European Journal of Pharmacology (v.613, #1-3)

BZYX was designed as a dual-binding-site acetylcholinesterase (AChE) inhibitor and selected from series of indanone derivatives. The present study was designed to examine the cognition-enhanced, anti-cholinesterase, and neuroprotective effects of BZYX. In the passive avoidance performance and radial arm maze, BZYX showed a comparable effect to donepezil and rivastigmine on memory deficits in different stages induced by scopolamine, NaNO2 and ethanol, respectively. Ellman's assay indicated BZYX exhibited high inhibition on AChE activity. IC50 values for BZYX: 0.058 ± 0.022 μM; donepezil: 0.019 ± 0.004 μM; rivastigmine: 3.81 ± 2.81 μM; glantamine: 3.01 ± 1.85 μM and huperzine A: 0.053 ± 0.016 μM. BZYX also presented great neuroprotecive function from apoptosis induced by hydrogen peroxide(H2O2) in PC12 cells. MTT assay and Annexin V-FITC Apoptosis Detection showed the viability of PC12 cells remarkably decreased with 400 μM H2O2, while it significantly increased when the cells were pretreated with 0.1–1.0 μM BZYX. BZYX pretreatment remarkably reversed the loss of mitochondria membrane potential (ΔΨm), scavenged reactive oxygen species formation induced by H2O2 and resulted in up-regulation of procaspase3 and xIAP protein level and down-regulation of phosphorylated JNK protein, p53 protein level and cleavage of caspase 3. It is speculated that the mitochondrial pathway, mediated by Bcl-2 family and Mitogen-Activated Protein Kinases (MAPKs), might involved in the neuroprotection of BZYX. These results first demonstrated that BZYX had neuroprotective effects as well as cognition enhancement and acetylcholinesterase inhibition. It is hopeful that BZYX becomes a potential candidate for use in the intervention for neurodegenerative diseases.
Keywords: Alzheimer's disease; Acetylcholinesterase inhibitor; Rodents; PC12 cells; Oxidative stress; Apoptosis;

Dipyridamole inhibits cobalt chloride-induced osteopontin expression in NRK52E cells by Tso-Hsiao Chen; Chia-Fang Chang; Shu-Chuan Yu; Jiueng-Chueng Wang; Cheng-Hsien Chen; Paul Chan; Horng-Mo Lee (10-18).
Osteopontin plays a pivotal role in the progression of interstitial fibrosis in renal ischemia. In the present study, rat renal tubular NRK52E cells treated with hypoxia mimetic cobalt chloride (CoCl2) increased osteopontin production, and are associated with increased phosphorylation of Akt/PKB (protein kinase B) and p38 mitogen-activated protein kinase (p38MAPK). Furthermore, pretreatment of cells with l-N-acetylcysteine (an antioxidant) inhibited CoCl2-stimulated osteopontin protein expression and p38MAPK phosphorylation, but not Akt/PKB phosphorylation. Pretreatment of cells with anti-inflammatory agents celecoxib, tanshinone IIA, and dipyridamole inhibited CoCl2-induced osteopontin production paralleled by heme oxygenase-1 (HO-1) induction. Pretreatment of cells with tin protoporphyrin (a HO-1 inhibitor) or hemoglobin (a carbon monoxide scavenging agent) reversed dipyridamole inhibition of osteopontin expression. Moreover, transfection of HO-1 small interfering RNA (siRNA) reduced dipyridamole-stimulated mitogen-activated protein kinase phosphatase-1 (MKP-1) phosphorylation. Conversely, MKP-1 knockdown reversed dipyridamole inhibition of osteopontin expression. Taken together, these data suggest that dipyridamole may inhibit CoCl2-induced osteopontin expression through HO-1 induction. Increased HO-1 may catalyze the conversion of heme into carbon monoxide, in turn carbon monoxide activates MKP-1. MKP-1 activation inhibits the p38MAPK signaling pathway that mediates CoCl2-induced osteopontin production.
Keywords: Osteopontin; Dipyridamole; Heme oxygenase-1; NRK52E tubular cell;

Morroniside protects human neuroblastoma SH-SY5Y cells against hydrogen peroxide-induced cytotoxicity by Wen Wang; Fangling Sun; Yi An; Houxi Ai; Li Zhang; Wenting Huang; Lin Li (19-23).
Oxidative stress-induced cell damage has been implicated in a variety of neurodegenerative disorders. Morroniside, an iridorid glycoside isolated from Cornus officinalis Sieb. Et Zucc., has shown potent antioxidant properties. The present study investigated the protective actions of morroniside against the cytotoxicity produced by exposure to H2O2 (300–500 μM) in SH-SY5Y cells. Intracellular accumulation of Ca2+, and decreases in mitochondrial membrane potential (MMP) caused by added H2O2 were reduced by morroniside. Incubation of cells with H2O2 caused a marked decrease in superoxide dismutase (SOD) activity; this decrease was significantly inhibited by morroniside. In addition, the percentage of cells undergoing H2O2-induced apoptosis was decreased, dose dependently, in the presence of morroniside. These results suggest that morroniside has protective effects against oxidative stress-induced neurotoxic processes.
Keywords: Morroniside; Cytotoxicity; Neurodegenerative disease; Oxidative stress; Cornus officinalis;

Methotrexate induces apoptosis in CaSki and NRK cells and influences the organization of their actin cytoskeleton by Antonina Joanna Mazur; Dorota Nowak; Hans Georg Mannherz; Maria Malicka-Błaszkiewicz (24-33).
Methotrexate is a widely used drug in treatments of various types of malignancies and in the therapy of rheumatoid arthritis. The goal of our study was to look at the effect of this dihydrofolate reductase inhibitor on the actin cytoskeleton, since actin plays an important role in cancer transformation and metastasis. For this reason we compared results obtained from experiments on CaSki (human uterine cervix cancer) and NRK (normal fibroblastic rat kidney) cells treated with methotrexate. It has been shown previously that methotrexate can induce apoptosis. Therefore we first examined whether methotrexate induces apoptosis in our model cells. For this aim we applied several assays like Caspase Glo™ 3/7, DNA fragmentation and binding of phosphatidylserine by annexin V-fluorescein. The data obtained indicated that methotrexate induces programmed cell death in CaSki and NRK cells. However, differences between CaSki and NRK cells were observed in the morphological alterations and dynamics of apoptosis induced by methotrexate. It seemed that cancer cells were more sensitive towards the cell death inducing activity at lower concentrations of methotrexate. Analysis by confocal microscopy of methotrexate-treated cells demonstrated that treatment with this folate antagonist affected the actin cytoskeleton, although the dis-organization of the actin cytoskeleton after treatment with methotrexate differed between cancer and normal cells.
Keywords: Actin cytoskeleton; Apoptosis; Caspase-3; Methotrexate;

