Acute Angiotensin II Research Articles

Angiotensin II-Type-1a Receptor and Renal K + Wasting during Overnight Low-Na + Intake.

Chronic Angiotensin-II (Ang-II) perfusion stimulates Kir4.1/Kir5.1 of the DCT via angiotensin-II-type-1a-receptor (AT1aR) and low-sodium-intake also stimulates Kir4.1/Kir5.1. However, it is not explored the role of AT1aR in mediating the effect of LS on Kir4.1/Kir5.1. We used patch-clamp-technique to examine Kir4.1/Kir5.1 activity of the DCT, employed immunoblotting to examine NCC expression/activity, and used in vivo perfusion-technique to measure renal-Na+ and renal-K+-excretion in control, kidney-tubule-specific-AT1aR-knockout (Ks-AT1aR-KO) and DCT-specific-AT1aR-knockout mice (DCT-AT1aR- KO). Ang-II acutely stimulated 40-pS-K+ channel (Kir4.1/Kir5.1-heterotetramer), increased whole-cell Kir4.1/Kir5.1-mediated K+-currents and the negativity of DCT-membrane-potential only in late-DCT2 but not in early-DCT. Acute Ang-II increased thiazide-induced renal Na+-excretion (ENa). The effect of Ang-II on Kir4.1/Kir5.1 and HCTZ-induced-ENa was absent in Ks-AT1aR-KO mice. Overnight-low-salt stimulated the expression of Agtr1a mRNA in DCT, increased whole-cell Kir4.1/Kir5.1-mediated K+-currents in late-DCT, hyperpolarized late-DCT membrane, augmented the expression of phosphor-Na-Cl-cotransporter (pNCC) and enhanced thiazide-induced renal-ENa in the control mice. However, the effect of overnight-low-salt on Kir4.1/Kir5.1-activity, DCT membrane potential and NCC activity/expression was abolished in DCT-AT1aR-KO or Ks-AT1aR-KO mice. Overnight-low-salt had no effect on baseline renal K+-excretion (EK) and plasma K+-concentrations in the control mice but it increased baseline renal-EK and decreased plasma K+-concentrations in DCT-AT1aR-KO or in Ks-AT1aR-KO mice. Acute Ang-II or overnight-LS stimulated Kir4.1/Kir5.1 in late-DCT and that AT1aR was responsible for acute Ang-II or overnight-low-salt-induced stimulation of Kir4.1/Kir5.1 and NCC. AT1aR of the DCT plays a role in maintaining adequate baseline renal-EK and plasma K+ concentrations during overnight-LS.

Pkd1 is necessary for full cardiac AngII-induced hypertrophy but not for full acute AngII-induced calcium handling changes.

The renin-angiotensin system (RAS) regulates blood pressure, extracellular fluid and electrolyte balance, cardiac inotropy, and vascular resistance. In the heart, angiotensin II (AngII) can induce hypertrophy directly by activating Protein Kinase D (PKD), a signaling kinase known to contribute to cardiac remodeling. Little is known, however, about the acute and chronic functional effects of AngII-induced PKD activation on calcium (Ca) handling. To investigate this, we used hearts from cardiac-specific PKD1 knockout (cKO) mice and their WT littermates. We measured Ca transients (CaT) and Ca spark (CaSp) frequencies in cardiomyocytes at 0.5 and 1 Hz pacing under acute and chronic AngII stimulation (100 nM in vitro and 3 mg/kg/day in osmotic minipump, respectively). Sarcoplasmic reticulum (SR) Ca load was assessed by rapid caffeine-induced Ca release. At baseline and after acute AngII treatment, CaT amplitude and tau decay, CaSp frequency and SR Ca load were similar in WT vs. PKD1 cKO. This was also the case for chronic exposure to AngII except that the tau decay was significantly slower in PKD1 cKO myocytes. In agreement with previous reports, echocardiographic measurements revealed that PKD1 cKO mice were also more resistant to chronic AngII-induced hypertrophy (LVPW increase 24.69 ± 2.74% vs. 54.73 ± 7.73 %) and ejection fraction reduction (4.08 ± 1.5% vs. 12.67 ± 0.52%). We conclude that despite PKD being necessary for the remodeling changes induced by chronic AngII, it does not alter the amplitude and kinetics of CaT and spontaneous Ca release, except for the slowed decay kinetics in the PKD1 cKO after chronic AngII treatment and decreased acute SR Ca load.

Chronic angiotensin II signaling drives sustained PIP2 deficits and altered sarcolemmal Cav1.2 expression and function in cardiomyocytes.

