European Journal of Pharmacology (v.498, #1-3)

Cancer gene therapy is the most studied application of gene therapy. Many genetic alterations are involved in the transformation of a normal cell into a neoplastic one. The two main gene groups involved in cancer development are oncogenes and tumor suppressor genes. While the latter eliminates cancerous cells via apoptosis, the former enhances cell proliferation. Therefore, apoptotic genes and anti-oncogenes are widely used in cancer gene therapy. In addition to oncogenes and tumor suppressor genes, chemotherapy and gene therapy can be combined through suicide gene strategy. A suicide gene encodes for a non-mammalian enzyme; this enzyme is used to convert a non-toxic prodrug into its active cytotoxic metabolite within the cancerous cells. Tumor suppressor genes, anti-oncogenes and suicide genes target cancer cells on the molecular level. On the other hand, cancer is immunogenic in nature; therefore, it can also be targeted on the immunological level. Boosting the immune response against cancerous cells is usually achieved via genes encoding for cytokines. Interleukin-12 gene, for example, is one of the most studied cytokine genes for cancer gene therapy applications. DNA vaccines are also used after conventional treatments to eliminate remnant malignant cells. All these therapeutic strategies and other strategies namely anti-angiogenesis and drug resistant genes are briefly reviewed and highlighted in this article.
Keywords: Cancer gene therapy; Anti-oncogene; Suicide gene; p53 gene; DNA vaccine; Cytokine gene;

Antiangiogenic versus cytotoxic therapeutic approaches to human pancreas cancer: an experimental study with a vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor and gemcitabine by Guido Bocci; Romano Danesi; Gabriele Marangoni; Anna Fioravanti; Ugo Boggi; Irene Esposito; Alessandro Fasciani; Elena Boschi; Daniela Campani; Generoso Bevilacqua; Franco Mosca; Mario Del Tacca (9-18).
Pancreatic adenocarcinoma is a leading cause of cancer death in the United States and represents a challenging chemotherapeutic problem. The pharmacological control of angiogenesis might represent a novel approach to the management of pancreas cancer, since the pathological development of vascular supply is a critical step for tumor growth and may affect its prognosis. In order to test this hypothesis, SU5416 ([3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one]) a selective inhibitor of the vascular endothelial growth factor receptor-2 tyrosine kinase, and gemcitabine (2′, 2′-difluorodeoxycytidine) were tested on endothelial (HUVEC) and pancreatic tumor cells (MIA PaCa-2) in vitro and in vivo alone and in simultaneous association. SU5416 inhibited HUVEC cells stimulated to proliferate by vascular endothelial growth factor but not MIA PaCa-2 cells; the drug concentration that decreased cell growth by 50% (IC50) was 0.14 μM. Furthermore, SU5416 reduced the development of microvessels from placental explants (IC50, 0.23 μM). Gemcitabine inhibited the growth of both HUVEC and MIA PaCa-2 cells with an IC50 of 0.08 and 0.1 μM, respectively. A synergistic effect (combination index <1 and dose reduction index >1) on anti-proliferative and pro-apoptotic activity was calculated with the simultaneous combination of the two drugs on endothelial cells. A marked in vivo antitumor effect on MIA PaCa-2 xenografts was observed with SU5416 at a protracted schedules, as well as with gemcitabine; furthermore, the combination between the two drugs resulted in an almost complete suppression of tumor growth and relapse. In conclusion, the present results provide the evidence of an effective anti-endothelial/antitumor activity of protracted administration of SU5416 on human pancreas cancer xenografts, which is comparable with the one obtained by gemcitabine; moreover, the synergistic combination between these drugs on endothelial cells and the promising association in pancreatic cancer xenografts could be used in future studies and translated into the clinical setting.
Keywords: SU5416; Gemcitabine; Pancreas cancer cell; Endothelial cell; VEGF; Angiogenesis;

Inhibition of UVA irradiation-modulated signaling pathways by rutaecarpine, a quinazolinocarboline alkaloid, in human keratinocytes by Sung-Mok Beak; Seung-Hwan Paek; Yurngdong Jahng; Yong Soo Lee; Jung-Ae Kim (19-25).
Matrix metalloproteinases (MMPs), a key component in photoaging of the skin due to exposure to ultraviolet A, appear to be increased by ultraviolet A irradiation-associated generation of reactive oxygen species. In this study, we investigated the effects of synthetic rutaecarpine, which is also found in Evodia rutaecarpa, on the ultraviolet A-induced changes in the expression of gelatinases: matrix metalloproteinase (MMP)-2 and MMP-9 using HaCaT human keratinocytes as a model cellular system. Ultraviolet A irradiation of HaCaT cells increased the gelatinolytic activities of MMP-2 and MMP-9, which was significantly suppressed by the pretreatment with rutaecarpine. In addition, rutaecarpine significantly suppressed the ultraviolet A-induced enhanced expression of MMP-2 and MMP-9 proteins and mRNAs. Rutaecarpine also inhibited the H2O2-induced increase in the expression of MMP-2 and MMP-9. Furthermore, rutaecarpine decreased the ultraviolet A-induced increased generation of reactive oxygen species. Taken together, these results suggest that rutaecarpine inhibited ultraviolet A-induced reactive oxygen species generation, resulting in the enhanced expression of MMP-2 and MMP-9 in human skin cells. These results further suggest that ruetaecarpine may be useful in the prevention of ultraviolet A-induced photoaging.
Keywords: Rutaecarpine; Reactive oxygen species; Matrix metalloproteinase; HaCaT human keratinocyte;

Ghrelin and des-acyl ghrelin both inhibit isoproterenol-induced lipolysis in rat adipocytes via a non-type 1a growth hormone secretagogue receptor by Giampiero Muccioli; Nicoletta Pons; Corrado Ghè; Filomena Catapano; Riccarda Granata; Ezio Ghigo (27-35).
Besides possessing a strong growth hormone (GH)-releasing activity, the gastrointestinal octanoylated peptide ghrelin has been reported to antagonize lipolysis in rat adipocytes. It is not yet clear whether this inhibitory activity on lipolysis is also shared by the major circulating isoform, des-acyl ghrelin, that does not activate the ghrelin receptor, namely the type 1a GH secretagogue-receptor (GHS-R1a) and lacks the endocrine effects of the acylated form. Here we show that des-acyl ghrelin, like ghrelin and some synthetic GHS (hexarelin and MK0677) and carboxy-terminally ghrelin fragments such as ghrelin-(1-5) and ghrelin-(1-10), all significantly reduced, over concentrations ranging from 1 to 1000 nM, the stimulation of glycerol release caused in rat epididymal adipocytes by the nonselective β-adrenoceptor agonist isoproterenol in vitro. The order of potency on stimulated-lipolysis was: des-acyl ghrelin=ghrelin>MK0677=hexarelin>ghrelin-(1-5)=ghrelin-(1-10). This ranking was consistent with the binding experiments performed on membranes of epididymal adipose tissue or isolated adipocytes that did not express mRNA for GHS-R1a. A common high-affinity binding site was recognized in these cells by both acylated and des-acylated ghrelin and also by hexarelin, MK0677, ghrelin-(1-5) and ghrelin-(1-10). In conclusion, these findings provide the first evidence that des-acyl ghrelin, as well as ghrelin, short ghrelin fragments and synthetic GHS, may act directly as antilipolytic factors on the adipose tissue through binding to a specific receptor which is distinct from GHS-R1a.
Keywords: Des-acyl ghrelin; Ghrelin; Growth hormone secretagogue receptor; Rat adipocyte; Isoproterenol-induced lipolysis;

Vanilloid receptor 1 was recently reported to play an important role in hyperalgesia, but the mechanisms by which this receptor is activated by endogenous inflammatory mediators, such as bradykinin and nerve growth factor, are not yet fully understood. Here, we investigated whether bradykinin, which is a pain-producing inflammatory mediator, sensitizes vanilloid receptor 1 by inducing the activation of cyclooxygenases, phospholipase C and phospholipase A2 in rat dorsal root ganglion cells. We demonstrated this using 45Ca2+ uptake and inositol phosphates accumulation assays, bradykinin activates phospholipase C and cyclooxygenase-1 through the bradykinin B2 receptor. The bradykinin B2 receptor then sensitizes vanilloid receptor 1 activity by facilitating non-selective Ca2+ channel activity, increasing the intracellular Ca2+ concentration from the extracellular pool. These methods would be useful for screening new drugs for activity at vanilloid receptor 1. These data suggest that endogenous substances produced by several enzymes may be capable of producing a synergistic response involving the vanilloid receptor 1.
Keywords: Bradykinin; Cyclooxygenase; Phospholipase A2; Phospholipase C; Vanilloid receptor 1;

