Atrial mRNA Expression Research Articles

Hyperoxia-induced airflow restriction and Renin-Angiotensin System expression in a bronchopulmonary dysplasia mouse model.

Mechanisms underlying hyperoxia-induced airflow restriction in the pediatric lung disease Bronchopulmonary dysplasia (BPD) are unclear. We hypothesized a role for Renin-Angiotensin System (RAS) activity in BPD. RAS is comprised of a pro-developmental pathway consisting of angiotensin converting enzyme-2 (ACE2) and angiotensin II receptor type 2 (AT2), and a pro-fibrotic pathway mediated by angiotensin II receptor type 1 (AT1). We investigated associations between neonatal hyperoxia, airflow restriction, and RAS activity in a BPD mouse model. C57 mouse pups were randomized to normoxic (FiO2 = 0.21) or hyperoxic (FiO2 = 0.75) conditions for 15 days (P1-P15). At P15, P20, and P30, we measured airflow restriction using plethysmography and ACE2, AT1, and AT2 mRNA and protein expression via polymerase chain reaction and Western Blot. Hyperoxia increased airflow restriction P15 and P20, decreased ACE2 and AT2 mRNA, decreased AT2 protein, and increased AT1 protein expression. ACE2 mRNA and protein remained suppressed at P20. By P30, airflow restriction and RAS expression did not differ between groups. Hyperoxia caused high airflow restriction, increased pulmonary expression of the pro-fibrotic RAS pathway, and decreased expression of the pro-developmental in our BPD mouse model. These associated findings may point to a causal role for RAS in hyperoxia-induced airflow restriction.

Abstract 11649: Serum Adiponectin Levels Correlate With Atrial PPARγ and TGFβ1 mRNA Expression in Patients Receiving Cardiac Surgery: Involvement of PPARγ on Atrial Fibrotic Remodeling

Introduction: The peroxisome proliferator-activated receptor gamma (PPAR γ) plays important roles in regulating processes related to fibrogenesis. On the other hand, adiponectin, a marker for PPARγ activity, is a hormone involved in maintaining energy homeostasis, and exerts anti-profibrotic effects in various organs. However, the roles of PPARγ and adiponectin on atrial fibrotic remodeling are still unknown. Hypothesis: We investigated the association of serum adiponectin levels and atrial gene expression of adiponectin, PPARγ and fibrosis-related genes in cardiovascular surgery patients including heart valve disease. Methods: Atrial mRNA expression levels including adiponectin and PPARγ were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) in 59 cases (sinus rhythm (SR) 24 patients, atrial fibrillation (AF) 35 patients) in which left atrium (LA) tissues were removed from the appendage during surgery. The immunohistochemical studies were used to identify the protein expression. The serum levels of adiponectin were measured by ELISA. Results: The immunohistochemical staining showed the expression of adiponectin, PPARγ and PPARα. The significant correlations were observed among the mRNA expression levels of extracellular matrix (ECM) (COL1A1, COL3A1, fibronectin) and the fibrosis-related genes such as TGFβ1, TIMP1, MMP2, NOX2, CTGF and PDGFD attributed to the fibrosis of myocardium. The mRNA expression level of PPARγ, but not PPARα, positively correlated with that of the ECM and the fibrosis-related gene expression level. The mRNA expression level of adiponectin also positively correlated with that of ECM. Patients with AF had higher atrial expression level of adiponectin, PPARγ, COL1A1 and TGFβ than those with SR. Serum adiponectin levels significantly correlated with atrial mRNA expression level of PPARγ and TGFβ1. Patients with AF had serum higher levels of adiponectin than those with SR. Conclusions: Atrial PPARγ and adiponectin associate with atrial fibrotic remodeling in cardiovascular surgery patients. Serum adiponectin levels correlate with atrial PPARγ and TGFβ1 mRNA expression, and might have potential utility as a biomarker in atrial fibrotic remodeling.

