European Journal of Pharmacology (v.408, #2)
Loreclezole as a simple functional marker for homomeric ρ type GABAC receptors by Urs Thomet; Roland Baur; Robert H Dodd; Erwin Sigel (R1-R2).
GABAC receptors are expressed in the whole brain, but predominantly in the retina. They can be identified by their unique pharmacology. The establishment of the entire pharmacology is, however, quite tedious. We show here that loreclezole dose dependently inhibits ionic currents elicited by GABA (γ-aminobutyric acid) with an IC50 of about 0.5 μM in homomeric ρ1 GABAC receptors expressed in Xenopus oocytes. Thus, loreclezole may constitute a functional marker for these receptors.
Keywords: GABA (γ-aminobutyric acid); GABAC receptor; ρ1 subunit; Loreclezole;
Effect of betulinic acid on intracellular-free Ca2+ levels in Madin Darby canine kidney cells by Kang-Ju Chou; Hua-Chang Fang; Hsiao-Min Chung; Jin-Shiung Cheng; Kam-Chung Lee; Li-Ling Tseng; Kwong-Yui Tang; Chung-Ren Jan (99-106).
The effect of betulinic acid, an anti-tumor and apoptosis-inducing natural product, on intracellular-free levels of Ca2+ ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was examined by using fura-2 as a Ca2+ dye. Betulinic acid caused significant increases in [Ca2+]i concentration dependently between 25 and 500 nM with an EC50 of 100 nM. The [Ca2+]i signal was composed of an initial gradual rise and a plateau. The response was decreased by removal of extracellular Ca2+ by 45±10%. In Ca2+-free medium, pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) abolished 250 μM betulinic acid-induced [Ca2+]i increases. Conversely, pretreatment with betulinic acid only partly inhibited thapsigargin-induced [Ca2+]i increases. Addition of 3 mM Ca2+ induced a [Ca2+]i increase after pretreatment with 250 nM betulinic acid in Ca2+-free medium for 5 min. This [Ca2+]i increase was not altered by the addition of 20 μM SKF96365 and 10 μM econazole. Inhibiting inositol 1,4,5-trisphosphate formation with the phospholipase C inhibitor U73122 (2 μM) abolished 250 nM betulinic acid-induced Ca2+ release. Pretreatment with 10 μM La3+ inhibited 250 nM betulinic acid-induced [Ca2+]i increases by 85±3%; whereas 10 μM of verapamil, nifedipine and diltiazem had no effect. In Ca2+ medium, pretreatment with 2.5 nM betulinic aid for 260 s potentiated 10 μM ATP and 1 μM thapsigargin-induced [Ca2+]i increases by 33±3% and 45±3%, respectively. Trypan blue exclusion revealed that acute exposure of 250 nM betulinic acid for 2–30 min decreased cell viability by 6±2%, which could be prevented by pretreatment with 2 μM U731222. Together, the results suggest that betulinic acid induced significant [Ca2+]i increases in MDCK cells in a concentration-dependent manner, and also induced mild cell death. The [Ca2+]i signal was contributed by an inositol 1,4,5-trisphosphate-dependent release of intracellular Ca2+ from thapsigargin-sensitive stores, and by inducing Ca2+ entry from extracellular medium in a La3+-sensitive manner.
Keywords: Betulinic acid; Ca2+; Ca2+ store; Fura-2; MDCK cell; Thapsigargin;
Phosphorylation of protein phosphatase-1 inhibitors, inhibitor-1 and DARPP-32, in renal medulla by Emi Higuchi; Akinori Nishi; Hideho Higashi; Yuhei Ito; Hirohisa Kato (107-116).
