AT1A Receptor Research Articles

Impact of High K Intake on Thiazide‐Sensitive Cation Excretion in Male and Female Wild‐type and AT1a Receptor Knockout Mice

Proximal tubule Na reabsorption is a critical determinant of both fluid flow and Na+ delivery to the distal nephron. Beyond that, perturbation of transport by the thiazide‐sensitive Na‐Cl co‐transporter (NCC), within the apical membrane of distal tubule, alters Na+ availability to the Ksecretory machinery of the connecting segment and collecting duct. Potassium intake blunts both proximal Na+ reabsorption via effects on the Na/H‐exchanger (NHE3) and also distal Na+ reabsorption via decreased NCC expression and function. Angiotensin II (Ang II) also impacts both proximal and distal nephron segments, mainly through the AT1a receptor, but its effect is to enhance Na+ reabsorption. Previously we reported that higher NCC expression and activity in female wild‐type (WT) mice, and these gender‐specific differences were absent in AT1aknockout (KO) mice. We have now studied the gender differences in response to high K (HK) intake in WT and AT1a KO mice. In the current work, renal clearance experiments were performed to examine NCC activity on mice fed with control (CK) and HK (5% KCl, 7 days) diets. Hydrochlorothiazide (HCTZ, 30mg/kg iv)‐induced changes in urine volume (UV), glomerular filtration rate (GFR), absolute (ENa, EK) and fractional (FENa, FEK) excretion were measured and compared between male and female WT and AT1a KO mice. HK induced changes in NCC and Na/H‐exchanger isoform 3 (NHE3) expressions were examined by Western blotting. In WT animals, under control diet, HCTZ‐induced cation excretion is greater in females, than in males (ENa+EK was 7.2±1.1 vs. 3.5±0.6 μEq/min/100gBW, P<0.05). This reflects a larger increase in Na+ excretion, as there is little gender difference in HCTZ‐induced K+ excretion (EK/ENa+EK was 20±2 in female and 28±3% in male, P>0.05). Under HK diet, the gender difference in HCTZ‐induced cation excretion is reduced (ENa+EK was 7.0±0.7 in female and 5.2±0.3 μEq/min/100gBW in male P<0.05) accompanied by a larger increment in K+ excretion in males (EK/ENa+EK was 57±5 in male and 36±4% in female P<0.05). With AT1a KO, male mice tend to resemble female mice, with respect to both HCTZ‐induced Na+ and K+ excretion. In turn, the female KO mice are similar to their WT controls. Both NCC and NHE3 transporter abundance were significantly reduced by HK in all groups of mice. In summary, compared with female mice, males appear to rely on greater proximal tubule Na+ reabsorption, showing reduced thiazide‐dependent Na+ excretion. Under control conditions, comparable HCTZ‐dependent K+ excretion by WT males and females – despite reduced Na+ excretion in males ‐ suggests greater sensitivity of K+ secretion to changes in Na+ delivery in the males. HK diet likely increases distal Na+ delivery in both genders, so that HCTZ‐dependent cation excretion is maintained despite reduced NCC abundance. In males, a larger fraction of Na+ escaping the DCT is exchanged for K+, leading to less Na+ excretion and greater K+ excretion than in females. In AT1a KO mice, gender differences in thiazide effect are largely erased, suggesting a role for Ang II in modulating these processes.Support or Funding InformationNIH/NIDDK, RO1 DK099284 and P01 DK 17433This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Whole body sodium depletion modifies AT1 mRNA expression and serotonin content in the dorsal raphe nucleus.

Angiotensin II (Ang II) acts on Ang II type 1 (AT1) receptors located in the organum vasculosum and subfornical organ (SFO) of the lamina terminalis as a main facilitatory mechanism of sodium appetite. The brain serotonin (5-HT) system with soma located in the dorsal raphe nucleus (DRN) provides a main inhibitory mechanism. In the present study, we first investigated the existence of Ang II AT1 receptors in serotonergic DRN neurones. Then, we examined whether whole body sodium depletion affects the gene expression of the AT1a receptor subtype and the presumed functional significance of AT1 receptors. Using confocal microscopy, we found that tryptophan hydroxylase-2 and serotonin neurones express AT1 receptors in the DRN. Immunofluorescence quantification showed a significant reduction in 5-HT content but no change in AT1 receptor expression or AT1/5-HT colocalisation in the DRN after sodium depletion. Whole body sodium depletion also significantly increased Agtr1a mRNA expression in the SFO and DRN. Oral treatment with the AT1 receptor antagonist losartan reversed the changes in Agtr1a expression in the SFO but not the DRN. Losartan injection into either the DRN or the mesencephalic aqueduct had no influence on sodium depletion-induced 0.3molL-1 NaCl intake. The results indicate the expression of Agtr1a mRNA in the DRN and SFO as a marker of sodium depletion. They also suggest that serotonergic DRN neurones are targets for Ang II. However, the function of their AT1 receptors remains elusive.