Sexual Dimorphism in the Expression of Cardiac and Hippocampal Renin-Angiotensin and Kallikrein–Kinin Systems in Offspring from Mice Exposed to Alcohol during Gestation

Prenatal alcohol exposure (PAE) impairs fetal development. Alcohol consumption was shown to modulate the renin-angiotensin system (RAS). This study aimed to analyze the effects of PAE on the expression of the renin-angiotensin system (RAS) and kallikrein-kinin system (KKS) peptide systems in the hippocampus and heart of mice of both sexes. C57Bl/6 mice were exposed to alcohol during pregnancy at a concentration of 10% (v/v). On postnatal day 45 (PN45), mouse hippocampi and left ventricles (LV) were collected and processed for messenger RNA (mRNA) expression of components of the RAS and KKS. In PAE animals, more pronounced expression of AT1 and ACE mRNAs in males and a restored AT2 mRNA expression in females were observed in both tissues. In LV, increased AT2, ACE2, and B2 mRNA expressions were also observed in PAE females. Furthermore, high levels of H2O2 were observed in males from the PAE group in both tissues. Taken together, our results suggest that modulation of the expression of these peptidergic systems in PAE females may make them less susceptible to the effects of alcohol.

Abstract 9449: Increased Neprilysin Expression Links Atrial Fibrotic Remodeling in Cardiovascular Surgery Patients

Introduction: Deposition of extracellular matrix (ECM) such as collagen leads to fibrosis, which plays essential roles in structural remodeling in atrial fibrillation (AF). Neprilysin (NEP) is an integral membrane-bound metallopeptidase with a wide spectrum of substrates and physiological functions. Hypothesis: We investigated the association of NEP and the atrial fibrosis-related genes in cardiovascular surgery patients including heart valve disease. Methods: Atrial mRNA expression including collagen and NEP were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) in 59 cases (sinus rhythm (SR) 24 patients, AF 35 patients) in which LA tissues were removed from the appendage during surgery. NEP mRNA expression was also analyzed in adipose tissues (AT) obtained from both epicardial and subcutaneous adipose tissues. The NEP protein expression was measured by western blotting analysis and immunohistochemistry staining, and serum level of NEP was measured by ELISA. Results: The immunohistochemical staining showed the expression of NEP in both AT and LA. Higher expression of NEP mRNA and protein in adipose tissues was observed, compared with LA. Serum NEP concentration did not significantly correlate with NEP mRNA and protein expression of AT and LA. The significant correlations were observed among ECM (COL1A1, COL3A1, fibronectin) and the five selected fibrosis-related genes (TGFβ1, CTGF, miR21, NOX2 and BAX) attributed to the fibrosis of myocardium in LA. NEP mRNA expression of LA also positively correlated with the ECM and the fibrosis-related gene expression level. The multiple regression analysis revealed TGFβ1, NOX2, and NEP as independent predictors of atrial COL1A1 mRNA expression level. In addition, it revealed NEP as an independent predictor of COL1A1 mRNA expression level, even adjusted by age, sex, and BMI. Serum NEP concentration did not correlate with the ECM and the fibrosis-gene expression level. Patients with AF had higher atrial expression of NEP protein, compared to those with SR. Conclusions: Increased atrial expression of NEP links atrial fibrotic remodeling and developing AF in cardiovascular surgery patients. However, serum NEP did not reflect atrial NEP expression in LA.

Long-term If current blockade increases right atrial mRNA expression of HCN4, encoding proteins for If

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This work was supported by a grant of the Romanian Ministry of Education and Research, CNCS - UEFISCDI, within PNCDI III Background Numerous long-term pharmacologic manipulations of cardiac ion channels have shown potential to modify the expression of genes encoding proteins for those channels. Purpose We aimed to investigate whether long-term pharmacologic inhibition of the hyperpolarization-activated inward current (If) using ivabradine affects the right atrial expression of genes encoding for hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, responsible for If. Methods The right atrial mRNA expression of HCN1, HCN2, and HCN4, and of the neuron-specific HCN3 isoform was quantified in 6 control (Control) and 12 ivabradine-treated (IVA; 10 mg/kg, 4 weeks) adult male Wistar rats. The expression levels of the target genes were normalized with GAPDH housekeeping gene levels and compared between the two groups. Results Right atrial HCN1 and HCN2 expression levels were both similar (both p >0.05) between the IVA and the Control rats. There was also no significant difference in the right atrial expression of the neuron-specific HCN3 isoform between the two groups (p = 0.22). However, the right atrial expression of HCN4, the most highly expressed HCN isoform in the sinus node, was significantly higher in the ivabradine-treated compared to the non-treated rats (p = 0.02). Conclusions Our study shows that chronic If blockade using ivabradine significantly up-regulates right atrial HCN4 expression. Although the examined samples contained both nodal and non-nodal tissue, since HCN4 is highly expressed in the nodal pacemaker cells and only sparsely in the remaining atrial myocardium, it is likely that the HCN4 changes observed in our study reflect sinus node alterations. HCN4 and a consequent If up-regulation could render the sinus node less sensitive to acute vagal inputs and protect against excessive vagal-induced bradycardia. Further studies will have to evaluate this hypothesis.

