European Journal of Pharmacology (v.679, #1-3)

Together with undernutrition and, on the opposite, overeating and obesity, sudden tissue hypoperfusion is the most important cause of mortality and disability worldwide. Tissue hypoperfusion/hypoxia rapidly triggers an unrestrained inflammatory cascade that is the main responsible for the severity of the eventual outcome. The brain plays a key role in inflammation, either through activation of the hypothalamic-pituitary-adrenal humoral response or through activation of the vagal “cholinergic anti-inflammatory pathway”. Both humoral and nervous brain responses to inflammation are under the regulatory control of melanocortins, which have moreover a direct anti-inflammatory effect on inflammatory cells. Abundant experimental and clinical evidence indicates that MC3/MC4 melanocortin receptor agonists and cholinergic receptor agonists (mainly at the α7-nicotinic subtype) should by now be considered as completely innovative, effective drugs for the treatment of hypoxic conditions; melanocortin agonists being practically devoid of harmful side effects.
Keywords: Ischemic disease; Tissue hypoxia; Ischemia–reperfusion; Brain control of inflammation; Cholinergic anti-inflammatory pathway; Melanocortins; α7-Nicotinic receptor agonists; Muscarinic receptor agonists; Shock; Stroke; Myocardial infarction;

Angiotensin AT1 receptor blockers suppress oxidized low-density lipoprotein-derived formation of foam cells by Mayuko Osada-Oka; Haruna Kita; Seiko Yagi; Takashi Sato; Yasukatsu Izumi; Hiroshi Iwao (9-15).
Although angiotensin II potently affects blood pressure and fluid balance, it is also involved in deterioration in atherosclerotic cardiovascular disease. Recently, angiotensin AT1 receptor blockers have been demonstrated to be effective in patients with atherosclerotic disease, but the exact mechanisms of these blockers are still controversial. Atherosclerotic plaques are characterized by cholesterol ester accumulation and acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) expression, which are both parameters of degeneration of macrophage-derived foam cells. We examined the effects of angiotensin AT1 receptor blockers on the formation of foam cells from macrophages.When macrophages from a human cell line were stimulated with oxidized low-density lipoprotein (oxLDL), the angiotensin AT1 receptor blockers candesartan and losartan attenuated the intracellular accumulation of cholesterol ester and the increases in mRNA and protein levels of ACAT-1. Moreover, the increase in oxLDL-induced ACAT-1 was reduced by AG1478, an inhibitor of the epidermal growth factor (EGF) receptor. Additionally, oxLDL up-regulated the protein level of heparin-binding EGF-like growth factor (HB-EGF), a ligand of the EGF receptor. Inhibitors of angiotensin-converting enzyme affected neither cholesterol ester accumulation nor the expression of ACAT-1. Although oxLDL itself increased the secretion of angiotensin II, the amount of secreted angiotensin II was insufficient to induce expression of ACAT-1 protein.Thus, we first demonstrated that angiotensin AT1 receptor blockers suppress ACAT-1 expression and cholesterol ester accumulation through an oxLDL-activated EGF receptor, but it is unclear how oxLDL activates angiotensin AT1 receptor in an angiotensin II-independent manner. The therapeutic mechanism of angiotensin AT1 receptor blockers for atherosclerosis may be at least partially explained by our present results.
Keywords: Angiotensin AT1 receptor; Oxidized LDL; Macrophage;

In the present study, adrenocorticotropic hormone (ACTH) release and intracellular calcium ([Ca2+]i) increase induced by arginine vasopressin (AVP) were characterized in collagenase-dispersed and 3-day cultured rat anterior pituitary cells. AVP and the selective vasopressin V1b receptor agonist, [1-deamino-4-cyclohexylalanine]AVP (d[Cha4]AVP) induced ACTH release with nanomolar potencies in both cell preparations, and produced a maximal stimulation that was about 1.5 fold greater in the 3-day cultured cells, indicating that the vasopressin V1b receptor–ACTH release pathway is enhanced over time in culture. In dispersed cells, AVP, oxytocin and d[Cha4]AVP induced [Ca2+]i increases with nanomolar potencies. The selective vasopressin V1a receptors antagonist, SR49059 (100 nM), together with the selective oxytocin receptors antagonist (d(CH2)5 1Tyr(Me)2,Thr4,Orn8,Tyr-NH2 9-vasotocin (100 nM), inhibited the maximal AVP response by ~ 70%, without affecting the response to d[Cha4]AVP, suggesting that the V1b receptor was only partially responsible for the AVP-induced [Ca2+]i increase. In contrast, in 3-day cultures, AVP induced an increase in [Ca2+]i, while oxytocin and d[Cha4]AVP did not. The response to AVP was completely antagonized by SR49059, whereas the vasopressin V1b receptor antagonists, SSR149415 and (d(CH2)5 1Tyr(Me)2,Thr4,Orn8,Tyr-NH2 9)-vasotocin had no effect, indicating that the [Ca2+]i increase was mediated exclusively by vasopressin V1a receptors. In conclusion, the enhancement of vasopressin V1b receptor-mediated ACTH release and the lack of a detectable vasopressin V1b receptor coupling to [Ca2+]i increase in cultured cells suggests the activation of a different/additional signaling pathway in the molecular mechanism of ACTH release.
Keywords: ACTH; Rat pituitary cell; Calcium response; Vasopressin V1a receptor; Vasopressin V1b receptor;

