ETB Receptor Expression Research Articles

Antiaging gene Klotho regulates endothelin-1 levels and endothelin receptor subtype B expression in kidneys of spontaneously hypertensive rats.

Klotho is an antiaging gene and is predominately expressed in kidneys. The endothelin system is critical in the regulation of kidney function. The objective of this study is to assess whether klotho affects the renal endothelin system in spontaneously hypertensive rats (SHRs). Four groups of male SHRs and one group of male Wistar-Kyoto (WKY) rats were used. In-vivo expression of klotho was achieved by AAV2 delivery of mouse klotho full-length cDNA (AAV.mKL). Four groups of SHRs were given (intravenously) AAV.mKL, AAV.LacZ, AAV.GFP, and phosphate-buffered saline, respectively. The WKY group was given phosphate-buffered saline and served as a control. At the end of week 12 after gene delivery, all animals were euthanized. Plasma endothelin-1 (ET-1) and renal ET-1 levels were increased in SHRs vs. WKY rats. In-vivo expression of klotho reversed the elevated ET-1 levels in SHRs. ETB receptor protein expression was decreased in both kidney cortex and medulla of SHRs. Interestingly, in-vivo expression of klotho abolished the downregulation of ETB protein expression in SHRs, suggesting that klotho regulates ETB receptor expression. Klotho gene delivery also eliminated the increase in the ratio of ETA/ETB in SHRs. Mitochondrial superoxide dismutase (Mn-SOD) protein expression was decreased in kidneys of SHRs, which was rescued by in-vivo expression of klotho. Klotho gene delivery abolished the upregulation of ET-1 levels and the downregulation of ETB and Mn-SOD expression in kidneys of SHRs. These findings revealed a previously unidentified role of klotho in the regulation of the renal ET system and Mn-SOD in SHRs.

Ontogeny of endothelin receptors in the brain, heart, and kidneys of neonatal rats.

Endothelin (ET) plays an important role in many physiological functions. It has been demonstrated that endogenous ET-1 concentration in the central nervous system (CNS) changes with age; however the ontogeny of ETA and ETB receptors in the brain, heart, and kidneys during postnatal development has not been studied. Brains, hearts and kidneys of rats at postnatal days 1, 7, 14 and 28 were evaluated for the expression of ETA and ETB receptors via Western blot. ETB receptors within the developing brain were further accessed via immunofluorescence. The mean organ and body weights increased proportionally with advancing age demonstrating normal growth. The expression of ETA receptors in the brain, heart, and kidneys and ETB receptor expression in the heart and kidneys was similar in these rats at postnatal ages 1, 7, 14 and 28days. However, brain ETB receptor expression significantly (P<0.001) decreased by 72% on day 28 compared to the levels on postnatal day 1. Upon immunofluorescent analysis, the intensity of ETB staining in the cerebral cortex and subventricular zones of the developing rat brain decreased significantly from day 1 to day 7 (P<0.001) and from day 7 to day 14 (P<0.0001). There was no further decrease in ETB intensity noted in the cerebral cortex and subventricular zones between day 14 and day 28 of postnatal age. The intensity of ETB receptor staining within the cerebrovasculature, on the other hand, increased significantly (P<0.05) from days 1 and 7 to day 14. These results demonstrate that expression of ETA receptors does not change with postnatal development. On the other hand ETB receptors in the cerebral cortex and subventricular zones of the brain decrease with age, while ETB receptors in the cerebrovasculature increase with age, implicating ETB receptor involvement in the structural maturity and development of the CNS.