We now evaluated the anti-inflammatory mechanisms of andrographolide on complement 5a (C5a)-induced macrophage recruitment in vitro. Andrographolide concentration dependently inhibited cell migration toward C5a with an IC50 of 5.6±0.7 μM. With relatively specific kinase inhibitors (PD98059, SB203580, SP600125, wortmannin and LY294002, respectively) the results showed that extracellular signal-regulated kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol-3-kinase (PI3K) were necessary for C5a-induced migration, whereas c-Jun N-terminal kinase (JNK) was nonessential. Andrographolide significantly attenuated C5a-stimulated phosphorylation of ERK1/2, and of its upstream activator, MAP kinase–ERK kinase (MEK1/2). C5a-activated ERK1/2 phosphorylation was 86±9% inhibited by 30 μM andrographolide. Under the same conditions, however, andrographolide failed to affect C5a-stimulated p38 MAPK and JNK phosphorylation. Andrographolide also strongly abolished C5a-stimulated Akt phosphorylation, a downstream target protein for PI3K. These results indicate that inhibition of cell migration by interfering with ERK1/2 and PI3K/Akt signal pathways may contribute to the anti-inflammatory activity of andrographolide.
Keywords: Andrographolide; Chemotactic migration; MAPK; ERK1/2; Akt;

κ-Opioid receptor stimulation inhibits growth of neonatal rat ventricular myocytes by Guijun Wang; Hongxin Wang; Yanling Yang; Tak Ming Wong (53-58).
The effects of trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate salt (U50,488H), a selective κ-opioid receptor agonist, on growth in neonatal ventricular myocytes were determined. In 15% serum culture medium, U50,488H at 0.1–1 μM significantly reduced the protein content, [3H]leucine uptake and cell size of the myocytes. The effect of U50,488H on protein content was abolished in the presence of 1 μM nor-binaltorphimine (nor-BNI), a selective κ-opioid receptor antagonist. In a 0.4% serum medium, U50,488H at 0.1–1 μM had no effect on myocyte growth. Interestingly, 1 μM U50,488H abolished the stimulatory effects of 1 μM norepinephrine on protein content, [3H]leucine uptake and cell size of the myocytes in the low serum medium. The effect of U50,488H was abolished by 1 μM nor-BNI. With the exception of cell size, the effects of norepinephrine were completely abolished by blockade of both α- and β-adrenoceptors, but only partially blocked by blockade of either adrenoceptors. These results provide first evidence that κ-opioid receptor stimulation inhibits growth of the neonatal ventricular myocyte as a result of direct action as well as by inhibiting sympathetic stimulation of the heart. The stimulatory effects of sympathetic activity on growth occurs via both α- and β-adrenoceptors.
Keywords: U50,488H; Neonatal rat ventricular myocyte; Protein content; [3H]Leucine; Cell size; Adrenoceptor;  ;

Regulation of serotonin 5-HT2C receptors by chronic ligand exposure by Mark G. Devlin; Nicola J. Smith; Olivia M. Ryan; Elizabeth Guida; Patrick M. Sexton; Arthur Christopoulos (59-69).
The effect of ligand pretreatment on human 5-hydroxytryptamine2C (5-HT2C) receptors was examined in CHO cells expressing high (CHO-1C7; 67±3 pmol/mg) or low (CHO-1C19; 72±10 fmol/mg) levels of the receptor. Seventy-two hours pretreatment of CHO-1C7 cells with various ligands did not affect receptor expression. Pretreatment with inverse agonists enhanced 5-HT-mediated inositol phosphate accumulation with no change in constitutive receptor activity. The enhanced agonist responsiveness was inversely correlated with the intrinsic activity of the pretreatment ligand. Seventy-two hours of pretreatment with the weak agonist, 5-methoxygramine, caused an elevation in constitutive activity but no alteration in 5-HT-mediated signaling. In CHO-1C19 cells, 24 but not 72 h of pretreatment with the inverse agonist mianserin enhanced 5-HT-mediated signaling, with no effect on basal signaling; pretreatment with 5-methoxygramine had no significant effect. These findings highlight differences in the pattern of chronic regulation of 5HT2C receptor signaling between high and low receptor expression levels in a common cellular background.
Keywords: Constitutive activity;

Apoptotic activity of betulinic acid derivatives on murine melanoma B16 cell line by Wing-Keung Liu; Joyce C.K. Ho; Florence W.K. Cheung; Bonnie P.L. Liu; Wen-Cai Ye; Chun-Tao Che (71-78).
The mitochondrion plays a crucial role in the process of apoptosis and has thus become one of the targets for the search for potential chemotherapeutic agents. Betulinic acid [3β-hydroxy-lup-20(19)lupaen-28-carbonic acid], a lupane-type triterpene which is abundant in many plant species, has been shown to exert a direct effect on the mitochondria and subsequent apoptosis in melanoma cells. Chemical synthesis and modification of betulinic acid are being explored to develop more potent derivatives. We present here the apoptotic activity of several natural derivatives of betulinic acid which were isolated from the roots of a Chinese medicinal herb, Pulsatilla chinensis (Bge) Regel [Ye, W., Ji, N.N., Zhao, S.X., Liu, J.H., Ye, T., McKervey, M.A., Stevenson, P., 1996. Triterpenoids from Pulsatilla chinensis. Phytochemistry 42, 799–802]. Of the five compounds tested, 3-oxo-23-hydroxybetulinic acid was the most cytotoxic on murine melanoma B16 cells (IC50=22.5 μg/ml), followed by 23-hydroxybetulinic acid and betulinic acid (IC50=32 and 76 μg/ml, respectively), with lupeol and betulin exhibiting the weakest cytotoxicity (IC50≥100 μg/ml). Exposure of B16 cells to betulinic acid, 23-hydroxybetulinic acid and 3-oxo-23-hydroxybetulinic acid caused a rapid increase in reactive oxidative species production and a concomitant dissipation of mitochondrial membrane potential in a dose- and time-dependent manner, which resulted in cell apoptosis, as demonstrated by fluorescence microscopy, gel electrophoresis and flow-cytometric analysis. Cell cycle analysis further demonstrated that both 3-oxo-23-hydroxybetulinic acid and 23-hydroxybetulinic acid dramatically increased DNA fragmentation at the expense of G1 cells at doses as low as 12.5 and 25 μg/ml, respectively, thereby showing their potent apoptotic properties. Our results showed that hydroxylation at the C3 position of betulinic acid is likely to enhance the apoptotic activity of betulinic acid derivatives (23-hydroxybetulinic acid and 3-oxo-23-hydroxybetulinic acid) on murine melanoma B16 cells.
Keywords: Melanoma; Betulinic acid derivative; Apoptosis; Reactive oxidative species; Mitochondrial membrane potential;

The appropriate method of etiologic therapy for gingival overgrowth is yet unknown. In this study drug-induced proliferation of Gin-1 cells, a normal human gingival fibroblast cell line, was examined by using the reagent water-soluble tetrazolium-1. Tranilast (100 μM) inhibited the nifedipine (10 μM)-induced proliferation of gingival fibroblasts. The level of basic fibroblast growth factor (bFGF) was determined by using an enzyme-linked immunosorbent assay kit. Tranilast inhibited the release of bFGF from the cells. In conclusion, tranilast depresses the nifedipine-induced proliferation of gingival fibroblasts by inhibiting the release of bFGF. Administration of tranilast may thus be clinically effective for the treatment of gingival overgrowth.
Keywords: Tranilast; Gingival fibroblast; bFGF; Proliferation;

Urantide mimics urotensin-II induced calcium release in cells expressing recombinant UT receptors by Valeria Camarda; Wei Song; Erika Marzola; Martina Spagnol; Remo Guerrini; Severo Salvadori; Domenico Regoli; Jonathan P. Thompson; David J. Rowbotham; David J. Behm; Stephen A. Douglas; Girolamo Calo'; David G. Lambert (83-86).
Urotensin-II is the natural ligand of the UT receptor. This novel system is involved in the regulation of cardiovascular functions. Recently, a urotensin-II analog ([Pen5,DTrp7,Orn8]urotensin-II(4–11)) named urantide, has been proposed as a selective and potent UT receptor antagonist. In order to pharmacologically characterize this new compound, urantide was tested on the native UT receptors of the rat aorta and on the human recombinant receptors expressed in CHO cells (CHOhUT). Indeed, urantide behaves as a competitive, potent (pA 2 8.24), and pure antagonist in the rat aorta bioassay, while as an agonist (pEC50 8.11) in a calcium mobilization assay performed in CHOhUT cells. Urantide should be considered a low efficacy partial agonist.
Keywords: Urotensin-II; Urantide; UT receptor; Ca2+; CHO cell; Aorta, rat;

