European Journal of Pharmacology (v.485, #1-3)

Cerivastatin inhibits proliferation of interleukin-1β-induced rat mesangial cells by enhanced formation of nitric oxide by Cornelia Blume; Danuta Sabuda-Widemann; Josef Pfeilschifter; Jörg Plum; K. Schrör; Bernd Grabensee; Karl-Friedrich Beck (1-10).
The antiproliferative effect of statins on mesangial cells could represent a new therapeutic approach in glomerulonephritis. We studied in rat mesangial cells whether the antiproliferative action of cerivastatin on mesangial cells may be mediated by mesangial nitric oxide (NO) formation due to the inducible NO synthase (iNOS) or by induction of cyclooxygenase-2. Mesangial cells were stimulated with interleukin-1β and treated with cerivastatin for 24 h. Cell proliferation was examined by bromodeoxy-uridine (BrdU) incorporation, and nitrite and prostaglandin production was measured in supernatants as a means for iNOS or cyclooxygenase-2 activity. iNOS and cyclooxygenase-2 expression was quantified by Northern and Western blot analyses. Cerivastatin (0.0625 μM) significantly inhibited DNA synthesis in interleukin-1β-stimulated mesangial cells without altering cell viability. Interleukin-1β-induced nitrite production was twofold increased by 0.05 μM cerivastatin, and this effect could be reversed by addition of 100 μM mevalonate. iNOS mRNA levels increased sixfold (33% of maximum) in cerivastatin-treated mesangial cells as compared with vehicle-treated controls (3.5% of maximum). iNOS and cyclooxygenase-2 protein expression increased threefold (iNOS: 2.77±0.53/cyclooxygenase-2: 3.49±1.25). The NOS inhibitors N-methyl-l-arginine (l-NMMA) and l-N6-(1-iminoethyl)lysine (l-NIL) reversed the antiproliferative effect of cerivastatin. The cyclooxygenase-2 inhibitor celecoxib did not alter DNA synthesis and iNOS or cyclooxygenase-2 expression, but blocked prostacyclin production in interleukin-1β and cerivastatin-treated mesangial cells. In conclusion, cerivastatin increased cytokine-induced iNOS and cyclooxygenase-2 expression, thus constituting NO-regulated growth inhibition of mesangial cells.
Keywords: Mesangial cell, rat; Interleukin 1-β; Statin; Cerivastatin; NO (nitric oxide); NO (nitric oxide) synthase, inducible; Cyclooxygenase-2;

Yohimbine, an indole alkaloid, is a natural α2-adrenoceptor antagonist and is frequently used to assess the mechanism of a drug's effect on α-adrenoceptors. Recently, several studies showed that yohimbine exhibited analgesic effects in in vivo animal models. However, the underlying mechanism is not known. We investigated the effects of yohimbine on Na+ channels and vanilloid VR1 receptors in dorsal root ganglion cells. We found that yohimbine inhibited tetrodotoxin-sensitive Na+ channels (NaV1.2), the tetrodotoxin-resistant Na+ channels, including both slow inactivating (NaV1.8) and persistent (NaV1.9) Na+ channels, and capsaicin-sensitive vanilloid VR1 receptors. Action potential firing activities of dorsal root ganglion neurons evoked by current injection or capsaicin were eliminated by yohimbine. The blocking effects of yohimbine on nociceptive-related ion channels and firing activities of dorsal root ganglion neurons may underlie the ionic mechanism of yohimbine's analgesic effects observed in in vivo studies.
Keywords: Yohimbine; Na+ channel; Vanilloid VR1 receptor; Dorsal root ganglion; Action potential; Firing activity;

Differentiation-inducing factor-1-induced growth arrest of K562 leukemia cells involves the reduction of ERK1/2 activity by Emi Akaishi; Torao Narita; Shinjiro Kawai; Yoshikazu Miwa; Toshiyuki Sasaguri; Kohei Hosaka; Yuzuru Kubohara (21-29).
The differentiation-inducing factor-1 (DIF-1) is a signal molecule that induces stalk cell differentiation in the cellular slime mold Dictyostelium discoideum. In addition, DIF-1 is a potent antileukemic agent that induces growth arrest in K562 cells. In this study, we investigated the mechanism of action of DIF-1 in K562 cells in the light of cell-cycle regulators such as cyclins, retinoblastoma protein (pRb), and the mitogen-activated protein kinase (MAPK) family. DIF-1 down-regulated cyclins D/E and a phosphorylated form of pRb (p-pRb), and thereby induced G1 arrest of the cell cycle. DIF-1 inactivated the extracellular signal-regulated kinase (ERK) in a biphasic manner but did not affect the c-Jun N-terminal kinase (JNK) or p38 MAPK. The MEK (MAPK kinase) inhibitor, U0126, which has been shown to induce growth arrest, inactivated ERK and down-regulated cyclins D and E. Although DIF-1 activated the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway, neither wortmannin nor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002; PI-3K inhibitors) cancelled DIF-1-induced growth arrest. The present results suggest that ERK inactivation may be involved in DIF-1-induced growth arrest and that PI-3K activity is not required for DIF-1-induced growth arrest in K562 cells.
Keywords: Dictyostelium; DIF-1 (differentiation-inducing factor-1); K562; ERK (extracellular signal-regulated kinase); Akt;

Electrophysiologic properties of lidocaine, cocaine, and n-3 fatty-acids block of cardiac Na+ channels by Yong-Fu Xiao; Qingen Ke; Sho-Ya Wang; Yinke Yang; Yu Chen; Ging Kuo Wang; James P. Morgan; Benjamin Cox; Alexander Leaf (31-41).
Lidocaine and cocaine, two local anesthetics, and n-3 polyunsaturated fatty acids in fish oils, inhibit the voltage-gated Na+ channels of cardiomyocytes. This inhibition by lidocaine and n-3 fish oil is associated with antiarrhythmic effects, whereas with cocaine lethal arrhythmias may occur. These electrophysiologic studies show that at the concentrations tested, the n-3 fish oil fatty acids and lidocaine share three actions on I Na: a potent inhibition of I Na; a strong voltage-dependence of this inhibition; and a large shift of the steady-state inactivation to hyperpolarized potentials. By contrast cocaine shares only the potent inhibition of I Na. The voltage-dependence of the inhibition is much decreased with cocaine, which produces only a very small leftward shift of the voltage-dependence of inactivation. The large leftward shift of the steady-state inactivation seems very important in the prevention of fatal arrhythmias by the n-3 fatty acids. Thus, we suggest that it is lack of this effect by cocaine, which is one factor, that eliminates its ability to prevent fatal cardiac arrhythmias. Further we report that in cultured neonatal rat cardiomyocytes n-3 fish oil fatty acids terminate the tachycardia induced by the α1 adrenergic agonist, phenylephrine, whereas cocaine accelerates the tachycardia and causes bouts of tachyarythmias.
Keywords: Fish oil; Polyunsaturated fatty acid; Arrhythmias; Adrenoceptor agonists; Eicosapentaenoic acid; Docosahexaenoic acid;

The aim of this study was to investigate the couplings between various binding sites on the GABAA receptor complex. We investigated combinations of three test compounds: (1) GABA (γ-aminobutyric acid), (2) Org 20549 [(2β3α5α)-21hydroxy-3Hydroxy-2(4morpholinyl)pregnan-20one methane-sulphonate)], a neuroactive steroid and (3) retigabine (D-23129, N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester), a new antiepileptic drug. Receptor-binding assays were conducted using rat brain membranes. [3H]TBOB ([3H]-t-butyl-bicyclo-ortho-benzoate) was the tracer ligand. All three test compounds inhibited the binding of [3H]TBOB with EC50's of 4.0, 98 and 23 μM, respectively. Isobolic analysis of the combination data showed that the three compounds act in synergy in displacing [3H]TBOB. These interactions could be described and quantified by a hypercube model in which each of the three test compounds and [3H]TBOB bind to different, allosterically coupled sites such that each of the test compounds allosterically displaces the tracer [3H]TBOB and allosterically enhances the affinity of any other test compound by a factor 4.4. The simultaneous binding of any two ligands enhances the affinity of the third by a factor 9. These results may contribute to the understanding of individual variability in drug responses and to the discussion about rational polytherapy.
Keywords: GABAA receptor complex; TBOB; Neuroactive steroid; Retigabine; Synergy; Isobole method; Molecular model;

