European Journal of Pharmacology (v.496, #1-3)

Characterisation of GEA 3175 on human platelets; by Anna K Asplund Persson; Louise Palmér; Peter Gunnarsson; Magnus Grenegård (1-9).
By comparing the effect of two nitric oxide (NO)-containing compounds, we found that S-nitroso-N-acetyl-d,l-penicillamine (SNAP), but not GEA 3175 (1,2,3,4-Oxatriazolium,3-(3-chloro-2-metylphenyl)-5-[[(4-methylphenyl)sulfonyl]amino]-, hydroxide inner salt), released NO. Despite this, both drugs elevated cyclic guanosine 3′,5′-monophosphate (cGMP) levels in human platelets. However, SNAP was more effective after short exposure times (5 and 20 s). The compounds also inhibited thrombin-induced rises in cytosolic Ca2+. Time studies revealed that the action of SNAP rapidly declined by increasing the length of incubation (from 5 s to 30 min). This desensibilisation phenomenon mainly involved the release of Ca2+ from intracellular stores. In comparison, GEA 3175-induced inhibition of cytosolic Ca2+ signalling was much more long-lasting. The soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reversed the effect of GEA 3175 on cytosolic Ca2+. Consequently, this inhibition depends solely on the increase in cGMP. In summary, differences between GEA 3175 and SNAP were observed in NO releasing, cGMP elevating and Ca2+ suppressive properties.
Keywords: NO (Nitric Oxide); cGMP (cyclic guanosine 3′,5′-monophosphate); Calcium; Aggregation; Platelet; SNAP (S-nitroso-N-acetyl-d,l-penicillamine); GEA 3175;

Terfenadine induces thymocyte apoptosis via mitochondrial pathway by Riyo Enomoto; Tomoe Komai; Yukari Yoshida; Chiyoko Sugahara; Emi Kawaguchi; Keiko Okazaki; Hiroki Kinoshita; Hiroto Komatsu; Yasuo Konishi; Eibai Lee (11-21).
The treatment of rat thymocytes with 10 μM terfenadine resulted in a significant increase in DNA fragmentation. The DNA fragmentation induced by terfenadine was dependent on its concentration and incubation time. In terfenadine-treated cells, the translocation of phosphatidylserine from the inside of plasma membrane to the outside, an early event of the apoptotic process, and chromatin condensation, the morphological characterization of apoptotic cell death, were observed. Terfenadine stimulated caspase-8, -9 and -3-like activities in an incubation time-dependent manner in thymocytes. The active forms of caspase-3 and -9 were detected in the extract from terfenadine-treated cells by immunoblotting analysis using specific antibodies to caspases, but active caspase-8 was not found in this fraction. Decrease in mitochondrial membrane potential and the release of cytochrome c from mitochondria to cytosol were observed in terfenadine-treated thymocytes. These results suggest that terfenadine induces apoptosis in rat thymocytes via mitochondrial pathway.
Keywords: Terfenadine; Apoptosis; Thymocyte; Mitochondria; Fexofenadine; Voltage-dependent K+ channel;

Penicillin blocks human α1β1 and α1β1γ2S GABAA channels that open spontaneously by Catarina E.L Lindquist; Julie E Dalziel; Brett A Cromer; Bryndis Birnir (23-32).
We used the open-channel blocker, penicillin (10 mM), as a tool to investigate if the human α1β1 or α1β1γ2S γ-aminobutyric acid type A (GABAA) receptor channels opened in the absence of GABA. Application of penicillin to cells expressing the receptors resulted in a transient inward whole-cell current, the off-current, upon penicillin removal. The amplitude of the off-current was dependent on the duration of the penicillin application, it reversed in polarity at depolarized potentials and exhibited “run-down” similar to the GABA-activated currents. Bicuculline (100 μM) blocked the off-current response. Pentobarbital (50 μM) enhanced the peak off-current amplitude by 2.8 and 3.4 in α1β1 and α1β1γ2S receptors, respectively. Diazepam (1 μM) only enhanced the off-current peak response in α1β1γ2S receptors (1.6) and induced the development of an inward current when applied alone. The results are consistent with that the α1β1 or α1β1γ2S GABAA receptors can open in the absence of GABA and raise the question of what role spontaneous channel openings have in the function of GABAA receptors.
Keywords: GABAA receptor; Benzodiazepine; Barbiturate; Inhibition; γ-aminobutyric acid; Spontaneous activity; Channel block; Tonic current;

N-(hydroxyphenyl)-arachidonamide (AM404) is an inhibitor of endocannabinoid transport. We examined the effects of AM404 on glutamatergic synaptic transmission using network-driven increases in intracellular Ca2+ concentration ([Ca2+] spikes) as an assay. At a concentration of 1 μM AM404 inhibited [Ca2+]i spiking by 73±8%. The cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), the vanilloid VR1 receptor antagonist capsazepine (CPZ), and treatment with pertussis toxin failed to block AM404-mediated inhibition. AM404 (3 μM) inhibited action-potential-evoked Ca2+ influx by 58±3% but failed to affect calcium influx evoked by depolarization with 30 mM K+, suggesting that the inhibition of electrically evoked [Ca2+]i increases and that [Ca2+]i spiking was due to inhibition of Na+ channels. Palmitoylethanolamide (PMEA), capsaicin (CAP) and (5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide (VDM11), compounds structurally similar to AM404, inhibited [Ca2+]i spiking by 34±10%, 42±18% and 67±12%, respectively. Thus, AM404 and related compounds inhibit depolarization-induced Ca2+ influx independent of cannabinoid receptors, suggesting caution when using these agents as pharmacological probes to study synaptic transmission.
Keywords: AM404; Excitatory synaptic transmission; N-acetyl ethanolamide; CB1 receptor; Cannabinoid; Hippocampus;

