ETA Blockade Research Articles

The Role of Endothelin Receptors on Sensory Nerve Mediated Dilation in Postmenopausal Women

Rapid local heating of the skin elicits biphasic vasodilation. The initial peak in skin blood flow (SkBF) is mediated by sensory nerves (i.e., the axon reflex). Both endothelin (ETA and ETB) receptors are located on axon terminals on nociceptor fibers, and therefore may be involved in the sensory nerve mediated vasodilation. Although prior work shows a blunted axon reflex with aging, this has yet to be examined in women, and the role of ETA and ETB receptors in this initial sensory nerve‐mediated vasodilatory response remains unclear.PURPOSEWe tested the hypothesis that the initial peak is blunted in postmenopausal women (POST) compared to young women (YW), and that blockade of ETA and ETB receptors would improve sensory nerve‐mediated dilation in POST.METHODSWe retrospectively analyzed data in 25 healthy YW (27±10 yr) and 16 healthy POST (56±1 yr). Cutaneous vasodilation to local heating was measured using laser doppler flowmetry during microdialysis perfusions of lactated Ringer’s (control), ETA receptor blockade (BQ‐123, 500nM), and ETB receptor blockade (BQ‐788, 300nM). Cutaneous vascular conductance (CVC) was calculated as SkBF/MAP at baseline and during the initial peak of local heating (42°C); CVC were normalized to maximal vasodilation achieved by perfusion of sodium nitroprusside (28mM) and heating to 43°C.RESULTSAll women were normotensive (MAP: YW 83±8 mmHg; POST 94±2 mmHg, p < 0.001) and not obese (YW 23±3 kg/m2; POST 24±1 kg/m2, p = 0.153). Sensory nerve‐mediated dilation at the control site was reduced in POST (YW 63 ±14 vs POST 49 ±17 %CVCmax, p=0.004). Blockade of ETA (YW 54 ±10 vs POST 55 ±9 %CVCmax, p=0.579) and ETB (YW 62 ±17 vs POST 59 ±13 %CVCmax, p=0.476) receptors did not impact the initial peak of local heating in either group.CONCLUSIONSThese preliminary data suggest the sensory nerve‐mediated vasodilation during local heating is attenuated with aging in women, but endothelin receptors do not contribute to this age‐related decrease. Additional research is needed to further understand the interactions between sex hormones and aging in regulating sensory nerve mediated vasodilation.

The acute pressure natriuresis response is suppressed by selective ETA receptor blockade.

Hypertension is a major risk factor for cardiovascular disease. In a significant minority of people, it develops when salt intake is increased (salt-sensitivity). It is not clear whether this represents impaired vascular function or disruption to the relationship between blood pressure (BP) and renal salt-handling (pressure natriuresis, PN). Endothelin-1 (ET-1) regulates BP via ETA and ETB receptor subtypes. Blockade of ETA receptors reduces BP but promotes sodium retention by an unknown mechanism. ETB blockade increases both BP and sodium retention. We hypothesized that ETA blockade promotes sodium and water retention by suppressing PN. We also investigated whether suppression of PN might reflect off-target ETB blockade. Acute PN was induced by sequential arterial ligation in male Sprague Dawley rats. Intravenous atrasentan (ETA antagonist, 5 mg/kg) halved the normal increase in medullary perfusion and reduced sodium and water excretion by >60%. This was not due to off-target ETB blockade because intravenous A-192621 (ETB antagonist, 10 mg/kg) increased natriuresis by 50% without modifying medullary perfusion. In a separate experiment in salt-loaded rats monitored by radiotelemetry, oral atrasentan reduced systolic and diastolic BP by ∼10 mmHg, but additional oral A-192621 reversed these effects. Endogenous ETA stimulation has natriuretic effects mediated by renal vascular dilation while endogenous ETB stimulation in the kidney has antinatriuretic effects via renal tubular mechanisms. Pharmacological manipulation of vascular function with ET antagonists modifies the BP set-point, but even highly selective ETA antagonists attenuate PN, which may be associated with salt and water retention.

High Glucose Induced Changes in Human VEC Phenotype in a 3D Hydrogel Derived From Cell-Free Native Aortic Root.

Background: Valvular endothelial cells (VEC) have key roles in maintaining valvular integrity and homeostasis, and dysfunctional VEC are the initiators and major contributors to aortic valve disease in diabetes. Previous studies have shown that HG stimulated an inflammatory phenotype in VEC. Inflammation was shown to induce endothelial to mesenchymal transition (EndMT), a process extensively involved in many pathologies, including calcification of the aortic valve. However, the effect of HG on EndMT in VEC is not known. In addition, there is evidence that endothelin (ET) is a proinflammatory agent in early diabetes and was detected in aortic stenosis, but it is not known whether HG induces ET and endothelin receptors and whether endothelin modulates HG-dependent inflammation in VEC. This study aims to evaluate HG effects on EndMT, on endothelin and endothelin receptors induction in VEC and their role in HG induced VEC inflammation.Methods and Results: We developed a new 3D model of the aortic valve consisting of a hydrogel derived from a decellularized extracellular cell matrix obtained from porcine aortic root and human valvular cells. VEC were cultured on the hydrogel surface and VIC within the hydrogel, and the resulted 3D construct was exposed to high glucose (HG) conditions. VEC from the 3D construct exposed to HG exhibited: attenuated intercellular junctions and an abundance of intermediate filaments (ultrastructural analysis), decreased expression of endothelial markers CD31 and VE–cadherin and increased expression of the mesenchymal markers α-SMA and vimentin (qPCR and immunocytochemistry), increased expression of inflammatory molecules ET-1 and its receptors ET-A and ET-B, ICAM-1, VCAM-1 (qPCR and Immunocytochemistry) and augmented adhesiveness. Blockade of ET-1 receptors, ET-A and ET-B reduced secretion of inflammatory biomarkers IL-1β and MCP-1 (ELISA assay).Conclusions: This study demonstrates that HG induces EndMT in VEC and indicates endothelin as a possible target to reduce HG-induced inflammation in VEC.

