European Journal of Pharmacology (v.669, #1-3)

The promise of EPC-based therapies on vascular dysfunction in diabetes by Adriana Georgescu; Nicoleta Alexandru; Andrei Constantinescu; Irina Titorencu; Doina Popov (1-6).
Diabetes mellitus is one of the most common metabolic diseases in the world and the vascular dysfunction represents a challenging clinical problem. In diabetes, endothelial cells (ECs), lining the inner wall of blood vessels, do not function properly and contribute to impaired vascular function. Circulating endothelial progenitor cells (EPCs), the precursor of mature EC, actively participate in endothelial repair, by moving to the vascular injury site to form mature EC and new blood vessels. Knowing that the therapeutic interventions can improve only a part of EC dysfunction in diabetes, this review addresses recent findings on the use of EPCs for cell therapy. The strategies proposed in review are based on in vivo and in vitro studies and, thus, their physiological relevance is confirmed. EPC therapy shows great promise for the prevention and cure of diabetes-induced vascular dysfunction.
Keywords: Diabetes; Vascular dysfunction; Circulating endothelial progenitor cell;

Discovery of a novel neuroprotective compound, AS1219164, by high-throughput chemical screening of a newly identified apoptotic gene marker by Takao Yamazaki; Masakazu Muramoto; Osamu Okitsu; Noriyuki Morikawa; Yasuhiro Kita (7-14).
We have reported that tacrolimus (FK506), an immunosuppressive drug, and diclofenac, a non-steroidal anti-inflammatory drug, possess different modes of neuroprotective action. FK506 suppresses only thapsigargin-induced apoptosis in neuroblastoma SH-SY5Y cells while diclofenac reverses tunicamycin-induced as well as thapsigargin-induced apoptosis. The aim of this study is to discover novel compounds that exert neuroprotective properties by using the transcriptional response of a newly identified gene, which was regulated by both FK506 and diclofenac, as a surrogate screening marker in high-throughput chemical screening and characterize the compounds in comparison with FK506 and diclofenac. Using a microarray with 4504 human cDNAs and quantitative RT-PCR, two genes as apoptotic markers, transmembrane protein 100 (TMEM100) and limb-bud and heart (LBH), were identified because the thapsigargin-induced elevations in their mRNA levels were reversed by both FK506 and diclofenac. A luciferase reporter assay with a TMEM100 promoter region was applied to high-throughput chemical screening. AS1219164, {3-[(E)-2-{5-[(E)-2-pyridin-4-ylvinyl]pyridin-3-yl} vinyl]aniline}, suppressed thapsigargin-induced transactivation of the TMEM100 gene and reversed thapsigargin-induced increases in TMEM100 and LBH mRNA levels in SH-SY5Y cells, similar to the effects of FK506 and diclofenac. Furthermore, AS1219164 protected against SH-SY5Y cell death induced by four apoptotic agents including thapsigargin, similar to diclofenac, but was more potent than diclofenac, while FK506 only showed protective effects against thapsigargin-induced cell death. In conclusion, a novel neuroprotecitve compound, AS1219164, was discovered by high-throughput chemical screening using a reporter assay with the TMEM100 gene promoter regulated by both FK506 and diclofenac. Reporter assay using the promoter region of a gene under pharmacological and physiological transcriptional regulation would be well suit for use in high-throughput chemical screening.
Keywords: AS1219164; FK506; Diclofenac; Neuroprotection; TMEM100 gene; LBH gene;

Novel electropharmacological activity of amiodarone on human HCN channels heterologously expressed in the Xenopus oocytes by Xinrong Fan; Yongjun Chen; Pan Wu; Junlian Xing; Hui Chen; Tao Song; Jing Yang; Jun Zhang; Congxin Huang (15-23).
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the pacemaker currents (If) in cardiac cells. The objectives of this study were to investigate the electropharmacological activity of amiodarone on human HCN channels heterologously expressed in Xenopus laevis oocytes. hHCN channels were expressed in oocytes and studied using the standard two-electrode voltage-clamp techniques. The results show that amiodarone blocks hHCN channels heterologously expressed in the Xenopus oocytes in a concentration- and use-dependent manner, but doesn't modify the voltage dependence of activation and reversal potentials. And the removal of blockage of HCN channels by amiodarone was favored by inward current flow, not by hyperpolarizing potential. Characteristics of blockage on hHCN channels were consistent with those of amiodarone as “trapped” drugs on human ether-a-go-go-related gene (HERG) channels. These results will be useful for elucidating the potentially antiarrhythmic mechanism of amiodarone.
Keywords: Amiodarone; hHCN channel; Voltage-clamp; Xenopus oocyte;