Concentration-dependent differential effects of quercetin on rat aortic smooth muscle cells by Chun-Ming Shih; Heng Lin; Yu-Chih Liang; Wen-Sen Lee; Wei-Fung Bi; Shu-Hui Juan (41-48).
Quercetin is one of the most ubiquitous bioflavonoids in foods of plant origin. Although quercetin is generally considered to provide protection against oxidative injury and inflammation, recent studies have demonstrated that its cytoprotective effects occur within a narrow concentration range. We attempted to examine the concentration-dependent effect on proliferation and inflammation in the primary culture of rat aortic smooth muscle cells. We demonstrate that quercetin inhibited [3H]thymidine incorporation into rat aortic smooth muscle cells only at concentrations ≦50 μM in a concentration-dependent manner. Nevertheless, quercetin, at concentrations ≧100 μM, reduced cell viability; this was further characterized as being due to apoptosis, which occurred through the proteolytic activation of pro-caspase-3. Additionally, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) substantially increased in rat aortic smooth muscle cells exposed to 100 μM quercetin, results which differ from observations by others and ourselves of cells exposed to ≦50 μM quercetin. Unlike P-JNK and P-p38, the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/ERK2) was not significantly affected by the concentration-dependent effects of quercetin. Surprisingly, the adverse effects of higher concentrations of quercetin could be ameliorated by adding the antioxidants, catalase, and N-acetylcysteine (NAC). Furthermore, the electrophoretic mobility shift assay (EMSA) showed that rat aortic smooth muscle cells exposed to quercetin at concentrations of ≦50 μM caused concentration-dependent inhibition of nuclear factor kappa B (NF-κB) activity, whereas concentrations of ≧100 μM resulted in increased NF-κB binding activity. We demonstrate for the first time that quercetin at low concentrations has antiproliferative and antiinflammatory effects, but at concentrations of ≧100 μM, is likely to induce the opposite effects on rat aortic smooth muscle cells.
Keywords: Aortic smooth muscle cell; Quercetin; Mitogen-activated protein kinase; Extracellular signal-regulated kinase; c-Jun N-terminal kinase;

Effects of amiodarone on mutant Na+/Ca2+ exchangers expressed in CCL 39 cells by Yasuhide Watanabe; Takahiro Iwamoto; Isao Matsuoka; Tomoyuki Ono; Munekazu Shigekawa; Junko Kimura (49-54).
Using the whole cell voltage clamp, we reported previously that amiodarone acutely inhibits Na+/Ca2+ exchange current (I NCX) in guinea pig cardiac ventricular myocytes. Intracellular application of trypsin via the patch pipette attenuated the blocking effect of amiodarone, suggesting that amiodarone affects the Na+/Ca2+ exchanger (NCX) from the cytoplasmic side. Here, we attempted to detect the site of amiodarone inhibition using wild type NCX1, mutants, and NCX3 expressed in CCL39 fibroblasts. I NCX was recorded by ramp pulses. Amiodarone at 30 μM inhibited I NCX by 80% in cells expressing wild type NCX1. However, 30 μM amiodarone inhibited I NCX by about 55% in cells expressing mutant NCX1 with amino acids 217-671 (ΔXIP) or 247-671 (Δ247-671) deleted in the long intracellular loop between the transmembrane segments (TM) 5 and 6. I NCXs from NCX mutants deleted of cytoplasmic TM1-2, TM3-4 or the C-terminus were inhibited by amiodarone to a similar extent as the wild type. Amiodarone also inhibited I NCX of NCX3 by 76%. These results suggest that a long intracellular loop may be involved in the inhibition of NCX1 by amiodarone, but that other intracellular loops, XIP region or C terminus are not involved in the amiodarone inhibition of NCX1.
Keywords: Antiarrhythmic drug; Amiodarone; Na+/Ca2+ exchange current; NCX1; CCL39 fibroblast;

The study of the interaction of non-steroidal anti-inflammatory drugs (NSAIDs) with several reactive oxygen species is of great interest in inflammatory conditions where an uncontrolled release of these potentially damaging intermediates has been documented. This study focused on the scavenging of three species (hydroxyl radical, hydrogen peroxide and hypochlorous acid) with several members of the oxicam family and with the sulfoanilide nimesulide. Reaction with hydroxyl radical was assessed by the modified deoxyribose assay, and rate constants were calculated showing values between 0.8 and 1.1×1010 M−1 s−1 for oxicams and of about 0.9×1010 M−1 s−1 for nimesulide and ibuprofen. These were consistent with those of the literature but in the same range as those for other NSAIDs and for several thiol-containing molecules. The study of hydrogen peroxide scavenging by the horseradish peroxidase (HRP) assay lacked specificity but no interaction could be evidenced by the glutathione peroxidase assay. The scavenging of hypochlorous acid was finally investigated by the recently developed para-aminobenzoic acid assay which demonstrated better performances for meloxicam (1.7×104 M−1 s−1) as compared to the other oxicams (tenoxicam: 4.0×104 M−1 s−1, piroxicam: 3.6×104 M−1 s−1, lornoxicam: 4.3×104 M−1 s−1) and nimesulide (2.3×103 M−1 s−1). These rate constants were, however, lower than those for thiol-containing molecules and ascorbate. These results suggest that the antioxidant properties of NSAIDs could be influenced by a proper pharmacomodulation as far as the scavenging of hypochlorous acid is concerned while the interest is quite limited for the scavenging of hydroxyl radical.
Keywords: Antioxidant activity; Non-steroidal anti-inflammatory drug; Hydroxyl radical; Hydrogen peroxide; Hypochlorous acid;

