AT1 mRNA Research Articles

Hypertension induced by peri-pubertal protein restriction depends on renin-angiotensin system dysfunction in adult male rats

Background and aimsHypertension depends on renin-angiotensin system dysfunction; however, little is known about its implications in the outcomes of neurogenic hypertension induced by peri-pubertal insults. This study aimed to evaluate whether hypertension induced by a peri-pubertal low-protein diet is related to renin-angiotensin system dysfunction in adult male Wistar rats. Methods and resultsThirty-day-old male Wistar rats were fed a low-protein diet (4% casein) for 30 days and subsequently fed a 20.5% normal protein diet for a 60-day dietary recovery (LP group). Control animals (NP group) were fed a 20.5% protein diet throughout their lives. Cardiovascular and renin–angiotensin system functions were evaluated on postnatal day 120 (6-24 animals per group). Statistical analyses were performed using the Student’s t-test. Animals with LP show increased arterial blood pressure. The angiotensin 2 dose-response curve of LP animals showed an increase in the pressor response at a lower dose (50 ng/kg) and a reduction in the pressor response at a higher dose (400 ng/kg) compared with NP animals. Angiotensin 2 type 1 receptor mRNA levels were increased in the hearts of LP animals; however, angiotensin 2 type 2 receptor and MAS receptor mRNA levels were reduced. In the aorta, AT1 and AT2 mRNA levels were increased in LP animals, whereas MAS receptor mRNA levels were decreased in comparison to NP animals. ConclusionThe renin-angiotensin system is disrupted in hypertension induced by protein restriction exposure during peri-pubertal life.

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.

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.

Fish protein hydrolysate supplementation in plant protein based diets for tiger puffer (Takifugu rubripes) is an effective strategy of fish meal sparing

This study aimed to evaluate effects of fish protein hydrolysate (FPH) supplementation in plant protein based diets for tiger puffer (Takifugu rubripes) on growth performance, intestinal peptide and amino acid transporters, and the expression of protein synthesis-related genes and texture parameters in muscle. Tiger puffer were fed negative and positive control diets with 280 and 400 g/kg fish meal (LFM and HFM) or test diets containing 52 and 104 g/kg FPH supplement as 60 and 120 g/kg fish meal (FPH6 and FPH12) substitute for 56 days. Each diet was randomly fed to triplicate groups of 30 fish per tank. Growth parameters (final body weight, weight gain and specific growth rate) in the LFM group were significantly lower than that of the HFM group (p < 0.05), while no differences in those growth results were observed among the FPH6, FPH12 and HFM groups (p > 0.05). Compared with the LFM group, the expressions of intestinal PepT1, EAAT3, PAT1 and y+LAT2 were up-regulated in the FPH12 and HFM groups, while intestinal CAT1 and B°AT1 mRNA levels showed opposite trends. To protein synthesis-related gene expression, compared with the LFM group, mRNA levels of TOR and S6k1 was not significantly affected by dietary treatments, but mRNA level of 4E-BP1 increased in the FPH12 group. Lower chewiness and higher adhesiveness were observed when FPH was supplemented to the diets. In conclusion, FPH supplementation in plant protein based diets demonstrated a sparing effect on fish meal, without the negative effect on the growth of tiger puffer.

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...

ROLE OF SPINAL AT1, IONOTROPIC GLUTAMATERGIC AND GABAERGIC RECEPTORS ON CONTROL OF RENAL SYMPATHETIC NERVE ACTIVITY IN 2K‐1C RATS

