AT2 Receptor Antagonist PD123319 Research Articles

Angiotensinergic neurotransmission in the bed nucleus of the stria terminalis is involved in cardiovascular responses to acute restraint stress in rats.

The brain angiotensin II acting via AT1 receptors is a prominent mechanism involved in physiological and behavioral responses during aversive situations. The AT2 receptor has also been implicated in stress responses, but its role was less explored. Despite these pieces of evidence, the brain sites related to control of the changes during aversive threats by the brain renin-angiotensin system (RAS) are poorly understood. The bed nucleus of the stria terminalis (BNST) is a limbic structure related to the cardiovascular responses by stress, and components of the RAS system were identified in this forebrain region. Therefore, we investigated the role of angiotensinergic neurotransmission present within the BNST acting via local AT1 and AT2 receptors in cardiovascular responses evoked by an acute session of restraint stress in rats. For this, rats were subjected to bilateral microinjection of either the angiotensin-converting enzyme inhibitor captopril, the selective AT1 receptor antagonist losartan, or the selective AT2 receptor antagonist PD123319 before they underwent the restraint stress session. We observed that BNST treatment with captopril reduced the decrease in tail skin temperature evoked by restraint stress, without affecting the pressor and tachycardic responses. Local AT2 receptor antagonism within the BNST reduced both the tachycardia and the drop in tail skin temperature during restraint. Bilateral microinjection of losartan into the BNST did not affect the restraint-evoked cardiovascular changes. Taken together, these data indicate an involvement of BNST angiotensinergic neurotransmission acting via local AT2 receptors in cardiovascular responses during stressful situations.

Estrogen normalizes maternal HFD-induced vascular dysfunction in offspring by regulating ATR.

Previous studies have shown that female offspring are resistant to fetal high-fat diet (HFD)-induced programming of heightened vascular contraction; however, the underlying mechanisms remain unclear. The present study tested the hypothesis that estrogen plays a key role in protecting females from fetal programming of increased vascular contraction induced by maternal HFD exposure. Pregnant rats were fed a normal diet (ND) or HFD (60% kcal from fat). Ovariectomy (OVX) and 17β-estradiol (E2) replacement were performed on 8-week-old female offspring. Aortas were isolated from adult female offspring. Maternal HFD exposure increased angiotensin II (Ang II)-induced contractions of the aorta in adult OVX offspring, which was abrogated by E2 replacement. The AT1 receptor (AT1R) antagonist losartan (10 μM), but not the AT2 receptor (AT2R) antagonist PD123319 (10 μM), completely blocked Ang II-induced contractions in both ND and HFD offspring. In addition, HFD exposure caused a decrease in endothelium-dependent relaxations induced by acetylcholine (ACh) in adult OVX but not OVX-E2 offspring. However, it had no effect on sodium nitroprusside (SNP)-induced endothelium-independent aorta relaxation in any of the six groups. Maternal HFD feeding increased AT1R, but not AT2R, leading to an increased AT1R/AT2R ratio in HFD-exposed OVX offspring, associated with selective decreases in DNA methylation at the AT1aR promoter, which was ameliorated by E2 replacement. Our results indicated that estrogen play a key role in sex differences of maternal HFD-induced vascular dysfunction and development of hypertensive phenotype in adulthood by differently regulating vascular AT1R and AT2R gene expression through a DNA methylation mechanism.

Sexually dimorphic role of smooth muscle cell estrogen receptor alpha in aging‐associated arterial stiffness