Invasion and metastasis are the major causes of treatment failure in patients with cancer. Here, we investigated the effects of ginsenoside Rh1 on tumor invasion and metastasis in human hepatocellular carcinoma HepG2 cells and its possible mechanism of action. Rh1 showed concentration- and time-dependent inhibition of HepG2 cell migration and invasion. Matrix metalloproteinase-1 (MMP-1) gene expression and its promoter activity were also concentration-dependently inhibited by Rh1 treatment. The inhibitory effect of Rh1 on MMP-1 expression was due to inactivation of the mitogen-activated protein kinases (MAPKs) ERK, JNK, and p38 MAPK. By transient transfection analysis with the MMP-1 promoter (− 2846 to − 29 nt) and AP-1 promoter, MMP-1 and AP-1 promoter activities were induced by phorbol myristate acetate (PMA) but were significantly inhibited by PD98059 (ERK1/2 inhibitor) or SP600125 (JNK inhibitor). The induction of MMP-1 and AP-1 promoters by PMA was attenuated by Rh1, and both promoter activities were synergistically inhibited by co-treatment with PD98059. To evaluate the effects of Rh1 on AP-1 dimers, expression analysis and electrophoretic mobility shift (EMSA) assay using radiolabeled AP-1-specific oligomers at proximal site (− 73 nt) and distal site (− 1600 nt) of the MMP-1 promoter were performed. The results showed that Rh1 inhibited the expression of c-Jun and c-Fos but did not affect the DNA binding ability of AP-1-specific oligomers. However, Rh1 attenuated the stability of c-Jun. Therefore, Rh1 has potential for development of novel chemotherapeutic agents for treatment of malignant cancers, including early hepatocellular carcinoma related to MMP-1 expression.
Keywords: Anti-metastasis; Ginsenoside Rh1; Invasion; Migration; AP-1; MMP-1;

Salidroside stimulates the accumulation of HIF-1α protein resulted in the induction of EPO expression: A signaling via blocking the degradation pathway in kidney and liver cells by Ken Yu-Zhong Zheng; Zhen-Xia Zhang; Ava Jiang-Yang Guo; Cathy Wen-Chuang Bi; Kevin Yue Zhu; Sherry Li Xu; Janis Ya-Xian Zhan; David Tai-Wei Lau; Tina Ting-Xia Dong; Roy Chi-Yan Choi; Karl Wah-Keung Tsim (34-39).
Rhodiolae Crenulatae Radix et Rhizoma (Rhodiola), the root and rhizome of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba, has been used as a traditional Chinese medicine (TCM) to increase the body resistance to mountain sickness in preventing hypoxia; however, the functional ingredient responsible for this adaptogenic effect has not been revealed. Here, we have identified salidroside, a glycoside predominantly found in Rhodiola, is the chemical in providing such anti-hypoxia effect. Cultured human embryonic kidney fibroblast (HEK293T) and human hepatocellular carcinoma (HepG2) were used to reveal the mechanism of this hematopoietic function mediated by salidroside. The application of salidroside in cultures induced the expression of erythropoietin (EPO) mRNA from its transcription regulatory element hypoxia response element (HRE), located on EPO gene. The application of salidroside stimulated the accumulation of hypoxia-inducible factor-1α (HIF-1α) protein, but not HIF-2α protein: the salidroside-induced HIF-1α protein was via the reduction of HIF-1α degradation but not the mRNA induction. The increased HIF-1α could account for the activation of EPO gene. These results supported the notion that hematopoietic function of Rhodiola was triggered, at least partially, by salidroside.A proposed mechanism for salidroside-induced the expressions of EPO gene in kidney and liver cells.Salidroside-induced EPO expression is mediated by HIF-1α, but not HIF-2α, that triggers the transcription of EPO gene via HRE regulatory element. Salidroside can accumulate HIF-1α protein via blocking the protein degradation pathway, possibly at the stage of blocking the hydroxylation of HIF-1α. However, salidroside cannot affect the transcriptional level of HIF-1α.Display Omitted
Keywords: Salidroside; Hypoxia-inducible factor; Erythropoietin; Hypoxia responsive element;

