European Journal of Pharmacology (v.582, #1-3)

The plastoquinones sargaquinoic acid and sargachromenol are major components of brown alga Sargassum sagamianum and are known to be involved in neurite growth and survival. In this study, we describe their novel pro-apoptotic activities in vitro and in vivo. In vitro, treatment with sargaquinoic acid or sargachromenol promoted cell death and activation of caspase-3, caspase-8, caspase-9 and poly (ADP-ribose) polymerase (PARP) in a concentration-dependent manner. Sargaquinoic acid- or sargachromenol-induced apoptosis was enhanced by co-treatment with UVB irradiation. It showed much higher levels of cleaved caspase-3, caspase-8, caspase-9, and PARP than with sargaquinoic acid and sargachromenol alone, while it had no effect on Bcl-2 and Bax expression level. Examination by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) and immunohistochemistry showed that topical application of sargaquinoic acid (1 mg/ml) before UVB irradiation (2.5 kJ/m2) of hairless mice also enhanced apoptosis including activation of caspase-3. Since a combination of phototherapy using UVB with topical reagents has been clinically applied to treat hyperproliferative skin disease, these results suggest that sargaquinoic acid and sargachromenol could be effective therapeutic agents.
Keywords: Sargaquinoic acid; Sargachromenol; UVB irradiation; Apoptosis; Keratinocytes;

The effects of huwentoxin-V, an insect neurotoxic peptide from Chinese tarantula Ornithoctonus huwena venom, were studied on neuronal voltage-gated ion channels. Whole-cell patch-clamp configuration indicated that huwentoxin-V specifically inhibited high-voltage-activated calcium channels in adult cockroach dorsal unpaired median neurons (IC50  ≈ 219 nM) while having no evident effect on voltage-gated potassium and sodium channels. Omega-conotoxin GVIA is a well-known neuronal N-type calcium channel blocker from the venom of the sea snail Conus geographus and it also can partially block calcium currents in cockroach dorsal unpaired median neurons. In our study, huwentoxin-V inhibited omega-conotoxin GVIA-sensitive, diltiazem-sensitive and partial omega-conotoxin GVIA and diltiazem-resistant calcium currents elicited from insect neurons. Based on the sensitivity of calcium currents to these toxins, insect neuronal HVA calcium channels might be classified into four types: Type I, omega-conotoxin GVIA-sensitive and huwentoxin-V-sensitive; type II, diltiazem-sensitive and huwentoxin-V-sensitive; type III, huwentoxin-V-sensitive but omega-conotoxin GVIA and diltiazem-resistant; type IV, omega-conotoxin GVIA and diltiazem-resistant and huwentoxin-V-resistant. Its specificity, incomplete inhibition and insect-toxic effects make it an interesting tool for investigating insect voltage-gated calcium channels and development of new insecticidal compounds.
Keywords: Huwentoxin-V; Insect; Calcium channel; Whole-cell; Patch-clamp;

Growth inhibitory effects of obovatol through induction of apoptotic cell death in prostate and colon cancer by blocking of NF-κB by So Yong Lee; Dong Yeon Yuk; Ho Seub Song; Do Young Yoon; Jae Kyung Jung; Dong Cheul Moon; Byung Seok Lee; Jin Tae Hong (17-25).
Biphenolic components in Magnolia obovata including magnolol and honokiol have shown several pharmacological activities such as anti-tumor, anti-oxidant and anti-inflammatory effects. Previously in cultured macrophage Raw264.7 cells and fibroblast, we found that obovatol, an active compound isolated from M. obovata inhibited NF-κB activity which has been known to be a significant transcriptional factor to control of cancer cell growth. We investigated here whether obovatol could inhibit NF-κB activity, and thereby inhibit cancer cell growth in prostate (LNCaP and PC-3) and colon cancer (SW620 and HCT116) cells. Treatment of obovatol (10, 15, 20, 25 μM) inhibits cancer cell growth in the absence or the presence of tumor necrosis factor-α (TNF-α , 10 ng/ml) and tetradecanoyl phorbol acetate (TPA 10 or 50 nM) in a concentration-dependent manner through induction of apoptotic cell death. Cytotoxic activity was not observed in normal cells with up to 50 μM obovatol. It was also found that obovatol inhibited TNF-α and TPA-induced transcriptional and DNA binding activities of NF-κB. In further study, obovatol decreased translocation p65 and p50 into nucleus via decrease of phosphorylation of IκB. Correlated well with the induction of apoptosis, obovatol increased the expression of the apoptotic genes; Bax, caspase-3, caspase-9, whereas inhibited expression of anti-apoptotic genes; Bcl-2, inhibitor of apoptosis protein (IAP-1) and X chromosome IAP (XIAP) as well as the cell proliferation marker genes; Cox-2, c-Fos, c-Jun and cyclin D1. These results suggest that obovatol inhibits prostate and colon cancer cell growth via induction of apoptotic cell death, and that inhibition of NF-κB may be a significant as its action mechanism.
Keywords: Obovatol; NF-κB; Apoptotic cell death; Colon cancer; Prostate cancer;