Previous studies performed in our laboratory showed a higher glutamate and angiotensin receptors activation, as well as an alteration in GABAergic pathways in central nuclei involved in the cardiovascular control that may lead to sympathoexcitation in renovascular hypertensive rats (2K‐1C). However, the role of the spinal cord neurons on the renal sympathoexcitation remains unclear in this model. Thus, we aimed to assess the influence of spinal glutamatergic and GABAergic (ionotropic receptors), as well as angiotensin II type 1 receptors (AT1) receptors on the renal sympathetic nerve activity (rSNA) in the 2K‐1C rats. Male Wistar rats (250–300 gr) were used to induce renovascular hypertension by clipping the renal artery with a silver clip (0.2 mm). After six weeks, a polyethylene catheter (PE‐10) was inserted into the subarachnoid space and advanced to the T10–11 vertebrae level in urethane‐anaesthetized rats (1.2 g/Kg, iv). All experiments were approved by the Institutional Ethical Committee of UNIFESP. The effects of intrathecal (i.t.) injection of kynurenic acid (KYN) (160 nmol in 2 μl), losartan (Los) (2nmol in 2 μl) or bicuculline (Bic) (8 mM in 2 μl) on blood pressure (BP) and rSNA were investigated. KYN i.t. injection induced a larger and significant fall on rSNA in the 2K‐1C compared to control rats. Intrathecally Los evoked a larger and significant fall in rSNA in the 2K‐1C compared to control group. KYN decreased BP similarly in both, control and 2K‐1C groups, however, Los significantly decreased BP in the 2K‐1C group only. Intrathecally Bic triggered a more intense BP and rSNA reponses in 2K‐1C compared to control rats. Gene expression analysis by qRT‐PCR showed a spinal (T11–12) AT1 mRNA upregulation in the 2K‐1C rats (1.71‐fold change), while no changes were observed in AT2, ionotropic glutamate and GABA receptors subunits. Therefore, our data show that ionotropic spinal cord excitatory amino acid, GABAergic and AT1 receptors play a significant role in the control of rSNA in the 2K‐1C model that may contribute to the maintenance of hypertension. The participation of spinal AT1 receptors seems to be more important in the establishment of sympathoexcitation in this model. The origin of those projections remains unclear.Support or Funding InformationCNPq, FAPESP (2018/01898‐4) and CAPES

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.

Angiotensin II Receptor 1 Blockage Limits Brain Damage and Improves Functional Outcome After Brain Injury in Aged Animals Despite Age-Dependent Reduction in AT1 Expression.

Traumatic brain injury (TBI) is a frequent pathology associated with poor neurological outcome in the aged population. We recently observed accelerated cerebral inflammation in aged mice in response to TBI. Candesartan is a potent specific inhibitor of angiotensin II receptor type 1 (AT1) which limits cerebral inflammation and brain damage in juvenile animals after experimental TBI. In the present study, we show significantly lower posttraumatic AT1 mRNA levels in aged (21 months) compared to young (2 months) mice. Despite low cerebral At1 expression, pharmacologic blockade by treatment with candesartan [daily, beginning 30 min after experimental TBI by controlled cortical impact (CCI)] was highly effective in both young and aged animals and reduced histological brain damage by −20% after 5 days. In young mice, neurological improvement was enhanced by AT1 inhibition 5 days after CCI. In older animals, candesartan treatment reduced functional impairment already on day 3 after TBI and post-traumatic body weight (BW) loss was attenuated. Candesartan reduced microglia activation (−40%) in young and aged animals, and neutrophil infiltration (−40% to 50%) in aged mice, whereas T-cell infiltration was not changed in either age group. In young animals, markers of anti-inflammatory microglia M2a polarization [arginase 1 (Arg1), chitinase3-like 3 (Ym1)] were increased by candesartan at days 1 and 5 after insult. In older mice 5 days after insult, expression of Arg1 was significantly higher independently of the treatment, whereas Ym1 gene expression was further enhanced by AT1 inhibition. Despite age-dependent posttraumatic differences in At1 expression levels, inhibition of AT1 was highly effective in a posttreatment paradigm. Targeting inflammation with candesartan is, therefore, a promising therapeutic strategy to limit secondary brain damage independent of the age.

Expression of mutant Sftpc in murine alveolar epithelia drives spontaneous lung fibrosis.

Epithelial cell dysfunction is postulated as an important component in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mutations in the surfactant protein C (SP-C) gene (SFTPC), an alveolar type II (AT2) cell-restricted protein, have been found in sporadic and familial IPF. To causally link these events, we developed a knockin mouse model capable of regulated expression of an IPF-associated isoleucine-to-threonine substitution at codon 73 (I73T) in Sftpc (SP-CI73T). Tamoxifen-treated SP-CI73T cohorts developed rapid increases in SftpcI73T mRNA and misprocessed proSP-CI73T protein accompanied by increased early mortality (days 7-14). This acute phase was marked by diffuse parenchymal lung injury, tissue infiltration by monocytes, polycellular alveolitis, and elevations in bronchoalveolar lavage and AT2 mRNA content of select inflammatory cytokines. Resolution of alveolitis (2-4 weeks), commensurate with a rise in TGF-β1, was followed by aberrant remodeling marked by collagen deposition, AT2 cell hyperplasia, α-smooth muscle actin-positive (α-SMA-positive) cells, and restrictive lung physiology. The translational relevance of the model was supported by detection of multiple IPF biomarkers previously reported in human cohorts. These data provide proof of principle that mutant SP-C expression in vivo causes spontaneous lung fibrosis, strengthening the role of AT2 cell dysfunction as a key upstream driver of IPF pathogenesis.

