European Journal of Pharmacology (v.595, #1-3)

Endocannabinoids as positive or negative factors in hematopoietic cell migration and differentiation by Deborah Patinkin; Garry Milman; Aviva Breuer; Ester Fride; Raphael Mechoulam (1-6).
The ethanolamides of arachidonic, myristic and linoleic acids reduce bone marrow cell migration, while the 2-glyceryl esters of these acids enhance migration. Thus the 2 major endocannabinoids, anandamide (arachidonoyl ethanolamide) and 2-AG (2-arachidonoyl glycerol), whose structural difference lies in the nature of the end-group alone, work in opposite directions. The endocannabinoid arachidonoyl serine, a vasodilator, also reduces migration. The effect of 2-AG is mediated, in part at least, through the cannabinoid receptors, while the effect of anandamide, as well as the rest of the compounds assayed, are not mediated through them. Almost all cannabinoids tested, including anandamide and 2-AG, lead to approximate doubling of CFU-GEMM (colony-forming unit: granulocyte, erythrocyte, macrophage, megakaryocyte) colonies. The effect of anandamide is considerably more potent than that of 2-AG. A surprising dose–response increase of erythroid cells is noted in cultures with the ester cannabinoids (in the absence of the cytokine erythropoietin), while a considerable dose–response augmentation of megakaryocytes is noted in cultures with the ethanolamide cannabinoids (in the presence of erythropoietin). This is suggestive of some cross-talk between two different regulatory systems, one governed by glycoprotein ligands and the other by endocannabinoids.
Keywords: Ethanolamide cannabinoids; Glycerol cannabinoids; Cannabinoid receptor CB1; Cannabinoid receptor CB2;

Calbindin-D28K is a calcium-binding protein in neuronal cytoplasm, which has the capability to protect neurons from degeneration. It was reported that glial cell line-derived neurotrophic factor (GDNF) increased calbindin-D28K expression in dopaminergic neurons in vitro. It was observed in our research that GDNF also enhanced the expression of calbindin-D28K in adult rat substantia nigra neurons in vivo. To investigate the intracellular signaling pathways underlying the calbindin-D28K expression induced by GDNF, immunoblot and immunoprecipitation analyses were performed in our present study. Our results showed that injection of GDNF alone into substantia nigra of an adult rat brain increased the calbindin-D28K expression; meanwhile, the phosphorylation level of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) increased. However, the calbindin-D28K expression induced by GDNF was specifically blocked by the inhibitor of phosphatidylinositol 3-kinase (PI3K), but the inhibitor of ERK1/2 did not block the calbindin-D28K expression. Furthermore, GDNF administration also caused the nuclear factor κB (NF-κB/p65), to translocate from cytoplasm into the nucleus, and the inhibitor of PI3K effectively blocked the translocation. Immunoprecipitation assay results further demonstrated that it was the p65/p52 complex of NF-κB, rather than the p65/p50 complex that translocated into the neuronal nucleus. The calbindin-D28K expression induced by GDNF was also inhibited when the NF-κB signaling pathway was blocked by Helenalin. These results described a novel mechanism by which the activation of PI3K/Akt→NF-κB (p65/p52) signaling pathway could play a role in the calbindin-D28K expression induced by GDNF.
Keywords: Glial cell line-derived neurotrophic factor; Calbindin-D28K; Phosphatidylinositol 3-kinase; Protein kinase B; Extracellular signal-regulated kinase 1/2; Nuclear factor κB;