Interaction between the Renin–Angiotensin System and Enteric Neurotransmission Contributes to Colonic Dysmotility in the TNBS-Induced Model of Colitis

Angiotensin II (Ang II) regulates colon contraction, acting not only directly on smooth muscle but also indirectly, interfering with myenteric neuromodulation mediated by the activation of AT1 /AT2 receptors. In this article, we aimed to explore which mediators and cells were involved in Ang II-mediated colonic contraction in the TNBS-induced rat model of colitis. The contractile responses to Ang II were evaluated in distinct regions of the colon of control animals or animals with colitis in the absence and presence of different antagonists/inhibitors. Endogenous levels of Ang II in the colon were assessed by ELISA and the number of AT1/AT2 receptors by qPCR. Ang II caused AT1 receptor-mediated colonic contraction that was markedly decreased along the colons of TNBS-induced rats, consistent with reduced AT1 mRNA expression. However, the effect mediated by Ang II is much more intricate, involving (in addition to smooth muscle cells and nerve terminals) ICC and EGC, which communicate by releasing ACh and NO in a complex mechanism that changes colitis, unveiling new therapeutic targets.

Effect of ischemic postconditioning on cell apoptosis and expression of relevant genes in non-culprit coronary arteries

This study was performed to determine the effect of ischemic postconditioning on cell apoptosis and angiotensin II receptor type 1 (AT1), connexin 43 (Cx43), and β-tubulin mRNA expression in non-culprit...

Angiotensin type 2 receptors: Role in aging and neuroinflammation in the substantia nigra

Overactivity of the angiotensin-type-1 receptor (AT1)/NADPH-oxidase axis enhances aging processes, neuroinflammation and neurodegeneration. The role of AT2 receptors in the above-mentioned AT1-related effects in the aged brain, particularly substantia nigra, was investigated in this study. In the nigra, we observed a progressive decrease in AT2 mRNA expression with aging, and AT2 deletion led to changes in spontaneous motor behavior, dopamine receptors, renin-angiotensin system, and pro-oxidative and pro-inflammatory markers similar to those observed in aged wild type (WT) mice. Both aged WT mice and young AT2 KO mice showed an increased AT1, decreased MAS receptor and increased angiotensinogen mRNA and/or protein expression, as well as upregulation of pro-oxidative and pro-inflammatory markers. In cultures of microglial cells, activation of AT2 receptors inhibited the LPS-induced increase in AT1 mRNA and protein expression and neuroinflammatory markers. Both in AT2 KO microglial cultures and microglia obtained from adult AT2 KO mice, an increase in AT1 mRNA expression was observed. In cultured dopaminergic neurons, AT2 activation down-regulated AT1 mRNA and protein, and dopaminergic neurons from adult AT2 KO mice showed upregulation of AT1 mRNA expression. Both in microglia and dopaminergic neurons the pathway AT2/nitric oxide/cyclic guanosine monophosphate mediates the regulation of the AT1 mRNA and protein expression through downregulation of the Sp1 transcription factor. MAS receptors are also involved in the regulation of AT1 mRNA and protein expression by AT2. The results suggest that an aging-related decrease in AT2 expression plays a major role in the aging-related AT1 overexpression and AT1-related pro-inflammatory pro-oxidative effects.

High salt diet decreases reproductive performance in rams and down-regulates gene expression of some components of the renin-angiotensin system in the testis