Contractile dysfunction, hypertrophy, and cell death during heart failure are linked to altered Ca2+ handling and elevated levels of angiotensin II (AngII). This hormone signals through Gq-coupled AT1 receptors, initiating hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Previous work revealed acute AngII signaling and PIP2 depletion destabilizes CaV1.2 clusters in cardiomyocytes, triggering their internalization, and subsequent reduction of whole-cell Ca2+ current (ICa) and contractility. However, the effects of chronically elevated AngII on the phospholipid landscape and associated effects on ICa remain unknown. We subjected mice to sustained delivery of AngII to test the impact on phosphoinositides, ICa, and excitation-contraction coupling. We hypothesized that chronic AngII signaling leads to sustained PIP2 and CaV1.2 deficits, contributing to the diminished functional output associated with disease development. Phospholipid mass spectrometry revealed extensive alteration of phosphoinositide species, including a ∼44% reduction in PIP2 and ∼37% reduction in cardioprotective PIP3 in AngII-infused hearts compared to controls. To study the effects of this phospholipid imbalance on CaV1.2, we examined ICa, finding similar current density in both groups. However, super-resolution imaging revealed a ∼54% reduction in t-tubular CaV1.2 expression and a ∼23% reduction in cluster area. Conserved ICa density despite reduced t-tubular CaV1.2 suggests that either the channels have relocated to the sarcolemmal crest, or their Po has increased. We examined crest CaV1.2 and observed no change with AngII, thus the Po of the remaining channels must increase to maintain ICa. Western blots revealed enhanced phosphorylation of CaV1.2, phosphorylation of RyR2, and an increased expression of the catalytic subunit of PKA, suggesting a compensatory sympathetic activation underlying conserved ICa. These findings support a model wherein chronic elevation of AngII disrupts phosphoinositide and CaV1.2 levels, elucidating the role of chronic AngII in disease progression.

Abstract P323: Direct Vascular Endothelial Actions Of Angiotensin II Via At 1 R And No Signaling Revealed By Intravital Multiphoton Microscopy.

The renin-angiotensin system (RAS) controls blood pressure and body fluid balance. The classic vasoactive effects of RAS are mediated by the binding of angiotensin II (ANGII) to ANGII type 1 receptors (AT 1 R) in vascular smooth muscle cells leading to vasoconstriction, or to opposing ANGII type 2 receptors (AT 2 R). However, only few studies investigated the direct effects of ANGII on vascular endothelial cells (VEC) in vivo and the mechanisms how VECs may mitigate ANGII-mediated vasoconstriction. The present study aimed to explore the molecular mechanisms and pathophysiological relevance of the direct actions of ANGII on VEC in the kidney. VEC calcium dynamics and NO synthesis were visualized by using intravital multiphoton microscopy (MPM) of Cdh5-G6 mice that selectively express the ratiometric Ca 2+ reporter GCaMP6f/tdTomato in VECs, or the endothelial uptake of iv injected nitric oxide (NO) sensitive dye DAF-FM, respectively. Bolus injection of ANGII (400 ng/kg, ia.) triggered a >4-fold increase in VEC calcium in afferent (AA) arterioles and glomerular capillaries, but not in peritubular capillaries. These responses were blocked by pretreatment with the AT 1 R inhibitor Losartan (60mg/kg ip.), but not by the AT 2 R inhibitor PD123319 (25mg/kg ip.)(1.2+/-0.4 p<0.001 and 5.6+/-0.9-fold, p=0.3, respectively). VEC responded to acute ANGII by increased NO synthesis as indicated by >1.5-fold increase in DAF-FM fluorescence intensity as compared to baseline (p<0.0001). In mice with unilateral ureteral obstruction (UUO), a well-known high ANGII and vasoconstrictor state associated with endothelial dysfunction, the ANGII induced calcium increase in VECs was significantly reduced (2.8+/-0.3-fold) as compared to control (4.6+/-0.4-fold, p<0.01). These results correlated with the significantly reduced AA diameter in UUO compared to control (8.2+/-0.4 and 10.43+/-0.3 μm, respectively, p<0.001). In summary, ANGII has major direct effects on VECs NO production that counterbalances agonist-induced vasoconstriction. These effects are diminished in conditions with endothelial dysfunction leading to increased vascular contractility. Cell-specific targeting of ANGII actions may represent exciting future therapeutic opportunities.

Adipocyte deficiency of ACE2 increases systolic blood pressures of obese female C57BL/6 mice

BackgroundObesity increases the risk for hypertension in both sexes, but the prevalence of hypertension is lower in females than in males until menopause, despite a higher prevalence of obesity in females. We previously demonstrated that angiotensin-converting enzyme 2 (ACE2), which cleaves the vasoconstrictor, angiotensin II (AngII), to generate the vasodilator, angiotensin-(1-7) (Ang-(1-7)), contributes to sex differences in obesity-hypertension. ACE2 expression in adipose tissue was influenced by obesity in a sex-specific manner, with elevated ACE2 expression in obese female mice. Moreover, estrogen stimulated adipose ACE2 expression and reduced obesity-hypertension in females. In this study, we hypothesized that deficiency of adipocyte ACE2 contributes to obesity-hypertension of females.MethodsWe generated a mouse model of adipocyte ACE2 deficiency. Male and female mice with adipocyte ACE2 deficiency or littermate controls were fed a low (LF) or a high fat (HF) diet for 16 weeks and blood pressure was quantified by radiotelemetry. HF-fed mice of each sex and genotype were challenged by an acute AngII injection, and blood pressure response was quantified. To translate these findings to humans, we performed a proof-of-principle study in obese transwomen in which systemic angiotensin peptides and blood pressure were quantified prior to and after 12 weeks of gender-affirming 17β-estradiol hormone therapy.ResultsAdipocyte ACE2 deficiency had no effect on the development of obesity in either sex. HF feeding increased systolic blood pressures (SBP) of wild-type male and female mice compared to LF-fed controls. Adipocyte ACE2 deficiency augmented obesity-induced elevations in SBP in females, but not in males. Obese female, but not obese male mice with adipocyte ACE2 deficiency, had an augmented SBP response to acute AngII challenge. In humans, plasma 17β-estradiol concentrations increased in obese transwomen administered 17β-estradiol and correlated positively with plasma Ang-(1-7)/AngII balance, and negatively to SBP after 12 weeks of 17β-estradiol administration.ConclusionsAdipocyte ACE2 protects female mice from obesity-hypertension, and reduces the blood pressure response to systemic AngII. In obese transwomen undergoing gender-affirming hormone therapy, 17β-estradiol administration may regulate blood pressure via the Ang-(1-7)/AngII balance.