BackgroundAging is associated with rising arterial stiffness (AS), an independent risk factor for cardiovascular disease. The development of aging‐associated AS follows a sexually dimorphic pattern with women developing AS later in life. Estrogen and estrogen receptor alpha (ERα) are protective in the premenopausal state, potentially due to the regulation of the vasoprotective angiotensin II type 2 receptor (AT2R), however, the role of estrogen/ERα in aging‐associated AS has not been defined and the mechanisms of the sexually‐dimorphic development of aging‐associated AS are unknown.Methods/ResultsAll studies were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. We generated novel smooth muscle cell‐specific ERα‐KO mice and measured aortic pulse wave velocity (PWV), an index of AS, in 3, 12, and 18 month old male and female intact and KO mice. Male mice develop increases in AS from 3 to 12 months compared to 3 to 18 months in females (p<0.05). SMC‐ERα‐KO protected females from the aging‐associated increase in AS (p<0.05). Circulating estrogens also declined in 18 month old females vs. 3 mo. (p<0.05), suggesting a role for unliganded SMC‐ERα in aging‐associated AS. SMC‐ERα‐KO females were also protected from the aging‐associated decline in aortic AT2R expression observed in intact females (p<0.05). To further investigate the potential role of unliganded SMC‐ERα in AS, we performed ovariectomy (OVX) or sham surgeries in young female SMC‐ERα‐Intact and KO mice and measured PWV. Results demonstrated an OVX‐driven increase in AS that was prevented by SMC‐ERα‐KO (p<0.05). This was also associated with an OVX‐mediated decrease in aortic expression of the AT2R, which was prevented by SMC‐ERα‐KO. To determine the in vivo role of AT2R in the vascular protection observed in 12 month old female mice, we treated 12 month old male and female mice with AT2R antagonist PD‐123319 (3mg/kg/day) or vehicle via osmotic minipumps for 4 weeks. Chronic inhibition of the AT2R increased AS in 12 month old females (p<0.05), but not males, suggesting that AT2R‐mediated protection contributes to the delay in aging‐associated AS in female mice. In vitro studies also demonstrated that estrogen upregulates AT2R mRNA and protein in female mouse aortic SMC, supporting an estrogen‐mediated mechanism of AT2R driven vascular protection in females.ConclusionThese data indicate sex differences in the time course of the development of aging‐associated arterial stiffness and suggest the presence of unliganded SMC‐ERα contributes specifically to aging‐associated arterial stiffness in females via reductions in vascular AT2R. Further, these results implicate that AT2R agonist therapy may improve cardiovascular outcomes in aging women to retain the beneficial downstream effects of estrogen signaling, without the potential detrimental effects of hormone replacement therapy.

Angiotensinergic Neurotransmissions in the Medial Amygdala Nucleus Modulate Behavioral Changes in the Forced Swimming Test Evoked by Acute Restraint Stress in Rats.

We investigated the role of angiotensin II type 1 (AT1 receptor) and type 2 (AT2 receptor) and MAS receptors present in the medial amygdaloid nucleus (MeA) in behavioral changes in the forced swimming test (FST) evoked by acute restraint stress in male rats. For this, rats received bilateral microinjection of either the selective AT1 receptor antagonist losartan, the selective AT2 receptor antagonist PD123319, the selective MAS receptor antagonist A-779, or vehicle 10 min before a 60 min restraint session. Then, behavior in the FST was evaluated immediately after the restraint (15 min session) and 24 h later (5 min session). The behavior in the FST of a non-stressed group was also evaluated. We observed that acute restraint stress decreased immobility during both sessions of the FST in animals treated with vehicle in the MeA. The decreased immobility during the first session was inhibited by intra-MeA administration of PD123319, whereas the effect during the second session was not identified in animals treated with A-779 into the MeA. Microinjection of PD123319 into the MeA also affected the pattern of active behaviors (i.e., swimming and climbing) during the second session of the FST. Taken together, these results indicate an involvement of angiotensinergic neurotransmissions within the MeA in behavioral changes in the FST evoked by stress.

Angiotensin AT2 Receptor is Anti-inflammatory and Reno-Protective in Lipopolysaccharide Mice Model: Role of IL-10.

Acute kidney injury (AKI) due to endotoxemic insult is predicted by the infiltration of neutrophils, monocytes and macrophages, and the release of pro-and anti-inflammatory cytokines to the site of injury. Earlier, we have demonstrated the role of angiotensin-II type 2 receptor (AT2R) stimulation in reno-protection in lipopolysaccharide (LPS)-induced inflammation and AKI in C57BL6/NHsd mice. Moreover, AT2R activation has been shown to increase the anti-inflammatory cytokine interleukin-10 (IL-10), its role in AT2R-mediated anti-inflammation and reno-protection is unknown. To address this question, in the present study mice were treated with the AT2R agonist C21 (0.3 mg/kg, intraperitoneally), LPS (5 mg/kg, intraperitoneally), or LPS with C21 pre-treatment with or without neutralizing IL-10 antibody. Treatment with C21 alone caused an increase in the plasma and kidney IL-10 levels, which peaks at 2-h, and returned to baseline at 6-h. The C21-induced IL-10 increase was blocked by the AT2R antagonist PD123319 suggesting AT2R’s involvement. LPS treatment caused a profound increase in tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and the LPS-induced increase in these cytokines was attenuated by the C21 pre-treatment (1-h prior LPS) both in the plasma and kidney. Neutralizing IL-10 antibody treatment abrogated the C21-lowering of TNF-α and IL-6 in the kidney but not in the plasma. Similar results as related to the cytokines profiles in all the groups were also observed in the heart and spleen. The alteration in early cytokine profile prompted us to measure the markers of renal function (blood urea nitogen and urinary creatinine) in order to analyze the effect of IL-10 neutralization. However, it was too early to observe changes in renal function. Therefore, the renal function and injury markers were again measured at 24 h. Treatment with neutralizing IL-10 antibody attenuated the C21-mediated improvement in indices of the kidney function, but not the biomarkers of renal injury (kidney injury molecule-1 and neutrophil-gelatinase associated lipocalin). Collectively, our data suggest that the involvement of IL-10 in AT2R-mediated anti-inflammation and reno-protection against LPS is complex, mediating the renal cytokine profile and kidney filtration function, but not the plasma cytokine profile and renal injury markers.