Deglycosylation of Fas receptor and chronic morphine treatment up-regulate high molecular mass Fas aggregates in the rat brain by Marı́a Julia Garcı́a-Fuster; Marcel Ferrer-Alcón; Antonio Miralles; Jesús Andrés Garcı́a-Sevilla (63-69).
This study was designed to immunodetect and characterize Fas receptor aggregates (oligomerization) in the brain and to assess its possible modulation in opiate addiction. High molecular mass, sodium dodecyl sulfate (SDS)- and β-mercaptoethanol-resistant Fas aggregates (∼110/120 and ∼203 kDa specific peptides) were immunodetected with a cytoplasmic domain-specific antibody in brain tissue (rat, mouse and human) and SH-SY5Y cells by Western blot analysis. Preincubation of rat cortical membranes with N-ethylmaleimide (NEM; 1 mM for 1 h at 37 °C) reduced the immunodensity of ∼203 kDa Fas aggregates (51%) and increased that of 35 kDa native Fas (172%) and 51/48 kDa glycosylated Fas (47%), indicating that disulfide bonds are involved in Fas dimerization. Enzymatic N-deglycosylation of Fas receptor increased the content of Fas aggregates (∼110/120 kDa: five- to sixfold, and ∼203 kDa: two- to threefold), suggesting that Fas glycosylation is involved in regulating receptor dimerization. Chronic (10–100 mg/kg for 5 days), but not acute (30 mg/kg for 2 h), treatment with morphine (a μ-opioid peptide receptor agonist) induced up-regulation of Fas aggregates in the brain (∼110/120 kDa: 39%, and ∼203 kDa: 89%). The acute and/or chronic treatments with δ- and κ-opioid peptide receptor agonists and with a σ1-receptor agonist did not readily alter the content of Fas aggregates in the rat brain. The results indicate that Fas aggregates are natively expressed in the brain and that its density is regulated by the state of Fas glycosylation. These forms of Fas (receptor homodimerization) are functionally relevant because they were up-regulated in the brain of morphine-dependent rats.
Keywords: Fas receptor aggregate; Fas protein; Deglycosylation; Opiate drug; Morphine addiction; Brain;

Xenon suppresses nociceptive reflex in newborn rat spinal cord in vitro; comparison with nitrous oxide by Ippei Watanabe; Makoto Takenoshita; Tadashi Sawada; Ichiro Uchida; Takashi Mashimo (71-76).
Although analgesic action of xenon has been reported, little is known about the effect of xenon at the spinal cord, which plays a crucial role in nociceptive transmission. We studied the effect of xenon on nociceptive reflex (the slow ventral root potential) and the monosynaptic reflex in neonatal rat spinal cord in vitro in comparison with nitrous oxide. Xenon (30%) and nitrous oxide (30%) were applied for 17 min through superfusing artificial cerebrospinal fluid. Xenon and nitrous oxide significantly reduced the amplitude of nociceptive reflex by ∼70% and ∼25%, respectively. Xenon and nitrous oxide also significantly reduced the amplitude of the monosynaptic reflex by ∼35% and ∼15%, respectively. These results indicate that xenon suppressed the synaptic transmission at the spinal cord, especially those of the slow ventral root potential, which reflect nociceptive transmission.
Keywords: General anesthetic; Inhalation; Monosynaptic reflex; Nociceptive reflex; Slow ventral root potential; Neonatal spinal cord;

Endothelin ETB receptors inhibit articular nociception and priming induced by carrageenan in the rat knee-joint by Josélia B Daher; Glória E.P Souza; Pedro D'Orléans-Juste; Giles A Rae (77-85).
The participation of the endothelin system on nociception and priming induced by carrageenan in the knee-joint was investigated. Intra-articular (i.a.) carrageenan (300 μg) caused long-lasting nociceptive effects (i.e., increases in paw elevation time [PET]), which were potentiated by endothelin-1 (dual endothelin ETA/ETB receptor agonist) and inhibited by sarafotoxin S6c (endothelin ETB receptor agonist; both at 30 pmol, i.a., 24 h beforehand). Priming the naive joint with carrageenan augmented nociceptive responses to a second carrageenan challenge, 72 h later. Carrageenan-induced priming, but not nociception, was potentiated by local BQ-788 (10 nmol, i.a., 15 min before priming; endothelin ETB receptor antagonist; N-cis-2,6-dimethylpiperidinocarbonyl-l-γ-methylleucyl-d-1-methoxycarbonyl-tryptophanil-d-norleucine), but BQ-123 (endothelin ETA receptor antagonist; cyclo [d-Asp-Pro-d-Val-Leu]) was ineffective. Sarafotoxin S6c markedly suppressed carrageenan-induced priming to nociception triggered by carrageenan, endothelin-1 or sarafotoxin S6c, and BQ-788 prevented this action. Thus, selective endothelin ETB receptor agonists inhibit carrageenan-induced nociception and priming in the naive joint. This priming effect of carrageenan to nociception evoked by subsequent inflammatory insults is limited by an endothelin ETB receptor-operated mechanism.
Keywords: Articular incapacitation; Hyperalgesia; Analgesia; Arthritic pain; IRL 1620; Endothelin receptor;