Altered Antioxidant and Inflammatory Signaling by Fibroblasts in the Hypertensive Heart

In response to hypertensive stimuli, like angiotensin II (AngII), cardiac fibroblasts (CFs) transform into an activated phenotype, which triggers increased collagen deposition and pro‐inflammatory signals. Transforming growth factor beta 1 (TGF‐β1) and reactive oxygen species are important modulators of AngII‐induced cardiac fibrosis and CF activation. The extent to which the AngII‐induced changes in CF phenotype persist in the absence of ongoing stimulation is not known. The objective of the present study was to determine the persistent impact of chronic AngII infusion on CF phenotype. Specifically, we performed an in vitro characterization of CFs isolated from rats chronically treated with AngII (in vivo) or saline (vehicle) and investigated acute (in vitro) responses to AngII thereafter. Adult male SD rats were administered AngII (200 ng/kg/min) or saline for 4 weeks. In half of the rats in each group, we measured mean arterial pressure (MAP), weighed the left ventricle (LV), and fixed a section of LV for assessment of collagen deposition (Sirius Red). In remaining rats, CFs were isolated and passaged to P1. CFs were treated in vitro with AngII (1uM) or vehicle for 72 hr. TGF‐β1 and monocyte chemoattractant protein 1 (MCP‐1) were assessed in culture media. Expression of pro‐/anti‐oxidant proteins NADPH oxidase 2 (Nox2), catalase and superoxide dismutase 1 (SOD1) were determined by western blot. Ang II increased MAP (43%, p<0.05), LV/BW (30%, p<0.05), and LV collagen deposition. TGF‐β1 release was not different from CFs isolated from saline vs. AngII infused rats. However, in vitro incubation with AngII significantly increased TGF‐β1 secretion in all CFs. CFs isolated from AngII‐infused rats secreted elevated levels of MCP‐1 (p<0.05), when compared to control CFs. MCP‐1 levels were not further modified by acute AngII treatment. Basal expression of Nox2, SOD1, and catalase was similar between CFs isolated from control vs. AngII‐infused rats. Acute AngII tended to increase Nox2 expression in CFs isolated from vehicle and AngII‐treated rats, whereas AngII only increased SOD1 expression (46%, p<0.05) in CFs from AngII‐infused rats. In vitro stimulation with AngII tended to increase catalase in CFs isolated from control rats, but reduced (20%, p<0.05) expression in CFs isolated from AngII infused rats. Taken together, these data demonstrate that CFs isolated from an actively remodeling myocardium continue to secrete elevated levels of MCP‐1, but not TGF‐β1, in vitro. Moreover, CFs from AngII‐infused rats displayed altered expression of antioxidant enzymes following AngII in vitro, which may play a role in worsened inflammatory signaling and resultant pro‐fibrotic activity. Future studies will further elucidate the fibrotic and inflammatory characteristics of CFs from the remodeling myocardium and determine the extent to which this persistent phenotype alters the future responsiveness to a pathogenic stimulus.Support or Funding InformationSpringboard Grant, UA College of Medicine – PhoenixThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nuclear translocation of calmodulin in pathological cardiac hypertrophy originates from ryanodine receptor bound calmodulin

In cardiac myocytes Calmodulin (CaM) bound to the ryanodine receptor (RyR2) constitutes a large pool of total myocyte CaM, but the CaM-RyR2 affinity is reduced in pathological conditions. Knock-in mice expressing RyR2 unable to bind CaM also developed hypertrophy and early death. However, it is unknown whether CaM released from this RyR2-bound pool participates in pathological cardiac hypertrophy.We found that angiotensin II (AngII) or phenylephrine (PE) both cause CaM to dissociate from the RyR2 and translocate to the nucleus. To test whether this nuclear CaM accumulation depends on CaM released from RyR2, we enhanced CaM-RyR2 binding affinity (with dantrolene), or caused CaM dissociation from RyR2 (using suramin). Dantrolene dramatically reduced AngII- and PE-induced nuclear CaM accumulation. Conversely, suramin enhanced nuclear CaM accumulation. This is consistent with nuclear CaM accumulation coming largely from the CaM-RyR2 pool. CaM lacks a nuclear localization signal (NLS), but G-protein coupled receptor kinase 5 (GRK5) binds CaM, has a NLS and translocates like CaM in response to AngII or PE. Suramin also promoted GRK5 nuclear import, and caused nuclear export of histone deacetylase 5 (HDAC5). Dantrolene prevented these effects. After 2–8 weeks of pressure overload (TAC) CaM binding to RyR2 was reduced, nuclear CaM and GRK5 were both elevated and there was enhanced nuclear export of HDAC5.Stress (acute AngII or TAC) causes CaM dissociation from RyR2 and translocation to the nucleus with GRK5 with parallel HDAC5 nuclear export. Thus CaM dissociation from RyR2 may be an important step in driving pathological hypertrophic gene transcription.