NFAT5 regulates renal gene expression in response to angiotensin II through Annexin-A2-mediated posttranscriptional regulation in hypertensive rats

The aim was to identify new targets that regulate gene expression at the posttranscriptional level in angiotensin II (ANGII)-mediated hypertension. Heparin affinity chromatography was used to enrich nucleic acid-binding proteins from kidneys of two-kidney, one-clip (2K1C) hypertensive Wistar rats. The experiment was repeated with 14-day ANGII infusion using Alzet osmotic mini pumps, with or without ANGII receptor AT1a inhibition using losartan in the drinking water. Mean arterial pressure increased after 2K1C or ANGII infusion and was inhibited with losartan. Heparin affinity chromatography and mass spectrometry were used to identify Annexin-A2 (ANXA2) as having differential nucleic acid-binding activity. Total Annexin-A2 protein expression was unchanged, whereas nucleic acid-binding activity was increased in both kidneys of 2K1C and after ANGII infusion through AT1a stimulation. Costaining of Annexin-A2 with α-smooth muscle actin and aquaporin 2 showed prominent expression in the endothelia of larger arteries and the cells of the inner medullary collecting duct. The nuclear factor of activated T cells (NFAT) transcription factor was identified as a likely Annexin-A2 target using enrichment analysis on a 2K1C microarray data set and identifying several binding sites in the regulatory region of the mRNA. Expression analysis showed that ANGII increases NFAT5 protein but not mRNA level and, thus, indicated that NFAT5 is regulated by posttranscriptional regulation, which correlates with activation of the RNA-binding protein Annexin-A2. In conclusion, we show that ANGII increases Annexin-A2 nucleic acid-binding activity that correlates with elevated protein levels of the NFAT5 transcription factor. NFAT signaling appears to be a major contributor to renal gene regulation in high-renin states.

Distinct roles of angiotensin receptors in autonomic dysreflexia following high-level spinal cord injury in mice

Autonomic dysreflexia (AD), a syndrome caused by loss of supraspinal control over sympathetic activity and amplified vascular reflex upon sensory stimuli below injury level, is a major health problem in high-level spinal cord injury (SCI). After supraspinal sympathetic control of the vasculature below the lesion is lost, the renin-angiotensin system (RAS) is thought to be involved in AD by regulating blood pressure and vascular reactivity. In this study, we aimed to assess the role of different RAS receptors during AD following SCI. Therefore, we induced AD by colorectal distention (CRD) in wild-type mice and mice deficient in the RAS components angiotensin (Ang) II type 1a receptor (AT1a) (Agtr1a−/−) and Ang-(1–7) receptor Mas (Mas−/−) four weeks after complete transection of spinal cord at thoracic level 4 (T4). Systemic blood pressure measurements and wire myography technique were performed to assess hemodynamics and the reactivity of peripheral arteries, respectively. CRD increased mean arterial blood pressure (MAP) and decreased heart rate (HR) in all three animal groups. However, we found less increases in MAP in Mas−/− mice compared to control mice after CRD, whereas AT1a deficiency did not affect the hemodynamic response. We found that the reactivity of wild-type and Mas−/− mesenteric arteries, which are innervated from ganglia distal but close to thoracic level T4, was diminished in response to Ang II in AD after T4-SCI, but this difference was not observed in the absence of AT1a receptors. CRD did not influence the reactivity of femoral arteries which are innervated from ganglia more distal to thoracic level T4, in response to Ang II in AD. In conclusion, we identified a specific role of the Ang-(1–7) receptor Mas in regulating the systemic blood pressure increase in AD in T4-SCI mice. Furthermore, AT1a signaling is not involved in this hemodynamic response, but underlies increased vascular reactivity in mesenteric arteries in response to Ang II, where it may contribute to adaptive changes in regional blood flow.

A18558 Proximal tubule-specific deletion of AT1a receptors in the kidney attenuates angiotensin II-induced hypertension

Objectives:To test the hypothesis that proximal tubule-specific deletion of angiotensin II (ANG II) AT1a receptors in the kidney attenuates ANG II-induced hypertension in mice, and that this effect involves superoxide.Methods:Proximal tubule-specific AT1a receptor-knockout mice (PT-AT1a-KO) were gen

A15268 Genetic deletion of AT1a Receptor selectively in the proximal tubule of the kidney attenuates renal ischemia-reperfusion injury and angiotensin II-induced hypertension in mice