Opioid agonists and antagonists can regulate the density of μ-opioid receptors in whole animal and in cell culture. High intrinsic efficacy agonists (e.g., etorphine), but not lower intrinsic efficacy agonists (e.g., morphine), produce μ-opioid receptor down-regulation and can alter the abundance of μ-opioid receptor mRNA. Conversely, opioid antagonists substantially increase the density of μ-opioid receptors without changing its mRNA. μ-Opioid receptor up-regulation has been associated with decreases in the trafficking protein dynamin-2, whereas μ-opioid receptor down-regulation produces an increase in dynamin-2 abundance. To probe the differences between opioid agonist and antagonist-induced μ-opioid receptor regulation, the current study determined changes in μ-opioid receptor density using a combined radioligand binding ([3H] DAMGO) and quantitative Western blotting approach in mouse spinal cord. Furthermore, the differences between intermittent and continuous dosing protocols were evaluated. Continuous (7–8 days) s.c. infusions of naloxone (5 mg/kg/day) or naltrexone (15 mg s.c. implant pellet) increased μ-opioid receptor density in radioligand binding assays (≈+80%) in mouse spinal cord and down-regulated dynamin-2 abundance (≈−30%), but had no effect on the abundance of immunoreactive μ-opioid receptor. Continuous (7 days) s.c. infusion of etorphine (200 μg/kg/day) decreased immunoreactive μ-opioid receptor (≈−35%) and [3H] DAMGO binding (≈−30%), and concurrently increased dynamin-2 abundance (≈+40%). Continuous (7 days) morphine infusion (40 mg/kg/day plus 25 mg s.c. implant pellet) had no effect on any outcome measure. Delivery of the same daily dose of etorphine or naloxone using intermittent (every 24 h for 7 days) s.c. administration had no effect on immunoreactive μ-opioid receptor, [3H] DAMGO binding or dynamin-2 abundance. These data indicate that μ-opioid receptor density, determined in radioligand binding assays, and immunoreactive dynamin-2 abundance are regulated by continuous, but not intermittent, opioid ligand treatment. Furthermore, the differential regulation of μ-opioid receptor abundance by agonists and antagonists in immunoblotting assays contrasts with changes in [3H] DAMGO binding. Taken together, these results suggest that etorphine-induced down-regulation may depend upon μ-opioid receptor degradation and changes in dynamin-2-mediated receptor trafficking. Conversely, antagonist-induced up-regulation does not require an increase in μ-opioid receptor synthesis and may entail conversion of receptors to an appropriate conformation to bind ligand, as well as changes in receptor trafficking.
Keywords: μ-Opioid receptor; Up-regulation; Down-regulation; Intermittent and continuous dosing;

Dopamine D4 receptors inhibit depolarization-induced [3H]GABA release in the rat subthalamic nucleus by Benjamín Floran; Leonor Floran; David Erlij; Jorge Aceves (97-102).
We explored the role of dopamine D4 receptors on [3H]GABA release in the subthalamic nucleus. [3H]GABA release was evoked by high K+ in slices of the nucleus. The selective dopamine D4 receptor agonist PD168,077 (N-[[4-(2-cyanophenyl)-1-piperazynil]methyl]-3-methyl-benzamide) inhibited GABA release with greater potency (EC50=3.2 nM) than quinpirole (EC50=200 nM). SKF 21297 (6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide), a dopamine D1-like receptor agonist, had no effect. L-745,870 (3-{[4-(4-chlorophenyl)piperazin-1-yl]methyl}-1-1H-pyrollo[2,3-b] pyridine), a selective dopamine D4 receptor antagonist, reverted the quinpirole inhibition with greater potency (IC50=8.7 nM) than that of the dopamine D2/D3 receptor antagonist sulpiride and raclopride (IC50=4804 and 788 nM, respectively). Both methylphenidate and methamphetamine, dopamine reuptake blockers, inhibited by 30% high K+-evoked GABA release; the inhibition was blocked by L-745,870. These results show that dopamine D4 receptors modulate GABA release in the subthalamic nucleus. The results would explain how agents that increase interstitial dopamine like methylphenidate and amphethamine might control locomotor hyperactivity seen in disorders of dopamine D4 receptors.
Keywords: Dopamine receptor; GABA transmission; Hyperactivity disorder;

Analgesic effect of TT-232, a heptapeptide somatostatin analogue, in acute pain models of the rat and the mouse and in streptozotocin-induced diabetic mechanical allodynia by János Szolcsányi; Kata Bölcskei; Árpád Szabó; Erika Pintér; Gábor Pethő; Krisztián Elekes; Rita Börzsei; Róbert Almási; Tamás Szűts; György Kéri; Zsuzsanna Helyes (103-109).
Somatostatin released from capsaicin-sensitive sensory nerves exerts systemic anti-inflammatory and antinociceptive actions. TT-232 is a stable, peripherally acting heptapeptide (D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH2) somatostatin analogue with highest binding affinity for somatostatin sst4 receptors. It has been shown to inhibit acute and chronic inflammatory responses and sensory neuropeptide release from capsaicin-sensitive nociceptors. In the present study the antinociceptive effects of TT-232 were analysed using both acute and chronic models of nociception. Formalin-induced pain behaviour, noxious heat threshold and streptozotocin-induced diabetic neuropathic mechanical allodynia were examined in rats and phenylquinone-evoked abdominal constrictions were tested in mice. TT-232 (80 μg/kg i.p.) inhibited both early (0–5 min) and late phases (25–45 min) of formalin-induced nociception as revealed by determination of the composite pain score. The minimum effective dose to elevate the noxious heat threshold and diminish the heat threshold drop (heat allodynia) evoked by resiniferatoxin (0.05 nmol intraplantarly) was 20 and 10 μg/kg i.p., respectively, as measured by an increasing-temperature hot plate. TT-232 (10–200 μg/kg s.c.) significantly inhibited phenylquinone-evoked writhing movements in mice, but within this dose range no clear dose–response correlation was found. Five weeks after streptozotocin administration (50 mg/kg i.v.) the diabetes-induced decrease in the mechanonociceptive threshold was inhibited by 10–100 μg/kg i.p. TT-232. These findings show that TT-232 potently inhibits acute chemical somatic/visceral and thermal nociception and diminishes chronic mechanical allodynia associated with diabetic neuropathy, thereby it could open new perspectives in the treatment of various pain syndromes.
Keywords: Somatostatin; Analgesic effect; Formalin test; Writhing test; Thermal nociception; Diabetic neuropathy;

The effects of neurosteroids, 17β-estradiol and dehydroepiandrosterone-sulfate (DHEA-S), were investigated on retinal degeneration using a rat model of ischemia–reperfusion injury. The animals were anaesthetized and retinal ischemia was induced by elevating the intraocular pressure to 120 mm Hg for 45 min. Neurosteroids were injected intraperitoneally before ischemia and immediately after reperfusion. Retinal biochemical changes such as increase of lactate content and decrease of glucose and ATP were significantly inhibited by neurosteroids compared to the control ischemic group. The effects of 17β-estradiol and DHEA-S were antagonized by pre-treatment with the σ1 site antagonist. These findings suggest that 17β-estradiol and dehydroepiandrosterone-sulfate may affect the metabolic state of surviving neurons and glial cells after ischemic injury and that they act, at least in part, through involvement of σ1 recognition sites.
Keywords: Neurosteroid; σ1 Recognition site; Retina; Neuroprotection;

Neuroprotective and neurotoxic effects of cyclosporine A on transient focal ischemia in mdr1a knockout mice by Michihiro Murozono; Shohei Matsumoto; Eriko Matsumoto; Atsushi Isshiki; Yasuo Watanabe (115-118).
The proper dose of cyclosporine A as a neuroprotective agent was investigated using the middle cerebral artery occlusion model of mdr1a knockout mice. After a 30-min occlusion period, reperfusion was performed and the vehicle or cyclosporine A (1 mg/kg or 10 mg/kg×2) was intraperitoneally administered to each animal model group. Forty eight hours after reperfusion, infarction volume in the 1 mg/kg cyclosporine A group was significantly less than that seen in the vehicle group, although, in the high dose cyclosporine A group, infarction volumes were significantly higher than those seen in the vehicle group. These results demonstrate that cyclosporine A shows not only anti-ischemic effects, but also neurotoxic effects depending on the dosage penetrating into the brain.
Keywords: Cyclosporine A; mdr1a; (Knockout mouse); Brain ischemia; NeuN;

