AT1 Antagonist Losartan Research Articles

Impact of neonatal hyperoxia on adult cardiac autonomic function in rats: Role of angiotensin II type 1 receptor activation

Individuals born preterm present altered cardiac autonomic function, a risk factor to heart diseases. Neonatal renin-angiotensin-system activation contributes to adult cardiomyopathy in rats exposed to neonatal hyperoxia, a well-established model of preterm birth-related conditions. Central angiotensin II receptor activation is a key modulator of the autonomic drive to the heart. Whether neonatal hyperoxia leads to alteration of the cardiac autonomic function through activation of the angiotensin II receptor type 1 (AT1) is unknown and was examined in the present study.Sprague-Dawley pups were exposed to hyperoxia or room air from postnatal days 3–10. AT1 antagonist losartan or water was given orally postnatal days 8–10. Blood pressure, autonomic function, left ventricular sympathetic innervation, β-adrenergic-receptors expression, and AT1 expression in the solitary-tract-nucleus were examined in adult rats.Neonatal hyperoxia led to loss of day-night blood pressure variation, decreased heart rate variability, increased sympathovagal balance, increased AT1 expression in the solitary-tract, decreased left ventricle sympathetic innervation, and increased β1-adrenergic-receptor protein expression. Losartan prevented the autonomic changes and AT1 expression in the solitary-tract but did not impact the loss of circadian blood pressure variation nor the changes in sympathetic innervation and in β1-adrenergic-receptor expression.In conclusion, neonatal hyperoxia leads to both central autonomic and cardiac sympathetic changes, partly programmed by neonatal activation of the renin-angiotensin system.

Frequency-coded patterns of sympathetic vasomotor activity are differentially evoked by the paraventricular nucleus of the hypothalamus in the Goldblatt hypertension model.

The paraventricular nucleus of the hypothalamus (PVN) contains premotor neurons involved in the control of sympathetic vasomotor activity. It is known that the stimulation of specific areas of the PVN can lead to distinct response patterns at different target territories. The underlying mechanisms, however, are still unclear. Recent evidence from sympathetic nerve recording suggests that relevant information is coded in the power distribution of the signal along the frequency range. In the present study, we addressed the hypothesis that the PVN is capable of organizing specific spectral patterns of sympathetic vasomotor activation to distinct territories in both normal and hypertensive animals. To test it, we investigated the territorially differential changes in the frequency parameters of the renal and splanchnic sympathetic nerve activity (rSNA and sSNA, respectively), before and after disinhibition of the PVN by bicuculline microinjection. Subjects were control and Goldblatt rats, a sympathetic overactivity-characterized model of neurogenic hypertension (2K1C). Additionally, considering the importance of angiotensin II type 1 receptors (AT1) in the sympathetic responses triggered by bicuculline in the PVN, we also investigated the impact of angiotensin AT1 receptors blockade in the spectral features of the rSNA and sSNA activity. The results revealed that each nerve activity (renal and splanchnic) presents its own electrophysiological pattern of frequency-coded rhythm in each group (control, 2K1C, and 2K1C treated with AT1 antagonist losartan) in basal condition and after bicuculline microinjection, but with no significant differences regarding total power comparison among groups. Additionally, the losartan 2K1C treated group showed no decrease in the hypertensive response triggered by bicuculline when compared to the non-treated 2K1C group. However, their spectral patterns of sympathetic nerve activity were different from the other two groups (control and 2K1C), suggesting that the blockade of AT1 receptors does not totally recover the basal levels of neither the autonomic responses nor the electrophysiological patterns in Goldblatt rats, but act on their spectral frequency distribution. The results suggest that the differential responses evoked by the PVN were preferentially coded in frequency, but not in the global power of the vasomotor sympathetic responses, indicating that the PVN is able to independently control the frequency and the power of sympathetic discharges to different territories.

Beneficial effects of AT1 antagonists treatment, losartan, on cardiovascular and cognitive function on aged male Dahl salt sensitive rats