Multiple effects of arginine vasopressin on prostaglandin E2 synthesis in fibroblasts by Fleisher-Berkovich Sigal; Kagan Elena; Grossman Nili; Danon Abraham (53-59).
Recent evidence supports the viewpoint that vasopressin, a neurohypophyseal peptide, should be also considered as a neuroendocrine modulator of immune and inflammatory responses. In this work we investigated the role of vasopressin in the regulation of prostaglandin E2 synthesis by human dermal fibroblasts. Recombinant human interleukin-1β increased prostaglandin E2 synthesis in fibroblasts about sixfold. The prostaglandin E2 response to interleukin-1β was attenuated by lower concentrations of vasopressin (10−10–10−9 M). By contrast, higher concentrations (10−8–10−7 M) of vasopressin effected significant enhancement of the interleukin-1β-induced prostaglandin E2 synthesis. In a similar way, vasopressin (10−8–10−7 M), in the absence of interleukin-1, significantly increased prostaglandin E2 production. An inhibitory effect of lower concentrations of vasopressin was also observed on basal production of prostaglandin E2. The effects of vasopressin on basal and interleukin-1β-induced prostaglandin E2 synthesis were antagonized by selective vasopressin receptor antagonists. The findings presented here disclose a novel modulatory role of vasopressin on prostaglandin E2 synthesis in human dermal fibroblasts and suggest a possible role of vasopressin in the regulation of inflammation.
Keywords: Interleukin-1; Vasopressin; Prostaglandin; Inflammation;

Peroxovanadate induces α1B-adrenoceptor phosphorylation and association with protein kinase C by Rocı́o Alcántara-Hernández; Luz del Carmen Medina; J.Adolfo Garcı́a-Sáinz (61-67).
Peroxovanadate induced a marked increase in the phosphorylation state of α1B-adrenoceptors. The effect was dose-dependent (EC50≈2 μM) and rapid, reaching its maximum in 5 min and remaining at this level for 30 min. Hydrogen peroxide also increased α1B-adrenoceptor phosphorylation but to a lesser extent, in an ephemeral fashion, and only at high (millimolar) concentrations. The effect of peroxovanadate was blocked by inhibitors of protein kinase C such as staurosporine and rottlerin and only partially reduced by genistein and inhibitors of phosphoinositide 3-kinase. Protein kinase C α, δ and ε are associated with the α1B-adrenoceptor under basal conditions, as reflected by coimmunoprecipitation. Such association was increased by peroxovanadate for all isoforms. In contrast, hydrogen peroxide increased only the association of the ε isoform to the adrenoceptor. Peroxovanadate decreased the ability of noradrenaline to increase intracellular calcium, indicating that the receptor phosphorylation induced has functional consequences.
Keywords: α1B-Adrenoceptor; Peroxovanadate; Phosphorylation receptor; Protein kinase C;

Cytochrome P450 isoenzymes involved in rat liver microsomal metabolism of californine and protopine by Liane D Paul; Dietmar Springer; Roland F Staack; Thomas Kraemer; Hans H Maurer (69-79).
Studies are described on the cytochrome P450 (CYP) isoenzyme dependence of the main metabolic steps of the Eschscholtzia californica alkaloids californine and protopine using rat liver microsomes. Preparations of E. californica are in use as phytopharmaceuticals and as herbal drugs of abuse. CYP isoenyzme dependences were studied using specific chemical inhibitors for CYP1A2, CYP2D1, and CYP3A2 (α-naphthoflavone, quinine, and ketoconazole, respectively). CYP2C11 was inhibited by specific antibodies for lack of specific chemical inhibitors. Californine N-demethylation was mainly catalyzed by CYP3A2 and to a minor extent by CYP1A2 and CYP2D1, but not by CYP2C11. CYP2D1 and CYP2C11 were shown to be mainly involved in demethylenation of both, californine and protopine, while CYP1A2 and CYP3A2 showed only minor contribution. Kinetic parameters of the reactions were established. K m and V max values for the californine N-demethylation were 4.5±4.7 μM and 22.9±13.7 min/mg protein (high affinity) and 161.3±16.7 μM and 311.8±39.4 min/mg protein (low affinity), respectively. Californine demethylenation and protopine demethylenation showed substrate inhibition and K m and V max values were 5.0±0.5 and 7.1±0.6 μM and 83.3±2.6 and 160.7±4.0 min/mg protein, respectively.
Keywords: Californine; Protopine; Metabolism; Cytochrome P450; Liver microsome, rat;

5-Aza-2′-deoxycytidine and paclitaxel inhibit inducible nitric oxide synthase activation in fibrosarcoma cells by Djordje Miljkovic; Ivana Cvetkovic; Marija Sajic; Olivera Vuckovic; Ljubica Harhaji; Milos Markovic; Vladimir Trajkovic (81-88).
Given the important role of gaseous free radical nitric oxide (NO) in tumor cell biology, we investigated the ability of the anti-cancer drugs 5-Aza-2′-deoxycytidine (ADC) and paclitaxel to modulate NO production in mouse L929 fibrosarcoma cells. Both drugs reduced IFN-γ-stimulated NO release in cultures of L929 and primary fibroblasts, but not in mouse peritoneal macrophages. The inhibitory effect was due to the reduced expression of inducible NO synthase (iNOS), the enzyme responsible for cytokine-induced intracellular NO synthesis, as both agents markedly suppressed the interferon-gamma (IFN-γ)-triggered increase in iNOS concentration in L929 cells. In addition, ADC and paclitaxel prevented the IFN-γ-triggered activation of p44/p42 mitogen-activated protein (MAP) kinase in L929 fibroblasts, suggesting a possible mechanism for the observed inhibition of iNOS expression. These results might have important implications for the therapeutic effect of ADC and paclitaxel, since their inhibitory action on NO release partly neutralized the NO-dependent toxicity of IFN-γ on L929 fibrosarcoma cells.
Keywords: 5-Aza-2′-deoxycytidine; Paclitaxel; Nitric oxide (NO); iNOS (nitric oxide (NO) synthase, inducible); L929; Fibrosarcoma;

Quercetin, a flavonoid, inhibits the proliferation, differentiation, and mineralization of osteoblasts in vitro by Michitaka Notoya; Yu Tsukamoto; Hiroyuki Nishimura; Je-Tae Woo; Kazuo Nagai; In-Sun Lee; Hiromi Hagiwara (89-96).
It is possible that the flavonoids that are found in many foods might have a protective effect against osteoclastic activity. However, little information is available about the effects of flavonoids on osteoblastogenesis. Therefore, we investigated the effects of quercetin, a flavonoid, on the metabolism of rat calvarial osteoblast-like cells (ROB cells) in culture. The proliferation of cells was markedly inhibited upon exposure of cells to quercetin at 5×10−6 to 1×10−5 M. Quercetin at 1×10−5 M did not induce apoptosis in ROB cells but arrested cells at the G1 phase of the cell cycle. In addition, quercetin stimulated the expression of mRNA for p21waf1/cip1, which inhibits the activity of cyclin-dependent kinases, and inhibited the phosphorylation of histone H1. Furthermore, after cells had ceased to proliferate, quercetin reduced the activity of alkaline phosphatase, the level of expression of mRNA for osteocalcin, the rate of deposition of Ca2+, and the formation of mineralized nodules, all of which are markers of osteoblast differentiation. These findings indicate that quercetin inhibits the proliferation, differentiation, and mineralization of osteoblastic cells.
Keywords: Quercetin; Flavonoid; Osteoblast; Proliferation; Differentiation; Cell cycle;

Allicin (from garlic) induces caspase-mediated apoptosis in cancer cells by Suby Oommen; Ruby John Anto; Gopal Srinivas; Devarajan Karunagaran (97-103).
Garlic (Allium sativum) has been used for centuries for treating various ailments, and its consumption is said to reduce cancer risk and its extracts and components effectively block experimentally induced tumors. Allicin, the major component present in freshly crushed garlic, is one of the most biologically active compounds of garlic. We found that allicin inhibited the growth of cancer cells of murine and human origin. Allicin induced the formation of apoptotic bodies, nuclear condensation and a typical DNA ladder in cancer cells. Furthermore, activation of caspases-3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase were induced by allicin. The present results demonstrating allicin-induced apoptosis of cancer cells are novel since allicin has not been shown to induce apoptosis previously. Our results also provide a mechanistic basis for the antiproliferative effects of allicin and partly account for the chemopreventive action of garlic extracts reported by earlier workers.
Keywords: Allicin; Apoptosis; SiHa; HeLa; SW480; L-929; Garlic;