TRPC channels are involved in endothelin 1 mediated inhibition of renin release from juxtaglomerular cells (1095.12)

Renin is produced by, stored in and released by juxtaglomerular (JG) cells. Increases in intracellular calcium (Ca) decrease renin release. I’ve found that JG cells contain multiple isoforms of canonical transient receptor potential (TRPC) Ca‐permeable channels. Among those, TRPC1, 4 and 5, are known to function as store‐operated Ca channels. Endothelin (ET)1 binds to G‐coupled protein receptors whose activation leads to a phospholipase C (PLC)/ IP3 signaling pathway, potentially stimulating store‐operated Ca channels. I hypothesized that endothelin inhibits renin release by activating TRPC store‐operated Ca channels. RT‐ PCR revealed expression of both ETA and ETB receptors in freshly isolated mouse JG cells. Incubation of primary cultures of JG cells with ET1 (10nM) decreased renin release by 28% from 0.84±0.07 to 0.60±0.07 µg ANGI/ml/hr/mg prot. (p&lt;0.001). An IP3 blocker (2APB, 0.1mM) returned renin release back to control. Addition of the TRPC blocker (SKF 96365, 50µM) completely reversed the Ca‐mediated inhibition of renin release by ET1 to 0.99±0.07 µg ANGI/ml/hr/mg prot. These results suggest that 1) JG cells express ETA and ETB receptors, 2) ET1 mediates inhibition of renin release in JG cells by activation of ETA or ETB receptors and subsequent PLC/IP3 signaling cascade activation, and 3) ET receptors link ET1 and TRPC store‐operated Ca channels to mediate ET1‐mediated Ca inhibition of renin release.Grant Funding Source: NIH 5PO1HL090550‐03

Early MEK1/2 Inhibition after Global Cerebral Ischemia in Rats Reduces Brain Damage and Improves Outcome by Preventing Delayed Vasoconstrictor Receptor Upregulation

BackgroundGlobal cerebral ischemia following cardiac arrest is associated with increased cerebral vasoconstriction and decreased cerebral blood flow, contributing to delayed neuronal cell death and neurological detriments in affected patients. We hypothesize that upregulation of contractile ETB and 5-HT1B receptors, previously demonstrated in cerebral arteries after experimental global ischemia, are a key mechanism behind insufficient perfusion of the post-ischemic brain, proposing blockade of this receptor upregulation as a novel target for prevention of cerebral hypoperfusion and delayed neuronal cell death after global cerebral ischemia. The aim was to characterize the time-course of receptor upregulation and associated neuronal damage after global ischemia and investigate whether treatment with the MEK1/2 inhibitor U0126 can prevent cerebrovascular receptor upregulation and thereby improve functional outcome after global cerebral ischemia. Incomplete global cerebral ischemia was induced in Wistar rats and the time-course of enhanced contractile responses and the effect of U0126 in cerebral arteries were studied by wire myography and the neuronal cell death by TUNEL. The expression of ETB and 5-HT1B receptors was determined by immunofluorescence.ResultsEnhanced vasoconstriction peaked in fore- and midbrain arteries 3 days after ischemia. Neuronal cell death appeared initially in the hippocampus 3 days after ischemia and gradually increased until 7 days post-ischemia. Treatment with U0126 normalised cerebrovascular ETB and 5-HT1B receptor expression and contractile function, reduced hippocampal cell death and improved survival rate compared to vehicle treated animals.ConclusionsExcessive cerebrovascular expression of contractile ETB and 5-HT1B receptors is a delayed response to global cerebral ischemia peaking 3 days after the insult, which likely contributes to the development of delayed neuronal damage. The enhanced cerebrovascular contractility can be prevented by treatment with the MEK1/2 inhibitor U0126, diminishes neuronal damage and improves survival rate, suggesting MEK1/2 inhibition as a novel strategy for early treatment of neurological consequences following global cerebral ischemia.

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.

Tumour necrosis factor-alpha participates on the endothelin-1/nitric oxide imbalance in small arteries from obese patients: role of perivascular adipose tissue