We previously reported that both nitric oxide (NO) generated from NO synthase by bombesin and NO generated from SIN-1 (NO donor) activate the brain cyclooxygenase (COX) (COX-1 for bombesin), thereby eliciting the secretion of both catecholamines (CA) from the adrenal medulla by brain thromboxane A2-mediated mechanisms in rats. NO exerts its effects via not only soluble guanylate cyclase, but also protein S-nitrosylation, covalent modification of a protein cysteine thiol. In this study, we clarified the central mechanisms involved in the bombesin-induced elevation of plasma CA with regard to the relationship between NO and COX-1 using anesthetized rats. Bombesin (1 nmol/animal, i.c.v.)-induced elevation of plasma CA was attenuated by carboxy-PTIO (NO scavenger) (0.5 and 2.5 μmol/animal, i.c.v.), but was not influenced by ODQ (soluble guanylate cyclase inhibitor) (100 and 300 nmol/animal, i.c.v.). The bombesin-induced response was effectively reduced by dithiothreitol (thiol-reducing reagent) (0.4 and 1.9 μmol/kg/animal, i.c.v.) and by N-ethylmaleimide (thiol-alkylating reagent) (0.5 and 2.4 μmol/kg/animal, i.c.v.). The doses of dithiothreitol also reduced the SIN-1 (1.2 μmol/animal, i.c.v.)-induced elevation of plasma CA, but had no effect on the U-46619 (thromboxane A2 analog) (100 nmol/animal, i.c.v.)-induced elevation of plasma CA even at higher doses (1.9 and 9.7 μmol/kg/animal, i.c.v.). Immunohistochemical studies demonstrated that the bombesin increased S-nitroso-cysteine-positive cells co-localized with COX-1 in the spinally projecting neurons of the hypothalamic paraventricular nucleus (PVN). Taken together, endogenous NO seems to mediate centrally administered bombesin-induced activation of adrenomedullary outflow at least in part by S-nitrosylation of COX-1 in the spinally projecting PVN neurons in rats.
Keywords: Brain; Nitric oxide; S-Nitrosylation; Bombesin; Adrenomedullary outflow; Cyclooxygenase-1;

Asiatic acid protects primary neurons against C2-ceramide-induced apoptosis by Xiangnan Zhang; Jiaying Wu; Yuan Dou; Binbin Xia; Weibo Rong; Gerald Rimbach; Yijia Lou (51-59).
Ceramides derived from sphingosine contribute to the apoptotic processes of neuronal cells in neurodegenerative disorders including Alzheimer's disease. This study investigates the potential neuroprotective effects of Asiatic acid, a triterpenoid derived from Centella asiatica, against C2-ceramides-induced cell death in primary cultured rat cortical neuronal cells. In primary neurons, Asiatic acid (0.01 to 1.0 μmol/l) reduced C2-ceramide-induced cell death and mitochondria membrane potential loss in a concentration-dependent manner. Furthermore, Asiatic acid decreased cellular production of reactive oxygen species following C2-ceramide treatment. At a maximal concentration of 1.0 μmol/l, Asiatic acid partly counteracted the pro-apoptotic effects of the C2-ceramide by reducing the cytosolic release of HtrA2/Omi, the upregulation of Bax and caspase 3, as well as the dephosphorlyation of ERK1/2. Taken together, these data suggest that Asiatic acid protects neurons from C2-ceramide-induced cell death by antagonizing mitochondria-dependent apoptosis.
Keywords: Asiatic acid; Neuron; C2-ceramide; Apoptosis; Mitochondrion;

Acute and chronic methylphenidate alters prefrontal cortex neuronal activity recorded from freely behaving rats by R. Layla Salek; Catherine M. Claussen; Adriana Pérez; Nachum Dafny (60-67).
Today's students around the world are striking deals to buy and sell the drug methylphenidate (MPD) for cognitive enhancement. Our knowledge on the effects of MPD on the brain is very limited. The present study was designed to investigate the acute and chronic effect of MPD on the prefrontal cortex (PFC) neurons. On experimental day 1 (ED1) recordings were obtained following saline injections and after 2.5 mg/kg MPD. On ED2 through ED6, daily single 2.5 mg/kg MPD was given followed by 3 washout days (ED7 to 9). On ED10, neuronal recordings were resumed from the same animal after saline and MPD injection similar to that obtained at ED1. Ninety PFC units were recorded, all responded to the initial MPD injection, 66 units (73%) increased their activity at ED10. Recordings were resumed for the 66 units that increased their firing rate at ED1, and following MPD injection 54 units (82%) exhibited significant increases in their baseline firing rates compared to ED1 baseline. When these 54 units were rechallenged (chronic effect) with MPD, 39/54 (72%) exhibited reduction in their firing rate which can be interpreted as tolerance. From the 24 (27%) units that responded to MPD at ED1 by decreasing their activity, 14 units (58%) exhibited a decrease in their baseline firing rates at ED10 compared to ED1 baseline. However, following MPD rechallenge of these 14 units, 11 units (79%) exhibited an increase in their firing rate which is interpreted as sensitization. In conclusion, all PFC units modified their neural baseline activity.
Keywords: Ritalin; Neuronal activity; Prefrontal cortex; Freely behaving rats;