The Expression of MAS1, an Angiotensin (1–7) Receptor, in the Eutopic Proliferative Endometria of Endometriosis Patients

Background/Aim: We demonstrated that AT1 and AT2 are expressed and both pathways balance the renin-angiotensin system in endometriosis. MAS1, a specific receptor of angiotensin (1–7), opposes AT1 pathway-associated tissue remodelling. It is not known whether MAS1 has an effect on the pathogenesis of endometriosis or not. Materials and Methods: Ovarian endometriotic tissues (endo-Ov) and eutopic endometrial tissues (endo-Em) were obtained from 29 patients with endometrial cysts. Normal endometrial tissues (cont-Em) were obtained from patients without endometriosis. Immunohistochemical staining was performed for MAS1, AT1 and AT2 in the endometriosis-associated tissues. The mRNA levels of these receptors were examined by quantitative reverse transcription PCR. Results: MAS1 was immune-positive at the apical side of the glandular epithelium in the endometriotic lesions. The MAS1 mRNA levels in endo-Ov were increased significantly, irrespective of the menstrual cycle phase. The MAS1 mRNA levels were significantly higher in the proliferative-tissues of the endometriosis patients than in those of the controls. The ratio of the MAS1 to the AT1 mRNA in the proliferative tissues was increased predominantly in the endometriosis patients compared with that in the controls. Conclusion: High MAS1 expression in the endometrium might promote the initiation of endometriosis via migration of proliferative tissue.

Expression of human monolysocardiolipin acyltransferase-1 improves mitochondrial function in Barth syndrome lymphoblasts

The mitochondrial polyglycerophospholipid cardiolipin (CL) is remodeled to obtain specific fatty acyl chains. This is predominantly accomplished by the transacylase enzyme tafazzin (TAZ). Barth syndrome (BTHS) patients with TAZ gene mutations exhibit impaired TAZ activity and loss in mitochondrial respiratory function. Previous studies identified monolysocardiolipin acyltransferase-1 (MLCL AT-1) as a mitochondrial enzyme capable of remodeling CL with fatty acid. In this study, we analyzed what relationship, if any, exists between TAZ and MLCL AT-1 with regard to CL remodeling and whether transfection of BTHS lymphoblasts with an MLCL AT-1 expression construct improves mitochondrial respiratory function. In healthy lymphoblasts, reduction in TAZ expression through TAZ RNAi transfection resulted in a compensatory increase in MLCL AT-1 mRNA, protein, and enzyme activity, but CL mass was unaltered. In contrast, BTHS lymphoblasts exhibited decreased TAZ gene and protein expression but in addition decreased MLCL AT-1 expression and CL mass. Transfection of BTHS lymphoblasts with MLCL AT-1 expression construct increased CL, improved mitochondrial basal respiration and protein leak, and decreased the proportion of cells producing superoxide but did not restore CL molecular species composition to control levels. In addition, BTHS lymphoblasts exhibited higher rates of glycolysis compared with healthy controls to compensate for reduced mitochondrial respiratory function. Mitochondrial supercomplex assembly was significantly impaired in BTHS lymphoblasts, and transfection of BTHS lymphoblasts with MLCL AT-1 expression construct did not restore supercomplex assembly. The results suggest that expression of MLCL AT-1 depends on functional TAZ in healthy cells. In addition, transfection of BTHS lymphoblasts with an MLCL AT-1 expression construct compensates, but not completely, for loss of mitochondrial respiratory function.

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.

Alleviation of Cardiac Damage by Dietary Fenugreek (Trigonella foenum-graecum) Seeds is Potentiated by Onion (Allium cepa) in Experimental Diabetic Rats via Blocking Renin-Angiotensin System.