We assessed the impact of vascular and perivascular tumour necrosis factor-alpha (TNF-α) on the endothelin (ET)-1/nitric oxide (NO) system and the molecular pathways involved in small arteries from visceral fat of obese patients (Obese) and Controls. Isolated small arteries from 16 Obese and 14 Controls were evaluated on a pressurized micromyograph. Endogenous ET-1 activity was assessed by the ETA blocker BQ-123. TNF-α and NO were tested by anti-TNF-α infliximab (IFX) and N(ω)-nitro-l-arginine methylester (L-NAME). Gene and protein expression of TNF-α, ET-1, ETA, and ETB receptors were determined by RT-PCR and IHC on arterial wall and in isolated adipocytes. Obese showed a blunted L-NAME-induced vasoconstriction, which was potentiated by IFX, and an increased relaxation to BQ-123, unaffected by L-NAME but attenuated by IFX. Perivascular adipose tissue (PVAT) removal reversed these effects. Obese showed intravascular superoxide excess, which was decreased by apocynin (NAD(P)H oxidase inhibitor), L-NAME, and BQ-123 incubations, and abolished by IFX. An increased vascular expression of ET-1, ETA, and ETB receptors, and higher vascular/perivascular TNF-α and TNF-α receptor expression were also detected. The arterial expression and phosphorylation of c-Jun N-terminal kinase (JNK) were higher in Obese vs. Controls, and downregulated by IFX. In small arteries of Obese, PVAT-derived TNF-α excess, and an increased vascular expression of ET-1 and ETA receptor, contribute to the ET-1/NO system imbalance, by impairing tonic NO release. Reactive oxygen species excess, via NAD(P)H oxidase activation, induces the endothelial nitric oxide synthase uncoupling, which in turn generates superoxide and impairs NO production. The up-regulated JNK pathway represents a crucial molecular signalling involved in this process.

Orchidectomy enhances the expression of endothelin-1 and ETB receptors in rat portal vein.

Functional studies have shown that orchidectomy increases the effects of phenylephrine on rat portal veins, but that it is completely prevented in the presence of both ETA and ETB receptor antagonists. Although it suggests the involvement of endothelin-1 (ET-1), the local production of this vasoactive peptide has not been directly quantified in portal veins. Therefore, the aim of the present study was to verify if orchidectomy increases the local expression of ET-1 as well as ETA and ETB receptors in the rat portal vein. Indeed, the genic expression of ET-1, ETA and ETB receptors in rat portal veins taken from control (CONT), orchidectomized (ORX) and ORX plus testosterone-replacement therapy (ORX + T) animals were determined by Real Time RT-PCR. The results showed that orchidectomy induced a significant increment in genic expression of ET-1and ETB receptors in the rat portal veins, which was completely reversed by testosterone replacement treatment. In conclusion, the results suggest that orchidectomy increases the production of ET-1 in the rat portal vein and that, at least partially, it may be related to the previously reported elevation of responses to phenylephrine.

Abstract 644: The Endothelin B Receptor Attenuates Endothelin Contraction Via An Endothelium Independent Mechanism In Mesenteric Arteries Of (mRen2)27 Rats

Endothelin-1 (ET-1) and Angiotensin II (Ang II) are important modulators of vascular tone. Contraction to ET-1 is dependent on the activation of ETA and ETB receptors. The contractile receptor ETA is localized in the smooth muscle cells whereas ETB receptors are predominantly found in the endothelium. We investigated the role of ETB receptors in ET-1 contraction in a model of Ang II-dependent hypertension, the (mRen2)27 rat. Third branch mesenteric arteries were isolated from 12 weeks old male Sprague-Dawley (SD) control and (mRen2)27 hypertensive rats and mounted on a wire Multi Myograph (Model 620, DMT) for determination of isometric force. Dose response curves to ET-1 (10 -11 -10 -7 M) were performed in arterial segments that were either intact, endothelium denuded or preincubated with the ETB specific antagonist BQ788 10 -6 M. Endothelial denudation was performed by passing a human hair through the arterial lumen and functional loss of the endothelium was confirmed by the absence of vasodilatation to acetylcholine. Maximal responses were expressed as percent of maximal response to 75mM KCl (%K MAX ) and the sensitivity as pD 2 (pD 2 = -Log[EC 50 ]). In SD control rats, addition of BQ788 significantly increased ET-1 sensitivity in intact arteries (8.9±0.1 vs 9.3±0.1, p&lt;0.05) and the increase was abolished by endothelial denudation with no change in maximal response. However, the ETB blocker BQ788 increased the maximal tension in both intact (127±2 vs 154±13 %K MAX , p&lt;0.05); and denuded (156±9 vs 188±12 %K MAX , p&lt;0.05) arteries from (mRen2)27 rats with no change in sensitivity. In summary, the present results demonstrate that endothelial ETB receptors influence vasodilatory function in control SD rats, but apparently attenuate the contraction to ET-1 in the (mRen2)27 rats. We conclude that the expression of ETB receptors in the endothelium and smooth muscle of resistance vessels in the (mRen2)27 rats may function as a compensatory mechanism to the increased blood pressure or activated angiotensin system found in this model of hypertension.