The effect of vinca alkaloid anticancer drug, vinorelbine, on chromatin and histone proteins in solution by Azra Rabbani-Chadegani; Elham Chamani; Zahra Hajihassan (34-38).
Vinorelbine (navelbin) belongs to vinca alkaloid anticancer drugs family with a broad spectrum of selective activity against mitotic microtubules. The present study is the first report demonstrating chromatin components as a novel target for vinorelbine in hepatocytes. The interaction was carried out in solution, employing fluorescence, UV spectroscopy and thermal denaturation techniques. Fluorescence emission spectra represented quenching of DNA chromospheres with drug and decreased fluorescence emission intensity in a dose-dependent manner. Binding of vinorelbine to chromatin induced very high hypochromicity and shifted DNA melting temperature to lower Tm. Vinorelbine binds to histone proteins with very high affinity when compared with the interaction of DNA intercalator anticancer drug, daunomycin, and the globular domain of the histones is considered as a main drug binding site. The results also showed that in the presence of vinorelbine, the absorbance of chromatin at 260 nm was decreased and the binding pattern was similar to daunomycin–chromatin complex. The results for the first time suggest that apart from tubulins, chromatin components can also be considered as a new target for this anticancer drug.
Keywords: Vinorelbine; Chromatin; Histone protein; DNA;

Pharmacological modulation of movement-evoked pain in a rat model of osteoarthritis by Prasant Chandran; Madhavi Pai; Eric A. Blomme; Gin C. Hsieh; Michael W. Decker; Prisca Honore (39-45).
This study was conducted to characterize movement-induced pain in a rat model of knee joint osteoarthritis and validate this behavioral assessment by evaluating the effects of clinically used analgesic compounds. Unilateral intra-articular administration of a chondrocyte glycolytic inhibitor monoiodoacetate, was used to induce knee joint osteoarthritis in Sprague–Dawley rats. In this osteoarthritis model, histologically erosive disintegration of the articular surfaces of the ipsilateral joint are observed which closely mimic the clinical picture of osteoarthritis. Movement-induced pain behavior was measured using hind limb compressive grip force evaluation. The animals exhibited pain behaviors epitomized by a long-lasting decrement in bilateral compressive hind limb grip force following unilateral knee injury. The effects of clinically used reference analgesics were evaluated 20 days following i.a. injection of monoiodoacetate. Full analgesic activity was observed for tramadol, celecoxib and diclofenac; moderate effects for indomethacin, duloxetine and gabapentin but weak or no effects for acetaminophen, ibuprofen and lamotrigine. As morphine reduced grip force in naïve rats, its analgesic effects could not be accurately evaluated in this model. Finally, the effects of celecoxib were maintained following chronic dosing. The results indicate that this in vivo model utilizing a movement-induced pain behavior spawned by knee joint osteoarthritis may provide a valuable tool in examining the role of potential analgesic targets in osteoarthritic pain. As the model is clinically relevant, it will further enhance the mechanistic understanding of chronic arthritic joint pain and help in developing newer and better therapeutic strategies to manage osteoarthritis pain.
Keywords: Animal model; Osteoarthritis; Pain;

Spinal mechanisms of antinociceptive action caused by guanosine in mice by André P. Schmidt; Ana Elisa Böhmer; Cristhine Schallenberger; Catiele Antunes; Mery Stéfani L. Pereira; Renata Leke; Susana T. Wofchuk; Elaine Elisabetsky; Diogo O. Souza (46-53).
It is well known that adenine-based purines exert multiple effects on pain transmission. Recently, we have demonstrated that intracerebroventricular (i.c.v.) administered guanine-based purines are antinociceptive against chemical and thermal pain models in mice. The present study was designed to further investigate the antinociceptive effects of guanosine in mice. Animals received an intrathecal (i.t.) injection of vehicle (0.1 mN NaOH) or guanosine (10 to 400 nmol). Measurements of cerebrospinal fluid (CSF) purine levels and spinal cord glutamate uptake were performed. Guanosine produced dose-dependent antinociceptive effects against tail-flick, hot-plate, intraplantar (i.pl.) capsaicin, and i.pl. glutamate tests. Additionally, i.t. guanosine produced significant inhibition of the biting behavior induced by i.t. injection of glutamate (175 nmol/site), AMPA (135 pmol/site), kainate (110 pmol/site), trans-ACPD (50 nmol/site), and substance P (135 ng/site), with mean ID50 values of 140 (103–190), 136 (100–185), 162 (133–196), 266 (153–461) and 28 (3–292) nmol, respectively. However, guanosine failed to affect the nociception induced by NMDA (450 pmol/site) and capsaicin (30 ng/site). Intrathecal administration of guanosine (200 nmol) induced an approximately 120-fold increase on CSF guanosine levels. Guanosine prevented the increase on spinal cord glutamate uptake induced by i.pl. capsaicin. This study provides new evidence on the mechanism of action of guanosine presenting antinociceptive effects at spinal sites. This effect seems to be at least partially associated with modulation of glutamatergic pathways by guanosine.
Keywords: Guanosine; Pain; Nociception; Spinal cord; Purine; Glutamate;

Central administration of palmitoylethanolamide reduces hyperalgesia in mice via inhibition of NF-κB nuclear signalling in dorsal root ganglia by Giuseppe D'Agostino; Giovanna La Rana; Roberto Russo; Oscar Sasso; Anna Iacono; Emanuela Esposito; Giuseppina Mattace Raso; Salvatore Cuzzocrea; Jesse LoVerme; Daniele Piomelli; Rosaria Meli; Antonio Calignano (54-59).
Despite the clear roles played by peroxisome proliferators-activated receptor α (PPAR-α) in lipid metabolism, inflammation and feeding, the effects of its activation in the central nervous system (CNS) are largely unknown. Palmitoylethanolamide (PEA), a member of the fatty-acid ethanolamide family, acts peripherally as an endogenous PPAR-α agonist, exerting analgesic and anti-inflammatory effects. Both PPAR-α and PEA are present in the CNS, but the specific functions of this lipid and its receptor remain to be clarified. Using the carrageenan-induced paw model of hyperalgesia in mice, we report here that intracerebroventricular administration of PEA (0.1–1 µg) 30 min before carrageenan injection markedly reduced mechanical hyperalgesia up to 24 h following inflammatory insult. This effect was mimicked by GW7647 (1 µg), a synthetic PPAR-α agonist. The obligatory role of PPAR-α in mediating PEA's actions was confirmed by the lack of anti-hyperalgesic effects in mutant mice lacking PPAR-α. PEA significantly reduced the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in sciatic nerves and restored carrageenan–induced reductions of PPAR-α in the L4-L6 dorsal root ganglia (DRG). To investigate the mechanism by which PEA attenuated hyperalgesia, we evaluated inhibitory kB-α (IkB-α) degradation and p65 nuclear factor kB (NF-κB) activation in DRG. PEA prevented IkB-α degradation and p65 NF-κB nuclear translocation, confirming the involvement of this transcriptional factor in the control of peripheral hyperalgesia. These results add further support to the broad-spectrum of biological and pharmacological effects induced by PPAR-α agonists, suggesting a centrally mediated component for these drugs in controlling inflammatory pain.
Keywords: Acylethanolamide; Peroxisome proliferator-activated receptor; Central nervous system; Sciatic nerve; Inflammation; Pain;