Introduction and objectiveCardiovascular diseases and hypertension are the major contributors to the pathogenesis of vascular dementia. The Dahl salt‐sensitive (DSS) rat model is characterized by age‐associated cardiovascular remodeling and the development of central arterial stiffness (CAS) in the presence of renin‐angiotensin system disbalance, accompanied by cognitive decline. We hypothesized, that the anti‐hypertensive treatment, antagonist for angiotensin II receptors losartan (LOS), via reducing CAS, will improve cardiovascular parameters and cognitive function in aged DSS male rats.MethodsMale DSS rats (n=30) were kept on a normal salt diet (0.5% NaCl) for the duration of the study. At 6 months of age, rats were given either LOS in drinking water (30mg/kg/day, n=14) or control treatment (n=16) for 6 months. Measurements, including systolic and diastolic BP (SBP, DBP) and pulse wave velocity (PWV), a measure of CAS (by echocardiology), were taken after 6 months of LOS treatment. Rats were tested in an operant chambers (OCh) attention challenge to assess attention and impulsivity; elevated plus maze (EPM) was performed to estimate anxiety‐like behavior. Statistical analyses were performed using 1‐way ANOVA, t‐test and Pearson correlation. Data are presented as mean ± SEM.Resultsfollowing 6 months of treatment, at 12‐mo of age, LOS treated rats had lower BP and PWV vs. control (Table 1). Performance on the OCh indicated that LOS rats had more correct responses vs. control rats (Fig. 1). The LOS group exhibited lower anxiety levels, because they spent less time on the closed shoulder in EPM (Table 1) and demonstrate 1.5‐fold increase of time in the open shoulder (p < 0.01). PWV and SBP was negatively correlated with time on open shoulder, total distance traveled and total correct answers at 12‐mo of age (Fig.2).ConclusionAged male DSS rats show beneficial effects of LOS on cardiovascular and cognitive function. The CAS and BP improvement by LOS was associated with improved attention and lower anxiety levels. The mechanistic basis of LOS effects on cognition via cerebrovascular and brain changes will be further investigated.

Angiotensin II type 1 receptor is involved in hypertension and vascular alterations caused by environmental toxicant hexachlorobenzene.

Environmental hexachlorobenzene (HCB) increases blood pressure (BP) in female rats, causing alterations in arterial structure and function. Here we study the role of Angiotensin II receptor type 1 (AT1) in HCB-induced hypertension through the use of AT1 antagonist losartan. HCB-treated male rats showed a 22.7% increase in BP which was prevented by losartan. Losartan blocked HCB-induced changes in arterial morphology (decreased aorta cell number and increased wall thickness). Losartan also prevented HCB-induced alterations in artery relaxation by acetylcholine and nitroprusside but not the reduction in the maximum contraction by phenylephrine. Losartan rescued arterial molecular alterations caused by HCB (i.e. an increase in TGF-β1 and AT1 expression and a decrease in eNOS expression and nitrite levels) and reduced hydrogen sulfide plasma concentration. In conclusion: in this work we demonstrate that AT1 activity is involved in HCB effects on the vascular system leading to hypertension.

Increased endothelial nitric oxide production after low level lead exposure in rats involves activation of angiotensin II receptors and PI3K/Akt pathway

BackgroundLead induces endothelial dysfunction and hypertension in humans and animals. Seven-day exposure to a low dose in rats reduces vasocontractile responses and increases nitric oxide (NO) bioavailability. We hypothesized that this occurs by angiotensin II receptors (AT1/AT2) activation. Materials and ResultsWistar rats were exposed to lead acetate (1 st dose 4 μg/100 g, subsequent dose 0.05 μg/100 g/day i.m., 7 days) or saline (control group). Lead acetate exposure reduced the phenylephrine vascular response. Pre-incubations with NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) or phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin) increased the contractile response in aortas from lead-treated rats. Pre-incubation with AT2 antagonist (PD123319) restored normal vascular contraction, and both PD123319 or AT1 antagonist (losartan) impeded the potentiated effects of L-NAME and wortmannin. Reinforcing those findings, increased NO bioavailability was blunted by AT1 and AT2 antagonists without summative effect when co-incubated. Finally, to test whether activation of AT1 could upregulate AT2 to increase NO bioavailability rats were simultaneously exposed to lead acetate and treated with losartan (15 mg/kg/day, orally given). Losartan prevented changes on vascular reactivity and endothelial modulation in lead-exposed group. Moreover, incubation with PD123319 had no more effects in aortic from losartan-treated rats. ConclusionOur results suggest that low-dose lead acetate exposure induces an increase of NO involving mainly AT2 receptor activation and the PI3K/Protein Kinase B (PI3K/Akt) pathway. Additionally, we suggest that AT1 activation plays a role in AT2 upregulation, probably as a protective mechanism. Altogether, these effects might contribute to preserving endothelial function against the harmful effects by lead in the vascular system.

Persistent cytosolic Ca2+ increase induced by angiotensin II at nanomolar concentrations in acutely dissociated subfornical organ (SFO) neurons of rats

It is known that angiotensin II (AII) is sensed by subfornical organ (SFO) to induce drinking behaviors and autonomic changes. AII at picomolar concentrations have been shown to induce Ca2+ oscillations and increase in the amplitude and frequency of spontaneous Ca2+ oscillations in SFO neurons. The present study was conducted to examine effects of nanomolar concentrations of AII using the Fura-2 Ca2+-imaging technique in acutely dissociated SFO neurons. AII at nanomolar concentrations induced an initial [Ca2+]i peak followed by a persistent [Ca2+]i increase lasting for longer than 1 hour. By contrast, [Ca2+]i responses to 50 mM K+, maximally effective concentrations of glutamate, carbachol, and vasopressin, and AII given at picomolar concentrations returned to the basal level within 20 min. The AII-induced [Ca2+]i increase was blocked by the AT1 antagonist losartan. However, losartan had no effect when added during the persistent phase. The persistent phase was suppressed by extracellular Ca2+ removal, significantly inhibited by blockers of L and P/Q type Ca2+ channels , but unaffected by inhibition of Ca2+ store Ca2+ ATPase. The persistent phase was reversibly suppressed by GABA and inhibited by CaMK and PKC inhibitors. These results suggest that the persistent [Ca2+]i increase evoked by nanomolar concentrations of AII is initiated by AT1 receptor activation and maintained by Ca2+ entry mechanisms in part through L and P/Q type Ca2+ channels, and that CaMK and PKC are involved in this process. The persistent [Ca2+]i increase induced by AII at high pathophysiological levels may have a significant role in altering SFO neuronal functions.