Endothelin-1 and -3 induce choleresis in the rat through ETB receptors coupled to nitric oxide and vagovagal reflexes

We have reported previously that centrally applied ET (endothelin)-1 and ET-3 induce either choleresis or cholestasis depending on the dose. In the present study, we sought to establish the role of these endothelins in the short-term peripheral regulation of bile secretion in the rat. Intravenously infused endothelins induced significant choleresis in a dose-dependent fashion, ET-1 being more potent than ET-3. Endothelins (with the exception of a higher dose of ET-1) did not affect BP (blood pressure), portal venous pressure or portal blood flow. ET-1 and ET-3 augmented the biliary excretion of bile salts, glutathione and electrolytes, suggesting enhanced bile acid-dependent and -independent bile flows. ET-induced choleresis was mediated by ET(B) receptors coupled to NO and inhibited by truncal vagotomy, atropine administration and capsaicin perivagal application, supporting the participation of vagovagal reflexes. RT (reverse transcription)-PCR and Western blot analysis revealed ETA and ET(B) receptor expression in the vagus nerve. Endothelins, through ET(B) receptors, augmented the hepatocyte plasma membrane expression of Ntcp (Na⁺/taurocholate co-transporting polypeptide; Slc10a1), Bsep (bile-salt export pump; Abcb11), Mrp2 (multidrug resistance protein-2; Abcc2) and Aqp8 (aquaporin 8). Endothelins also increased the mRNAs of these transporters. ET-1 and ET-3 induced choleresis mediated by ET(B) receptors coupled to NO release and vagovagal reflexes without involving haemodynamic changes. Endothelin-induced choleresis seems to be caused by increased plasma membrane translocation and transcriptional expression of key bile transporters. These findings indicate that endothelins are able to elicit haemodynamic-independent biological effects in the liver and suggest that these peptides may play a beneficial role in pathophysiological situations where bile secretion is impaired.

Endothelin receptors in augmented vasoconstrictor responses to endothelin‐1 in chronic intermittent hypoxia

Chronic intermittent hypoxia (CIH) contributes to the development of cardiovascular diseases in patients with obstructive sleep apnoea. Many studies have shown an association between increased circulating endothelin (ET)-1 levels and CIH. The aim of the present study was to determine the role of ET receptors in altered aortic function in an animal model of CIH. Rats were subjected to CIH (Fi o2 9% for 1min, repeated every 2min for 8h/day, 7days/week) for 3weeks. After 3weeks, the rats were killed and their aortas retrieved for use in in vitro experiments (isometric force measurement), histological analysis, immunohistochemistry and western blotting. Aortas from rats subjected to CIH exhibited marked endothelial dysfunction and increased responsiveness to ET-1. Furthermore, CIH induced increased ET-1 and ETA receptor expression, whereas ETB receptor expression was decreased. Aortic contractile responses to ET-1 were inhibited by the ETA and ETB receptor antagonists BQ-123 and BQ-788, respectively. Acetylcholine-induced relaxation responses were significantly attenuated in aortas from rats subjected to CIH, whereas CIH had no significant effect on aortic responses to sodium nitroprusside. The results of the present study suggest that increased expression of ETA receptors, which mediate a potent vasoconstrictor response, plays an important role in the pathogenesis of CIH. In addition, decreased endothelial ETB receptor expression, which is associated with the functional decline of endothelium-dependent vasodilation, also contributes to the pathogenesis of CIH. It appears that the ETB receptor-induced buffering of ET-1 responsiveness is mediated via a nitric oxide-dependent mechanism.