Gold(I) complexes determine apoptosis with limited oxidative stress in Jurkat T cells by Maria Pia Rigobello; Alessandra Folda; Barbara Dani; Roberta Menabò; Guido Scutari; Alberto Bindoli (26-34).
In Jurkat T cells, S-triethylphosphinegold(I)-2,3,4,6-tetra-O-acetyl-1-thio-β-d-glucopyranoside (auranofin) and triethylphosphine gold(I) chloride (TepAu) induced apoptosis, as estimated by DNA fragmentation and visualised by fluorescence microscopy. Apoptosis was characterised by mitochondrial cytochrome c release which was not prevented by cyclosporin A. Apoptosis appeared to be triggered by inhibition exerted by gold(I) compounds on the cytosolic and mitochondrial isoforms of thioredoxin reductase, which determined a definite increase in hydrogen peroxide, whereas glutathione and its redox state were not modified. Total thiols showed a slight decrease, particularly in the presence of auranofin. However, no significant lipid peroxidation or nitric oxide formation were observed after incubation with gold(I) complexes, indicating that the cells had not been subjected to extensive oxidative stress. Interestingly, the gold(I) compound aurothiomalate was poorly effective, both in inhibiting thioredoxin reductase and in inducing apoptosis. These results demonstrate that the increased production of hydrogen peroxide determines an oxidative shift responsible for the occurrence of apoptosis and not involving lipid peroxidation.
Keywords: Apoptosis; Gold(I) complexes; Jurkat T cells; Mitochondria; Reactive oxygen species; Thiols; Thioredoxin reductase;

The effect of the selective inhibitor of Na+/Ca2+ exchanger (NCX), KB-R7943, on large-conductance Ca2+-activated K+ (BKCa) channels was examined in cultured human umbilical vein endothelial cells (HUVECs) and freshly isolated mouse aortic smooth muscle cells (MASMCs). In voltage-clamped cells, KB-R7943 reversibly activated BKCa currents in HUVECs and MASMCs. The EC50 of KB-R7943 for BKCa current activation in HUVECs was determined to be 6.78 ± 0.7 μM. In inside-out and outside-out patches, KB-R7943 markedly increased BKCa channel activity and slightly decreased single channel current amplitudes. In inside-out patches, KB-R7943 shifted the relationship between [Ca2+] i and open probability (P o) to the left; the [Ca2+] i required to evoke half-maximal activation changed from 1220 ± 68 nM (in the absence of KB-R7943) to 620 ± 199 nM (in the presence of 10 μM KB-R7943). In addition, KB-R7943 shifted the relationship between membrane potential and P o to the left; the membrane potential to evoke half-maximal activation changed from 76.86 ± 1.09 mV (in the absence of KB-R7943) to 49.62 ± 2.55 mV (in the presence of 10 μM KB-R7943). In conclusion, KB-R7943 was found to act as a potent BKCa channel activator, which increases the sensitivity of BKCa channels to cytosolic free Ca2+ and membrane potential, and thereby BKCa channel activity. These results should be considered when KB-R7943 is used as NCX blocker.
Keywords: KB-R7943; Large-conductance Ca2+-activated K+ channels; Endothelial cells;

Volatile anesthetics and endogenous cannabinoid anandamide have additive and independent inhibitory effects on α7-nicotinic acetylcholine receptor-mediated responses in Xenopus oocytes by Shelley N. Jackson; Sachin K. Singhal; Amina S. Woods; Marisela Morales; Toni Shippenberg; Li Zhang; Murat Oz (42-51).
In earlier studies, the volatile anesthetics and the endogenous cannabinoid anandamide have been shown to inhibit the function of α7-nicotinic acetylcholine receptors. In the present study, interactions between the effects of volatile anesthetics and anandamide on the function of α7-nicotinic acetylcholine receptors expressed in Xenopus oocytes were investigated using the two-electrode voltage-clamp technique. Anandamide and volatile anesthetics isoflurane and halothane inhibited currents evoked with acetylcholine (100 μM) in a reversible and concentration-dependent manner. Coapplication of anandamide and volatile anesthetics caused a significantly greater inhibition of α7-nicotinic acetylcholine receptor function than anandamide or volatile anesthetics alone. Analyses of oocytes by matrix-assisted laser desorption/ionization mass spectroscopy indicated that volatile anesthetics did not alter the lipid profile of oocytes. Results of studies with chimeric α7-nicotinic acetylcholine-5-HT3 receptors comprised of the N-terminal domain of the α7-nicotinic acetylcholine receptor and the transmembrane and carboxyl-terminal domains of 5-HT3 receptors suggest that while isoflurane inhibition of the α7-nicotinic acetylcholine receptor is likely to involve the N-terminal region of the receptor, the site of action for anandamide involves transmembrane and carboxyl-terminal domains of the receptors. These data indicate that endocannabinoids and isoflurane have additive inhibitory effects on α7-nicotinic acetylcholine receptor function through allosteric binding sites located on the distinct regions of the receptor.
Keywords: Anandamide; Volatile anesthetic; Nicotinic receptor; Xenopus oocyte;