Food restriction and experimentally-induced diabetes (streptozotocin) can modify serotonin (5-HT) neurotransmission and sensitivity to drugs acting on 5-HT systems. This study examined the effects of food restriction and streptozotocin on the hypothermic effects of the 5-HT1A receptor agonist (+)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT), the 5-HT2 receptor agonist (±)-2,5-dimethoxy-4-methylamphetamine hydrochloride (DOM), the 5-HT releaser fenfluramine, and the selective 5-HT reuptake inhibitor (SSRI) fluoxetine. All four drugs significantly decreased body temperature in free feeding rats. Limiting rats to 10 g/day of food for 7 days decreased body weight and sensitivity to 8-OH-DPAT induced hypothermia, without affecting sensitivity to DOM, fenfluramine, or fluoxetine induced hypothermia. Subsequently, 7 days of free feeding restored body weight and sensitivity to 8-OH-DPAT. Sensitivity to all drugs was significantly decreased 7 days after 50 mg/kg streptozotocin; subsequently, 10 days of insulin replacement restored sensitivity to all drugs. These results extend to body temperature the observation that food restriction and experimentally-induced diabetes differentially modify sensitivity to drugs acting on 5-HT systems and they further suggest that the clinical response to therapeutic drugs acting on 5-HT systems might be impacted by nutritional and insulin status.
Keywords: Serotonin; Body temperature; Rat; Streptozotocin; Food restriction;

The l-arginine/nitric oxide/cyclic GMP pathway has been proposed as the mechanism of action for peripheral antinociception concerning several groups of drugs, including opioids and nonsteroidal analgesics. The aim of the present study was to investigate the involvement of the l-arginine/NO/cGMP pathway on antinociception induced by xylazine, an α2-adrenoceptor agonist extensively used in veterinary medicine and animal experimentation. The rat paw pressure test was used by inducing hyperalgesia via intraplantar injection of prostaglandin E2 (2 μg). Xylazine was administered locally into the right hind paw (25, 50 and 100 μg) and either NO synthase inhibitor L-NOarg (12, 18 and 24 μg/paw), soluble guanylyl cyclase inhibitor ODQ (25, 50 and 100 µg/paw) or cGMP-phosphodiesterase inhibitor zaprinast (50 μg/paw) were previously administered to the right hind paw of Wistar rats. Xylazine administration elicited a local antinociceptive effect, since only much higher doses produce a systemic effect in the contralateral paw. The peripheral antinociceptive effect induced by xylazine (100 µg/paw) was antagonized by L-NOarg and by ODQ; however, zaprinast potentiated the antinociceptive effect of xylazine at 25 µg/paw. The results provide evidence that xylazine probably induces peripheral antinociceptive effect by l-arginine/NO/cGMP pathway activation.
Keywords: Xylazine; α2-Adrenoceptor; Nitric oxide; Cyclic GMP; Peripheral antinociception;

Antinociceptive effects of systemic lidocaine: Involvement of the spinal glycinergic system by Uta Muth-Selbach; Henning Hermanns; Jens Ulrich Stegmann; Kathrin Kollosche; Rainer Freynhagen; Inge Bauer; Peter Lipfert (68-73).
Beside their action on voltage-gated Na+ channels, local anesthetics are known to exert a variety of effects via alternative mechanisms. The antinociceptive effect of lidocaine is well documented, yet the exact mechanism is not fully understood. Whether glycinergic mechanisms, which play a pivotal role in pain modulation, are involved in lidocaine-induced antinociception is hitherto unclear. In the present study, lidocaine was injected intravenously in rats using the formalin test for acute pain and the chronic constriction injury model for neuropathic pain. The effect of intrathecally administered d-serine (an agonist at the glycine-binding site at the NMDA-receptor), its inactive isomer l-serine, CGP 78608 (antagonist at the glycineB-site of the NMDA-receptor) and strychnine (antagonist at inhibitory glycine-receptors) on lidocaine-induced antinociception was examined. Systemically administered lidocaine was antinociceptive in both acute and chronic pain model. In the formalin test, the effect of lidocaine was antagonized by d-serine, but not by l-serine or strychnine. In the chronic constriction injury model, antinociception evoked by lidocaine was reduced by d-serine, strychnine and CGP 78608, while l-serine had no effect. These results indicate a modulatory effect of lidocaine on the NMDA-receptor. Additionally, since in our study lidocaine-induced antinociception was antagonized by both glycineB-site modulators and strychnine our results may favor the hypothesis of a general glycine-like action of lidocaine or some of its metabolites on inhibitory strychnine-sensitive receptors and on strychnine-insensitive glycine receptors.
Keywords: Lidocaine; Pathological pain; Nociception; Chronic constriction injury; Formalin test; Glycine;