Endothelin systems in the brain: involvement in pathophysiological responses of damaged nerve tissues.

In addition to their potent vasoconstriction effects, endothelins (ETs) show multiple actions in various tissues including the brain. The brain contains high levels of ETs, and their production is stimulated in many brain disorders. Accumulating evidence indicates that activation of brain ET receptors is involved in several pathophysiological responses in damaged brains. In this article, the roles of brain ET systems in relation to brain disorders are reviewed. In the acute phase of stroke, prolonged vasospasm of cerebral arteries and brain edema occur, both of which aggravate brain damage. Studies using ET antagonists show that activation of ETA receptors in the brain vascular smooth muscle induces vasospasm after stroke. Brain edema is induced by increased activity of vascular permeability factors, such as vascular endothelial growth factor and matrix metalloproteinases. Activation of ETB receptors stimulates astrocytic production of these permeability factors. Increases in reactive astrocytes are observed in neurodegenerative diseases and in the chronic phase of stroke, where they facilitate the repair of damaged nerve tissues by releasing neurotrophic factors. ETs promote the induction of reactive astrocytes through ETB receptors. ETs also stimulate the production of astrocytic neurotrophic factors. Recent studies have shown high expression of ETB receptors in neural progenitors. Activation of ETB receptors in neural progenitors promotes their proliferation and migration, suggesting roles for ETB receptors in neurogenesis. Much effort has been invested in the pursuit of novel drugs to induce protection or repair of damaged nerve tissues. From these studies, the pharmacological significance of brain ET systems as a possible target of neuroprotective drugs is anticipated.

Male-Female Differences in Upregulation of Vasoconstrictor Responses in Human Cerebral Arteries

Background and purposeMale-female differences may significantly impact stroke prevention and treatment in men and women, however underlying mechanisms for sexual dimorphism in stroke are not understood. We previously found in males that cerebral ischemia upregulates contractile receptors in cerebral arteries, which is associated with lower blood flow. The present study investigates if cerebral arteries from men and women differ in cerebrovascular receptor upregulation.Experimental approachFreshly obtained human cerebral arteries were placed in organ culture, an established model for studying receptor upregulation. 5-hydroxtryptamine type 1B (5-HT1B), angiotensin II type 1 (AT1) and endothelin-1 type A and B (ETA and ETB) receptors were evaluated using wire myograph for contractile responses, real-time PCR for mRNA and immunohistochemistry for receptor expression.Key resultsVascular sensitivity to angiotensin II and endothelin-1 was markedly lower in cultured cerebral arteries from women as compared to men. ETB receptor-mediated contraction occurred in male but not female arteries. Interestingly, there were similar upregulation in mRNA and expression of 5-HT1B, AT1, and ETB receptors and in local expression of Ang II after organ culture.Conclusions and ImplicationsIn spite of receptor upregulation after organ culture in both sexes, cerebral arteries from women were significantly less responsive to vasoconstrictors angiotensin II and endothelin-1 as compared to arteries from men. This suggests receptor coupling and/or signal transduction mechanisms involved in cerebrovascular contractility may be suppressed in females. This is the first study to demonstrate sex differences in the vascular function of human brain arteries.