Role of angiotensin receptors in the medial amygdaloid nucleus in autonomic, baroreflex and cardiovascular changes evoked by chronic stress in rats.

This study investigated the role of AT1 , AT2 and Mas angiotensinergic receptors within the MeA in autonomic, cardiovascular and baroreflex changes evoked by a 10-day (1hr daily) repeated restraint stress (RRS) protocol. Analysis of cardiovascular function after the end of the RRS protocol indicated increased values of arterial pressure, without heart rate changes. Arterial pressure increase was not affected by acute MeA treatment after the RRS with either the selective AT1 receptor antagonist losartan, the selective AT2 receptor antagonist PD123319 or the selective Mas receptor antagonist A-779. Analysis of heart rate variability indicated that RRS increased the sympathetic tone to the heart, which was inhibited by MeA treatment with either losartan, PD123319 or A-779. Baroreflex function assessed using the pharmacological approach via intravenous infusion of vasoactive agents revealed a facilitation of tachycardia evoked by blood pressure decrease in chronically stressed animals, which was inhibited by MeA treatment with losartan. Conversely, baroreflex responses during spontaneous fluctuations of blood pressure were impaired by RRS, and this effect was not affected by injection of the angiotensinergic receptor antagonists into the MeA. Altogether, the data reported in the present study suggest an involvement of both angiotensinergic receptors present in the MeA in autonomic imbalance evoked by RRS, as well as an involvement of MeA AT1 receptor in the enhanced baroreflex responses during full range of blood pressure changes. Results also indicate that RRS-evoked increase in arterial pressure and impairment of baroreflex responses during spontaneous variations of arterial pressure are independent of MeA angiotensinergic receptors.

Angiotensinergic receptors in the medial amygdaloid nucleus differently modulate behavioral responses in the elevated plus-maze and forced swimming test in rats

The brain renin-angiotensin system (RAS) has been implicated in anxiety and depression disorders, but the specific brain sites involved are poorly understood. The medial amygdaloid nucleus (MeA) is involved in expression of behavioral responses. However, despite evidence of the presence of all angiotensinergic receptors in this amygdaloid nucleus, regulation of anxiety- and depressive-like behaviors by angiotensinergic neurotransmissions within the MeA has never been reported. Thus, the present study aimed to investigate the role angiotensin II (AT1 and AT2 receptors) and angiotensin-(1–7) (Mas receptor) receptors present within the MeA in behavioral responses in the elevated plus-maze (EPM) and forced swimming test (FST). For this, male Wistar rats had cannula-guide bilaterally implanted into the MeA, and independent sets of animals received bilateral microinjections of either the selective AT1 receptor antagonist losartan, the selective AT2 receptor antagonist PD123319, the selective Mas receptor antagonist A-779 or vehicle into the MeA before the EPM and FST. Treatment of the MeA with either PD123319 or A-779 decreased the EPM open arms exploration, while losartan did not affect behavioral responses in this apparatus. However, intra-MeA microinjection of losartan decreased immobility in the FST. Administration of either PD123319 or A-779 into the MeA did not affect the immobility during the FST, but changed the pattern of the active behaviors swimming and climbing. Altogether, these results indicate the presence of different angiotensinergic mechanisms within the MeA controlling behavioral responses in the FST and EPM.

Maternal high-fat diet increases vascular contractility in adult offspring in a sex-dependent manner.