Although salt-tolerant plants can be used to combat dryland salinity, these plants contain high concentrations of salt (NaCl), which may have deleterious effects if fed to livestock. Twenty-four Merino rams (9 mo of age) with similar body weight were equally allocated to two groups and fed a normal- or high-salt diet (0.5 and 12% NaCl, respectively) for 3 mo. Rams fed the high-salt diet had lower live weight gains, higher water intake, smaller testes (466 ± 48.4 vs 604 ± 51.1 g) and reductions (P <0.05) in spermatogenesis, ejaculate volumes (0.89 ± 0.18 vs 1.24 ± 0.15 mL/ejaculate), sperm concentration (14.3 ± 2.05 vs 22.3 ± 2.33 × 109 sperm/mL) and DNA integrity (DNA fragmentation rate: 5.85 ± 1.09 vs 1.13 ± 0.14%) compared to controls. For in vitro fertilization, although cleavage percentage was not significantly affected, hatching rate was lower (30.8 ± 3.81 vs 52.8 ± 4.08%, P <0.05) for sperm from rams on the high-salt diet. Furthermore, the 12% salt diet decreased plasma concentrations of metabolic (leptin and insulin) and sex (T, FSH and LH) hormones, but did not affect the plasma renin-angiotensin system (RAS) component (REN, Ang II, ACE and ALD). Regarding components of the testicular RAS, the 12% salt diet decreased (P <0.05) expression of REN, Ang II and AT2 mRNA, although ACE and AT1 were unaffected. Furthermore, the 12% salt diet decreased (P <0.05) mRNA expression of key genes for spermatogenesis (Hsp70, c-kit and Cyclin A), and sex hormone receptors (AR, FSHR, LHR, CYP11A1 and CYP17A1), but there were no significant effects on key enzymes (LDH, SDH, AKP and ATPase) in the testis. In conclusion, the high-salt diet reduced ram reproductive performance; we inferred that changes in testis RAS may have had an important role in these reproductive defects.

Relaxin reduces susceptibility to post-infarct atrial fibrillation in mice due to anti-fibrotic and anti-inflammatory properties

BackgroundRelaxin-2 (RLX) is a peptide hormone that exerts beneficial anti-fibrotic and anti-inflammatory effects in diverse models of cardiovascular disease. The goal of this study was to determine the effects of RLX treatment on the susceptibility to atrial fibrillation (AF) after myocardial infarction (MI). MethodsMice with cryoinfarction of the left anterior ventricular wall were treated for two weeks with either RLX (75 μg/kg/d) or vehicle (sodium acetate) delivered via subcutaneously implanted osmotic minipumps. ResultsRLX treatment significantly attenuated the increase in AF-inducibility following cryoinfarction and reduced the mean duration of AF episodes. Furthermore, epicardial mapping of both atria revealed an increase in conduction velocity. In addition to an attenuation of atrial hypertrophy, chronic application of RLX reduced atrial fibrosis, which was linked to a significant reduction in atrial mRNA expression of connective tissue growth factor. Transcript levels of the pro-inflammatory cytokines interleukin-6 and interleukin-1β were reduced in RLX treated mice, but macrophage infiltration into atrial myocardium was similar in the vehicle and RLX treated groups. ConclusionTreatment with RLX in mice after MI reduces susceptibility to AF due to anti-inflammatory and anti-fibrotic properties. Because to these favorable actions, RLX may become a new therapeutic option in the treatment of AF, even when complicating MI.

Propofol inhibits expression of angiotensin II receptor type 2 in dorsal root ganglion neurons.

The renin-angiotensin system (RAS) is involved in nociception and has functions in the cardiovascular system. The primary role of the RAS is to mediate the effect of angiotensin II (Ang II) through Ang II receptor type 2 (AT2). Due to this, AT2 has become a novel therapeutic target for the relief of peripheral neuropathic pain in humans. As it is one of the most popular induction agents of general anesthesia, propofol also exerts peripheral antinociceptive effects. The present study assessed the effect of propofol on the expression of AT2 in cultured dorsal root ganglion (DRG) neurons. The results indicate that propofol decreases AT2 mRNA expression in a statistically significant dose- and time-dependent manner (P<0.05). This resulted in a marked decrease in AT2 protein expression and the density of Ang II-binding AT2 on the cell membrane of DRG neurons. The effect of propofol was reversed by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Although propofol exhibited no significant effect on AT2 gene promoter activity, it significantly decreased the stability of AT2 mRNA (P<0.05). However, this effect was reversed by LY294002. In addition, propofol increased PI3K activity in a concentration-dependent manner in DRG neurons. In conclusion, to the best of our knowledge, the current study provides the first evidence suggesting that propofol inhibits the expression of AT2 in DRG neurons by decreasing the stability of AT2 mRNA through a PI3K-dependent mechanism. The present study provides novel insights into the mechanisms of the peripheral antinociceptive action of propofol and suggests a potential means of regulating Ang II/AT2 signaling in the peripheral nervous system.