Regulation of intracellular dopamine levels by dopaminergic drugs: Involvement of vesicular monoamine transporter by Yasuhiko Izumi; Noriyuki Yamamoto; Toshiaki Kume; Hiroshi Katsuki; Hideyuki Sawada; Akinori Akaike (52-61).
Endogenous dopamine could serve as a susceptibility factor for dopaminergic neuronal death. Our previous study demonstrated that depletion of dopamine content induced by dopamine receptor agonist was relevant to neuroprotection. In the current study, we have investigated the mechanisms underlying the dopamine-lowering effect of dopaminergic drugs using pheochromocytoma (PC12) cells. The majority of agonistic or antagonistic ligands for the dopamine receptor reduced intracellular dopamine levels in PC12 cells. The reduction of dopamine content induced by (−)-pramipexole, a dopamine D2/D3 receptor agonist, was not mediated by the activation of dopamine D2-like receptors. (−)-Pramipexole subtly suppressed the dopamine synthesis, but did not facilitate its metabolism. Dopamine was released just after stimulation with (−)-pramipexole, whereas the accumulative amount of released dopamine for 24 h was not increased. Furthermore, (−)-pramipexole prevented the uptake of [3H] dopamine into vesicles in a competitive manner. The dopaminergic drugs which gave rise to reduction in dopamine content also interfered with vesicular dopamine transport. These results suggest that dopaminergic drugs can reduce intracellular dopamine via inhibition of vesicular monoamine uptake.
Keywords: Dopamine; Dopaminergic drug; Parkinson disease; Pramipexole; VMAT;

Previously, we reported that intracerebroventricularly (i.c.v.) administered arginine-vasopressin evokes the secretion of noradrenaline and adrenaline from adrenal medulla through the brain phospholipase C- and diacylglycerol-mediated and cyclooxygenase-mediated mechanisms in rats. Diacylglycerol can be hydrolyzed by diacylglycerol lipase to 2-arachidonoyl-sn-glycerol, which may be further degradated by monoacylglycerol lipase to free arachidonic acid, a representative substrate of cyclooxygenase. Recently, 2-arachidonoyl-sn-glycerol has been recognized as a major endocannabinoid, which can modulate synaptic transmission in the brain. In the present experiment, therefore, we examined (1) a role of the brain 2-arachidonoyl-sn-glycerol as a precursor of arachidonic acid in the centrally administered vasopressin-induced elevation of plasma noradrenaline and adrenaline, and (2) a regulatory role of the brain 2-arachidonoyl-sn-glycerol as an endocannabinoid on the vasopressin-induced response, using urethane-anesthetized rats. The vasopressin (0.2 nmol/animal, i.c.v.)-induced elevation of plasma catecholamines was reduced by RHC-80267 (diacylglycerol lipase inhibitor) (1.3 and 2.6 μmol/animal, i.c.v.) and also reduced by MAFP (monoacylglycerol lipase inhibitor) (0.7 and 1.4 μmol/animal, i.c.v.). MAFP (1.4 μmol/animal, i.c.v.) also attenuated the 2-arachidonoyl-sn-glycerol (0.5 μmol/animal, i.c.v.)-induced elevation of plasma catecholamines. AM 251 (cannabinoid CB1 receptor antagonist) (90 and 180 nmol/animal, i.c.v.) potentiated the vasopressin (0.2 nmol/animal, i.c.v.)-induced response, while AM 630 (cannabinoid CB2 receptor antagonist) (198 and 793 nmol/animal, i.c.v.) was largely ineffective. In addition, WIN 55212-2 (cannabinoid CB receptor agonist) (188 and 470 nmol/animal, i.c.v.) dose-dependently reduced the vasopressin-induced response. These results suggest that the brain 2-arachidonoyl-sn-glycerol generated from diacylglycerol plays a role as a precursor of arachidonic acid in the centrally administered vasopressin-induced activation of the adrenomedullary outflow, and also negatively regulates the peptide-induced central response through the brain cannabinoid CB1 receptors in rats.
Keywords: Vasopressin; Adrenal medulla; Brain; 2-Arachidonoyl-sn-glycerol; Cannabinoid CB1 receptor;