Suppressing effect of the cannabinoid CB1 receptor antagonist, SR 141716, on alcohol's motivational properties in alcohol-preferring rats by Giancarlo Colombo; Giovanni Vacca; Salvatore Serra; Mauro A.M. Carai; Gian Luigi Gessa (119-123).
Administration of the cannabinoid CB1 receptor antagonist, SR 141716 [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide], has been reported to reduce alcohol intake and alcohol self-administration in different models of excessive alcohol consumption, including the selectively bred Sardinian alcohol-preferring (sP) rats. The present study investigated whether SR 141716 was also capable of decreasing, in this rat line, alcohol's motivational properties. Extinction responding for alcohol, defined as the maximal number of lever responses reached in the absence of alcohol in rats trained to lever-press for alcohol, was used as index of alcohol's motivational properties. Rats were initially trained to lever-press for oral alcohol (15%, v/v) under a fixed ratio (FR) schedule of FR4. Once self-administration behavior was established, extinction sessions were conducted. SR 141716 (0, 0.3, 1 and 3 mg/kg; i.p.) was acutely administered before extinction sessions. In order to assess the specificity of SR 141716 action on extinction responding for alcohol, a separate group of sP rats was trained to lever-press for a 3% (w/v) sucrose solution under an FR4 schedule. SR 141716 administration produced a dose-dependent, virtually complete suppression of extinction responding for alcohol. In contrast, extinction responding for sucrose was not significantly altered by treatment with SR 141716. Further to the consummatory aspects, these results also extend the suppressing effect of SR 141716 to the appetitive aspects of alcohol drinking behavior in sP rats. The results also implicate the cannabinoid CB1 receptor in the neural substrate mediating alcohol's motivational properties in this rat line.
Keywords: Cannabinoid CB1 receptor antagonist; Extinction responding for alcohol; Sardinian alcohol-preferring (sP);

Higher levels of cognition, such as executive functions, are known to be disrupted in psychiatric disorders such as schizophrenia. As a potential model of executive function, rats were trained in a three-lever operant conditioning chamber to respond on two of the three levers in one of six possible correct sequences. When the rat completed a two-response sequence correctly for 10 consecutive trials, the correct sequence was randomly changed to another two-response sequence without signaling the rat. Rats readily acquired the behavioral baseline and completed all six response-sequences within a 60-min session. Phencyclidine, MK-801 ((5S,10R)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine), apomorphine, scopolamine and triazolam all produced dose-related decreases in the total number of sequences completed. Phencyclidine and MK-801 markedly increased all errors while scopolamine produced modest increases; triazolam increased only total and intrarule errors, while apomorphine had no significant effect on errors. The present results suggest that within-session repeated acquisition of response sequences has the potential to be a useful model for studying executive function in rats.
Keywords: MK-801; Phencyclidine; Triazolam;

The brain cAMP regulating system and its downstream elements play a pivotal role in the therapeutic effects of antidepressants. We previously reported the increase in activities of phosphodiesterase 4, a major phosphodiesterase isozyme hydrolyzing cAMP, in the frontal cortex and hippocampus of learned helplessness rats, an animal model for depression. The present study was undertaken to examine the combination of effects of rolipram, a phosphodiesterase 4 inhibitor, with imipramine, a typical tricyclic antidepressant, on depressive behavior in learned helplessness rats. Concurrently, cAMP-response element (CRE)-binding activity and brain-derived neurotrophic factor (BDNF) levels related to the therapeutic effects of antidepressants were determined. Repeated administration of imipramine (1.25–10 mg/kg, i.p.) or rolipram (1.25 mg/kg, i.p.) reduced the number of escape failures in learned helplessness rats. Imipramine could not completely ameliorate the escape behavior to a level similar to that of non-stressed rats even at 10 mg/kg. However, repeated coadministration of rolipram with imipramine (1.25 and 2.5 mg/kg, respectively) almost completely eliminated the escape failures in learned helplessness rats. The reduction of CRE-binding activities and BDNF levels in the frontal cortex or hippocampus in learned helplessness rats were ameliorated by treatment with imipramine or rolipram alone. CRE-binding activities and/or BDNF levels of the frontal cortex and hippocampus were significantly increased by treatment with a combination of rolipram and imipramine compared to those in imipramine-treated rats. These results indicated that coadministration of phosphodiesterase type 4 inhibitors with antidepressants may be more effective for depression therapy and suggest that elevation of the cAMP signal transduction pathway is involved in the antidepressive effects.
Keywords: Phosphodiesterase 4; Rolipram; Antidepressant; Learned helplessness; cAMP response element; Brain-derived neurotrophic factor;

Opioid and monoamine systems mediate the discriminative stimulus of tramadol in rats by Małgorzata Filip; Karolina Wydra; Salim Yalcin Inan; Marta Dziedzicka-Wasylewska; Edmund Przegaliński (143-151).
We analyzed the ability of the mu opioid peptide receptor ligands morphine and naloxone and several antidepressant drugs that are serotonin (fluoxetine), noradrenaline (reboxetine), mixed serotonin and noradrenaline (milnacipram and venlafaxine), dopamine (nomifensine) reuptake inhibitors, as well as roxindole (a nonselective drug) to substitute for, or alter, tramadol discrimination. Male Wistar rats were trained to discriminate tramadol (20 mg/kg) from saline in a two-choice water-reinforced paradigm. Out of the drugs studied, only morphine substituted for tramadol. In combination experiments, naloxone (0.1–1 mg/kg) attenuated the stimulus effects of tramadol (20 mg/kg) and the substitution evoked by morphine (2 mg/kg). Milnacipram (10 mg/kg) or reboxetine (10 mg/kg) enhanced the effects of tramadol (2.5–10 mg/kg); the other antidepressant drugs used failed to modulate tramadol discrimination. Our results indicate that tramadol can be used as a stimulus cue in rats, and that mu opioid peptide mechanisms are involved in its effects, while noradrenergic uptake inhibitors can enhance tramadol discrimination.
Keywords: Tramadol; Morphine; Naloxone; Antidepressant drug; Discrimination;

Anxiolytic-like effects of PHCCC, an allosteric modulator of mGlu4 receptors, in rats by Katarzyna Stachowicz; Kinga Kłak; Aleksandra Kłodzińska; Ewa Chojnacka-Wojcik; Andrzej Pilc (153-156).
We examined the potential anxiolytic-like activity of (−)-N-phenyl-7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxamide (PHCCC), an allosteric modulator of metabotropic glutamate4 receptors (mGlu4), after administration into the basolateral amygdala, using the conflict drinking Vogel test in rats as a model. The results indicate that PHCCC, but not 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), the selective antagonist of group mGlu1 receptors, showed significant, dose-dependent anticonflict effects without affecting the threshold current or water intake. The results indicate that positive allosteric modulation of mGlu4 receptors may be a useful therapeutic approach to anxiety.
Keywords: Allosteric mGlu4 receptor modulator; mGlu1 receptor antagonist; Anxiety; Amygdala;

Behavioral sensitization to the discriminative stimulus effects of methamphetamine in rats by Tsutomu Suzuki; Yoshie Fukuoka; Tomohisa Mori; Mayumi Miyatake; Minoru Narita (157-161).
Sensitization to the discriminative stimulus effects of psychostimulants is not fully understood. Therefore, the present study was designed to investigate the development of sensitization to the discriminative stimulus of methamphetamine in rats. A dose–response curve for methamphetamine and a generalization test for cocaine were recorded in rats trained to discriminate between 1.0 mg/kg methamphetamine and saline. A significant leftward shift of the dose–response curve for methamphetamine and of the dose–generalization curve for cocaine was observed following repeated administration of methamphetamine (2.0 mg/kg) instead of saline. These findings suggest that repeated administration of methamphetamine can produce behavioral sensitization to the discriminative stimulus effects of methamphetamine in rats.
Keywords: Drug discrimination; Sensitization; Methamphetamine;

The mediators involved in the hyperpolarizing effects of lipopolysaccharide and of the bradykinin B1 receptor agonist des-Arg9-bradykinin on the rat aorta were investigated by comparing the responses of aortic rings of spontaneously hypertensive and normotensive Wistar rats. Endothelized rings from hypertensive rats were hyperpolarized by des-Arg9-bradykinin and lipopolysaccharide, whereas de-endothelized rings responded to lipopolysaccharide but not to des-Arg9-bradykinin. In endothelized preparations, the responses to des-Arg9-bradykinin were inhibited by N ω-nitro-l-arginine and iberiotoxin. De-endothelized ring responses to lipopolysaccharide were inhibited by iberiotoxin, glibenclamide and B1 antagonist Lys-[Leu8,des-Arg9]-bradykinin. This antagonist also inhibited hyperpolarization by des-Arg9-bradykinin and by the á2-adrenoceptor agonist, brimonidine. Our results indicate that Ca2+-sensitive K+ channels are the final mediators of the responses to des-Arg9-bradykinin, whereas both Ca2+- and ATP-sensitive K+ channels mediate the responses to lipopolysaccharide. The inhibitory effects of Lys-[Leu8,des-Arg9]-bradykinin is due to a direct action on Ca2+- and ATP-sensitive potassium channels.
Keywords: Des-Arg9-bradykinin; Lipopolysaccharide; Rat, spontaneously hypertensive; Aorta; K+ channel;