Inhibitor-1 and DARPP-32 (dopamine and cAMP-regulated phosphoprotein, Mr 32 kDa) are each phosphorylated by cAMP-dependent protein kinase, resulting in their conversion to potent inhibitors of protein phosphatase-1. Protein phosphatase-1 is involved in the regulation of Na+ reabsorption from renal tubule by modulating the activity of Na+,K+-ATPase. In this study, we have investigated the regulation of inhibitor-1 and DARPP-32 phosphorylation in slices of renal medulla. Activation of cAMP-dependent protein kinase by forskolin and 8-bromo-cAMP increased the level of phosphorylated inhibitor-1. Okadaic acid (1 μM), used to inhibit protein phosphatase-2A, increased the level of phosphorylated inhibitor-1, but cyclosporin A had no effect. DARPP-32, like inhibitor-1, was phosphorylated by cAMP-dependent protein kinase and dephosphorylated only by protein phosphatase-2A. These data demonstrate that the phosphorylation of inhibitor-1 and DARPP-32 is regulated by the balance of phosphorylation by cAMP-dependent protein kinase and dephosphorylation by protein phosphatase-2A in renal medulla. Furthermore, the phosphorylation step is regulated by pharmacological stimuli such as activation of β1-adrenoceptors and dopamine D1 receptors.
Keywords: cAMP-dependent protein kinase; Adrenoceptor; Dopamine; Loop of Henle; (Mouse);
Evidence that the σ1 receptor is not directly coupled to G proteins by Weimin Hong; Linda L Werling (117-125).
Sigma (σ) receptors have been implicated in psychosis, cognition, neuroprotection, and locomotion in the central nervous system. The signal transduction mechanisms for σ receptors have not been fully elucidated. In this study, we examined the possible coupling between σ1 receptors and heterotrimeric guanine nucleotide-binding proteins (G proteins) in rodent brain. In σ1 receptor-rich cerebellar membrane preparations, the competitive binding curves of two σ1 agonists, (+)pentazocine and 1S,2R-(−)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)cyclohexylamine (BD737), were unaffected by the addition of 10 μM guanosine-5′-O-(γ-thio)-triphosphate (GTPγS). Neither (+)pentazocine (1–100 μM) nor BD737 (0.01–10 μM) stimulated GTPase activities significantly above basal levels in agonist-stimulated GTPase activity assays in cerebellar membranes. Furthermore, when using the method of agonist-stimulated [35S]GTPγS binding as assessed by autoradiography, we did not observe significant stimulation of [35S]GTPγS binding in rat brain sections by either (+)pentazocine or BD737. The above results demonstrate that the σ1 receptor is not likely be directly coupled to G proteins.
Keywords: σ Receptor; (+)Pentazocine; Haloperidol; Cerebellum; GTPase; [35S]GTPγS autoradiography;
Astroglial cell death induced by excessive influx of sodium ions by Shinichi Takahashi; Mamoru Shibata; Jun Gotoh; Yasuo Fukuuchi (127-135).
Na+ influx has been implicated to play an important role in the mechanisms of neuronal cell damage under ischemia as well as in neurodegenerative disorders. Thus far, however, the effects of Na+ influx on astrocytic damage have not been studied extensively. In the present study, we have examined the effects of Na+ influx induced by veratridine (Na+ channel opener), monensin (Na+ ionophore), and glutamate (co-transportation with Na+) on rat cultured astroglial damage. Cells were incubated with bicarbonate buffer with 25 mM glucose containing either 100 μM veratridine, 10 μM monensin, or 1 mM glutamate with or without 1 mM ouabain for 20 h. Cellular damage was evaluated quantitatively by lactate dehydrogenase (LDH) release or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Veratridine, monensin, or glutamate alone did not induce significant astroglial damage. Veratridine and monensin co-incubated with ouabain, which inhibits active extrusion of Na+ by Na+,K+-ATPase, thereby enhances intracellular Na+ accumulation, caused significant cell death (P<0.001, approximately 50% cell damage), whereas glutamate did not. Na+-free solution substituted by choline (impermeable cation) attenuated cell damage induced by veratridine and monensin markedly, while Li+ substitution (permeable cation) rather exacerbated. Nifedipine (100 μM), a blocker of l-type Ca2+ channel, reduced veratridine-induced glial damage by 50%. Neither bepridil nor benzamil, a blocker of Na+–Ca2+ exchanger, had any protection. Cyclosporin A (1 or 10 μM), an inhibitor of mitochondrial permeability transition or 10 μM N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethyl ketone (zVAD-fmk), which inhibits a broad range of caspases, did not show protective effects.
