Angiotensin-(1–7) in Paraventricular Nucleus Modulates Sympathetic Activity and Cardiac Sympathetic Afferent Reflex in Renovascular Hypertensive Rats

Background

Excessive sympathetic activity contributes to the pathogenesis and progression of hypertension. Enhanced cardiac sympathetic afferent reflex (CSAR) is involved in sympathetic activation. This study was designed to determine the roles of angiotensin (Ang)-(1–7) in paraventricular nucleus (PVN) in modulating sympathetic activity and CSAR and its signal pathway in renovascular hypertension.

Methodology/Principal Findings

Renovascular hypertension was induced with two-kidney, one-clip method. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in sinoaortic-denervated and cervical-vagotomized rats with anesthesia. CSAR was evaluated with the RSNA and MAP responses to epicardial application of capsaicin. PVN microinjection of Ang-(1–7) and cAMP analogue db-cAMP caused greater increases in RSNA and MAP, and enhancement in CSAR in hypertensive rats than in sham-operated rats, while Mas receptor antagonist A-779 produced opposite effects. There was no significant difference in the angiotensin-converting enzyme 2 (ACE2) activity and Ang-(1–7) level in the PVN between sham-operated rats and hypertensive rats, but the Mas receptor protein expression in the PVN was increased in hypertensive rats. The effects of Ang-(1–7) were abolished by A-779, adenylyl cyclase inhibitor SQ22536 or protein kinase A (PKA) inhibitor Rp-cAMP. SQ22536 or Rp-cAMP reduced RSNA and MAP in hypertensive rats, and attenuated the CSAR in both sham-operated and hypertensive rats.

Conclusions

Ang-(1–7) in the PVN increases RSNA and MAP and enhances the CSAR, which is mediated by Mas receptors. Endogenous Ang-(1–7) and Mas receptors contribute to the enhanced sympathetic outflow and CSAR in renovascular hypertension. A cAMP-PKA pathway is involved in the effects of Ang-(1–7) in the PVN.     

Central bombesin possibly induces S-nitrosylation of cyclooxygenase-1 in pre-sympathetic neurons of rat hypothalamic paraventricular nucleus

Aims

Cyclooxygenase (COX) can be activated by nitric oxide-induced (NO-induced) conversion of cysteine thiol group of COX into S-nitrosothiol. We previously reported the involvement of brain COX/NO synthase (NOS) in centrally administered bombesin-, a stress-related neuropeptide, induced secretion of rat adrenal noradrenaline and adrenaline. To examine a possible involvement of the NO-induced modification of COX in bombesin-induced response, we investigated whether bombesin induces close proximity of COX-1 and neuronal NOS (nNOS) or S-nitroso-cysteine in pre-sympathetic spinally projecting neurons in the rat hypothalamic paraventricular nucleus (PVN), a regulatory center of adrenomedullary outflow.

Main methods

In twelve-week-old male Wistar rats, pre-sympathetic spinally projecting neurons in the PVN were labeled with a retrograde tracer Fluoro-Gold (FG). After intracerebroventricular administration of bombesin, we performed double immunohistochemical analysis for Fos and COX-1 or nNOS in FG-labeled PVN neurons. We also performed a fluorescent in situ proximity ligation assay (PLA) for visualizing of close proximity (< 40 nm) of COX-1 with nNOS or S-nitroso-cysteine.

Key. findings

Bombesin significantly increased the number of Fos-immunoreactive cells in FG-labeled PVN neurons with COX-1 or nNOS immunoreactivity. 7-Nitroindazole, a selective nNOS inhibitor, abolished Fos-immunoreactivity induced by bombesin in COX-1-immunoreactive FG-labeled PVN neurons. Bombesin also induced PLA-positive signals indicating close proximity of COX-1/nNOS and COX-1/S-nitroso-cysteine in FG-labeled PVN neurons.

Significance

Centrally administered bombesin possibly induces S-nitrosylation of COX-1 through close proximity of COX-1 and nNOS in pre-sympathetic spinally projecting PVN neurons, thereby activating COX-1 during the bombesin-induced activation of central adrenomedullary outflow in the rat.     