A maternal high-fat diet (HFD) is a risk factor for cardiovascular diseases in offspring. The aim of the study was to determine whether maternal HFD causes the epigenetic programming of vascular angiotensin II receptors (ATRs) and leads to heightened vascular contraction in adult male offspring in a sex-dependent manner. Pregnant rats were treated with HFD (60% kcal fat). Aortas were isolated from adult male and female offspring. Maternal HFD increased phenylephrine (PE)-and angiotensin II (Ang II)-induced contractions of the aorta in male but not female offspring. NG-nitro-L-arginine (ʟ-NNA; 100 μM) abrogated the maternal HFD-induced increase in PE-mediated contraction. HFD caused a decrease in endothelium-dependent relaxations induced by acetylcholine in male but not female offspring. However, it had no effect on sodium nitroprusside-induced endothelium-independent relaxations of aortas regardless of sex. The AT1 receptor (AT1R) antagonist losartan (10 μM), but not the AT2 receptor (AT2R) antagonist PD123319 (10 μM), blocked Ang II-induced contractions in both control and HFD offspring in both sexes. Maternal HFD increased AT1R but decreased AT2R, leading to an increased ratio of AT1R/AT2R in HFD male offspring, which was associated with selective decreases in DNA methylation at the AT1aR promoter and increases in DNA methylation at the AT2R promoter. The vascular ratio of AT1R/AT2R was not significantly different in HFD female offspring compared with the control group. Our results indicated that maternal HFD caused a differential regulation of vascular AT1R and AT2R gene expression through a DNA methylation mechanism, which may be involved in HFD-induced vascular dysfunction and the development of a hypertensive phenotype in adulthood in a sex-dependent manner.

AT2 and MAS (but not AT1) angiotensinergic receptors in the medial amygdaloid nucleus modulate the baroreflex activity in rats.

The medial amygdaloid nucleus (MeA) is a limbic structure that has been demonstrated to be part of the central circuitry regulating baroreflex function. However, the local neurochemical mechanisms involved in baroreflex control by this forebrain structure is poorly understood. Thus, in the present study, we investigated the specific role of AT1, AT2, and MAS angiotensinergic receptors within the MeA in baroreflex responses in unanesthetized rats. For this, the baroreflex function was assessed using both the pharmacological approach via intravenous infusion of vasoactive agents and the sequence analysis technique. Using the pharmacological approach, we observed that bilateral microinjection of the selective AT2 receptor antagonist PD123319 into the MeA increased the tachycardia evoked by blood pressure decrease, but without affecting the reflex bradycardia caused by blood pressure increase. Besides, bilateral microinjection of the selective MAS receptor antagonist A-779 decreased both tachycardic and bradycardic responses of the baroreflex. The sequence analysis technique indicated that PD123319 into the MeA increased baroreflex effectiveness index while A-779 had an opposite effect. Treatment of the MeA with the selective AT1 receptor antagonist losartan did not affect baroreflex function assessed by either the pharmacological approach or sequence analysis technique. Overall, these findings provide evidence that MAS receptor within the MeA plays a facilitatory role in baroreflex function, whereas local AT2 receptor inhibits cardiac baroreflex responses. Results also indicate that AT1 receptor within the MeA is not involved in the control of baroreflex function.

Prevention of lipopolysaccharide-induced CD11b+ immune cell infiltration in the kidney: role of AT2 receptors.

Immune cell infiltration plays a central role in mediating endotoxemic acute kidney injury (AKI). Recently, we have reported the anti-inflammatory and reno-protective role of angiotensin-II type-2 receptor (AT2R) activation under chronic low-grade inflammatory condition in the obese Zucker rat model. However, the role of AT2R activation in preventing lipopolysaccharide (LPS)-induced early infiltration of immune cells, inflammation and AKI is not known. Mice were treated with AT2R agonist C21 (0.3 mg/kg), with and without AT2R antagonist PD123319 (5 mg/kg) prior to or concurrently with LPS (5 mg/kg) challenge. Prior-treatment with C21, but not concurrent treatment, significantly prevented the LPS-induced renal infiltration of CD11b+ immune cells, increase in the levels of circulating and/or renal chemotactic cytokines, particularly interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) and markers of renal dysfunction (blood urea nitrogen and albuminuria), while preserving anti-inflammatory interleukin-10 (IL-10) production. Moreover, C21 treatment in the absence of LPS increased renal and circulating IL-10 levels. To investigate the role of IL-10 in a cross-talk between epithelial cells and monocytes, we performed in vitro conditioned media (CM) studies in human kidney proximal tubular epithelial (HK-2) cells and macrophages (differentiated human monocytes, THP-1 cells). These studies revealed that the conditioned-media derived from the C21-treated HK-2 cells reduced LPS-induced THP-1 tumor necrosis factor-α (TNF-α) production via IL-10 originating from HK-2 cells. Our findings suggest that prior activation of AT2R is prophylactic in preventing LPS-induced renal immune cell infiltration and dysfunction, possibly via IL-10 pathway.