Ferulic acid exerts antidepressant-like effect in the tail suspension test in mice: Evidence for the involvement of the serotonergic system by Ana Lúcia B. Zeni; Andréa Dias E. Zomkowski; Marcelo Maraschin; Ana Lúcia S. Rodrigues; Carla I. Tasca (68-74).
Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is a phenolic compound present in several plants with claimed beneficial effects in prevention and treatment of disorders linked to oxidative stress and inflammation. In this study, we aimed to verify the possible antidepressant-like effect of acute oral administration of ferulic acid in the forced swimming test (FST) and tail suspension test (TST) in mice. Additionally, the mechanisms involved in the antidepressant-like action and the effects of the association of ferulic acid with the antidepressants fluoxetine, paroxetine, and sertraline in the TST were investigated. Ferulic acid produced an antidepressant-like effect in the FST and TST (0.01–10 mg/kg, p.o.), without accompanying changes in ambulation. The pretreatment of mice with WAY100635 (0.1 mg/kg, s.c., a selective 5-HT1A receptor antagonist) or ketanserin (5 mg/kg, i.p., a 5-HT2A receptor antagonist) was able to reverse the anti-immobility effect of ferulic acid (0.01 mg/kg, p.o.) in the TST. The combination of fluoxetine (5 mg/kg, p.o.), paroxetine (0.1 mg/kg, p.o.) or sertraline (1 mg/kg, p.o.) with a sub-effective dose of ferulic acid (0.001 mg/kg, p.o.) produced a synergistic antidepressant-like effect in the TST, without causing hyperlocomotion in the open-field test. Taken together, these results demonstrate that ferulic acid exerts antidepressant-like effect in the FST and TST in mice through modulation of the serotonergic system.
Keywords: Depression; Ferulic acid; Tail suspension test; Antidepressant; Serotonergic system;

The glial cell modulators, ibudilast and its amino analog, AV1013, attenuate methamphetamine locomotor activity and its sensitization in mice by Sarah E. Snider; Sarah A. Vunck; Edwin J.C.G. van den Oord; Daniel E. Adkins; Joseph L. McClay; Patrick M. Beardsley (75-80).
Over 800,000 Americans abuse the psychomotor stimulant, methamphetamine, yet its abuse is without an approved medication. Methamphetamine induces hypermotor activity, and sensitization to this effect is suggested to represent aspects of the addiction process. Methamphetamine's regulation of 3′–5′-cyclic adenosine monophosphate (cAMP) levels may be partially responsible for its behavioral effects, and compounds that inhibit phosphodiesterase (PDE), the enzyme that degrades cAMP, can alter methamphetamine-induced behaviors. Methamphetamine also activates glial cells and causes a subsequent increase in pro-inflammatory cytokine levels. Modulation of glial cell activation is associated with changes in behavioral responses, and substances that oppose inflammatory activity can attenuate drug-induced behaviors. Ibudilast (aka AV411; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine), inhibits both PDE and glial pro-inflammatory activity. Ibudilast's amino analog, AV1013, modulates similar glial targets but negligibly inhibits PDE. The present study determined whether ibudilast and AV1013 would attenuate methamphetamine-induced locomotor activity and its sensitization in C57BL/6J mice. Mice were treated b.i.d. with ibudilast (1.8–13 mg/kg), AV1013 (10–56 mg/kg) or their vehicles intraperitoneally for 7 days, beginning 48 h before 5 days of daily 1-h locomotor activity tests. Each test was initiated by either a methamphetamine (3 mg/kg) or a saline injection. Ibudilast significantly (P < 0.05) reduced the acute, chronic, and sensitization effects of methamphetamine's locomotor activity without significantly affecting activity by itself. AV1013 had similar anti-methamphetamine effects, suggesting that glial cell activity, by itself, can modulate methamphetamine's effects and perhaps serve as a medication target for its abuse.
Keywords: Ibudilast; AV411; AV1013; Methamphetamine; Glial; Relapse; Sensitization; Locomotor activity;