Angiotensin ll Type 1 Receptor (AT1) Induces Altered Vascular Responses in Allergic Mice via NOX4 and HIF‐a

Airway diseases like asthma leads to cardiovascular disease (CVD). We have shown that in a mouse model of asthma, airway hypersensitivity is exacerbated by Angiotensin II (Ang II) via AT1 receptor activation (Am J Respir Crit Care Med. 2015;191:A5663). In this study (see graphic for hypothesis), we investigated the contribution of NOX4 to Ang II-mediated aortic responses in a mouse model of allergic asthma developed in this laboratory. The effects of NOX4 inhibitor GKT137831, HIF-1α inhibitor FM19 and thioredoxin (TRX) activator auranofin were studied on Ang-ll induced-contraction in isolated aortic rings from asthmatic mice using ex-vivo organ bath. Mice were divided into control (CON) and allergen sensitized-challenged (SEN) groups. Mice were sensitized (i.p.) on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. AT1 antagonist losartan was given i.p. as a single bolus dose (20mg/kg) on day 14 to SEN mice. Organ bath studies were done on day 14 and Western Blot analyses were done to determine aortic expression of AT1, NOX4 as well as p22phox, the subunits of NOX4. In the SEN group, Ang II (10μM) produced higher aortic contraction (33.48±3.26% vs vs. 18.06±3.07% in CON; p<0.05) that was lowered significantly with the use of inhibitors for HIF-1α (10μM; 13.70%±7.43%) and NOX4 (3.54%±6.61%) respectively. Studies have shown that TRX can inhibit Ang ll, HIF-1α sand NOX 4 in the vascular system. Our data showed that TRX activator (auranofin) inhibited Ang ll-mediated contraction to a greater extent in CON mice than SEN. Aortic AT1 receptor expression was significantly higher in SEN mice (1.74±0.08) than CON (1.00±0.13). The expression of NOX4 (1.27±0.06 vs. 0.99±0.09) and p22phox (3.45±0.35 vs. 1.35±0.62) were significantly higher in SEN mice compared to CON. Treatment with losartan significantly attenuated the increases in NOX4 (from1.27±0.06 to 0.67±0.11) and p22phox (from 3.45±0.35 to 0.26±0.11) in SEN mice. These data suggest that most Ang ll through HIF-1α and upregulation by Nox 4, may contribute to altered vascular responses in allergic mice, while TRX antioxidant effect is lower in SEN mice. AT1 antagonist losartan reverses all of these effects, and thus may prevent altered vascular responses to Ang ll in asthma. Support or Funding Information Institutional Research Grant Long Island University SCHEMATIC REPRESENTATION OF HYPOTHESIS TESTED AND PROPOSED PATHWAY FOR ANG ll-MEDIATED VASCULAR CONTRACTION IN ASTHMA This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Local activation of the pulmonary extravascular angiotensin system induces epithelial apoptosis and lung fibrosis.

Previous work suggests that a local extravascular angiotensin system plays an important role in the development of pulmonary fibrosis through stimulation of alveolar epithelial cell (AEC) apoptosis and collagen deposition. To demonstrate a causative role for the local tissue angiotensin (ANG) system in lung fibrosis, we hypothesize that overexpression of the angiotensinogen (AGT) gene or pharmacologic elevation of lung tissue ANG II levels might cause apoptosis of AECs and lung fibrosis. ANGII levels were elevated in rat or mouse lung tissue by intratracheal instillation of either purified ANGII or an adenovirus expressing AGT, or by ubiquitous overexpression of AGT in transgenic mice. Intratracheal instillation of purified ANGII caused significant collagen accumulation in lung tissue, both ex vivo and in vivo. Ubiquitous overexpression of AGT enhanced the profibrotic effect of bleomycin given at suboptimal doses. Intratracheal delivery of an adenoviral vector expressing mouse AGT (Ad-AGT) overexpressed AGT primarily in AECs and caused both apoptosis of AECs and pulmonary fibrosis. The lung collagen accumulation and AEC apoptosis caused by Ad-AGT was blocked by the caspase inhibitor ZVAD-fmk, by the ANG receptor AT1 antagonist Losartan or by the non-selective ANGII receptor antagonist Saralasin. Together, these data support the hypothesis that elevated pulmonary expression of AGT and its conversion to angiotensin II plays a causative role in the development of lung fibrosis through its induction of AEC apoptosis.