Meperidine, remifentanil and tramadol but not sufentanil interact with α2-adrenoceptors in α2A-, α2B- and α2C-adrenoceptor knock out mice brain by Jan Höcker; Bernd Weber; Peter H. Tonner; Jens Scholz; Philipp-Alexander Brand; Henning Ohnesorge; Berthold Bein (70-77).
α2-adrenoceptor agonists like clonidine or dexmedetomidine increase the sedative and analgesic actions of opioids. Furthermore opioids like meperidine show potent anti-shivering effects like α2-adrenoceptor agonists. The underlying molecular mechanisms of these effects are still poorly defined. The authors therefore studied the ability of four different opioids (meperidine, remifentanil, sufentanil and tramadol) to interact with different α2-adrenoceptor subtypes in mice lacking individual α2A-, α2B- or α2C-adrenoceptors (α2-adrenoceptor knock out (α2-AR KO) mice)). The interaction of opioids with α2-adrenoceptors was investigated by quantitative receptor autoradiography in brain slices of α2A-, α2B- or α2C-adrenoceptor deficient mice. Displacement of the radiolabelled α2-adrenoceptor agonist [125I]-paraiodoclonidine ([125I]-PIC) from α2-adrenoceptors in different brain regions by increasing opioid concentrations was measured, and binding affinity of the analysed opioids to α2-adrenoceptor subtypes in different brain regions was quantified. Meperidine, remifentanil and tramadol but not sufentanil provoked dose dependent displacement of specifically bound [125I]-PIC from all α2-adrenoceptor subtypes in cortex, cerebellum, medulla oblongata, thalamus, hippocampus and pons. Required concentrations of meperidine and remifentanil for [125I]-PIC displacement from α2B- and α2C-adrenoceptors were lower than from α2A-adrenoceptors, indicating higher binding affinity for α2B- and α2C-adrenoceptors. In contrast, [125I]-PIC displacement by tramadol indicated higher binding affinity to α2A-adrenoceptors than to α2B- and α2C-adrenoceptors. Our results indicate that meperidine, remifentanil and tramadol interact with α2-adrenoceptors in mouse brain showing different affinity for α2A-, α2B- and α2C-adrenoceptors. In contrast, the µ-agonist sufentanil did not show any α2-adrenoceptor interaction. These effects may have an impact on the pharmacologic actions of these opioids.
Keywords: α2-adrenoceptor subtypes; Knock out mice; Opioid displacement; [125I]-paraiodoclonidine; Receptor autoradiography;

Morphine sex-dependently induced place conditioning in adult Wistar rats by Manizheh Karami; Mohammad Reza Zarrindast (78-87).
The present study was conducted to investigate the potential sex-differences in morphine-induced conditioned place preference. A 3-day unbiased conditioning procedure was used to establish conditioned place preference in adult male and female Wistar rats (weighing 200–250 g). The effect of morphine on locomotor activity of subjects was also studied. Naloxone (0.5–2 mg/kg, i.p.), a selective antagonist of mu-opioid receptor or sulpiride (0.5–2 mg/kg, s.c.), a selective antagonist of dopamine D2 receptor was administered, during conditioning, to indicate the receptor-mediated mechanisms governing upon possible sex-differences to the opioid response. Results show that morphine (0.5–10 mg/kg, s.c.) differently produced a significant place preference in female and male Wistar rats. Although, the opioid maximum response in both sexes was observed at 7.5 mg/kg, but, it was found that female rats acquired conditioned place preference at a lower dose (0.5 mg/kg, s.c.) of morphine compared to male rats. Moreover, the increase in morphine-induced response at higher doses (5–10 mg/kg, s.c.) was more pronounced in females than the males, indicating that female Wistar rats are more sensitive to the place conditioning induced by morphine. Also, the females were more sensitive to locomotor activation induced by morphine at least at one dose (7.5 mg/kg). Animals' body-weight at 10 mg/kg of opioid was increased, the effect that was not dependent to sex. The results also demonstrate that naloxone (1 and 2 mg/kg, i.p.) induced a significant place preference in two sexes with no significant effect on animals' locomotor activity. The antagonist in males but not in females showed a significant effect on animals' body-weight. Naloxone (0.5–2 mg/kg, i.p.) prior-administration to morphine, during conditioning, attenuated the opioid response in two sexes. The attenuation of the morphine response was more pronounced in males than the other sex at the higher dose (2 mg/kg) of the antagonist. In addition, the preadministration of naloxone, during morphine conditioning, both attenuated the drug-induced hyperactivity in females and decreased the animals' body-weight, albeit more effectively in females than the males. Sulpiride injections (1 and 2 mg/kg s.c.), during the conditioning period, induced a significant aversion in males but not in females with no significant effect either on locomotor activity or body-weight in both sexes. When sulpiride (0.5–2 mg/kg, s.c.), during conditioning, was morphine pre-injected, the antagonist at higher doses significantly attenuated the opioid response in males, reflecting the involvement of dopamine D2 receptor in sex-dependent morphine-conditioned place preference. Prior-injections of sulpiride to morphine produced a significant effect on locomotor activity of females. The effect of the antagonist preinjections on body-weight was also observed in males. Present results indicate sex-differences both in reinforcing and locomotor activity effects of morphine in Wistar rats.
Keywords: Sex-difference; Morphine; Naloxone; Sulpiride; Place conditioning; Locomotor activity; Body-weight; (Wistar rat);

The aim of the present study was to investigate the effect of antidepressant drugs (characterized by a different mechanism of action), administered jointly with the selective 5-HT6 receptor antagonist N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide (SB-399885), in the forced swim test in rats. All the compounds under study were given intraperitoneally in doses which did not shorten the immobility time of rats. Co-administration of SB-399885 (3 mg/kg) and imipramine (20 mg/kg), desipramine (20 mg/kg), bupropion (5 mg/kg) or moclobemide (20 mg/kg), produced significant anti-immobility action, whereas SB-399885 (3 mg/kg) given jointly with citalopram (20 mg/kg) did not affect immobility time. None of the compounds studied, given alone or jointly, increased the general activity of rats measured in the open field test. The obtained results indicate that the blockade of 5-HT6 receptors may facilitate the anti-immobility effect of imipramine, desipramine, bupropion or moclobemide in the forced swim test.
Keywords: Antidepressants; SB-399885; 5-HT6 receptor antagonist; Forced swim test; (Rat);