Hyperglycemia is one of the metabolic and homeostatic abnormalities that increase the cardiovascular mortality in diabetic patients by increased oxidative stress. We have recently reported amelioration of oxidative stress in cardiac tissue by dietary fenugreek (Trigonella foenum-graecum) seeds and onion (Allium cepa) in streptozotocin-induced diabetic rats. The mechanistic aspects of the cardio-protective influence of dietary fenugreek seeds (10%) and onion (3% powder) both individually and in combination on hyperglycemia-mediated cardiac damage was further investigated in this study on streptozotocin-induced diabetic rats. Cardio-protective influence of these dietary spices was evidenced by their blocking potential on renin-angiotensin system. This might be the consequence of reduced activation of angiotensin-converting enzyme (ACE) and angiotensin type 1 receptor (AT1) in cardiac tissue. The combination produced an additive effect on ACE and AT1 protein and mRNA expressions. Increased expression of type IV collagen, fibronectin, Bax, 4-hydroxynonenal, iNOS and metabolites of nitric oxide (nitrate/nitrite) along with disturbed PUFA-to-SFA ratio and activities of cardiac marker enzymes in blood confirmed the myocardial damage. Dietary fenugreek seed, onion and fenugreek+onion were found to ameliorate these pathological changes in the cardiovascular system. The beneficial effect being higher with the combination sometime amounting to additive (iNOS expression) or even a synergistic (cardiac Bax and type IV collagen expression and circulatory marker enzymes) in diabetic rats. Thus, the results of present investigation suggested that the combination of fenugreek seeds and onion offers higher beneficial influence in ameliorating cardiac damage accompanying diabetes.

Egg white-derived peptides prevent cardiovascular disorders induced by mercury in rats: Role of angiotensin-converting enzyme (ACE) and NADPH oxidase

The study aimed to investigate the effects of egg white hydrolysate (EWH) on vascular disorders induced by mercury (Hg). For this, male Wistar rats were treated for 60days: Untreated (saline, i.m.); Mercury (HgCl2, i.m., 1st dose 4.6μg/kg, subsequent doses 0.07μg/kg/day); Hydrolysate (EWH, gavage, 1g/kg/day); Hydrolysate-Mercury. Systolic (SBP) and diastolic (DBP) blood pressure measurement and vascular reactivity experiments in aorta were performed. We analyzed endothelial dependent and independent vasodilator responses and vasoconstrictor response to phenylephrine (Phe) in absence and presence of endothelium, a NOS inhibitor, a NADPH oxidase inhibitor, the superoxide dismutase, a non-selective COX inhibitor, a selective COX-2 inhibitor, an AT-1 receptors blocker. In situ superoxide anion production, SOD-1, NOX-4, p22phox, COX-2 and AT-1 mRNA levels and NOX-1 protein expression were performed in aorta while the determination of angiotensin converting enzyme (ACE) activity was measured in plasma. As results, EWH prevented the increase in SBP and Phe responses and the endothelial dysfunction elicited by Hg, which was related to decreased ACE activity and NOX activation by EWH and, subsequently, alleviated ROS production and improved NO bioavailability in aorta. In conclusion, EWH could be considered as alternative or complementary treatment tools for Hg-induced cardiovascular damage.

Gene expression of amino acid transporter in pigeon (Columbia livia) intestine during post-hatch development and its correlation with amino acid in pigeon milk

This study was conducted to evaluate gene expression of the amino acid transporter in post-hatch pigeon small intestine and the association of pigeon milk amino acid with the above transporter's gene expression. A total of 48 pigeon breeding families were randomly allocated to 8 groups of 6 replicates of one parental pigeon pair and 2 squabs. Samples of pigeon milk and duodenum, jejunum, and ileum were collected on d 1, 2, 3, 4, 6, 8, 10, and 14 post hatch. The results showed that levels of crude protein (8.93 to 15.56%) were highest in pigeon milk on an air-dry basis. Amino acid content in pigeon milk remained constant in the first 4 d, declined abruptly at d 6, then increased dramatically from d 8 to 14. There was a significant effect of interaction between age and intestinal segments on those amino acid transporters gene expression. mRNA abundance of ATB0’+, SNAT-2, LAT-4, rBAT, b0’+ AT, EAAT-3 and PAT-1 was highest in the ileum; B0 AT1, asc-1, and IMINO were predominate in the jejunum; and CAT-1 and y+ LAT2 were greatest in the duodenum. Age-related changes of amino acid transporter mRNA was inconsistent. mRNA levels of SNAT-2, rBAT, y+ LAT2, b0’+ AT, and EAAT-3 ascended with age, whereas that of asc-1, CAT-1, and IMINO diminished significantly. Levels of B0 AT1 and PAT-1 mRNA abundance were minimized at d 6. However, few correlations were found between pigeon milk amino acid and the amino acid transporter gene expressions in squab small intestine. Our findings provide a comprehensive elaboration on ontogeny of the amino acid transporter in post-hatch pigeon intestine.