Prevention of Lipopolysaccharide‐Induced CD11b+ Leukocytes Infiltration in the Kidney: Role of AT2 Receptors

Leukocyte infiltration plays a central role in mediating endotoxemic acute kidney injury (AKI). We have recently shown the anti‐inflammatory and reno‐protective role of angiotensin‐II type‐2 receptor (AT2R) activation under chronic low‐grade inflammatory condition using the obese Zucker rat model. The present study tests the hypothesis that prior activation of AT2R attenuates LPS‐mediated early infiltration of leukocytes, inflammation, and AKI. Mice were pre‐treated with AT2R agonist C21 (0.3 mg/kg), with and without AT2R antagonist PD123319 (5 mg/kg) prior to or concurrently with lipopolysaccharide (LPS, 5 mg/kg) challenge. Pre‐treatment with C21, but not concurrent treatment, significantly prevented the LPS‐induced renal infiltration of CD11b+ leukocytes, increase in the levels of circulating and renal chemotactic cytokines, particularly interleukin‐6 (IL‐6) and markers of renal dysfunction (blood urea nitrogen and albuminuria), while preserving anti‐inflammatory interleukin‐10 (IL‐10) production. Moreover, C21 treatment in the absence of LPS increases renal and circulating IL‐10 levels. To investigate the role of IL‐10 in a cross talk between epithelial cells and monocytes, we performed in vitro conditioned media studies in human kidney proximal tubular epithelial (HK‐2) cells and human monocytes (THP‐1 cells). These studies revealed that the conditioned‐media derived from the C21‐treated HK‐2 cells reduced LPS‐induced THP‐1 TNF‐α production via IL‐10 originating from HK‐2 cells. Our findings suggest that activation of AT2R prior to LPS challenge prevents monocyte infiltration and inflammatory renal injury, possibly via an increase in renal IL‐10 production.Support or Funding InformationThis study was supported by NIH grants R01 DK117495 and R01 DK61578.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vascular Action of Insulin in Pregnancy

Physiological insulin resistance associated with pregnancy is attributed to the increase of anti‐insulin hormones such as placental lactogen and p85 (potent negative regulator of PI3K), human growth hormone, TNFα, as well as adipokines. Besides, AT1 receptor for angiotensin II has a clear role in insulin resistance while the role of AT2 receptors is not well understood. On the other hand, it has been described an increased vascular expression of AT2 receptor during pregnancy. On the other hand, insulin has shown a PI3K‐AKT NO mediated vasodilator effect that may contribute to its metabolic actions. Therefore, the aim of this study was to evaluate if pregnancy decrease in the vasodilatory action of insulin and AT2 receptors participation in this change. Using a conventional isolated organ preparation, the vasodilator effect of cumulative concentrations of insulin was evaluated in rat aortic rings pre‐contracted with phenylephrine. Vasorelaxation of aorta rings was compared at day 14 and day 20 (term) of pregnancy in the presence and absence of the AT2 receptor antagonist PD123319. Pregnancy produced resistance to insulin induced vasodilatation in the thoracic aorta while the vasodilator effect increased in the abdominal aorta only in rings with intact endothelium. AT2 receptors participation on the insulin induced vasodilation was more evident on day 14 (half) gestation. These results suggest that the vascular action of insulin is enhanced by the participation of AT2 receptors for angiotensin II. Also, that both insulin action and AT2 receptor participation during pregnancy depend on the vascular territory studied.Support or Funding InformationSIP Instituto Politécnico Nacional, COFFAAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiontensinergic Receptors in the Medial Amygdaloid Nucleus Is Involved in Anxiogenic‐Like Effect Evoked by Emotional Stress in Rats