Angiotensin ll Type 2 (AT2) Receptors: Novel Target in Asthma

Angiotensin ll (Ang ll) is produced locally in the lungs and may potentiate airway responsiveness in asthmatic patients. Ang ll induces its effects by activating two GPCR subtypes: AT1 and AT2. This lab has previously found that AT1 antagonist losartan reduces airway inflammation and reactivity. The AT2 receptors counterbalance the effects of AT1 and are known to be anti‐inflammatory. However, it is not known if AT2 receptors have any effects in asthma. We studied the effects of AT2 agonist novokinin and antagonist PD 12339 administered in vivo in a mouse model of asthma. Mice were divided into control (non‐allergic; CON) and allergen sensitized‐challenged (SEN) groups, and were sensitized i.p. on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. Methacholine (MCh; 48mg/ml) produced significantly higher airway responsiveness in allergic mice compared to control (563.71±40% in SEN vs. 208.79±20.84% in CON, p&lt;0.05). However, novokinin (i.p. 0.3mg/kg) treated SEN mice had significantly attenuated MCh‐indcued airway responsiveness (563.71±40% in SEN vs. 252±11% in SEN+Novokinin, p&lt;0.001). The AT2 antagonist PD 12339 (i.p.5mg/kg) significantly increased MCh airway responsiveness (563.71±40% in SEN vs. 757±30% in SEN+PD 12239, p&lt;0.05). Differential BAL cell analysis showed novokinin also decreased eosinophils (221±21 in SEN vs. 122±18 in SEN+PD 12239, p&lt;0.001), while PD 12239 had no effect on eosinophils (245± 16 cells in SEN+PD 12339). Our data suggests that activation of AT2 receptors may attenuate airway reactivity and inflammation in our model of asthma.Support or Funding InformationLong Island University start‐up funds (DSP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Beta-Arrestin1 Prevents Preeclampsia by Down-Regulation of Mechanosensitive AT1-B2 Receptor Heteromers

Preeclampsia is the most frequent pregnancy-related complication worldwide with no cure. In search for underlying pathomechanisms, we found that transgenic AT1-B2 receptor expression under control of the smooth muscle-specific SM22alpha promoter was sufficient to trigger symptoms of preeclampsia in pregnant mice. Vascular smooth muscle cells from AT1-B2-transgenic mice showed hypersensitive calcium signaling and increased mechanosensitivity. This phenotype was attributed to AT1-B2 heteromerization because down-regulation of AT1- B2 by the beta-arrestin-biased AT1 antagonist, SII, reversed AT1-B2mediated effects in vascular smooth muscle cells whereas the unbiased AT1 antagonist losartan was ineffective. In vivo, down-regulation of AT1B2 by lentiviral transduction of ARRB1-S412A inhibited preeclampsia. Likewise, induction of Arrb1 by the small-molecule drug amlodipine, promoted AT1-B2 down-regulation, and prevented AT1-B2-induced mechanosensitivity and symptoms of preeclampsia. The deduced treatment strategy could improve the therapy of human preeclampsia because vascular AT1-B2 heteromerization was also identified as a characteristic feature of placental biopsies from pregnancies complicated by preeclampsia.

Aberrant Angiotensin Signaling in a Mouse Model of Post‐Traumatic Stress Disorder (PTSD)

Independent of their beneficial effects on hypertension and cardiovascular related disease, angiotensin receptor type 1 (AT1R) blockers can improve stress-related symptoms. AT1R receptor-mediated actions can be counteracted directly or indirectly by the angiotensin receptor type 2 receptor (AT2R). Our recent studies in a mouse model of PTSD have shown that AT1R blockade increases the extinction (learned inhibition) of a traumatic fear memory and that AT1R mRNA expression is reduced in fear related brain regions of losartan treated animals. These data imply that downstream AT1 signaling events maybe important in consolidation of fear memory extinction. Therefore we investigated the acute effects of AT2R inhibition on fear memory and baseline anxiety. We performed classical Pavlovian fear conditioning pairing auditory cues with foot shocks and examined fear extinction behavior and cardiovascular responses in the presence of the AT2R antagonist PD 123319. Twenty-four hours following fear conditioning, PD 123319 (15 mg/kg IP) was administered prior to fear memory extinction. The PD treated group exhibited significantly less freezing behavior (F10, 300 = 1.9; p<0.05) during fear expression and contrary to our previous results with the AT1 antagonist losartan, there was no effect during extinction retention, an index of long-term fear memory. These data indicate that AT1R and AT2R may have divergent effects on short and long-term fear memory formation. Further studies are required to understand the differential regulation of angiotensin receptor signaling in PTSD.