The present study evaluated effects of wogonin (5,7-dihydroxy-8-methoxyflavone) on excitotoxic and oxidative stress-induced neuronal damage in primary cultured rat cortical cells. Wogonin was shown to inhibit the excitotoxicity induced by glutamate or N-methyl-D-aspartic acid, whereas it showed no effects on the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- or kainate-induced toxicity. In addition, wogonin inhibited the oxidative neuronal damage induced by H2O2, xanthine/xanthine oxidase, and by a glutathione depleting agent d,l-buthionine [S,R]-sulfoximine. Furthermore, wogonin dramatically inhibited lipid peroxidation initiated by Fe2+ and l-ascorbic acid in rat brain homogenates. It also exhibited 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Taken together, these results demonstrate that wogonin exhibits neuroprotective actions in cultured cortical cells by inhibiting excitotoxicity and various types of oxidative stress-induced damage, and that its antioxidant actions with radical scavenging activity may contribute, at least in part, to the neuroprotective effects.
Keywords: Wogonin; Neuroprotection; Cortical neuron; Antioxidant; Excitotoxicity; Oxidative damage;

Herein, we examined the direct coupling of human dopamine D1 receptors to Gs proteins using an antibody capture assay together with a detection technique employing scintillation proximity assay beads. Using a specific antibody, dopamine (DA) and the selective dopamine D1 receptor agonists, 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF81297) and 3-allyl-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF82958), behaved as high-efficacy agonists (∼100%) in stimulating guanosine-5′-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding to Gs in L-cells, whereas 2,3,4,5,-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF38393) displayed partial agonist properties (70%). The action of dopamine was specifically mediated by human dopamine D1 receptors inasmuch as the selective human dopamine D1 receptor antagonist, (R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-benzazepine-7-ol (SCH23390), blocked dopamine-induced [35S]GTPγS binding to Gs with a pK B (9.29) close to its pK i (9.33). The antipsychotic agents, clozapine and haloperidol, displayed no intrinsic activity when tested alone and inhibited dopamine-stimulated Gs activation with pK B's of 6.7 and 7.3, respectively, values close to their pK i values at these sites. In conclusion, the use of an anti-Gs protein immunoprecipitation assay coupled to scintillation proximity assays allows direct evaluation of the functional activity of dopamine D1 receptors ligands at the G protein level. Employing this novel technique, the typical and atypical antipsychotics, clozapine and haloperidol, respectively, both exhibited antagonist properties at dopamine D1 receptors.
Keywords: Dopamine D1 receptor; Gs protein; Scintillation proximity assay; Clozapine; Haloperidol;

κ-Opioid receptors are differentially labeled by arylacetamides and benzomorphans by Daniela E. Rusovici; S.Stevens Negus; Nancy K. Mello; Jean M. Bidlack (119-125).
Using Chinese Hamster Ovary cell membranes that stably expressed the human κ-opioid receptor, we investigated the hypothesis that κ1- and κ2-opioid receptors, historically defined by their phrmacological selectivity for either arylacetamides or benzomorphans are, in fact, different affinity states or binding sites on the same κ-opioid receptors. Receptor binding studies showed that GTPγS potently inhibited [3H](5α,7α,8β)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro [4.5]dec-8-yl)-benzeneacetamide (U69,593) binding, compared to virtually no inhibition of [3H]bremazocine binding. Saturation binding experiments showed a three-fold decrease in [3H]U69,593 affinity in the presence of GTPγS, but GTPγS had no effect on [3H]bremazocine affinity. The κ-opioid receptor antagonist nor-binaltorphimine had a four-fold higher affinity for [3H]U69,593-labeled receptors than for [3H]bremazocine-labeled receptors. Functional selectivity studies, measuring the stimulation of [35S]GTPγS agonist-induced binding, showed a significantly higher U69,593-induced G protein-receptor activation in comparison to the stimulation observed with bremazocine. These results suggest that pharmacologically defined 1κ-opioid receptor subtypes may be different affinity states of the same receptor.
Keywords: Benzomorphan; Arylacetamide; Nor-binaltorphimine; κ-Opioid receptor;

Ebselen inhibits p38 mitogen-activated protein kinase-mediated endothelial cell death by hydrogen peroxide by Nermin Ali; Masanori Yoshizumi; Koichiro Tsuchiya; Moe Kyaw; Yoshiko Fujita; Yuki Izawa; Shinji Abe; Yasuhisa Kanematsu; Shoji Kagami; Toshiaki Tamaki (127-135).
Ebselen (2-phenly-1, 2-benzisoselenazol-3[2H]-one) is a seleno-organic compound exhibiting both glutathione peroxidase and antioxidant activity. Although it has been reported that ebselen is effective against hydrogen peroxide (H2O2)-induced cell death in several cell types, its effect on endothelial cell damage has not yet been elucidated. In the present study, we examined the effect of ebselen on H2O2-induced human umbilical vein endothelial cells (HUVECs) death, and its intracellular mechanism. Our findings showed that pretreatment of HUVECs with ebselen resulted in a significant recovery from H2O2-induced cell death in a concentration-dependent manner. In addition to the inhibition of lactate dehydrogenase (LDH) leakage, ebselen inhibited H2O2-induced cytochrome c release and caspase-3 activation and the resultant apoptosis in HUVECs. Moreover, it was observed that H2O2 significantly stimulated activation of mitogen-activated protein (MAP) kinases, i.e., p38 MAP kinase, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2). Ebselen inhibited H2O2-induced p38 MAP kinase, but not JNK or ERK1/2 activation. Furthermore, SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]-1H-imidazole), a specific p38 MAP kinase inhibitor, inhibited H2O2-induced p38 MAP kinase phosphorylation, cytochrome c release, caspase-3 activation, as well as cell death in HUVECs. These findings suggest that ebselen attenuates H2O2-induced endothelial cell death through the inhibition of signaling pathways mediated by p38 MAP kinase, caspase-3, and cytochrome c release. Thus, inhibition of p38 MAP kinase by ebselen may imply its usefulness for prevention and/or treatment of endothelial cell dysfunction, which was suggested to be the first step in the development of atherosclerosis.
Keywords: Hydrogen peroxide; Ebselen; p38 MAP (mitogen-activated protein) kinase; Apoptosis; HUVEC (human umbilical vein endothelial cell);

Effect of the ecto-ATPase inhibitor, ARL 67156, on the bovine chromaffin cell response to ATP by Desinee A. Drakulich; Calvin Spellmon; Terry D. Hexum (137-140).
Bovine chromaffin cells contain an ecto-ATPase (K m=1.57±0.27×10−4 M) which can hydrolyze ATP present in the culture media. ARL 67156 is a competitive inhibitor of this ATPase (K i=2.55±1.36×10−7 M). A small increase in potency (threefold) is seen when ARL 67156 is included during measurement of ATP-stimulated inositol phosphate formation. ARL 67156 also acts on chromaffin cell P2Y receptors to increase inositol phosphate formation (EC50=4.9×10−5 M). It is useful as an ecto-ATPase inhibitor in studies with bovine chromaffin cells since it exhibits a 300-fold selectivity for the ecto-ATPase versus the P2Y receptor.
Keywords: Ecto-ATPase; Chromaffin cell; ARL 67156; Inositol phosphate; P2Y receptor;

Protein kinase C (PKC)-β and other PKC isozymes have been implicated in the loss of endothelial barrier function in diabetic microangiopathy. The effects of a PKC-β-specific inhibitor, LY379196, on hyperpermeability responses to high-glucose, angiotensin II, α-thrombin and endothelin-1 were evaluated using an in vitro model of human pulmonary artery endothelial cell monolayers. LY379196 attenuated the increase in transendothelial albumin flux induced by glucose 40 mM (e.g. 411±160% [high-glucose] vs. 167+37% [high-glucose+LY379196], P<0.001) and angiotensin II 10 μM (e.g. 121±12% vs. 246±35%, P<0.01); endothelin-1 had no significant effect on monolayer permeability. LY379196 had no significant effect on the marked hyperpermeability response to α-thrombin 1 μM. Thus, two major pathways involved in vascular leakage in diabetic microangiopathy are amenable to therapeutic blockade by PKC-β inhibition.
Keywords: Protein kinase C-β (PKC-β) inhibitor; Protein kinase C (PKC); Endothelial permeability; Angiotensin II; Diabetic retinopathy;