Superiority of YM598 over atrasentan as a selective endothelin ETA receptor antagonist by Hironori Yuyama; Yukiko Noguchi; Akira Fujimori; Masashi Ukai; Noriko Fujiyasu; Akiyoshi Ohtake; Shuichi Sato; Katsumi Sudoh; Masao Sasamata; Keiji Miyata (171-177).
The binding affinities of (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2,2′-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598) for native human endothelin ETA and ETB receptors expressed in human coronary artery smooth muscle cells (CASMC) and a human melanoma cell line, SK-Mel-28, respectively, were examined, and the results compared with those for the endothelin receptor antagonists atrasentan and bosentan. The in vivo endothelin ETA receptor inhibitory activities of YM598 and atrasentan were also compared through the suppression of the big endothelin-1-induced pressor response in pithed rats. K i values of YM598, atrasentan, and bosentan for native human endothelin ETA receptors were 0.772, 0.0551, and 4.75 nM, while those for native human endothelin ETB receptors were 143, 4.80, and 40.9 nM, respectively. The calculated selectivity ratios of YM598, atrasentan, and bosentan for endothelin ETA versus ETB receptors were 185, 87 and 8.6, respectively. In pithed rats, YM598 and atrasentan inhibited the big endothelin-1 (1 nmol/kg)-induced pressor response in a dose-dependent manner on both intravenous and oral administration. The inhibitory effect of YM598 was less potent than that of atrasentan when these agents were intravenously administered, but closely similar on oral administration. These results suggest that YM598 has high selectivity for native human ETA against ETB receptors, and that YM598 is superior to atrasentan as an ETA receptor antagonist with regard to pharmacological bioavailability in rats.
Keywords: YM598; Atrasentan; Bosentan; Antagonist; Endothelin receptor;

Restoration of middle cerebral artery thrombosis by novel glycoprotein IIb/IIIa antagonist FK419 in guinea pig by Akira Moriguchi; Kayoko Mihara; Toshiaki Aoki; Masashi Maeda; Nobuteru Tojo; Nobuya Matsuoka; Seitaro Mutoh (179-188).
We compared the antithrombotic efficacy of FK419 [(S)-2-acetylamino-3-[(R)-[1-[3-(piperidin-4-yl)propionyl]piperidin-3-ylcarbonyl]amino] propionic acid trihydrate], a novel nonpeptide glycoprotein IIb/IIIa antagonist, with recombinant tissue plasminogen activator (rt-PA) and other antithrombotic agents (aspirin, ozagrel, argatroban and heparin). FK419 not only inhibited ADP- and collagen-induced guinea pig platelet aggregation, but also induced disaggregation for ADP-induced aggregated platelets in vitro. In the photochemically induced middle cerebral artery thrombosis model in guinea pigs, FK419 dose-dependently shortened the time to first reperfusion and the total middle cerebral artery occlusion time and reduced ischemic brain damage and ameliorated neurological deficits measured 24 h after middle cerebral artery occlusion. Rt-PA similarly improved the middle cerebral artery patency, brain damage and neurological deficits. Neither aspirin, ozagrel, argatroban nor heparin restored the middle cerebral artery blood flow and improved the brain damage or neurological deficits. These results demonstrated that novel glycoprotein IIb/IIIa antagonist FK419 could disperse thrombus and ameliorated ischemic brain damage, suggesting that FK419 would be an attractive intervention for stroke patients.
Keywords: FK419; Platelet glycoprotein IIb/IIIa; Stroke; (Guinea pig); Thrombolysis;

Atorvastatin enhances sildenafil-induced vasodilation through nitric oxide-mediated mechanisms by Michele M. Castro; Elen Rizzi; Ricardo R. Rascado; Sabrina Nagassaki; Lusiane M. Bendhack; Jose E. Tanus-Santos (189-194).
Statins have cholesterol-independent effects including an increased vascular nitric oxide (NO) activity and are commonly used by patients with cardiovascular disease. Such patients frequently have erectile dysfunction, which may be treated with sildenafil, a selective inhibitor of phosphodiesterase type 5. Since statins and sildenafil can activate the NO-cGMP pathway, we investigated whether pre-treatment with atorvastatin (0, 5 and 30 mg/kg/day) for 2 weeks affects sildenafil (1 pM–100 mM)-induced relaxation of aortic rings isolated from Wistar rats. We also examined the hemodynamic consequences of this interaction in Wistar rats. Plasma nitrite/nitrate (NO x ) concentrations were determined using an ozone-based chemiluminescence assay. While pre-treatment with atorvastatin increased the potency of sildenafil-induced vasorelaxation (P<0.01), no differences were observed in the maximum sildenafil-induced relaxation. Pre-incubation of aortic rings with N G-nitro-l-arginine methyl ester (l-NAME) reversed atorvastatin-induced increase in the potency of sildenafil relaxation. In addition, pre-treatment with atorvastatin enhanced plasma NO x concentrations and sildenafil-induced hypotension and tachycardia (all P<0.05). These results suggest that atorvastatin increases the vascular sensitivity to sildenafil through NO-mediated mechanisms.
Keywords: Atorvastatin; Nitric oxide; Phosphodiesterase-5 inhibitor; Sildenafil; Statins; Vascular reactivity;

Synergistic interaction of endogenous platelet-activating factor and vasopressin in generating angina in rats by János Nemcsik; Krisztina Kordás; József Egresits; Ferenc László; Ferenc A. László; Imre Pávó; Éva Morschl (195-202).
We examined the involvement of endogenous vasopressin and platelet-activating factor (PAF) in the pathogenesis of two types of experimental angina in urethane-anaesthetised male Wistar rats. In the first model, epinephrine (10 μg kg−1) was injected into the tail vein, followed at the development of the maximum blood pressure response, i.e., 30 s later, by phentolamine (15 mg kg−1). In the second model, the vasopressin V1 receptor agonist ornithine-vasopressin (ornipressin; 0.5 IU kg−1, i.v.) was administered. The heart rate, mean arterial blood pressure and surface electrocardiogram (ECG, standard lead II) were registered simultaneously. As a measure of myocardial ischaemia, at 1 min after phentolamine or ornipressin administration, we found significant ST-segment depression, lasting for more than 10 or 5 min, respectively. Pretreatment (15 min, s.c.) with the vasopressin V1 receptor antagonist Mca1,Tyr(Me)2AVP (the Manning peptide; 0.02–0.2 μg kg−1) or the platelet-activating factor receptor antagonist ginkgolide B (BN 52021; 0.25–2.5 mg kg−1) alone caused a dose-dependent reduction of the ST-segment depression. Concurrent administration of the two antagonists in their threshold doses (0.02 μg kg−1 and 0.25 mg kg−1) also attenuated the ST-segment depression in both models. Neither antagonist affected the blood pressure or heart rate changes throughout the studies. Our results suggest that endogenous vasopressin and platelet-activating factor interact synergistically in provoking myocardial ischaemia in vivo in experimental angina in the rat.
Keywords: Platelet-activating factor; Vasopressin; Experimental angina; Synergistic interaction; Pathogenesis of myocardial ischaemia;

Differences in the vascular selectivity and tolerance between the NO donor/β-blocker PF9404C and nitroglycerin by Ana Ruiz-Nuño; Arancha Rosado; Antonio G. García; Manuela G. López; Mercedes Villarroya (203-210).
The vascular selectivity of PF9404C ((2′S),(2S)-3-isopropylamine,1-[4-(2,3-dinitroxy)propoxymethyl]-phenoxy-2′-propanol), a new β-blocker with nitric oxide (NO)-donor and vasodilator properties, was studied in different rabbit arteries and veins. Phenylephrine (10−6 M) or 35 mM K+ were used to pre-contract the arteries and veins prior to study the relaxant effects of PF9404C and nitroglycerin. The potency of both drugs to depress the phenylephrine-induced contraction was greater than that shown in the blockade of the K+-evoked contraction in most of the vessels studied, with the exception of the central ear artery. PF9404C exhibited about three-fold higher potency than nitroglycerin to relax the majority of the vessels studied, especially when they were contracted with K+, and showed a certain selectivity of action for the renal artery. PF9404C produced autotolerance but this effect was about 20-fold less pronounced than that observed with nitroglycerin. Cross-tolerance in those preparations pre-exposed to PF9404C that were relaxed later on with nitroglycerin was much greater than autotolerance. The tolerance for nitroglycerin was practically abolished in the presence of N-acetylcysteine. However, this was not the case for PF9404C. These results indicate that, although sharing the property of being NO donors, PF9404C and nitroglycerin show a different profile in causing vasodilation; furthermore, the tolerance to this effect is lesser for PF9404C and seems to be mediated by a mechanism different to that of nitrates. This makes PF9404C a nice pharmacological tool to further develop novel NO-donor compounds with a lesser degree of vascular tolerance than those now available.
Keywords: PF9404C; NO donor; Vascular selectivity; Tolerance;