The present study was aimed to evaluate the antihypertensive effect of diosmin in deoxycorticosterone acetate (DOCA)-salt induced hypertension in male Wistar rats. Hypertension was induced in uninephrectomized rats by weekly twice subcutaneous injection of DOCA (25 mg/kg body weight) and 1% NaCl in the drinking water for six consecutive weeks. The important pathological events that occurred in DOCA-salt treated rats were significant increase in systolic, diastolic blood pressure, sodium and chloride in serum and lipid peroxidation products (thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes) in plasma and tissues (liver, kidney, heart and aorta) and significant decrease in serum potassium, total nitrite and nitrate levels in plasma. The activities of hepatic aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma-glutamyl transpeptidase and the levels of renal urea, uric acid, creatinine in serum, water intake, and organ weight (kidney and heart) were significantly increased in DOCA-salt hypertensive rats. DOCA-salt treated rats also showed a significant decrease in body weight, activities of superoxide dismutase, catalase and glutathione peroxidase in erythrocyte and tissues and the levels of reduced glutathione, vitamin C and vitamin E in plasma and tissues. Treatment with diosmin (25, 50 and 100 mg/kg body weight) brings back all the above parameters to near normal level, in which 50 mg/kg body weight showed the highest effect than that of other two doses. Histopathology of heart and kidney also confirmed the protective effect of diosmin. Thus the experiment clearly showed that diosmin acts as an antihypertensive agent against DOCA-salt induced hypertension.
Keywords: Diosmin; Antioxidant; DOCA; Nitric oxide;

We have recently shown that responses to pressor nerve stimulation in the pithed rat are mediated by α1A- and α1D-adrenoceptors, with no evidence for α2-adrenoceptor involvement, and that responses previously identified as α2-adrenoceptor mediated are actually α1D-adrenoceptor mediated. We have now re-examined the subtypes of α-adrenoceptor involved in pressor responses produced by exogenous agonists in the pithed rat preparation to confirm whether α2-adrenoceptors are involved in these responses. The α2-adrenoceptor and α1D-adrenoceptor antagonist yohimbine (1 mg/kg) and the α2A-adrenoceptor antagonist methoxy-idazoxan (5 mg/kg) significantly shifted, but the α1D-adrenoceptor antagonist BMY 7378 (8-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspir o[4.5]decane-7,9-dione dihydrochloride) (1 mg/kg) did not affect, the pressor potency of the α2-adrenoceptor agonist xylazine. α1-adrenoceptor antagonists showed low potency against pressor responses to xylazine. The pressor potency of the α1-adrenoceptor agonist amidephrine was not affected by BMY 3778 (1 mg/kg) but significantly shifted by prazosin (0.01 mg/kg) and by yohimbine (1 mg/kg). In contrast, the pressor potency of phenylephrine was significantly shifted by both yohimbine and BMY 7378 (1 mg/kg), but to a greater extent by the α1A-adrenoceptor antagonist RS 100329 (5-Methyl-3-[3-[3-[4-[2-(2,2,2,trifluroethoxy) phenyl]-1-piperazinyl]propyl]-2,4-(1H,3H)-pyrimidinedione] hydrochloride) (0.1 mg/kg). In conclusion, we have identified and separated α1A-, α1D- and α2A-adrenoceptor antagonist actions of yohimbine against pressor responses. Pressor responses to exogenous agonists in the pithed rat involve both α1A- and α1D-adrenoceptors and in addition, α2A-adrenoceptors.
Keywords: α1A-adrenoceptor; α1D-adrenoceptor; α2-adrenoceptor; α2A-adrenoceptor; Pithed rat; Vasopressor responses;

Our previous studies showed that the hypotensive effect of chronic ethanol in female rats is reduced by ovariectomy (OVX) rats and was restored after estrogen replacement (OVXE2). Further, in randomly cycling rats, chronic ethanol increased cardiac parasympathetic dominance and subsequently reduced myocardial contractility and blood pressure (BP). In this study, we tested the hypothesis that alterations in myocardial contractility and sympathovagal control account for the E2 exacerbation of the hemodynamic effects of ethanol. BP, myocardial contractility (+ dP/dtmax), and spectral cardiovascular autonomic profiles were evaluated in radiotelemetered OVX, and OVXE2 rats receiving liquid diet with or without ethanol (5%, w/v) for 13 weeks. In OVX rats, ethanol caused modest hypotension along with significant increases in + dP/dtmax during weeks 2–5. The high-frequency (IBIHF, 0.75–3 Hz) and low-frequency (IBILF, 0.25–0.75 Hz) bands of interbeat intervals were briefly increased and decreased, respectively, by ethanol. Compared with its effects in OVX rats, chronic treatment of OVXE2 rats with ethanol elicited significantly greater and more sustained reductions in systolic (SBP) and diastolic (DBP) blood pressures and + dP/dtmax. Altered sympathovagal balance and parasympathetic overactivity were more evident in ethanol-treated OVXE2 rats as suggested by the sustained: (i) increases in high-frequency bands of interbeat intervals (IBIHF, 0.75–3 Hz), and (ii) decreases in low-frequency IBI bands (IBILF, 0.25–0.75 Hz), IBILF/HF ratio and + dP/dtmax. The plasma ethanol concentration was not affected by changes in the hormonal milieu. These findings suggest that estrogen exacerbates the ethanol-evoked reductions in myocardial contractility and BP and the associated parasympathetic overactivity in female rats.
Keywords: Ethanol; Estrogen; Blood pressure; Myocardial contractility; Hemodynamic variability; Female rat; (Rat);