Anxiolytic- and antidepressant-like profile of a new CRF1 receptor antagonist, R278995/CRA0450 by Shigeyuki Chaki; Atsuro Nakazato; Ludo Kennis; Masato Nakamura; Claire Mackie; Masayuki Sugiura; Petra Vinken; David Ashton; Xavier Langlois; Thomas Steckler (145-158).
1-[8-(2,4-dichlorophenyl)-2-methylquinolin-4-yl]-1,2,3,6-tetrahydropyridine-4-carboxamide benzenesulfonate (R278995/CRA0450) is a newly synthesized corticotropin-releasing factor subtype 1 (CRF1) receptor antagonist. In the present study, in vitro and in vivo pharmacological profiles of R278995/CRA0450 were investigated. R278995/CRA0450 showed high affinity for recombinant and native CRF1 receptors without having affinity for the CRF2 receptor. R278995/CRA0450 attenuated CRF-induced cyclic AMP formation in AtT-20 cells and CRF-induced forepaw treading in gerbils, indicating that R278995/CRA0450 is an antagonist of the CRF1 receptor. In addition to CRF1 receptor antagonism, R278995/CRA0450 showed high affinity for the σ1 receptor, and attenuated (+)-SKF10,047-induced head-weaving behavior, suggesting σ1 receptor antagonism. R278995/CRA0450 showed dose-dependent in vivo occupancy when assessed by ex vivo receptor binding, indicating good brain penetration. R278995/CRA0450 did not alter spontaneous anxiety when tested in the rat elevated plus maze (up to 3 mg/kg, p.o.) or lick suppression test (up to 10 mg/kg, i.p.). However, potent anxiolytic-like properties were observed in rats subjected to swim stress prior to testing on the elevated plus-maze, indicating activity primarily in tests taxing stress-induced anxiety. R278995/CRA0450 was inactive in mouse tail suspension, rat forced swim and rat differential-reinforcement-of-low-rate 72-s (DRL72), while it showed dose-dependent antidepressant-like effects in the rat learned helplessness paradigm and the olfactory bulbectomy model, demonstrating activity in a subset of animal models of depression associated with subchronic stress exposure. No or only mild effects were seen in tests of locomotor activity, motor coordination and sedation. These results indicate that R278995/CRA0450 is an orally active CRF1 and σ1 receptor antagonist with potent anxiolytic-like and antidepressant-like activities.
Keywords: CRF (corticotropin-releasing factor); CRF1 receptor; Anxiety; Depression; Stress-related behavior; R278995/CRA0450;

Levetiracetam improves choreic levodopa-induced dyskinesia in the MPTP-treated macaque by Erwan Bezard; Michael P. Hill; Alan R. Crossman; Jonathan M. Brotchie; Anne Michel; Renee Grimée; Henrik Klitgaard (159-164).
l-3,4 dihydroxyphenylalanine (levodopa)-induced dyskinesia in Parkinson's disease patients is characterized by a mixture of chorea and dystonia. Electrophysiological studies suggest that chorea is associated with abnormal synchronization of firing of basal ganglia neurons while dystonia is not. Levetiracetam is a novel anti-epileptic drug known to exhibit unique desynchronizing properties in contrast to other anti-epileptic drugs. We assessed the anti-dyskinetic efficacy of levetiracetam (13, 30 and 60 mg/kg, p.o.) administered in combination with an individually tailored dose of levodopa (Levodopa/carbidopa, 4:1 ratio, 19±1.8 mg/kg, p.o.), in six dyskinetic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned macaques. Levetiracetam (60 mg/kg) significantly reduced levodopa-induced chorea during the first hour post-treatment but had no effect on dystonia. Levetiracetam, at all doses tested, had no effect on the anti-parkinsonian action of levodopa. These results suggest that levetiracetam may provide a novel therapeutic approach specifically aimed at the choreic form of levodopa-induced dyskinesia.
Keywords: Parkinson disease; Dystonia; Synchronization; Anti-epileptic drug;

Profile of spinal and supra-spinal antinociception of (−)-linalool by Alessandra T Peana; M.Graziella De Montis; Eugenio Nieddu; M.Teresa Spano; Paolo S D'Aquila; Proto Pippia (165-174).
We previously reported that administration of (−)-linalool, the naturally occurring enantiomer in essential oils, induced a significant reduction in carrageenin-induced oedema and in acetic acid-induced writhing. The latter effect was completely antagonised by the muscarinic receptor antagonist atropine and by the opioid receptor antagonist naloxone. To further characterise the antinociceptive profile of (−)-linalool, we studied its effect in the hot plate and the formalin in tests. In addition, to determine the possible involvement of the cholinergic, opioidergic and dopaminergic systems, we tested the effects of atropine, pirenzepine, a muscarinic M1 receptor antagonist, naloxone, sulpiride, a dopamine D2 receptor antagonist and (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH-23390), a dopamine D1 receptor antagonist on (−)-linalool-induced antinociception. Moreover, since K+ channels seem to play an important role in the mechanisms of pain modulation, we examined the effect of glibenclamide, an ATP-sensitive K+ channel inhibitor on (−)-linalool-induced antinociception. The administration of (−)-linalool (100 and 150 mg/kg, s.c.) increased the reaction time in the hot-plate test. Moreover, (−)-linalool (50 and 100 mg/kg) produced a significant reduction in the early acute phase of the formalin model, but not in the late tonic phase. The highest dose (150 mg/kg) caused a significant antinociceptive effect on both phases. The antinociceptive effects of (−)-linalool were decreased by pre-treatment with atropine, naloxone, sulpiride and glibenclamide but not by pirenzepine and SCH-23390. These results are in agreement with the demonstrated pharmacological properties of linalool, mainly its cholinergic, local anaesthetic activity and its ability to block NMDA receptors. Furthermore, a key role seems to be played by K+ channels, whose opening might be the consequence of a stimulation of muscarinic M2, opioid or dopamine D2 receptors.
Keywords: (−)-Linalool; Antinociception; Cholinergic; Opioid; Dopamine; K+ channel; ATP-sensitive channel;

Catalepsy induced by intra-striatal administration of nitric oxide synthase inhibitors in rats by Elaine A. Del Bel; Célia A. da Silva; Francisco S. Guimarães; Marcela Bermúdez-Echeverry (175-181).
Systemic administration of nitric oxide synthase (NOS) inhibitors induces catalepsy in a dose-dependent manner in male Albino-Swiss mice. The objective of the present work was to investigate if similar effects occur in rats and if these effects are centrally mediated. The results showed that systemic administration of N G-nitro-l-arginine (l-NOARG, 40–160 mg/kg, i.p.), a non-selective NOS inhibitor, induced catalepsy in rats. Similar effects were found after intracerebroventricular (i.c.v.) injection of l-NOARG (50–200 nmol) or N G-nitro-l-arginine methylester (l-NAME, 100–200 nmol). The dose–response curve of the former compound, however, had an inverted U shape. The effect of l-NOARG (100 nmol, i.c.v.) was completely prevented by pre-treatment with l-arginine (300 nmol, i.c.v.) but not by d-arginine (300 nmol, i.c.v.). Intra-striatal injection of N G-monomethyl-l-arginine (l-NMMA, 100 nmol), 7-nitroindazole (7-NIO, 100 nmol), l-NOARG (25–100 nmol) or l-NAME (50–200 nmol) also induced catalepsy. Similar to i.c.v. administration, the latter two compounds produced bell-shaped dose–response curves. The cataleptic effect of intra-striatal administration of l-NAME (100 nmol) was reversed by local treatment with l-arginine (100 nmol). These results suggest that interference with the striatal formation of nitric oxide may induce significant motor effects in rats.
Keywords: Striatum; Motor control; Glutamate; Dopamine;

Effect of late treatment with γ-hydroxybutyrate on the histological and behavioral consequences of transient brain ischemia in the rat by Alessandra Ottani; Anna Valeria Vergoni; Sabrina Saltini; Chiara Mioni; Daniela Giuliani; Marta Bartiromo; Davide Zaffe; Annibale R Botticelli; Anna Ferrari; Alfio Bertolini; Susanna Genedani (183-191).
It has been previously described that γ-hydroxybutyrate (GHB) provides significant protection against transient global cerebral ischemia in the rat (four vessel occlusion model), when given 30 min before or 10 min after artery occlusion. Here, we show that in the same rat model, significant protection can also be obtained when treatment is started 2 h after the ischemic episode. In saline-treated animals, 30 min of global ischemia followed by reperfusion caused a massive loss of neurons in the hippocampal CA1 subfield (examined 63 days after the ischemic episode), and an impairment of sensory-motor performance (tested on the 51st and 63rd days after ischemia) and of spatial learning and memory (evaluated starting 46 days after the ischemic episode). Treatment with GHB—300 mg/kg intraperitoneally (i.p.) 2 h after the ischemia–reperfusion episode, followed by 100 mg/kg i.p. twice daily for the following 10 days—afforded a highly significant protection, against both histological damage and sensory-motor and learning-memory impairments. These data further suggest the possible therapeutic effectiveness of GHB in brain ischemia, and indicate that the underlying mechanism of action involves non-immediate steps of the ischemia-induced cascade of events.
Keywords: Cerebral ischemia; GHB (γ-hydroxybutyrate); Immunohistochemistry; (Rat); Sensory-motor test; Spatial learning;