Effects of bacterial lipopolysaccharide (Escherichia coli serotype, 055:B5, 20 mg kg−1, i.p., for 6 h) and a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate, Y-27632 (10−9–10−5 M) were investigated on the contractile responses of the rat mesenteric artery to phenylephrine (10−9–3x10−5 M), angiotensin-2 (10−10–10−6 M) and endothelin-1 (10−10–10−7 M). Moreover, alteration in the level of Rho-kinase (ROCK-2) expression was examined in the superior mesenteric artery obtained from saline- and lipopolysaccharide-treated rats by Western blotting. Endotoxemic rat mesenteric rings exhibited no different contractions to phenylephrine and angiotensin-2 but augmented contractile activity to endothelin-1. In the mesenteric artery obtained from the endotoxemic rats, acetylcholine-induced vasorelaxation did not differ; pD2 value for acetylcholine was 7.85±0.12 in the endotoxemic rings; however, it was 7.81±0.15 in the control rings (P>0.05). Y-27632 induced relaxation, which was the same in the control arteries as in endotoxemic ones when contracting agent was phenylephrine. However, when endothelin-1 was used to precontract the rings, Y-27632 produced enhanced relaxation in endotoxemic vessels. pD2 values for Y-27632 were, respectively, 7.69±0.12 and 8.20±0.10 in control and endotoxemic rings precontracted by endothelin-1 (10−8 M) (P<0.01). Moreover, Y-27632 (10−5 M) suppressed the contraction induced by angiotensin-2 (10−10–10−6 M). Western blot analysis revealed that Rho-kinase was upregulated significantly in the mesenteric artery obtained from the rats treated with LPS for 6 h. In addition, serum NO2 /NO3 level, which was detected by Griess method, was 10.0±1.4 μM in endotoxemic rats; however, it was 6.6±0.5 μM in control (P<0.05). Taken together, these results show that the expression of the contractile protein Rho-kinase could be upregulated in endotoxemic mesenteric artery and this upregulation may be coincided with an enhanced contraction to endothelin-1 but not phenylephrine and angiotensin-2.
Keywords: Endothelin-1; Lipopolysaccharide; Mesenteric artery; Nitric oxide; Rho-kinase; Y-27632;

Pharmacological characterisation of the thermogenic effect of bupropion by Yong-Ling Liu; Ian P. Connoley; David J. Heal; Michael J. Stock (219-225).
The pharmacological mechanism of bupropion's thermogenic effect has been investigated in female Wistar rats by measuring oxygen consumption at thermoneutrality (29 °C). Bupropion (30 mg/kg) rapidly increased oxygen consumption (VO2) with a maximum effect at 30 min, and VO2 remained elevated throughout the 4-h experimental period. The nonselective 5-hydroxytryptamine (5-HT or serotonin) receptor antagonist, metergoline (1 mg/kg), and the α1-adrenoceptor antagonist, prazosin (1 mg/kg), had no effect on the VO2 response to bupropion, whereas the α2-adrenoceptor antagonist, RS79948 [(8aR, 12aS, 13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]-naphthyridine hydrochloride] (1 mg/kg), potentiated the response. The VO2 response to bupropion during the first 60 min was significantly inhibited by a high dose of the nonselective β-adrenoceptor antagonist, propranolol (20 mg/kg), but it had no effect at a low dose (1 mg/kg). Pretreatment with the dopamine D2/D1 receptor antagonist, (+)butaclamol (200 μg/kg), caused a partial, but significant, inhibition (P<0.01) of the VO2 response to bupropion during the first 60 min, and this antagonist abolished the effect of bupropion between 90 and 240 min. Pretreatment with a combination of a high dose of propranolol (20 mg/kg) and (+)butaclamol (200 μg/kg) prevented any increase in VO2 induced by bupropion. It is concluded that the β3-adrenoceptor subtype, as well as dopamine D2/D1 receptors, is responsible for the increase in oxygen consumption induced by bupropion. We have previously demonstrated that bupropion did not significantly reduce food intake in rats. Hence, in this species, its weight-reducing action predominantly results from thermogenesis mediated via activation of β3-adrenergic and dopamine D2/D1 receptors. Because bupropion has also been reported not to alter food intake in the clinic, thermogenesis may also contribute to its antiobesity effect in man.
Keywords: Thermogenesis; Oxygen consumption; Bupropion; Brown adipose tissue; Brown fat;

We have investigated the effect of mast cell activation induced by immunological and non-immunological stimuli on the sensitivity to adenosine of parenchymal strips prepared from lungs removed from Brown Norway (BN) rats actively sensitized to ovalbumin. Strips responded to ovalbumin with a biphasic contractile response. Responses to adenosine were markedly increased 30 min after ovalbumin. The first phase of the response to ovalbumin was abolished by the 5-hydroxytryptamine (5-HT)2 receptor antagonist, methysergide and unaffected by the cysteinyl leukotriene receptor antagonist, iralukast. The second phase was abolished by iralukast and unaffected by methysergide. The response to adenosine was markedly reduced by methysergide but not significantly altered by iralukast. Compound 48/80 (condensation product of N-methyl-p-methoxyphenylethylamine with formaldehyde) induced methysergide-sensitive contractions of the parenchymal strip and potentiated adenosine; the augmented response to adenosine was blocked by methysergide. Thus, activation of mast cells in the lung by either immunological or non-immunological stimuli results in augmentation of the mast cell-mediated contractile response to adenosine.
Keywords: Adenosine; Airway hyperresponsiveness; Allergen challenge; Ovalbumin; Compound 48/80; Rat, Brown Norway; Lung parenchymal strip;

Galantamine is efficacious for vascular dementia and Alzheimer's disease. Its application leads to some negative gastrointestinal side effects. The present study observes galantamine-induced influence on gastrointestinal motility of rats and its effects on isolated gastrointestinal smooth muscles. The gastrointestinal tract was studied by X-ray contrast examination. Functional disturbances were observed: hypertonia, increased stomach and ileal peristalsis activity, accelerated intestinal passage. In vitro, the drug caused tonic contractions in smooth muscle preparations and increased the gastric and ileal phasic amplitude. The jejunal smooth muscle strips demonstrated an opposite tendency. The reactions were a result of the interaction of galantamine-accumulated endogenic acetycholine with M- and N-acetylcholine receptors. The tonic effects were influenced in varying degree by atropine and ipratropium, whereas the phasic by atropine, ipratropium, hexametonium and methysergide. In conclusion, the in vitro effects registered satisfactorily explain in vivo examined galantamine-induced changes in the gastrointestinal tract of the treated rats and can be considered as main cause for development of such changes.
Keywords: Galantamine; Gastrointestinal tract; Acetylcholine; Acetylcholine receptor;

Mechanisms of postjunctional synergism between adenosine 5′-triphosphate (ATP) and noradrenaline were studied in isolated guinea pig vas deferens. Whereas prior exposure to ATP had no significant effect on noradrenaline-mediated contractions, noradrenaline concentration-dependently enhanced ATP-induced contractions. Similarly to noradrenaline, histamine, which also acts via phospholipase-coupled receptors, induced contractions of the vas deferens and enhanced subsequent responses to ATP. Although phorbol-12, 13-dibutyrate (PDBu), a stimulant of protein kinase C (PKC), failed to induce contractions, it significantly potentiated ATP-induced contractions. The PKC inhibitor, Calphostin C, prevented this effect and the noradrenaline-mediated enhancement of ATP-induced contractions. The phosphatase inhibitor cantharidin induced a time- and concentration-dependent tonic contraction and markedly increased subsequent contractions to ATP. It is suggested that noradrenaline potentiates the contractile response of the vas deferens to ATP via a PKC-mediated mechanism. This may involve the inhibition of myosin light chain phosphatase (MLCP) and subsequent calcium sensitisation.
Keywords: Noradrenaline; ATP; Vas deferens; Synergism; PKC;