Binge ethanol during chronic ethanol abuse augments liver injury but the underlying mechanism remains unknown. CREB (cyclic AMP response element binding protein) is implicated as a key transcription factor in liver regeneration and hepatic glucose and lipid metabolism. We examined the effects of ethanol on the phosphorylation of CREB in hepatocytes, and in vivo in rat liver after chronic ethanol binge. For in vivo studies, rats were fed ethanol in liquid diet for 4 weeks followed by single binge administration of ethanol (intragastric, 5 g/kg body weight). Four hours after binge administration, liver samples were collected and analyzed. Treatment of hepatocytes with ethanol caused increased phosphorylation of p38 MAPK (mitogen activated protein kinase), MSK-1 (mitogen and stress activated kinase) and CREB in the nuclear compartment without activation of ERK1/2 (extracellular regulated kinase); whereas angiotensin II induced activation of CREB was accompanied by activation of ERK1/2. In chronic ethanol-binge studies, analysis of the whole cell extracts showed increased phosphorylation of CREB, with no effect on CREB protein levels; increased phospho-ERK1/2, and decreased phospho-p38 MAPK. In contrast, the nuclear levels of phospho-CREB and CREB protein were reduced. Reduction in phospho-CREB and CREB proteins in the nuclear extracts was accompanied by suppression of mRNA levels for CPT-1 (carnitine palmitoyl transferase-1) and increase in hepatic steatosis after binge. It is concluded that binge ethanol causes defect in the nuclear accumulation of CREB protein, phospho-CREB, and an exaggerated hepatic steatosis. These in vivo effects are distinct from the effects of ethanol on hepatocytes in vitro.
Keywords: Binge; Chronic ethanol; CREB; MAPK; MSK-1; Nuclear translocation; Steatosis;

Fisetin, a bioactive flavonol, attenuates allergic airway inflammation through negative regulation of NF-κB by Fera Y. Goh; Nadine Upton; Shouping Guan; Chang Cheng; Muthu K. Shanmugam; Gautam Sethi; Bernard P. Leung; W.S. Fred Wong (109-116).
Persistent activation of nuclear factor-κB (NF-κB) has been associated with the development of asthma. Fisetin (3,7,3′,4′-tetrahydroxyflavone), a naturally occurring bioactive flavonol, has been shown to inhibit NF-κB activity. We hypothesized that fisetin may attenuate allergic asthma via negative regulation of the NF-κB activity. Female BALB/c mice sensitized and challenged with ovalbumin developed airway inflammation. Bronchoalveolar lavage fluid was assessed for total and differential cell counts, and cytokine and chemokine levels. Lung tissues were examined for cell infiltration and mucus hypersecretion, and the expression of inflammatory biomarkers. Airway hyperresponsiveness was monitored by direct airway resistance analysis. Fisetin dose-dependently inhibited ovalbumin-induced increases in total cell count, eosinophil count, and IL-4, IL-5 and IL-13 levels recovered in bronchoalveolar lavage fluid. It attenuated ovalbumin-induced lung tissue eosinophilia and airway mucus production, mRNA expression of adhesion molecules, chitinase, IL-17, IL-33, Muc5ac and inducible nitric oxide synthase in lung tissues, and airway hyperresponsiveness to methacholine. Fisetin blocked NF-κB subunit p65 nuclear translocation and DNA-binding activity in the nuclear extracts from lung tissues of ovalbumin-challenged mice. In normal human bronchial epithelial cells, fisetin repressed TNF-α-induced NF-κB-dependent reporter gene expression. Our findings implicate a potential therapeutic value of fisetin in the treatment of asthma through negative regulation of NF-κB pathway.
Keywords: Airway hyperresponsiveness; Flavonol; Lung epithelium; Ovalbumin; TNF-α;