Acute, local infusion of angiotensin II impairs microvascular and metabolic insulin sensitivity in skeletal muscle.

Angiotensin II (AngII) is a potent vasoconstrictor implicated in both hypertension and insulin resistance. Insulin dilates the vasculature in skeletal muscle to increase microvascular blood flow and enhance glucose disposal. In the present study, we investigated whether acute AngII infusion interferes with insulin's microvascular and metabolic actions in skeletal muscle. Adult, male Sprague-Dawley rats received a systemic infusion of either saline, AngII, insulin (hyperinsulinaemic euglycaemic clamp), or insulin (hyperinsulinaemic euglycaemic clamp) plus AngII. A final, separate group of rats received an acute local infusion of AngII into a single hindleg during systemic insulin (hyperinsulinaemic euglycaemic clamp) infusion. In all animals' systemic metabolic effects, central haemodynamics, femoral artery blood flow, microvascular blood flow, and skeletal muscle glucose uptake (isotopic glucose) were monitored. Systemic AngII infusion increased blood pressure, decreased heart rate, and markedly increased circulating glucose and insulin concentrations. Systemic infusion of AngII during hyperinsulinaemic euglycaemic clamp inhibited insulin-mediated suppression of hepatic glucose output and insulin-stimulated microvascular blood flow in skeletal muscle but did not alter insulin's effects on the femoral artery or muscle glucose uptake. Local AngII infusion did not alter blood pressure, heart rate, or circulating glucose and insulin. However, local AngII inhibited insulin-stimulated microvascular blood flow, and this was accompanied by reduced skeletal muscle glucose uptake. Acute infusion of AngII significantly alters basal haemodynamic and metabolic homeostasis in rats. Both local and systemic AngII infusion attenuated insulin's microvascular actions in skeletal muscle, but only local AngII infusion led to reduced insulin-stimulated muscle glucose uptake. While increased local, tissue production of AngII may be a factor that couples microvascular insulin resistance and hypertension, additional studies are needed to determine the molecular mechanisms responsible for these vascular defects.

Impact of Chronic Angiotensin Treatment on the Responses of Isolated Cardiac Fibroblasts to Acute Pro‐Fibrotic Stimuli

Pathological cardiac remodeling involving fibrosis can lead to heart failure in people with hypertension. We have recently demonstrated that angiotensin II (AngII)‐induced increases in left ventricular collagen content and macrophage infiltration are attenuated by concomitant bradykinin‐B1 receptor (B1R) antagonism in Sprague‐Dawley (SD) rats. Given that AngII has been shown to upregulate B1R, the goal of the present study was to determine the impact of B1R stimulation on fibroblast physiology following chronic (in vivo) or acute (in vitro) treatment with AngII. Adult male SD rats were implanted with osmotic mini‐pumps that delivered AngII (200 ng/kg/min) or saline for 4 weeks. Cardiac fibroblasts were isolated from the left ventricle of saline and AngII‐treated rats and passaged to P1. Cells were treated in vitro with AngII (1uM), a selective B1R agonist (Lys‐ [Des‐Arg9], 100 nM), AngII+B1R agonist or vehicle for 48 hr. Transforming growth factor beta 1 (TGF‐β1) and monocyte chemoattractant protein 1 (MCP‐1) were assessed in culture media. Expression of the matricellular protein periostin and the pro‐oxidant NADPH oxidase 2 (Nox2) were determined in cell lysate by western blot. Fibroblasts isolated from rats chronically treated with AngII secreted elevated levels of MCP‐1 in vitro (p<0.05), when compared to control fibroblasts. Although MCP‐1 secretion was not further modified by acute AngII treatment, stimulation with the B1R agonist tended to reduce MCP‐1 levels. TGF‐β1 release was not different from cells isolated from saline or AngII infused rats. However, in vitro incubation with AngII significantly increased secretion of TGF‐β1 to a similar degree in fibroblasts isolated from saline or AngII treated rats. B1R stimulation had no impact on TGF‐β1 levels. Periostin expression tended to be increased in fibroblasts isolated from AngII infused rats, while Nox2 expression was not different. Acutely, AngII tended to increase Nox2 expression in vitro to a greater degree in fibroblasts that were isolated from saline‐infused rats. Selective B1R stimulation in vitro resulted in a significant 1.77‐fold upregulation of Nox2 only in fibroblasts isolated from saline treated rats (p<0.05). Combined AngII+B1R stimulation did not further increase Nox2 expression. Taken together, these data demonstrate that upregulation of MCP‐1 production and periostin induced by chronic AngII persist at least to P1 in isolated cardiac fibroblasts. Moreover, chronic AngII resulted in an attenuated increase in Nox2 in response to acute AngII stimulation. Notably, we demonstrate for the first time that selective B1R activation results in a marked increase in Nox2 expression in cardiac fibroblasts. Although B1R antagonism has been shown to offset fibrotic and inflammatory effects of AngII in vivo, acute effects of AngII stimulation are not enhanced in the presence of B1R stimulation. Future studies will determine the extent to which in vivo or in vitro B1R antagonism prevents AngII‐induced fibroblast activation. Investigation of oxidative stress and the nature of Nox2 regulation by B1R activation may provide greater insight into the impact of bradykinin on fibroblast physiology.Support or Funding InformationSpringboard, UA COM‐PThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deletion of the EGF receptor in vascular smooth muscle cells prevents chronic angiotensin II-induced arterial wall stiffening and media thickening.