Opioid profiles of Cys2-containing enkephalin analogues by Nevena Pencheva; Peter Milanov; Lubomir Vezenkov; Tamara Pajpanova; Emilia Naydenova (249-256).
To elucidate the structural features determining δ-opioid receptor properties of enkephalin analogues containing Cys(O2NH2) in position 2, a series of Cys2-containing derivatives were synthesized and tested for their effectiveness in depressing electrically evoked contractions of the mouse vas deferens (predominantly enkephalin-selective δ-opioid receptors) and the guinea-pig ileum (μ- and κ-opioid receptors). The peptidase resistance of the compounds was also tested. The ratio IC50 in the guinea-pig ileum/IC50 in the mouse vas deferens, indicating selectivity for δ-opioid receptors, was high for Cys(O2NH2)2-containing analogues and especially for [Cys(O2NH2)2, Leu5]enkephalin, which was about seven times more selective than δ-opioid receptor selective ligand cyclic [d-Pen2, d-Pen5]enkephalin (DPDPE). The dissociation constant (K A) and relative efficacy (e rel) of the compounds in the mouse-isolated vas deferens were determined using explicit formulae derived by fitting of the data points with two-parametric hyperbolic function. The obtained values for K A and e rel suggest that: (i) incorporation of Cys(O2NH2)2 in the molecule of [Leu5]enkephalin highly increases the efficacy and does not change significantly the affinity of the respective analogues to δ-opioid receptors; [Cys(O2NH2)2, Leu5]enkephalin has higher affinity than DPDPE, but is less resistant to enzyme degradation; the effect of this modification on the efficacy is decreased when methionine is in position 5; (ii) d-configuration of Cys(O2NH2)2-containing analogues increases their peptidase resistance, but reduces efficacy and affinity of the peptides towards δ-opioid receptors; (iii) the substitution of Cys(O2NH2) with Hcy(O2NH2) reduces the efficacy, affinity and potency of the respective analogues and maintains their sensitivity to endogenous peptidases; (iv) the substitution of the sulfonamide group with benzyl group in the molecule of Cys in position 2 decreases their efficacy and affinity toward δ-opioid receptors, but attaches resistance to enzyme degradation. The results obtained in this study allow: (i) to involve the receptor affinity and agonist efficacy as drug-design consideration for δ-opioid receptor properties of newly synthesized compounds and (ii) to characterize some of the structural features, which set the pattern for their opioid profiles.
Keywords: Enkephalin; Cysteine; δ-Opioid receptor; Affinity; Efficacy;

Effect of pioglitazone on endotoxin-induced decreases in hepatic drug-metabolizing enzyme activity and expression of CYP3A2 and CYP2C11 by Jun Ueyama; Kiyoyuki Kitaichi; Masayuki Nadai; Mitsunori Iwase; Nao Tomyo; Hiroaki Kanazawa; Ryujiro Suzuki; Kenji Takagi; Kenzo Takagi; Takaaki Hasegawa (257-265).
It has been reported that peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands ameliorate the expression of inducible nitric oxide synthase (iNOS) by endotoxin. In the present study, we investigated the effect of pioglitazone, a potent PPAR-γ ligand, on the endotoxin-induced reduction of hepatic drug-metabolizing enzyme activity and on the down-regulation of the expression of hepatic cytochrome P450 (CYP) 3A2 and CYP2C11 proteins in rats. Endotoxin (1 mg/kg) significantly decreased hepatic drug-metabolizing enzyme activity in vivo, as represented by the systemic clearance of antipyrine and protein levels of CYP3A2 and CYP2C11 24 h after intraperitoneal injection. Pretreatment with pioglitazone (10 mg/kg, 4 times at 10-min intervals) significantly protected the endotoxin-induced decreases in the systemic clearance of antipyrine and protein levels of CYP3A2, but not CYP2C11, with no biochemical and histopathological changes in the liver. Pioglitazone alone had no effect on the systemic clearance of antipyrine and protein levels of CYP3A2 or CYP2C11. Pioglitazone significantly protected endotoxin-induced overexpression of iNOS in the liver, but not the overproduction of nitric oxide (NO) in plasma. It is unlikely that the protective effect of pioglitazone against endotoxin-induced decreases in the hepatic drug-metabolizing enzyme activity and protein levels of CYP3A2 in the liver is due to the inhibition of the overproduction of NO.
Keywords: Endotoxin; CYP3A2; CYP2C11; Pioglitazone; PPAR-γ; Antipyrine clearance; (Rat);

Blood flow-dependent changes in intrarenal nitric oxide levels during anesthesia with halothane or sevoflurane by Kazuhito Kusudo; Kaori Ishii; Matlubur Rahman; Yasuharu Aki; Akira Miyatake; Hiroaki Kosaka; Shoji Kimura; Tatsuhiko Komatsu; Masataka Yokoyama; Kiyoshi Morita; Youichi Abe; Akira Nishiyama (267-273).
We previously demonstrated that intrarenal nitric oxide (NO) levels and renal blood flow are reduced during halothane anesthesia. Studies were performed to determine if volatile anesthetics-induced reductions in renal NO levels are associated with blood flow changes. Halothane and sevoflurane at 0.8 and 2.4 Mac were administered by inhalation to dogs, and cGMP and NOx concentrations in the renal interstitial fluid were measured by a microdialysis method. Neither halothane nor sevoflurane at 0.8 Mac altered renal blood flow and renal interstitial cyclic guanosine monophosphate (cGMP) and NOx levels, but both anesthetics significantly decreased these values at 2.4 Mac. Using an adjustable aortic clamp, renal perfusion pressure was reduced in 2 steps without halothane and sevoflurane anesthesia. Renal blood flow as well as cGMP and NOx concentrations in the renal interstitial fluid were unchanged within the autoregulatory range, but significantly decreased below the autoregulatory range. Changes in cGMP and NOx concentrations in the renal interstitial fluid were highly correlated with renal blood flow changes during halothane or sevoflurane anesthesia, and during stepwise reductions in renal perfusion pressure. The results suggested that halothane- and sevoflurane-induced decreases in intrarenal NO levels result from reductions in blood flow.
Keywords: Halothane; Sevoflurane; Nitric oxide (NO); Renal blood flow; Microdialysis;

Inhibition of cyclooxygenase-2, but not cyclooxygenase-1, reduces prostaglandin E2 secretion from diabetic rat retinas by Surya P. Ayalasomayajula; Aniruddha C. Amrite; Uday B. Kompella (275-278).
Up-regulation of cyclooxygenase-2 occurs in retinal cells during the early onset of diabetic retinopathy. Under these conditions, prostaglandin production is elevated, which in turn leads to an increased expression of vascular endothelial growth factor (VEGF)—a growth factor implicated in vascular leakage and neovascularization. In this ex vivo study, we tested whether cyclooxygenase-1 or cyclooxygenase-2 is responsible for diabetes-induced secretion of prostaglandin E2 from isolated rat retinas. Celecoxib, a selective cyclooxygenase-2 inhibitor, significantly inhibited prostaglandin E2 secretion, whereas SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole], a selective cyclooxygenase-1 inhibitor, had no inhibitory effect. These results suggests that the enzymatic activity of cyclooxygenase-2, but not cyclooxygenase-1, results in prostaglandin E2 secretion under diabetic conditions.
Keywords: Cyclooxygenase-2; Celecoxib; Prostaglandin E2; Diabetic retinopathy; VEGF;

Inhibition of interleukin-1β reduces mouse lung inflammation induced by exposure to cigarette smoke by Paulo Castro; Alexandre Legora-Machado; Larissa Cardilo-Reis; Samuel Valença; Luis Cristóvão Porto; Christoph Walker; Claudia Zuany-Amorim; Vera Lucia Gonçalves Koatz (279-286).
We examined nuclear factor κB activation, release of inflammatory mediators and cellular infiltration in acute cigarette smoke inflammation models. One hour after exposure to one puff of cigarette smoke, alveolar macrophages from bronchoalveolar lavage (BAL) fluid of C57BL/6J mice showed an increased activity of nuclear factor κB-DNA binding but similar numbers as compared to that of BAL fluid from mice exposed to ambient air. Exposure to 1 cigarette/day for 1, 4 or 7 days led to an increase in interleukin-1β and monocyte chemoattractant protein-1 levels and to a progressive influx of nuclear factor κB-activated alveolar macrophages into the BAL fluid and lung tissue. Exposure to 2 cigarettes/day for 7 days led to a significant increase in interleukin-1β levels accompanied by a massive alveolar macrophage influx into the BAL fluid. Tumor necrosis factor-α levels and subsequent neutrophil influx were only detected after exposure to 4 or 8 cigarettes/day for 7 days. Treatment of mice with an antibody anti-interleukin-1β during cigarette smoke exposure for 7 days significantly reduced both interleukin-1β levels and alveolar macrophage influx. These data show that a single exposure to cigarette smoke rapidly activates alveolar macrophages, inducing the production of interleukin-1β, which may play an important role in triggering chronic cigarette smoke-mediated lung inflammation.
Keywords: Cigarette smoke; Lung, mouse; Inflammation;