Evidence that GABAergic neurons in the spinal trigeminal nucleus are involved in the transmission of inflammatory pain in the rat: a microdialysis and pharmacological study by Andrea Viggiano; Marcellino Monda; Alessandro Viggiano; Maria Chiefari; Caterina Aurilio; Bruno De Luca (87-92).
The aim of this experiment was to investigate the role of the γ-aminobutyric acid (GABA)-ergic transmission in the nociception within the spinal trigeminal nucleus.The formalin test was used as an animal model of inflammatory pain. Two groups of six rats were used. The behavioural response to the labial injection of formaldehyde (50 μl of a 5% solution) (group 1) or saline (group 2) was evaluated by recording the time spent in facial grooming during a period of 8 min (one period before and seven consecutive periods after the injection). The extracellular concentration of GABA in the trigeminal caudalis nucleus was evaluated, during the formalin test, on samples of 30 μl each (one sample before and three samples after the labial injection) obtained by microdialysis and analysed by HPLC with electrochemical detection of the o-phtalaldeyde pre-column derivate. Subsequently, three more groups of six rats each were injected with saline, muscimol (GABAa receptor agonist), or bicuculline (GABAa receptor antagonist) in the trigeminal caudalis nucleus, before performing the formalin test.The injection of formaldehyde induced a biphasic behavioural response and an increase of the GABA levels at 15–45 min. The injection of bicuculline, but not muscimol or saline, strongly decreased the behavioural response of the formalin test.These findings suggest that GABAergic neurons in the trigeminal caudalis nucleus are involved in the transmission of nociceptive information.
Keywords: GABAergic neuron; Spinal trigeminal nucleus; Microdialysis;

α2-Adrenoceptors and 5-HT receptors mediate the antinociceptive effect of new pyrazolines, but not of dipyrone by Maria Celoni M Godoy; Michele R Fighera; Fabiane R Souza; Ariane E Flores; Maribel A Rubin; Marlı́ R Oliveira; Nilo Zanatta; Marcos A.P Martins; Helio G Bonacorso; Carlos F Mello (93-97).
In this study, we investigated whether spinal noradrenergic and serotonergic systems are involved in the antinociception induced by the novel pyrazolines 3-methyl- and 3-phenyl-5-hydroxy-5-trichloromethyl-4,5-dihydro-1H-1-pyrazole-1-carboxyamide (MPCA and PPCA, respectively), and the pyrazolinone dipyrone in the acetic acid writhing (stretching) test in mice. Intrathecal (i.t.) administration of methysergide (3 and 10 μg) and yohimbine (3 μg), but not of prazosin (0.3 and 1 μg) prevented the antinociceptive action of MPCA and PPCA (500 μmol/kg, s.c.). Dipyrone-induced antinociception (500 μmol/kg, s.c.) was not affected by methysergide or adrenoceptor antagonists. These results suggest that spinal 5-HT receptors and α2-adrenoceptors are involved in the antinociception induced by MPCA and PPCA, but not in that elicited by dipyrone.
Keywords: Dipyrone; Antinociception; Serotonin; Methysergide; Yohimbine; Pyrazoline;

Reversal of Δ9-tetrahydrocannabinol-induced tolerance by specific kinase inhibitors by Caroline E Bass; Sandra P Welch; Billy R Martin (99-108).
Tolerance develops to the pharmacological effects of Δ9-tetrahydrocannabinoid (THC) following repetitive administration. Adaptations in signaling pathways involved in tolerance to THC-induced behaviors are not understood. The objective of our study was the evaluation of kinase involvement in the expression of tolerance to the above four THC-induced behaviors. Kinase inhibitors that specifically inhibit cyclic AMP-dependent protein kinase (PKA), cyclic GMP-dependent protein kinase (PKG), calmodulin-dependent protein kinase (PKC) and src tyrosine kinase were tested for reversal of tolerance to THC's effects. PKG and PKC inhibitors did not reverse tolerance in any behavioral measure. Src tyrosine kinase inhibition reversed tolerance to only the hypoactive effects of THC. PKA inhibition reversed tolerance to all measures, although the doses of inhibitor and time-course of inhibition varied among behaviors. Thus, our data suggest that PKA activity plays a major role in THC-induced tolerance, and that THC produces its multiple effects through different signaling pathways.
Keywords: Cannabinoid; Kinase; Antinociception; Tolerance;

Spontaneous contractions of the rat portal vein were potentiated in magnitude by phenylephrine, cyclopiazonic acid, ryanodine or caffeine. All these drugs can deplete Ca2+ from intracellular stores, which stimulates store-operated cation entry in some tissues. The possibility that depletion of Ca2+ from intracellular stores potentiates the spontaneous contractions was therefore investigated using functional experiments. Phenylephrine or cyclopiazonic acid was added to tissues in Ca2+-free Krebs solution, followed by a 30-min washout. After addition of extracellular Ca2+, the spontaneous contractions were potentiated. This showed the stimulus for potentiating the contractions remained so long as intracellular Ca2+ stores were depleted. Following phenylephrine washout in normal Krebs solution, potentiation of the spontaneous contractions was attenuated with time. This attenuation was abolished by the protein kinase C inhibitor calphostin C. These results show depletion of Ca2+ from intracellular stores potentiates spontaneous contractions of the portal vein. Protein kinase C may inhibit this mechanism.
Keywords: Portal vein, rat; Spontaneous contraction; Store-operated Ca2+ entry; Cyclopiazonic acid; Ryanodine; Protein kinase C;