Mo1885 Role of the Hippo-YAP Pathway in the Regulation of DNA Synthesis by cAMP in Intestinal Epithelial Cells and Swiss 3T3 Fibroblasts

Background: The mammalian Hippo pathway, comprised by the Mst1/2 and Lats1/2 kinase cascade, has been implicated in the regulation of organ size in response to cell-cell contact and diffusible molecules, including those that act via G protein-coupled receptors (GPCRs). The transcription co-activator Yes-associated protein (YAP) is a major downstream effector of the Hippo pathway. The precise role of YAP in intestinal epithelial cell proliferation, regeneration and cancer remains controversial, as evidence for both growth-stimulatory and growth-suppressive roles of YAP in the gut have been presented. Given the fundamental role of the Hippo-YAP pathway in cell regulation, it is vital to define the function of YAP in intestinal epithelial cells. Results: In order to identify a model system to explore GPCRmediated regulation of the Hippo pathway in intestinal epithelial cells, we determined the localization of endogenous YAP1/2 in response to the Gq-coupled receptor agonist angiotensin II (ANGII) in IEC-6 and IEC-18 cells. ANGII is a regulatory peptide generated in the GI tract increasingly recognized to play a role in GI mucosal inflammation and carcinogenesis. In un-stimulated IEC-18 or IEC-6 cells, YAP1/2 was located in the nucleus and cytoplasm. Stimulation of these cells with ANG II induced a rapid and dramatic cytoplasmic relocalization of YAP1/2. Pretreatment with the selective AT1 antagonist Losartan completely prevented ANG II-induced YAP1/2 re-localization, verifying that this unexpected effect was mediated by the high affinity Gq-coupled ANG II receptor. The re-localization of YAP1/2 in response to ANG II was dose-dependent and could be detected as early 15 min after stimulation and subsided after 2-4 h of stimulation. Similar findings were made with IEC6 cells. These novel results indicate that ANG II induces biphasic re-distribution of YAP1/ 2 in intestinal epithelial cells characterized by strong and transient cytoplasmic localization followed by subsequent nuclear re-localization. We next determined whether ANG II induces YAP phosphorylation at Ser127 and Ser391 in IEC-18 cells, highly conserved residues located within a consensus Lats1/2 sequence (HXRXXS). Lysates of IEC-18 cells stimulated with ANGII for various times were analyzed by Western blotting with antibodies that detect the phosphorylated state of YAP1 at Ser127 and at Ser391. We found that ANGII induced a rapid (detectable within 15 min) and transient (persisted for 2 h) increase in YAP phosphorylation in intestinal epithelial IEC-18 cells. Conclusion: These novel results demonstrated that induces YAP re-localization and phosphorylation at residues targeted by Lats1/2 in intestinal epithelial cells. As ANG II is a potent mitogen for IEC-18 cells, we hypothesize that YAP acts sequentially in the cytoplasm and nucleus to promote proliferation in intestinal epithelial cells.

Mo1884 Regulation of YAP Localization and Phosphorylation in Intestinal Epithelial Cells

Background: The mammalian Hippo pathway, comprised by the Mst1/2 and Lats1/2 kinase cascade, has been implicated in the regulation of organ size in response to cell-cell contact and diffusible molecules, including those that act via G protein-coupled receptors (GPCRs). The transcription co-activator Yes-associated protein (YAP) is a major downstream effector of the Hippo pathway. The precise role of YAP in intestinal epithelial cell proliferation, regeneration and cancer remains controversial, as evidence for both growth-stimulatory and growth-suppressive roles of YAP in the gut have been presented. Given the fundamental role of the Hippo-YAP pathway in cell regulation, it is vital to define the function of YAP in intestinal epithelial cells. Results: In order to identify a model system to explore GPCRmediated regulation of the Hippo pathway in intestinal epithelial cells, we determined the localization of endogenous YAP1/2 in response to the Gq-coupled receptor agonist angiotensin II (ANGII) in IEC-6 and IEC-18 cells. ANGII is a regulatory peptide generated in the GI tract increasingly recognized to play a role in GI mucosal inflammation and carcinogenesis. In un-stimulated IEC-18 or IEC-6 cells, YAP1/2 was located in the nucleus and cytoplasm. Stimulation of these cells with ANG II induced a rapid and dramatic cytoplasmic relocalization of YAP1/2. Pretreatment with the selective AT1 antagonist Losartan completely prevented ANG II-induced YAP1/2 re-localization, verifying that this unexpected effect was mediated by the high affinity Gq-coupled ANG II receptor. The re-localization of YAP1/2 in response to ANG II was dose-dependent and could be detected as early 15 min after stimulation and subsided after 2-4 h of stimulation. Similar findings were made with IEC6 cells. These novel results indicate that ANG II induces biphasic re-distribution of YAP1/ 2 in intestinal epithelial cells characterized by strong and transient cytoplasmic localization followed by subsequent nuclear re-localization. We next determined whether ANG II induces YAP phosphorylation at Ser127 and Ser391 in IEC-18 cells, highly conserved residues located within a consensus Lats1/2 sequence (HXRXXS). Lysates of IEC-18 cells stimulated with ANGII for various times were analyzed by Western blotting with antibodies that detect the phosphorylated state of YAP1 at Ser127 and at Ser391. We found that ANGII induced a rapid (detectable within 15 min) and transient (persisted for 2 h) increase in YAP phosphorylation in intestinal epithelial IEC-18 cells. Conclusion: These novel results demonstrated that induces YAP re-localization and phosphorylation at residues targeted by Lats1/2 in intestinal epithelial cells. As ANG II is a potent mitogen for IEC-18 cells, we hypothesize that YAP acts sequentially in the cytoplasm and nucleus to promote proliferation in intestinal epithelial cells.