Cloning and pharmacological characterization of the dog cannabinoid CB2 receptor by Christian Ndong; Dajan O'Donnell; Sultan Ahmad; Thierry Groblewski (24-31).
Comparison of human, rat and mouse cannabinoid CB2 receptor primary sequences has shown significant divergence at the mRNA and protein sequence level, raising the possibility of species specific pharmacological properties. Additionally, given the importance of the dog as a non-rodent species for predicting human safety during the drug development process, we cloned the dog CB2 receptor gene and characterized its in-vitro pharmacological properties in a recombinant expression system. A 1.1 kb dog peripheral cannabinoid receptor (dCB2) fragment encoding a 360 amino acid protein was cloned from dog spleen cDNA. Analysis of the cloned dCB2 polypeptide sequence revealed that it shares between 76 and 82% homology with rat, mouse, human and predicted chimpanzee cannabinoid CB2 receptors. The dog CB2 receptor expressed in CHO cells displayed similar binding affinities for various synthetic and endogenous cannabinoids as compared to those measured for the human and rat cannabinoid CB2 receptors. However, these ligands exhibited altered functional potencies and efficacies for the dog cannabinoid CB2 receptor, which was also found to be negatively coupled to adenylate cyclase activity. These complex pharmacological differences observed across species for the cannabinoid CB2 receptor suggest that caution should be exerted when analyzing the outcome of animal efficacy and safety studies, notably those involving cannabinoid CB2 receptor targeting molecules tested in the dog.
Keywords: Cannabinoid receptor; Cloning; Radioligand binding; Species selectivity; Pharmacology; GPCR; cAMP assay;

Interaction between ciprofloxacin and melanin: The effect on proliferation and melanization in melanocytes by Artur Beberok; Ewa Buszman; Dorota Wrześniok; Michał Otręba; Janina Trzcionka (32-37).
There have been described serious adverse events caused by ciprofloxacin in pigmented tissues. It is known that some fluoroquinolones bind well to melanin rich tissues, but the relation between their affinity to melanin and the skin or eye toxicity is not well documented. The aim of this study was to examine whether ciprofloxacin binds to melanin, and how this interaction affects the proliferation and melanization in melanocytes. We have demonstrated that complexes which ciprofloxacin forms with melanin possess at least two classes of independent binding sites. Their association constants are K1  ~ 105  M− 1 and K2  ~ 102  M− 1, respectively. Ciprofloxacin has induced evident concentration-dependent loss in melanocytes viability. The value of ED50 was found to be ~ 0.5 mM. It has also been shown that ciprofloxacin reduces melanin content, and decreases tyrosinase activity in human skin melanocytes. The ability of ciprofloxacin to interact with melanin and its inhibitory effect on melanization in melanocytes in vitro may explain a potential role of melanin in the mechanisms of ciprofloxacin toxic effects in vivo.The demonstrated effect of ciprofloxacin on cell viability and melanization process in pigmented cells in vitro can imply the role of melanocytes and melanin in vivo in the mechanisms of undesirable side effects of this drug on pigmented tissues.Display Omitted
Keywords: Ciprofloxacin; Melanin; Melanocyte; Melanization; Tyrosinase activity;

A novel indirubin derivative PHII-7 potentiates adriamycin cytotoxicity via inhibiting P-glycoprotein expression in human breast cancer MCF-7/ADR cells by Ruizan Shi; Wei Li; Xiuli Zhang; Yanjun Zhang; Hongwei Peng; Yinliang Xie; Dongmei Fan; Rong Liu; Xuyi Liu; Dongsheng Xiong (38-44).
Multidrug resistance (MDR) is a major impediment to the effective chemotherapy of many human malignancies, and novel MDR reversal agents are desirable for combination therapy to reduce MDR, enhance anti-tumor activity and reduce side effects. Overexpression of P-glycoprotein (P-gp) is the most prevalent cause of MDR in cancer tissues, and resistance to apoptosis is a common characteristic for the multidrug resistant cancer cells. Our group has synthesized a novel potent anti-tumor indirubin derivative, PHII-7. In this study, MCF-7/ADR cells, an adriamycin (ADR)-selected human breast tumor cell line with the MDR phenotype, were used to investigate the anticancer properties of this novel indirubin derivative. Cytotoxicity and apoptosis assays showed that PHII-7 significantly inhibited cell growth, induced apoptosis, potentiated ADR cytotoxicity and restored chemotherapy sensitivity in the MDR cancer cells. Further studies indicated that by down-regulation of P-gp expression, PHII-7 partially inhibited P-gp efflux pump function and increased intracellular accumulation of Rhodamine 123, a P-gp substrate. These results provide a biochemical basis for possible clinical application of PHII-7 alone or in combination with conventional antineoplastic agents in the treatment MDR tumors.
Keywords: PHII-7; MDR (multidrug resistance); P-gp (P-glycoprotein); Reversal effect;