In vivo vascular smooth muscle cell (VSMC) EGF receptor (EGFR) contributes to acute angiotensin II (AII) effects on vascular tone and blood pressure. The ubiquitously expressed EGFR has been implicated in vascular remodelling preceding end-organ damage by pharmacological inhibition, and AII signalling in cultured vascular cells is partly EGFR-dependent. However, the role of VSMC-EGFR invivo during AII-induced pathophysiological processes is not known. This study assesses the invivo relevance of VSMC-EGFR during chronic AII challenge without further stressors, using a mouse model with inducible, VSMC-specific EGFR knock out (VSMC-EGFR-KO). In these mice functional and structural vascular, renal and cardiac effects or biomarkers were investigated invivo and exvivo. Vascular smooth muscle cell-EGFR-KO prevented AII-induced media hypertrophy of mesenteric arteries, renal arterioles and the aorta, VSMC ERK1/2-phosphorylation as well as the impairment of vascular compliance. Furthermore, induction of vascular fibrosis, creatinineamia, renal interstitial fibrosis as well as the increase in fractional water excretion was prevented. AII-induced increase in systolic blood pressure was mitigated. By contrast, endothelial dysfunction, induction of vascular inflammatory marker mRNA and albuminuria were not inhibited. Cardiac and cardiomyocyte hypertrophy were also not prevented by VSMC-EGFR-KO. Vascular smooth muscle cell-EGFRs are relevant for pathological AII action invivo. Our data show invivo and exvivo the necessity of VSMC-EGFR for AII-induced structural and functional vascular remodelling, not including endothelial dysfunction. Hereby, VSMC-EGFR gains importance for complete AII-induced renal end-organ damage succeeding vascular remodelling.

Angiotensin II Modulates IKs by Multiple Mechanisms with Varying Outcomes

Previous studies showed that acute angiotensin II (AngII) application decreases IKs in ventricular myocytes, but increases IKs in atrial myocytes from guinea-pig (GPV and GPA). Chronic AngII application (released by implanted mini-pump, 4 weeks) also decreases IKs in GPV but increases IKs in GPA. Extrapolating these observations to human heart suggests that AngII may increase the risk for long QT and atrial fibrillation by differentially remodeling ventricular and atrial IKs, respectively. Recently we identify a fundamental difference in IKs between GPV and GPA: KCNQ1 and KCNE1 (Q1 and E1, pore-forming and auxiliary IKs subunits) are largely separated in GPV (Q1 intracellular while E1 on surface) but better colocalized on cell surface in GPA. Furthermore, AngII incubation induces significant Q1 translocation to cell surface in GPV but not in GPA. We hypothesize that AngII modulation of IKs involves multiple signaling pathways, which develop with different time courses and exert differential effects on IKs. We test this hypothesis by coexpressing Q1, E1, and AngII-receptor (AT1R) in COS-7 cells, and monitor AngII (1 uM) effects on IKs using patch clamp recording, confocal imaging, and protein fractionation/biochemical quantification. Acute AngII exposure reduces IKs (some with early transient increase) and right shifts V0.5 of activation. Overexpressing Ca-binding-deficient calmodulin reduces these effects. Incubation with AngII for 1 or 24 hr followed by patch clamp in the absence of AngII shows that IKs is increased over time-control. Cell surface Q1 protein level is higher after 1-hr AngII incubation. After 24-hr AngII incubation, cell surface Q1 level is the same as time-control, but overall Q1 protein level is higher. These data suggest that AngII modulation of IKs involves [Ca]i elevation/PKC activation/PIP2 depletion (acute), Q1 translocation to cell surface (1 hr), changes in Q1 biosynthesis and/or degradation (24 hr).

Vitamin D Levels Are Associated with Cardiac Autonomic Activity in Healthy Humans