Involvement of different types of potassium channels in the antidepressant-like effect of tramadol in the mouse forced swimming test by Cristiano R. Jesse; Ethel A. Wilhelm; Nilda B.V. Barbosa; Cristina W. Nogueira (74-78).
Administration of tramadol elicited an antidepressant-like effect in the rat forced swimming test (FST) by a mechanism dependent on the inhibition of the L-arginine-nitric oxide (NO)-guanylate cyclase pathway. Since it has been reported that NO can activate different types of potassium (K+) channels in several tissues, the present study investigated the possibility of synergistic interactions between different types of K+ channel inhibitors and tramadol in the mouse FST. Intracerebroventricular pretreatment of mice with tetraethylammonium (TEA, a non-specific inhibitor of K+ channels, 25 pg/site), glibenclamide (an ATP-sensitive K+ channel inhibitor, 0.5 pg/site) or charybdotoxin (a large- and intermediate conductance calcium-activated K+ channel inhibitor, 25 pg/site) was able to produce a synergistic action of a subeffective dose of tramadol (1 mg/kg, p.o.). Conversely, pretreatment with apamin (a small-conductance calcium-activated K+ channel inhibitor, 10 pg/site) did not modify the action of a subeffective dose of tramadol (1 mg/kg, p.o.). Administration of tramadol and the K+ channel inhibitors, alone or in combination, did not affect the number of crossings and rearings in the open field test (OFT). Reduction in the immobility time elicited by an active dose of tramadol (40 mg/kg, p.o.) in the FST was prevented by pretreatment of mice with cromakalim (a K+ channel opener, 10 µg/site, i.c.v.), without affecting the number of crossings and rearings in the OFT. Thus, our findings clearly suggest that oral acute administration of tramadol produces antidepressant-like effect on the FST in mice by a mechanism that involves the K+ channels.
Keywords: Tramadol; Potassium channels; Antidepressant; Mice (Mouse); Forced swimming test;

Effect of metoprolol on vulnerable plaque in rabbits by changing shear stress around plaque and reducing inflammation by Chen Liang; Li Xiaonan; Cai Xiaojun; Li Changjiang; Xu Xinsheng; Jiang Guihua; Hu Xiaobo; Zhang Yanen; Su Runyi; Lu Huixia; Zhang Yun; Zhang Mei (79-85).
The β-adrenoceptor antagonists are known to reduce cardiovascular events, but less is known about their effects on vulnerable plaque. The purpose of this study is to explore the role of metoprolol on vulnerable plaque and the possible mechanism. Vulnerable plaque model was established by local transfection with p53 gene in New Zealand Rabbits. Metoprolol treatment attenuated vessel positive remodeling and reduced vulnerability index (1.61 ± 0.58 vs. 2.33 ± 0.12, P  < 0.01). Although the difference did not reach statistical significance, the rate of rupture of atherosclerotic plaque (31% vs. 75%) and intima-media thickness (0.05 ±0.01 vs. 0.08 ± 0.01 cm) were less in the metoprolol group than in the control group. The level of shear stress-related inflammatory cytokines such as intercellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), matrix metalloproteinase 1 (MMP-1), were lower in the metoprolol group than in the control group (P  < 0.01). Compared with control group, total cholesterol and low-density lipoprotein cholesterol were lower (P  < 0.01) in the metoprolol group. After metoprolol treatment, shear stress increased, and was not different to baseline (physiological shear stress, P  > 0.05). Shear stress and vulnerability index showed a negative correlation. These findings suggest that metoprolol could inhibit the development of atherosclerosis and stabilize vulnerable plaque by regulation of lipid and reduction of inflammation, in which the change from low shear stress to physiological shear stress around plaque may play an important role.
Keywords: Metoprolol; Vulnerable plaque; Inflammation; Lipid; Shear stress;

Phenylephrine contracts porcine pulmonary veins via α1B-, α1D-, and α2-adrenoceptors by Tilo Görnemann; Carlos M. Villalón; David Centurión; Heinz H. Pertz (86-92).
We have recently shown that the postjunctional α2-adrenoceptor mediating contraction of porcine pulmonary veins is of the α2C-subtype. We could also demonstrate that α1-adrenoceptors might contribute to the contraction in that blood vessel. In the present study, we aimed at characterising the α1-adrenoceptor subtype(s) involved using pharmacological and molecular biological methods. In isolated rings of porcine pulmonary veins the typical α1-adrenoceptor agonist phenylephrine caused a concentration-dependent contraction that was inhibited by the α1B-adrenoceptor selective antagonists 1-[4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl]-2-[2-(isopropyl)-6-methoxyphenoxy]ethan-1-one (Rec15/2615; pA2 8.96 ± 0.13) and 4-amino-2-[4-[1-(benzyloxycarbonyl)-2(S)-[[(1,1-dimethylethyl)amino]carbonyl]-piperazinyl]-6,7-dimethoxyquinazoline (L-765,314; pA2 7.22 ± 0.05), as well as the α1D-adrenoceptor selective antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY7378; pA2 8.29 ± 0.15, slope of the Schild plot 0.75 ± 0.09, significantly different from unity, P  < 0.05), but not by the α1A-adrenoceptor selective antagonists (±)-1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl]amino]-2,4(1H,3H)-pyrimidinedione (B8805-033) and N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethanamine (RS-17053). These findings suggest that phenylephrine activates both α1B- and α1D-adrenoceptors. The observation was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) in porcine pulmonary veins, where mRNA signals for α1B- and α1D-adrenoceptors could be detected. However, the antagonist properties of rauwolscine and yohimbine (non-subtype selective α2-adrenoceptor antagonists) against phenylephrine showed that this agonist also activates α2-adrenoceptors in pulmonary veins. This was strengthened in experiments using tissues that were stimulated with forskolin (cell permeable activator of adenylyl cyclase). Phenylephrine mimicked the effect of the selective α2-adrenoceptor agonist UK14304 by causing an inhibition of forskolin-stimulated cAMP accumulation that was blocked by rauwolscine. It is concluded that, in addition to α1B- and α1D-adrenoceptors, phenylephrine can stimulate α2-adrenoceptors in porcine pulmonary veins.
Keywords: Pulmonary vein; Phenylephrine; α1-Adrenoceptor subtypes; α2-Adrenoceptors; (Pig);

Cardioprotection of salidroside from ischemia/reperfusion injury by increasing N-acetylglucosamine linkage to cellular proteins by Tiejun Wu; Heping Zhou; Zhenxiao Jin; Shenghui Bi; Xiuling Yang; Dinghua Yi; Weiyong Liu (93-99).
The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is increasingly recognized as an important posttranslational modification that modulates cellular function. Recent studies suggested that augmentation of O-GlcNAc levels increase cell survival following stress. Salidroside, one of the active components of Rhodiola rosea, shows potent anti-hypoxia property. In the present study, we reported the cardioprotection of salidroside from ischemia and reperfusion. Cardiomyocytes were exposed to 4 h of ischemia and 16 h of reperfusion, and then cell viability, apoptosis, glucose uptake, ATP levels and cytosolic Ca2+ concentration were determined, and O-GlcNAc levels were assessed by Western blotting. Salidroside (80 uM) was added 24 h before ischemia/reperfusion was induced. Treatment with salidroside markedly improved cell viability from 64.7 ± 4.5% to 85.8 ± 3.1%, decreased lactate dehydrogenase (LDH) release from 38.5 ± 2.1% to 21.2 ± 1.7%, reduced cell apoptosis from 27.2 ± 3.2% to 12.2 ± 1.9%, significantly improved cardiomyocytes glucose uptake by 1.7-fold and increased O-GlcNAc levels by 1.6-fold, as well as reducing cytosolic Ca2+ concentration compared to untreated cells following ischemia/reperfusion. Furthermore, the improved cell survival and the increase in O-GlcNAc with salidroside were attenuated by alloxan, an inhibitor of O-GlcNAc transferase. These results suggested that salidroside significantly enhances glucose uptake and increases protein O-GlcNAc levels and this is associated with decreased cardiomyocytes injury following ischemia/reperfusion.
Keywords: Salidroside; Ischemia; Reperfusion; Acetylglucosamine;