Prenatal endothelin or thromboxane receptor antagonism surpasses sympathoinhibition in improving cardiorenal malfunctions in preeclamptic rats

Current therapies for preeclampsia (PE) and its complications are limited and defective. Considering the importance of endothelin (ET) and thromboxane A2 (TXA2) signaling in PE pathophysiology, we tested the hypothesis that prenatal blockade of endothelin ETA or thromboxane TXA2 receptors favorably reprograms preeclamptic cardiovascular and renal insults. PE was induced by daily oral administration of L-NAME (50 mg/kg) to pregnant rats for 7 consecutive days starting from gestational day 14. The effects of co-exposure to atrasentan (ETA receptor blocker, 10 mg/kg/day) or terutroban (TXA2 receptor blocker, 10 mg/kg/day) on cardiovascular and renal anomalies induced by PE were assessed on gestational day 20 (GD20) and at weaning time and compared with those evoked by the sympatholytic drug α-methyldopa (α-MD, 100 mg/kg/day), a prototypic therapy for PE management. Among all drugs, terutroban was basically the most potent in ameliorating PE-evoked increments in blood pressure and decrements in creatinine clearance. Cardiorenal tissues of PE rats exhibited significant increases in ETA and TXA2 receptor expressions and these effects disappeared after treatment with atrasentan and to a lesser extent by terutroban or α-MD. Atrasentan was also the most effective in reversing the reduced ETB receptor expression in renal tissues of PE rats. Signs of histopathological damage in cardiac and renal tissues of PE rats were mostly improved by all therapies. Together, pharmacologic elimination of ETA or TXA2 receptors offers a relatively better prospect than α-MD in controlling perinatal cardiorenal irregularities sparked by PE.

Prenatal Endothelin or Thromboxane Receptor Antagonism Surpasses Sympathoinhibition in Managing Cardiovascular and Renal Malfunctions in Preeclamptic Rats

Current therapies for hypertension and end-organ damage caused by preeclampsia (PE) are limited and defective. Considering the importance of endothelin (ET) and thromboxane A2 (TXA2) signaling in PE pathophysiology, we tested the hypothesis that pharmacologic blockade of ETA or TXA2 receptors improves preeclamptic cardiovascular and renal insults. PE was induced by daily oral administration of L-NAME (50 mg/kg) to pregnant rats for 7 consecutive days starting from gestational day 14. The effects of co-exposure to atrasentan (ETA receptor blocker, 10 mg/kg/day) or terutroban (TXA2 receptor blocker, 10 mg/kg/day) on cardiovascular and renal anomalies induced by PE were assessed at gestational day 20 (GD20) and weaning time and compared to those evoked by α-methyldopa (10 mg/kg/day), the prototypic drug for PE management. Among all 3 drugs, terutroban was basically the most potent in ameliorating PE-evoked increments in blood pressure and decrements in urine sodium and creatinine clearance. The gene expression of ETA, but not TXA2, receptors was significantly increased in cardiac and renal tissues of PE rats both at GD20 and weaning and these effects disappeared after co-treatment with individual protective drugs. By contrast, ETB receptor expression was reduced in PE renal tissues and restored back to non-PE levels by atrasentan, but not α-methyldopa or terutroban. Signs of histopathological damage in cardiac and renal tissues of PE rats were indiscriminately improved by all therapies. Together, pharmacologic elimination of ETA or TXA2 receptors offers a relatively better prospect than α-methyldopa in controlling perinatal cardiovascular and renal complications sparked by PE.

HYPOTENSIVE EFFECTIVENESS OF THE INTRARENAL MEDULLARY ENDOTHELIN ETA RECEPTOR BLOCKADE IN THE TWO ANIMAL MODELS OF HYPERTENSION