Keywords: Astrocyte; Ca2+; LDH (lactate dehydrogenase); Mitochondrion; MTT; Na+,K+-ATPase; Ouabain;
Inhibition of inducible nitric oxide synthase gene expression by indomethacin or ibuprofen in β-amyloid protein-stimulated J774 cells by Osamu Ogawa; Hiroyuki Umegaki; Daigo Sumi; Toshio Hayashi; Akira Nakamura; Navin Kumar Thakur; Juri Yoshimura; Hidetoshi Endo; Akihisa Iguchi (137-141).
Recent studies show that a mononuclear phagocyte lineage, including microglia, plays a possible role in the pathogenesis of Alzheimer's disease through nitric oxide (NO)-mediated neurotoxicity. Epidemiological studies show that nonsteroidal anti-inflammatory drugs (NSAIDs) have a protective effect against Alzheimer's disease. Based on these observations, it has been hypothesized that an anti-Alzheimer's disease effect of NSAIDs could result from the inhibition of NO synthesis. We report here that indomethacin or ibuprofen dose-dependently reduce β-amyloid protein and interferon-γ-induced NO production, accompanied by an inhibition of inducible nitric oxide synthase mRNA expression in J774 cells, a murine macrophage cell line. Aspirin, however, does not produce such an effect, suggesting that the cyclooxygenases pathway is not involved in the inhibitory effects of NSAIDs on β-amyloid protein and interferon-γ-induced NO production in J774 cells.
Keywords: Alzheimer's disease; Microglia; NSAIDs; Aspirin; Interferon-γ;
Potentiation of formalin-evoked adenosine release by an adenosine kinase inhibitor and an adenosine deaminase inhibitor in the rat hind paw: a microdialysis study by Xue Jun Liu; Thomas D White; Jana Sawynok (143-152).
The present study examined the effects of local subcutaneous administration of formalin on adenosine release from the rat hind paw, and the effects of inhibitors of adenosine metabolism on such release. Microdialysis probes were inserted into the subcutaneous tissue of the plantar surface of rat hind paws. Samples were collected every 10 min at a perfusion rate of 2 μl/min and high performance liquid chromatography was used to measure adenosine levels. At lower concentrations of formalin (0.5–2.5%), a significant increase in adenosine levels was observed in the first 10 min after formalin injection, while at the highest concentration of formalin (5%), the increase in adenosine release was observed over 60 min. Co-administration of the adenosine kinase inhibitor 5′-amino-5′-deoxyadenosine (100 nmol) with formalin, significantly increased adenosine release evoked by 0.5–1.5% formalin, but did not produce a further enhancement of release evoked by 5% formalin. The adenosine deaminase inhibitor 2′-deoxycoformycin (100 nmol) significantly increased adenosine levels at 5% formalin but had no effect at lower concentrations of formalin. In confirmation of previous studies, subcutaneous injection of formalin (0.5–5%) produced a characteristic biphasic concentration-related expression of nociceptive behaviours and an increase in paw volume. This study directly demonstrates that formalin can evoke a concentration-dependent local release of adenosine from the rat hind paw. The ability of an adenosine kinase inhibitor and an adenosine deaminase inhibitor to modulate this release is dependent on substrate adenosine concentrations.
Keywords: Adenosine; Adenosine kinase; Adenosine deaminase; Formalin; Microdialysis; Pain; Inflammation;
κ-Opioid receptor-mediated analgesia: hotplate temperature and sex differences by Frans van Haaren; Sara Scott; Laura B Tucker (153-159).