Cancer cell lines derived from hepatocytes have an altered phenotype and they lack hepatocyte-specific functions. It is at least partly due to the under-expression of transcription factors such as hepatocyte nuclear factor 4α (HNF4α), steroid receptor co-activator 1 (SRC1) etc. Recently, a strategy of transient transfection of human hepatic cells with HNF4α revealed improved hepatospecific functions, including the expression of drug-metabolizing enzymes. In the current study we established a human cell line derived from HepG2 cells stably transfected with human HNF4α, and we examined this line for hepatospecific markers. Of the 9 clones analyzed, we found an increased secretion of fibrinogen (9 clones), albumin (5 clones) and plasminogen (3 clones), while secretion of alpha1-antitrypsin was not changed. The expression of pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) proteins but not mRNAs was slightly increased. TCDD-dependent induction of CYP1A1 mRNA and protein was augmented in 50% of clones, but there was no correlation between the CYP1A1 inducibility and expression levels of AhR and HNF4α. Induction of CYP3A4 mRNA by rifampicin was about 1.5–2.5 fold (clones 2, 4, 6, 7) and it was not significantly different from CYP3A4 mRNA induction in parent HepG2. The basal expression of CYP3A4 protein was increased in all clones, but rifampicin-induced expression of CYP3A4 protein was in all clones lower than in parent HepG2. Overall, the stable over-expression of HNF4α in HepG2 cells restores some of the hepatospecific functions, but it has a minor effect on the expression of xenobiotic-metabolizing enzymes and their regulators.
Keywords: Drug metabolism; Cell line; Hepatocytes; Cytochrome P450;

The clozapine metabolite N-desmethylclozapine displays variable activity in diverse functional assays at human dopamine D2 and serotonin 5-HT1A receptors by Peter Heusler; Liesbeth Bruins Slot; Amélie Tourette; Stéphanie Tardif; Didier Cussac (51-58).
N-desmethylclozapine (NDMC or norclozapine) is the major active metabolite of the antipsychotic clozapine in humans. The activity of NDMC differs from clozapine at a number of neurotransmitter receptors, probably influencing the pharmacological effects of clozapine treatment. Here, we tested the properties of NDMC in comparison with clozapine at recombinant human dopamine D2 and serotonin 5-HT1A receptors, using a panel of functional assays implicating diverse signalling pathways. At dopamine D2 receptors, NDMC as well as clozapine did not display agonist activity in measures of G protein activation by [35S]GTPγS binding and in the sensitive Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) phosphorylation assay. In contrast, there were weak partial agonist actions of NDMC (but not of clozapine) for dopamine D2-dependent activation of Ca2+ liberation via coexpressed chimeric Gαq/o proteins and for G protein-coupled inward rectifier potassium channel (GIRK) current induction in Xenopus oocytes. Intriguingly, GIRK currents induced by NDMC via dopamine D2 receptors showed a rapid and transient time course, strikingly different from currents recorded with other receptor agonists. At serotonin 5-HT1A receptors, NDMC was a more efficacious partial agonist than clozapine for [35S]GTPγS binding, ERK1/2 phosphorylation and GIRK activation. Respective low and moderate partial agonist properties of NDMC at dopamine D2 and serotonin 5-HT1A receptors thus differentiate the metabolite from its parent drug and may contribute to the overall effects of clozapine pharmacotherapy.
Keywords: N-desmethylclozapine; Clozapine; Antipsychotics; Dopamine D2 receptor; Serotonin 5-HT1A receptor;

Pain remains a significant clinical challenge and currently available analgesics are not adequate to meet clinical needs. Emerging evidence suggests the role of imidazoline I2 receptors in pain modulation primarily from studies of the non-selective imidazoline receptor ligand, agmatine. However, little is known of the generality of the effect to selective I2 receptor ligands. This study examined the antinociceptive effects of two selective I2 receptor ligands 2-BFI and BU224 (> 2000-fold selectivity for I2 receptors over α2 adrenoceptors) in a hypertonic (5%) saline-induced writhing test and analyzed their interaction with morphine using a dose-addition analysis. Morphine, 2-BFI and BU224 but not agmatine produced a dose-dependent antinociceptive effect. Both composite additive curve analyses and isobolographical plots revealed a supra-additive interaction between morphine and 2-BFI or BU224, whereas the interaction between 2-BFI and BU224 was additive. The antinociceptive effect of 2-BFI and BU224 was attenuated by the I2 receptor antagonist/α2 adrenoceptor antagonist idazoxan but not by the selective α2 adrenoceptor antagonist yohimbine, suggesting an I2 receptor-mediated mechanism. Agmatine enhanced the antinociceptive effect of morphine, 2-BFI and BU224 and the enhancement was prevented by yohimbine, suggesting that the effect was mediated by α2 adrenoceptors. Taken together, these data represent the first report that selective I2 receptor ligands have substantial antinociceptive activity and produce antinociceptive synergy with opioids in a rat model of acute pain. These data suggest that drugs acting on imidazoline I2 receptors may be useful either alone or in combination with opioids for the treatment of pain.
Keywords: Imidazoline I2 receptor; Morphine; Antinociception; Writhing test;