Isobolographic and behavioral characterizations of interactions between vigabatrin and gabapentin in two experimental models of epilepsy by Jarogniew J. Luszczki; Neville Ratnaraj; Philip N. Patsalos; Stanislaw J. Czuczwar (13-21).
The aim of this study was to characterize the pharmacodynamic, pharmacokinetic and adverse-effect profiles of vigabatrin and gabapentin. Isobolographic analysis was used in two mouse experimental models of epilepsy: the maximal electroshock seizure threshold test and pentylenetetrazole-induced seizures. In the maximal electroshock seizure threshold test, electroconvulsions were produced by a current with various intensities whilst in the pentylenetetrazole test a CD97 dose (100 mg/kg) was used. Potential adverse-effect profiles of interactions of vigabatrin with gabapentin at three fixed-ratios of 1:3, 1:1 and 3:1 from both seizure tests were evaluated in the chimney (motor performance) and grip-strength (skeletal muscular strength) tests. Vigabatrin and gabapentin total brain concentrations were determined with high performance liquid chromatography. Vigabatrin and gabapentin administered singly increased the electroconvulsive threshold (TID20 — 226.2 and 70.0 mg/kg, respectively). With isobolography, the combination of vigabatrin with gabapentin at the fixed-ratio of 1:3 exerted supra-additive (synergistic) interactions whilst at 1:1 and 3:1 additivity occurred. Similarly, vigabatrin and gabapentin administered singly suppressed the pentylenetetrazole-induced seizures (ED50 values — 622.5 and 201.1 mg/kg, respectively). Isobolography revealed that vigabatrin with gabapentin in combination at the fixed-ratio of 1:1 produced supra-additive (synergistic) interaction whilst at 1:3 and 3:1 additivity occurred. In combination neither motor coordination nor skeletal muscular strength was affected. Total vigabatrin and gabapentin brain concentrations revealed that neither drug affected the pharmacokinetics of the other. Vigabatrin and gabapentin have a favorable pharmacodynamic interaction in animal seizure models in the absence of acute adverse effects or concurrent pharmacokinetic changes.
Keywords: Drug interaction; Gabapentin; Isobolographic analysis; Maximal electroshock seizure threshold test; Pentylenetetrazole-induced seizure test; Vigabatrin;

We previously reported the synthesis of three new opioid agonists as well as their in vitro and in vivo activity [Girón, R., Abalo, R., Goicoechea, C., Martín, M.I., Callado, L.F., Cano, C., Goya, P., Jagerovic, N. 2002. Synthesis and opioid activity of new fentanyl analogs. Life Sci. 71, 1023–1034]. One of them, N-[1-phenylpyrazol-3-yl]-N-[1-(2-phenethyl)-4-piperidyl)] propenamide (IQMF-4), showed an interesting antinociceptive activity. Intraperitoneally (i.p.) administered, it was as effective as fentanyl or morphine, being less potent than fentanyl but more so than morphine. The aim of the present work was to evaluate its antinociceptive effect by different routes of administration, using the hot plate test, and to investigate possible side effects, such as tolerance and withdrawal, in vitro, using the myenteric plexus-longitudinal muscle strip preparation from guinea pig ileum, and in vivo, using the hot plate test. IQMF-4 was more potent than morphine when administered per os (p.o.), but less potent when administered intracerebroventricularly (i.c.v.). By both routes, fentanyl is more potent that IQMF-4. When IQMF-4 was administered i.p., naloxone methiodide, a peripherally acting antagonist, was able to completely block its antinociceptive effect, whereas, after i.c.v. administration, the blockade was only partial. An interesting feature of the new compound is that it induces tolerance in vitro but not in vivo. Moreover, though in vitro withdrawal was not different from fentanyl or morphine, in vivo withdrawal symptoms were significantly less frequent in mice treated with IQMF-4 than in those treated with morphine or fentanyl. Although more assays are required, these results show that IQMF-4 appears to be a potent analgesic compound with an interesting peripheral component, and reduced ability to induce dependence.
Keywords: Opioid; Peripheral effect; Analgesia; Tolerance; Dependence;

Lead (Pb2+) exposure in children can induce long-lasting deficits in cognitive function and has been modeled in experimental animals. Based on previous studies which demonstrated that S-adenosyl-l-methionine (SAM) is beneficial in the treatment of lead intoxication, here, we asked the question if SAM treatment could rescue the impaired cognition and synaptic plasticity induced by lead. Rats drank 1500 ppm lead acetate (PbAc) solution or distilled water throughout gestation and lactation. After weaning at postnatal day 22, one half of the control and lead-exposed male offspring were intraperitoneally injected 20 mg SAM/kg daily over a period of 20–22 days. Electrophysiological and Morris water maze test were performed at 44–54 days of age. The result showed that the impaired learning ability induced by lead could be improved significantly by SAM. Furthermore, our results revealed that long-term potentiation (LTP) of excitatory postsynaptic potential and population spike impairments induced by lead were also ameliorated by SAM treatment.
Keywords: Lead; S-adenosyl-l-methionine; Morris water maze; Long-term potentiation;