Vitamin D deficiency (≤50nmol/L 25-hydroxy vitamin D) is a cardiovascular (CV) risk factor that affects approximately one billion people worldwide, particularly those affected by chronic kidney disease (CKD). Individuals with CKD demonstrate abnormal cardiac autonomic nervous system activity, which has been linked to the significant rates of CV-related mortality in this population. Whether vitamin D deficiency has a direct association with regulation of cardiac autonomic activity has never been explored in humans. Methods: Thirty-four (34) healthy, normotensive subjects were studied and categorized based on 25-hydroxy vitamin D deficiency (deficient vs. non-deficient, n = 7 vs. 27), as well as 1,25-dihydroxy vitamin D levels (above vs. below 25th percentile, n = 8 vs. 26). Power spectral analysis of electrocardiogram recordings provided measures of cardiac autonomic activity across low frequency (LF) and high frequency (HF, representative of vagal contribution) bands, representative of the sympathetic and vagal limbs of the autonomic nervous system when transformed to normalized units (nu), respectively, as well as overall cardiosympathovagal balance (LF:HF) during graded angiotensin II (AngII) challenge (3 ng/kg/min × 30 min, 6 ng/kg/min × 30 min). Results: At baseline, significant suppression of sympathovagal balance was observed in the 25-hydroxy vitamin D-deficient participants (LF:HF, p = 0.02 vs. non-deficient), although no other differences were observed throughout AngII challenge. Participants in the lowest 1,25-dihydroxy VD quartile experienced significant withdrawal of inhibitory vagal control, as well as altered overall sympathovagal balance throughout AngII challenge (HF, mean difference = −6.98 ± 3 nu, p = 0.05; LF:HF, mean difference = 0.34 ± 0.1, p = 0.043 vs. above 25th percentile). Conclusions: Vitamin D deficiency is associated with suppression of resting cardiac autonomic activity, while low 1,25-dihydroxy vitamin D levels are associated with unfavourable cardiac autonomic activity during an acute AngII stressor, offering a potential pathophysiological mechanism that may be acting to elevate CV risk in in populations with low vitamin D status.

204 ALPHA CALCITONIN GENE-RELATED PEPTIDE PLAYS A PROTECTIVE ROLE IN BOTH AN ACUTE AND SUSTAINED MODEL OF HYPERTENSION, A MECHANISM WHICH MAY BE LINKED TO THE LOSS OF ENDOTHELIAL NITRIC OXIDE SYNTHASE (ENOS) AS THE HYPERTENSION PROGRESSES

Calcitonin gene-related peptide (CGRP) is a sensory nerve-derived neuropeptide, and its potent microvascular vasodilator activities have been suggested to be protective in a range of murine models of hypertension using...

1008 ANGIOTENSIN TYPE-2 RECEPTOR CONTRIBUTES TO REDUCED VASCULAR RESPONSE TO ANGIOTENSIN-II IN YOUNG RATS

Background: The angiotensin type-2 receptor (AT2R) is known to oppose the vasoconstrictive actions of angiotensin II (AngII). During fetal development AT2R expression is very high, which upon maturation regresses. The present study investigates the sensitivity to acute AngII in young rats and the contribution of the AT2R in attenuating the vasoconstrictor response to AngII. Methods: Male Sprague-Dawley rats at 3 and 6 weeks of age were anesthetized. Mean arterial pressure (MAP) and renal blood flow (RBF; transonic flowmeter), was measured in response to i.v. administration of AngII at 0 (basal) 10 (10AngII) and 100ng/kg/hr (100AngII). The AT2R inhibitor, PD-123319, was given and responses to 100AngII were repeated. Results: In the 6wk rats, 10 and 100AngII increased MAP (89 ± 4 mmHg) by 10 ± 7 and 18 ± 5 mmHg, respectively. Likewise, RBF (4.0 ± 0.6 ml/min/gkw) was reduced by 8 ± 3% and 34 ± 3%. In the 3wk rats basal MAP and RBF were 72 ± 4 mmHg and 2.9 ± 0.3 ml/min/gkwt, The 10AngII dose had no effect on MAP, while 100AngII increased MAP by 6 ± 2 mmHg. RBF in the 3wk rats was not was not affected by the 10ng AngII dose (-2 ± 1% change), while the higher dose produced a 10 ± 3% decrease. PD-123319 potentiated the change in RBF to 100AngII in the 3wk animals (23 ± 4% reduction), but had no additional effect in the 6wk rats. Conclusions: These data indicate that during development, the presence of the AT2R in young rodents contributes to the attenuated vasoconstrictor response to AngII. These findings may have implication for the use of drugs that inhibit the renin-angiotensin system for the treatment of pediatric hypertension and renal failure.

In vivo MEMRI reveals persistent activation of the brain autonomic areas by an acute systemic angiotensin II injection

IntroductionAngiotensin II (ANGII) acts centrally to activate the sympathetic nervous system in hypertension. Here, we show that even a single systemic ANGII injection causes long‐lasting neuronal activation in the autonomic brain areas.MethodsIn vivo mapping of basal and ANGII‐evoked neuronal activity was carried out in the Wistar‐Kyoto (WKY) rat using manganese‐enhanced MRI (MEMRI) at 4.7Tesla. Rats were treated with manganese chloride (MnCl2 30 mM solution, i.p.;16–20 hrs prior to the MRI), and T1‐weighted images were obtained 16–20 hrs after a single ANGII injection (0.32μg/kg i.p.). Coronal slice scans (positioned caudally from end of the cerebellum/medulla rostrally towards the hypothalamus) were manually segmented using itkSNAP, and data analyzed for differences in normalized signal intensity.ResultsAcute ANGII injection caused an immediate pressor response (ÄSBP=~20mmHg) which normalized 2 hours following the injection. Despite this, ANGII evoked persistent neuronal activation in the central amygdala, hypothalamus (PVN, preoptic and lateral hypothalamic areas), and brainstem (NTS and RVLM).ConclusionEven a single systemic ANGII injection results in a lasting effect on the brain autonomic areas. Thus, repeated pro‐hypertensive stimuli in these brain areas may lead to pre‐sympathetic neuronal plasticity, resulting in heightened sympathetic drive and hypertension.