Objectives: To test the hypothesis that genetic deletion of angiotensin II (ANG II) AT1a receptors selectively in the proximal tubule of the kidney attenuates renal ischemia-reperfusion injury and ANG II-induced hypertension in mice. Methods: Proximal tubule-specific AT1a receptor-knockout mice (PT-AT1a-KO) were generated using the standard Cre/LoxP recombination approach. Adult male C57BL/6J (WT), global AT1a-KO, and PT-AT1a-KO mice were subjected to sham surgery or 45 min bilateral renal ischemia, followed by reperfusion for 24 h or 7 days (n = 8–12 per group). ANG II-dependent hypertension was induced by infusing ANG II at 1.5 mg/kg/day, i.p., for 2 weeks. Systolic blood pressure (SBP), renal function, glomerular, tubuointerstital, and perivascular injury were determined and compared, respectively. Results: Basal systolic blood pressure was 22 ± 5 mmHg lower in global AT1a-KO or 11 ± 3 mmHg lower in PT-AT1a-KO mice (p < 0.05 vs. WT). WT mice developed significant glomerular, cortical tubulointerstitial and perivascular injury 24 h or 7 days after renal ischemia-reperfusion was induced (p < 0.01). Ischemia and reperfusion renal injury was not prevented in global AT1a-KO mice (n.s. vs. WT), but it was significantly attenuated in PT-AT1a-KO mice 24 h or 7 days after renal ischemia-reperfusion was induced (p < 0.05 vs. WT or AT1a-KO). WT mice developed severe ANG II-induced hypertension, as expected, but it was significantly attenuated in PT-AT1a-KO mice (p < 0.01 vs. WT). Conclusion: AT1a receptors in the proximal tubule of the kidney play a key role in the pathogenesis of renal ischemia-reperfusion injury and ANG II-dependent Hypertension (supported by 2R01DK067299 and 2R01DK102429 to Zhuo, and by National Natural Science Foundation of China #81360290 and Guangxi Natural Science Foundation #2014GXNSFAA118195 to Zhang).

NF-κB inhibitor on Toll-like receptor 4 signal-induced expression of angiotensinogen and AT1a receptor in neonatal rat left ventricular myocytes.

Effects of toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway on expression of angiotensinogen and AT1a receptor were investigated, to explore the role of TLR4/NF-κB signaling pathway in cardiovascular disease. Neonatal rat left ventricular myocytes (NRVMs) were cultured and cardiomyocytes were identified by immunocytochemical staining of sarcomeric α-actin. NRVMs were treated with lipopolysaccharide (LPS) at a dose of 10, 100 and 1,000 ng/ml, and RT-PCR was performed 24 h later to detect the expression of TLR4, angiotensinogen (ATG) and AT1a at mRNA level. NRVMs were cultured and pretreated with caffeic acid phenethylester (CAPE) for 30 min. Then NRVMs were stimulated with LPS (1,000 ng/ml) for 24 h. Nuclear translocation of NF-κB p65 was detected by immunocytochemistry. Expression of TLR4, angiotensinogen and AT1a receptor after CAPE stimulation was detected by RT-PCR. TLR4 mRNA was highly expressed in in vitro cultured NRVMs, and the expression level was significantly increased by LPS (10–1,000 ng/ml) stimulation in a dose-dependent manner (P<0.05). LPS stimulation also significantly increased the expression levels of angiotensinogen and AT1a receptor in a dose-dependent manner (P<0.05). NF-κB was activated and nuclear translocation of NF-κB p65 occurred after stimulation with LPS (1,000 ng/ml) for 24 h, while CAPE (20 µg/ml) inhibited the nuclear translocation of NF-κB p65 and inhibited LPS-induced expression of angiotensinogen and AT1a receptor. With LPS stimulation, TLR4 signaling positively regulates the expression of TLR4 and upregulates the expression of angiotensinogen and AT1a receptor in NRVMs. CAPE, an inhibitor of NF-κB, inhibited NF-κB p65 activation and inhibited the upregulation of TLR4, angiotensinogen and AT1a receptors induced by LPS. These results suggest that NF-κB plays a key regulatory role in the above-mentioned effects induced by LPS. Intervention with TLR4/NF-κB signaling may become a new target for prevention and treatment of cardiovascular diseases.