Loss of amitriptyline analgesia in α2A-adrenoceptor deficient mice by Ümit Kazim Özdoğan; Janne Lähdesmäki; Heikki Mansikka; Mika Scheinin (193-196).
Tricyclic antidepressants have analgesic and sedative effects in addition to their antidepressive properties. We tested the acute analgesic and locomotor inhibitory effects of the tricyclic antidepressant amitriptyline and the α2-adrenoceptor agonist clonidine in wild-type control and in α2A-adrenoceptor knockout mice in hot-plate and tail-flick tests. Amitriptyline-induced analgesia was lost in α2A-adrenoceptor knockout mice. The locomotor inhibitory effect of amitriptyline was reduced, but not fully abolished in α2A-adrenoceptor knockout mice. Similar results were obtained with clonidine. We conclude that α2A-adrenoceptors appear to have a significant role in amitriptyline-induced acute analgesia in mice, and that α2A-adrenoceptors also participate in the sedative effects of amitriptyline.
Keywords: Locomotor activity; Analgesia; Sedation; Amitriptyline; α2A-Adrenoceptor; Clonidine; (Mouse);

Tonic inhibition by orphanin FQ/nociceptin of noradrenaline neurotransmission in the amygdala by Yukie Kawahara; Mayke B. Hesselink; Guus van Scharrenburg; Ben H.C. Westerink (197-200).
The present microdialysis study investigated whether nociceptin/orphanin FQ exerts a tonic inhibition of the release of noradrenaline in the basolateral nucleus of the amygdala in awake rats. The non-peptide competitive nociceptin/orphanin FQ (N/OFQ) peptide receptor antagonist J-113397 (20 mg/kg i.p.) induced an increase in the release of noradrenaline to about 150–200%. The increase was strongly suppressed by local infusion of an endogenous N/OFQ peptide receptor agonist, nociceptin/orphanin FQ (1 μM) via retrograde microdialysis, into the basolateral nucleus of the amygdala. Local infusion of nociceptin/orphanin FQ (1 μM) itself reduced noradrenaline release in the basolateral nucleus of the amygdala to about 70% of basal levels. These results indicate that a large part of basal release of noradrenaline in the basolateral nucleus of the amygdala is under tonic inhibitory control by endogenous nociceptin/orphanin FQ through the N/OFQ peptide receptors localized within the basolateral nucleus of the amygdala.
Keywords: J-113397; Microdialysis; Orphanin FQ; Nociceptin; Amygdala; (Rat);

There is a body of evidence implying the involvement of the central glutamatergic system in morphine dependence. In this study, we examined the effect of intracerebroventricular (i.c.v.) administration of a potent glutamate transporter inhibitor, dl-threo-β-benzyloxyaspartate (dl-TBOA), on acute morphine-induced antinociception, expression of somatic and negative affective components of morphine withdrawal, and acquisition of morphine-induced conditioned place preference in rats. I.c.v administration of dl-TBOA (10 nmol) to naive rats did not affect the acute antinociceptive effect of morphine. I.c.v. administration of dl-TBOA (10 nmol) to morphine-dependent rats significantly facilitated the expression of naloxone-precipitated somatic signs and conditioned place aversion. dl-TBOA (3 and 10 nmol) significantly facilitated acquisition of morphine-induced conditioned place preference. dl-TBOA itself produced neither conditioned place aversion nor place preference in naive rats. These results suggest that central glutamate transporters play inhibitory roles in the expression of somatic and negative affective components of morphine withdrawal and the reinforcing effect of morphine.
Keywords: Morphine dependence; Morphine withdrawal; Glutamate transporter; dl-threo-β-benzyloxyaspartate; Conditioned place aversion; Conditioned place preference;

Intravenous self-administration of abused solvents and anesthetics in mice by Elena A Blokhina; Olga A Dravolina; Anton Y Bespalov; Robert L Balster; Edwin E Zvartau (211-218).
Volatile organic solvents, fuels and anesthetics are subject to abuse. The aim of the present study was to evaluate i.v. self-administration of several of these chemicals in drug- and experiment-naive mice using a commercially available vehicle, intralipid. Two strains of mice (DBA/2 and Swiss) were allowed to self-administer toluene (0.0017–0.17 μmol/infusion), 1,1,1-trichloroethane (0.006–0.19 μmol/infusion), ethanol (0.32–1.6 μmol/infusion), cyclohexane (0.0017–0.052 μmol/infusion), propofol (0.01–0.53 μmol/infusion) and flurothyl (0.00042–0.072 μmol/infusion) or their vehicles during 30-min tests. During the test, each nose-poke of the master mouse resulted in a 1.88-μl i.v. infusion to the master mouse and a yoked control mouse. When the delivery line was loaded with a reinforcing drug solution, the number of nose-pokes of the master mice significantly exceeded that for yoked control mice. In the present experiments, significant differences in rates of nose-poking were observed between mice receiving response-contingent and response-noncontingent deliveries of ethanol and toluene in both strains of mice and of 1,1,1-trichloroethane in Swiss mice. These data suggest that the reinforcing effects of abused inhalants can be studied using i.v. self-administration procedures.
Keywords: Ethanol; Self-administration; (Mouse);

Mechanisms underlying endothelium-dependent flow increase in perfused rat mesenteric vascular bed by Hideyuki Fujioka; Kazuhide Ayajiki; Kazuya Shinozaki; Tomio Okamura (219-225).
The isolated rat mesenteric vasculature was perfused at constant pressures of 40, 80 or 120 mm Hg and the change in flow rate was measured. In the presence of phenylephrine, treatment with 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS) or N G-nitro-l-arginine (l-NA) significantly inhibited the pressure-dependent flow rate increase, but treatment with indomethacin or charybdotoxin plus apamin did not. Acetylcholine, bradykinin and ADP increased the flow rate, which had been markedly suppressed by CHAPS. At 80 mm Hg, the flow rate increase induced by these agonists was not affected by indomethacin plus l-NA, but was suppressed by subsequent treatment with charybdotoxin plus apamin. Changes in the perfusion pressure did not significantly affect the flow rate increases induced by the agonists. In conclusion, the opening of charybdotoxin plus apamin-sensitive Ca2+-dependent K+ channels may be mainly involved in the endothelium-dependent flow rate increase induced by the agonists, whereas nitric oxide (NO) may be responsible for the endothelium-dependent, pressure-induced flow rate increase.
Keywords: Constant-pressure perfusion; Mesenteric vascular bed; (Rat); Endothelium; NO (nitric oxide); K+ channel, Ca2+-dependent;

Blockade of β1- and desensitization of β2-adrenoceptors reduce isoprenaline-induced cardiac fibrosis by Fazia Brouri; Naima Hanoun; Odile Mediani; Françoise Saurini; Michel Hamon; Paul M Vanhoutte; Philippe Lechat (227-234).
The aim of the present study was to analyse the role of β1- and β2-adrenoceptors in the catecholamine-induced myocardial remodeling, especially the interstitial fibrosis. Wistar rats were subjected to a 2-week chronic isoprenaline administration (30 μg/kg/h). Rats received a concomitant treatment with the selective β1-adrenoceptor antagonist, bisoprolol (50 mg/kg/day p.o.) or were chronically pretreated with the selective β2-adrenoceptor agonist salbutamol (40 μg/kg/h) for 1 week to induce β2-adrenoceptor desensitization. The pretreatment with salbutamol induced a 59% down-regulation of left ventricular β2-adrenoceptors compared to control. The extent of the isoprenaline-induced left ventricular fibrosis was significantly reduced in both the bisoprolol and salbutamol groups compared with the control isoprenaline-treated group especially in the apical region (1.7±0.6% and 1.4±0.3% versus 6.0±1.3%, respectively, P<0.005). β1-adrenoceptor blockade and β2-adrenoceptors down-regulation provided similar protection against isoprenaline-induced cardiac interstitial fibrosis suggesting that both β-adrenoceptors are involved in such cardiac remodeling process.
Keywords: β-Adrenoceptor; β-Adrenoceptor antagonist; Catecholamine; Fibrosis;