Five different profiles of dihydropyridines in blocking T-type Ca2+ channel subtypes (Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3 (α1I)) expressed in Xenopus oocytes by Taiji Furukawa; Toshihide Nukada; Yoshiko Namiki; Yoriko Miyashita; Kento Hatsuno; Yasunari Ueno; Takeshi Yamakawa; Takaaki Isshiki (100-107).
1,4-dihydropyridine (DHP) Ca2+ antagonists have recently been shown to block T-type Ca2+ channels, which may render favorable actions on cardiovascular systems. However, this evaluation remains to be done systematically for each T-type Ca2+ channel subtype except for the Cav3.1 (α1G) subtype. To address this issue at the molecular level, blocking effects of 14 kinds of DHPs (amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, efonidipine, felodipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nitrendipine), which are clinically used for treatments of hypertension, on 3 subtypes of T-type Ca2+ channels [Cav3.2 (α1H), Cav3.3 (α1I), and Cav3.1 (α1G)] were investigated in the Xenopus oocyte expression system using the two-microelectrode voltage-clamp technique. These 3 kinds (α1H, α1I and α1G) of T-type channels were blocked by amlodipine, manidipine and nicardipine. On the other hand, azelnidipine, barnidipine, benidipine and efonidipine significantly blocked α1H and α1G, but not α1I channels, while nilvadipine and nimodipine apparently blocked α1H and α1I, but not α1G channels. Moreover, aranidipine blocked only α1H channels. By contrast, cilnidipine, felodipine, nifedipine and nitrendipine had little effects on these subtypes of T-type channels. The result indicates that the blockade of T-type Ca2+ channels by derivatives of DHP Ca2+ antagonist was selective for the channel subtype. Therefore, these selectivities of DHPs in blocking T-type Ca2+ channel subtypes would provide useful pharmacological and clinical information on the mode of action of the drugs including side-effects and adverse effects.
Keywords: T-type calcium channel; Dihydoropyridine; Calcium antagonist; Xenopus oocyte;

Portal hypertension is a hemodynamic syndrome due to pathological increase in portal flow and portal pressure. These pathological changes in external flow loads will inevitably cause vascular remodeling in the portal vein, which is usually measured by an opening angle. The present study showed that carbon tetrachloride (CCl4)-induced portal hypertension fully developed at 8 weeks and the opening angle of portal vein increased progressively in the pathogenesis of intrahepatic portal hypertension which was significantly augmented at 10 weeks. Although portal pressure and portal flow were reduced, treatment with either propranolol or nifedipine alone for 3 weeks did not decrease the augmented opening angle of the portal vein, while combined treatment with propranolol and nifedipine markedly reduced the increased opening angle of the portal vein and endothelial nitric oxide synthase (eNOS) mRNA expression but not inducible nitric oxide synthase (iNOS) mRNA expression. The decreasing effect of propranolol plus nifedipine on the elevated opening angle was significantly weakened by L-arginine and markedly reinforced by N-nitro-l-arginine-mythel-ester (L-NAME). These results indicate that combined use of propranolol and nifedipine ameliorates portal vein remodeling in portal hypertension at least by the nitric oxide-dependent way.
Keywords: Portal hypertension; Portal vein; Opening angle; Remodeling; Propranolol; Nifedipine; Carbon tetrachloride;

Diabetes modulates capacitative calcium entry and expression of transient receptor potential canonical channels in human saphenous vein by Ada W.Y. Chung; Karen Au Yeung; Elliott Chum; Elena B. Okon; Cornelis van Breemen (114-118).
Diabetes is associated with a perturbation of signaling pathways in vascular tissue, which causes vasomotor dysfunction such as hypertension. We have previously demonstrated that vessels from diabetic patients were more contractile than those from non-diabetic. However, in human vessels, the receptor-stimulated contraction is mainly due to enzymatic, rather than calcium signaling pathway. In this study, we hypothesized that the differential contractile response between diabetic and non-diabetic human vessels could be due to the receptor signaling to sarcoplasmic reticulum and the regulation of capacitative calcium entry. In saphenous vein samples (n  = 20) collected from diabetic patients undergoing bypass surgery, the contraction initiated by the addition of the sarco-endoplasmatic reticulum calcium ATPase blocker, cyclopiazonic acid, was significantly higher than that in the vessels from non-diabetic patients (n  = 26) (84.0 ± 14.9% vs 44.2 ± 9.2%), and this contraction was inhibited by SKF-96365, an inhibitor of store-operated calcium channels. Pre-incubation with indomethacin reduced the cyclopiazonic acid-induced contraction in the non-diabetic veins, but had no effect on the diabetic ones. The gene expression of transient receptor potential canonical channels (TRPC)4 was upregulated by 22% in the diabetic vessels compared with the non-diabetic ones. However, the protein expression of TRPC1 and TRPC6 was downregulated in the diabetic group by 50%. We concluded that diabetes would modulate the capacitative calcium entry likely through the store-operated calcium channel specifically via the regulation of TRPC.
Keywords: Diabetes; Capacitative calcium entry; Transient receptor potential canonical channel; Vasomotor function; Store-operated calcium channel;