Objective: Endothelin-1 (ET-1) is involved in the pathogenesis of arterial hypertension and chronic kidney disease, however, its mechanism of action is unclear. ET-1 is known to act via ETA and ETB receptors; their activation leads, respectively, to constriction or NO-mediated dilation of the systemic and renal vasculature. ET-1 levels are elevated in experimental and in human hypertension. Here we examined the effects of ETA receptor blockade with atrasentan, administered intravenously (i.v.) or directly to the renal medullary interstitium (i.med.) in two models of rat hypertension. Design and method: In anaesthetized rats with hypertension induced by uninephrectomy followed by high-salt (4% Na w/w) diet (HS/UNX) and in spontaneously hypertensive rats (SHR) we measured mean arterial pressure (MAP), total renal blood flow (RBF, renal artery probe), renal medullary blood flow (MBF, laser-Doppler flux), and renal medullary nitric oxide (NO) signal. Atrasentan doses were: 0.6 mg/kg/h i.v. or 0.12 mg/kg/h i.med. Results: In HS/UNX intravenous atrasentan reduced MAP (142 ± 7 to 117 ± 8 mmHg, p < 0.001) and increased NO signal by 12% (p < 0.05). RBF fell from 25.6 ± 1.7 to 22.3 ± 1.6 ml/min (p < 0.03). Intramedullary atrasentan decreased MAP (127 ± 6 to 115 ± 6, p < 0.04) and increased NO signal by 9.7% (p < 0.03). RBF and MBF did not change significantly. In SHR intravenous atrasentan decreased MAP (171 ± 3 to 152 ± 9 mmHg, p < 0.03) and RBF (6.4 ± 0.5 to 5.0 ± 0.4 ml/min, p < 0.02); NO signal increased by 9.1% (p < 0.01). Intramedullary atrasentan decreased MAP (176 ± 4 to 167 ± 7 mmHg, p < 0.001); NO increased by 10% and MBF by 26% (p < 0.02–0.05). Evidently, in both hypertension models atrasentan always decreased MAP and increased tissue NO. Conclusions: To summarise, in both hypertension models blockade of ETA receptors decreased MAP, reflecting systemic vasodilation. The pressure decrease was followed by a decrease in RBF, probably the result of reduced renal perfusion pressure and imperfect autoregulation. Remarkably, MAP decrease was also seen with intramedullary ETA blockade but then without impairment of renal circulation. Of particular interest was a regular increase in tissue NO: we propose that the blockade of ETA unmasked the action of unbound ET-1 via ETB receptors, leading to NO release.

POS-378 SELECTIVE ETA ANTAGONIST ATRASENTAN, RAPIDLY REDUCES ALBUMINURIA AND DOWNREGULATES INTRA-RENAL PRO-INFLAMMATORY AND PRO-FIBROTIC TRANSCRIPTIONAL NETWORKS IN THE GDDY MOUSE MODEL OF SPONTANEOUS IGA NEPHROPATHY

Endothelin pathway activation has been observed in kidney biopsies from IgA nephropathy (IgAN) patients which may be an important driver of disease progression by promoting proteinuria along with kidney inflammation and fibrosis via ETA receptor activation. Atrasentan is a potent and selective ETA antagonist which has demonstrated rapid and sustained reductions in proteinuria, preservation of kidney function and improved kidney outcomes in diabetic kidney disease patients. However, the effects of atrasentan have not been previously investigated in IgAN. The objective of this study was to evaluate the effect of short-term treatment of varying doses of atrasentan in the gddY mouse model of spontaneous IgAN, with a focus on dynamic changes in the intra-renal transcriptional profile. 6-week-old male gddY mice were administered atrasentan (10, 20 or 30 mg/kg/day, n=3/group or 0 mg/kg/day, n=2/group) in drinking water for 5 days. Urine albumin to creatinine ratio (UACR) was measured at baseline and on Day 4 of treatment. On Day 5, a terminal plasma sample was collected for the measurement of atrasentan plasma concentrations and kidney cortex was flash-frozen for RNA-seq which was analyzed by pairwise differential gene expression using edgeR’s quasi-likelihood F-test (FDR adjusted p-value < 0.05) and gene pathway analysis (FDR adjusted p-value filter of < 0.001). At baseline, gddY mice had substantial albuminuria, with a mean UACR of 293.1 ± 16 mg/g. Atrasentan reduced UACR from baseline by 28 ± 44 % (p=ns), 62 ± 8 % (p=0.0498) and 63 ± 6 % (P=0.029) at 10, 20 and 30 mg/kg/day, respectively. Free terminal atrasentan plasma concentrations were 90 pM, 100 pM and 207 pM at the 10, 20 and 30 mg/kg/day doses respectively, providing free atrasentan concentrations 2.6-fold, 2.9-fold and 6.1-fold above the atrasentan ETA Ki (34 pM), respectively and in the same range as observed clinically at the optimized atrasentan dose of 0.75 mg. High quality RNA sequencing reads revealed dose-dependent effects of atrasentan on differential gene expression compared to untreated control ddY mice, including reductions in known ETA target genes ET-1 and ETA. Ingenuity Pathway Analysis to calculate enrichment scores showed that atrasentan produced prominent, dose-dependent downregulation of inflammatory, proliferative, and fibrotic gene networks. Atrasentan was observed to downregulate signaling pathways previously implicated in IgAN pathogenesis including the PDGF, NF-κB, IL-6 and TNF pathways. In the gddY mouse model of spontaneous IgAN, selectively blocking ETA with atrasentan leads to rapid reductions in albuminuria and results in intra-renal transcriptional downregulation of inflammatory and fibrotic signaling. The dynamic transcriptional changes in the kidney, including downregulation of known direct ETA receptor target genes, following only 5 days of treatment and prior to sustained long-term reductions in albuminuria and blood pressure that could mediate this benefit, is consistent with direct anti-inflammatory and antifibrotic effects of ETA blockade in IgAN. These results support further characterization of the effects of long-term treatment of atrasentan in the gddY mouse model and also support the therapeutic potential of atrasentan in IgAN patients to reduce proteinuria and kidney inflammation and fibrosis, key drivers of IgAN progression.