Inhibitory effect of calcitonin gene-related peptide on hypoxia-induced rat pulmonary artery smooth muscle cells proliferation: Role of ERK1/2 and p27 by Xian-Wei Li; Chang-Ping Hu; Wei-Hua Wu; Wei-Fang Zhang; Xiao-Zhou Zou; Yuan-Jian Li (117-126).
Calcitonin gene-related peptide (CGRP) inhibits angiotensin II-induced proliferation of aortic smooth muscle cells via inactivation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). ERK1/2 is necessary for the degradation or down-regulation of the cell cycle inhibitor p27, and is also crucial in mediating proliferation of pulmonary artery smooth muscle cells (PASMCs). Whether ERK1/2/p27 signal pathway is involved in CGRP-mediated pathogenesis of pulmonary hypertension and vascular remodeling remains unknown. Pulmonary hypertension was induced by hypoxia in rats, and capsaicin (50 mg/kg, s.c.) was used to deplete endogenous CGRP. Proliferation of cultured PASMCs was determined by BrdU incorporation method and flow cytometry. The expression/level of CGRP, p27, ERK1/2, c-fos and c-myc was analyzed by radioimmunoassay, immunohistochemistry, real-time PCR or Western blot. Sensory CGRP depletion by capsaicin exacerbated hypoxia-induced pulmonary hypertension in rats, as shown by an increase in right ventricle systolic pressure, mean pulmonary artery pressure and vascular hypertrophy, accompanied with decreased p27 expression and increased expression of phosphorylated ERK1/2, c-fos and c-myc. Exogenous application of CGRP significantly inhibited hypoxia-induced proliferation of PASMCs concomitantly with increased p27 expression and decreased expression of phosphorylated ERK1/2, c-fos and c-myc. These effects of CGRP were abolished in the presence of CGRP8–37. Knockdown of p27 also reversed the inhibitory effect of CGRP on proliferation of PASMCs and expression of c-fos and c-myc, but not on ERK1/2 phosphorylation. These results suggest that CGRP inhibits hypoxia-induced proliferation of PASMCs via ERK1/2/p27/c-fos/c-myc pathway. Down-regulation of CGRP may contribute to remodeling of pulmonary arteries in hypoxia-induced pulmonary hypertension.
Keywords: Calcitonin gene-related peptide; Pulmonary hypertension; Pulmonary artery smooth muscle cell; p27; ERK1/2; Proliferation;

Effects of α1-adrenoceptor antagonists on phenylephrine-induced salivary secretion and intraurethral pressure elevation in anesthetized rats by Hiroko Yanai-Inamura; Akiyoshi Ohtake; Yukiko Noguchi; Toshiki Hatanaka; Masanori Suzuki; Koji Ueshima; Shuichi Sato; Masao Sasamata (127-131).
α1-Adrenoceptor antagonists are widely used for the treatment of voiding dysfunction associated with benign prostatic hyperplasia. Activation of α1-adrenoceptors is reported to induce salivary secretion in rats and humans. However, the effects of α1-adrenoceptor antagonists on salivary secretion remain unknown. Here, we investigated the effects of the α1-adrenoceptor antagonists prazosin, silodosin, tamsulosin and urapidil on phenylephrine-induced salivary secretion and compared the results with the effects on phenylephrine-induced intraurethral pressure (IUP) elevation in anesthetized rats. All antagonists inhibited phenylephrine-induced salivary secretion and IUP elevation in a dose-dependent fashion. Comparison of DR10 values (the dose required to shift the dose–response curve 10-fold to the right) in both tissues showed that the inhibitory effect of silodosin was significantly more potent in the salivary gland than in the urethra (18-fold), but tamsulosin (2.3-fold), prazosin (1.7-fold) and urapidil (1.1-fold) did not show comparable tissue selectivity. These results suggest that α1-adrenoceptor antagonists inhibit not only urethral contraction but also salivary secretion, and that high tissue selectivity for the salivary gland over the urethra as shown by silodosin may contribute to the incidence of dry mouth.
Keywords: α1-Adrenoceptor antagonist; Intraurethral pressure; Salivary secretion;