Pleiotrophin prevents cocaine-induced toxicity in vitro by Esther Gramage; Luis F. Alguacil; Gonzalo Herradon (35-38).
Pleiotrophin is a cytokine involved in differentiation, survival and repair processes in the central nervous system. Pleiotrophin is upregulated in the brain after administration of different drugs of abuse, thus suggesting a protective role of this cytokine on drug-induced toxicity. We have tested this hypothesis in vitro using NG108-15 cells, a line widely used for neurotoxicity studies. It was found that pleiotrophin (3 and 6 μM) significantly prevents cocaine (5 mM)-induced cytotoxicity as measured by the neutral red test. Similar results were obtained in PC12 cells, which were found to endogenously express both pleiotrophin and its main target, receptor protein tyrosine phosphatase (RPTP) β/ζ. Blockade of pleiotrophin signaling using anti-pleiotrophin antibodies (2 μg/ml) did not potentiate cocaine-induced toxicity; interestingly, incubation of PC12 cells only with anti-pleiotrophin antibodies significantly reduced cellular viability, thus suggesting an important role of endogenous pleiotrophin signaling in cell survival. The data suggest that pleiotrophin overexpression in response to drugs of abuse may be relevant to prevent drug-induced toxicity.
Keywords: Pleiotrophin; Cocaine; Neurotoxicity; Survival; Dopaminergic;

AMP-activated protein kinase and hypoxic pulmonary vasoconstriction by Tom P. Robertson; Kirsteen J.W. Mustard; Tristan H. Lewis; Jill H. Clark; Christopher N. Wyatt; Elisa A. Blanco; Chris Peers; D. Grahame Hardie; A. Mark Evans (39-43).
Hypoxic pulmonary vasoconstriction is a vital homeostatic mechanism that aids ventilation–perfusion matching in the lung, for which the underlying mechanism(s) remains controversial. However, our most recent investigations strongly suggest that hypoxic pulmonary vasoconstriction is precipitated, at least in part, by the inhibition of mitochondrial oxidative phosphorylation by hypoxia, an increase in the AMP/ATP ratio and consequent activation of AMP-activated protein kinase (AMPK). Unfortunately, these studies lacked the definitive proof that can only be provided by selectively blocking AMPK-dependent signalling cascades. The aim of the present study was, therefore, to determine the effects of the AMPK inhibitor compound C upon: (1) phosphorylation in response to hypoxia of a classical AMPK substrate, acetyl CoA carboxylase, in rat pulmonary arterial smooth muscle and (2) hypoxic pulmonary vasoconstriction in rat isolated intrapulmonary arteries. Acetyl CoA carboxylase phosphorylation was increased approximately 3 fold in the presence of hypoxia (pO2  = 16–21 mm Hg, 1 h) and 5-aminoimidazole-4-carboxamide riboside (AICAR; 1 mM; 4 h) and in a manner that was significantly attenuated by the AMPK antagonist compound C (40 μM). Most importantly, pre-incubation of intrapulmonary arteries with compound C (40 μM) inhibited phase II, but not phase I, of hypoxic pulmonary vasoconstriction. Likewise, compound C (40 μM) inhibited constriction by AICAR (1 mM). The results of the present study are consistent with the activation of AMPK being a key event in the initiation of the contractile response of pulmonary arteries to acute hypoxia.
Keywords: AMP-activated protein kinase; Hypoxic pulmonary vasoconstriction; Compound C; AICAR;

We have previously shown that the β-blocking drug metoprolol improves cardiac function in the streptozotocin-diabetic rat partly by inducing parallel improvements in cardiac metabolism and gene expression. β-blockers have been previously reported to increase the expression of β1 and β2-adrenoceptors, but their effects on the expression of β3-adrenoceptors are unknown. The aim of the present study was to investigate whether metoprolol increases β3-adrenoceptor expression and downstream Akt-mediated signaling. Left ventricular function was measured in paced isolated working hearts. β1, β2 and β3 adrenoceptor-expression levels were measured using Western blotting. Protein kinase A (PKA) and calcium/calmodulin dependent protein kinase II (CAMK-II) activities, as well as Akt phosphorylation, were measured as indices of downstream target activation. Chronic metoprolol treatment improved cardiac function and produced a marked increase in the expression of all three β-adrenoceptor subtypes which was associated with a decrease in PKA activity and an increase in Akt phosphorylation. Akt-mediated phosphorylation of endothelial nitric oxide synthase (eNOS) was not altered, but phosphorylation of the transcription factor FOXO-3 was increased. Metoprolol increased the expression of β1, β2 and β3 adrenoceptors, associated with repression of FOXO-3 expression. β-adrenoceptor signaling shifted from PKA to Akt-mediated signaling, associated with phosphorylation of FOXO-3 but not eNOS.
Keywords: Metoprolol; β-adrenoceptor; Diabetic cardiomyopathy; Nitric oxide; Forkhead transcription factor;