The medial amygdaloid nucleus (MeA) is part of limbic circuit implicated in responses to stress. Besides, angiotensinergic neurotransmission has been described as a prominent neurochemical mechanism involved in stress‐evoked physiological and behavioral responses. However, the involvement of angiotensinergic neurotransmission within the MeA in behavioral responses evoked by exposure to aversive threats has never been investigated. Therefore, the aim of this study was to investigate the involvement of angiotensinergic neurotransmission within the MeA in anxiogenic‐like effect evoked by an acute session of restraint stress in rats. For this, male Wistar rats (240g–260g) had cannula‐guide bilaterally implanted into the MeA. The restraint stress was performed by placing the animals in a plastic cylindrical tube for 60 minutes. Independent group of animals received bilateral microinjections into the MeA of angiotensin II (0.05nmol/100nL), the selective AT1receptor antagonist losartan (1nmol/100nL), the selective AT2receptor antagonist PD123319 (0.05nmol/100nL), angiotensin 1–7 (0.05nmol/100nL), the selective Mas receptor antagonist A‐779 (0.1nmol/100nL) or vehicle (saline, 100nl)10 min before the onset of the restraint stress session. Immediately after the end of the restraint session, the anxiety‐like behavior was assessed in the elevated plus maze (EPM). Animals of naïve group (non‐stressed) were tested in the EPM together with stressed animals. We identified that restraint stress decreased the percentage of time spent (F(6,63)=7.98; P<0.0001) and number of entries (F(6,63)=7.40; P<0.0001) in the open arms of the EPM in the vehicle‐treated animals, when compared with naïve animals. Restraint did not affect the number of entries in the enclosed arms (F(6,63)=0.30; P>0.05) and the time spent in the center (F(6,63)=0.85; P>0.05). MeA treatment with either losartan, angiotensin II or angiotensin 1–7 inhibited the restraint‐evoked decrease in the percentage of time spent (P<0.05) and number of entries (P<0.05) in the open arms. Microinjection of either PD123319 (time: P<0.001; entries: P<0.0007) or A‐779 (time: P<0.008; entries: P<0.01) did not affect the decrease in the exploration of the EPM open arms evoked by restraint stress. In conclusion, our results indicate that AT1receptors within the MeA is involved in anxiogenic‐like effect of emotional stress. Besides, present data suggest that activation of the Mas receptor within the MeA inhibits this behavioral response.Support or Funding InformationFAPESP, CNPq and PADC‐FCF/UNESPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiotensin II Type 2 Receptor and Receptor Mas Are Colocalized and Functionally Interdependent in Obese Zucker Rat Kidney.

The actions of angiotensin II type 2 receptor (AT2R) and the receptor Mas (MasR) are complex but show similar pronatriuretic function; particularly, AT2R expression and natriuretic function are enhanced in obese/diabetic rat kidney. In light of some reports suggesting a potential positive interaction between these receptors, we tested hypothesis that renal AT2R and MasR physically interact and are interdependent to stimulate cell signaling and promote natriuresis in obese rats. We found that infusion of AT2R agonist C21 in obese Zucker rats (OZR) increased urine flow and urinary Na excretion which were attenuated by simultaneous infusion of the AT2R antagonist PD123319 or the MasR antagonist A-779. Similarly, infusion of MasR agonist Ang-(1-7) in OZR increased urine flow and urinary Na excretion, which were attenuated by simultaneous infusion of A-779 or PD123319. Experiment in isolated renal proximal tubules of OZR revealed that both the agonists C21 and Ang-(1-7) stimulated NO which was blocked by either of the receptor antagonists. Dual labeling of AT2R and MasR in OZR kidney sections and human proximal tubule epithelial cells showed that AT2R and MasR are colocalized. The AT2R also coimmunoprecipitated with MasR in cortical homogenate of OZR. Immunoblotting of cortical homogenate cross-linked with zero-length oxidative (sulfhydryl groups) cross-linker cupric-phenanthroline revealed a shift of AT2R and MasR bands upward with overlapping migration for their complexes which were sensitive to the reducing β-mercaptoethanol, suggesting involvement of -SH groups in cross-linking. Collectively, the study reveals that AT2R and MasR are colocalized and functionally interdependent in terms of stimulating NO and promoting diuretic/natriuretic response.

Diminazene aceturate prevents nephropathy by increasing glomerular ACE2 and AT2 receptor expression in a rat model of type1 diabetes.