Experimental non-alcoholic fatty liver disease results in decreased hepatic uptake transporter expression and function in rats by Craig D. Fisher; Andrew J. Lickteig; Lisa M. Augustine; Ronald P.J. Oude Elferink; David G. Besselsen; Robert P. Erickson; Nathan J. Cherrington (119-127).
Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of diagnoses ranging from simple fatty liver (SFL), to non-alcoholic steatohepatitis (NASH). This study aimed to determine the effect of moderate and severe NAFLD on hepatic transporter expression and function in vivo. Rats were fed a high-fat diet (SFL model) or a methionine-choline-deficient diet (NASH model) for eight weeks. Hepatic uptake transporter function was determined by bromosulfophthalein (BSP) disposition. Transporter expression was determined by branched DNA signal amplification assay and western blotting; inflammation was identified by immunostaining of liver slices for interleukin 1 beta (IL-1β). MC− rats showed significant retention of BSP in the plasma when compared to control rats. Hepatic NTCP, OATP1a1, 1a4, 1b2 and 2b1; and OAT 2 and 3 mRNA levels were significantly decreased in high-fat and MC− diet rats when compared to control. Protein expression of OATP1a1 was significantly decreased in high-fat animals, while OATP1a1 and OATP1b2 expressions were significantly lower in MC− rats when compared to control. Liver tissue from high-fat and MC− rats stained positive for IL-1β, a pro-inflammatory cytokine known to decrease expression of NTCP, OATP and OAT transporters, suggesting a plausible mechanism for the observed transporter alterations. These data suggest that different stages of NAFLD result in altered hepatic uptake transporter expression that can lead to a functional impairment of xenobiotic uptake from the blood. Furthermore, NAFLD may alter the plasma retention time of clinically relevant drugs that are reliant on these transporters and may increase the potential drug toxicity.
Keywords: Fatty liver disease; Hepatic uptake transport; Inflammation;

Role of sensory innervation in the rat pulmonary neutrophil recruitment induced by staphylococcal enterotoxins type A and B by Ivani A. Desouza; Enilton A. Camargo; Nadia S. Mariano; João B. Optiz-Neto; Jorge S. Resende; Gláucia C. Mello; Soraia K.P. Costa; Gilberto De Nucci; Edson Antunes (128-134).
Rat airways exposure to Staphylococcal enterotoxin A (SEA) and B (SEB) induces marked neutrophil influx. Since sensory neuropeptides play important roles in cell infiltration, in this study we have investigated its contribution in triggering SEA- and SEB-induced pulmonary neutrophil infiltration. Male Wistar rats were exposed intratracheally with SEA (3 ng/trachea) or SEB (250 ng/trachea). Animals received different in vivo pretreatments, after which the neutrophil counts and levels of substance P and IL-1 in bronchoalveolar lavage fluid were evaluated. Alveolar macrophages and peritoneal mast cells were incubated with SEA and SEB to determine the IL-1 and TNF-α levels. Capsaicin pretreatment significantly reduced SEA- and SEB-induced neutrophil influx in bronchoalveolar lavage fluid, but this treatment was more effective to reduce SEA responses. Treatments with SR140333 (tachykinin NK1 receptor antagonist) and SR48968 (tachykinin NK2 receptor antagonist) decreased SEA-induced neutrophil influx, whereas SEB-induced responses were inhibited by SR140333 only. Cyproheptadine (histamine/5-hydroxytriptamine receptor antagonist) and MD 7222 (5-HT3 receptor antagonist) reduced SEA- and SEB-induced neutrophil influx. The substance P and IL-1 levels in bronchoalveolar lavage fluid of SEA-exposed rats were significantly higher than SEB. In addition, SEA (but not SEB) significantly released mast cell TNF-α. Increased production of TNF-α and IL-1 in alveolar macrophages was observed in response to SEA and SEB. In conclusion, sensory neuropeptides contribute significantly to SEA- and SEB-induced pulmonary neutrophil recruitment, but SEA requires in a higher extent the airways sensory innervation, and participation of mast cells and alveolar macrophage products.
Keywords: Enterotoxin; C-fiber; Airways; Neurogenic inflammation; Substance P; Cytokines;

Effects of silodosin and tamsulosin on the urethra and cardiovascular system in young and old dogs with benign prostatic hyperplasia by Shinya Kobayashi; Yoshitaka Tomiyama; Satoshi Tatemichi; Yuji Hoyano; Mamoru Kobayashi; Yoshinobu Yamazaki (135-140).
We examined whether the effects (efficacy on the urethra and hypotension) of silodosin (α1A-adrenoceptor antagonist) and tamsulosin (α1A + 1D-adrenoceptor antagonist) in dogs with benign prostatic hyperplasia altered with age. We used young and old dogs, diagnosed as having benign prostatic hyperplasia by veterinarian's palpation. Under anesthesia, the increase in intraurethral pressure evoked by hypogastric nerve stimulation was measured, together with the level of systemic mean blood pressure. Each drug was administered intravenously in progressively increasing doses. At the end of the experiment, the prostate was isolated from each dog, then weighed and investigated pathologically to confirm benign prostatic hyperplasia. The wet weight of the prostate was greater in old dogs with benign prostatic hyperplasia than in young dogs with benign prostatic hyperplasia. By light microscopy, hyperplasia in the prostatic epithelium was confirmed in both groups. Silodosin (0.3–300 μg/kg) dose-dependently inhibited the hypogastric nerve stimulation-induced increase in intraurethral pressure (without significant hypotensive effects) in both young and old dogs with benign prostatic hyperplasia. Tamsulosin (0.3–300 μg/kg) also dose-dependently inhibited the intraurethral pressure increase in both groups, but it had a hypotensive effect that was significantly greater in old than in young dogs with benign prostatic hyperplasia. In conclusion, as regards the effect of silodosin on intraurethral pressure, potency was similar between young and old dogs with benign prostatic hyperplasia, and it was without significant hypotensive effects. We therefore suggest that silodosin might be a good medication for lower urinary tract symptoms in patients with benign prostatic hyperplasia in all age groups.
Keywords: α1-Adrenoceptor; Benign prostatic hyperplasia; Silodosin; Aging;

IL-6-trans-signalling increases rapid-eye-movement sleep in rats by Ulrike May; Thomas Schiffelholz; Paul Christian Baier; James M. Krueger; Stefan Rose-John; Jürgen Scheller (141-145).
Interleukin 6 (IL-6), a cytokine of the gp130-signalling-family, plays an important role in immediate immunological functions, in metabolism and in the central nervous system. IL-6-signalling is mediated by classic-signalling via the membrane bound IL-6 receptor or by IL-6-trans-signalling via the soluble IL-6 receptor. Whereas the receptor subunit gp130 is ubiquitously expressed within the body, IL-6 receptor expression is restricted to distinct cell populations. Within the brain parenchyma the IL-6 receptor is sparsely expressed, and therefore the brain is mostly dependent on IL-6-trans-signalling in its response to IL-6. Recently we have shown that IL-6-trans-signalling but not classic-signalling plays a pivotal role in the establishment and maintenance of chronic inflammation and cancer, whereas its role in sleep regulation has not been studied so far. We reasoned that the IL-6-trans-signalling mimetic Hyper-IL-6 which in contrast to IL-6 alone can activate almost all cells of the brain might have a profound effect on sleep regulation and performed sleep recordings with rats injected with recombinant Hyper-IL-6. In the present study, the i.c.v. administration of the designer cytokine Hyper-IL-6 into rats at dark onset increased the amount of rapid-eye-movement sleep (REM sleep) but did not affect non-rapid-eye-movement sleep (non-REM sleep). Our data define a new role of IL-6-trans-signalling in sleep regulation.
Keywords: IL-6-trans-signalling; Sleep; Hyper-IL-6; Cytokine; (Rat);

