European Journal of Pharmacology (v.532, #1-2)

Previously, we demonstrated that human serotonin (5-HT) 5-HT7 receptors display marked constitutive activity. Here, we tested if the constitutive activation of adenylyl cyclase by 5-HT7 receptors influenced both the desensitization properties of transfected 5-HT7 receptors and the ability of endogenous Gs-coupled receptors to activate adenylyl cyclase. Using membranes from stably transfected HEK293 cells expressing the recombinant human 5-HT7 receptor splice variants (5-HT7(a), 5-HT7(b) and 5-HT7(d)), we compared the effects of 1-h or 24-h preincubation of the agonist 5-HT, partial inverse agonists mesulergine and SB269970, and full inverse agonists clozapine and methiothepin on subsequent activation of adenylyl cyclase by both 5-HT through transfected 5-HT7 receptors and the endogenous Gs-coupled β-adrenoceptors and prostaglandin receptors of HEK293 cells. The data show that stable expression of 5-HT7 receptors is sufficient to attenuate adenylyl cyclase activation by endogenous Gs-coupled receptors. Interestingly, preincubation with inverse agonists not only failed to result in the predicted resensitization of all receptor mediated adenylyl cyclase activation, but some inverse agonists further attenuated (desensitized) β-adrenoceptor and prostaglandin-stimulated adenylyl cyclase activation similar to long-term agonist exposure by 5-HT. These effects were not correlated with inverse agonist efficacy, were not accompanied by receptor down-regulation and appear to be mediated by a protein kinase A (PKA) independent mechanism. It is concluded that the human 5-HT7 receptor mediates heterologous desensitization of endogenous Gs-coupled receptors through an unknown and potentially novel mechanism.
Keywords: Adenylyl cyclase; Inverse agonist; Constitutive activity; Splice variant; Resensitization;

Stimulation of erythrocyte phosphatidylserine exposure by chlorpromazine by Ahmad Akel; Tobias Hermle; Olivier M. Niemoeller; Daniela S. Kempe; Philipp A. Lang; Philipp Attanasio; Marlis Podolski; Thomas Wieder; Florian Lang (11-17).
Side effects of treatment with chlorpromazine include anaemia which could result from decreased formation or accelerated clearance of circulating erythrocytes. Recently, a novel mechanism leading to erythrocyte clearance has been disclosed. Osmotic shock, oxidative stress and glucose deprivation lead to activation of cation channels, Ca2+ entry, activation of a Ca2+-sensitive erythrocyte scramblase and subsequent exposure of phosphatidylserine at the erythrocyte surface. As macrophages are equipped with phosphatidylserine receptors, they bind, engulf and degrade phosphatidylserine exposing cells. The present experiments have been performed to explore whether chlorpromazine triggers phosphatidylserine exposure of erythrocytes. The phosphatidylserine exposure was estimated from annexin binding as determined in fluorescence activated cell sort (FACS) analysis. A 24 h exposure to glucose-free medium decreased cytosolic ATP levels, decreased cellular levels of reduced glutathione (GSH) and increased annexin binding. The effect on annexin binding and ATP but not on GSH was significantly enhanced in the presence of chlorpromazine (10 μM). Higher concentrations of chlorpromazine (40 μM) increased cytosolic Ca2+ activity. Osmotic shock and Cl removal similarly increased annexin binding, effects again being enhanced in the presence of chlorpromazine. In conclusion, the present observations point to a novel side effect of chlorpromazine, i.e. increased sensitivity of erythrocytes to glucose deprivation. The effect could well contribute to the known anaemia observed in the treatment with this antipsychotic drug.
Keywords: Cell volume; Cytosolic ATP; GSH (glutathione); Apoptosis; Phosphatidylserine; Calcium;

Naturally occurring glucagon-like peptide-2 (GLP-2) receptors in human intestinal cell lines by Anette Sams; Sven Hastrup; Marie Andersen; Lars Thim (18-23).
Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor.Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC50  = 9.7 ± 0.04 (mean ± S.E.M., n  = 4)).In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells.The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.
Keywords: GLP-2 receptor; Gα protein signalling; Intestinal growth; RT-PCR; Western blot; cAMP;