Role of Endothelin‐1 in Regulating Basal Macrovascular Cerebral Blood Flow: Healthy versus Metabolic Syndrome

Endothelin‐1 (ET‐1) is a potent vasoconstrictor that regulates vascular tone by binding to the ETA receptors on vascular smooth muscle. In healthy humans, blockade of ETA receptors increases resting forearm blood flow. ET‐1 appears to contribute to poor outcomes in animal models of cerebral ischemia, but the role of ET‐1 signaling in control of basal cerebral blood flow (CBF) in healthy adults is unknown. Interestingly, ET‐1 is upregulated in insulin resistance (IR) and resting CBF is reduced primarily in the anterior circulation, which may be due to enhanced ET‐1 signaling. We hypothesized: 1) ETA receptor blockade would increase CBF more in adults with IR, but play little to no role in regulating CBF in healthy adults and 2) ETA blockade would increase CBF more in the anterior than posterior circulation. Healthy normal weight adults (Control; n=6; BMI 23±2 kg/m2; age 24±4 yrs) and adults with Metabolic Syndrome (MetSyn; n=4; BMI 34±8 kg/m2; age 25±12 yrs) underwent magnetic resonance imaging (MRI) study visits. A subset of subjects completed a second MRI study visit (Control, n=6; MetSyn, n=2) with ETA blockade (10mg oral Ambrisentan). Phase‐contrast vastly undersampled isotropic projection reconstruction (PCVIPR) MRI was used to quantify total and regional CBF (CBF= blood velocity• artery cross sectional area). CBF in the anterior circulation was represented as the sum of CBF in the left and right internal carotid arteries (ICA) whereas CBF in the posterior circulation was represented by the basilar artery (BA). Total CBF was taken as the sum of flow in the left and right ICAs and BA. Heart rate (HR), mean arterial pressure (MAP), and end‐tidal CO2 (ETCO2) were monitored. Significance was determined using a mixed two‐way ANOVA or one‐way ANOVA and significance was set at p≤0.05. Results are mean±SD. HR, MAP, and ETCO2 were similar between Control and MetSyn (p&gt;0.05) and did not change with Ambrisentan in either group (p&gt;0.05). Resting total CBF was higher in Control compared to MetSyn (Control 704±87 vs. MetSyn 487±44 mL/min, p&lt;0.05). Ambrisentan did not change total CBF (p&gt;0.05). Resting anterior CBF was similar between groups (Control 535±82 vs. MetSyn 460±26 mL/min, p&gt;0.05), and was unchanged by Ambrisentan (p&gt;0.05). Resting posterior CBF was higher in Control compared to MetSyn (Control 168±33 vs. MetSyn 127±18 mL/min, p&lt;0.05) and Ambrisentan increased posterior CBF (main effect of Ambrisentan, p=0.05); however, the absolute and relative change in posterior CBF was similar between groups (%ΔCBF Control 9±15 vs. MetSyn 11±8% p&gt;0.05). IR adults exhibited lower resting total and posterior CBF compared to healthy adults. Contrary to our hypothesis, Ambrisentan did not increase total or anterior CBF in IR. Surprisingly, Ambrisentan increased CBF in the posterior circulation of healthy and IR adults similarly. Therefore, ET‐1 signaling appears to play a role in regulating vascular tone in the posterior region, however, IR adults do not exhibit enhanced sensitivity to ET‐1. A longer duration or increased severity of IR may be needed to alter cerebrovascular responses to ET‐1 signaling.Support or Funding InformationADA1‐16‐ICTS‐099

Role of endothelin receptor type A on catecholamine regulation in the olfactory bulb of DOCA-salt hypertensive rats: Hemodynamic implications

Increasing evidence shows that the olfactory bulb is involved in blood pressure regulation in health and disease. Enhanced noradrenergic transmission in the olfactory bulb was reported in hypertension. Given that endothelins modulate catecholamines and are involved in the pathogenesis of hypertension, in the present study we sought to establish the role of the endothelin receptor type A on tyrosine hydroxylase, the rate limiting enzyme in catecholamine biosynthesis, in the olfactory bulb of DOCA-salt hypertensive rats.Sprague-Dawley male rats, randomly divided into Control and DOCA-Salt hypertensive groups, were used to assess endothelin receptors by Western blot and confocal microscopy, and their co-localization with tyrosine hydroxylase in the olfactory bulb. Blood pressure and heart rate as well as tyrosine hydroxylase expression and activity were assessed following BQ610 (ETA antagonist) applied to the brain. DOCA-Salt hypertensive rats showed enhanced ETA and decreased ETB expression. ETA co-localized with tyrosine hydroxylase positive neurons. Acute ETA blockade reduced blood pressure and heart rate and decreased the expression of total tyrosine hydroxylase and its phosphorylated forms. Furthermore, it also diminished mRNA tyrosine hydroxylase expression and accelerated the enzyme degradation through the proteasome pathway as shown by pretreatment with MG132, (20s proteasome inhibitor) intracerebroventricularly applied.Present findings support that the brain endothelinergic system plays a major role through ETA activation in the increase of catecholaminergic activity in the olfactory bulb of DOCA-Salt hypertensive rats. They provide rationale evidence that this telencephalic structure contributes in a direct or indirect way to the hemodynamic regulation in salt dependent hypertension.