774: AT1-AA enhances renal sensitivity and blood pressure in response to ANGII during pregnancy

pressure in response to ANGII during pregnancy Justin Brewer, Ruisheng Liu, Kedra Wallace, Florian Herse, Janae Moseley, Ralf Dechend, Gerd Walluk, James Martin, Babbette LaMarca University of Mississippi Medical Center, Maternal Fetal Medicine, Jackson, MS, University of Mississippi Medical Center, Department of Physiology, Jackson, MS, University of Mississippi Medical Center, Obstetrics and Gynecology, Jackson, MS, HELIOS Clinic, Max-DelbruckCenter for Molecular Medicine, Berlin, Germany OBJECTIVE: Agonistic autoantibodies to the angiotensin II (ANGII) type I receptor (AT1-AA) are proposed to be important links between hypertension and increased maternal vascular sensitivity to ANGII during preeclampsia. This study was designed to determine effects of AT1-AA to enhance blood pressure (MAP) and renal vascular sensitivity to ANGII. STUDY DESIGN: First, we examined acute administration of ANGII on MAP in AT1-AA induced hypertensive pregnant rats. ANGII (100ng/ kg/min) was administered via jugular infusion to normal pregnant (NP) (n 6) controls and AT1-AA (1:50, n 10) infused pregnant rats. We examined the direct effects of ANGII on renal vasoconstriction in the presence and absence of the AT1-AA on isolated afferent arterioles (Af-Art) from NP rats. A dose response of increased constriction of the Af-Art with ANGII alone (10-11-10-8) or increasing AT1-AA alone (1:200, 1:150, and 1:100) was established. We subsequently measured Af-Art constriction to ANGII (10-11, 10-10, 10-9) with nonconstrictor dose of AT1-AA (1:200). Second, we examined chronic ANGII on MAP and renal artery resistive index (RI) in AT1-AA induced hypertensive pregnant rats. Power Doppler velocimetry was performed, bilaterally at two levels, using Vevo 770 unit with 30 Hz transducer and insonating angle 30. Statistical analysis was performed using STATA. RESULTS: MAP increased 30mmHg with acute ANGII in NP rats but markedly increased 47mmHg with ANGII in AT1-AA infused rats. Increasing ANGII from (10-11-10-8) decreased Af-Art diameter 1520% but sharply decreased Af-Art diameter 80% in AT1-AA pretreated vessels. MAP was 95 / 1 mmHg in NP and increased to 116 / 3 mmHg with chronic ANGII, to 120 / 3 mmHg with AT1-AA and was 112 / 3 mmHg, with ANGII AT1-AA (P 0.05). RI in the renal arteries was 0.67 in NP, 0.70 in AT1-AA infused pregnant rats, and was 0.74 (P 0.002) in ANGII AT1-AA pregnant rats. CONCLUSION: These data support the hypothesis that AT1-AA may be an important mechanism whereby MAP and renal vascular responses are greatly enhanced during preeclampsia.

Inactivation of Adenosine A2A Receptor Attenuates Basal and Angiotensin II-induced ROS Production by Nox2 in Endothelial Cells

Endothelial cells (ECs) express a Nox2 enzyme, which, by generating reactive oxygen species (ROS), contributes to EC redox signaling and angiotensin II (AngII)-induced endothelial dysfunction. ECs also express abundantly an adenosine A2A receptor (A2AR), but its role in EC ROS production remains unknown. In this study, we investigated the role of A2AR in the regulation of Nox2 activity and signaling in ECs with or without acute AngII stimulation. In cultured ECs (SVEC4–10), AngII (100 nm, 30 min) significantly increased Nox2 membrane translocation and association with A2AR. These were accompanied by p47phox, ERK1/2, p38 MAPK, and Akt phosphorylation and an increased ROS production (169 ± 0.04%). These AngII effects were inhibited back to the control levels by a specific A2AR antagonist (SCH58261), or adenosine deaminase, or by knockdown of A2AR or Nox2 using specific siRNAs. Knockdown of A2AR, as determined by Western blotting, decreased Nox2 and p47phox expression. In wild-type mouse aorta, SCH58261 significantly reduced acute AngII-induced ROS production and preserved endothelium-dependent vessel relaxation to acetylcholine. These results were further confirmed by using aortas from A2AR knock-out mice. In conclusion, A2AR is involved in the regulation of EC ROS production by Nox2. Inhibition or blockade of A2AR protects ECs from acute AngII-induced oxidative stress, MAPK activation, and endothelium dysfunction.

BAS/BSCR28 Blockade of adenosine A2A receptor attenuates angiotensin II-induced reactive oxygen species production and impairment of endothelium-dependent vessel relaxation in mouse aortas

The endothelium expresses abundantly an adenosine A2A receptor (A2AR) which has important roles in the regulation of vascular function. The endothelium expresses also the AT1-type receptor for angiotensin II (AngII)...