390 Characterization of the Renin-Angiotensin System Regulation in Cavernosal Tissue of Aged Rats

Cardiovascular system plays fundamental role in the maintenance of male sexual function. Renin-Angiotensin system (RAS) is a major hormonal system governing cardiovascular homeostasis. Regulation of RAS is realized by multiple components exerting either deleterious or beneficial effects through ACE-AT1 and ACE-AT2/ACE2-Mas signaling, respectively. Local RAS activity within penile tissue has been documented, hence we have hypothesized that disturbance in its regulation can potentially contribute to the erectile dysfunction (ED) onset. In the current study, we aim to characterize local RAS regulation in rat model of aging-associated ED. RNA was isolated from corpus cavernosum (CC) of 12-week, 12-month and 24-month-old Wistar rats. Local expression of RAS components and several ED markers has been measured by RT-PCR. Angiotensinogen, precursor of effector RAS peptides, was highly expressed within CC, suggesting intense local regulation of the system. AT1 mRNA expression in the 12-month- and 24-month-old rats was decreased by 22 % (p=0.0160) and 35 % (p=0.0005), respectively. AT1 expression was positively correlated with ACE expression (p<0.0001), which was decreased by 29% (p=0.0130) in 24-month-old animals. Presence of AT2 mRNA in the CC tissue was negligible in comparison with AT1 receptor mRNA (103-fold difference). ACE2 and Mas mRNA expression was not altered in the 12-month- and 24-month-old rats. Additionally, 2-fold (p=0.0002) and 1.5 fold (p=0.0234) upregulation of eNOS was detected in 12-months- and 24-months-old rats, respectively, while arginase II mRNA levels was reduced by 50% (p<0.0001) in both groups of aged animals.

Expression of Angiotensin II Types 1 and 2 Receptors in Endometriotic Lesions

Background/Aim: The aim of this study was to evaluate the gene and protein expression of angiotensin type (AT) 1, AT2 receptors in endometriotic lesions and its relation to prostaglandin (PG) synthases. Materials and Methods: Endometriosis samples were obtained from 32 patients with endometriotic cysts. Endometrial tissues were obtained during operations for benign gynecological conditions. The expression of the AT1 and AT2 receptor mRNA and that of PG-endoperoxide synthase 2 and microsomal PGE2 synthase-1 (mPGES-1) was examined by quantitative RT-PCR. Immunohistochemical staining was performed for these receptors. Results: AT1 and AT2 receptor proteins were mostly located in endometrial glandular epithelium and some stromal cells. Immunoreactivity of the receptor proteins was observed in both the eutopic endometrium and endometriotic lesions. The AT1/AT2 ratio in endometriotic cysts (median 7.29, range 1.88-187.60) was significantly increased compared with that in the eutopic endometrium in the proliferative-phase in controls (median 1.01, range 0.37-2.09, p < 0.001). There was a relationship between the AT1 mRNA expression and that of mPGES-1 mRNA in the endometriotic cysts (r = 0.394089, p < 0.05). There was a significant relationship between the mRNA expression of the AT2 receptor and that of mPGES-1 in eutopic endometrium of non-endometriotic control (r = 0.610714, p < 0.05). Conclusion: Renin-angiotensin system may play an important role in the pathophysiology of endometriosis.

Protective effect of an angiotensin-converting-enzyme inhibitor on neurogenic pulmonary edema in rabbits