Contractile responses of human thyroid arteries to vasopressin

Aims

In the present study we investigated the intervention of nitric oxide and prostacyclin in the responses to vasopressin of isolated thyroid arteries obtained from multi-organ donors.

Main methods

Paired artery rings from glandular branches of the superior thyroid artery, one normal and the other deendothelised, were mounted in organ baths for isometric recording of tension. Concentration–response curves to vasopressin were determined in the absence and in the presence of either the vasopressin V₁ receptor antagonist d(CH₂)₅Tyr(Me)AVP (10^− 8 M), the nitric oxide synthase inhibitor N^G-monomethyl-l-arginine (L-NMMA, 10^− 4 M), or the inhibitor of prostaglandins indomethacin (10^− 6 M).

Key findings

In artery rings under resting tension, vasopressin produced concentration-dependent, endothelium-independent contractions. The vasopressin V₁ receptor antagonist d(CH₂)₅Tyr(Me)AVP (10^− 8 M) displaced the control curve to vasopressin 19-fold to the right in a parallel manner. The contractile response to vasopressin was unaffected by L-NMMA or by indomethacin.

Significance

Vasopressin causes constriction of human thyroid arteries by stimulation of V₁ vasopressin receptors located on smooth muscle cells. These effects are not linked to the presence of an intact endothelium or to the release of nitric oxide or prostaglandins. The constriction of thyroid arteries may be particularly relevant in certain pathophysiological circumstances in which vasopressin is released in amounts that could interfere with the blood supply to the thyroid gland.     

Angiotensin II and Angiotensin-(1-7) in Paraventricular Nucleus Modulate Cardiac Sympathetic Afferent Reflex in Renovascular Hypertensive Rats

Background

The enhanced cardiac sympathetic afferent reflex (CSAR) is involved in the sympathetic activation that contributes to the pathogenesis and progression of hypertension. Activation of AT₁ receptors by angiotension (Ang) II in the paraventricular nucleus (PVN) augments the enhanced CSAR and sympathetic outflow in hypertension. The present study is designed to determine whether Ang-(1-7) in PVN plays the similar roles as Ang II and the interaction between Ang-(1-7) and Ang II on CSAR in renovascular hypertension.

Methodology/Principal Findings

The two-kidney, one-clip (2K1C) method was used to induce renovascular hypertension. The CSAR was evaluated by the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to epicardial application of capsaicin in sinoaortic-denervated and cervical-vagotomized rats with urethane and α-chloralose anesthesia. Either Ang II or Ang-(1-7) in PVN caused greater increases in RSNA and MAP, and enhancement in CSAR in 2K1C rats than in sham-operated (Sham) rats. Mas receptor antagonist A-779 and AT₁ receptor antagonist losartan induced opposite effects to Ang-(1-7) or Ang II respectively in 2K1C rats, but losartan had no effects in Sham rats. Losartan but not the A-779 abolished the effects of Ang II, while A-779 but not the losartan blocked the effects of Ang-(1-7). PVN pretreatment with Ang-(1-7) dose-dependently augmented the RSNA, MAP, and CSAR responses to the Ang II in 2K1C rats. Ang II level, AT₁ receptor and Mas receptor protein expression in PVN increased in 2K1C rats compared with Sham rats but Ang-(1-7) level did not.

Conclusions

Ang-(1-7) in PVN is as effective as Ang II in enhancing the CSAR and increasing sympathetic outflow and both endogenous Ang-(1-7) and Ang II in PVN contribute to the enhanced CSAR and sympathetic outflow in renovascular hypertension. Ang-(1-7) in PVN potentiates the effects of Ang II in renovascular hypertension.     

Stimulation of ANP by angiotensin-(1-9) via the angiotensin type 2 receptor

Aims

Angiotensin-(1-9) [Ang-(1-9)] and Ang-(1-7) are cleaved by Ang converting enzyme 2 forming Ang I and Ang II, respectively, and the truncated Angs play a role in regulating atrial natriuretic peptide (ANP) secretion. Previously, we found that Ang-(1-7) stimulates ANP secretion via the Mas receptor. However, the effect of Ang-(1-9) on ANP secretion is still unknown. The aim of the present study is to determine whether Ang-(1-9) stimulates ANP secretion and to characterize the signaling pathway involved in stimulating secretion.