We aimed to further define the pathway mediating the inhibitory effects of κ-opioid receptor stimulation on Ca2+ transients and hypertrophic responses to β1-adrenoceptor stimulation. We determined the effects of Trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid methanesulfonate salt (U50,488H), a selective κ-opioid receptor agonist, on the enhancement of spontaneous Ca2+ transients and the induction of hypertrophy by isoprenaline, a β-adrenoceptor agonist, in cultured neonatal ventricular myocytes. The results were compared with those found with KN93, a selective Ca2+/calmodulin-dependent kinase (CaMKII) inhibitor, propranolol, a β-adrenoceptor antagonist, and verapamil, a L-type Ca2+ channel antagonist. Hypertrophy of cardiomyocytes was characterized by increases in (i) total protein content; (ii) cell size; and (iii) [3H]leucine incorporation. 10 μmol/l isoprenaline increased all three parameters. We also determined the expression of nuclear CaMKIIδ in response to U50,488H in the presence or absence of isoprenaline. To determine whether the effects of U50,488H were receptor-mediated, its effects were also measured following blockade of the κ-opioid receptor with nor-binaltorphimine. κ-Opioid receptor stimulation suppressed the stimulatory effect of isoprenaline on Ca2+ transients and cardiac hypertrophy, as did KN93, propranalol and verapamil. U50,488H also suppressed the expression of nuclear CaMKIIδB in the presence, but not in the absence of isoprenaline. These results suggest that the inhibitory effect of κ-opioid receptor stimulation on β1-adrenoceptor stimulation may also involve CaMKIIδ.
Keywords: U50,488H; κ-Opioid receptor; Isoprenaline; Neonatal ventricular myocyte; Cardiac hypertrophy; Ca2+; CaMKIIδ;

Physiological levels of glutamine prevent morphine-induced preconditioning in the isolated rat heart by André Heinen; Ragnar Huhn; Markus W. Hollmann; Benedikt Preckel; Coert J. Zuurbier; Wolfgang Schlack; Jan Fräßdorf; Nina C. Weber (58-64).
Morphine induces cardioprotection against ischaemia–reperfusion injury. While aiming to investigate the underlying signal transduction cascade of morphine preconditioning in isolated Langendorff-perfused rat hearts, the expected cardioprotection was not detectable. Thus, we investigated the influence of different preconditioning protocols and substrate conditions on cardioprotection in this experimental model. Isolated rat hearts underwent 35 min global ischaemia followed by 60 min reperfusion. Morphine PC was initiated by 3 cycles of 5 min 1 μM morphine with either 5 min washout [3PC5 (5)] or 15 min washout [3PC5 (15)] before ischaemia; by 15 min morphine with 15 min washout before ischaemia [PC15 (15)]; or by 15 min 10 μM morphine with 15 min washout [PC15 (15)-10 μM]. Ischaemic preconditioning was initiated by 3 cycles of 3 min ischaemia; in another group, hearts received 1 μM morphine continuously for 10 min before ischaemia until the end of reperfusion [continued morphine]. To investigate the effects of glutamine, two groups received a glutamine-free perfusate: a control group, and a morphine preconditioning group [3PC5 (15)]. Ischaemic preconditioning reduced infarct size by 75%, and continued morphine by 46% compared to control group. With the glutamine containing perfusate, none of the morphine PC pretreatments had an effect on infarct size. In glutamine-free perfusate, 3 cycles of 5 min 1 μM morphine with 15 min washout reduced infarct size from 45% ± 8% (control) to 20% ± 5% (3PC5 (15). Cardioprotection by morphine-induced preconditioning is model dependent: in the isolated rat heart, morphine preconditioning is prevented by a glutamine containing perfusate.
Keywords: Morphine; Preconditioning; Glutamine; Isolated heart;