The effect of morphine sensitization on extracellular concentrations of GABA in dorsal hippocampus of male rats by Maryam Farahmandfar; Mohammad-Reza Zarrindast; Mehdi Kadivar; Seyed Morteza Karimian; Nasser Naghdi (66-70).
Repeated, intermittent exposure to drugs of abuse, such as morphine results in response enhancements to subsequent drug treatments, a phenomenon referred to as behavioral sensitization. As persistent neuronal sensitization may contribute to the long-lasting consequences of drug abuse, characterizing the neurochemical mechanisms of sensitization is providing insights into addiction. Although it has been shown that GABAergic systems in the CA1 region of dorsal hippocampus are involved in morphine sensitization, the alteration of extracellular level of GABA in this area in morphine sensitization has not been investigated. In the present study, using the in vivo microdialysis technique, we investigated the effect of morphine sensitization on extracellular GABA concentration in CA1 region of dorsal hippocampus of freely moving rats. Sensitization was induced by subcutaneous (s.c.) injection of morphine, once daily for 3 days followed by 5 days free of the opioid treatment. The results showed that extracellular GABA concentration in CA1 was decreased following acute administration of morphine in non-sensitized rats. However, morphine-induced behavioral sensitization significantly increased the extracellular GABA concentration in this area. The enhancement of GABA in morphine sensitized rats was inhibited by administration of naloxone 30 min before each of three daily doses of morphine. These results suggest an adaptation of the GABAergic neuronal transmission in dorsal hippocampus induced by morphine sensitization and it is implied that opioid receptors may play an important role in this effect.
Keywords: Morphine; Behavioral sensitization; GABA; Hippocampus; Microdialysis;

Brain regions mediating α3β4 nicotinic antagonist effects of 18-MC on nicotine self-administration by Stanley D. Glick; Elizabeth M. Sell; Sarah E. McCallum; Isabelle M. Maisonneuve (71-75).
18-Methoxycoronaridine (18-MC), a putative anti-addictive agent, has been shown to decrease the self-administration of several drugs of abuse in rats. 18-MC is a potent antagonist at α3β4 nicotinic receptors. Consistent with high densities of α3β4 nicotinic receptors being located in the medial habenula and the interpeduncular nucleus, 18-MC has been shown to act in these regions to decrease both morphine and methamphetamine self-administration. The present study was conducted to determine if 18-MC's effect on nicotine self-administration is mediated by acting in these same brain regions. Because moderate densities of α3β4 receptors occur in the dorsolateral tegmentum, ventral tegmental area, and basolateral amygdala, these brain areas were also examined as potential sites of action of 18-MC. Local administration of 18-MC into either the medial habenula, the basolateral amygdala or the dorsolateral tegmentum decreased nicotine self-administration. Surprisingly, local administration of 18-MC into the interpeduncular nucleus increased nicotine self-administration while local administration of 18-MC into the ventral tegmental area had no effect on nicotine self-administration. Similar effects were produced by local administration of either mecamylamine or conotoxin AuIB. These data are consistent with the hypothesis that 18-MC decreases nicotine self-administration by indirectly modulating the dopaminergic mesolimbic pathway via blockade of α3β4 nicotinic receptors in the medial habenula, basolateral amygdala, and dorsolateral tegmentum. The data also suggest that an action of 18-MC in the interpeduncular nucleus may attenuate aversive and/or depressive effects of nicotine.Display Omitted
Keywords: 18-Methoxycoronaridine; α-Conotoxin AuIB; Mecamylamine; Nicotine; Medial habenula; Interpeduncular nucleus; Basolateral amygdala; Ventral tegmental area; Dorsolateral tegmentum; α3β4 nicotinic receptors; Drug self-administration; Drug addiction;