Endothelin Receptor‐dependent Responses in Overweight/Obese Postmenopausal Women

Age‐related weight gain is greater in women compared to men, such that postmenopausal women are more likely to be overweight or obese (OW/Ob). Previous studies have demonstrated that OW/Ob older adults have impaired endothelial function in part due to endothelin‐1 mediated vasoconstriction via the ETA receptor. In addition, our laboratory has recently demonstrated an important role for ETB receptors and its contribution to vascular endothelial dysfunction in postmenopausal women.PURPOSEThe purpose of this study was to test the hypothesis that ETB and ETA receptors contribute to impaired vasodilatory function in OW/Ob postmenopausal women.METHODSFourteen postmenopausal women have completed the study: 8 normal weight (NW; 57±5 years, 22±2 kg/m2) and 6 OW/Ob (57±8 years, 28±3 kg/m2). We measured vasodilatory responses to local heating of the skin (laser Doppler flowmetry) during microdialysis perfusions of lactated Ringer's (Control), ETB receptor blockade (BQ‐788, 300nM), and ETA receptor blockade (BQ‐123, 500 nM). Cutaneous vascular conductance (CVC) was calculated during the plateau phase of local heating (42°C), and normalized to maximal vasodilation achieved by perfusion of sodium nitroprusside (28mM) and heating to 43°C. A two‐way ANOVA with post hoc Bonferroni correction was performed to compare the impact of weight on vascular responses to ETA and ETB mediated vasodilation compared to control. Data are presented as mean ± standard deviation.RESULTSETB receptor blockade increased vasodilation in NW women (Control: 88±4 vs. BQ‐788: 94±4 %CVC max; P=0.03), whereas ETA receptor blockade did not significantly alter vasodilation (BQ‐123: 88±3 %max CVC; P&gt;0.90 vs. Control). In OW/Ob, both ETB and ETA blockade increased vasodilation (Control: 89±4 vs. BQ‐788: 95±4, BQ‐123: 94±5 %max CVC; P&lt;0.05). Furthermore, the vasodilatory response to ETA receptor blockade was greater in OW/Ob compared to NW (P=0.03).CONCLUSIONThese preliminary data suggest that while blockade of ETB receptors improves vasodilatory function in postmenopausal women, there is a larger contribution of ETA receptors impacting endothelial function in OW/Ob postmenopausal women.Support or Funding InformationSupported by: NIH GRANT P20 GM 113125 and AHA 16SDG30700015This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A6061 Renoprotection provided by additional diuretic treatment on top of RAS and ETA blockade in subtotally nephrectomized Ren-2 transgenic rats

A6061 Renoprotection provided by additional diuretic treatment on top of RAS and ETA blockade in subtotally nephrectomized Ren-2 transgenic rats

Activation of G Protein‐coupled Estrogen Receptor Promotes Endothelin‐dependent Natriuresis in Female Rats

The novel estrogen receptor, G protein‐coupled estrogen receptor (GPER), is a membrane‐bound receptor linked to acute signaling pathways. GPER activation elicits cardiovascular and nephroprotective effects against salt‐induced complications. However, its specific role in renal sodium handling is not yet defined. We hypothesized that activation of GPER in the renal medulla facilitates sodium excretion. Isosmotic saline was infused into the renal medullary interstitium (500 μl/h) of anesthetized male and female Sprague‐Dawley rats for an 80 min equilibration period and 20 min baseline urine collection period. Then, G1 (GPER agonist, 50 pmol/kg/min) or vehicle was infused into the renal medulla for an additional 20 min period. Compared with vehicle, G1 significantly increased urinary sodium excretion in females (from 0.6 ± 0.1 to 1.0 ± 0.1 μmol/min n=9, p&lt;0.05). G1 did not change blood pressure or urinary potassium excretion in comparison with vehicle control (106 ± 3 vs. 105 ± 4 mmHg, 0.5 ± 0.1 vs. 0.4 ± 0.1 μmol/min, n=9). Medullary blood flow remained unchanged during these experiments. In males, G1 did not change urinary sodium excretion (from 0.6 ± 0.1 to 0.7 ± 0.1 μmol/min, n=9). No sex‐differences in medullary GPER mRNA expression was observed, suggesting that sex‐differences in G1 effects on sodium excretion could be related to GPER downstream signaling cascade. Given that endothelin‐1 (ET‐1) is a well‐established natriuretic factor that is regulated by sex and sex steroids, we hypothesized that GPER activation promotes natriuresis via an ET‐1‐dependent pathway. To test this idea, we studied the effect of medullary infusion of G1 after dual blockade of ETA and ETB receptors achieved by i.v. bolus injection of ABT‐627 (5 mg/kg) and A‐192621 (10 mg/kg), respectively. We found that the natriuretic response to medullary GPER activation in females was inhibited by dual blockade of ET‐1 receptors (from 0.5 ± 0.2 to 0.5 ± 0.2 μmol/min n=5). These data uncover a novel role for renal medullary GPER in promoting sodium excretion via an ET‐1‐dependent pathway in females, but not in males.Support or Funding InformationFunded by AHA 15POST25090329 to EYG and P01 HL136267 to DMP and JSPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 037: Exaggerated Placental Ischemia-induced Hypertension in Endothelin Receptor Type B (ETB)-deficient Pregnant Rats s Independent of Increased sFlt-1 or ROS Levels