Involvement of nitric oxide in amiodarone- and dronedarone-induced coronary vasodilation in guinea pig heart by Pierre Guiraudou; Sylvie Cosnier Pucheu; Regine Gayraud; Patrick Gautier; Alain Roccon; Jean Marc Herbert; Dino Nisato (119-127).
Amiodarone, a powerful antiarrhythmic compound, possesses coronary and peripheral vasodilator properties. The mechanisms responsible for these effects remain incompletely understood. In the present study, the coronary effects of amiodarone and dronedarone, a non-iodinated amiodarone-like compound, were investigated in isolated guinea pig hearts perfused at constant flow with high K+ solution (40 mM). Amiodarone (0.01–10 μM), dronedarone (0.01–1 μM) and verapamil (0.01–10 μM) induced concentration-dependent decreases in coronary perfusion pressure. Amiodarone- and dronedarone-mediated reductions in coronary perfusion pressure were not modified by a cyclooxygenase inhibitor, indomethacin (3 μM). l-Nitro-l-arginine (l-NOARG; 3–100 μM) caused a rightward shift of concentration–response curves to amiodarone and dronedarone in a dose-dependent way; l-arginine, a nitric oxide (NO) precursor, reversed this effect. Furthermore, when guinea pigs were treated with N G-nitro-l-arginine methyl ester (l-NAME; 20 mg/kg), amiodarone could not reduce coronary perfusion pressure. NO synthase inhibition did not affect the coronary perfusion pressure response to verapamil. 1H-[1,2,4]Oxadiazole (4,3-a) quinoxalin-1-one (ODQ), a specific inhibitor of the guanylyl cyclase, inhibited the effects of amiodarone but not those of verapamil. In the presence of l-NOARG and ODQ, and in hearts from animals treated with l-NAME, a decrease in coronary perfusion pressure was still observed at the highest concentration of dronedarone. These results show that, in guinea pig hearts, the coronary vasodilation induced by amiodarone highly depends on nitric oxide. Dronedarone differs from amiodarone by a remaining relaxant effect, refractory to inhibition of NO synthase pathway, probably due to its Ca+ antagonist properties.
Keywords: Amiodarone; Dronedarone; l-NOARG; Nitric oxide; (Guinea pig); Coronary pressure;

The orally active nonpeptide selective endothelin ETA receptor antagonist YM598 prevents and reverses the development of pulmonary hypertension in monocrotaline-treated rats by Hironori Yuyama; Akira Fujimori; Masanao Sanagi; Akiko Koakutsu; Katsumi Sudoh; Masao Sasamata; Keiji Miyata (129-139).
We investigated the preventive and therapeutic effects of the selective endothelin ETA receptor antagonist potassium(E)-N-[6-methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl)pyrimidin-4-yl]-2-phenylenthenesulfonamidate (YM598) on the development of pulmonary hypertension in monocrotaline-induced pulmonary hypertensive and hypoxemic rats. In the prevention study, oral administration of YM598 (0.1 and 1 mg/kg) or bosentan (30 mg/kg) for 4 weeks was started on the day following monocrotaline (60 mg/kg) injection. In the therapeutic study, oral administration of YM598 (0.1, 0.3 and 1 mg/kg) for 2 weeks was started 3 weeks after monocrotaline injection. In the prevention study, a marked increase in pulmonary arterial pressure and right ventricular hypertrophy, a decrease in right cardiac function and hypoxemia were observed. Histopathological examination indicated the presence of pulmonary remodeling, including medial wall thickening of the pulmonary microvasculature and alveolar disorders. YM598 suppressed the increase in pulmonary arterial pressure, right ventricular hypertrophy and systemic congestion, and improved the hypoxemia, but bosentan had only modest effects. Histopathological disorders were also ameliorated by YM598. In the therapeutic study, YM598 also ameliorated the pulmonary hypertension and hypoxemia in monocrotaline-treated rats. These results suggest that YM598 effectively prevented and reversed the development of pulmonary hypertension, and reduced the pulmonary vascular remodeling and parenchymal injury in monocrotaline-treated rats. YM598 also improved hypoxemia which accompanied with the severe pulmonary hypertension in these rats.
Keywords: Endothelin; Heart failure; Hypertension; Hypoxia;