Atrap Deficiency Increases Arterial Blood Pressure and Plasma Volume

The angiotensin receptor-associated protein (Atrap) interacts with angiotensin II (AngII) type 1 (AT1) receptors and facilitates their internalization in vitro, but little is known about the function of Atrap in vivo. Here, we detected Atrap expression in several organs of wild-type mice; the highest expression was in the kidney where it localized to the proximal tubule, particularly the brush border. There was no Atrap expression in the renal vasculature or juxtaglomerular cells. We generated Atrap-deficient (Atrap-/-) mice, which were viable and seemed grossly normal. Mean systolic BP was significantly higher in Atrap-/- mice compared with wild-type mice. Dose-response relationships of arterial BP after acute AngII infusion were similar in both genotypes. Plasma volume was significantly higher and plasma renin concentration was markedly lower in Atrap-/- mice compared with wild-type mice. (125)I-AngII binding showed enhanced surface expression of AT1 receptors in the renal cortex of Atrap-/- mice, accompanied by increased carboanhydrase-sensitive proximal tubular function. In summary, Atrap-/- mice have increased arterial pressure and plasma volume. Atrap seems to modulate volume status by acting as a negative regulator of AT1 receptors in the renal tubules.

Is renal Na+,K+‐ATPase a new target for renin–angiotensin blocking agents in diabetic nephropathy?

Diabetic nephropathy is a clinical syndrome characterized by increasing arterial blood pressure, albuminuria and a relentless decline in renal function. It is the leading cause of end-stage renal failure in the western world and the incidence of end-stage renal disease due to diabetes has been increasing dramatically during the past 20 years. Furthermore, patients suffering from diabetic nephropathy have an increased risk of cardiovascular morbidity and mortality. It has been demonstrated that optimized glycaemic control and aggressive antihypertensive treatment with particular focus on the renin–angiotensin system (RAS) blockers have reduced the individual patient risk for development and progression of diabetic nephropathy (Rossing, 2005). An important aspect of the development of diabetic nephropathy is the inappropriately activated renin–angiotensin system, and fluid and sodium retention contributing to the development of hypertension and renal damage (Feldt-Rasmussen et al. 1987). An understanding of the physiology leading to these changes could potentially improve the prevention and treatment of this important complication. In this context Fekete and colleagues (Fekete et al. 2008) demonstrate, in this issue of The Journal of Physiology, that STZ-diabetes in rats increases the mRNA expression, protein level, Ser23 phosphorylation and enzyme activity of renal Na+,K+-ATPase (NKA) which is further elevated by a pressor dose of angiotensin II (ANGII) for 24 h. The NKA is important for the regulation of the fluid and sodium potassium balance by extruding sodium from the basolateral membrane in proximal tubuli. It is, however, also demonstrated in their study that although the abundance and activity of renal NKA is increased, the channels may no longer be functional as they are to some extent redistributed from the basolateral membrane to the cytosol in diabetic animals and even more so when ANGII is added. Whether this is the result of an adaptive response to increased filtered Na+ or a cause of the disturbed Na+ balance is not studied. Thus, a potentially important effect of both chronic hyperglycaemia and acute ANGII on NKA activity and location is demonstrated. The authors speculate if this is a new mechanism contributing to the development of renal disease in diabetes and furthermore, it is suggested that prevention of the ANGII-induced changes described in the study could be a new explanation of part of the beneficial effect of RAS blocking pharmacological intervention in diabetic nephropathy which has been demonstrated in many clinical trials. So far this has been attributed to ANGII effects on haemodynamic (systemic and intrarenal blood pressure) and non-haemodynamic factors (such as effects on growth factors, PAI-1, nephrin, inflammation, glomerular pore size and charge selectivity). The mechanisms for the hyperglycaemia and ANGII-induced effects are not studied, and although they may act through different pathways, a common pathway is also possible. Recently Peti-Peterdi et al. (2008) described how elevated glucose metabolized to elevated levels of succinate, which activates the GPR91 receptor in the juxtaglomerular apparatus (JGA) leading to subsequent release of renin in the JGA. Thus, a link between hyperglycaemia and activation of the renin–angiotensin system was demonstrated. Therefore, RAS blockade could potentially block the effect of hyperglycaemia as well as the effect of non-hyperglycaemia-related RAS activation. Additional studies of the effect of RAS blockade on NKA activation and redistribution in diabetes could help us understanding if hyperglycaemia induces the changes through activation of the renin–angiotensin system, and further evaluation of diabetes with additional stress activating the RAS would help us to understand if the observation by Fekete and colleagues (Fekete et al. 2008) is indeed a new mechanism explaining the beneficial effect of RAS intervention in preventing and treating diabetic nephropathy. It would also be important to evaluate the effect on renal NKA activity and localization of RAS activation through dietary sodium restriction or diuretic treatment, demonstrated to enhance the clinical efficacy of RAS blocking agents. Thus, as with any good study, the paper by Fekete et al. provides new and interesting knowledge related to the problem of sodium and fluid retention in diabetic nephropathy, potentially contributing to the development of diabetic glomerulosclerosis, and thus a potential target for intervention. However, the study also raises new questions which need to be explored before we will know if an effect on renal NKA by RAS blocking agents affects the development of diabetic nephropathy.