Enhanced expression of contractile endothelin ETB receptors in rat coronary artery after organ culture by Evelina Johnsson; Aida Maddahi; Angelica Wackenfors; Lars Edvinsson (94-101).
Endothelin-1 is a potent vasoconstrictor mediating its effects via two receptor subtypes, the endothelin type A (ETA) preferentially situated on smooth muscle cells, mediating vasoconstriction and endothelin type B (ETB) mainly located on endothelial cells, mediating vasodilatation. In cardiovascular disease and in organ culture in vitro, endothelin ETB receptors are up-regulated on smooth muscle cells. The objectives of the present study were to characterise the endothelin receptor-induced vasoconstriction and quantify the endothelin receptor mRNA levels and immunoreactivity in fresh and cultured rat coronary arteries. We demonstrate that endothelin-1 induces strong and equal concentration-dependent contractions in fresh and cultured segments from the left anterior descending coronary artery. Sarafotoxin 6c, an endothelin ETB receptor agonist, had negligible effect in fresh arteries but produced significant vasoconstriction after organ culture. The endothelin ETB receptor mRNA level and the receptor protein immunoreactivity were increased, whereas the level of endothelin ETA receptor mRNA was down-regulated but not its receptor protein immunoreactivity after organ culture. Pharmacological inhibition of endothelium-derived dilatory mediators did not influence endothelin ETA or ETB receptor-mediated vasoconstriction in fresh segments. In cultured arteries, inhibition of endothelial vasodilators potentiated the effect of sarafotoxin 6c. In conclusion, endothelin ETB receptor stimulation in cultured coronary arteries elicits vasoconstriction. This is likely not related to endothelial dysfunction with putative loss of its vasodilator components, but rather explained by the up-regulation of contractile endothelin ETB receptors on smooth muscle cells.
Keywords: BQ788; FR139317; Cardiovascular disease; Gene expression;

PDE4 and PDE5 regulate cyclic nucleotides relaxing effects in human umbilical arteries by António José Santos-Silva; Elisa Cairrão; Manuel Morgado; Ezequiel Álvarez; Ignacio Verde (102-109).
Cyclic nucleotides (cAMP and cGMP) are the main second messengers linked to vasodilatation. They are synthesized by cyclases and degraded by different types of phosphodiesterases (PDE). The effect of PDE inhibition and cyclases stimulation on 5-hydroxytryptamine (5-HT; 1 μM) and histamine (10 μM) contracted arteries was analysed. Stimulation of guanylate cyclase or adenylate cyclase relaxed the histamine- and 5-HT-induced contractions indicating that intracellular increase of cyclic nucleotides leads to vasodilatation of the human umbilical artery. We investigated the role of different PDE families in the regulation of this effect. The presence of the different PDE types in human umbilical artery smooth muscle was analysed by RT-PCR and the expression of PDE1B, PDE3A, PDE3B, PDE4C, PDE4D and PDE5A was detected. The unspecific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 50 μM) relaxed histamine-contracted human umbilical artery on 47.4 ± 7.2%. This effect seems to be due to PDE4 and PDE5 inhibition because among the selective PDE inhibitors used only the PDE4 inhibitor (rolipram; 1 μM) and the PDE5 inhibitors (dipyridamole and T0156; 3 μM and 1 μM respectively) induced significant relaxation (39.0 ± 8.7, 30.4 ± 6.0 and 36.3 ± 2.8 respectively). IBMX, dipyridamole and T0156 produced similar relaxation on 5-HT-induced contraction. After forskolin, the addition of IBMX or rolipram increased the effect of the adenylate cyclase stimulator and almost completely relaxed the human umbilical artery contracted by histamine (92.5 ± 4.9 and 90.9 ± 4.7 respectively), suggesting a main role of PDE4. The data obtained with 5-HT contracted arteries confirmed this, because only rolipram and IBMX significantly increased the forskolin vasodilator effect. The administration of dipyridamole and T0156 after sodium nitroprusside (SNP) induced a significant increase of the SNP relaxant effect on histamine-contracted arteries, but PDE1 and PDE3 inhibition did not increase the effect of the guanylate cyclase stimulator. Similar effects were obtained in 5-HT contracted arteries, the SNP induced relaxation was increased by the PDE5 inhibition, but not by PDE1 or PDE3 inhibition. In summary, our results demonstrate that: 1) the increase of cAMP and/or cGMP levels induces relaxation of the human umbilical vascular smooth muscle; 2) four families of PDE are expressed in this smooth muscle: PDE1, PDE3, PDE4 and PDE5; 3) between these families, PDE4 and PDE5 are the key enzymes involved in the regulation of the relaxation associated to cAMP and cGMP, respectively.
Keywords: Cyclic nucleotide; Phosphodiesterase; Human umbilical artery; Smooth muscle;