Aldehyde dehydrogenase, nitric oxide synthase and superoxide in ex vivo nitrate tolerance in rat aorta by Ivan S de la Lande; Jacqueline M Stepien; Andrew C Philpott; Patrick A Hughes; Irene Stafford; John D Horowitz (141-149).
The role of aldehyde dehydrogenase (ALDH) in ex vivo tolerance to transdermal glyceryl trinitrate was explored in rat aorta. ALDH activity, measured by aldehyde-induced NADH formation, was strongly depressed in the tolerant arteries. ALDH inhibitors, chloral hydrate (0.3 mM) and cyanamide (0.1–1 mM) inhibited relaxation to glyceryl trinitrate in non-tolerant and tolerant arteries. The inhibition differed from tolerance in that (a) the glyceryl trinitrate concentration–response curve was sigmoidal cf. biphasic in tolerance, (b) the potentiating effect of nitric oxide synthase (eNOS) inhibition was unchanged cf. increased in tolerance and (c) superoxide inhibited the response cf. no significant effect in tolerant or non-tolerant arteries. Hence, reduced ALDH activity does not account fully for ex vivo tolerance. The discrepancies are consistent with evidence that (a) organic nitrates, unlike chloral and cyanamide, irreversibly inactivate ALDH (hence reduced enzyme saturability can explain the biphasic curve) and (b) eNOS contributes to tolerance by a mechanism independent of glyceryl trinitrate metabolism.
Keywords: Glyceryl trinitrate; Tolerance, ex vivo; Aldehyde dehydrogenase; Superoxide; Nitric oxide synthase;

Cocaine HCl is well known for its toxic effects on the cardiovascular system, but little is known about its effects on different regional blood vessels. We designed experiments to determine if cocaine HCl could influence the tension of isolated aortic rings, i.e., induce contraction or relaxation. Surprisingly, cocaine HCl (1×10−5 to 6×10−3 M) relaxed isolated aortic rings precontracted by phenylephrine in a concentration-dependent manner. No significant differences were found between intact or denuded isolated aortic rings (P>0.05). The maximal % relaxations of intact vs. denuded isolated aortic rings were 108.9±24.3% vs. 99.5±8.3% (P>0.05). Cocaine HCl, 2×10−3 M, was found to inhibit contractions by phenylephrine; EC50s were increased (P<0.01) and Emax's were decreased (51.3±16.4% vs. 89.8±10.6%, P<0.01). A variety of amine antagonists could not inhibit the relaxant effects of cocaine HCl (P>0.05). The cyclooxygenase-1 inhibitor, indomethacin, also failed to inhibit relaxations induced by cocaine HCl (P>0.05). Neither l-arginine, N(G)-monomethyl-l-arginine (l-NMMA), nor methylene blue could inhibit the relaxations induced by cocaine HCl (P>0.05), suggesting cocaine HCl does not relax isolated aortic rings by inducing the synthesis or release of nitric oxide (NO) or prostanoids from either endothelial or vascular muscle cells. Inhibitors of cAMP, cGMP and protein kinase G (PKG) also failed to inhibit cocaine-induced relaxations. Cocaine HCl (1×10−5 to 6×10−3 M) could also relax isolated aortic rings precontracted by phenylephrine in high K+ depolarizing buffer. Surprisingly, calyculin A, an inhibitor of myosin light chain (MLC) phosphatase, inhibited cocaine-induced relaxations in a concentration-dependent manner, suggesting the probable importance of cocaine-induced MLC phosphatase activation in rat aortic smooth muscle cells. It was also found that cocaine HCl could dose-dependently inhibit Ca2+-induced contractions of isolated aortic rings in high K+–Ca2+-free buffer, suggesting that cocaine HCl may inhibit Ca2+ influx and/or intracellular release.
Keywords: Aortic ring; Cocaine; Relaxation; Phosphatase; Ca2+ entry; Aortic dissection;

Role of endothelin ETB receptor in the pathogenesis of monocrotaline-induced pulmonary hypertension in rats by Masahiro Nishida; Yuka Okada; Kenji Akiyoshi; Keiko Eshiro; Masanori Takaoka; Cheryl E. Gariepy; Masashi Yanagisawa; Yasuo Matsumura (159-165).
We investigated the role of endothelin ETB receptor in the development of monocrotaline-induced pulmonary hypertension, by using the spotting-lethal (sl) rat, which carries a naturally occurring deletion in the endothelin ETB receptor gene. Three weeks after injection of saline or monocrotaline (60 mg/kg, s.c.), hemodynamics, cardiac hypertrophy and endothelin-1 levels in right ventricle were determined. Monocrotaline produced a marked pulmonary hypertension associated with increases in right ventricular pressure and hypertrophy, pulmonary arterial medial thickening and the endothelin-1 levels. These monocrotaline-induced alterations tended to be enhanced in ETB-deficient homozygous rats, compared with cases in wild-type rats. The treatment with the selective ETA receptor antagonist ABT-627 [2R-(4-methoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid] for 3 weeks (10 mg/kg/day, twice daily) almost completely suppressed the monocrotaline-induced pulmonary hypertension and related organ damage both in ETB-deficient and wild-type animals to the same levels. Thus, we suggest that the antagonism of the ETA receptor is essential for the protection from monocrotaline-induced pulmonary hypertension, irrespective of the presence of the ETB receptors, although a protective role of ETB receptor-mediated action in the pathogenesis of this disease model cannot be ruled out.
Keywords: Endothlin-1; ETA receptor; ETB receptor; ETB receptor-deficient rat; Pulmonary hypertension; Monocrotaline;