Abstract P324: Role of GABAergic Signaling in AgRP Neurons in the Dipsogenic and Metabolic Responses to Brain Angiotensin

Obesity is a major risk factor for development of hypertension, and evidence supports a role for the suppression of resting metabolic rate (RMR) in the development and maintenance of obesity. Angiotensin II (ANG) AT1A receptors, localized to cells expressing the leptin receptor and Agouti-related peptide (AgRP) within the arcuate nucleus of the hypothalamus (ARC), are involved in transcriptional control of AgRP and γ-aminobutyric acid (GABA) synthetic genes ( Gad65 , Gad67 , Slc32a1 ) within the ARC, as well as the integrative control of thermogenic sympathetic nerve activity (SNA) and RMR responses in vivo to various stimuli including leptin and high fat diet. These data led us to hypothesize that AT1A functions to suppress GABA signaling in AgRP neurons, ultimately to disinhibit SNA and RMR. Treatment of cultured mouse hypothalamic N43/5 cells (which express endogenous Agtr1a , AgRP and Gad67 ) with 100 nM ANG for 1 hour (n=6) resulted in reduced AgRP (0.57-fold of control, p=0.03) and Gad67 (0.72-fold of control, p=0.09) mRNA. Mice with targeted disruption of Slc32a1 (vesicular GABA transporter; VGAT) in AgRP neurons (VGAT AgRP-KO mice) were generated on a mixed background. Compared to littermates (male n=9-17, female n=7-8), VGAT AgRP-KO mice (male n=8-13, female n=2-3) at 8 weeks of age exhibited normal body mass (genotype p=ns, sex p&lt;0.05, interaction p=ns), no changes in food intake (genotype p=ns, sex p=ns, interaction p=ns), but a sex-dependent genotype effect on fluid intake was observed (male con 4.5±0.3 vs KO 5.1±0.4; fem con 5.1±0.4 vs KO 3.7±0.6 mL/d; interaction p&lt;0.05). A preliminary study of male mice instrumented for chronic infusion of ANG (5 ng/hr, 21 days icv) was also performed. This caused modest increases in drinking (con+aCSF n=7, 3.3±0.8, con+ANG n=4, 5.2±1.0, KO+ANG n=3, 7.9±1.1 mL/d, p&lt;0.05) but no change in energy efficiency (11.2±2.9, 11.3±3.5, 10.1±4.1 mg/kcal), indicating no change in energy expenditure, possibly due to the mixed background of this strain. We conclude that ANG acts at AgRP cells of the ARC to suppress GABA and AgRP signaling, and this contributes to energy and fluid homeostasis in a complex, strain- and sex-dependent manner.

Alteration at transcriptional level of cardiac renin–angiotensin system by letrozole treatment

Introduction: The use of aromatase inhibitors (AIs) for breast cancer led to a marked change in ventricular function. Since accumulating evidence indicates that overactivation of the cardiac renin-angiotensin system (RAS) plays an important role in the development of cardiovascular diseases such as hypertrophy and remodelling, we aimed to investigate whether letrozole alters the transcription level of RAS related genes in the cardiac tissue.Methods: Twenty four rats were randomly divided into four groups (n = 6 per group): two groups were letrozole treated (1 and 2 mg/kg/day orally), one group was vehicle treated (DMSO) and one group was the control group without any treatment. 12 weeks after beginning treatment with letrozole, we examined the rate of transcription of renin, angiotensinogen, AngII type 1a and 1b (AT1a and AT1b) and type 2 receptors (AT2) in the rat heart using real-time polymerase chain reaction.Results: The cardiac mRNA levels of several components of the RAS in the rats treated with letrozole were significantly increased including AT1a receptor (80%), renin (51%), and angiotensinogen (33%). Though not significant, AT2 receptor levels were observed to decrease with increasing doses of letrozole.Conclusions: Letrozole can induce significant changes in some RAS related genes. These alterations are important to understand the pathways and consequences beyond cardiac events induced by breast cancer treatments.

AT1A Receptors on Vasopressin‐Producing Cells are Important for Vasopressin Secretion but not Blood Pressure Responses to Chronic Intracerebroventricular Angiotensin in Mice