The present experiment was designed to investigate the dose–effect and time–effect functions of the κ−opioid receptor agonist [5α,7α,8β)-(−)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]benzeneacetamide] (U69,593) in intact and gonadectomized male and female rats as a function of hotplate temperature (45°C or 51°C). At 45°C baseline lick latencies were longer in female rats than in male rats. Lick latencies increased dose-dependently in all subjects except intact female rats. U69,593 increased lick latencies as a function of time since its administration in all subjects. At 51°C baseline lick latencies did not differ between groups and they increased dose-dependently in all subjects. The effectiveness of U69,593 decreased as a function of time since its administration, but not in castrated male rats. These observations suggest that gonadal hormones could play a role in modulating the behavioral effects of U69,593 when subjects are tested at different hotplate temperatures.
Keywords: Opioid analgesia; U69,593; Hotplate; Sex differences; Lick latency; (Rat);
Elevated circulating levels of anandamide after administration of the transport inhibitor, AM404 by Andrea Giuffrida; Fernando Rodriguez de Fonseca; Felice Nava; Patrick Loubet-Lescoulié; Daniele Piomelli (161-168).
The biological actions of the endogenous cannabinoid anandamide are terminated by carrier-mediated transport into neurons and astrocytes, followed by enzymatic hydrolysis. Anandamide transport is inhibited by the compound N-(4-hydroxyphenyl)arachidonylamide (AM404). AM404 potentiates several responses elicited by administration of exogenous anandamide, suggesting that it may also protect endogenous anandamide from inactivation. To test this hypothesis, we studied the effects of AM404 on the plasma levels of anandamide using high-performance liquid chromatography/mass spectrometry (HPLC/MS). Systemic administration of AM404 (10 mg kg−1 intraperitoneal, i.p.) caused a gradual increase of anandamide in rat plasma, which was significantly different from untreated controls at 60 and 120 min after drug injection. In plasma, both AM404 and anandamide were associated with a plasma protein, which we identified as albumin by non-denaturing polyacrylamide gel electrophoresis. AM404 (10 mg kg−1, i.p.) caused a time-dependent decrease of motor activity, which was reversed by the cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide·hydrochloride (SR141716A, 0.5 mg kg−1, i.p). These results are consistent with the hypothesis that AM404 inhibits anandamide inactivation in vivo.
Keywords: Cannabinoid; Anandamide; Fatty acid ethanolamides; High-performance liquid chromatography; Mass spectrometry; Anandamide transport;
Failure of baclofen to modulate discriminative–stimulus effects of cocaine or methamphetamine in rats by Patrik Munzar; Scott W Kutkat; Cori R Miller; Steven R Goldberg (169-174).
The effects of baclofen, an agonist at GABAB receptors, were evaluated in rats trained to discriminate 10.0 mg/kg of cocaine or 1.0 mg/kg of methamphetamine from saline under a fixed-ratio 10 schedule of food delivery. Baclofen (0.56–5.6 mg/kg) did not attenuate the discriminative–stimulus effects of the training dose of cocaine or methamphetamine and did not produce any shift in the cocaine and methamphetamine dose–response curves. Higher baclofen doses (3.0–5.6 mg/kg), however, markedly depressed or completely eliminated food-maintained responding. This suggests that previous reports of baclofen-induced decreases in cocaine self-administration behavior are connected, in some way, with either a general suppression of appetitive behaviors or with sedation and locomotor depression, rather than with any pharmacologically specific effect, and not accompanied by changes in subjective response to cocaine, as assessed by discriminative–stimulus measures.
Keywords: Baclofen; Cocaine; Methamphetamine; Drug-discrimination; (Rat);
Effects of aminoguanidine administration on vascular hyporeactivity in thoracic aorta from endotoxaemic rats by Ayşe Erol; Sezen Koşay (175-181).