The present paper is to examine whether liquiritigenin is able to attenuate the Alzheimer's-like learning and memory deficits in a transgenic (Tg) mouse model that over-expresses amyloid protein precursor (APP), and explores the underlying mechanisms. Consistent with our previous observations, we found that treatment with liquiritigenin improved the behavioral performance of Tg mice and it attenuated the protein expression of oligomeric form of amyloid β-peptide (Aβ). Furthermore, treatment with liquiritigenin inhibited astrocytosis in the hippocampus, and it may through its inhibitory activities on Notch-2, an important molecular regulating neural proliferation and differentiation. These findings provide evidence for beneficial activity of liquiritigenin in a mouse model of Alzheimer's disease and support the continued investigation of Notch signaling pathway as a target for treatment of Alzheimer's disease.
Keywords: Alzheimer's disease; Liquiritigenin; β-amyloid peptide; Behavior; Astrogliosis; Notch-2;

Antihypertensive, insulin-sensitising and renoprotective effects of a novel, potent and long-acting angiotensin II type 1 receptor blocker, azilsartan medoxomil, in rat and dog models by Keiji Kusumoto; Hideki Igata; Mami Ojima; Ayako Tsuboi; Mitsuaki Imanishi; Fuminari Yamaguchi; Hiroki Sakamoto; Takanobu Kuroita; Naohiro Kawaguchi; Nobuhiro Nishigaki; Hideaki Nagaya (84-93).
The pharmacological profile of a novel angiotensin II type 1 receptor blocker, azilsartan medoxomil, was compared with that of the potent angiotensin II receptor blocker olmesartan medoxomil. Azilsartan, the active metabolite of azilsartan medoxomil, inhibited the binding of [125I]–Sar1–I1e8-angiotensin II to angiotensin II type 1 receptors. Azilsartan medoxomil inhibited angiotensin II-induced pressor responses in rats, and its inhibitory effects lasted 24 h after oral administration. The inhibitory effects of olmesartan medoxomil disappeared within 24 h. ID50 values were 0.12 and 0.55 mg/kg for azilsartan medoxomil and olmesartan medoxomil, respectively. In conscious spontaneously hypertensive rats (SHRs), oral administration of 0.1–1 mg/kg azilsartan medoxomil significantly reduced blood pressure at all doses even 24 h after dosing. Oral administration of 0.1–3 mg/kg olmesartan medoxomil also reduced blood pressure; however, only the two highest doses significantly reduced blood pressure 24 h after dosing. ED25 values were 0.41 and 1.3 mg/kg for azilsartan medoxomil and olmesartan medoxomil, respectively. In renal hypertensive dogs, oral administration of 0.1–1 mg/kg azilsartan medoxomil reduced blood pressure more potently and persistently than that of 0.3–3 mg/kg olmesartan medoxomil. In a 2-week study in SHRs, azilsartan medoxomil showed more stable antihypertensive effects than olmesartan medoxomil and improved the glucose infusion rate, an indicator of insulin sensitivity, more potently (≥ 10 times) than olmesartan medoxomil. Azilsartan medoxomil also exerted more potent antiproteinuric effects than olmesartan medoxomil in Wistar fatty rats. These results suggest that azilsartan medoxomil is a potent angiotensin II receptor blocker that has an attractive pharmacological profile as an antihypertensive agent.
Keywords: Angiotensin II type 1 receptor; Azilsartan medoxomil; TAK-491; Hypertension; Insulin resistance; Proteinuria;

Vasodilation of retinal arterioles induced by activation of BKCa channels is attenuated in diabetic rats by Asami Mori; Sachi Suzuki; Kenji Sakamoto; Tsutomu Nakahara; Kunio Ishii (94-99).
The large-conductance Ca2+-activated K+ (BKCa) channels modulate the retinal vascular tone, but question of whether the impairment of the channel function contributes to abnormalities of retinal circulation has not yet been completely elucidated. The purpose of this study was to examine effects of diabetes on the vasodilation induced by activation of BKCa channels. Male Wistar rats were treated with streptozotocin and experiments were performed 2 weeks later. The streptozotocin-treated animals were given drinking water containing 5% d-glucose to shorten the term in the development of retinal vascular dysfunction. The retinal vascular responses were assessed by measuring diameter of retinal arterioles in the fundus images that were captured with an original fundus camera system. In non-diabetic rats, vasodilator effects of acetylcholine on retinal arterioles were significantly reduced by iberiotoxin, an inhibitor of BKCa channels. However, the inhibitory effect of iberiotoxin was not observed in diabetic rats, and the responses to the BKCa channel opener BMS-191011 were almost completely abolished. The retinal vasodilator response to acetylcholine, possibly an endothelium-derived hyperpolarizing factor-mediated response, observed after treatment with NG-nitro-l-arginine methyl ester and indomethacin was markedly reduced in diabetic rats. The responses to pinacidil, an opener of ATP-sensitive K+ channels, were unchanged. These results suggest that the retinal vasodilator response mediated through mechanisms involving activation of BKCa channels is diminished at the early stage of diabetes in rats. The impairment of BKCa channel function may contribute to abnormal retinal hemodynamics in diabetes and consequently play an important role in the pathogenesis of diabetic retinopathy.
Keywords: Acetylcholine; Endothelium-derived hyperpolarizing factor; Large-conductance Ca2+-activated K+ channel; Retinal circulation;