Vasopressin (AVP) plays an important role in cardiometabolic physiology by elevating blood pressure and water reabsorption. This hormone is produced in the magnocellular neurosecretory cells (MNCs) within the supraoptic nucleus (SON) and periventricular nucleus (PVN) of the hypothalamus. Angiotensin II (ANG) has been shown to act at AT1A receptors in the brain and cause AVP‐dependent elevations in blood pressure. Critically, the contribution of AT1A receptors specifically on AVP‐producing cells to blood pressure remains unclear. We hypothesized that AT1A receptor knockout only from AVP‐expressing cells would prevent blood pressure elevations in response to chronic intracerebroventricular (ICV) ANG infusion. Mice with AT1A knockout from AVP‐producing cells (AT1AAVP‐KO) were generated by breeding AVP‐Cre mice with AT1Aflox mice. Subcutaneous osmotic mini‐pumps were used to infuse artificial cerebrospinal fluid (aCSF) or ANG (50 ng/hr) into the lateral cerebral ventricle. Blood pressure was measured daily by tail‐cuff plethysmography for three weeks before and two weeks following implantation surgery. Water intake and urine output were measured using metabolic cages. Baseline systolic blood pressure (SBP) was indistinguishable among groups (Control aCSF 113±2, Control ANG 112±2, AT1AAVP‐KO aCSF 114±3, AT1AAVP‐KO ANG 113±2 mmHg; p=ns). ANG increased SBP similarly in control and KO mice (Control aCSF 107±3, Control ANG 121±2, AT1AAVP‐KO aCSF 111±2, AT1AAVP‐KO ANG 119±4 mmHg; ANG p&lt;0.05, genotype p=ns, interaction p=ns). Water intake (Control aCSF 2.8±0.3, Control ANG 13.3±3.3, AT1AAVP‐KO aCSF 2.2±0.3, AT1AAVP‐KO ANG 11.3±2.9 mL/day; ANG p&lt;0.05, genotype p=ns, interaction p=ns) and urine output (Control aCSF 0.7±0.2, Control ANG 7.7±2.6, AT1AAVP‐KO aCSF 0.4±0.1, AT1AAVP‐KO ANG 6.4±1.9 mg/day; ANG p&lt;0.05, genotype p=ns, interaction p=ns) were also increased by ANG but not different between genotypes. Interestingly, urine copeptin was elevated by ANG in controls but not AT1AAVP‐KO mice (Control aCSF 27.6±6.1, Control ANG 137.9±35.6, AT1AAVP‐KO aCSF 78.3±15.7, AT1AAVP‐KO ANG 60.0±15.7 mL/day; ANG p&lt;0.05, genotype p=ns, interaction p&lt;0.05). For all studies, Control aCSF n=14, Control ANG n=7, AT1AAVP‐KO aCSF n=14, AT1AAVP‐KO ANG n=10. These data support the conclusion that AT1A receptors on AVP‐expressing cells are important for the control of AVP secretion in response to ICV ANG, but that this specific sub‐population of receptors are of minor importance for SBP responses to ICV ANG.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AT1aR Dependent GABAa Inhibition in the MnPO Following Chronic Intermittent Hypoxia

Chronic intermittent hypoxia (CIH) is an animal model that simulates the hypoxemia seen in obstructive sleep apnea (OSA). Rats exposed to CIH exhibit an increase in sympathetic tone and reactivity and a persistent elevation in blood pressure during periods of normoxia, similar to that observed in patients with OSA. The median preoptic nucleus (MnPO) exhibits increases in Angiotensin type 1a receptor (AT1aR) mRNA following CIH and blocking this increase in AT1aR mRNA prevents the sustained increase in blood pressure. Our lab has previously reported that AT1aR activation influences GABAa mediated inhibition within the MnPO. Here we investigate the role of Angiotensin II (Ang II) signaling via the AT1a receptor in the MnPO and its influence on excitatory/inhibitory balance in rats subjected to CIH. Under isoflurane (2–3%) anesthesia, male Sprague‐Dawley rats (250–350 g) received microinfusions (0.4 μL) of recombinant AAV construct containing GFP reporter and shRNA against AT1aR (AT1a KD) or an AAV construct containing the GFP reporter and a shRNA scramble (Scr) targeted to the MnPO. After recovery, rats were subjected to 7 days of CIH (0800–1600 hrs). The CIH protocol consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10×/hr for 8 consecutive hrs (during the normal inactive/sleep phase) on 7 consecutive days. After 7 days CIH, the rats were anesthetized with isoflurane (2–3%) and coronal slices (300 μm) containing the MnPO were cut using standard in vitro slice procedures. Loose patch recordings were obtained from GFP labeled neurons using borosilicate glass micropipettes containing aCSF as the internal solution (1–3 MΩ). Spontaneous action potential firing was recorded in response to focal application of Ang II (100 nM, 30s) or muscimol (100 uM, 30s). Brief focal application of Ang II produced a transient increase in spontaneous action potential frequency of MnPO neurons from both CIH/Scr (n = 11) and normoxic/Scr (n = 9) rats. The Ang II dependent increase in activity was blocked in neurons from AT1a KD rats from both CIH (n = 12) and normoxic (n = 10) groups. The GABAa agonist muscimol decreased action potential activity (n = 10) of neurons from normoxic/Scr rats. This GABAa mediated inhibition was blunted in neurons from CIH/Scr (n = 13) and normoxic/AT1a KD (n = 10) rats. However, GABAa activation from neurons in the CIH/AT1a KD (n = 15) group produced an increase in spontaneous activity. The current study shows AT1aR KD mediated reduction in GABAa inhibition is exacerbated to the point where GABAa activation is excitatory following CIH. Future studies will address the influence of reduced AT1a signaling combined with reduced GABAa mediated inhibition on downstream targets of the MnPO in AT1aKD from both normoxic and CIH rats.Support or Funding InformationSupported by P01 HL088052This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lack of contribution of nitric oxide synthase to cholinergic vasodilation in murine renal afferent arterioles.