Class Ic antiarrhythmics block human skeletal muscle Na channel during myotonia-like stimulation by Futoshi Aoike; Masanori P. Takahashi; Saburo Sakoda (24-31).
Flecainide, a class Ic antiarrhythmic drug, has been anecdotally reported to improve myotonia, but little is known about its kinetics on human skeletal muscle sodium channels applicable in vivo. Here we explored the anti-myotonic action of flecainide for human skeletal muscle sodium channels heterologously expressed in cultured cells. Flecainide blocked sodium channels in a highly state-dependent manner with 20-fold difference in IC50 between use-dependent and tonic blocks. When pulses of brief depolarization simulating myotonia were applied from a holding potential of − 90 mV, flecainide at therapeutic concentrations significantly blocked sodium currents. Flecainide slowed the time course of recovery but most channels recovered from block within 10–20 s. In contrast to mexiletine, flecainide did not markedly block sodium current during prolonged depolarization, suggesting an open-channel blocking action. Considering the slow recovery from block and the specific action against repetitive depolarization, flecainide may represent a potent therapeutic agent for myotonia.
Keywords: Myotonia; Flecainide; Anti-arrhythmia agent; Sodium channel; Patch–clamp technique;

Mechanisms of antiplatelet activity of PC-09, a newly synthesized pyridazinone derivative by Shiou-Chi Cherng; Wen-Hsin Huang; Chia-Yang Shiau; An-Rong Lee; Tz-Chong Chou (32-37).
In this study, we examined whether PC-09, a new pyridazinone derivative, has antiplatelet activity in vitro and further investigated the possible mechanisms involved. Pretreatment with PC-09 resulted in an inhibition on rabbit platelet aggregation and ATP release induced by arachidonic acid, collagen or thrombin, with the IC50 values of 5.4 to 76.8 μM. The thromboxane B2 formation caused by collagen or thrombin was markedly inhibited by PC-09, but there was no alteration in that caused by arachidonic acid. The rise of platelet intracellular calcium level stimulated by aggregation agonists and collagen-induced platelet membrane surface glycoprotein IIb/IIIa expression was also reduced by PC-09. In addition, PC-09 itself significantly increased the cyclic AMP level through inhibiting cyclic AMP phosphodiesterase activity. These findings demonstrate that PC-09 is an inhibitor of platelet aggregation, which may be associated with mechanisms including inhibition of thromboxane A2 formation, intracellular calcium mobilization and platelet surface GPIIb/IIIa expression accompanied by increasing cyclic AMP level.
Keywords: PC-09; Platelet aggregation; Cyclic AMP; Thromboxane B2; Glycoprotein IIb/IIIa;

Effects of drug combinations on smooth muscle cell proliferation: An isobolographic analysis by Tom J. Parry; Rathna Thyagarajan; Dennis Argentieri; Robert Falotico; John Siekierka; Ronald J. Tallarida (38-43).
Although sirolimus is a potent inhibitor of vascular smooth muscle cell (VSMC) proliferation and is effective at preventing restenosis in the majority of clinical revascularization procedures employing sirolimus-eluting stents, some VSMC may escape the antiproliferative effects of sirolimus. The present study examines the effects of combining sirolimus with other known cell cycle-specific antiproliferative agents (cladribine, topotecan or etoposide) on cultured coronary artery VSMC proliferation and utilizes a novel isobolographic approach to determine whether sirolimus/antiproliferative agent combinations produce subadditive, additive or supraadditive potentiation of antiproliferative activity. All agents were found to inhibit coronary artery VSMC proliferation in a dose-dependent manner. Cladribine was found to potentiate the antiproliferative activity of sirolimus in either an additive or supraadditive manner, depending upon the cladribine concentration. Topotecan potentiated the sirolimus antiproliferative activity by simple additivity while etoposide yielded subadditive potentiation. The present results demonstrate the utility of isobolographic analysis for identifying and optimizing antiproliferative drug combinations.
Keywords: Vascular smooth muscle; Drug combination; Sirolimus; Isobolographic analysis;