Main methods

We examined the effects of Ang-(1-9) on ANP secretion and atrial contractility with and without inhibitors in isolated perfused atria.

Key findings

Ang-(1-9) stimulated ANP secretion and concentration without change in atrial contractility. Ang-(1-9)-induced-ANP secretion was increased from 5% to 60% by 3 μM Ang-(1-9) during the low-stretch state of the atrium. This stimulatory effect of Ang-(1-9) on ANP secretion was attenuated by pretreatment with an Ang II type 2 receptor (AT₂R) antagonist but not by AT₁R or Mas receptor antagonist. In addition, pretreatment with inhibitors of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) blocked Ang-(1-9)-induced ANP secretion. In the high-stretch atrial state, Ang-(1-9)-induced ANP secretion was increased more than in the low-stretch state following addition of 1 μM Ang-(1-9) (from 108% to 170%). In an in vivo experiment, acute infusion of Ang-(1-9) increased plasma ANP level without altering arterial blood pressure. This effect was attenuated by pretreatment with AT₂R antagonist but not by Mas receptor antagonist.

Significance

These results suggest that Ang-(1-9) stimulates ANP secretion via the AT₂R-PI3K-Akt-NO-cGMP pathway.     

C-type natriuretic peptide (CNP) signal peptide fragments are present in the human circulation

Background

Signal peptides may be novel biomarkers in cardiovascular diseases.

Methods

We developed a novel immunoassay to the signal peptide of preproCNP (CNPsp) and used this to document circulating venous concentrations of CNPsp in normal healthy volunteers (n = 109), regional plasma CNPsp concentrations in patients undergoing clinically indicated catheterisation (n = 24) and temporal CNPsp concentrations in patients with ST-elevation myocardial infarction (STEMI) <4 h after symptom onset (n = 8). The structure/sequence of circulating CNPsp was confirmed by tandem mass spectrometry (MS/MS).

Results

In normal human plasma, CNPsp was detectable at levels higher than NT-proCNP (74 ± 17 vs. 20 ± 5.5 pmol/L). There was no correlation between NTproCNP and CNPsp, but plasma concentrations of sibling signal peptides – CNPsp and BNPsp – were strongly correlated (r = 0.532, P < 0.001). In patients undergoing catheterisation, there were significant arterio-venous step-ups in CNPsp concentrations across the heart (P < 0.01) and kidney (P < 0.01). Arterial concentrations of CNPsp significantly correlated with heart rate (r = 0.446, P < 0.05). In STEMI patients, plasma concentrations of CNPsp showed a biphasic elevation pattern between 6 and 12 h after symptom onset, with 12 h values significantly elevated (∼3-fold) compared with levels at presentation (P < 0.05). MS/MS verified circulating CNPsp to be preproCNP(14–23) and preproCNP(16–23) peptides.

Conclusions

This is the first report of a circulating preproCNP derived signal peptide. Given the clear cardiac and renal secretion profiles of CNPsp and its response in STEMI patients, further studies on potential biological functions and biomarker applications of CNPsp in cardiovascular disease are warranted.     

Plasma ADMA concentrations associate with aerobic fitness in postmenopausal women

Aims

Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase, an enzyme responsible for the generation of NO. Plasma concentrations of ADMA increase in the elderly and in postmenopausal women. In fact, an elevated ADMA level is a risk factor of cardiovascular disease. Aerobic exercise has a beneficial effect on cardiovascular disease. However, the relationship between ADMA and aerobic fitness is unknown. The aim of this study was to determine whether plasma ADMA concentrations correlate with aerobic fitness levels in postmenopausal women.

Main methods

Thirty healthy postmenopausal women aged 50–76 years participated in this study. We measured plasma concentrations of ADMA and oxygen consumption at the ventilatory threshold (VO_2VT) as an index of aerobic fitness. Subjects were divided into the low aerobic fitness (Low fitness) and high aerobic fitness (High fitness) groups, and the dividing line was set at the median VO_2VT value.