Overproduction of nitric oxide has been implicated in the pathogenesis of the vascular hyporesponsiveness of endotoxic shock. In this study, we investigated the effects of aminoguanidine, an inducible nitric oxide synthase inhibitor, on the decreased vascular responsiveness in endotoxic shock. Male albino rats were administered intraperitoneally aminoguanidine (25, 50 or 75 mg kg−1) 1 h after they received saline or lipolysaccharide (Escherichia coli serotype 055:B5). The thoracic aortas were removed 18 h after lipopolysaccharide administration and suspended in organ baths containing Krebs solution, and tested for vascular reactivity. Contractile responses to phenylephrine and potassium chloride, and relaxant responses to acetylcholine were reduced in endotoxaemic animals. Aminoguanidine was ineffective in improving the vascular hypocontractility at 25 and 75 mg kg−1 doses; but at 50 mg kg−1 dose, it restored the decreased contractile responses toward normal values. Diminished relaxant responses to acetylcholine were restored by aminoguanidine at all three different doses. There were no significant differences in sodium nitroprusside induced relaxant responses between all groups. Administration of aminoguanidine in control animals did not change vascular responses to any agent. These data suggest that aminoguanidine treatment improves the vascular hyporesponsiveness to contractile- and endothelium-dependent relaxant agents observed in endotoxic shock.
Keywords: Endotoxic shock; Aminoguanidine; Nitric oxide (NO); (Rat);
CGRP and adrenomedullin receptor populations in human cerebral arteries: in vitro pharmacological and molecular investigations in different artery sizes by Anette Sams; Elizabeth Knyihár-Csillik; Jan Engberg; Délia Szok; János Tajti; István Bodi; Lars Edvinsson; László Vécsei; Inger Jansen-Olesen (183-193).
The aim of the present study was to determine functional and molecular characteristics of receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin in three different diameter groups of lenticulostriate arteries. Furthermore, the presence of perivascular neuronal sources of CGRP was evaluated in these arteries. In the functional studies, in vitro pharmacological experiments demonstrated that both CGRP and adrenomedullin induce α-CGRP-(8-37) sensitive vasodilation in artery segments of various diameters. The maximal amounts of vasodilation induced by CGRP and adrenomedullin were not different, whereas the potency of CGRP exceeded that of adrenomedullin by 2 orders of magnitude. Significant negative correlations between artery diameters and maximal responses were demonstrated for CGRP and adrenomedullin. In addition, the potency of both peptides tended to increase in decreasing artery diameter. In the molecular experiments, levels of mRNAs encoding CGRP receptors and receptor subunits were compared using reverse transcriptase polymerase chain reactions (RT-PCR). The larger the artery, the more mRNA encoding receptor activity-modifying proteins 1 and 2 (RAMP1 and RAMP2) was detected relative to the amount of mRNA encoding the calcitonin receptor-like receptor. By immunohistochemistry, perivascular CGRP containing nerve fibres were demonstrated in all the investigated artery sizes. In conclusion, both CGRP and adrenomedullin induced vasodilation via CGRP receptors in human lenticulostriate artery of various diameter. The artery responsiveness to the CGRP receptor agonists increased with smaller artery diameter, whereas the receptor-phenotype determining mRNA ratios tended to decrease. No evidence for CGRP and adrenomedullin receptor heterogeneity was present in lenticulostriate arteries of different diameters.
Keywords: Lenticulostriate artery; RT-PCR; CGRP (calcitonin gene-related peptide); Adrenomedullin; CRLR (calcitonin receptor-like receptor); RAMP (receptor activity-modifying protein);
Redox regulation of S-nitrosocysteine-mediated vasodilation in vivo by Azizul Hoque; James N Bates; Stephen J Lewis (195-198).
This study examined the effects of the lipophobic electron acceptor, nitroblue tetrazolium (2×5 μmol/kg, i.v.) on the vasodilation produced by the putative endothelium-derived S-nitrosothiol, l-S-nitrosocysteine (400 nmol/kg, i.v.), and the nitric oxide (NO) donor, (Z)-1-|N-methyl-N-[6(N-methylammoniohexyl)amino]|diazen-1-ium-1,2-diolate (MAHMA NONOate, 25 nmol/kg, i.v.), in anesthetized rats. The administration of nitroblue tetrazolium resulted in delayed but long-lasting increases in vascular resistances. The l-S-nitrosocysteine-induced vasodilator responses were markedly diminished whereas the MAHMA NONOate-induced responses were not affected by nitroblue tetrazolium. These results support the possibility that l-S-nitrosocysteine interacts with membrane thiols that are subject to nitroblue tetrazolium-induced oxidation (i.e., disulfide-bridge formation) and that nitroblue tetrazolium-induced vasoconstriction may involve a loss of potency of endothelium-derived S-nitrosothiols.