Effects of estradiol on cardiac ion channel currents by Clemens Möller; Rainer Netzer (44-49).
Steroids are known to exert direct and indirect effects on cardiovascular functions, and women have been found to be more susceptible to QT prolongation than men. Although many clinical studies have been performed, the effects of steroids on cardiac repolarization are not yet fully understood. We examined the effects of 17-beta-estradiol (estradiol) on the major cardiac currents that are correlated to clinical observations of arrhythmias. Effects on the two major currents responsible for repolarization of the cardiac action potential (mediated by the human ether-à-gogo related gene (HERG) product), and by the potassium channel Q1 (KCNQ1) co-expressed with the potassium channel accessory subunit E1 (KCNE1) were examined, as well as effects on the sodium inward current (mediated by the sodium channel 5A (SCN5A) and generating the rapid upstroke of the action potential). A concentration-dependent effect of estradiol on the KCNQ1/KCNE1-mediated potassium current was observed. The half-maximal inhibition concentration (IC50) of estradiol on the KCNQ1/KCNE1 ion channel was calculated to 1.13 ± 0.23 μM. The HERG-mediated potassium and the SCN5A-mediated sodium currents, however, were only slightly reduced by estradiol at concentrations of up to 30 μM. This suggests that alterations of the cardiac action potentials by steroids may be mediated by interaction with the KCNQ1/KCNE1 ion channel.
Keywords: QT prolongation; HERG; KCNQ1; Patch-clamp; Estradiol;

The purpose of the present study was to investigate the influence of the time course of inflammation and the implication of spinal and supraspinal sites on the antihyperalgesic effects of the α2-adrenoceptor agonist medetomidine. Behavioral experiments showed a more intense antihyperalgesia in the phase of maintenance of inflammation than in the early or resolution stages. Maximum effect, without sedation, was observed with a dose of 40 μg/kg (66 ± 12% and 76 ± 15% reduction of mechanical and thermal hyperalgesia). No change was observed in the paw swelling, indicating that its effects were not secondary to a reduction of inflammation. In electrophysiological experiments, the effect was more pronounced in animals with an intact spinal cord than in spinalized animals (max. effects of 2 ± 0.7% vs. 48 ± 11% of control, noxious mechanical stimulation). We conclude that the antihyperalgesic effect of medetomidine depends on the time course of inflammation and that it is mainly located supraspinally.
Keywords: Pain; Hyperalgesia; Carrageenan; Single motor unit; Behavioral;

Several studies have shown that psychostimulants can induce differential immediate early gene and neuropeptide expression in the patch versus matrix compartments of dorsal striatum. The patch compartment contains a high density of mu opioid receptors and activation of these receptors may contribute to psychostimulant-induced gene expression in the patch versus matrix compartments of dorsal striatum. However, the contribution of mu opioid receptor activation to psychostimulant-induced changes in gene expression in the patch compartment of dorsal striatum has not been examined. The current study examined the role of mu opioid receptors in psychostimulant induction of preprodynorphin, c-fos and zif/268 messenger RNA expression in the patch versus matrix compartments of dorsal striatum. Male Sprague-Dawley rats were treated with the mu opioid receptor antagonist, clocinnamox (1 mg/kg, s.c.), 24 h prior to treatment with cocaine (30 mg/kg, i.p.) or methamphetamine (15 mg/kg, s.c.) and sacrificed 45 min or 3 h later. Mu opioid receptor antagonism blocked psychostimulant-induced preprodynorphin messenger RNA expression only in the rostral patch compartment, whereas psychostimulant-induced zif/268 messenger RNA expression in the patch and matrix compartments was attenuated throughout the dorsal striatum. Clocinnamox pretreatment had no effect on stimulant-induced increases in c-fos expression. These data suggest that mu opioid receptor activation plays a specific role in psychostimulant-induced preprodynorphin messenger RNA expression in the rostral patch compartment and zif/268 messenger RNA expression throughout dorsal striatum.
Keywords: Methamphetamine; Cocaine; Immediate early gene; Opioid peptide; Basal ganglia;