While the pathogenesis of preeclampsia is not fully understood, studies implicate placental ischemia. Reduced uterine perfusion pressure (RUPP)-induced placental ischemia/hypoxia in animal models stimulates release of factors like antiangiogenic sFlt-1 into the maternal circulation increasing vascular-renal ET-1. ET-1 promotes hypertension via reactive oxygen species (ROS). Blockade of vasoconstrictive ETA abolishes RUPP hypertension. Deficiency of vasodilatory ETB in rats leads to increased blood pressure in pregnancy. While ETB deficiency markedly enhances RUPP hypertension, it is unknown if there is exaggerated RUPP-induced sFlt-1, ET-1 or ROS levels in ETB-def rats. The hypothesis was tested that placental ischemia/hypoxia-induced release of sFlt-1 and circulating ET-1 and ROS are greater in ETB-def rats. Eighteen-week-old ETB-def and transgenic (Tg) control pregnant rats were generated with Wistar Hannover males. RUPP or Sham surgeries were on gestational day 14 and assessment of plasmas and placentas at day 19. RUPP increased placental sFlt-1 (pg/mg) similarly in RUPP ETB-def (781±113, N=5) vs Sham ETB-def (573±54, N=12) and RUPP Tg (631±62, N=5) vs Sham Tg (547±31, N=12) (P&lt;0.05). In placental explant cultures, acute hypoxia (48 h 1% O2 vs normoxia 6% O2) stimulated a comparable release of sFlt-1 (pg/mg) in Sham ETB-def (2577±135 vs 2070±78) and Sham Tg (3208±318 vs 2553±107) (P&lt;0.05). Unexpectedly, plasma sFlt-1 (pg/mL) was lower in RUPP ETB-def (153±48) vs Sham ETB-def (476±125) and RUPP Tg (238±32) vs Sham Tg (463±102) (P&lt;0.05). Plasma ET-1 (fmol/L) was exaggerated in RUPP ETB-def (954±70) and greater in Sham ETB-def (735±43) vs RUPP Tg (122±14) or Sham Tg (142±41) (P&lt;0.05). Plasma H2O2 (umol/L) was not exaggerated in RUPP ETB-def (5.4±1.2) or RUPP Tg (4.0±0.5) but was greater (P&lt;0.05) in Sham ETB-def (6.2±0.3) vs Sham Tg (3.6±0.3). In conclusion, these data suggest in 1) normal pregnancy, ETB is crucial for blood pressure control by regulating bioavailable ET-1 to prevent ROS production and 2) placental ischemia, ETB reduces excess ET-1 to buffer hypertension independently of sFlt-1 or ROS. These data support ETB physiology as important in controlling blood pressure in pregnancy and its loss in mediating hypertension in preeclampsia.

Dual NEP/ECE inhibition improves endothelial function in mesenteric resistance arteries of 32-week-old SHR.

Endothelin 1 (ET-1), a potent vasoconstrictor, pro-mitogenic and pro-inflammatory peptide, may promote development of endothelial dysfunction and arterial remodeling. ET-1 can be formed through cleavage of big-ET-1 by endothelin-converting enzyme (ECE) or neutral endopeptidase (NEP). We investigated whether chronic treatment with the novel dual NEP/ECE inhibitor SOL1 improves functional and structural properties of resistance-sized arteries of 32-week-old male spontaneously hypertensive rats (SHR). SHR received a chronic 4-week treatment with SOL1, losartan or hydralazine. We then compared effects of inhibition of NO synthase (NOS) (100 μM l-NAME), blockade of ETA- and ETB-receptors (10 μM bosentan) and stimulation of the endothelium with 0.001-10 μM acetylcholine (ACh) in isolated third-order mesenteric resistance arteries. Losartan and hydralazine significantly lowered blood pressure. Losartan decreased the media-to-lumen ratio of resistance arteries. l-NAME (1) increased arterial contractile responses to K+ (5.9-40 mM) in the losartan, SOL1 and vehicle group and (2) increased the sensitivity to phenylephrine (PHE; 0.16-20 μM) in the SOL1 group but not in the losartan, hydralazine and vehicle group. Relaxing responses to ACh in the absence or presence of l-NAME during contractions induced by either 10 μM PHE or 40 mM K+ were not altered by any in vivo treatment. Acute treatment with bosentan did, however, significantly improve maximal relaxing responses involving endothelium-derived nitric oxide and -hyperpolarizing factors in the SOL1 group but not in the losartan, hydralazine or vehicle group. Thus, chronic inhibition of NEP/ECE improved basal endothelial function but did not alter blood pressure, resistance artery structure and stimulated endothelium-dependent relaxing responses in 32-week-old SHR.