Keywords: l-S-Nitrosocysteine; Nitric oxide (NO); Redox; Vasodilation; (Rat);
Anti-inflammatory effects of peripheral benzodiazepine receptor ligands in two mouse models of inflammation by Sandra R. Torres; Tânia S. Fröde; Geisson M. Nardi; Natalio Vita; Rennée Reeb; Pascual Ferrara; Rosa M. Ribeiro-do-Valle; Roseli C. Farges (199-211).
In vivo treatment of mice with peripheral benzodiazepine receptor ligands exerts an inhibitory effect on the inflammatory response in two models of acute inflammation. In the first model, pretreatment of the animals (24 h) with 1-(2-chlorophenyl)-N-methyl-N(1-methylpropyl)-3-isoquinoline carboxamide (PK11195) and 7-chloro-5-(4-Chlorophenyl)-1,3-dihydro-1-methyl-2-H-1,4-benzodiazepin-2 (Ro5-4864), at different doses (0.00001–10 mg/kg, i.p.) dose dependently inhibited the formation of mouse paw oedema induced by carrageenan with mean ID50s of 0.009 (95% confidence limits=0.0076–0.013) and 0.04 (95% confidence limits=0.025–0.0086) mg/kg, respectively. Both ligands (0.1 mg/kg, i.p.) inhibited in the same way the mouse paw oedema induced by carrageenan in animals with and without adrenal glands. PK11195 and Ro5-4864 (0.1 mg/kg, i.p.) inhibited the mouse paw oedema induced by several inflammatory mediators. In the second model, the pretreatment (24 h) with peripheral benzodiazepine receptor ligands (0.1 mg/kg, i.p.) exerted an inhibitory effect on neutrophil influx and produce a marked inhibition of carrageenan-produced interleukin-13 and interleukin-6 in pleural exudation. Our results extend previous findings that peripheral benzodiazepine receptor is involved in the inflammatory response, and suggest that this action may be linked to the action of different inflammatory mediators, probably mainly by the inhibition of the release of pro-inflammatory cytokines.
Keywords: Benzodiazepine receptor; Paw oedema; Pleurisy;
Wound healing acceleration of a novel transforming growth factor-β inducer, SEK-1005 by Yoshiko Abe; Kouji Inagaki; Akihiko Fujiwara; Kiyoshi Kuriyama (213-218).
The studies were carried out to elucidate the effect of a novel cyclic peptide, SEK-1005 (C45H70N8O13), on wound healing. SEK-1005 (4–10 μg/wound) applied topically significantly accelerated the healing of a full-thickness wound on the dorsal skin of a rat. In a healing-impaired mouse, the peptide (2–10 μg/wound) had more potent activity, exerting an effect comparable to that of basic fibroblast growth factor (FGF). However, SEK-1005 (0.1–100 ng/ml) scarcely promoted the proliferation of cultured fibroblasts (NIH3T3 cells) while basic FGF (0.2–5 ng/ml) showed marked mitogenic activity. SEK-1005 (2–10 μg/wound) significantly increased the topical production of transforming growth factor (TGF)-β1, a cytokine that is known to accelerate wound healing. This activity was closely correlated with the wound-repairing effect. From the above, SEK-1005 can be considered as a new type of wound healing agent with potent TGF-β1-inducing activity.
Keywords: Wound healing accelerator; Healing-impaired; Cyclic peptide; SEK-1005; TGF-β1 (transforming growth factor-β1) inducer;