5-HT1A receptor agonist affects fear conditioning through stimulations of the postsynaptic 5-HT1A receptors in the hippocampus and amygdala by XiaoBai Li; Takeshi Inoue; Tomohiro Abekawa; ShiMin Weng; Shin Nakagawa; Takeshi Izumi; Tsukasa Koyama (74-80).
Evidence from preclinical and clinical studies has shown that 5-HT1A receptor agonists have anxiolytic actions. The anxiolytic actions of 5-HT1A receptor agonists have been tested by our previous studies using fear conditioning. However, little is known about the brain regions of anxiolytic actions of 5-HT1A receptor agonists in this paradigm. In the present study, we investigated the effects of bilateral microinjections of flesinoxan, a selective 5-HT1A receptor agonist, into the hippocampus, amygdala and medial prefrontal cortex on the expression of contextual conditioned freezing and the defecation induced by conditioned fear stress in rats. These results reveal that both intrahippocampal and intraamygdala injections of flesinoxan decreased the expression of conditioned freezing, while injections into the medial prefrontal cortex did not. In addition, intraamygdala injection of flesinoxan attenuated the increased defecation induced by conditioned fear, but injections into the hippocampus and medial prefrontal cortex failed. These results suggest that flesinoxan exerts its anxiolytic actions in the fear conditioning through stimulations of the postsynaptic 5-HT1A receptors in the hippocampus and amygdala.
Keywords: Flesinoxan; 5-HT1A receptor agonist; Fear conditioning; The hippocampus; The amygdala; The medial prefrontal cortex;

Effects of μ-, δ- and κ-opioid receptor agonists on methamphetamine-induced self-injurious behavior in mice by Tomohisa Mori; Shinobu Ito; Taizo Kita; Minoru Narita; Tsutomu Suzuki; Toshiko Sawaguchi (81-87).
Opioid receptor agonists can differentially modify the behavioral effects of direct/indirect dopamine receptor agonists, such as methamphetamine, cocaine and apomorphine. However, the effects of opioid receptor agonists on high-dose methamphetamine-induced behavior have not yet been clarified. Therefore, the present study was undertaken to investigate the effects of μ (morphine)-, δ (SNC80)- and κ (U50,488H)-opioid receptor agonists on methamphetamine-induced self-injurious behavior and locomotor activity in mice. Methamphetamine (20 mg/kg) induced severe self-injurious behavior. In a combination test, some opioid receptor agonists significantly attenuated methamphetamine-induced self-injurious behavior, with potencies in the order morphine > buprenorphine (μ-opioid and κ-opioid receptor agonist/antagonist) > U50,488H, as maximum effects. These results suggest that the stimulation of μ- and κ-opioid receptors plays an inhibitory role in high-dose methamphetamine-induced stereotypic self-injurious behavior in mice, without affecting locomotor activity.
Keywords: Methamphetamine; Self-injurious behavior; Opioid receptor agonists;

Role of CGRP and GABA in the hypotensive effect of intrathecally administered anandamide to anesthetized rats by María del Carmen García; Edda Adler-Graschinsky; Stella Maris Celuch (88-98).
In urethane-anesthetized rats the intrathecal (i.t.) injection of 100 nmol anandamide produced a hypotensive effect (− 19.3 ± 1.6 mm Hg; n  = 6) that was mimicked by i.t. administration of 0.25 nmol calcitonin gene-related peptide (CGRP; − 26.2 ± 1.8 mm Hg, n  = 4). Both effects were antagonized either by the CGRP receptor antagonist CGRP8–37 (5 nmol; i.t.) or by the γ-aminobutyric acid (GABA)A receptor antagonist bicuculline (8.8 nmol, i.t) or by the GABAB receptor antagonist 2-hydroxy saclofen (110 nmol; i.t.). On the contrary, blockade of spinal CGRP receptors by CGRP8–37 did not modify the hypotensive response to either the GABAA-receptor agonist muscimol (8.8 nmol; i.t.) or the GABAB-receptor agonist baclofen (100 nmol; i.t). This result suggests a unidirectional effect of CGRP on the GABAergic system. The response to anandamide remained unaltered after acute inhibition of nitric oxide (NO) synthase activity by either i.t. (1 μmol) or i.v. (10 mg/kg) injection of N G-nitro-l-arginine methyl ester (L-NAME), but increased significantly after long-term L-NAME administration (70 mg/kg/day; four weeks; p.o.), thus suggesting compensatory changes in cardiovascular homeostasis. It is proposed that the hypotensive effect of anandamide in urethane-anesthetized rats could involve the release of CGRP followed by the release of GABA in the spinal cord. NO does not appear to have a direct participation in the spinal mechanisms involved in the decrease of the blood pressure caused by anandamide.
Keywords: Anandamide; Calcitonin gene-related peptide; γ-aminobutyric acid; Nitric oxide; Spinal cord; Blood pressure;