We have shown previously that the concentration of Vasoactive Intestinal Peptide (VIP) in the heart is inversely correlated with the degree of fibrosis in a number of experimental models of early myocardial fibrosis. Vasopeptidase inhibition and angiotensin converting enzyme inhibition both decrease myocardial fibrosis. In this study, we sought to determine whether this myocardial protective effect might reflect increased VIP concentrations in the heart. We compared the effects of 4 weeks treatment of the vasopeptidase inhibitor omapatrilat and the angiotensin converting enzyme inhibitor enalapril on the degree of fibrosis and the concentration of VIP in the heart in salt sensitive hypertension induced by treatment with l-nitro-ω-methylarginine (l-NAME). Systolic blood pressure decreased in both treatment groups compared with control (omapatrilat P<0.005; enalapril P<0.001). Myocardial fibrosis was less for omapatrilat than control (P<0.0005) and enalapril (P<0.0005) groups. Myocardial VIP was greater in omapatrilat than in controls (P<0.005) and enalapril-treated rats (P<0.05). We conclude that vasopeptidase inhibition exerts a greater myocardial protective effect than angiotensin converting enzyme inhibition. Further, this myocardial protective effect is associated with increased VIP in the heart suggesting a pathogenetic role for VIP depletion in the development of fibrosis in the heart.
Keywords: Myocardial fibrosis; VIP (Vasopeptidase Intestinal Peptide); Vasopeptidase inhibition; Angiotensin converting enzyme; Neutral endopeptidase; Cardiac failure;

Angiotensin II stimulates superoxide production via both angiotensin AT1A and AT1B receptors in mouse aorta and heart by Matlubur Rahman; Shoji Kimura; Akira Nishiyama; Hirofumi Hitomi; Guoxing Zhang; Youichi Abe (243-249).
The present study was conducted to determine the roles of angiotensin AT1A and AT1B receptors in angiotensin II-induced superoxide anion production in mouse aorta and heart. Superoxide anion production in aorta was determined by the lucigenin chemiluminescence method, and thiobarbituric acid reactive substances in heart tissues were measured by biochemical assay. The basal production rate of superoxide anion in aorta of wild type (WT) mice was significantly higher than in angiotensin AT1A receptor knockout (AT1A KO) mice. Angiotensin II (2.8 mg/kg/day, s.c. for 13 days) significantly increased superoxide anion production in aorta of both AT1A KO and WT mice. However, the superoxide anion production rate in aorta of angiotensin II-infused AT1A KO mice was significantly lower than in angiotensin II-infused WT mice. Valsartan (40 mg/kg/day in drinking water) prevented angiotensin II-induced superoxide anion production in aorta of WT and AT1A KO mice. Similarly, thiobarbituric acid reactive substances levels in heart tissues of angiotensin II-treated WT and AT1A KO mice were significantly higher than those in vehicle-infused WT and AT1A KO mice, respectively. Valsartan prevented angiotensin II-induced increases of thiobarbituric acid reactive substances levels in heart tissue of both WT and AT1A KO mice. These results indicate that angiotensin II stimulates superoxide anion production via both angiotensin AT1A and AT1B receptors, and that angiotensin AT1A receptors appear to play a predominant role in angiotensin II-induced superoxide anion production in mouse aorta and heart.
Keywords: Angiotensin II; Superoxide anion; Angiotensin AT1A receptor; Angiotensin AT1B receptor; Aorta; Heart;

Our previous studies have demonstrated that acute ethanol administration counteracts imidazoline I1 receptor but not α2-adrenoceptor-mediated hypotension in spontaneously hypertensive rats (SHR). In the present study, we investigated the effect of chronic ethanol administration on hypotensive responses elicited by acute administration of selective imidazoline I1 receptor (rilmenidine) or α2-adrenoceptor (α-methyldopa) agonist along with ethanol effects on: (i) locomotor activity and (ii) time-domain indices of variability in blood pressure (standard deviation of mean arterial pressure) and heart rate (standard deviation of beat-to-beat intervals and root mean square of successive differences in R–R intervals). Hemodynamic and locomotor responses elicited by rilmenidine or α-methyldopa were assessed in radiotelemetered ethanol-fed (2.5% or 5% w/v, 12 week) and control SHR. In control SHR, i.p. rilmenidine (600 μg/kg) or α-methyldopa (100 mg/kg) significantly reduced blood pressure. Rilmenidine had no effect on heart rate whereas α-methyldopa elicited a biphasic response (tachycardia followed by bradycardia). Blood pressure and heart rate oscillations were also reduced by both drugs, which may conform to sympathoinhibition. The hypotensive effect of rilmenidine or α-methyldopa was significantly attenuated by ethanol feeding (2.5% or 5%) in a concentration-dependent manner. In addition, ethanol attenuated α-methyldopa-evoked reduction in heart rate, but not blood pressure, variability in marked contrast to attenuating rilmenidine-evoked reductions in blood pressure, but not heart rate, variability. These findings demonstrate that, unlike its acute effects, chronic ethanol attenuates both imidazoline I1 receptor and α2-adrenoceptor-mediated hypotension whereas its effect on hemodynamic variability depended on the nature of the hypotensive stimulus.
Keywords: Ethanol; Rilmenidine; α-Methyldopa; Hypotension; Hemodynamic variability; Time-domain analysis;

Rho-kinase expression and its contribution to the control of perfusion pressure in the isolated rat mesenteric vascular bed by Kansu Büyükafşar; Onur Arıkan; Mustafa Ark; Ata Seçilmiş; İsmail Ün; Ergin Şingirik (263-268).
Rho-kinase expression was investigated in the rat mesenteric artery and the effects of its inhibitors, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632) and fasudil (HA-1077), were examined on the increase in perfusion pressure induced by two different receptor agonists, namely the α-adrenoceptor agonist, phenylephrine and, the endothelin ETA and ETB receptor agonist, endothelin-1. Y-27632 and fasudil produced a concentration-dependent decrease in perfusion pressure. There was no difference between the concentration–response lines of these two inhibitors. The maximum decrease in the perfusion pressure induced by 10−5 M Y-27632 was 85.8±3.7% when the tone was increased by phenylephrine. However, it was 48.1±5.4% (P<0.001) when the perfusion pressure was elevated by endothelin-1. Saponin perfusion (100 mg l−1, for 10 min), which abolished acetylcholine-induced relaxation, did not significantly modify the Y-27632-elicited relaxation. Western blot analysis revealed that rat mesenteric artery expresses Rho-kinase protein with a molecular weight of approximately 160 kDa. These results show that Rho-kinase enzyme is expressed in rat mesenteric artery and that it contributes to the control of vascular resistance. Moreover, endothelium removal had no marked effect on the vasodilatation induced by Y-27632. In addition, the endothelin-1-induced vasoconstriction was more resistant to the Rho-kinase inhibitors than was that induced by phenylephrine, probably because excitatory endothelin receptors are associated with this signal transduction pathway at a different level from that of α-adrenoceptors.
Keywords: Endothelin-1; Mesenteric artery; Rho-kinase; Saponin; Y-27632;

In the present study, we investigated whether copper ions are involved in the decomposition of endogenous S-nitrosothiols by ultraviolet (UV) light irradiation in the mouse gastric fundus. The effects of copper ions and chelators of copper(I) and copper(II), neocuproine and cuprozine, respectively, were studied on relaxations in response to S-nitrosoglutathione, UV irradiation, exogenous nitric oxide (NO), added as acidified NaNO2, and isoproterenol. UV irradiation of smooth muscle strips induced fast and transient relaxations which were mimicked by exogenous NO. S-Nitrosoglutathione induced concentration-dependent relaxations, which were more sustained than those elicited by UV irradiation or NO. CuCl2 did not affect relaxations elicited by UV irradiation, exogenous NO and isoproterenol but enhanced those elicited by S-nitrosoglutathione. CuSO4 but not FeSO4 mimicked the effect of CuCl2 on relaxations elicited by S-nitrosoglutathione. Neocuproine, the copper(I)-specific chelator, inhibited both photorelaxation and S-nitrosoglutathione-induced relaxation, and this inhibition was prevented by CuCl2. In contrast, neocuproine significantly enhanced the relaxations in response to exogenous NO, without affecting the relaxations elicited by isoproterenol. Cuprizone, a specific copper(II) chelator, did not affect relaxations in response to S-nitrosoglutathione, UV irradiation, exogenous NO and isoproterenol. These results suggest that copper(I) and not copper(II) may play a role in the NO release evoked by the light-induced decomposition of endogenous S-nitrosothiols in mouse gastric fundus. Also, results with the selective copper(I) chelator, neocuproine, confirmed our recent findings that the endogenous “store” of S-nitrosoglutathione, rather than NO, acts as an intermediate in photorelaxation of the mouse gastric fundus, and that photorelaxation may be a suitable model to elucidate the nature of endogenous S-nitrosothiols.
Keywords: Gastric fundus; Photorelaxation; Nitric oxide (NO); S-nitrosoglutathione; CuCl2; Neocuproine; Cuprizone;