Key findings

VO_2VT was significantly higher in the High fitness group than in the Low fitness group (P < 0.01). The plasma ADMA concentrations in the High fitness group were significantly lower than those in the Low fitness group (P < 0.05). There was a negative correlation between plasma ADMA concentrations and VO_2VT (r = − 0.532, P < 0.01).

Significance

We found that plasma ADMA concentrations were associated with aerobic fitness in postmenopausal women. The results of this study suggest that habitual aerobic exercise may decrease plasma ADMA concentrations.     

Genetic variants of estrogen beta and leptin receptors may cause gynecomastia in adolescent

Objective

Gynecomastia is a benign breast enlargement in males that affects approximately one-third of adolescents. The exact mechanism is not fully understood; however, it has been proposed that estrogen receptors and aromatase enzyme activity may play important roles in the pathogenesis of gynecomastia. While many studies have reported that aromatase enzyme (CYP19) gene polymorphism is associated with gynecomastia, only one study has shown a relationship between estrogen receptor (ER) alpha and beta gene polymorphism and gynecomastia. Thus, the aim of this study was to evaluate the relationships between CYP19 (rs2414096), ER alpha (rs2234693), ER beta (rs4986938), leptin (rs7799039), and leptin receptor (rs1137101) gene polymorphisms and gynecomastia.

Methods

This study included 107 male adolescents with gynecomastia and 97 controls. Total serum testosterone (T) and estradiol (E2) levels were measured, and DNA was extracted from whole blood using the PCR–RFLP technique. The polymorphic distributions of CYP19, ER alpha, ER beta, leptin and leptin receptor genes were compared.

Results

The median E2 level was 12.41 (5.00–65.40) pg/ml in the control group and 16.86 (2.58–78.47) pg/ml in the study group (p < 0.001). The median T level was 2.19 (0.04–7.04) ng/ml in the control group and 1.46 (0.13–12.02) ng/ml in the study group (p = 0.714). There was a significant relationship between gynecomastia and leptin receptor rs1137101 (p = 0.002) and ER beta receptor rs4986938 gene polymorphisms (p = 0.002).

Conclusions

According to our results, increased E2 level and ER beta gene rs4986938 polymorphism might explain why some adolescents have gynecomastia. Leptin receptor gene rs1137101 polymorphism might affect susceptibility to gynecomastia.     

Polymorphisms of the TLR4 gene and risk of gastric cancer

Objective

Toll-like receptor 4 (TLR4) is an important lipo-polysaccharide (LPS) receptor in gastric epithelial cell signaling transduction and plays critical roles in the development and progression of gastric cancer (GC). We investigated the effects of TLR4 gene polymorphisms and gene–environmental interactions on the risk of GC in Northeastern China.

Methods

We genotyped two single-nucleotide polymorphisms (SNPs) in TLR4 (rs10116253 and rs1927911) in 217 GC patients and 294 cancer-free controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Odds ratio (OR) and 95% confidence intervals (CIs) were estimated by unconditional logistic-regression models.

Results

Individuals carrying CC genotype of rs10116253 and TT genotype of rs1927911 had a significantly decreased risk of GC (adjusted OR = 0.33, 95% CI 0.18–0.60, P < 0.001 and adjusted OR = 0.37, 95% CI 0.21–0.67, P = 0.001 respectively), compared with TT genotype of rs10116253 and CC genotype of rs1927911. In addition, the SNP effects were additive to the effects of some known environmental factors without any interaction between them in the susceptibility to GC.

Conclusion

Our data suggested that TLR4 gene polymorphisms may be associated with a decreased risk of GC in Chinese population. And these SNPs and their combined effects with environmental factors may be associated with the risk of GC.     

Anti-inflammatory effects of anethole in lipopolysaccharide-induced acute lung injury in mice

Aims

Anethole, the major component of the essential oil of star anise, has been reported to have antioxidant, antibacterial, antifungal, anti-inflammatory, and anesthetic properties. In this study, we investigated the anti-inflammatory effects of anethole in a mouse model of acute lung injury induced by lipopolysaccharide (LPS).