Influence of combinations of acetylsalicylic acid, acetaminophen, and diclofenac on platelet aggregation by Katja S. Galliard-Grigioni; Martin Fehr; Walter H. Reinhart (65-68).
Acetylsalicylic acid (aspirin) is often given together with other nonsteroidal anti-inflammatory drugs and acetaminophen. The latter have been accused in epidemiologic studies to cause an increased cardiovascular risk. We have, therefore, analysed the influence of various such drug combinations on platelet aggregation in vitro. Citrated blood was incubated with either 25 μg/ml acetaminophen, 0.5 μg/ml aspirin, 0.04 μg/ml diclofenac, or buffer; followed by a second of the above-mentioned solutions. After a 20 min incubation, platelet aggregation was assessed with a platelet function analyser (PFA-100®), which measures the pore closure time (CT) by aggregating platelets. The length of CT reflects the degree of platelet inhibition. Acetaminophen alone did not affect platelet aggregation. Aspirin and diclofenac both increased CT (184 ± 69 s, P  < 0.01 and 196 ± 54 s, P  < 0.001; control 120 ± 13 s). Combinations of either aspirin and diclofenac, aspirin and acetaminophen, or diclofenac and acetaminophen increased CT further (290 ± 22 s, 281 ± 36 s, 288 ± 25 s, respectively, P  < 0.001). The time sequence of drug application was important: when diclofenac or acetaminophen was added before aspirin, platelet aggregation was less inhibited than when given in opposite order, i.e. aspirin prior to diclofenac or acetaminophen. We conclude that acetaminophen by itself does not affect platelet aggregation, but potentiates the antiaggregatory effect of aspirin or diclofenac. Aspirin given before acetaminophen or diclofenac had a more potent antiaggregatory effect than vice versa. These observations may have clinical implications.
Keywords: Acetaminophen; Aspirin; Diclofenac; NSAID; Platelet aggregation;

Potent antioxidant role of Pirfenidone in experimental cirrhosis by Adriana Salazar-Montes; Luis Ruiz-Corro; Alberto López-Reyes; Eugenio Castrejón-Gómez; Juan Armendáriz-Borunda (69-77).
Three important features must be considered when proposing therapeutic strategies in liver cirrhosis: inflammation, oxidative stress and fibrogenesis. Pirfenidone is a synthetic molecule which oxidative action has not been tested in cirrhosis. Cirrhosis was induced in rats by ligation of the common bile duct or carbon tetrachloride (CCl4) chronic intoxication and treated with Pirfenidone or Diphenyleneiodonium (a potent known antioxidant) for the last two weeks for bile duct ligation model or for the last three weeks for CCl4 chronic intoxication. A 60% reduction in fibrosis index for bile duct ligation model and 42% for CCl4 along with reduced inflammation was observed. Considerable reduction on hepatic enzymes and total and direct bilirubins were detected with Pirfenidone in both models. Pirfenidone antioxidant capacity rendered a 28% and 30% reduction in nitrites and Malonyldealdehide concentration in bile duct ligation and 52% and 38% in CCl4. With respect to gene expression, fibrotic genes like transforming growth factor-β (TGF-β) and Collagen Iα (Col-1α) were down-regulated by Pirfenidone and increased expression of regenerative genes like hepatocyte growth factor (HGF) and c-met . superoxide dismutase (SOD), catalase (CAT) and inducible nitric oxide synthase (iNOS) gene expression were importantly down-regulated where nuclear factor kappa B (NF-κB) binding activity also decreased with Pirfenidone treatment. Also, SOD and CAT functional activity decreased after Pirfenidone action. On the other hand, Diphenyleneiodonium induced a drop in oxidative stress similar in extent to Pirfenidone, but it was not as effective as Pirfenidone in reducing fibrosis.In this work, we showed antioxidant properties of Pirfenidone beyond its well-known antifibrotic effect. These features make Pirfenidone an attractive drug for trying fibrotic diseases accompanied by oxidative stress processes.
Keywords: Pirfenidone; Experimental liver; Gastrointestinal and urogenital pharmacology;