We have previously reported significant increases in neuronal nitric oxide synthase (NOS) immunostaining in renal arterioles of angiotensin type 1A receptor (AT1A) knockout mice, and in arterioles and macula densa cells of AT1A/AT1B knockout mice. The contribution of nitric oxide derived from endothelial and macula densa cells in the maintenance of afferent arteriolar tone and acetylcholine-induced vasodilation was functionally determined in kidneys of wild-type, AT1A, and AT1A/AT1B knockout mice. Acetylcholine-induced changes in arteriolar diameters of in vitro blood-perfused juxtamedullary nephrons were measured during control conditions, in the presence of the nonspecific NOS inhibitor, Nω-nitro-l-arginine methyl ester (NLA), or the highly selective neuronal NOS inhibitor, N5-(1-imino-3-butenyl)-l-ornithine (VNIO). Acetylcholine (0.1 mM) produced a significant vasoconstriction in afferent arterioles of AT1A/AT1B mice (-10.9 ± 5.1%) and no changes in afferent arteriolar diameters of AT1A knockout mice. NLA (0.01-1 mM) or VNIO (0.01-1 μM) induced significant dose-dependent vasoconstrictions (-19.8 ± 4.0% 1 mM NLA; -7.8 ± 3.5% 1 μM VNIO) in afferent arterioles of kidneys of wild-type mice. VNIO had no effect on afferent arteriole diameters of AT1A knockout or AT1A/AT1B knockout mice, suggesting nonfunctional neuronal nitric oxide synthase. These data indicate that acetylcholine produces a significant renal afferent arteriole vasodilation independently of nitric oxide synthases in wild-type mice. AT1A receptors are essential for the manifestation of renal afferent arteriole responses to neuronal nitric oxide synthase-mediated nitric oxide release.

Blood pressure regulation by the angiotensin type 1 receptor in the proximal tubule.

The renin-angiotensin system (RAS) plays a critical role in the pathogenesis of hypertension. Homeostatic actions of the RAS, such as increasing blood pressure (BP) and vasoconstriction, are mediated via type 1 (AT1) receptors for angiotensin II. All components of the RAS are present in the renal proximal tubule, which reabsorbs the bulk of the glomerular filtrate, making this segment of the nephron a location of great interest for solute handling under RAS influence. This review highlights recent studies that illustrate the key role of renal proximal tubule AT1 receptors in BP regulation. A variety of investigative approaches have demonstrated that angiotensin II signaling via AT1a receptors, specifically in the renal proximal tubule, is a major regulator of BP and sodium homeostasis. Reduction of proximal tubule AT1a receptors led to lower BPs, whereas overexpression generally caused increased BPs. AT1a receptors in the proximal tubule are critical to the regulation of BP by the kidney and the RAS. The pattern of BP modulation is associated with alterations in sodium transporters. As a key site for sodium homeostasis, the renal proximal tubule could hence be a potential target in the treatment of hypertension.

Effects of Ginkgo biloba leaf extract on local renin-angiotensin system through TLR4/NF-κB pathway in cardiac myocyte.

The present study investigated the effects of Ginkgo biloba leaf extract (GBE50) on lipopolysaccharide (LPS) induced Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway and its effects on angiotensinogen (ATG) and AT1a receptor, so as to explore the mechanism of GBE50 in prevention and treatment of left ventricular remodeling. In vitro cultured neonatal rat ventricular myocytes (NRVMs) were divided into 4 groups including i) control group: DMEM medium; ii) LPS group: iii) LPS + GBE50 group; iv) LPS + caffeic acid phenethyl ester (CAPE, specific inhibitor of NF-κB) group. Nuclear translocation of NF-κB p65 was detected by immunocytochemical method after intervention for 24 h. Expression of TLR4, ATG, AT1a receptors and β-myosin heavy chain (β-MHC) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). Protein content of cardiomyocytes was measured by Coomassie Brilliant Blue method. Under LPS stimulation, expression level of TLR4 mRNA in NRVMs was significantly increased (P<0.01), nuclear translocation of NF-κB p65 was increased, expression levels of ATG, AT1a receptor and β-MHC mRNA and the protein content in cells were also increased significantly (P<0.01). GBE50 and CAPE significantly inhibited nuclear translocation of NF-κB p65. GBE50 and CAPE treatments also reduced the increased mRNA levels of TLR4, ATG, AT1a receptor and β-MHC and protein content in cell caused by LPS stimulation. We concluded that, GBE50 can inhibit the activation of local renin-angiotensin system by inhibiting the activation of TLR4/NF-κB and TLR4/NF-κB, signaling pathway inhibition may be one of the mechanisms of the role of Ginkgo biloba leaf extract in preventing myocardial remodeling.