Main methods

BALB/C mice were intraperitoneally administered anethole (62.5, 125, 250, or 500 mg/kg) 1 h before intratracheal treatment with LPS (1.5 mg/kg) and sacrificed after 4 h. The anti-inflammatory effects of anethole were assessed by measuring total protein and cell levels and inflammatory mediator production and by histological evaluation and Western blot analysis.

Key findings

LPS significantly increased total protein levels; numbers of total cells, including macrophages and neutrophils; and the production of inflammatory mediators such as matrix metalloproteinase 9 (MMP-9), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO) in bronchoalveolar lavage fluid. Anethole (250 mg/kg) decreased total protein concentrations; numbers of inflammatory cells, including neutrophils and macrophages; and the inflammatory mediators MMP-9, TNF-α and NO. In addition, pretreatment with anethole decreased LPS-induced histopathological changes. The anti-inflammatory mechanism of anethole in LPS-induced acute lung injury was assessed by investigating the effects of anethole on NF-κB activation. Anethole suppressed the activation of NF-κB by blocking IκB-α degradation.

Significance

These results, showing that anethole prevents LPS-induced acute lung inflammation in mice, suggest that anethole may be therapeutically effective in inflammatory conditions in humans.     

Influence of parafascicular thalamic input on neuronal activity within the nucleus accumbens is mediated by nitric oxide — An in vivo study

Aims

Thalamostriatal fibers are involved in cognitive tasks such as acquisition, learning, processing of sensory events, and behavioral flexibility and might play a role in Parkinson's disease. The aim of the present study was the in vivo electrochemical characterization of the projection from the lateral aspect of the parafascicular thalamus (Pfl) to the dorsolateral aspect of the nucleus accumbens (dNAc). Since nitric oxide (NO) plays a crucial role in striatal synaptic transmission, its implication in Pfl-evoked signaling within the dNAc was investigated.

Main methods

The Pfl was electrically stimulated utilizing paired pulses and extracellular potentials were recorded within the dNAc. Simultaneously, the dNAc was superfused using the push–pull superfusion technique for local application of compounds and for assessing the influence of NO on release of glutamate, aspartate and GABA.

Key findings

Stimulation of the Pfl evoked a negative-going component at 9–14 ms followed by a positive-going component at 39–48 ms. The early response was current-dependent and diminished by superfusion of the dNAc with tetrodotoxin, kynurenic acid or N^G-nitro-l-arginine methyl ester (L-NAME), while 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA/NO) increased this evoked potential. Transmitter release was inhibited by L-NAME and facilitated by PAPA/NO.

Significance

This study describes for the first time in vivo extracellular electrical responses of the dNAc on stimulation of the Pfl. Synaptic transmission within the dNAc on stimulation of the Pfl seems to be facilitated by NO.     

β-Glucan from Saccharomyces cerevisiae reduces lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages

Background

β-Glucans obtained from fungi, such as baker's yeast (Saccharomyces cerevisiae)-derived β-glucan (BBG), potently activate macrophages through nuclear factor κB (NFκB) translocation and activation of its signaling pathways. The mechanisms by which β-glucans activate these signaling pathways differ from that of lipopolysaccharide (LPS). However, the effects of β-glucans on LPS-induced inflammatory responses are poorly understood. Here, we examined the effects of BBG on LPS-induced inflammatory responses in RAW264.7 mouse macrophages.

Methods

We explored the actions of BBG in RAW264.7 macrophages.

Results

BBG inhibited LPS-stimulated nitric oxide (NO) production in RAW264.7 macrophages by 35–70% at concentrations of 120–200 μg/ml. BBG also suppressed mRNA and protein expression of LPS-induced inducible NO synthase (iNOS) and mitogen-activated protein kinase phosphorylation, but not NFκB activation. By contrast, a neutralizing antibody against dectin-1, a β-glucan receptor, did not affect BBG-mediated inhibition of NO production. Meanwhile, BBG suppressed Pam3CSK-induced NO production. Moreover, BBG suppressed LPS-induced production of pro-and anti-inflammatory cytokines, including interleukin (IL)-1α, IL-1ra, and IL-27.

