathway by Alfredo Vannacci; Roberto Baronti; Giovanni Zagli; Cosimo Marzocca; Simone Pierpaoli; Daniele Bani; Maria Beatrice Passani; Pier Francesco Mannaioni; Emanuela Masini (289-297).
We report the effects of exogenous and endogenous carbon monoxide (CO) on the immunological activation of human basophils. Hemin (1−100 μM), a heme oxygenase substrate analogue, significantly increased the formation of bilirubin from partially purified human basophils, thus indicating that these cells express heme oxygenase. This effect was reversed by preincubating the cells for 30 min with Zn-protoporphyrin IX (100 μM), a heme oxygenase inhibitor. Hemin (100 μM) also decreased immunoglobulin G anti-Fcε (anti-IgE)-induced activation of basophils, measured by the expression of a membrane granule-associated protein, identified as cluster differentiation protein 63 (CD63), and by histamine release. These effects were reversed by Zn-protoporphyrin IX (100 μM), by oxyhemoglobin (HbO2), a CO scavenger (100 μM), and by 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ), an inhibitor of the soluble guanylyl cyclase (100 μM). Exposure of basophils to exogenous CO (10 μM for 30 min) also decreased their activation, while nitrogen (N2) was ineffective. HbO2 and ODQ reversed the inhibition, reversing both membrane protein CD63 expression and histamine release to basal values. Both hemin and exogenous CO significantly raised cGMP levels in basophils and blunted the rise of calcium levels caused by immunological activation. This study suggests that CO increases cGMP formation, which in turn induces a fall in intracellular Ca2+ concentration, thereby resulting in the inhibition of human basophil activation.
Keywords: Heme oxygenase; Basophil; Histamine; CD63; cGMP;

Erratum to “CP8668, a novel orally active nonsteroidal progesterone receptor modulator with tetrahydrobenzindolone skeleton” [Eur. J. Pharmacol. 461 (2003) 73–78] by Yuji Tabata; Yumiko Iizuka; Rie Shinei; Ken-ichi Kurihara; Tsuneo Okonogi; Shigeru Hoshiko; Yasushi Kurata (299).

Author index (301-303).

Keyword index (305-310).