A structural modulator of tumor necrosis factor type 1 receptor promotes bone formation under lipopolysaccharide-induced inflammation in a murine tooth extraction model by Hiroyuki Nakachi; Kazuhiro Aoki; Nobuyoshi Tomomatsu; Neil Alles; Kenichi Nagano; Masashi Yamashiro; Hongtao Zhang; Ramachandran Murali; Mark I. Greene; Keiichi Ohya; Teruo Amagasa (132-138).
Recently an increase in the serum levels of a bone formation marker after anti-tumor necrosis factor (TNF)-α therapy in rheumatoid arthritis patients has been reported. However, there remains no direct evidence that TNF-α antagonist could accelerate bone formation under inflammatory condition. Cavity-induced allosteric modification (CIAM) compound, F002, is a newly developed-TNF-α antagonist, which was designed by using the structure of TNF type 1 receptor, thus preventing TNF-α-induced signaling. In this study, we examined whether the CIAM compound can promote bone formation under inflammatory condition induced by lipopolysaccharide (LPS). Thirty-six 10-week-old male mice (C57BL/6J) were used. Half of the mice received 10 mg/kg LPS, while the other half received PBS. Thereafter, incisor extraction was performed at 4 days after either LPS or PBS injection. Intraperitoneal injections of 2 mg/kg/day, 4 mg/kg/day CIAM, or vehicle (10% DMSO) were performed once a day from day 0 to day 7 after incisor tooth extraction. The mice were sacrificed at 21 days after the extraction. The regenerated bone mineral density (BMD) in the tooth socket, and the mineral apposition rate and the bone formation rate, direct evidences of bone formation, were significantly decreased in the LPS-injected group compared to the PBS-injected group. CIAM compound dose-dependently prevented the decrease of BMD under the LPS-injected condition, and promoted both the mineral apposition rate and the bone formation rate significantly compared to the LPS-injected group. We did not observe any significant differences among the PBS-injected groups. Taken together, TNF-α antagonist CIAM compound, was found to accelerate bone formation under LPS-induced inflammatory condition.Display Omitted
Keywords: TNF-α antagonist; Tooth extraction model; Bone formation; Inflammation; Bone destruction;

The constitutive androstane receptor (CAR, NR1I3) has a central role in detoxification processes, regulating the expression of a set of genes involved in metabolism. The dual role of NR1I3 as both a xenosensor and as a regulator of endogenous energy metabolism has recently been accepted. Here, we investigated the mechanism of transcriptional regulation of the glucose metabolising genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) by the cis isomer of 2,4,6-triphenyldioxane-1,3 (cisTPD), a highly effective NR1I3 activator in rat liver. It was shown that expression of the gluconeogenic genes PEPCK and G6Pase was repressed by cisTPD treatment under fasting conditions. Western-blot analysis demonstrated a clear reduction in the intensity of PEPCK and G6Pase immunobands from the livers of cisTPD-treated animals relative to bands from the livers of control animals. Chromatin immunoprecipitation assays demonstrated that cisTPD prevents the binding of FOXO1 to the insulin response sequences in the PEPCK and G6Pase gene promoters in rat liver. Moreover, cisTPD-activated NR1I3 inhibited NR2A1 (HNF-4) transactivation by competing with NR2A1 for binding to the NR2A1-binding element (DR1-site) in the gluconeogenic gene promoters. Thus, our results are consistent with the hypothesis that the cisTPD-activated NR1I3 participates in the regulation of the gluconeogenic genes PEPCK and G6Pase.
Keywords: NR1I3; CYP2B; PEPCK; G6Pase;

Protective effect of atorvastatin on bone tissue in orchidectomised male albino Wistar rats by Iveta Gradosova; Helena Zivna; Vladimir Palicka; Sona Hubena; Klara Svejkovska; Pavel Zivny (144-150).
Recent studies have shown that atorvastatin influences bone metabolism. We investigated its bone protective effect in orchidectomised rats after 12 weeks of treatment. Eight-week-old rats were divided into 3 groups: sham-operated group, control group after orchidectomy and experimental group after orchidectomy with atorvastatin administration (12 mg/kg/day). Bone mineral density and bone marker concentrations of aminoterminal propeptide of procollagen type I (PINP), osteoprotegerin (OPG), insulin-like growth factor 1 (IGF-1) in serum, and carboxy-terminal cross-linking telopeptide of type I collagen (CTX-I), bone alkaline phosphatase (BALP), bone morphogenetic protein 2 (BMP-2) in bone homogenate were measured. Total serum calcium and tibial calcium content was determined. Femurs were used for three-point bending test of the shaft and compression testing of the femoral neck. Bone markers (CTX-I, BALP, BMP-2) in control rats were higher vs. sham-operated rats. Atorvastatin reduced CTX-I, BMP-2 and OPG compared to controls. IGF-1 was decreased in control rats vs. sham-operated rats; atorvastatin increased IGF-1 vs. control rats. Atorvastatin exerts a positive effect on bone metabolism by increasing bone mineral density of the whole body, which had decreased under the effects of orchidectomy. Three-point bending test revealed an increase in maximal load values of the left femurs after atorvastatin administration compared to controls. The diameter of the left femur and length of both femurs were increased after atorvastatin administration compared to controls. Our findings suggest that atorvastatin has a beneficial effect on bone metabolism in orchidectomised rats by decreasing bone turnover, with resulting improvement in bone mineral density and bone biomechanical properties.
Keywords: Atorvastatin; Orchidectomised rat; Bone marker; Bone mineral density; Biomechanical testing;