Abstract P413: Vascular AT1 Receptors Control Blood Pressure by Augmenting Peripheral Vascular Resistance in Female Mice

Angiotensin (Ang) II is a major mediator of hypertension pathogenesis and end organ damage. Our understanding of how Ang II elicits these effects on a cellular level continues to evolve. However, the majority of these studies utilize male subjects, despite the awareness that males and females differ in mechanisms of BP regulation. Here we investigated the role of vascular smooth muscle cell (VSMC) AT1A receptors to BP control in female mice. Using Loxp technology and Cre transgenes, we created female mice with cell-specific deletion of AT 1A receptors in smooth muscle cells (SMKO mice). We found that elimination of these receptors led to a significant (8 mmHg) reduction in baseline BP in female SMKO mice (similar to male SMKO mice) compared with controls (108±2 versus 116±1 mmHg; P =0.004) with no effect on modulating sodium sensitivity in female SMKO mice (unlike male SMKO mice in which sodium sensitivity was enhanced). Over a 4-week Ang II infusion, the severity of Ang II - dependent hypertension was minimally affected during the first 2 weeks of Ang II infusion, corresponding to no difference in sodium excretion. However, female SMKO mice had a 35% less BP elevation compared to controls over the final 2 weeks of Ang II infusion (SMKO, 20±2 versus control, 30±3 Change in SBP mmHg; P =0.01). The acute vasoconstrictor responses to Ang II in the systemic vasculature were reduced (by approximately 50-75%) in female SMKO mice (0.1 μg/kg- SMKO, 3.6±1.2 mmHg versus control, 7.3±0.7 mmHg; P =0.01, 0.3 μg/kg- SMKO, 3.3±1.8 mmHg versus control, 12.1 ± 1.3 mmHg; P =0.0008, 1.0 μg/kg- SMKO, 8.5±1.9 mmHg versus control, 19.4±2.0 mmHg; P =0.002). In addition, the acute Ang II reduction in renal tissue perfusion was nearly eliminated in female SMKO mice. In contrast, we previously reported that in male SMKO mice have a much larger (50%) reduction in BP during chronic Ang II infusion associated with increased natriuresis, but relatively preserved vasoconstrictor response (25% reduction) during acute Ang II infusions. These findings suggest that in female mice, direct actions of AT 1A receptors in VSMCs are essential for regulation of basal BP and Ang II-dependent hypertension. However, this effect is mediated by reductions in peripheral vascular resistance rather than sodium retention.

Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα12/13-mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gαq/11-mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways.

Gender difference in kidney electrolyte transport. I. Role of AT1a receptor in thiazide-sensitive Na+-Cl- cotransporter activity and expression in male and female mice.

We studied gender differences in Na+-Cl- cotransporter (NCC) activity and expression in wild-type (WT) and AT1a receptor knockout (KO) mice. In renal clearance experiments, urine volume (UV), glomerular filtration rate, absolute Na+ (ENa) and K+ (EK), and fractional Na+ (FENa) and K+ excretion were measured and compared at peak changes after bolus intravenous injection of hydrochlorothiazide (HCTZ; 30 mg/kg). In WT, females responded more strongly than males to HCTZ, with larger fractional increases of UV (7.8- vs. 3.4-fold), ENa (11.7- vs. 5.7-fold), FENa (7.9- vs. 4.9-fold), and EK (2.8- vs. 1.4-fold). In contrast, there were no gender differences in the responses to the diuretic in KO mice; HCTZ produced greater effects on male KO than on WT but similar effects on females. In WT, total (tNCC) and phosphorylated (pNCC) NCC protein expressions were 1.8- and 4.6-fold higher in females compared with males (P < 0.05), consistent with the larger response to HCTZ. In KO mice, tNCC and pNCC increased significantly in males to levels not different from those in females. There were no gender differences in the expression of the Na+/H+ exchanger (NHE3) in WT; NHE3 protein decreased to similar extents in male and female KO animals, suggesting AT1a-mediated NHE3 expression in proximal tubules. The resulting increase in delivery of NaCl to the distal nephron may underlie increased NCC expression and activity in mice lacking the AT1a receptor.

Perinatal Exposure to Western Diet Programs Autonomic Dysfunction in the Male Offspring.