Curine, a bisbenzylisoquinoline alkaloid, blocks L-type Ca2+ channels and decreases intracellular Ca2+ transients in A7r5 cells by Marcos A.A. Medeiros; José F. Pinho; Daysiane P. De-Lira; José M. Barbosa-Filho; Demetrius A.M. Araújo; Steyner F. Cortes; Virgínia S. Lemos; Jader S. Cruz (100-107).
Curine is a novel bisbenzylisoquinoline alkaloid that has previously been reported as a vasodilator. The underlying mechanism(s) of the vasodilator effect of curine remains to be characterized. In this study, we investigated the cellular mechanism that is responsible for the vasodilator effect of curine in the rat aorta. The vasorelaxant activity of curine was recorded using a myograph. Ca2+ currents in A7r5 cells were measured using the whole-cell patch-clamp technique. Intracellular Ca2+ transients were determined using confocal microscopy. In a concentration-dependent manner, curine inhibited contractions elicited by high extracellular K+ and Bay K8644 in the rat aorta and reduced the rise in the intracellular Ca2+ concentration induced by membrane depolarization in response to an increase in extracellular K+ concentration in vascular smooth muscle cells. Moreover, curine decreased the peak amplitude of L-type Ca2+ currents (I Ca,L) in a concentration-dependent manner without changing the characteristics of the current density vs. voltage relationship and the steady-state activation of I Ca,L. Furthermore, curine shifted the steady-state inactivation curve of I Ca,L toward more hyperpolarized membrane potentials. None of the following modified the effect of curine on I Ca,L amplitude: 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterases; dibutyryl cyclic AMP, an activator of protein kinase A (PKA); or 8-Br-cyclic GMP, an activator of protein kinase G (PKG). Our results showed that curine inhibited the L-type voltage-dependent Ca2+ current in rat aorta smooth muscle cells, which caused a decrease in intracellular global Ca2+ transients that led to vasorelaxation.
Keywords: Curine; Rat aorta; A7r5 cells; Vasodilation; L-type calcium channel;

Experimentally-induced hyperthyroidism in rodents is associated with signs and symptoms of pulmonary hypertension. The main objective of the present study was to investigate the effect of thyroxine-induced pulmonary hypertension on the contractile response of the pulmonary artery to 5-HT and the possible underlying signaling pathway. 5-HT concentration-dependently contracted artery segments from control and thyroxine-treated rats with pD2 values of 5.04 ± 0.19 and 5.34 ± 0.14, respectively. The maximum response was significantly greater in artery segments from thyroxine-treated rats. Neither BW 723C86 (5-HT2B-receptor agonist) nor CP 93129 (5-HT1B-receptor agonist) contracted ring segments of the pulmonary artery from control and thyroxine-treated rats at concentrations up to 10− 4  M. There was no significant difference in the level of expression of 5-HT2A-receptor protein between the two groups. Ketanserin (3 × 10− 8  M) produced a rightward shift of the concentration–response curve to 5-HT in both groups with equal potency (− logKB values were 8.1 ± 0.2 and 7.9 ± 0.1 in control and thyroxine-treated rats, respectively). Nifedipine (10− 6  M) inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. The calcium-activated chloride channel blocker, niflumic acid (10− 4  M) also inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. It was concluded that hyperthyroidism enhanced 5-HT-induced contractions of the rat pulmonary artery by a mechanism involving increased activity of calcium-activated chloride channels.
Keywords: Pulmonary artery; Thyroxine; 5-HT; Niflumic acid; Nifedipine; Ketanserin;