Mo1907 A Cohort Study of Risk Factors for Developing Erosive Esophagitis Following Negative Index Endoscopy in a Large Health Check-Up Population

Background: The mammalian Hippo pathway, comprised by the Mst1/2 and Lats1/2 kinase cascade, has been implicated in the regulation of organ size in response to cell-cell contact and diffusible molecules, including those that act via G protein-coupled receptors (GPCRs). The transcription co-activator Yes-associated protein (YAP) is a major downstream effector of the Hippo pathway. The precise role of YAP in intestinal epithelial cell proliferation, regeneration and cancer remains controversial, as evidence for both growth-stimulatory and growth-suppressive roles of YAP in the gut have been presented. Given the fundamental role of the Hippo-YAP pathway in cell regulation, it is vital to define the function of YAP in intestinal epithelial cells. Results: In order to identify a model system to explore GPCRmediated regulation of the Hippo pathway in intestinal epithelial cells, we determined the localization of endogenous YAP1/2 in response to the Gq-coupled receptor agonist angiotensin II (ANGII) in IEC-6 and IEC-18 cells. ANGII is a regulatory peptide generated in the GI tract increasingly recognized to play a role in GI mucosal inflammation and carcinogenesis. In un-stimulated IEC-18 or IEC-6 cells, YAP1/2 was located in the nucleus and cytoplasm. Stimulation of these cells with ANG II induced a rapid and dramatic cytoplasmic relocalization of YAP1/2. Pretreatment with the selective AT1 antagonist Losartan completely prevented ANG II-induced YAP1/2 re-localization, verifying that this unexpected effect was mediated by the high affinity Gq-coupled ANG II receptor. The re-localization of YAP1/2 in response to ANG II was dose-dependent and could be detected as early 15 min after stimulation and subsided after 2-4 h of stimulation. Similar findings were made with IEC6 cells. These novel results indicate that ANG II induces biphasic re-distribution of YAP1/ 2 in intestinal epithelial cells characterized by strong and transient cytoplasmic localization followed by subsequent nuclear re-localization. We next determined whether ANG II induces YAP phosphorylation at Ser127 and Ser391 in IEC-18 cells, highly conserved residues located within a consensus Lats1/2 sequence (HXRXXS). Lysates of IEC-18 cells stimulated with ANGII for various times were analyzed by Western blotting with antibodies that detect the phosphorylated state of YAP1 at Ser127 and at Ser391. We found that ANGII induced a rapid (detectable within 15 min) and transient (persisted for 2 h) increase in YAP phosphorylation in intestinal epithelial IEC-18 cells. Conclusion: These novel results demonstrated that induces YAP re-localization and phosphorylation at residues targeted by Lats1/2 in intestinal epithelial cells. As ANG II is a potent mitogen for IEC-18 cells, we hypothesize that YAP acts sequentially in the cytoplasm and nucleus to promote proliferation in intestinal epithelial cells.

Abstract 575: Complement Activation in Placental Ischemia-induced Hypertension is not dependent on Angiotensin II Type 1 Receptor

Early onset preeclampsia is characterized by hypertension, reduced placental perfusion, intrauterine growth restriction and increased activation of complement, part of innate immunity. We reported that inhibition of complement activation attenuated reduced uterine perfusion pressure (RUPP)-induced hypertension in the pregnant rat. Although recent studies in pregnant mice indicate injection of autoantibodies to angiotensin II Type 1 receptor (AT1-AA) activate complement and increase blood pressure, the sequence of events following placental ischemia remains unknown. Thus, we hypothesized AT1-AA interaction with its antigen was responsible for complement activation critical to placental ischemia-induced hypertension. The AT1 antagonist losartan prevents AT1-AA interaction with the AT1 receptor. Thus, we determined the effect of losartan on complement activation following placental ischemia in rat. Dams received drinking water with or without 30 mg/kg/day losartan on gestation day (GD)13-19. On GD14, rats underwent Sham surgery or RUPP surgery with placement of clips on abdominal aorta and uterine arteries to decrease placental perfusion. On GD19, mean arterial pressure (MAP) via arterial catheter and serum C3a as an indicator of complement activation were measured. As expected, RUPP surgery increased complement activation (C3a) and MAP and decreased fetal weight compared to Sham. Importantly, losartan treatment did not significantly change RUPP-induced increase in C3a. These data indicate AT1-AA interaction with the AT1 receptor does not mediate complement activation and its subsequent involvement in hypertension following placental ischemia.