SDF-1alpha up-regulates interleukin-6 through CXCR4, PI3K/Akt, ERK, and NF-kappaB-dependent pathway in microglia by Dah-Yuu Lu; Chih-Hsin Tang; Wei-Lan Yeh; Kar-Lok Wong; Chih-Peng Lin; Yi-Hung Chen; Chih-Ho Lai; Yuh-Fung Chen; Yuk-Man Leung; Wen-Mei Fu (146-154).
Stromal cell-derived factor-1 (SDF-1), also known as CXCL12, and its receptor CXC chemokine receptor 4 (CXCR4) express in various kinds of cells in central nervous system. The SDF-1/CXCR4 signaling pathway is regulated by diverse biological effects. SDF-1 is up-regulated in the ischemic penumbra following stroke and has been known to be associated with the homing of bone marrow cells to injury. However, the effect of SDF-1α/CXCR4 on cytokine production in microglia is mostly unknown. Here, we demonstrated that SDF-1α enhanced IL-6 production in both primary cultured microglia and BV-2 microglia. We further investigated the signaling pathway involved in IL-6 production stimulated by SDF-1α in microglia. SDF-1α increased IL-6 production in both protein and mRNA levels. These effects were attenuated by ERK, phosphatidylinositol 3-kinase (PI3K), NF-κB inhibitors, and IκB protease inhibitor. Stimulation of microglia with SDF-1α also increased Akt and ERK1/2 phosphorylation. In addition, SDF-1α treatment also increased IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, IκBα degradation, p65 phosphorylation at Ser276, translocation of p65 and p50 from cytosol to nucleus and κB-luciferase activity. Moreover, SDF-1α-mediated increase of κB-luciferase activity was inhibited by pre-transfection of DN-p85, DN-Akt or DN-ERK2. Increase of IKK α/β phosphorylation and binding of p65 and p50 to the NF-κB element were both antagonized by PI3K and ERK inhibitors. Our results demonstrate a mechanism linking SDF-1α and IL-6, and provide additional support for the notion that SDF-1α plays a regulatory role in microglia activation.
Keywords: SDF-1α; Microglia; IL-6; NF-κB; ERK; Akt;

Inhibition of cathepsin K reduces bone erosion, cartilage degradation and inflammation evoked by collagen-induced arthritis in mice by Lena Svelander; Helena Erlandsson-Harris; Leif Astner; Urszula Grabowska; Lars Klareskog; Erik Lindstrom; Ellen Hewitt (155-162).
Cathepsin K (EC 3.4.22.38) is expressed by osteoclasts and synovial fibroblasts and its proteolytic activity is hypothesized to play a role in the pathology of rheumatoid arthritis. This study explored the effects of the cathepsin K inhibitor N-(1-{[(Cyanomethyl)amino]carbonyl}cyclohexyl)-4-[2-(4-methylpiperazin-1-yl)-1,3-thiazol-4-yl]benzamide (L-006235) in murine collagen-induced arthritis. L-006235 is a potent inhibitor of recombinant human and murine cathepsin K, enzymes (K i :0.073 nM and IC50: 2.4 nM, respectively) and at the cellular level in human osteoclasts (IC50: 28 nM) with ~ 1000-fold selectivity against cathepsin S. L-006235 did not result in splenic invariant chain p10 accumulation, a specific marker of cathepsin S inhibition. L-006235 was dosed daily (25 mg/kg, p.o.), either prophylactically (days 0–42) or therapeutically (14 days post onset of disease) to DBA/1J mice subjected to collagen-induced arthritis. Disease severity was scored during the course of the study. Histological evaluation of cartilage and bone degradation together with related biomarkers namely, deoxypyridinoline, cartilage oligomeric matrix protein and C-terminal telopeptide degradation product of type I collagen (CTX-I) were analyzed after the study. After prophylactic or therapeutic administration, L-006235 significantly reduced biomarkers reflecting bone and cartilage degradation. Pathological changes at the histological level were significantly reduced after prophylactic treatment (P  < 0.01), but not after therapeutic treatment. Prophylactic treatment with L-006235 delayed disease onset (P  < 0.01) and reduced the disease severity score (P  < 0.05). Inhibition of cathepsin K activity exerts beneficial effects on collagen-induced arthritis in mice and thus warrants further investigation as a therapeutic intervention in human rheumatoid arthritis.
Keywords: Cathepsin K; Rheumatoid arthritis; Collagen-induced arthritis; Bone; Cartilage; Inflammation;

ASB16165, a phosphodiesterase 7A inhibitor, reduces cutaneous TNF-α level and ameliorates skin edema in phorbol ester 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation model in mice by Kumiko Kadoshima-Yamaoka; Megumi Goto; Masao Murakawa; Ryosuke Yoshioka; Yoshitaka Tanaka; Hidekazu Inoue; Hidenobu Murafuji; Satomi Kanki; Yasuhiro Hayashi; Kazuhiro Nagahira; Atsuto Ogata; Takashi Nakatsuka; Yoshiaki Fukuda (163-166).
Possible role of phosphodiesterase 7A (PDE7A) in skin inflammation was examined using ASB16165, a specific inhibitor for PDE7A. Epicutaneous application of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse ear resulted in induction of skin edema, and topical treatment with ASB16165 inhibited the induction of skin edema in a dose-dependent manner. The TPA challenge also increased the level of TNF-α at the application site, and the ASB16165 treatment reduced the TNF-α level in the skin. In addition, ASB16165 suppressed the production of TNF-α by human keratinocytes stimulated in vitro with TPA and calcium ionophore. Forskolin, an activator of adenylyl cyclase, as well as dibutyryl cAMP also showed inhibitory effect on the TNF-α production in the cells, suggesting involvement of cAMP in TNF-α generation. These results demonstrate that PDE7A might regulate TNF-α production in keratinocytes in a cAMP-dependent fashion. As immunostaining analysis revealed that PDE7A is expressed in the epidermis and TNF-α is known to contribute to the TPA-induced edema, it is possible that the inhibitory effect of ASB16165 on skin edema in mouse TPA-induced dermatitis model is mediated by suppression of TNF-α production. This is the first report suggesting the association of PDE7A with the function of keratinocytes. ASB16165 will be useful as an agent for skin inflammation in which TNF-α plays a pathogenic role (e.g. psoriasis).
Keywords: Skin inflammation; Keratinocyte; TNF-α; Phosphodiesterase 7 (PDE7); Psoriasis;