The neurotrophin brain-derived neurotrophic factor (BDNF) occurs in elevated levels during airway inflammation, including asthma and hypoxic lung injury, and has been suggested to be associated with airway hyperresponsiveness in these conditions. The aim of the present study was to examine whether airway responses to histamine challenge and levels of exhaled nitric oxide (NO) in vivo might be altered upon BDNF treatment. Pulmonary resistance, lung compliance, insufflation pressure, and levels of exhaled NO were measured in anaesthetized guinea pigs exposed to BDNF prior to challenge with histamine and with intact or inhibited endogenous NO production. BDNF pretreatment significantly enhanced histamine-evoked increase in pulmonary resistance and insufflation pressure, as well as the decrease in lung compliance. BDNF markedly accentuated the reduction in exhaled NO following histamine challenge. In animals with inhibited endogenous NO production BDNF induced a significantly earlier histamine-evoked increase in airway responses. The present data show that BDNF can induce an augmentation of histamine-evoked airway responses and reduce levels of NO in exhaled air in vivo. Endogenous NO seems to exert a braking action on BDNF-induced enhancement of airway responses and a reduced ability to release NO may be one mechanism for increased airway response during elevated BDNF levels. Taken together this indicates that BDNF may be of importance for airway hyperresponsiveness in vivo. The interaction between BDNF and airway NO formation, and its relation to airway responses, merit further investigation.
Keywords: Airway; Asthma; Brain-derived neurotrophic factor; Bronchial hyperresponsiveness; Histamine; L-NAME (N ω L-nitro-L-arginine methyl ester); Lung compliance; Neurotrophin; Nitric oxide; Pulmonary resistance;

Our previous research showed that ATP and adenosine 5′-O-2-thiodiphosphate (ADPβS) induce contractile effects in the longitudinal muscle of mouse distal colon via activation of P2Y receptors which are not P2Y1 or P2Y12 subtypes. This study investigated the nature of the P2Y receptor subtype(s) and the mechanisms leading to the intracellular calcium concentration increase necessary to trigger muscular contraction. Motor responses of mouse colonic longitudinal muscle to P2Y receptor agonists were examined in vitro as changes in isometric tension. ATP or ADPβS induced muscular contraction, which was not affected by P2Y11 or P2Y13 selective antagonists. Calcium-free solution or the calcium channel blocker, nifedipine, failed to modify the contractile responses to ATP or ADPβS, which were virtually abolished by depletion of calcium intracellular stores after repetitive addition of carbachol in calcium-free medium with addition of cyclopiazonic acid. Neomycin or U-73122, phospholipase C inhibitors, or 2-aminoethoxy-diphenylborate (2-APB), membrane-permeant IP3 receptor inhibitor reduced the response to ATP, whilst ryanodine or ruthenium red, inhibiting calcium release from ryanodine-sensitive stores, abolished the response to ADPβS. Responses to maximally effective concentrations of ATP and ADPβS were not fully additive. Desensitisation with ADPβS antagonized the contractile effects of ATP, as desensitisation with ATP antagonized the response to ADPβS. In the longitudinal muscle of mouse distal colon, ATP and ADPβS induce muscular contraction via a P2Y receptor, coupled to differential signal pathways leading to intracellular calcium increase.
Keywords: ATP; P2Y purinoreceptor; Mouse distal colon; Muscular contraction; Intracellular calcium store;

The central or systemic administration of 3-carboxy-4-octyl-2-methylenebutyrolactone (C75), a synthetic inhibitor of fatty acid synthase (FAS), causes anorexia and profound weight loss in rodents. The amount of food intake and gastrointestinal mobility are closely related. In this study, an attempt has been made to investigate the effects and mechanisms of C75 on gastric emptying and gastrointestinal transit after intracerebroventricular (i.c.v.) injection in mice. Our data showed that C75 (1, 5, 10 µg/mouse) dose-dependently delayed gastric emptying and gastrointestinal transit in fasted mice. 10 µg C75 delayed gastric emptying by about 21.4% and reduced gastrointestinal transit by about 31.0% compared with vehicle control group. Administration (i.c.v.) of 5-(tetradecyloxy)-2-furoic acid (TOFA, an acetyl-CoA carboxylase (ACC) inhibitor) or ghrelin attenuated the delayed gastrointestinal mobility effect induced by 10 µg C75. Taken together, C75 is able to decrease gastrointestinal mobility and it seems possible that malonyl-CoA and ghrelin might play an intermediary role in these processes.
Keywords: C75; Gastric emptying; Gastrointestinal transit; Malonyl-CoA; Ghrelin;