Angiotensin Type 1 Receptor Antagonists—A Novel Approach to Augmenting Posttraumatic Stress Disorder and Phobia Therapies?

The renin-angiotensin system system, which includes the renin and angiotensin-converting enzyme pathways, and its primary effector peptide angiotensin II (AII) acting through its two cognate receptors, AT1 and AT2 receptors, has been known for 70 years as a major regulator of vascular tone and fluid metabolism (1). Angiotensin receptor blockers (ARBs), consisting of a group of orally active drugs such as losartan (the group is often referred to as the “sartans”), and angiotensinconverting enzyme inhibitors are currently mainstream treatments for essential hypertension and some aspects of diabetes. In addition to these peripheral effects, AII and AT1 receptors are widely distributed in the brain and are being implicated in the regulation of many brain functions, including cerebral circulation, inflammation, neuroendocrine responses, learning, and memory (2). Because many ARBs are already on the market and have a good safety record with long-term use, they would be particularly attractive candidates to be used as novel treatments for central nervous system disorders where current therapies are inadequate. The article by Marvar et al. (3) in this issue of Biological Psychiatry may provide a strong impetus to consider testing ARBs, particularly as an adjunct therapy to augment cognitive-behavioral therapy, in posttraumatic stress disorder (PTSD) and phobias. The idea that ARBs may have some beneficial effects for patients with PTSD first arose from a correlational finding in an epidemiologic study, where veterans treated with ARBs for their medical conditions appeared to develop a less severe form of PTSD compared with veterans who were not taking ARBs (4). Although such correlations may turn out to be spurious, Marvar et al. decided to test whether the beneficial effect of ARBs is mechanistically demonstrable using preclinical models. There are robust animal models that can be used to study the basic circuits and phenotypes involved in PTSD symptoms. Using a standard auditory-cue paired conditioned fear model in mice, Marvar et al. tested the effects of single and multiple doses of selective AT1 antagonist losartan on fear extinction behavior, gene expression changes in the brain, and neuroendocrine and cardiovascular responses. They found that losartan treatment enhanced the consolidation of fear extinction without affecting acquisition, baseline anxiety, blood pressure, and neuroendocrine stress measures. Mice treated with losartan for 2 weeks also demonstrated reduced amygdala AT1 receptor messenger RNA (mRNA) levels. The authors concluded that AT1 receptor antagonism does have potential beneficial effects in a preclinical model of PTSD symptoms and could enhance the extinction of fear memories in PTSD.

Insights into the molecular basis of action of the AT1 antagonist losartan using a combined NMR spectroscopy and computational approach

The drug:membrane interactions for the antihypertensive AT1 antagonist losartan, the prototype of the sartans class, are studied herein using an integrated approach. The pharmacophore arrangement of the drug was revealed by rotating frame nuclear Overhauser effect spectroscopy (2D ROESY) NMR spectroscopy in three different environments, namely water, dimethyl sulfoxide (DMSO), and sodium dodecyl sulfate (SDS) micellar solutions mimicking conditions of biological transport fluids and membrane lipid bilayers. Drug association with micelles was monitored by diffusion ordered spectroscopy (2D DOSY) and drug:micelle intermolecular interactions were characterized by ROESY spectroscopy. The localisation of the drug in the micellar environment was investigated by introducing 5-doxyl and 16-doxyl stearic acids. The use of spin labels confirmed that losartan resides close to the micelle:water interface with the hydroxymethyl group and the tetrazole heterocyclic aromatic ring facing the polar surface with the potential to interact with SDS charged polar head groups in order to increase amphiphilic interactions. The spontaneous insertion, the diffusion pathway and the conformational features of losartan were monitored by Molecular Dynamics (MD) simulations in a modeled SDS micellar aggregate environment and a long exploratory MD run (580ns) in a phospholipid dipalmitoylphosphatidylcholine (DPPC) bilayer with the AT1 receptor embedded. MD simulations were in excellent agreement with experimental results and further revealed the molecular basis of losartan:membrane interactions in atomic-level detail. This applied integrated approach aims to explore the role of membranes in losartan's pathway towards the AT1 receptor.