The development of nerve-induced activation, receptor properties and cellular signalling was examined by comparing the urinary bladder of new-born (0–2 days) and adult mice. Tissue strips were isolated and the effects of different neuromuscular agents on force were investigated during electrical field stimulation. The nerve-induced contractions of the urinary bladders from new-born mice were less influenced by desensitisation with α, β-methylene ATP and more sensitive to scopolamine compared with those of the adult bladder. There were no differences in α, β-methylene ATP or ATP responsiveness between adult and new-born tissue, showing that the lower purinergic component of the nerve-induced responses in the new-born mice was due to properties of the transmitter release rather than to a change in receptor function. Dose–response curves for carbachol revealed a lower peak response in new-born bladders compared with adults. The phasic component of the cholinergic contractions was pronounced and initiated at low carbachol concentrations in the new-born tissue. The carbachol contractions of both new-born and adult urinary bladder tissue were inhibited by the Rho kinase inhibitor Y27632 and by the protein kinase G activator 8-Br-cGMP. However, the sustained phase of carbachol contraction was significantly less sensitive to Y27632 and 8-Br-cGMP in new-born tissue. These results suggest that the receptor mediated calcium sensitisation mechanism is less prominent in new-born compared with adult mice and that the contractions of new-born bladders are less influenced by the nitric oxide (NO)-cGMP inhibitory pathway.
Keywords: Development; Cholinergic; Purinergic; Rho-kinase; cGMP;

Pharmacological characterization of urinary bladder smooth muscle contractility following partial bladder outlet obstruction in pigs by Ivan Milicic; Steven A. Buckner; Anthony Daza; Michael Coghlan; Thomas A. Fey; Michael E. Brune; Murali Gopalakrishnan (107-114).
Partial bladder outlet obstruction of the pig is considered as a valuable preclinical model for evaluating the profile of compounds for the treatment of bladder overactivity. In this study, we characterized the pharmacological properties of isolated bladder smooth muscle from pigs following partial outlet obstruction and its sensitivity to potassium channel openers. Bladder strips from obstructed animals showed significantly lower maximal efficacy (E max) and sensitivity to stimulation by ATP and carbachol, but not to those evoked by serotonin, compared to age-matched controls. Tissue strips from obstructed animals also showed a 2.5-fold increase in the potency and significantly reduced maximum response following K+ depolarization. With respect to spontaneous activity, bladder strips from control strips demonstrated little spontaneous phasic activity at all preloads examined. In contrast, bladder strips from obstructed animals showed large preload-dependent increases in spontaneous phasic activity at preload values of 16–32 g. The potencies of KATP channel openers to relax carbachol-evoked contractions showed a good 1 : 1 correlation (r 2  = 0.90) between obstructed and control bladder strips. These studies demonstrate that obstructed pig bladders show enhanced spontaneous phasic activity especially at elevated preloads, which may underlie unstable myogenic bladder contractions reported in cystometrographic measurements in vivo. The impaired responses to electrical field stimulation could be attributed to reduced efficacies and/or lower sensitivities of muscarinic and purinergic signaling pathways. KATP channel sensitivities remain essentially unimpaired in the obstructed bladder and could be effectively modulated by openers with potential for the treatment of overactive bladder secondary to outlet obstruction.
Keywords: Potassium channel opener; Detrusor smooth muscle; Smooth muscle relaxation; ATP-sensitive potassium channels; Sulfonylurea receptor; Outlet obstruction; Overactive bladder;

Functional inhibition of intestinal and uterine muscles by non-permeant triphenylethylene derivatives by Jorge Marrero-Alonso; Benito García Marrero; Tomás Gómez; Mario Díaz (115-127).
We have previously shown that the triphenylethylene antiestrogen tamoxifen reversibly inhibited spontaneous contractile activity in isolated duodenal muscle. Now, we have synthesized different quaternary ammonium salts of tamoxifen by changing the substituents on the nitrogen of the alkylaminoethoxy side-chain, to obtain plasma membrane impermeable compounds. Synthesized molecules were N-desmethyl-tamoxifen-hydrochloride, ethylbromide-tamoxifen and butylbromide-tamoxifen, which differed in the size of their ionic side-chain. All compounds rapidly and reversibly inhibited spontaneous and CaCl2-induced contractions in mouse duodenum and uterus. Dose-response analyses revealed a structure-activity relationship where the larger the side-chain the higher the inhibitory potency. Fourier analyses on triphenylethylene-relaxed duodenal tissues showed that harmonic components of contractile activity were readily recovered upon exposure to the L-type calcium channel agonist 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-pyridine-3-carboxilic acid methyl ester (BAY-K644). Likewise, BAY-K644 completely reversed triphenylethylene-induced effects on uterine tonic tension. Our experiments suggest that impermeant tamoxifen derivatives relax visceral smooth muscle through a membrane-mediated non-genomic mechanism that involves inhibition of L-type calcium channels.
Keywords: Tamoxifen derivatives; Impermeant triphenylethylene salts; Non-genomic effects; Uterine muscle; Intestinal muscle; Ca2+ channels;