Emodin is known to be used in the treatment of cholesterol stones and cholecystitis. This study sought to investigate the effects of emodin on the contraction of gallbladder smooth muscle (GBSM), intracellular Ca2+ concentration and L-type calcium current in GBSM cells. Gallbladder muscle strips were obtained from adult guinea pigs and the resting tension was recorded. Gallbladder smooth muscle cells were isolated by enzymatic digestion. Cells were loaded with fluo-3/AM and [Ca2+]i was determined by a laser confocal microscope. Calcium current was recorded by the whole-cell patch clamp method. Emodin increased the resting tension of GBSM strips in a dose-dependent manner. Emodin elevated [Ca2+]i in GBSM cells, and this effect was attenuated by pretreatment with nifedipine. In addition, Emodin increased L-type calcium current at concentrations of 1 to 30 µM (at + 10 mV, 10 µM, 45.1 ± 5.2% compared to control, EC50 = 3.11 µM). In the presence of protein kinase C (PKC) inhibitor, Staurosporine, emodin did not significantly affect the calcium current. However, phorbol 12, 13-dibutyrate mimicked emodin in enhancement of the calcium current. These results suggest that emodin promotes gallbladder contraction by increasing Ca2+ influx through L-type calcium channel via PKC pathway.
Keywords: Emodin; Gallbladder smooth muscle; L-type calcium current; Intracellular Ca2+ concentration;

Pyrrolidine dithiocarbamate, an antioxidant and a potent inhibitor of nuclear factor-kappa B (NF-κB), is known to have protective effect against ischemia and reperfusion injury. This study examined the cytoprotective mechanism of pyrrolidine dithiocarbamate against the microcirculatory failure caused by hepatic ischemia and reperfusion. Rats were subjected to 60 min of hepatic ischemia followed by 5 h of reperfusion. Pyrrolidine dithiocarbamate (100 mg/kg) or the vehicle was administered intraperitoneally 24 h before ischemia. The level of serum aminotransferases and hepatic lipid peroxides significantly increased, and the glutathione contents fell in the ischemia/reperfusion group. Pyrrolidine dithiocarbamate prevented the increase in the level of serum enzymes and hepatic lipid peroxides, and the decrease in the glutathione contents. The NF-κB DNA-binding activity was inhibited by a pre-treatment with pyrrolidine dithiocarbamate. Ischemia and reperfusion significantly increased the mRNA expression of the endothelin-1 and endothelin ETB receptor, which was prevented by pyrrolidine dithiocarbamate. There were significant increases in the mRNA expressions of inducible nitric oxide synthase, tumor necrosis factor-alpha, and cyclooxygenase-2, in the livers after ischemia and reperfusion. These increases were attenuated by the pyrrolidine dithiocarbamate treatment. In a rat model of hepatic ischemia and reperfusion, our results suggest that the hepatoprotective actions of pyrrolidine dithiocarbamate may be mediated in part through the modulation of imbalanced expression of vascular stress genes.
Keywords: Hepatic ischemia/reperfusion; Microcirculatory failure; Nuclear factor-kappa B; Oxidative stress; Pyrrolidine dithiocarbamate;

Treatment with N-tosyl-l-phenylalanine chloromethyl ketone after the onset of collagen-induced arthritis reduces joint erosion and NF-κB activation by Jin Choi; Kyung-Ho Ha; Mi-Sun Byun; So Youn Min; Min-Jung Park; Hyun-Sil Park; Hye-Joa Oh; Ji Hyeon Ju; Ho-Youn Kim; Dae-Myung Jue (108-113).
N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) is known to inhibit NF-κB activation and the expression of inflammation mediators in cultured cells. We measured the potential of TPCK to inhibit the pathogenesis of collagen-induced arthritis by blocking NF-κB activation. Arthritis was induced in DBA/1J mice by the injection of bovine type II collagen in adjuvant on days 0 and 14. Mice received either TPCK (3 or 10 mg/kg, i.p.) or vehicle three times a week for 3 weeks starting on day 21. TPCK moderately reduced clinical disease activity scores, whereas it markedly suppressed histological indications of joint destruction. In vitro production of tumor necrosis factor-α, interleukin-6, and monocyte chemotactic protein-1 from lipopolysaccharide-stimulated spleen cells was also reduced by in vivo treatment with TPCK. Proliferation of cells isolated from spleen or draining lymph nodes and production of interferon-γ and interleukin-17 in response to stimulation with type II collagen was decreased by TPCK. Moreover, nuclear NF-κB activity induced by collagen immunization was significantly reduced in mice treated with TPCK. Finally, osteoclast differentiation of bone marrow cells induced by macrophage colony-stimulating factor and receptor activator of NF-κB ligand was completely inhibited by TPCK. These results indicate that TPCK attenuates collagen-induced arthritis and bone erosion by suppressing NF-κB activation and thus expression of inflammatory and osteoclastogenic genes.
Keywords: Rheumatoid arthritis; Collagen-induced arthritis; Tosylphenylalanyl chloromethyl ketone; NF-κB; Cytokine; Inflammation;