Angiotensin II AT2receptor activation attenuates AT1receptor-induced increases in the glomerular filtration of albumin: a multiphoton microscopy study

In this study, we assessed the acute effects of angiotensin II on the albumin glomerular sieving coefficient (GSC) using intravital microscopy. The experiments were performed on Munich Wistar Froemter (MWF) rats. Alexa-Fluor-594 albumin was injected intravenously, and the fluorescence intensity in the glomerular capillaries and Bowman's space was determined to calculate the albumin GSC. The GSC was measured before and during the constant infusion of angiotensin II (10 ng·min(-1)·kg(-1) body wt). Baseline mean arterial pressure (MAP) was 99 ± 5 mmHg and stabilized at 137 ± 5 mmHg during angiotensin II infusion. The baseline GSC averaged 0.00044 ± 4.8 × 10(-5) and increased by 286 ± 44% after angiotensin II infusion (P < 0.0001). The proximal tubular Alexa-Fluor-594 albumin uptake was enhanced during angiotensin II infusion (518% of the baseline value during angiotensin II vs. 218% in controls; P < 0.0001). No change in GSC was observed when the AT1 antagonist losartan was injected before the start of angiotensin II infusion. The AT2 antagonist PD123319 increased the baseline GSC from 0.00052 ± 3.6 × 10(-5) to 0.00074 ± 8.2 × 10(-5) (P = 0.02) without altering the MAP. During angiotensin II infusion with losartan, PD123319 increased the albumin GSC from 0.00037 ± 5.8 × 10(-5) to 0.00115 ± 0.00015 (P = 0.001). When the renal perfusion pressure was mechanically controlled, the GSC increased from 0.0007 ± 0.00019 to 0.0025 ± 0.00063 during angiotensin II infusion (P = 0.047), similar to what was observed when the renal perfusion pressure was allowed to increase. In summary, AT1 activation acutely increases the albumin GSC. This effect appears to be largely independent of changes in the renal perfusion pressure. The AT2 receptor partially attenuates the proteinuric effects of the AT1 receptor.

Angiotensin AT1 and AT2 receptor antagonists modulate nicotine-evoked [3H]dopamine and [3H]norepinephrine release

Tobacco smoking is the leading preventable cause of death in the United States. A major negative health consequence of chronic smoking is hypertension. Untoward addictive and cardiovascular sequelae associated with chronic smoking are mediated by nicotine-induced activation of nicotinic receptors (nAChRs) within striatal dopaminergic and hypothalamic noradrenergic systems. Hypertension involves both brain and peripheral angiotensin systems. Activation of angiotensin type-1 receptors (AT1) release dopamine and norepinephrine. The current study determined the role of AT1 and angiotensin type-2 (AT2) receptors in mediating nicotine-evoked dopamine and norepinephrine release from striatal and hypothalamic slices, respectively. The potential involvement of nAChRs in mediating effects of AT1 antagonist losartan and AT2 antagonist, 1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid (PD123319) was evaluated by determining their affinities for α4β2* and α7* nAChRs using [3H]nicotine and [3H]methyllycaconitine binding assays, respectively. Results show that losartan concentration-dependently inhibited nicotine-evoked [3H]dopamine and [3H]norepinephrine release (IC50: 3.9±1.2 and 2.2±0.7μM; Imax: 82±3 and 89±6%, respectively). In contrast, PD123319 did not alter nicotine-evoked norepinephrine release, and potentiated nicotine-evoked dopamine release. These results indicate that AT1 receptors modulate nicotine-evoked striatal dopamine and hypothalamic norepinephrine release. Furthermore, AT1 receptor activation appears to be counteracted by AT2 receptor activation in striatum. Losartan and PD123319 did not inhibit [3H]nicotine or [3H]methyllycaconitine binding, indicating that these AT1 and AT2 antagonists do not interact with the agonist recognition sites on α4β2* and α7* nAChRs to mediate these effects of nicotine. Thus, angiotensin receptors contribute to the effects of nicotine on dopamine and norepinephrine release in brain regions involved in nicotine reward and hypertension.

Acute changes in glomerular albumin filtration during systemic infusion of angiotensin II – a multiphoton microscopy study

Antiproteinuric effects of ACE inhibitors have classically been assigned to their hemodynamic effects. Here we assess the acute effects of angiotensin II (AngII) on albumin glomerular sieving coefficient (GSC) in young anesthetized Munich Wister Froemter rats using intravital microscopy. Alexa‐Fluor‐594 albumin was injected i.v. and fluorescence intensities were determined in glomerular capillaries and in Bowman's space (BS). The albumin GSC was then calculated according to: GSC = (intensity BS – intensity background)/(intensity capillaries – intensity background). GSC was measured before and during constant infusion of Ang II (27 ng/min/g BW). Baseline MAP averaged 90±4 mm Hg and stabilized at 130+/−11 mm Hg during AngII infusion. Capillary flow velocity was reduced during AngII infusion. Albumin GSC averaged 0.00038+/−.00009 at baseline and increased by 263+/−34% within 10 min after constant AngII infusion. During AngII infusion, de novo fluorescence was observed in the proximal tubules indicating uptake of filtered Alexa‐albumin. These effects were abolished when a bolus of the AT1 antagonist losartan (9.9 μg/g BW) was injected prior to AngII. In control experiments (saline infusion) no significant changes in MAP, GSC or tubular fluorescence were observed. In summary, AngII causes rapid increases in glomerular albumin filtration, which are presumably mediated by angiotensin AT1 receptors.