Although the deleterious influence of protein deficiency on fetal programming is well documented, the impact of a Western diet on epigenetic mechanisms is less clear. We hypothesized that high-fat high-sucrose diet (HFHSD) consumption during pregnancy leads to epigenetic modifications within the progeny's compensatory renin-angiotensin system (RAS), affecting autonomic and metabolic functions. Dams were fed HFHSD (45% fat and 30% sucrose) or regular chow (RD) from mating until weaning of the pups (~7weeks). Offspring from both groups were then maintained on chow and studied in adulthood (3-7months). Offspring from HFHSD-exposed dams (OH) exhibited no difference in body weight or fasting blood glucose compared to controls (OR). In 3-month-old offspring, DNA methylation was significantly lower for the ACE2 gene (P<0.05) in the brainstem, kidney and cecum. Moreover, ACE2 activity in the hypothalamus was increased at 7months (OH: 91±1 vs. OR: 74±4 AFU/mg/min, P<0.05). Although baseline blood pressure was not different between groups, vagal tone in OH was significantly impaired compared to OR. At the same time, OH offspring had a 1.7-fold increase in AT1a receptor expression and a 1.3-fold increase in ADAM17 mRNA. DOCA-salt treatment further revealed and exacerbated hypertensive response in the OH progeny (OH: 130±6 vs. OR: 108±3mmHg, P<0.05). Taken together, our data suggest that perinatal exposure to HFHSD resulted in epigenetic modifications of the compensatory brain RAS, potentially affecting plasticity of neuronal networks leading to autonomic dysfunction in the male offspring.

Resistance to hypertension mediated by intercalated cells of the collecting duct.

The renal collecting duct (CD), as the terminal segment of the nephron, is responsible for the final adjustments to the amount of sodium excreted in urine. While angiotensin II modulates reabsorptive functions of the CD, the contribution of these actions to physiological homeostasis is not clear. To examine this question, we generated mice with cell-specific deletion of AT1A receptors from the CD. Elimination of AT1A receptors from both principal and intercalated cells (CDKO mice) had no effect on blood pressures at baseline or during successive feeding of low- or high-salt diets. In contrast, the severity of hypertension caused by chronic infusion of angiotensin II was paradoxically exaggerated in CDKO mice compared with controls. In wild-type mice, angiotensin II induced robust expression of cyclooxygenase-2 (COX-2) in renal medulla, primarily localized to intercalated cells. Upregulation of COX-2 was diminished in CDKO mice, resulting in reduced generation of vasodilator prostanoids. This impaired expression of COX-2 has physiological consequences, since administration of a specific COX-2 inhibitor to CDKO and control mice during angiotensin II infusion equalized their blood pressures. Stimulation of COX-2 was also triggered by exposure of isolated preparations of medullary CDs to angiotensin II. Deletion of AT1A receptors from principal cells alone did not affect angiotensin II-dependent COX2 stimulation, implicating intercalated cells as the main source of COX2 in this setting. These findings suggest a novel paracrine role for the intercalated cell to attenuate the severity of hypertension. Strategies for preserving or augmenting this pathway may have value for improving the management of hypertension.

AT1A Receptors in AgRP Neurons Disinhibit αMSH Neurotransmission

The brain renin‐angiotensin system (RAS) and leptin contribute to the control of resting metabolic rate (RMR) and their receptors are co‐expressed in areas of the brain critical for metabolic control, supporting the concept that the central interaction between angiotensin and leptin may be critical to regulate RMR and thus obesity. We have previously demonstrated that angiotensin AT1A receptors co‐localize to leptin‐sensitive cells in the arcuate nucleus (ARC), which also express agouti‐related peptide (AgRP). Moreover, mice lacking angiotensin AT1A receptors in AgRP neurons (AT1AAgRP‐KO) exhibit attenuated brown adipose tissue sympathetic nerve activity in response to leptin (4 hrs post‐leptin, control n=4, 221±18 vs AT1AAgRP‐KO n=5, 150±10 % baseline, p&lt;0.05). AT1AAgRP‐KO mice also exhibit suppressed stimulation of RMR in response to exogenous alpha‐melanocyte stimulating hormone (αMSH; control n=13, +0.032±0.005 vs AT1AAgRP‐KO n=9, +0.012±0.005 kcal/hr, p&lt;0.05), leading us to hypothesize that the activation of AT1A receptors in leptin‐sensitive AgRP neurons disinhibits αMSH neurotransmission and subsequent stimulation of RMR via suppression of inhibitory signaling within the ARC. We examined brain punches from the ARC of AT1AAgRP‐KO mice for the expression of AgRP, neuropeptide Y (NPY), and genes involved in gamma‐aminobutyric acid (GABA) signaling, including the glutamate decarboxylase enzymes, GAD1 and GAD2, and the vesicular GABA transporter VGAT, in response to selective ablation of the AT1A receptor. AT1AAgRP‐KO mice (n=5) exhibited significant (p&lt;0.05) inductions in AgRP (10.3‐fold), NPY (4.8‐fold), GAD1 (1.9‐fold), GAD2 (1.5‐fold) and VGAT (6.9‐fold) in the ARC compared to control littermates (n=10). Taken together, these data support a role for AT1A receptors within AgRP neurons of the ARC in the control of BAT SNA and RMR through suppression of inhibitory AgRP/NPY/GABA signaling and thus disinhibition of stimulatory αMSH neurotransmission.