Conclusions

Our results indicate that BBG is a powerful inhibitor of LPS-induced NO production by downregulating iNOS expression. The mechanism involves inactivation of mitogen-activated protein kinase and TLR2 pathway, but is independent of dectin-1.

General significance

BBG might be useful as a novel agent for the chemoprevention of inflammatory diseases.     

Combinational effects of farnesoid X receptor antagonist and statin on plasma lipid levels and low-density lipoprotein clearance in guinea pigs

Aims

We previously reported anti-dyslipidemic effects of a farnesoid X receptor antagonist in monkeys. In this study, we compared the cholesterol-lowering effects of single and combined administration of a farnesoid X receptor antagonist, compound-T8, and the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor atorvastatin in a guinea pig model.

Main methods

Plasma levels of 7α-hydroxy-4-cholesten-3-one, a marker of hepatic cholesterol 7α-hydroxylase activity, were measured after a single administration of compound-T8. The effects of compound-T8 or atorvastatin on plasma cholesterol levels and low-density lipoprotein (LDL) clearance were investigated after 14 or 16 days of repeated dosing, respectively. Fractional catabolic rate of plasma LDL was estimated by intravenous injection of DiI-labeled human LDL. The cholesterol-lowering effects of combination therapy were investigated after 7 days of repeated treatment.

Key findings

Compound-T8 (10 and 30 mg/kg) increased plasma 7α-hydroxy-4-cholesten-3-one levels in a dose-dependent manner. Single administration of compound-T8 (30 mg/kg) and atorvastatin (30 mg/kg) reduced plasma non-high-density lipoprotein (non-HDL) cholesterol levels by 48% and 46%, respectively, and increased clearance of plasma DiI-labeled LDL by 29% and 35%, respectively. Compound-T8 (10 mg/kg) or atorvastatin (10 mg/kg) reduced non-HDL cholesterol levels by 19% and 25%, respectively, and combination therapy showed an additive effect and lowered cholesterol levels by 48%.

Significance

Similar to atorvastatin, compound-T8 reduced plasma non-HDL cholesterol levels accompanied with accelerated LDL clearance in guinea pigs. Combination therapy additively decreased plasma non-HDL cholesterol levels. Therefore, monotherapy with a farnesoid X receptor antagonist and combination therapy of a farnesoid X receptor antagonist with atorvastatin would be attractive dyslipidemia treatment options.     

Evaluation of mutagenic and genotoxic activities of lobeline and its modulation on genomic instability induced by ethanol

Aim

Lobeline is a natural alkaloid derived from Lobelia inflata that has been investigated as a clinical candidate for the treatment of alcoholism. In a pre-clinical trial, lobeline decreased the preference for and consumption of ethanol, due to the modulation of the nicotinic acetylcholine receptor. However, the interaction between lobeline and ethanol is poorly known and thus there are safety concerns.The present study was conducted to evaluate the mutagenic and genotoxic effects of lobeline and assess its modulation of ethanol-induced toxicological effects.

Main methods

CF-1 male mice were divided into five groups. Groups received an intraperitoneal injection of saline solution, lobeline (5 or 10 mg/kg), ethanol (2.5 g/kg), or lobeline plus ethanol, once a day for three consecutive days. Genotoxicity was evaluated in peripheral blood using the alkaline comet assay. The mutagenicity was evaluated using both Salmonella/microsome assay in TA1535, TA97a, TA98, TA100, and TA102 Salmonella typhimurium strains and the micronucleus test in bone marrow. Possible liver and kidney injuries were evaluated using biochemical analysis.

Key findings

Lobeline did not show genotoxic or mutagenic effects and did not increase the ethanol-induced genotoxic effects in blood. Lobeline also protected blood cells against oxidative damage induced by hydrogen peroxide. Biochemical parameters were not altered, indicating no liver or kidney injuries or alterations in lipid and carbohydrate metabolisms.

Significance

These findings suggest that lobeline does not induce gene or chromosomal mutations, and that this lack of genetic toxicity is maintained in the presence of ethanol, providing further evidence of the safety of this drug to treat alcohol dependence.     