Dexamethasone delays ulcer healing by inhibition of angiogenesis in rat stomachs by Jiing C Luo; Vivian Y Shin; Edgar S.L Liu; Yi N Ye; William K.K Wu; Wallace H.L So; Full Y Chang; Chi H Cho (275-281).
Using the non-ulcerogenic doses of dexamethasone, we explored the action of glucocorticoids on ulcer healing and its relationship with angiogenic factors in the gastric mucosa. We applied dexamethasone (0.1 or 0.2 mg/kg/day) intragastrically in rats with acetic acid-induced gastric ulcer. The mucosal prostaglandin E2 level and protein expressions of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) at the ulcer margin were determined. Ulcer induction significantly increased protein expressions of bFGF, VEGF, and prostaglandin E2 level at the ulcer margin together with angiogenesis at the ulcer margin and base. The non-ulcerogenic doses of dexamethasone inhibited angiogenesis at the ulcer margin and ulcer base and delayed ulcer healing. These were associated with a significant decrease of prostaglandin E2 level and VEGF expression, but not the bFGF expression. Supplementation with prostaglandin E2 attenuated the inhibitory action of dexamethasone on VEGF expression and reversed the adverse effects of dexamethasone on angiogenesis and ulcer healing, without influencing bFGF expression. We concluded that dexamethasone given at non-ulcerogenic doses could decrease angiogenesis and delay acetic acid-induced ulcer healing; these actions were at least, in part, due to depletion of prostaglandin E2 level followed by down-regulation of VEGF at the ulcer margin of the stomach.
Keywords: Angiogenesis; bFGF (basic fibroblast growth factor); Dexamethasone; Gastric ulcer; Prostaglandin E2; VEGF (vascular endothelial growth factor);

T-0156 (2-(2-methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2-yl)methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride) is a newly synthesized phosphodiesterase type 5 inhibitor, and its potency and selectivity are higher than those of sildenafil in an enzyme assay. In the present study with anesthetized dogs, we examined the effects of intravenous T-0156 or sildenafil on the pelvic nerve stimulation-induced penile tumescence and light-adapted flicker stimulation-induced electroretinogram, parameters of which are reported to be indicators for inhibition of phosphodiesterase type 5 and type 6, respectively. Both compounds potentiated the penile tumescence in a dose-dependent manner. T-0156 at 10 μg/kg and sildenafil at 100 μg/kg showed almost the same potentiating percentage (181.5±31.1% and 190.0±37.9%) in spite of the plasma concentration of T-0156 being about five times lower than that of sildenafil (16.7±1.6 and 78.8±5.3 ng/ml), indicating that the effect of T-0156 on tumescence is more potent than that of sildenafil. While the high dose of T-0156 (1000 μg/kg) reduced the amplitude and increased the latency of the electroretinogram positive wave, the effects of T-0156 were conversely weaker than those of sildenafil (reduction of amplitude; T-0156: 41.1±8.0%, sildenafil: 71.7±3.9%, increase of latency; T-0156: 3.9±0.6%, sildenafil: 14.5±1.4%, at 1000 μg/kg). These results clearly showed the difference in the properties of T-0156 and sildenafil in pharmacological studies with anesthetized dogs, and the difference appeared to correspond with their inhibitory potencies for phosphodiesterase type 5 and type 6. It was concluded that T-0156 would be a useful pharmacological tool as a potent and highly selective phosphodiesterase type 5 inhibitor.
Keywords: T-0156; Sildenafil; Phosphodiesterase type 5 inhibitor; Phosphodiesterase type 6; Penile tumescence; Electroretinogram;

Comparison of effects of cyclooxygenase inhibitors on myometrial contraction and constriction of ductus arteriosus in rats by Baris Karadas; Tijen Kaya; Ihsan Bagcivan; Celal Kaloglu; Tevfik Guvenal; Ali Cetin; Ahmet Serdar Soydan (289-298).
The aim of this study was to compare the tocolytic effect of a selective cylooxygenase-2 inhibitor, DFU (5,5-dimethyl-3(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone), indomethacin and nimesulide on myometrial strips isolated from rats in both lipopolysaccharide-induced preterm labour and term labour. We also compared the constrictor effects of DFU and indomethacin on the fetal ductus arteriosus. Myometrial strips were obtained from preterm and term labour Wistar albino rats and were mounted in organ baths for the recording of isometric tension. DFU, nimesulide and indomethacin significantly inhibited KCl-, oxytocin-, prostaglandin E2- and prostaglandin F-stimulated contractions of myometrial strips isolated from rats in preterm and term labour. The E max value of indomethacin was significantly lower than those for DFU and nimesulide (P<0.05), with no change-log (10) EC50 values. There was no significant difference between in -log (10) EC50 and E max values of DFU and nimesulide for any of the tissues (P>0.05). In addition, there was no significant difference between −log (10) EC50 and E max values for each of these three agents in myometrial tissues isolated from rats in preterm and term labour (P>0.05). Fetal ductus arteriosus was significantly constricted by DFU (10 or 100 mg/kg) in preterm and term rats, although DFU (10 or 100 mg/kg)-induced constriction ratios were significantly lower than those for indomethacin (P<0.05). These data demonstrate that DFU, a specific cyclooxygenase-2 inhibitor, could be considered as a new therapeutic agent for preterm labour. However, careful attention should be given to constriction of the fetal ductus arteriosus.
Keywords: DFU; Nimesulide; Indomethacin; Myometrium; Ductus arteriosus;

Effects of TAK-802, a novel acetylcholinesterase inhibitor, on distension-induced rhythmic bladder contractions in rats and guinea pigs by Hiroshi Nagabukuro; Satoshi Okanishi; Shigemitsu Imai; Yuji Ishichi; Yuji Ishihara; Takayuki Doi (299-305).
In the present study, we investigated the effects of 8-[3-[1-[(3-fluorophenyl)methyl]-4-piperidinyl]-1-oxopropyl]-1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]quinolin-4-one (TAK-802), a novel acetylcholinesterase inhibitor, on distension-induced rhythmic bladder contractions in urethane-anesthetized rats and guinea pigs. TAK-802 potently inhibited human-erythrocyte-derived acetylcholinesterase activity with an IC50 value of 1.5 nM, which represented a potency 30 and 250 times greater than that of the two carbamate acetylcholinesterase inhibitors, neostigimine and distigmine, respectively. Unlike the carbamate acetylcholinesterase inhibitors, TAK-802 exhibits high selectivity for acetylcholinesterase inhibition over butyrylcholinesterase inhibition. In an assay conducted to measure the muscarinic and nicotinic actions, TAK-802 was found to exhibit higher selectivity for muscarinic actions over nicotinic actions in comparison to distigmine. Both TAK-802 and distigmine increased isovolumetric bladder contractions in rats and guinea pigs in a dose-dependent manner, with a minimum effective dose (MED) of 0.01 and 0.03 mg/kg i.v., respectively, in rats, and 0.01 and 0.1 mg/kg i.v., respectively, in guinea pigs. The effects of both the drugs were completely abolished by atropine. These results suggest that TAK-802 and other acetylcholinesterase inhibitors can effectively increase reflex bladder contractions by increasing the efficacy of acetylcholine released by nerve impulses. On the other hand, bethanechol, a muscarinic agonist, markedly changed the pattern of distension-induced bladder contractions when administered at the dose of 1 mg/kg i.v., and it did not necessarily augment well-coordinated bladder contractions. Thus, considering that it has some selectivity for muscarinic action, TAK-802 might be expected to be useful in the treatment of voiding dysfunction caused by impaired detrusor contractility.
Keywords: TAK-802; Acetylcholinesterase inhibitor; Bladder; Micturition; (Rat); (Guinea pig);