The cardiovascular and renal effects of a highly potent μ-opioid receptor agonist, cyclo[N ε,N β-carbonyl-d-Lys2,Dap5]enkephalinamide by Jolanta Gutkowska; Marek Jankowski; Danuta Pawlak; Suhayla Mukaddam-Daher; Jan Izdebski (167-174).
Investigation of the acute cardiovascular and renal effects of cyclo[N ε,N β-carbonyl-d-Lys2,Dap5]enkephalinamide (cUENK6), the most potent μ-opioid receptor agonist, revealed dose-related effects, but most pronounced during the first hour post i.v. injections. During first hour, cUENK6 (3 μg/rat) stimulated (P<0.001) excretion of urine (1.1±0.2 vs. 3.3±0.3 ml/h), sodium (60±10 vs. 124±12 μeq/h), potassium and cGMP (1.76±0.19 vs. 4.92±0.80 nmol/h). These effects were inhibited by naloxone (4 mg/kg i.v.), but not by naloxonazine (35 mg/kg s.c.), or 4 mg/kg i.v. naloxone methiodide. cUENK6 stimulated urinary atrial natriuretic peptide (ANP)-like activity (113±12 vs. 167±20 pg/h, P<0.02) and the effect was totally abolished by naloxone. cUENK6 also suppressed the transient stress-induced elevation in blood pressures and heart rate that occurred over the first 30-min post-injection, an effect attenuated by naloxone. Plasma ANP increased 2-h post-injection (123±11 vs. 192±21 pg/ml, P<0.005), and was associated with augmented ANP mRNA levels in right atria and left ventricles. Thus, cUENK6 evokes renal effects by enhancing activity of the renal natriuretic peptide system.
Keywords: Opioids; Enkephalin analogue cUENK6; Naloxonazine; Diuresis; Natriuresis; Blood pressure; Natriuretic peptides; Urodilatin; Vasopressin;

Urocortin does not reduce the renal injury and dysfunction caused by experimental ischaemia/reperfusion by Nimesh S.A Patel; Marika Collin; Christoph Thiemermann (175-180).
Recent evidence indicates that activators of the serine/threonine kinase pathway protect against ischaemia/reperfusion. Here, we investigate the effects of renal ischaemia/reperfusion on the degree of renal dysfunction and injury with urocortin in rats. Rats treated with urocortin or its vehicle (saline) were subjected to bilateral renal artery occlusion (45 min) and reperfusion (6 h). At the end of experiments, the following indicators and markers of renal injury and dysfunction were measured: plasma urea, creatinine and aspartate aminotransferase, urine flow and creatinine clearance. Urocortin (1 or 15 μg/kg i.v.), administered 5 min prior to reperfusion, was not able to significantly reduce plasma urea, creatinine and aspartate aminotransferase indicating a non-protective effect on the renal dysfunction and reperfusion-injury caused by ischaemia/reperfusion. In addition, 15 μg/kg urocortin significantly depressed urine flow and creatinine clearance, which was associated with a significant depression in mean arterial pressure, indicating reduced renal perfusion. Thus, we propose that the pharmacological application of urocortin does not reduce the renal injury caused by bilateral renal ischaemia/reperfusion.
Keywords: Kidney; Reperfusion-injury; Renal dysfunction; Urocortin;

Time-dependent reduction of acetylcholine-induced relaxation in corpus cavernosum of cholestatic rats: role of nitric oxide and cyclooxygenase pathway by Mehdi Dehghani; Hamed Sadeghipour; Hamed Shafaroodi; Hooman Honar; Kiarash Riazi; Mohammad Reza Ebrahimkhani; Amir Reza Hajrasouliha; Sina Tavakoli; Ahmad Reza Dehpour (181-187).
The endothelium-dependent relaxation of corpus cavernosum smooth muscle and the roles of nitric oxide (NO) and arachidonic acid products of cyclooxygenase were investigated in non-operated, SHAM-operated, and bile duct-ligated rats. We further investigated the time-dependent alterations of corpus cavernosum relaxation in 2-, 7-, and 14-day bile duct-ligated animals. Acetylcholine produced concentration-dependent relaxation in phenylephrine-precontracted strips of corpus cavernosum. A significant reduction in the acetylcholine-induced relaxation was observed 2 days after bile duct ligation, and a greater reduction was observed on subsequent days. Incubation with 20 μM indomethacin reduced the acetylcholine-induced relaxation of the corpus cavernosum of unoperated rats while it had no effect in the corpus cavernosum of bile duct-ligated rats. Chronic treatment with Nω-Nitro-l-Arginine Methyl Ester (L-NAME, 3 mg/kg/day, intraperitoneally) reduced the relaxation responses in the unoperated group while it had no effect in the bile duct-ligated group. These results show that acetylcholine-induced corporal relaxation is impaired in cholestatic rats, and this may be related to deficient nitric oxide production by the endothelium. The involvement of prostaglandins in this impairment seems unlikely.
Keywords: Corpus cavernosum relaxation; Cholestasis; Acetylcholine; Indomethacin; Nitric oxide; (Rat);