Insufficient hypothalamic angiotensin-converting enzyme 2 is associated with hypertension in SHR rats.

Angiotensin-Converting Enzyme 2 (ACE2) is a key enzyme in the renin-angiotensin system (RAS), which is implicated in the pathogenesis of hypertension and other cardiovascular diseases. In this study we investigated the expression of ACE2 in the hypothalamus and pituitary tissues and its relationship to hypertension by comparing them in male WKY and SHR rats. We observed that the plasma levels of corticotrophin releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and aldosterone (ALD) were all lower in SHR than WKY rats (P<0.05), whereas plasma angiotensin II (AngII) levels were higher in SHR rats (P<0.05). Levels of ACE mRNA and protein were higher in the hypothalamus of SHR than WKY rats (P<0.05). By contrast, hypothalamic expression of ACE2 protein was lower in SHR rats (P<0.05), despite comparable mRNA levels in SHR and WKY rats. There were no differences in the expression levels of ACE, ACE2, AT1 or Mas mRNA in the pituitaries of SHR and WKY rats (P>0.05). These results suggest that insufficiency of hypothalamic ACE2 is associated with hypertension in SHR rats.

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.

Abstract P613: Absolute Gene Quantification Of Intrarenal Renin-angiotensin System Components Using Droplet Digital PCR Analysis

The intrarenal renin-angiotensin system (RAS) has been shown to play crucial roles in the development of hypertension and RAS associated kidney injury including diabetic nephropathy. Although some circulating RAS components are filtered into kidneys and contribute to the regulation of intrarenal RAS activity, evaluating expression levels of RAS components in the kidney is important to elucidate the mechanisms underlying intrarenal RAS activation. Digital PCR is a new technique that has been established to quantify absolute target gene levels, which allows for comparisons of different gene levels. Thus, this study was performed to establish profiles of absolute gene copy numbers for intrarenal RAS components in wild-type (WT) rats, WT and streptozotocin (STZ)-induced diabetic mice. Male Sprague-Dawley rats (N=5) and male C57BL/6J mice were used in this study. The mice were subjected to either control (N=5) or STZ (200 mg/kg, N=4) injection. Seven days after STZ injection, copy numbers of renal cortical angiotensinogen (AGT), angiotensin-converting enzyme (ACE), ACE2, angiotensin type 1 receptor a (AT1a), and AT2 mRNA were determined by a droplet digital PCR. Since (pro)renin proteins produced by juxtaglomerular cells are secreted to circulating system, analysis of renin mRNA was excluded from this evaluation. In the renal cortex of WT rats, the copy number of AGT was higher than other measured RAS components (AGT: 719.2±46.6, ACE: 116.0±14.9, ACE2: 183.6±21.5, AT1a: 196.0±25.2 copies in 1 ng total RNA). AT2 levels were lower than other components (0.068±0.01 copies). In WT mice, ACE exhibited the highest copy number in the components (AGT: 447.2±29.0, ACE: 1662.4±61.2, ACE2: 676.8±41.5, AT1a: 867.0±16.8, AT2: 0.049±0.01 copies). Although STZ-induced diabetes did not change ACE2 and AT1a, ACE levels were reduced (765.5±98.1 copies) and AT2 levels were augmented (0.10±0.01 copies) as previously demonstrated. Accordingly, the absolute quantification by digital PCR established precise gene profiles of intrarenal RAS components, which will provide rationales for targeting the each component in future studies. Furthermore, the results indicate that the high sensitive assay accurately quantifies rare target genes including intrarenal AT2.