Both α2B- and α2C-adrenoceptor subtypes are involved in the mediation of centrally induced gastroprotection in mice by Zoltán S. Zádori; Nashwan Shujaa; Serena B. Brancati; Lutz Hein; Klára Gyires (115-120).
α2-adrenoceptors are known to mediate gastroprotective effect in both acid-dependent and acid-independent ulcer models. The aim of the present study was to determine, which of the three α2-adrenoceptor subtypes (α2A, α2B or α2C) is responsible for this protection. Various α2-adrenoceptor agonists and antagonists were administered intracerebroventricularly (i.c.v.) to C57BL/6 mice with deletion of genes encoding the different subtypes. The gastric mucosal damage was induced by orally injected acidified ethanol. Both the non-selective α2-adrenoceptor agonist clonidine (0.3–2.8 nmol) and the α2B/C-adrenoceptor subtype preferring agonist ST-91 (0.5–11.5 nmol) induced dose-dependent gastroprotective effect in wild type, α2A-, α2B- and α2C-KO mice. In contrast, the α2A-adrenoceptor subtype agonist oxymetazoline (0.07–84 nmol i.c.v.) reduced only slightly the development of ethanol-induced ulcers. The effect of clonidine was antagonized by the non-selective antagonist yohimbine (25 nmol) and the α2B/C-adrenoceptor antagonist ARC 239 (10.4 nmol), but not by the α2A-adrenoceptor antagonist BRL 44408 (7.5 nmol). ARC 239 also reversed the effect of clonidine in α2A-, α2B- and α2C-KO mice, while the selective α2C-adrenoceptor antagonist JP 1302 (52 nmol) antagonized that only in α2B-KO, but not in α2A- and α2C-KO mice. These results suggest that α2B- and α2C-adrenoceptor subtypes can equally contribute to the mediation of gastroprotective effect induced by α2-adrenoceptor agonists in mice.
Keywords: Gastroprotection; Ethanol-induced ulcer; α2-adrenoceptor subtype; Knock out mouse;

Selective histamine H3 and H4 receptor agonists exert opposite effects against the gastric lesions induced by HCl in the rat stomach by Gabriella Coruzzi; Maristella Adami; Cristina Pozzoli; Iwan J.P. de Esch; Rogier Smits; Rob Leurs (121-127).
The present study investigated the role of histamine H3 and H4 receptors in gastric mucosal defense, by the use of selective ligands. Firstly, the affinities of several histaminergic agonists for the rat histamine H3 and H4 receptors were checked in HEK 293 T cells transfected with either receptor subtype. Next, functional activities were determined in conscious rat against the ulcerogenic effect of 0.6 N HCl. Radioligand binding studies showed that immethridine and methimepip were the most selective agonists at rat H3 receptors, whereas VUF10460 displayed approximately a 50-fold selectivity for the rat H4 receptor over the H3 receptor. In conscious rats, immethridine and methimepip significantly reduced (66% and 48% inhibition, respectively) the gastric lesions induced by HCl; the effect of immethridine was antagonized by the H3 receptor antagonist A-331440, but not by the H4 receptor antagonist JNJ7777120. The mixed H3/H4 receptor agonist immepip induced a significant aggravation of HCl damage, which was prevented by JNJ7777120; HCl-induced lesions were also significantly enhanced by the H4 receptor agonists VUF10460 and VUF8430; however, this effect was not modified by JNJ7777120. Overall, this study indicates that, whereas the histamine H3 receptor is involved in the protection of rat stomach against concentrated HCl, the functional role of the H4 receptor is still to be defined, although selective agonists induce proulcerogenic effects under HCl challenge. Finally, the species-dependent variations in affinity and receptor selectivity observed for most ligands need to be carefully addressed in the pharmacological characterization of histamine H3 and H4 receptor functions in vivo.
Keywords: Histamine H3 receptor; Histamine H4 receptor; Binding; Gastric lesions; HCl;

Eicosapentaenoic acid regulates IκBα and prevents tubulointerstitial injury in kidney by Osamu Takase; Keiichi Hishikawa; Nozomu Kamiura; Masanori Nakakuki; Hiroyuki Kawano; Kiyoshi Mizuguchi; Toshiro Fujita (128-135).
Fish oil containing n-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is well known to prevent the progression of IgA nephropathy. However, the mechanism through which fish oil prevents the progression of renal injury remains uncertain. We tried to clarify the effects of EPA on tubulointerstitial injury in the kidney both in vivo and in vitro. We examined the effects of EPA, especially to focus on nuclear factor kappa B (NF-κB), using Thy-1 nephritis models. Also the mechanism of EPA was investigated using small-interfering RNA (siRNA) in lipopolysaccharide (LPS)-stimulated proximal tubular epithelial cells (PTECs). In Thy-1 nephritis models, EPA significantly inhibited tubulointerstitial injury and the infiltration of macrophages into tubulointerstitial lesions except severe glomerular injury at early stage. Compared with control animals, NF-κB activation was significantly augmented in the Thy-1 nephritic kidney. However, treatment with EPA significantly reduced NF-κB activation, down-regulated the expressions of NF-κB-dependent molecules. Also in LPS-stimulated PTECs, LPS augmented NF-κB activation and the expression of NF-κB-dependent molecules. As in the case with the Thy-1 nephritis models, treatment with EPA inhibited them, prevented the degradation of IκBα in LPS-stimulated PTECs. Pre-treatment with siRNA for IκBα abolished the inhibitory effect of EPA on LPS-induced NF-κB activation, suggesting that EPA inhibited NF-κB activation by regulating IκBα. Our results indicate that EPA prevents the early progression of tubulointerstitial injury in Thy-1 nephritis models, and the inhibitory effect of EPA on the expression of inflammatory molecules via the regulation of IκBα in cultured cells may explain this mechanism.
Keywords: Clusterin; Inflammation; Lipopolysaccharide; NF-κB; Thy-1;