Geldanamycin enhances hepatocyte growth factor stimulation of eNOS phosphorylation in endothelial cells by Kennedy Makondo; Akihiro Kamikawa; Mohamed Ahmed; Akira Terao; Masayuki Saito; Kazuhiro Kimura (110-115).
Previously, we demonstrated that hepatocyte growth factor (HGF) potently stimulates endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production through a calcium- and Akt-mediated phosphorylation at Ser-1179 (Ser-1177 human) in bovine aortic endothelial cells. The regulation of eNOS, however, also involves interaction with chaperone proteins such as heat shock protein (HSP) 90, which can be enhanced by agonist stimulation of the enzyme. In the present work, the role of HSP90 in HGF stimulation of eNOS was examined in an endothelial cell culture system. Treatment of endothelial cells with geldanamycin, a commonly used HSP90 inhibitor, augmented HGF-stimulated eNOS phosphorylation at Ser-1179, while it did not alter eNOS phosphorylation at Thr-497. However, other HSP90 inhibitors, namely 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and radicicol, did not possess similar effects. Neither HGF nor geldanamycin treatment, independently or in combination, altered HSP90/eNOS interaction in endothelial cells. In addition, geldanamycin treatment did not enhance the HGF-induced phosphorylation of Akt, ERK1/2 and p38MAPK. Src kinase inhibition by PP2 also failed to block the geldanamycin effects. These results suggest that geldanamycin, but neither 17-AAG nor radicicol, may enhance HGF-mediated eNOS Ser-1179 phosphorylation by some as yet unknown mechanisms independently of HSP90 inhibition.
Keywords: HSP90; Geldanamycin; Akt; HGF; Nitric oxide; Endothelial nitric oxide synthase;

Endothelial dependence of matrix metalloproteinase-mediated vascular hyporeactivity caused by lipopolysaccharide by Jonathan Cena; Manoj M. Lalu; Cory Rosenfelt; Richard Schulz (116-122).
Septic shock remains the leading cause of death in intensive care units in North America. Recent evidence implicates matrix metalloproteinases (MMP) in the pathogenesis of sepsis. MMP activity is upregulated in blood vessels exposed to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines and contributes to vascular hyporeactivity to vasoconstrictors. The exact mechanism of MMP-mediated vascular hyporeactivity is unknown. We investigated the contribution of the endothelium in the MMP response to LPS-mediated vascular hyporeactivity in vitro. Tone induced by phenylephrine in isolated rat aortic rings with either intact or denuded endothelium was measured in the presence of LPS for 6 h. These rings were incubated with the nitric oxide (NO) synthase inhibitor, N G-nitro-l-arginine methyl ester (l-NAME), to determine whether NO synthase was involved in the response, or the MMP inhibitors, doxycycline or GM6001. MMP activity was measured after 6 h. LPS caused a greater reduction of phenylephrine-induced tone in endothelium-intact rings versus endothelium-denuded rings, indicating both endothelium-independent and -dependent mechanisms for LPS-induced vascular hyporeactivity. l-NAME abolished the response to LPS in both endothelium-intact and endothelium-denuded rings. MMP inhibitors prevented the LPS-induced loss of tone in endothelium-intact but not endothelium-denuded rings. LPS caused significantly greater MMP-2 activity in endothelium-intact aortae which was attenuated by doxycycline. MMP-2 activity in endothelium-denuded aortae was unchanged by LPS. The vascular endothelium contributes to MMP-mediated vascular dysfunction induced by LPS. The protective effect of MMP inhibition is endothelium-dependent and is a novel mechanism by which MMPs contribute to vascular dysfunction.
Keywords: Matrix metalloproteinase; Lipopolysaccharide; Vascular biology; Endotoxemia; Septic shock;

Role of tachykinin NK1 and NK2 receptors in colonic sensitivity and stress-induced defecation in gerbils by Dorota Kakol-Palm; Mikael Brusberg; Elin Sand; Håkan Larsson; Vicente Martinez; Anders Johansson; Bengt von Mentzer; Ingrid Påhlman; Erik Lindström (123-131).
The pharmacology of tachykinin NK receptors varies greatly among species. The aim of the present study was to assess the role of NK1 and NK2 receptors in mediating colorectal distension-evoked nociception and psychological stress-induced defecation in gerbils, a species with human-like NK receptor pharmacology. The effects of the selective NK1 and NK2 receptor antagonists, aprepitant and saredutant, on acute (1 h) restraint stress-evoked defecation and plasma adenocorticotropin (ACTH) levels in gerbils were assessed. The effects of antagonists alone or in combination on colorectal distension-evoked visceral pain in conscious gerbils were evaluated using the visceromotor response as a surrogate marker of pain. Restraint stress increased fecal pellet output 2–3-fold and plasma ACTH levels 9-fold. Aprepitant inhibited the defecatory and endocrine responses to stress by 50%, while saredutant completely normalized the same parameters. Visceral pain responses during colorectal distension were attenuated by both compounds, but aprepitant (19 ± 6% inhibition, P  < 0.01) was slightly more effective than saredutant (10 ± 9% inhibition, P  < 0.05). A combination of both compounds resulted in an additive effect (30 ± 10% inhibition, P  < 0.01). The results demonstrate that NK1 and NK2 receptors are involved in stress-related colonic motor alterations and visceral pain responses in gerbils and that combined antagonism provides enhanced inhibition of visceral pain responses. This suggests that for therapeutic use in for instance functional gastrointestinal disorders, dual NK1/NK2 receptor antagonists may provide better clinical outcome than selective compounds.
Keywords: Aprepitant; Saredutant; Colorectal distension; Stress; Visceral pain; Tachykinin; NK receptor; Gerbil; Colonic motility;