Up-regulation of adenylylcyclases I and II induced by long-term adaptation of rats to morphine fades away 20 days after morphine withdrawal

Background

Activation of adenylyl cyclase (AC) by prolonged exposure of mammalian organism to morphine was demonstrated in previous studies of mechanism of action of this drug. However, expression level of individual AC isoforms was not analyzed in crucial cell structure, plasma membrane (PM).

Methods

Rats were adapted to morphine for 10 days and sacrificed 24 h (group + M10) or 20 days (+ M10/−M20) after the last dose. Control animals were sacrificed in parallel with morphine-treated (groups − M10 and (− M10/−M20)). Percoll®-purified PM were isolated from brain cortex and analyzed by immunoblotting and specific radioligand binding.

Results

ACI (ACII) was increased 8× (2.5×) in morphine-adapted rats (+ M10) when compared with controls (− M10). Increase of ACI and II by long-term adaptation to increasing doses of morphine represented a specific effect as the amount of ACIII–ACIX, of prototypical PM marker, Na, K-ATPase and of trimeric G protein α and β subunits was unchanged. Increase of ACI and II was not detected in PM isolated from group (+ M10/−M20). Thus, the marked increase of ACI and ACII faded away 20 days since the last dose of morphine.

Conclusions

We assume that the specific increase in expression level of ACI and ACII in brain cortex of morphine-adapted rats proceeds as a compensatory, homeostatic response to prolonged exposure to inhibitory drug, morphine.

General significance

Our findings demonstrate that the dramatic and specific change of the crucial component of the opioid receptor cascade in brain cortex, manifested as an increase in PM level of ACI and II, is reversible.     

Echinocystic acid,isolated from Gleditsia sinensis fruit,protects endothelial progenitor cells from damage caused by oxLDL via the Akt/eNOS pathway

Aims

Our previous studies revealed that echinocystic acid (EA) showed obvious attenuation of atherosclerosis in rabbits fed a high-fat diet. However, the underlying mechanisms remain to be elucidated. Considering the importance of endothelial progenitor cells (EPCs) in atherosclerosis, we hypothesise that EPCs may be one of the targets for the anti-atherosclerotic potential of EA.

Main methods

After in vitro cultivation, EPCs were exposed to 100 μg/mL of oxidised low-density lipoprotein (oxLDL) and incubated with or without EA (5 and 20 μM) for 48 h. An additional two groups of EPCs (oxLDL + 20 μM EA) were pre-treated with either wortmannin, an inhibitor of the phosphoinositide 3-kinase (PI3K) pathway, or nitro-l-arginine methyl ester (l-NAME), an endothelial nitric oxide synthase (eNOS)-specific inhibitor. Assessment of EPC apoptosis, adhesion, migration, and nitric oxide (NO) release was performed using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining, cell counting, caspase-3 activity assay, transwell chamber assay, and Griess reagent, respectively. The protein expression of protein kinase B (Akt) and eNOS was detected using Western blot.

Key findings

Treatment of EPCs with oxLDL induced significant apoptosis and impaired adhesion, migration, and NO production. The deleterious effects of oxLDL on EPCs were attenuated by EA. However, when EPCs were pre-treated with wortmannin or l-NAME, the effects of EA were abrogated. Additionally, oxLDL significantly down-regulated eNOS protein expression as well as repression of eNOS and Akt phosphorylation.

Significance

The inhibitory effect of oxLDL on Akt/eNOS phosphorylation was attenuated by EA. Taken together, the results indicate that EA protects EPCs from damage caused by oxLDL via the Akt/eNOS pathway.     

Methyl and isopropyl N-methylanthranilates attenuate diclofenac- and ethanol-induced gastric lesions in rats

Aims

Two natural alkaloids, methyl (M) and isopropyl (I) N-methylanthranilates, with recently demonstrated significant pharmacological activities, were assayed for their possible overall effect on intact gastric mucosa and their protective properties towards the onset of gastric lesions induced by diclofenac (a non-steroidal anti-inflammatory drug, NSAID) or ethanol.