One of the protective actions of angiotensin converting enzyme-2 (ACE2) is the inactivation of angiotensin II. Expression and activity of ACE2 was reduced in glomeruli of diabetic patients and in animal models of diabetes. Recently the potential role of recombinant ACE2 administration in preventing diabetic nephropathy (DN) has been shown. Here we have tested the effects of the ACE2 activator, diminazene aceturate (DIZE), in a model of DN. Male Wistar rats were rendered diabetic using a single dose of streptozotocin (55mg·kg-1 , i.p.). After 4weeks, diabetic animals were divided into experimental groups and treated with DIZE, at a low dose (5mg·kg-1 ·day-1 ), a high dose (15mg·kg-1 ·day-1 ) and the high dose with of the AT2 receptor antagonist PD123319 (10mg·kg-1 ·day-1 ). At the end of the treatment , kidneys from all the groups were collected and processed separately for glomerular isolation, protein isolation, mRNA extraction and for immunohistochemical studies. Treatment with DIZE restored ACE2 expression in glomeruli and increased expression of AT2 receptors in whole kidney and isolated glomeruli of diabetic animals. DIZE administration reduced angiotensin II levels and increased angiotensin-(1-7) levels in diabetic kidney. However, PD123319 treatment reversed all these actions of DIZE. DIZE treatment reduced diabetes-induced renal damage as shown by reduction of fibrosis and apoptosis. These protective actions of DIZE were blocked by the AT2 receptor antagonist. Taken together, these results suggest that DIZE protected against DN through the ACE2/angiotensin-(1-7)/ AT2 receptor axis.

Angiotensin II-AT1–receptor signaling is necessary for cyclooxygenase-2–dependent postnatal nephron generation

Deletion of cyclooxygenase-2 (COX-2) causes impairment of postnatal kidney development. Here we tested whether the renin angiotensin system contributes to COX-2-dependent nephrogenesis in mice after birth and whether arescue of impaired renal development and function in COX-2-/- mice was achievable. Plasma renin concentration in mouse pups showed a birth peak and a second peak around day P8 during the first 10 days post birth. Administration of the angiotensin II receptor AT1 antagonist telmisartan from dayP1 to P3 did not result in cortical damage. However, telmisartan treatment from day P3 to P8, the critical time frame of renal COX-2 expression, led to hypoplastic glomeruli, a thinned subcapsular cortex and maturational arrest of superficial glomeruli quite similar to that observed in COX-2-/- mice. In contrast, AT2 receptor antagonist PD123319 was without any effect on renal development. Inhibition of the renin angiotensin system by aliskiren and enalapril caused similar glomerular defects as telmisartan. Administration of the AT1 receptor agonist L162313 to COX-2-/- pups improved kidney growth, ameliorated renal defects, but had no beneficial effect on reduced cortical mass. L162313 rescued impaired renal function by reducing serum urea and creatinine and mitigated pathologic albumin excretion. Moreover, glomerulosclerosis in the kidneys ofCOX-2-/- mice was reduced. Thus, angiotensin II-AT1-receptor signaling is necessary for COX-2-dependent normal postnatal nephrogenesis and maturation.

Angiotensin Ⅱ induces the differentiation of mouse epicardial progenitor cells into vascular smooth muscle-like cells

Epicardial progenitor cells (EpiCs) have a crucial role in cardiac development and vasculature formation. Here we detected the expression of Angiotensin II (Ang II) receptors AT1 and AT2 on EpiCs and demonstrated that AngII could increase the expression of smooth muscle specific markers, including α-smooth muscle actin (α-SMA) and myosin heavy chain 11 (Myh11) in EpiCs. Moreover, the expression of α-SMA and Myh11 induced by Ang II was blocked by pretreatment of EpiCs with the AT1 receptor antagonist losartan, but not the AT2 receptor antagonist PD123319. We further showed that the AngII-induced cells showed significant contractile responses to carbachol. These results implied that AngII could effectively induce EpiCs to differentiate into vascular smooth muscle-like cells through the AT1 receptor.

Mitochondrial-dependent mechanisms are involved in angiotensin II-induced apoptosis in dopaminergic neurons.

Introduction:We recently demonstrated that angiotensin II (Ang II) was involved in the etiology of Parkinson’s disease (PD) via induction of apoptosis of dopaminergic neurons, but the mechanisms are not completely elucidated. Here, we asked whether mitochondrial-dependent mechanisms contributed to the Ang II-induced dopaminergic neuronal apoptosis.Materials and methods:CATH.a cells were incubated with Ang II in combination with mitochondrial permeability transition pore (mPTP) inhibitors or angiotensin receptor antagonists, and apoptosis rate, caspase-3 activity, cytochrome c levels, and mPTP opening were assessed.Results:We showed that Ang II triggered apoptosis via a mitochondrial-dependent pathway, as elevated cytochrome c levels were observed in the cytosol. By employing cyclosporin A and sanglifehrin A, two specific mPTP inhibitors, we revealed that cytochrome c release from mitochondria into cytoplasm was ascribed to mPTP opening. Meanwhile, the aforementioned effects could be abrogated by an AT1 receptor antagonist losartan rather than an AT2 receptor antagonist PD123319.Conclusion:This study demonstrates that Ang II triggers mitochondrial-dependent apoptosis via facilitating mPTP opening through an AT1 receptor-mediated fashion in dopaminergic neurons. These findings give insight into the effect of Ang II in the etiology of PD, and reinforce the application of AT1 receptor antagonists for PD treatment.