The purpose of this study was to evaluate the efficacy of rolipram, a phosphodiesterase (PDE) 4 inhibitor, in a mouse model of dermatitis induced by repeated application of 2,4,6-trinitro-1-chlorobenzene (TNCB). BALB/c mice were sensitized with 0.3% w  /  v TNCB applied to the ear on day − 7, followed by application three times a week from day 0. Rolipram, prednisolone and cyclosporine A were administered orally once daily from day 0 to 21. Rolipram at a dose of 10 mg/kg/day significantly inhibited the ear thickness and the increase in cytokine levels and enzyme activity in the ear. Interleukin (IL)-4 production was markedly decreased in cervical lymph node cells from animals treated with rolipram at a dose of 10 mg/kg/day. Prednisolone and cyclosporine A significantly reduced ear thickness. These compounds significantly decreased the total cell and lymphocyte number of the cervical lymph nodes. Furthermore, prednisolone markedly suppressed body weight gain, and cyclosporine A significantly increased the serum total IgE concentration compared with that in the vehicle-treated control. Rolipram, unlike prednisolone and cyclosporine A, did not influence body weight and the total IgE concentration in the serum. The present results suggest that the PDE4 inhibitor is a promising oral medicine for the treatment of chronic skin inflammatory diseases.
Keywords: 2,4,6-trinitro-1-chlorobenzene; Rolipram; Phosphodiesterase 4; Dermatitis;

The melanocortin peptide HP228 displays protective effects in acute models of inflammation and organ damage by Stephen J. Getting; Clara Di Filippo; Michele D'Amico; Mauro Perretti (138-144).
The clinically efficacious melanocortin peptide HP228 has here been investigated for its anti-inflammatory efficacy. In this study we have investigated the efficacy of HP228 in murine acute models of inflammation and myocardial ischaemia. Systemic treatment of mice with HP228 inhibited neutrophil accumulation in zymosan; urate crystal and carrageenan induced inflammatory models. In the urate model this was due to inhibition of pro-inflammatory chemokines and cytokines, whilst different mechanisms exist for zymosan peritonitis and carrageenan-induced air-pouch inflammation. HP228 was next evaluated in a model of myocardial ischaemia, another condition where cytokines and neutrophils are thought to play a causal role. HP228 caused a 50% reduction in myocardial damage following reperfusion. HP228 therefore inhibits the most important facet of the host inflammatory response namely leukocyte migration. These data show for the first time that the clinically efficacious peptide HP228 displays protective effects in models of inflammation and organ damage.
Keywords: Melanocortin; Cytokine; Chemokine; Inflammation; Myocardial ischaemia;

Dihydrocucurbitacin B, isolated from Cayaponia tayuya, reduces damage in adjuvant-induced arthritis by José Miguel Escandell; María-Carmen Recio; Salvador Máñez; Rosa-María Giner; Miguel Cerdá-Nicolás; José-Luis Ríos (145-154).
23,24-Dihydrocucurbitacin B, from the anti-rheumatic plant Cayaponia tayuya, was tested on arthritis induced by adjuvant to corroborate the anti-inflammatory properties of this plant. Arthritis was induced in Lewis rats; the resulting arthritic rats were then treated with dihydrocucurbitacin B (1 mg/kg orally, daily, 1 week). The effect of dihydrocucurbitacin B on the synthesis, release, and activity of pro-inflammatory enzymes (elastase, cyclooxygenase-2, and nitric oxide synthase-2) as well as its effect on different mediators (tumor necrosis factor-α and interleukin-1β) were determined. Dihydrocucurbitacin B modified the evolution of the clinical symptoms, reducing the swelling and bone and tissue damage along with the development of the disease, modifying the cell infiltration and the expression of both nitric oxide synthase-2 and cyclooxygenase-2. In addition, it decreased the tumor necrosis factor-α and interleukin-1β production in lymphocytes, but did not modify it in macrophages.
Keywords: Dihydrocucurbitacin B; Adjuvant-induced arthritis; Nitric oxide synthase; Cyclooxygenase; Tumor necrosis factor-α; Interleukin-1β;