The purpose of the present study was to examine whether cannabinoid receptor agonists influence spontaneous contractile activity of longitudinal muscle in mouse ileum in vitro. Isolated segments of mouse ileum displayed spontaneous contractions with an amplitude and frequency of about 300 mg and 30 cpm, respectively. The endocannabinoid anandamide (1–100 μM), the selective cannabinoid CB1 receptor agonist, ACEA (0.1 μM–10 μM), but not the selective cannabinoid CB2 receptor agonist, JWH 133 (0.1 μM–10 μM), reduced in a concentration-dependent manner the spontaneous mechanical activity. The inhibitory effect consisted in a decrease of the mean amplitude of longitudinal spontaneous contractions, without changes in the resting tone. The inhibitory effect induced by cannabinoids was significantly antagonized by the selective cannabinoid CB1 receptor antagonist, SR141716A (0.1 μM), but not by the selective cannabinoid CB2 receptor antagonist, AM630 (0.1 μM). None of the cannabinoid antagonists, at the concentration used, did affect the spontaneous mechanical activity. The ACEA-induced reduction of spontaneous contractions was almost abolished by tetrodotoxin, atropine or apamin and it was unaffected by hexamethonium or N ω-nitro-l-arginine methyl ester (l-NAME), inhibitor of nitric oxide synthase. The myogenic contractions evoked by carbachol were not affected by ACEA. In conclusion, the present results suggest that activation of neural cannabinoid CB1 receptors may play a role in the control of spontaneous mechanical activity through inhibition of acetylcholine release from cholinergic nerve. Activation of small conductance Ca2+-dependent K+ channels is involved in this action.
Keywords: Cannabinoids; CB1 receptors; Spontaneous mechanical activity; Mouse ileum; Acetylcholine;

Involvement of peroxynitrite in pollen-induced nasal blockage in guinea pigs by Nobuaki Mizutani; Takeshi Nabe; Masanori Fujii; Shin Yoshino; Shigekatsu Kohno (139-144).
Nitric oxide (NO) has been implicated in early and late phase nasal blockage in a Japanese cedar pollen-induced experimental allergic rhinitis guinea pig model. In this study, we investigated the role of peroxynitrite, which is formed by a rapid reaction of NO with superoxide anion, in the antigen-induced biphasic nasal blockage. Sensitized guinea pigs were repeatedly challenged by pollen inhalation once every week. The peroxynitrite scavenger, ebselen (30 mg/kg), or the xanthine oxidase inhibitor, allopurinol (50 mg/kg), was intraperitoneally administered 30 min before the antigen challenge. The late phase nasal blockage induced 4 h after the challenge was largely suppressed by ebselen (57% inhibition; P  < 0.05) and allopurinol (47% inhibition; P  < 0.05), but neither ebselen nor allopurinol influenced the early phase response. On the other hand, the intranasal instillation of peroxynitrite (10− 3 and 10− 2 M, 10 μl/nostril) caused a remarkable dose-dependent nasal blockage in the sensitized guinea pig. These results suggest that peroxynitrite plays a major role in the late phase nasal blockage induced by the antigen challenge in sensitized guinea pigs.
Keywords: Peroxynitrite; Allergic rhinitis; Nasal blockage; Nitric oxide; Superoxide; Leukotriene D4; Ebselen; N ω-Nitro-l-arginine methyl ester; Allopurinol; (Guinea pig);