PP.35.02] EFFECT OF ANGIOTENSIN RECEPTOR STIMULATION IN THE ROSTRAL VENTROLATERAL MEDULLA ON BLOOD PRESSURE IN NORMOTENSIVE RATS

Objective: There is increasing evidence that glutamate and gamma-aminobutyric acid (GABA) interact within the rostral ventrolateral medulla (RVLM) modulating the central regulation of blood pressure and sympathetic tone. Angiotensin II (Ang II) is the most important effector in the renin-angiotensin-aldosterone system (RAAS) mediating its actions through the angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R). The AT2R is part of the protective arm of the RAAS and mediates opposing effects counterbalancing the AT1R mediated actions. The selective and specific AT2R agonist Compound 21 (C21) is a useful research tool to investigate the AT2R mediated effects. Design and method: In the present study we assess through local stimulation and blockade of AT1R and AT2R the possible role of the brain RAAS at the level of the RVLM on the regulation of blood pressure. In vivo microdialysis, for measurement of extracellular glutamate and GABA levels, was carried out in the RVLM of conscious normotensive Wistar rats while locally infusing C21, Ang II, and PD123319. Glutamate and GABA levels were quantified by HPLC. Subsequently, the effect of central administration of C21, Ang II, and PD123319 on mean arterial pressure was assessed with a pressure transducer for continuous monitoring. Results: Infusion of Ang II into the RVLM significantly increased glutamate levels (P < 0.05) and significantly decreased GABA levels (P < 0.01). Infusion of the AT2R agonist C21 (0.05 μg/μl/h) tended to decrease glutamate and increase GABA levels, while absolutely no change was seen when co-infusing C21 together with the AT2 receptor antagonist PD123319. C21 infusion (0.05 μg/μl/h) significantly lowered blood pressure (P < 0.01). These responses were abolished by co-administration of C21 and PD123319. No changes in neurotransmitter levels or effects on blood pressure were seen with PD123319 infusion alone. Conclusions: These data demonstrate that acute specific stimulation of AT2R in the RVLM by local C21 infusion reduces blood pressure in normotensive rats, and suggests that decreases in local glutamate and increases in GABA levels may be involved in the sympatho-inhibitory action underlying this hypotensive response.

Abstract P184: Co-localization and Natriuretic Interdependence of Angiotensin AT2R and MasR in Obese Rat Kidney

The actions of angiotensin II type 2 receptor (AT 2 R) and receptor mas (MasR) are complex but show similar pro-natriuretic function; particularly AT 2 R expression and natriuretic function are enhanced in obese/diabetic rat kidney. In light of previous reports, we tested hypothesis that AT 2 R and MasR are interdependent to produce natriuresis in obese rats due to potential physical interaction. Infusion of AT 2 R agonist C21 (5 μg/kg/min) in obese Zucker rats (OZR) caused diuresis/natriuresis which were attenuated by simultaneous infusion of the AT 2 R antagonist PD123319 (50 μg/kg/min) or the MasR antagonist A-779 (50 μg/kg/min). Similarly, infusion of MasR agonist Ang-(1-7) (110 fmol/kg/min) in OZR caused diuresis/netriuresis, which were attenuated by simultaneous infusion of A-779 or PD123319. Dual labeling of AT 2 R and MasR in OZR kidney slices revealed four-fold co-localization of AT 2 R and MasR (9.83 vs. 2.50 dual labeled cells/1600 μm 2 ) compared with lean rats in which AT 2 R is not natriuretic. Moreover, the AT 2 R co-immunoprecipitates with MasR in cortical homogenate of OZR. Immunoblotting of AT 2 R and MasR with zero length oxidative (sulfhydryl groups) cross-linker cupric-phenanthroline in OZR cortical homogenate revealed a shift of AT 2 R (~62 kDa) and MasR (~54 kDa) bands upward with overlapping migration for their complexes (~160 kDa and 245 kDa) which were sensitive to the reducing β-mercaptoethanol. Similar observations were made in HK-2 cells, where glucose (25 mM) treatment enhanced the crosslinking. Collectively, the study reveals AT2R and MasR are co-localized and functionally interdependent in producing natriuretic response. Hyperglycemic oxidative stress affecting sulfhydryl groups present a potential mechanism of such physical interaction between these receptors. (Support: R01DK061578)