Role of substance P in allergic nasal symptoms in rats by Md. Ashequr Rahman; Toshio Inoue; Chiaki Kamei (155-161).
The present study was undertaken to investigate the pathological role of substance P in allergic nasal symptoms in rats. The topical application of substance P caused an increase in the incidence of sneezing and nasal rubbing in a dose-dependent fashion, and at a dose of 30 nM/site it showed a significant effect. L-732,138, a tachykinin NK1 receptor antagonist, at doses of 3 and 10 mg/kg showed a significant inhibition of the nasal signs induced by exogenous substance P in rats. In addition, L-732,138 also showed a significant inhibition of nasal behavior induced by antigen in actively sensitized rats at the same dose. On the other hand, histamine H1 receptor antagonists, such as cyproheptadine, epinastine and olopatadine had no effect on the nasal behaviors induced by exogenous substance P, even at higher doses, indicating that exogenous substance P does not cause the degranulation of mucosal mast cells in the rat. Moreover, all the histamine H1 receptor antagonists showed the dose-dependent inhibition of the nasal signs induced by antigen in actively sensitized rats, which revealed that the inhibition of these drugs was exhibited through the antagonistic effect on histamine H1 receptors. Therefore, from these results, it is reasonable to conclude that substance P released from the nasal mucosa through the activation of tachykinin NK1 receptors during the antigen antibody reaction plays an important role in allergic nasal symptoms.
Keywords: Tachykinin NK1 receptor antagonist; L-732,138; Substance P; Antigen; Sensitization; EEG (Electroencephalogram) spike; Olfactory bulb; Cyproheptadine; Olopatadine; Epinastine;

Modulation of iNOS expression by a nitric oxide-releasing derivative of the natural antioxidant ferulic acid in activated RAW 264.7 macrophages by Daniela Ronchetti; Francesco Impagnatiello; Massimiliano Guzzetta; Laura Gasparini; Monica Borgatti; Roberto Gambari; Ennio Ongini (162-169).
We have previously reported that NCX 2057, a new chemical entity bearing a nitric oxide (NO)-releasing moiety linked to the natural antioxidant ferulic acid, shows marked anti-inflammatory properties in a model of chronic brain inflammation. We have now studied the effects of NCX 2057 and its metabolic products, ferulic acid and NCX 2059, on inducible nitric oxide synthase (iNOS) expression and function in lipopolysaccharide/interferon-γ (LPS/IFNγ)-stimulated RAW 264.7 macrophages.NCX 2057 inhibited iNOS mRNA and protein expression (IC50  = 6.2 ± 1.0 μM) without altering iNOS protein degradation rate. NCX 2057 also decreased the levels of LPS/IFNγ-induced nitrite accumulation (IC50  = 4.3 ± 0.7 μM) in RAW 264.7 cells. Conversely, NCX 2059, which does not possess NO-donating properties, was only weakly effective (IC50  > 100 μM) and ferulic acid was inactive. To understand further the mechanisms underlying anti-inflammatory properties we studied the effects of NCX 2057 on selected transcription factors. Unlike ferulic acid, NCX 2057 inhibited LPS-induced translocation/activation of the nuclear factor, NF-κB, while other transcription factors, such as, Sp1, NF-IL2A and STAT-1 were not affected.The present data support the concept that NO adds important anti-inflammatory properties to ferulic acid. Thus, NCX 2057 represents a new prototype drug for the treatment of disorders associated with chronic inflammation and oxidative stress.
Keywords: Ferulic acid; NCX 2057(3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 4-(nitrooxy)butyl ester); Nitric oxide; NF-κB (nuclear factor kappa-B); Inflammation; RAW 264.7 macrophages;