Neurogenic pulmonary edema (NPE ) was indicative of poor prognosis in the epidemic of enterovirus 71 infections. The pathogenesis of NPE remains poorly understood. The objectives of this experimental study were to explore whether RAS is activated during NPE in rabbit models induced by fibrin and the effects of an angiotensin converting enzyme inhibitor (enalaprilat) on NPE. NPE models were induced by intracisternal injection of fibrinogen and thrombin. According to random number table method, 18 healthy adult New Zealand rabbits were assigned to three groups (with 6 in each) : normal control group (Con group), NPE group and enalaprilat treated (Ena) group. After establishment of NPE models, rabbits in Ena group were given intravenous enalaprilat 0.5 mg/kg. Expression of ACE,ACE2,AT1R mRNA of the lung tissue were evaluated by real-time polymerise chain reaction; and Ang II of the lung tissue was determined by enzyme linked immunosorbent assay ( ELISA ). Meanwhile, histopathological lung injury scores were evaluated. ACE mRNA expression level in NPE group ( 17.2 ± 3.3) appeared an increasing trend in contrast to Con group ( 12.6 ± 5.2 ) and Ena group ( 11.5 ± 2.4, both P > 0.05 ). Compared with Con group (81 ± 22 ), ACE2 mRNA expression levels of NPE group ( 52 ± 6 ) and Ena group ( 45 ± 13 ) both decreased ( both P < 0.05 ) . ACE mRNA/ACE2 mRNA expression levels of NPE group ( 0.33 ± 0.06 ) and Ena group ( 0.26 ± 0.04 ) were higher than those of Con group ( 0.16 ± 0.05, both P < 0.05 ), as well as the ratio of Ena group decreased compared with untreated NPE group ( 0.26 ± 0.04 vs. 0.33 ± 0.06, P < 0.05 ) . There were no statistically significant differences in expression of AT1 mRNA of the lung tissue among three groups, but Ena group ( 4.8 ± 1.1) in contrast to NPE group ( 6.7 ± 1.3) has no significant difference (P > 0.05). Lung AngII level of NPE group [(540 ± 147) pg/ml] was significantly higher than that of Con group [(253 ± 37 ) pg/ml] and Ena group [(309 ± 35 ) pg/ml, both P < 0.05 ]. Gross pathologic examination showed that pink foamy edema fluid appeared in the tracheal tubes in NPE group, but spontaneously appeared in neither Con group nor Ena group; and the level of pulmonary subpleural bleeding in Con group, 12 graded 0; in NPE group, 2 graded II, 10 graded III; in Ena group, 2 graded, 8 grade II, 2 grade III. The histopathologic lung injury scores in Ena group was decreased in contrast to NPE group (1.36 ± 0.26 vs.2.32 ± 0.49, P < 0.05) and mainly for the improvement of alveolar overdistension and interstitial edema. The present study showed that when NPE occurs, a high lung AngII concentration was associated with an imbalance between ACE mRNA to ACE2 mRNA expression level. Activated local RAS in lung tissue resulted in lung injury. Enalaprilat treatment may attenuate lung injury by interventing local RAS in lung tissue with decreased ratio of ACE mRNA to ACE2 mRNA and lung AngII concentration. The result will be significant for the angiotensin converting enzyme inhibitor used in the theatment of NPE.

Effect of angiotensin II infusion on rhythmic clock gene expression and local renin-angiotensin system in the aorta of Wistar rats.

Endogenous daily rhythms in physiology are regulated by the circadian system consisting of the central and peripheral components. The renin-angiotensin system, involved predominantly in water balance and blood pressure control, exerts 24 h rhythmicity in many of its parameters. The present study is aimed to study possible interactions between these two control systems. We analyzed effects induced by angiotensin II administration on clock gene expression in the aorta of rat and an ability of angiotensin II to influence the local tissue renin-angiotensin system. Angiotensin II was infused in a dose of 100 ng/kg/min by subcutaneously implanted osmotic minipumps for 28 days to male Wistar rats. Gene expression was measured by real time PCR. Angiotensin II administration resulted in an increase in blood pressure, heart weight/body weight index, and water intake in comparison with controls. We observed a significant phase advance in per2 and npas2 mRNA rhythms and decreased mesor of npas2 rhythmic expression in the aorta of angiotensin II-treated rats compared to control. Angiotensin II administration did not influence daily pattern and level of at1 mRNA expression. The ratio ace/ace2 showed a rhythmic pattern in the aorta of control rats with peak levels in the dark period. Angiotensin II infusion influenced clock gene expression and diminished a daily rhythm in ace/ace2 mRNA ratio indicating modulatory effect of angiotensin II on tissue renin-angiotensin system in the aorta.