Acetylcholine mediates the release of IL-8 in human bronchial epithelial cells by a NFkB/ERK-dependent mechanism by Mirella Profita; Anna Bonanno; Liboria Siena; Maria Ferraro; Angela M. Montalbano; Flora Pompeo; Loredana Riccobono; Michael P. Pieper; Mark Gjomarkaj (145-153).
Acetylcholine may play a role in cell activation and airway inflammation. We evaluated the levels of both mRNA and protein of muscarinic M1, M2, M3 receptors in human bronchial epithelial cell line (16HBE). 16HBE cells were also stimulated with acetylcholine and extracellular signal-regulated kinase1/2 (ERK1/2) and NFkB pathway activation as well as the IL-8 release was assessed in the presence or absence of the inhibitor of Protein-kinase (PKC) (GF109203X), of the inhibitor of mitogenic activated protein-kinase kinase (MAPKK) (PDO9805), of the inhibitor of kinaseB-α phosphorilation (pIkBα) (BAY11-7082), and of muscarinic receptor antagonists tiotropium bromide, 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), telenzepine, gallamine. Additionally, we tested the IL-8-mediated neutrophil chemotactic activity of 16HBE supernatants stimulated with acetylcholine in the presence or absence of tiotropium. 16HBE cells expressed both protein and mRNA for muscarinic M3, M2 and M1 receptors with levels of muscarinic M3 receptor > muscarinic M1 receptor > muscarinic M2 receptor. Acetylcholine (10 μM) significantly stimulated ERK1/2 and NFkB activation as well as IL-8 release in 16HBE cells when compared to basal values. Furthermore, while the use of tiotropium, 4-DAMP, GF109203X, PDO98059, BAY11-7082 completely abolished these events, the use of telenzepine and gallamine were only partially able to downregulate these effects. Additionally, acetylcholine-mediated IL-8 release from 16HBE cells significantly increased chemotaxis toward neutrophils and this effect was blocked by tiotropium. In conclusion, acetylcholine activates the release of IL-8 from 16HBE involving PKC, ERK1/2 and NFkB pathways via muscarinic receptors, suggesting that it is likely to contribute to IL-8 related neutrophilic inflammatory disorders in the airway. Thus, muscarinic antagonists may contribute to control inflammatory processes in airway diseases.
Keywords: Epithelial cells; Muscarinic receptors; MAP kinases; NFkB, Interleukin-8; Neutrophil chemotax;

The JAK-3 inhibitor CP-690550 is a potent anti-inflammatory agent in a murine model of pulmonary eosinophilia by Elizabeth Kudlacz; Maryrose Conklyn; Catharine Andresen; Carrie Whitney-Pickett; Paul Changelian (154-161).
Janus kinase 3 (JAK-3) is a tyrosine kinase that has been shown to participate in the signaling of several cytokines that are believed to play a role in allergic airway disease, e.g. IL-2, 4 and 9. The current study describes the immunosuppressive effects of CP-690550, a novel, small molecule inhibitor of JAK-3, in a murine model of allergic pulmonary inflammation. In vitro, CP-690550 potently inhibited IL-4 induced upregulation of CD23 (IC50  = 57 nM) and class II major histocompatibility complex (MHCII) expression (IC50  = 71 nM) on murine B cells. Repeat aerosol exposure to ovalbumin in wild-type mice sensitized to the antigen resulted in preferential recruitment of Th2-like cells (IL-4+ and IL-5+) into bronchoalveolar lavage fluid (BAL). The importance of IL-4 in the development of pulmonary eosinophilia was supported by a marked (90%) reduction in the influx of these cells in IL-4KO mice similarly sensitized and ovalbumin exposed. Animals dosed with CP-690550 (15 mg/kg/d) during the period of antigen sensitization and boost demonstrated marked reductions in BAL eosinophils and levels of IL-13 and eotaxin following ovalbumin aerosol exposure. The JAK-3 inhibitor (1.5–15 mg/kg/d) also effectively reduced the same parameters when administered during the period of antigen challenge. In contrast, the calcineurin inhibitor tacrolimus (10 mg/kg) was effective only when administered during the period of ovalbumin aerosol exposure. These data support the participation of JAK-3 in processes that contribute to pulmonary eosinophilia in the allergic mouse model. CP-690550 represents an intriguing novel therapy for treatment of allergic conditions associated with airway eosinophilia including asthma and rhinitis.
Keywords: JAK3; JAK inhibition; Murine asthma model; Pulmonary eosinophilia;

Antioxidant and pancreas-protective effect of aucubin on rats with streptozotocin-induced diabetes by Lei Jin; Hong-Yu Xue; Li-Ji Jin; Shu-Ying Li; Yong-Ping Xu (162-167).
Oxidative stress has been suggested as a contributory factor in development and complication of diabetes. The aim of the present study was to determine the protective effect of aucubin on lipid peroxidation and activities of antioxidant defense systems and to conduct immunohistochemical evaluation of pancreas in streptozotocin-induced diabetic rats. Lipid peroxidation was determined by assessing the concentration of malondialdehyde and activities of antioxidant enzymes — catalase, glutathione peroxidase and superoxide dismutase in liver and kidneys of rats were determined. Changes of blood glucose and immunohistochemical evaluation on pancreas were also investigated as part of the pathology of diabetes. In our study, aucubin treatment lowered blood glucose. Diabetic rats exhibited an increase in the level of lipid peroxidation and decrease in activities of antioxidant enzymes in liver and kidneys as compared to control rats. Administration of aucubin to diabetic rats for 15 days significantly reversed damage associated with diabetes. In addition, diabetic rats showed an obvious decrease in insulin immunoreactivity and the number of β cells in pancreas, but the pancreas of aucubin-treated rats were improved and the number of immunoreactive β cells were significantly increased. These results indicated that aucubin may have value as a safe preventive or therapeutic agent against diabetes mellitus.
Keywords: Diabetes mellitus; Blood glucose; Lipid peroxidation; Antioxidant enzymes; Pancreas;