Prostaglandin D2 induces contractions through activation of TP receptors in peripheral lung tissue from the guinea pig by Anna-Karin Larsson; Annika Hagfjärd; Sven-Erik Dahlén; Mikael Adner (136-142).
Prostaglandin D2 (PGD2), released through mast cell activation, is used as a non-invasive biomarker in patients with asthma. Since PGD2 can elicit opposing effects on airway tone via activation of the PGD2 receptors DP1 and DP2 as well as the thromboxane receptor TP, the aim of this study was to characterize the receptors that are activated by PGD2 in the guinea pig lung parenchyma. PGD2 and the thromboxane analog U46619 induced concentration-dependent contractions. U46619 was more potent and caused stronger effect than PGD2. The specific TP receptor antagonist SQ-29548 and the combined TP and DP2 receptor antagonist BAYu3405 concentration-dependently shifted the curves for both agonists to the right. The DP1 receptor agonist BW245 induced a weak relaxation at high concentrations, whereas the DP1 receptor antagonist BWA868C did not affect the PGD2 induced contractions. The specific DP2 receptor agonist 13,14-dihydro-15-keto-PGD2 showed neither contractile nor relaxant effect in the parenchyma. Furthermore, studies in precision-cut lung slices specified that airways as well as pulmonary arteries and veins contracted to both PGD2 and U46619. When the lung parenchyma from ovalbumin sensitized guinea pigs were exposed to ovalbumin, both thromboxane B2 and PGD2 were released. Ovalbumin also induced maximal contractions at similar level as PGD2 in the parenchyma, which was partly reduced by SQ-29548. These data show that PGD2 should be recognized as a TP receptor agonist in the peripheral lung inducing contraction on airways, arteries and veins. Therefore, a TP receptor antagonist can be useful in combination treatment of allergic responses in asthma.
Keywords: Guinea pig; Lung parenchyma; Ovalbumin; Precision cut lung slice; Prostaglandin D2; Thromboxane;

Inhibition of advanced glycation end products by aminoguanidine restores mast cell numbers and reactivity in alloxan-diabetic rats by Vinicius F. Carvalho; Luisa T. Florim; Emiliano de O. Barreto; Rafael C. Torres; Marcelo M. Batista; Fabio C. Amendoeira; Renato S.B. Cordeiro; Marco A. Martins; Patrícia M.R. e Silva (143-148).
Mast cell number and reactivity have been shown to be down-regulated under diabetic conditions. This study was undertaken in order to investigate the role of the advanced glycation end products in the reduction of mast cell number and reactivity in diabetic rats. The effect of aminoguanidine on mast cell apoptosis was also evaluated. Diabetes was induced by intravenous injection of alloxan into fasted rats and aminoguanidine was administered after 3 days of diabetes induction, once daily for 18 consecutive days. Mast cell apoptosis and levels of Bax, a pro-apoptotic member of Bcl-2 family, were evaluated by TUNEL and western blot, respectively. Diabetes led to increased levels of fructosamine and AGEs in the plasma, an effect prevented by aminoguanidine. Treatment with aminoguanidine restored mast cell numbers in the pleural cavity and in mesenteric tissue of diabetic rats. Aminoguanidine also significantly reversed the diabetes-induced reduction in histamine release, as measured by fluorescence, following activation with substance P or antigen in vitro. Increased apoptosis and levels of Bax in mast cells from diabetic rats were inhibited by aminoguanidine. In conclusion, our findings showed that aminoguanidine restored the number and reactivity of mast cells in diabetic rats, accompanied by suppression of apoptosis, evidencing that advanced glycation end product formation has a critical role in mast cell behavior of diabetic rats.
Keywords: Advanced glycation end-product; Diabetes; Mast cell; Apoptosis;