Involvement of Central Endothelin ETA and Cannabinoid CB1 Receptors and Arginine Vasopressin Release in Sepsis Induced by Cecal Ligation and Puncture in Rats.

We previously reported that endothelin-1 (ET-1) reduced the frequency of spontaneous excitatory currents in vasopressinergic magnocellular cells through the activation of endothelin ETA receptors in rat brain slices. This effect was abolished by a cannabinoid CB1 receptor antagonist, suggesting the involvement of endocannabinoids. The present study investigated whether the blockade of ETA or CB1 receptors during the phase of increased levels of ET-1 after severe sepsis increases the survival rate of animals concomitantly with an increase in plasma arginine vasopressin (AVP) levels. Sepsis was induced in male Wistar rats by cecal ligation and puncture (CLP). Treatment with the CB1 receptor antagonist rimonabant (Rim; 10 and 20 mg/kg, orally) 4 h after CLP (three punctures) significantly increased the survival rate compared with the CLP per vehicle group. Intracerebroventricular treatment with the ETA receptor antagonist BQ123 (100 pmol) or with Rim (2 μg) 4 and 8 h after CLP but not the ETB receptor antagonist BQ788 (100 pmol), also significantly improved the survival rate. Sham-operated and CLP animals that were treated with Rim had significantly lower core temperature than CLP animals. However, oral treatment with Rim did not change bacterial count in the peritoneal exudate, neutrophil migration to the peritoneal cavity, leucopenia or increased plasma interleukin-6 levels induced by CLP. Both Rim and BQ123 also increased AVP levels 12 h after CLP. The blockade of central CB1 and ETA receptors in the late phase of sepsis increased the survival rate, reduced body temperature and increased the circulating AVP levels.

Endothelin contributes to blunted renal autoregulation observed with a high-salt diet.

Autoregulation of renal blood flow (RBF) is an essential function of the renal microcirculation that has been previously shown to be blunted by excessive dietary salt. Endogenous endothelin 1 (ET-1) is increased following a high-salt (HS) diet and contributes to the control of RBF but the differential effects of ET-1 on renal microvessel autoregulation in response to HS remain to be established. We hypothesized that a HS diet increases endothelin receptor activation in normal Sprague-Dawley rats and blunts autoregulation of RBF. The role of ET-1 in the blunted autoregulation produced by a HS diet was assessed in vitro and in vivo using the blood-perfused juxtamedullary nephron preparation and anesthetized rats, respectively. Using highly selective antagonists, we observed that blockade of either ETA or ETB receptors was sufficient to restore normal autoregulatory behavior in afferent arterioles from HS-fed rats. Additionally, normal autoregulatory behavior was restored in vivo in HS-fed rats by simultaneous ETA and ETB receptor blockade, whereas blockade of ETB receptors alone showed significant improvement of normal autoregulation of RBF. Consistent with this observation, autoregulation of RBF in ETB receptor-deficient rats fed HS was similar to both ETB-deficient rats and transgenic control rats on normal-salt diets. These data support the hypothesis that endogenous ET-1, working through ETB and possibly ETA receptors, contributes to the blunted renal autoregulatory behavior in rats fed a HS diet.

Endothelin-a receptor antagonism after renal angioplasty enhances renal recovery in renovascular disease.

Percutaneous transluminal renal angioplasty/stenting (PTRAS) is frequently used to treat renal artery stenosis and renovascular disease (RVD); however, renal function is restored in less than one half of the cases. This study was designed to test a novel intervention that could refine PTRAS and enhance renal recovery in RVD. Renal function was quantified in pigs after 6 weeks of chronic RVD (induced by unilateral renal artery stenosis), established renal damage, and hypertension. Pigs with RVD then underwent PTRAS and were randomized into three groups: placebo (RVD+PTRAS), chronic endothelin-A receptor (ET-A) blockade (RVD+PTRAS+ET-A), and chronic dual ET-A/B blockade (RVD+PTRAS+ET-A/B) for 4 weeks. Renal function was again evaluated after treatments, and then, ex vivo studies were performed on the stented kidney. PTRAS resolved renal stenosis, attenuated hypertension, and improved renal function but did not resolve renal microvascular rarefaction, remodeling, or renal fibrosis. ET-A blocker therapy after PTRAS significantly improved hypertension, microvascular rarefaction, and renal injury and led to greater recovery of renal function. Conversely, combined ET-A/B blockade therapy blunted the therapeutic effects of PTRAS alone or PTRAS followed by ET-A blockade. These data suggest that ET-A receptor blockade therapy could serve as a coadjuvant intervention to enhance the outcomes of PTRAS in RVD. These results also suggest that ET-B receptors are important for renal function in RVD and may contribute to recovery after PTRAS. Using clinically available compounds and techniques, our results could contribute to both refinement and design of new therapeutic strategies in chronic RVD.