In vitro antibacterial and anti-inflammatory effects of honokiol and magnolol against Propionibacterium sp. by Junho Park; Jongsung Lee; Eunsun Jung; Yumi Park; Kukhyun Kim; Byunghwa Park; Kwangseon Jung; Eunkyung Park; Jieun Kim; Deokhoon Park (189-195).
Honokiol and magnolol, two major phenolic constituents of Magnolia sp., have been known to exhibit antibacterial activities. However, until now, their antibacterial activity against Propionibacterium sp. has not been reported. To this end, the antibacterial activities of honokiol and magnolol were detected using the disk diffusion method and a two-fold serial dilution assay. Honokiol and magnolol showed strong antibacterial activities against both Propionibacterium acnes and Propionibacterium granulosum, which are acne-causing bacteria. The minimum inhibitory concentrations (MIC) of honokiol and magnolol was 3–4 μg/ml (11.3–15 μM) and 9 μg/ml (33.8 μM), respectively. In addition, the killing curve analysis showed that magnolol and honokiol killed P. acnes rapidly, with 105 organisms/ml eliminated within 10 min of treatment with either 45 μg (169.2 μM) of magnolol or 20 μg (75.2 μM) of honokiol per ml. The cytotoxic effect of honokiol and magnolol was determined by a colorimetric (3-(4,5-dimetyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT) assay using two animal cell lines, human normal fibroblasts and HaCaT. In this experiment, magnolol exhibited lower cytotoxic effects than honokiol at the same concentration, but they showed similar cytotoxicity when triclosan was employed as an acne-mitigating agent. In addition, they reduced secretion of interleukin-8 and tumor necrosis factor α (TNF-α) induced by P. acnes in THP-1 cells indicating the anti-inflammatory effects of them. When applied topically, neither phenolic compound induced any adverse reactions in a human skin primary irritation test. Therefore, based on these results, we suggest the possibility that magnolol and honokiol may be considered as attractive acne-mitigating candidates for topical application.
Keywords: Antibacterial; Anti-inflammatory; MIC (Minimum inhibitory concentration); MBC (Minimum bactericidal concentration); Irritation;

Tumour necrosis factor-α mediates neutrophil migration to the knee synovial cavity during immune inflammation by Gustavo Bombini; Cláudio Canetti; Francisco A.C Rocha; Fernando Q Cunha (197-204).
Tumour necrosis factor (TNF)-α, interleukin-1β, interleukin-8 and leukotriene B4 have an important role on neutrophil recruitment during immune-inflammation. Here we evaluated the participation of several inflammatory mediators on ovalbumin-induced neutrophil recruitment in the knee articular space of immunized rats. Ovalbumin administration in immunized, but not in control, rats induced a dose- and time-dependent neutrophil accumulation, which was inhibited by dexamethasone, pentoxifylline or thalidomide, but not by selective inhibitors of nitric oxide (nitro-l-arginine), platelet-activating factor (BN50730 or UK74505), prostaglandins (indomethacin), histamine (meclisine) or leukotriene B4 (MK 886 and CP105,696). Anti-TNF-α antiserum, but not anti-interleukin-1β or anti-CINC-1 (cytokine-induced neutrophil chemoattractant 1) antisera, impaired ovalbumin-induced neutrophil accumulation. High amounts of TNF-α were detected in the exudates, which was inhibited by dexamethasone, pentoxifylline and thalidomide. These results suggest a specific role for TNF-α in this model, and the ability of pentoxifylline and thalidomide to inhibit both neutrophil influx and TNF-α release may have therapeutic implications in arthritis.
Keywords: TNF-α; Arthritis; Neutrophil; Immune inflammation; Pentoxifylline; Thalidomide;

Treatment of type IIb familial combined hyperlipidemia with the combination pravastatin-piperazine sultosilate by Lluı́s Masana; Jesús Villoria; Mariano Sust; Emilio Ros; Nuria Plana; Francisco Pérez-Jiménez; Miguel Franco; Josefina J Oliván; Xavier Pintó; Sebastián Videla (205-212).
The risk of coronary heart disease is increased for any given low-density lipoprotein (LDL) cholesterol level in patients with high levels of triglycerides because some triglyceride-rich lipoproteins are atherogenic. This paper reports the results of a pilot clinical trial aimed to evaluate a novel triglyceride-lowering drug in combination with pravastatin to treat combined hyperlipidemia. Twenty-six patients with type 2b hyperlipoproteinemia were randomized to receive pravastatin 40 mg/day or pravastatin 40 mg/day plus piperazine-sultosilate 1000 mg/day for 12 weeks if their cholesterol levels, but not triglyceride levels, had responded to therapeutic lifestyle changes and treatment with 40 mg/day of pravastatin. Concentrations of triglycerides, cholesterol and apolipoproteins A and B were measured in duplicate before and after the intervention. There were no significant differences between groups in the change from baseline in the concentration of serum triglycerides. Conversely, significant differences were found for LDL cholesterol, which increased slightly with pravastatin alone but decreased with the combination (12.605±22.777% vs. −6.396±13.157%, respectively; p=0.022). Apolipoprotein-B levels increased with pravastatin alone but remained stable with the combined treatment (10.464±8.446% vs. 0.767±12.335%; P=0.028). The increase in the pravastatin group was significant. Although sultosilate was not efficacious in reducing triglycerides, it helped to decrease the concentration of small, dense, atherogenic LDL particles that are less receptor-sensitive and which could accumulate during long-term statin therapy in patients with high levels of triglycerides.
Keywords: Benzenesulfonate; Familial combined hyperlipidemia; Hypercholesterolemia; Lipoprotein;

GLUT1-deficient mice exhibit impaired endothelium-dependent vascular relaxation by James L Park; Charles W Heilig; Frank C Brosius (213-214).
We tested the hypothesis that decreased glucose transporter 1 (GLUT1) expression alters endothelial function. Nitric oxide-dependent endothelial relaxation, but not endothelium-independent relaxation, was significantly reduced in aortas from transgenic mice expressing GLUT1 antisense mRNA, compared to aortas from nontransgenic littermates. These data suggest that GLUT1-dependent glucose metabolism may play an important role in regulating endothelial function.
Keywords: GLUT1 (glucose transporter1); Endothelium; Antisense;

Author index (215-216).

Keyword index (217-221).