The role of estradiol in adrenal insufficiency and its interaction with corticosterone on hydromineral balance

Estradiol (E2) plays an important role in controlling the homeostasis of body fluids. Several studies have reported the involvement of the hypothalamic pituitary adrenal axis (HPA) in the homeostatic control of hydromineral balance and the influence of estrogens on the modulation of this system. Nevertheless, until now, the physiological relevance of HPA axis activity on the hydromineral balance in females has not yet been fully elucidated. Therefore, the objective of the present study was to evaluate the effects of E2 (20μg/animal) pretreatment on neuroendocrine and hydroelectrolyte changes induced by adrenalectomy (ADX) with or without glucocorticoid hormone replacement (corticosterone, CORT; 10mg/kg) in ovariectomized rats (OVX). The results show that sodium appetite, natriuresis and the elevated plasma angiotensin II (ANG II) concentration induced by ADX were attenuated by E2 pretreatment. Additionally, a reduction of AT1 mRNA expression in the subfornical organ (SFO) and an increase in plasma atrial natriuretic peptide (ANP) concentrations by E2 pretreatment were observed. E2 pretreatment reversed the reduction in water intake induced by ADX in ADX CORT-replaced rats. Moreover, E2 pretreatment attenuated corticotropin releasing factor (CRF) mRNA expression in the paraventricular nucleus (PVN) induced by ADX. In contrast, E2 pretreatment increased CRF mRNA expression in the PVN in ADX CORT-replaced rats. Taken together, these results suggest that E2 has an important role in the modulation of behavioral and neuroendocrine responses involved in the maintenance of body fluid homeostasis in ADX rats with or without glucocorticoid replacement therapy.

Atrial endothelin-1 signalling in hypertensive rats

Purpose: Hypertension induces cardiac remodelling at the structural and functional level. Endothelin-1 (ET-1) may contribute to cardiac remodelling. It is not known, however, whether atrial ET-1 signalling is altered in hypertension. Here we tested the hypothesis that ET-1 signalling is augmented in the atria of spontaneously hypertensive rats (SHR) and that this may contribute to hypertension-induced atrial arrhythmias. Methods: SHR and control Wistar-Kyoto (WKY) rats were studied at 6-8 months of age. Hearts were removed for morphometric analysis. Atria were snap frozen and analyzed for mRNA and protein expression by qPCR and Western blotting. Isolated atrial myocytes were field stimulated in the presence of 50 nM ET-1, and Ca transients (CaTs) were characterized at the global or subcellular level by epifluorescence (Fura-2/AM) and line scan confocal microscopy (Fluo-4/AM). To mimic atrial tachycardia, atrial tissue slices were stimulated at 5 Hz for up to 20 h and used for Western blotting. Results: At 6-8 months of age, SHR exhibited compensated LV hypertrophy, but left atrial hypotrophy, despite increased atrial mRNA levels of BNP (2-fold) and β-MHC (3.6-fold), while α-MHC was unaffected. There were no differences in global CaTs between atrial myocytes from WKY and SHR. Exposure of atrial myocytes to 50 nM ET-1 caused a 35% increase in systolic Ca in SHR (n=14), but only a 16% increase in WKY (n=21; P<0.05). Moreover, in SHR atrial myocytes, ET-1 induced a larger fractional SR Ca release (P<0.05 vs WKY). The ET-1-induced increase in CaTs occurred in the subsarcolemmal as well as in the central cytoplasm. Notably, ET-1 also induced arrhythmogenic Ca waves both in SHR and WKY atrial myocytes. Further analysis at the molecular level indicated that the atrial mRNA expression of ET-1 was increased (2-fold) in SHR. ETA receptor expression was unaltered at the mRNA level but upregulated (4-fold) at the protein level in the left atria. Gαq and PLCβ (isoforms 1 and 4) mRNA expression were also upregulated in SHR. Simulated atrial tachycardia in atrial tissue slices caused a 2-fold upregulation of ETA receptor expression in both SHR and WKY. Conclusions: At 6-8 months, SHR exhibit increased atrial expression of ET-1, ETA receptors, Gαq and PLCβ. Atrial tachycardia further increases ETA receptor expression. At the functional level, atrial myocytes from SHR respond to ET-1 with increases in systolic Ca and the development of arrhythmogenic Ca waves. Thus, atrial ET-1 signalling is enhanced already at early stages of hypertension and may contribute to the initiation of atrial tachycardia in hypertension.