Involvement of enhanced neurokinin NK3 receptor expression in the severe asthma guinea pig model by Osamu Mukaiyama; Kiyoshi Morimoto; Emi Nosaka; Sakiko Takahashi; Makoto Yamashita (287-294).
In this study, we investigated the involvement of neurokinin NK3 receptors in a severe asthma model prepared by administering ovalbumin via inhalation three times to systemically sensitized guinea pigs. [3H]senktide, a neurokinin NK3 receptor ligand, showed significant specific binding to the lungs from the model animals, but not to those from negative control animals. The airway responsiveness to intravenous neurokinin B, a neurokinin NK3 receptor agonist, was increased in the model, indicating an increase in functional NK3 receptors. Furthermore, SB 223956 ((−)-3-methoxy-2-phenyl-N-[(1S)-phenylpropyl]quinoline-4-carboxamide), a selective neurokinin NK3 receptor antagonist, significantly inhibited the ovalbumin-induced airway hyperresponsiveness to inhaled methacholine, but it did not show significant effects on the ovalbumin-induced airway narrowing and eosinophil accumulation. These results suggest that the expressed neurokinin NK3 receptors in the severe asthma model are involved in the development of airway hyperresponsiveness.
Keywords: Airway hyperresponsiveness; Severe asthma model; (Guinea pig); Neurokinin NK3 receptor;

15d-prostaglandin J2 reduces multiple organ failure caused by wall-fragment of Gram-positive and Gram-negative bacteria by Laura Dugo; Marika Collin; Salvatore Cuzzocrea; Christoph Thiemermann (295-301).
Septic shock is still the major cause of death in surgical intensive care units. Both gram-positive (G+) and gram-negative (G−) bacteria have been isolated in the blood of a large portion of septic patients, and these polymicrobial infections often have a higher mortality than infections due to a single organism. Cell wall fragments from G+ and G− bacteria synergise to cause shock and multiple organ dysfunction in vivo (G+/G− shock).Male Wistar rats were anaesthetised and received a coadministration of wall fragments from G+ and G− bacteria, Staphilococcus aureus (S. aureus) peptidoglycan [0.3 mg/kg, intravenously (i.v.)] and Escherichia coli (E. coli) lipopolysaccharide (1 mg/kg, i.v.) or vehicle (saline, 1 ml/kg, i.v.). G+/G− shock for 6 h resulted in an increase in serum levels of creatinine (indicator of renal dysfunction), alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (γ-GT), bilirubin (markers for hepatic injury and dysfunction) and creatine kinase (CK, an indicator of neuromuscular, skeletal muscle or cardiac injury). Pretreatment of rats with the peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist 15d-prostaglandin J2 (0.3 mg/kg, i.v., 30 min prior to G+/G−) reduced the multiple organ injury/dysfunction caused by coadministration of peptidoglycan+lipopolysaccharide. The selective PPAR-γ antagonist GW9662 (2-Chloro-5-nitrobenzanilide) (1 mg/kg, i.v., given 45 min prior to G+/G−) abolished the protective effects of 15d-prostaglandin J2. 15d- prostaglandin J2 did not affect the biphasic fall in blood pressure or the increase in heart rate caused by administration of peptidoglycan+lipopolysaccharide. The mechanism(s) of the protective effect of this cyclopentenone prostaglandin are—at least in part—PPAR-γ dependent, as the protection afforded by 15d-prostaglandin J2 was reduced by the PPAR-γ antagonist GW9662. We propose that 15d-prostaglandin J2 or other ligands for PPAR-γ may be useful in the therapy of the organ injury associated with septic shock.
Keywords: Peroxisome-proliferator activated receptor-γ; Lipopolysaccharide; Peptidoglycan; GW9662; (Rat);

Benidipine hydrochloride (benidipine) is a dihydropyridine-Ca2+ channel blocker with antioxidant properties. We examined the effects of benidipine on cytokine-induced expression of adhesion molecules and chemokines, which play important roles in the adhesion of monocytes to endothelium. Pretreatment of human aortic endothelial cells (HAECs) with benidipine (0.3–10 μmol/l) for 24 h significantly suppressed cytokine-induced vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) mRNA and protein expression, resulting in reduced adhesion of THP-1 monocytes. Benidipine also suppressed induction of monocyte chemoattractant protein (MCP)-1 and interleukin-8. Benidipine inhibited redox-sensitive transcriptional nuclear factor-κB (NF-κB) pathway, as determined by Western blotting of inhibitory κB (IκB) phosphorylation and luciferase reporter assay. Results of analysis using optical isomers of benidipine and antioxidants suggested that these inhibitory effects were dependent on pharmacological effects other than Ca2+ antagonism such as antioxidant effects. Benidipine may thus have anti-inflammatory properties and benefits for in the treatment of atherosclerosis.
Keywords: Benidipine; Antioxidant; Endothelium; VCAM-1; Atherosclerosis;

Protective role of NF-κB1 (p50) in experimental pneumococcal meningitis by Stefan Kastenbauer; Uwe Koedel; Falk Weih; Löms Ziegler-Heitbrock; Hans-Walter Pfister (315-318).
Nuclear factor-kappaB (NF-κB) is a critical regulator of many genes involved in the pathogenesis of bacterial meningitis. Recently, activation of NF-κB was shown to be a key event in the inflammatory host response and the development of intracranial complications during experimental pneumococcal meningitis. Since the p50 subunit of NF-κB lacks a transactivation domain and can therefore act as a transcriptional repressor, we investigated whether NF-κB1 (p50) exerts anti-inflammatory effects in pneumococcal meningitis. p50-deficient mice had higher cerebellar pneumococcal titers (10.06±0.47 vs. 8.51±1.06 log colony-forming units [cfu]/cerebellum), cerebrospinal fluid (CSF) leukocyte counts (11,475±2340 vs. 8444±1405 cells/μl) and brain concentrations of interleukin-1β (125.9±50.3 vs. 58.5±52.2 pg/mg protein) than their wild-type littermates. With ceftriaxone therapy, none of the wild-type mice but 43% of the p50-deficient animals died. In conclusion, lack of NF-κB1 (p50) was associated with impaired bacterial clearing, enhanced inflammatory host response and increased mortality during pneumococcal meningitis.
Keywords: Meningitis; Streptococcus pneumoniae; NF-kappaB1; NF-kappaB p50 subunit; Host response;

To study the effect of phosphodiesterase (PDE) 3 inhibition on plasma insulin and glucose levels, the selective PDE 3 inhibitor milrinone (0.25, 1.0, and 2.5 mg/kg) was given orally to anesthetized CL57Bl/6J mice 10 min before a gastric glucose gavage (150 mg/mouse). It was found that milrinone augmented the glucose-mediated increase in plasma insulin at 1.0 and 2.5 mg/kg without, however, any improvement in glucose elimination. In contrast, when given 10 min before intravenous glucose (1 g/kg), milrinone (1 mg/kg) did not affect the insulin response to glucose. The increase in glucagon-like peptide-1 (GLP-1) levels after gastric glucose was not altered by milrinone. However, the PDE3 inhibitor augmented the insulin response to intravenous GLP-1 (2.8 nmol/kg). We therefore conclude that PDE3 inhibition by milrinone augments insulin secretion in vivo in mice after oral but not after intravenous glucose, which may be explained by enhanced response to the cAMP-dependent insulinotropic action of endogenously released GLP-1.
Keywords: Phosphodiesterase; Type 2 diabetes; Milrinone; Glucagon-like peptide-1; Insulin secretion;

Carbamazepine enhances brain production of kynurenic acid in vitro by Tomasz Kocki; Janusz Kocki; Marian Wielosz; Waldemar A. Turski; Ewa M. Urbanska (325-326).
Disturbed formation of kynurenic acid, an endogenous antagonist of glutamate ionotropic receptors, might contribute to the pathogenesis of seizures. Here, the effect of anticonvulsant drug, carbamazepine on the production of kynurenic acid was studied. Carbamazepine (0.5–3 mM) enhanced kynurenic acid synthesis in rat cortical slices and also increased the activity of kynurenine aminotransferase (KAT) I at 0.1–3.0 mM concentration. Thus, anticonvulsant drugs, such as carbamazepine, might act partially via stimulation of kynurenic acid production.
Keywords: Kynurenic acid; Carbamazepine;

Author Index (327-330).

Keyword Index (331-337).