The purpose of this study was to determine whether cyclooxygenase expression in arteries is affected by diabetes. Streptozotocin-injected rats and Goto-Kakizaki rats were used as animal models for type 1 and type 2 diabetes, respectively. Cyclooxygenase-2 expression was induced by lipopolysaccharide. Lipopolysaccharide-induced cyclooxygenase-2 expression was significantly lower in aortas isolated from streptozotocin-injected rats and Goto-Kakizaki rats than in aortas of control rats, while expression level of cyclooxygenase-1 was not affected by lipopolysaccharide and was not different in aortas of the three groups of rats. The level of 6-keto-prostaglandin F that accumulated in the presence of lipopolysaccharide as well as the basal accumulation level in the absence of lipopolysaccharide was significantly lower in aortas of streptozotocin-injected rats and Goto-Kakizaki rats than in aortas of control rats. The net increase in 6-keto-prostaglandin F level in response to stimulation with lipopolysaccharide, which was calculated by subtracting the basal accumulation level from the total accumulation level, was also significantly lower in aortas of streptozotocin-injected rats and Goto-Kakizaki rats than in aortas of control rats. There were no significant differences in the accumulated 6-keto-prostaglandin F levels in the absence or presence of lipopolysaccharide and the levels of basal and lipopolysaccharide-induced cyclooxygenase-2 expression in control or Goto-Kakizaki rat aortas under the conditions of different glucose concentrations in the medium. These results suggest that lipopolysaccharide-induced cyclooxygenase-2 expression and subsequent prostacyclin production are decreased in aortas isolated from both type 1 and type 2 diabetes rats.
Keywords: Atherosclerosis; Cyclooxygenase; Diabetes mellitus; Prostacyclin; Vascular smooth muscle;

PAR-5359, a well-balanced PPARα/γ dual agonist, exhibits equivalent antidiabetic and hypolipidemic activities in vitro and in vivo by Mi-Kyung Kim; Yu Na Chae; Moon Ho Son; Soon Hoe Kim; Jin Kwan Kim; Ho Sang Moon; Chan Sun Park; Myung-Ho Bae; Eunkyung Kim; Taedong Han; Hyun-ho Choi; Young Ah Shin; Byung-Nak Ahn; Chun Ho Lee; Joong In Lim; Chang Yell Shin (119-125).
Peroxisome proliferator-activated receptor (PPAR) α and γ are key regulators of lipid homeostasis and insulin resistance. In this study, we characterize the pharmacological profiles of PAR-5359, a dual agonist of PPARα and γ with well-balanced activities. In transient transactivation assay, PAR-5359 (3-(4-{2[4-(4chloro-phenyl)-3,6-dihydro-2H-pyridin-1-yl]-ethoxy}-phenyl)-(2S)-ethoxy-propionic acid) significantly activated human and mouse PPARα and γ without activating PPARδ. In functional assays using human mesenchymal stem cells and human hepatoma HepG2 cells, PAR-5359 significantly induced adipocyte differentiation and human ApoA1 secretion, which coincided with its transactivation potencies against the corresponding human receptor subtypes. Interestingly, PAR-5359 showed equivalent potencies against the mouse receptor subtypes (α and γ; 2.84 μM and 3.02 μM, respectively), which suggests the possibility that PAR-5359 could simultaneously activates each subtype of receptors subtype in under physiological conditions. In an insulin-resistant ob/ob mouse model, PAR-5359 significantly reduced plasma insulin levels, improved insulin sensitivity (HOMA-IR), and completely normalized plasma glucose levels. In a severe diabetic db/db mouse model, PAR-5359 dose-dependently reduced the plasma levels of glucose (ED30  = 0.07 mg/kg). Furthermore, it lowered plasma levels of non HDL- (ED30  = 0.13 mg/kg) and total cholesterol (ED30  = 0.03 mg/kg) in high cholesterol diet-fed rats for 4 days treatment. These results suggest that PAR-5359 has the balanced activities for PPARα and PPARγ in vivo as well as in vitro. And its balanced activities may render PAR-5359 as a pharmacological tool in elucidating the complex roles of PPARα/γ dual agonists.
Keywords: PPARalpha/gamma dual agonist; Insulin resistance; Dyslipidemia; PAR-5359; Rosiglitazone; Gemfibrozil;