Evaluation of the brain and kidney renin-angiotensin system and oxidative stress in neonatal handled rats

The aim of this study was to test the hypothesis that the renin-angiotensin system (RAS) components, as well as the oxidative stress system, would respond to early environmental changes. Thus, we have evaluated the effects of neonatal handling on both brain and kidney RAS and oxidative stress. Pups were divided into two groups: nonhandled and handled. The procedure consisted of handling them for 1 min/day in the first 10 days of life. On days 1, 5, and 10, animals were killed by decapitation. Blood samples were collected and the brain and kidneys were removed. Renin, AT(1), and AT(2) mRNA expression were evaluated through RT-PCR. Angiotensin II (ANG II) serum concentration was also measured. An increased ANG II concentration, brain and kidney AT(2) mRNA expression were demonstrated. The kidney mRNA AT(1) expression was decreased. There was also a kidney lipid peroxidation increase and a brain superoxide dismutase and catalase decrease. In conclusion, handling in the neonatal period induces the activation of the angiotensinergic system, as well as modulates its mRNA receptor expression. The oxidative stress balance system seems not to be involved.

The relationship between gene expression of cationic and neutral amino acid transporters in the small intestine of chick embryos and chick breed, development, sex, and egg amino acid concentration

This study was conducted to investigate the gene expression of cationic and neutral amino acid (AA) transporters in the small intestine of chick embryos with different genetic backgrounds [Wenshi Yellow-Feathered chick (WYFC) and White Recessive Rock chick (WRRC)]. The study also investigated the correlation between the abundance of AA transporter mRNA and the AA content of fertilized eggs. Intestinal samples were collected on embryonic d 9, 12, 14, 17, and 19 and the day of hatch. The results showed that, before incubation, the AA content of WRRC eggs was lower (P < 0.05) than the AA content of WYFC eggs. In WYFC, the mRNA abundance of CAT-1 [solute carrier (SLC) family 7 member 1], CAT-4 (SLC family 7 member 4), rBAT (SLC family 3 member 1), y(+)LAT-1 (SLC family 7 member 7), y(+)LAT-2 (SLC family 7 member 6), LAT-4 (SLC family 43 member 2), and SNAT-2 (SLC family 38 member 2), as detected by real-time reverse transcriptase PCR, was greater (P < 0.05) than the mRNA abundance detected in the WRRC samples. The mRNA abundance of all measured AA transporters was affected (P < 0.05) by embryonic age. Sex had the largest effect (P < 0.05) on the mRNA expression of CAT-1, CAT-4, y(+)LAT-2, and LAT-4 in WYFC and on CAT-4 and B(0)AT-1 (SLC family 6 member 19) mRNA expression in WRRC. In WYFC, only CAT-1 mRNA expression was negatively correlated (r = -0.68 to -0.84, P < 0.05) with all AA content. However, few correlations were detected between AA content and the mRNA expression of multiple transporters in WRRC. These findings provide a comprehensive profile of the temporal and spatial mRNA expression of AA transporters in the small intestine of chick embryos. Few correlations were detected between the AA content of the eggs and mRNA expression of specific AA transporters in the small intestine.

Effects of Pioglitazone on High-Fat-Diet–Induced Ventricular Remodeling and Dysfunction in Rats

To investigate the effect of pioglitazone on high-fat (HF)-diet-induced left ventricular (LV) hypertrophy and dysfunction in rats. A total of 36 male Sprague-Dawley rats were randomly divided into 3 groups, namely, control, HF diet, and pioglitazone treatment group. High-fat diet group (HF group) animals were treated with HF diet for 30 weeks, whereas pioglitazone group was treated with HF diet for 30 weeks and pioglitazone in the last 6 weeks of the 30-week treatment. Fasting plasma free fatty acids (FFAs), serum, and myocardial triglyceride were measured. Left ventricular function was assessed by echocardiography. Renin, angiotensin II, and angiotensin types 1 and 2 (AT1/AT2) receptors in the myocardium were analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR). Systolic blood pressure, plasma FFA, serum, and myocardial triglyceride concentrations in HF group were higher than in control and pioglitazone groups (P < .01). There was no significant difference in LV weight index and LV posterior wall thickness between HF and pioglitazone groups; both were higher than in the control group (P < .01). Left ventricular ejection fraction, fraction of shortening, and cardiac index in HF group were lower than in the control and pioglitazone groups (P < .05). Myocardial expression of angiotensin II and AT1 receptor protein in HF group was higher when compared with the control and pioglitazone groups (P < .01). Myocardial renin and angiotensin II messenger RNA (mRNA) in HF group was also higher when compared with the control and pioglitazone groups, whereas the expression of AT2 mRNA was lower (P < .01). Pioglitazone diminished HF-diet-induced LV dysfunction. These effects may be related to a reduction in blood pressure, myocardial triglycerides sedimentary, and suppression of renin-angiotensin system.