α-Helical structure in the C-terminus of vasoactive intestinal peptide: functional and structural consequences by Satomi Onoue; Asami Matsumoto; Yumiko Nagano; Keiichi Ohshima; Yuki Ohmori; Shizuo Yamada; Ryohei Kimura; Takehiko Yajima; Kazuhisa Kashimoto (307-316).
The conformational properties of vasoactive intestinal peptide (VIP) include the N-terminal randomized structure and the C-terminal long α-helical structure. We have previously observed that the N-terminal random coil structure plays a crucial role in the receptor-selectivity. Here, to clarify how the formation of the α-helix plays a role in its biological functions, we chemically synthesized VIP analogues modified at the C-terminus, mid-chain, and N-terminus of the α-helical region, and evaluated the relationship between their α-helical contents and their biological activities including relaxant effects on murine stomach and receptor-binding activities. VIP and VIP-(1–27) showed equipotent biological activities with 48% and 50% α-helical content, respectively, each of which corresponds to 14 amino acid residues. VIP-(1–26) was 10% and threefold less potent in relaxant and binding activities, respectively, compared with VIP, and its 49% α-helical content resulted in 13 residues involved in the α-helix. Further truncation from 25 to 21 resulted in decrease in the α-helical content from 43% to 29%, corresponding residues from 11 to 6, the relaxant activity from 72% to 4%, and the affinity to the membrane from 60-fold to over 104-fold less potency. In addition, disruption of the mid-chain and the N-terminus in the α-helical stretch by oxidation of Met17 and deletion of Thr11 also inhibited biological activities. These findings suggest that the presence of α-helical structure forming in 14 amino acid residues between position 10 and 23 in VIP is essential to its biological functions and the C-terminal amino acid residues between position 24 and 27 are requisite for this α-helical formation.
Keywords: Binding activity; Chemical synthesis; CD (Circular dichroism); Helical structure; Relaxant activity; VIP (vasoactive intestinal peptide) derivative;

Thromboxane A2 (TP) receptor in the non-pregnant porcine myometrium and its role in regulation of spontaneous contractile activity by Jinshan Cao; Akira Wakatsuki; Munenori Yoshida; Takio Kitazawa; Tetsuro Taneike (317-327).
Although there are species-related differences in uterine prostanoid receptor subtypes, functional prostanoid receptors in the porcine uterus are similar with those in the human uterus (FP, TP, EP1, EP2, EP3, DP and IP) except for the TP receptor. These similarities promoted us to determine whether TP receptors are present in the non-pregnant porcine uterus. For this purpose, the effects of TP receptor agonists and antagonists were investigated by a contraction study and by a binding study. 9,11-Dideoxy-9α, 11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619, 1 nM–10 μM), a stable thromboxane A2 mimetic, caused tetrodotoxin-resistant contraction in both longitudinal and circular muscles of the uterine cornu. The pEC50 value in the longitudinal muscle (6.69) was lower than that in the circular muscle (7.62), but the maximum response in the longitudinal muscle was two times larger than that in the circular muscle. The longitudinal and circular muscles of other regions (corpus and cervix) also responded to U46619, and region-related difference in contractile responses was observed only in the longitudinal muscles. 4(Z)-6-(2-o-Chlorophenyl-4-o-hydroxyphenyl-1,3-dioxan-cis-5-yl) hexenoic acid (ICI192605) and 7-[3-[[2-[(phenylamino)carbonyl] hydrazino]methyl]7-oxabicyclo[2.2.1]hept-2-yl]-,[1S-[1α,2α(Z),3α,4α]]-]5-heptenoic acid (SQ29548) inhibited the contractile responses to U46619 competitively. The longitudinal and circular muscles in the cornu contained a single class of [3H]SQ29548 binding site with similar K d values (30 nM), but B max in the circular muscle (90.9±8.6 fmol/mg protein) was two times higher than that in the longitudinal muscle (58.2±8.6 fmol/mg protein). The ranking order of competition by TP receptor agonists and antagonists (with pK i values in parentheses) was [1S-[1,2(Z),3(1E,3S*),4]]-7-[3-[3-Hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP, 7.70)>SQ29548 (7.39)>7-[3-(3-Hydroxy-1-octenyl)bicycle[3.1.1]hept-2-yl]-,[2S-[2α(Z),3β(1E,3R*)]]-5-heptenoic acid (CTA2, 6.55)>7-[3-(3-hydroxy-1-octenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-yl-,[1S-[1α,2β(Z),3α(1E,3R*),5α]]-5-heptenoic acid (PTA2, 6.50)>U46619 (6.41)>7-[5-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1] hept-6yl]-,[1S-[1α,4α,5α(1E,3R*),6β(Z)]]-5-heptenoic acid (U44069, 6.34), and this order is consistent with current TP receptors. Treatment with indomethacin (100 nM) and N-tert-butyl-N¢-[(2-cyclohexylamino-5-nitrobenzene) sulfonyl] urea (BM-531, 10 μM) inhibited the spontaneous contractile activities of both longitudinal and circular muscles. The present results indicate that contractile TP receptors are present in the non-pregnant porcine uterus. Therefore, the prostanoid receptor subtypes that exist in the porcine uterus (TP, IP, DP, FP, EP1, EP2 and EP3) are the same as those present in the human uterus. The distribution of TP receptors in the porcine uterus differed depending on the type of myometrium (longitudinal and circular muscles) and region of the uterus. The endogenous thromboxane A2-TP receptor pathway is thought to play a physiological role in regulation of spontaneous contractile activity in the porcine uterus.
Keywords: U46619; Prostanoid TP receptor; Uterus; Thromboxane A2; Contractility;

Antitussive activity of the tachykinin NK1 receptor antagonist, CP-99994, in dogs by Richard W. Chapman; Aileen House; Fei Liu; Chander Celly; Hong Mei; John A. Hey (329-332).
CP-99994 [(+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine] is a selective tachykinin NK1 receptor antagonist that inhibits cough in guinea pigs and cats. This study examined the antitussive effects of CP-99994 in dogs produced by mechanical stimulation of the intrathoracic trachea. CP-99994 (10 mg/kg, p.o.) inhibited cough frequency by 52% at 2 h, 31% at 6 h and by 21% at 24 h. Cough amplitude was inhibited by 45% at 6 h but unchanged at 2 and 24 h after CP-99994. Plasma levels of CP-99994 were highest at 2 h (75±26 ng/ml) and fell to 22±6 ng/ml at 6 h. These results demonstrate antitussive activity of CP-99994 in dogs at a dose proven to antagonize tachykinin NK1 receptors in this species.
Keywords: Cough; Tachykinin NK1 receptor; CP-99994; (Dog);

Discovery of a novel protein tyrosine phosphatase-1B inhibitor, KR61639: potential development as an antihyperglycemic agent by Hyae Gyeong Cheon; Sun-Mee Kim; Sung-Don Yang; Jae Du Ha; Joong-Kwon Choi (333-339).
Protein tyrosine phosphatase-1B (PTP-1B), a negative regulator of insulin signaling, may be an attractive therapeutic target for type 2 diabetes mellitus. High throughput screening (HTS) for PTP-1B inhibitors using compounds from the Korea Chemical Bank identified several hits (active compounds). Among them, a hit with 1,2-naphthoquinone scaffold was chosen for lead development. KR61639, {4-[1-(1H-indol-3-yl)-3,4-dioxo-3,4-dihydro-naphthalen-2-ylmethyl]-phenoxy}-acetic acid tert-butyl ester, inhibited human recombinant PTP-1B with an IC50 value of 0.65 μM in a noncompetitive manner. KR61639 showed modest selectivity over several phosphatases and increased insulin-stimulated glycogen synthesis in HepG2 cells and stimulated 2-deoxyglucose uptake in 3T3/L1 adipocytes. In addition, in vivo study using ob/ob mouse demonstrated that KR61639 exerted a hypoglycemic action when given orally. Thus, KR61639 may be a good starting point for lead optimization in developing a novel antidiabetic agent.
Keywords: PTP-1B (protein tyrosine phosphatase-1B); 1,2-Naphthoquinone; Deoxyglucose uptake; Glycogen incorporation; ob/ob mouse;

Mutation S363A in the human δ-opioid receptor selectively reduces down-regulation by a peptide agonist by Edita Navratilova; Eva V. Varga; Dagmar Stropova; Janelle C. Jambrosic; William R. Roeske; Henry I. Yamamura (341-343).
Chemically distinct opioid agonists have different abilities to down-regulate opioid receptors. The present study investigated the role of Ser363 in human δ-opioid receptor down-regulation by a δ-selective peptide- and non-peptide agonist. Cyclic[d-Pen2,d-Pen5]enkephalin (DPDPE)-mediated down-regulation was significantly attenuated by a S363A mutation. In contrast, this mutation had no effect on down-regulation by (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide (SNC80). These results demonstrate that the molecular mechanism of the human δ-opioid receptor down-regulation is agonist-specific.
Keywords: δ-Opioid receptor; Trafficking, agonist-directed; Down-regulation;

Author index (347-349).

Keyword index (351-357).