Effects of selective endothelin (ET)-A receptor antagonist versus dual ET-A/B receptor antagonist on hearts of streptozotocin-treated diabetic rats

AimsThe aim was to study the differences in the effectiveness of two types of endothelin (ET) receptor antagonists (selective ET-A or dual ET-A/B antagonists) on the hearts of streptozotocin (STZ)-induced diabetic rats (type I diabetes) at functional and biochemical/molecular levels. Main methodsCitrate saline (vehicle) or STZ was injected into rats. The ET-A/B dual receptor antagonist (SB209670, 1mg/kg/day) and the ET-A receptor antagonist (TA-0201, 1mg/kg/day) were then administered to these rats. One week after injection, the animals were separated into those receiving SB209670, TA-0201 or vehicle by 4-week osmotic mini-pump. Key findingsThe VEGF level and percent fractional shortening in the diabetic heart were significantly decreased compared to the non-diabetic heart, whereas SB209670 and TA-0201 treatments greatly and comparably prevented this decrease. SB209670 treatment was more effective in reversing decreased expressions of KDR and phosphorylated AKT, downstream of VEGF angiogenic signaling, than TA-0201 treatment. The eNOS levels in hearts were significantly higher in diabetic rats than in healthy rats, and this increase was significantly reduced by TA-0210 but not by SB209670 treatment. SignificanceImprovement of KDR mRNA and pAKT levels by SB209670 but not TA-0201 suggests that dual ET-A/-B blockade may be effective in improving intracellular systems of these components in the diabetic rat heart. However, the present study also showed that TA-0201 or SB209670 improved percent fractional shortening and VEGF levels of the diabetic hearts to a similar extent, suggesting that ET-A blockade and dual ET-A/-B blockade are similarly effective in improving cardiac dysfunction in the diabetic rats.

Functional angiotensin and endothelin, but not thromboxane, pathways are necessary for the hypertension and ‎baroreflex dysfunction caused by cyclosporine in rats (LB565)

We investigated the effects of pharmacologic interventions that selectively interrupt angiotensin II (Ang II), endothelin (ET), or ‎thromboxane (TXA2) signaling on CSA‐evoked hypertension and arterial baroreflex impairment in conscious rats. Baroreflex ‎curves relating changes in heart rate (HR) to increases (phenylephrine, PE) or decreases (sodium nitroprusside, SNP) in blood ‎pressure (BP) were constructed and slopes of the curves were taken as a measure of baroreflex sensitivity (BRSPE and BRSSNP). ‎CSA (25 mg/kg/day i.p., 7 days) significantly increased BP and HR and attenuated reflex HR responses and slopes of the ‎baroreflex curves generated by PE and SNP. Concurrent blockade of ETA (atrasentan) or AT1 receptors (losartan), or inhibition of ‎angiotensin converting enzyme (captopril) reduced the hypertensive, but not the tachycardic, effect of CSA. Further, atrasentan ‎reversed the CSA‐evoked impairment in both BRSPE and BRSSNP whereas losartan or captopril selectively improved BRSPE. The ‎blockade of TXA2 receptors by terutroban failed to alter the detrimental effects of CSA on BP or baroreflex gain. It is concluded ‎that the facilitation of Ang II and ETA, but not TXA2, signaling contributes, at least partly, to the rise in BP caused by CSA ‎possibly via the inhibition of reflex HR control. The study highlights some potential therapeutic modalities for the control of the ‎adverse effects of CSA on hemodynamics.‎

Selective endothelin ETA and dual ETA/ETB receptor blockade improve endothelium-dependent vasodilatation in patients with type 2 diabetes and coronary artery disease

AimsEndothelin-1 contributes to endothelial dysfunction in patients with atherosclerosis and type 2 diabetes. In healthy arteries the ETA receptor mediates the main part of the vasoconstriction induced by endothelin-1 whilst the ETB receptor mediates vasodilatation. The ETB receptor expression is upregulated on vascular smooth muscle cells in atherosclerosis and may contribute to the increased vasoconstrictor tone and endothelial dysfunction observed in this condition. Due to these opposing effects of the ETB receptor it remains unclear whether ETB blockade together with ETA blockade may be detrimental or beneficial. The aim was therefore to compare the effects of selective ETA and dual ETA/ETB blockade on endothelial function in patients with type 2 diabetes and coronary artery disease. Main methodsForearm endothelium-dependent and endothelium-independent vasodilatation was assessed by venous occlusion plethysmography in 12 patients before and after selective ETA or dual ETA/ETB receptor blockade. Key findingsDual ETA/ETB receptor blockade increased baseline forearm blood flow by 30±14% (P<0.01) whereas selective ETA blockade did not (14±8%). Both selective ETA blockade and dual ETA/ETB blockade significantly improved endothelium-dependent vasodilatation. The improvement did not differ between the two treatments. There was also an increase in endothelium-independent vasodilatation with both treatments. Dual ETA/ETB blockade did not significantly increase microvascular flow but improved transcutaneous pO2. SignificanceBoth selective ETA and dual ETA/ETB improve endothelium-dependent vasodilatation in patients with type 2 diabetes and coronary artery disease. ETB blockade increases basal blood flow but does not additionally improve endothelium-dependent vasodilatation.