Pyrrolidine dithiocarbamate, a NF-κB inhibitor, upregulates MMP-1 and MMP-13 in IL-1β-stimulated rheumatoid arthritis fibroblast-like synoviocytes by Kyoung Soo Kim; Da Hee Oh; Hyun Mi Choi; Jun Soo Bang; Chun Jeih Ryu; Jung Hoe Kim; Myung Chul Yoo; Hyung-In Yang (167-175).
Activated NF-κB plays an important role in the expression of matrix metalloproteinase (MMP)-1 and MMP-13 in rheumatoid arthritis and osteoarthritis. The objective of this study was to determine the effects of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) on the expression of MMPs in IL-1β-stimulated fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis patients. FLSs were treated with IL-1β (10 ng/ml) for 24 h in the presence or absence of PDTC. The level of MMP-1 and MMP-13 increased in response to PDTC in time- and dose-dependent manners in IL-1β-stimulated FLSs; the expressions of IL-6 and vascular endothelial growth factor (VEGF) decreased in a PDTC concentration-dependent manner. However, PDTC-mediated repression of IL-6 and VEGF expression was not observed in TNF-α-stimulated rheumatoid arthritis FLSs. In contrast, other NF-κB inhibitors, such as fenofibrate, N-acetylcysteine and MG132, decreased MMP expression in IL-1β-stimulated FLSs. The stimulatory effect of PDTC on MMP expression was not mimicked by specific inhibitors of the mitogen-activated protein kinase (MAPK) signaling pathway. Treatments with 100 μM PDTC did not inhibit the phosphorylation of p-ERK1/2, p-P38, and p-JNK, or the transnuclear migration of NF-κB through degradation of IκB-α in IL-1β-stimulated FLSs. These results suggest that the increase of MMP expression may occur in a stimuli-specific manner or by an NF-κB independent mechanism. Therefore, therapeutic NF-κB inhibitors should be thoroughly studied before their clinical use in treating rheumatoid arthritis, as undesirable genes may be upregulated through unknown mechanisms, possibly resulting in worse symptoms.
Keywords: Pyrrolidine dithiocarbamate (PDTC); Fibroblast-like synoviocytes (FLS); Arthritis; Matrix metalloproteinase (MMP); NF-κB inhibitor;

Expression of the epithelial Na+ channel and other components of an aldosterone response pathway in human adrenocortical cells by Timothy J. Burton; Georgina Cope; Jing Wang; Joalice C. Sim; Elena A.B. Azizan; Kevin M. O'Shaughnessy; Morris J. Brown (176-181).
We have unexpectedly found expression of the epithelial Na+ channel (ENaC) in human adrenocortical cells and tested the hypothesis that these cells contain the components of an aldosterone response pathway. Tissue was obtained from patients undergoing adrenalectomy and mRNA and protein expression of recognised components of an aldosterone-response pathway were determined by RT-PCR and Western blotting. The effects of mineralocorticoid receptor agonists and antagonists, amiloride analogues, and extracellular Na+ on basal and stimulated aldosterone release from immortalised (H295R) cells were determined by radioimmunoassay. Expression of mRNA for α-, β- and γ-subunits of ENaC, the mineralocorticoid receptor, Nedd4L, Sgk1 and 11β hydroxysteroid dehydrogenase type II was confirmed in human adrenal cortex. Using Western blotting α-, β- and γ-ENaC expression was demonstrated in adrenocortical cells. Measurements of 24 h aldosterone release from H295R cells showed stimulation by K+ and angiotensin II, suppression by both Na+ and high-concentration 5-(N-ethyl-N-isopropyl) amiloride (EIPA, blocker of Na+–H+ exchange) and no change with benzamil (ENaC blocker). 22Na-uptake into H295R cells was inhibited by EIPA, but not by benzamil. Our experiments suggest that the components of an aldosterone response pathway are present in human adrenal cortex. Studies in H295R cells, however, suggest that ENaC is not an important mediator of 22Na-uptake or aldosterone production. Further studies are required to determine the importance of an adrenal aldosterone response pathway.
Keywords: Epithelial Na+ channel; Aldosterone; Adrenal cortex;

Both V1A and V1B vasopressin receptors deficiency result in impaired glucose tolerance by Kazuaki Nakamura; Toshinori Aoyagi; Masami Hiroyama; Shinji Kusakawa; Reiko Mizutani; Atsushi Sanbe; Junji Yamauchi; Masazumi Kamohara; Kazuhiro Momose; Akito Tanoue (182-188).
[Arg8]-vasopressin (AVP) is involved in the regulation of glucose homeostasis via vasopressin V1A and vasopressin V1B receptor. Our previous studies have demonstrated that vasopressin V1A receptor deficient (V1AR−/−) mice exhibited hyperglycemia, vasopressin V1B receptor deficient (V1BR−/−) mice, in contrast, exhibited hypoglycemia with hypoinsulinemia. These findings indicate that vasopressin V1A receptor deficiency results in decreased insulin sensitivity, whereas vasopressin V1B receptor deficiency results in increased insulin sensitivity. In our previous and present studies, we used the glucose tolerance test to investigate glucose tolerance in mutant mice, lacking either the vasopressinV1A receptor, the vasopressin V1B receptor, or both receptors, that were kept on a high-fat diet. Glucose and insulin levels were lower in V1BR−/− mice than in wild type (WT) mice when both groups were fed the high-fat diet, which indicates that the insulin sensitivity of the V1BR−/− mice was enhanced. V1AR−/− mice on the high-fat diet, on the other hand, exhibited overt obesity, along with an impaired glucose tolerance, while WT mice on the high-fat diet did not. Next, in order to assess the effect of vasopressin V1B receptor deficiency on the development of glucose intolerance caused by vasopressin V1A receptor deficiency, we generated mice that were deficient for both vasopressin V1A receptor and vasopressin V1B receptor (V1ABR−/−), fed them a high-fat diet, and examined their glucose tolerances using the glucose tolerance test. Glucose tolerance was impaired in V1ABR−/− mice, suggesting that the effects of vasopressin V1B receptor deficiency could not influence the development of hyperglycemia promoted by vasopressin V1A receptor deficiency, and that blockade of both receptors could lead to impaired glucose tolerance.
Keywords: Vasopressin; Glucose homeostasis; Vasopressin V1 receptor;