Main methods

The influence of I and M on gastric mucosa integrity was assessed by oral administration in doses of 200 mg/kg. The gastroprotective action of I and M in doses of 50, 100 and 200 mg/kg was analyzed in the diclofenac and ethanol-induced gastric lesion models in rats. After the treatment, the stomachs of the animals were analyzed (captured by a digital camera). Ulcer scoring, morphometric and histopathological analyses of the stomachs were done.

Key findings

The oral application of these compounds on their own, even in quite high doses (200 mg/kg) did not induce gastric lesions. Both alkaloids exerted a very strong antiulcer activity, even in low doses (50 mg/kg), by decreasing the number of lesions caused by the application of either diclofenac or ethanol, eliminating them completely or reducing them to a form of mucosal hyperemia.

Significance

Their possible mechanism of action was discussed and due to their many positive properties including anxiolytic, antidepressant, antinociceptive, anti-inflammatory and gastroprotective activities, as well as a cheap and simple synthetic route for their preparation, methyl and isopropyl N-methylanthranilates, both alike, might represent a cost effective alternative sought for in the treatment of peptic ulcers and/or new safer NSAIDs for pain management.     

Interleukin-23 receptor genetic variants contribute to susceptibility of multiple cancers

Aim

Interleukin-23 (IL-23) and IL-23 receptor (IL23R) play an important role during the T-helper 17 (Th17) cell-mediated inflammatory process as well as pathogenesis of multiple cancers. Several IL-23R single nucleotide polymorphisms (SNPs), especially rs6682925, rs10889677 and rs1884444 polymorphisms, are considered to have significant impacts on susceptibility of multiple cancers. A number of case-control studies have explored the role these genetic polymorphisms in development of carcinogenesis, but the conclusions are inconsistent. Therefore, we conducted this meta-analysis to systematically investigate the associations between the three genetic variants and multiple cancer risk.

Methods

A total of ten studies are eligible (12,211 patients and 14,650 controls). Pooled odds ratios (ORs) and the 95% confidence interval (95% CI) were appropriately calculated using either fixed-effect model or random-effect model.

Results

Significant associations between rs6682925 or rs10889677 polymorphism and cancer risk were found (OR = 1.11, 95% CI = 1.03–1.21, P = 0.007; or OR = 0.85, 95% CI = 0.71–0.92, P = 0.001). However, there was no such association between rs1884444 genotypes and cancer susceptibility (P > 0.05).

Conclusion

These findings reveal that the IL-23R rs6682925 and rs10889677 genetic variants play a more important part in pathogenesis of multiple cancers.     

Association study of polymorphisms in the receptor for advanced glycation end-products (RAGE) gene with susceptibility and prognosis of heart failure

Background

Functional polymorphisms in the receptor for advanced glycation end-products (RAGE) gene have been implicated in several vascular diseases. However, to date, no study investigated the association of RAGE polymorphisms with heart failure (HF).

Objective

In this study we tested the hypothesis that the 63-bp insertion/deletion, the − 374T > A (rs1800624) and the − 429T > C (rs1800625) polymorphisms in the RAGE gene might be associated with susceptibility to HF and could predict all-cause mortality in Brazilian outpatients with left ventricular systolic dysfunction.

Methods

A total of 273 consecutive HF patients (196 Caucasian- and 77 African-Brazilians) and 334 healthy blood donors (260 Caucasian- and 74 African-Brazilians) were enrolled in a tertiary care university hospital. Genotyping of RAGE polymorphisms was done by polymerase chain reaction (PCR) or PCR followed by enzyme restriction analysis.

Results

The allele, genotype and haplotype frequencies of − 374T > A and − 429T > C polymorphisms were not significantly different between HF patients and healthy blood donors in both ethnic groups. However, among African-Brazilians, the frequency of carriership of the del allele was lower in HF patients than in blood donors (2.6% vs 12.2%, respectively, p = 0.008). Patients were followed-up for a median of 38 months and the survival analysis did not reveal a consistent association between RAGE polymorphisms and all-cause death in both ethnic groups.

Conclusion

The − 374T > A and − 429T > C polymorphisms in the RAGE gene were not associated with the susceptibility and prognosis of HF. Notwithstanding, the 63-bp ins/del polymorphism might be involved in the susceptibility to HF in African-Brazilians.