Delta-9-tetrahydrocannabinol (THC) injection suppresses serum interleukin-12 (IL-12) levels in Legionella pneumophila-infected mice. Dendritic cells are a major producer of IL-12 and mouse, bone marrow-derived dendritic cell cultures produced high levels of the IL-12p40 following L. pneumophila infection. Treatment with THC suppressed this cytokine response in a concentration-dependent manner and the endocannabinoid, 2-arachidonoyolglycerol, less potently suppressed cytokine production. Dendritic cells expressed mRNA for cannabinoid receptor 1 (CB1), cannabinoid CB2 receptor, and vanilloid receptor 1 (TRPV1) and the addition of the Gi inhibitor, pertussis toxin, completely attenuated suppression induced by 3 and 6 μM THC but not by 10 μM THC. Furthermore, THC suppression was partially attenuated in dendritic cells from cannabinoid CB1 receptor and CB2 receptor knockout mice and in dendritic cells co-treated with THC and cannabinoid receptor antagonists. Cytokine suppression was not attenuated by pretreatment with the TRPV1 antagonist, capsazepine. These results suggest that THC-induced suppression of serum IL-12 is partly due to a suppression of IL-12 production by dendritic cells and that Gi signaling and cannabinoid receptors, but not TRPV1, are involved in this suppressive effect.
Keywords: IL-12; Cannabinoids; Vanilloid receptor; 2-arachidonoylglycerol; Dendritic cells;

Heme oxygenase 1 mediates anti-inflammatory effects of 2′,4′,6′-tris(methoxymethoxy) chalcone by Sung Hee Lee; Geom Seog Seo; Ji Yeong Kim; Xing Yu Jin; Hee-Doo Kim; Dong Hwan Sohn (178-186).
We report that the synthetic chalcone 2′,4′,6′-tris(methoxymethoxy) chalcone (TMMC) is an anti-inflammatory compound that reduces nitric oxide (NO) production by inhibiting of inducible NO synthase (iNOS) expression, and that TMMC decreases the degradation of the inhibitory factor κB, leading to inhibition of nuclear factor-κB translocation into the nucleus in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. We also demonstrate that TMMC by itself is a potent inducer of heme oxygenase 1 (HO-1). Inhibition of HO-1 activity or scavenging of carbon monoxide, a byproduct of heme degradation, significantly attenuated this anti-inflammatory action. Treating cells with the specific p42/44 MAPK inhibitor, PD98059, blocked the TMMC-mediated induction of HO-1 and the inhibition of LPS-stimulated expression of iNOS. TMMC also depleted intracellular GSH. Our data suggest that TMMC exerts an anti-inflammatory effect in macrophages through a mechanism that involves the induction of HO-1, which is mediated by activation of p42/44 MAPK and GSH depletion.
Keywords: 2′,4′,6′-Tris(methoxymethoxy) chalcone; Nitric oxide; Nuclear factor-κB; Heme oxygenase 1; p42/44 MAPK; GSH;

Beneficial effect of resveratrol on cholecystokinin-induced experimental pancreatitis by Annamária Szabolcs; Ilona S. Varga; Csaba Varga; Anikó Berkó; József Kaszaki; Tamás Letoha; László Tiszlavicz; Réka Sári; János Lonovics; Tamás Takács (187-193).
Resveratrol is a phytoalexin with strong antioxidant and anti-inflammatory effects reaching high concentrations in red wine. The aim of our study was to test the effects of resveratrol pretreatment on cholecystokinin-octapeptide (CCK-8)-induced acute pancreatitis in rats. Animals were divided into a control group, a group treated with CCK-8 and a group receiving 10 mg/kg resveratrol prior to CCK-8 administration. Resveratrol ameliorated the CCK-8-induced changes in the laboratory parameters, and reduced the histological damage in the pancreas. The drug failed to improve the pancreatic antioxidant state, but increased the amount of hepatic reduced glutathione and prevented the reduction of hepatic catalase activity. Resveratrol-induced inhibition of nuclear factor kappa B (NF-κB) activation or reduction of the pancreatic tumor necrosis factor-alpha (TNF-α) concentration could not be demonstrated. In conclusion, the beneficial effects of resveratrol on acute pancreatitis seem to be mediated by the antioxidant effect of resveratrol or by an NF-κB-independent anti-inflammatory mechanism.
Keywords: Resveratrol; Pancreatitis; Inflammation; Scavenger; Rat;