1329 GENOME-WIDE EFFECTS OF PROSTATE CANCER ON DENDRITIC CELLS IN THE MURINE MODEL

You have accessJournal of UrologyProstate Cancer: Basic Research (VI)1 Apr 20131329 GENOME-WIDE EFFECTS OF PROSTATE CANCER ON DENDRITIC CELLS IN THE MURINE MODEL Blake Moore, P. Joseph Yannie, Ekaterine Goliadze, MaryEllen Dolat, Alberic Rogman, Jeffrey Wolters, and Georgi Guruli Blake MooreBlake Moore Richmond, VA More articles by this author , P. Joseph YannieP. Joseph Yannie Richmond, VA More articles by this author , Ekaterine GoliadzeEkaterine Goliadze Richmond, VA More articles by this author , MaryEllen DolatMaryEllen Dolat Richmond, VA More articles by this author , Alberic RogmanAlberic Rogman Richmond, VA More articles by this author , Jeffrey WoltersJeffrey Wolters Richmond, VA More articles by this author , and Georgi GuruliGeorgi Guruli Richmond, VA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.2683AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The apoptotic and suppressive effects of prostate cancer on immune-competent cells are well known. The aim of this study was to evaluate the genome-wide effects of prostate cancer on dendritic cells (DC), the most effective antigen-presenting cells, in a murine prostate cancer model. METHODS Dendritic cells were compared to DC incubated with RM-1 murine prostate cancer cells. Affymetrix Mouse 430 v2 Array was used for analysis containing 45000 probes. Quantitative polymerase chain reaction (qPCR) was performed to validate changes in the endothelin axis. RNAs from murine prostate cancer and normal murine prostate were also compared. RESULTS There was a significant increase in the expression of cell death inducing genes in DC incubated with prostate cancer cells, including multiple caspases. There was also a down regulation of proinflammatory genes including a number of interleukins and interferons. A change in the expression of endothelin receptors was seen as well. Considering the role of the endothelin axis in DC functioning, we used qPCR to further evaluate the condition of the endothelin axis in DC under prostate cancer influence. There was a significant decrease in the expression of ET-1 (4.50±1.33 fold) and ETA receptor (2.67±0.13), which are involved in DC survival and pro-inflammatory function. There was also an increase in the expression of ETB receptor (3.42±0.14) involved in DC apoptosis. Genome-wide comparison of RNAs derived from murine prostate cancer and murine prostate further demonstrated decreased expression of apoptotic genes by prostate cancer cells. CONCLUSIONS Prostate cancer induces a wide variety of changes in DC including decreased expression of genes responsible for pro-inflammatory function and survival. Alterations of the endothelin axis may be one reason why tumor escapes immune monitoring. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e543 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Blake Moore Richmond, VA More articles by this author P. Joseph Yannie Richmond, VA More articles by this author Ekaterine Goliadze Richmond, VA More articles by this author MaryEllen Dolat Richmond, VA More articles by this author Alberic Rogman Richmond, VA More articles by this author Jeffrey Wolters Richmond, VA More articles by this author Georgi Guruli Richmond, VA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effects of Cyclic Intermittent Hypoxia on ET-1 Responsiveness and Endothelial Dysfunction of Pulmonary Arteries in Rats

Obstructive sleep apnoea (OSA) is a risk factor for cardiovascular disorders and in some cases is complication of pulmonary hypertension. We simulated OSA by exposing rats to cyclic intermittent hypoxia (CIH) to investigate its effect on pulmonary vascular endothelial dysfunction. Sprague-Dawley Rats were exposed to CIH (FiO2 9% for 1 min, repeated every 2 min for 8 h/day, 7 days/wk for 3 wk), and the pulmonary arteries of normoxia and CIH treated rats were analyzed for expression of endothelin-1 (ET-1) and ET receptors by histological, immunohistochemical, RT-PCR and Western Blot analyses, as well as for contractility in response to ET-1. In the pulmonary arteries, ET-1 expression was increased, and ET-1 more potently elicited constriction of the pulmonary artery in CIH rats than in normoxic rats. Exposure to CIH induced marked endothelial cell damage associated with a functional decrease of endothelium-dependent vasodilatation in the pulmonary artery. Compared with normoxic rats, ETA receptor expression was increased in smooth muscle cells of the CIH rats, while the expression of ETB receptors was decreased in endothelial cells. These results demonstrated endothelium-dependent vasodilation was impaired and the vasoconstrictor responsiveness increased by CIH. The increased responsiveness to ET-1 induced by intermittent hypoxia in pulmonary arteries of rats was due to increased expression of ETA receptors predominantly, meanwhile, decreased expression of ETB receptors in the endothelium may also participate in it.

Inositol 1,4,5-Trisphosphate Receptor and Calcium Calmodulin-Dependent Protein Kinase are Involved in Endothelin Receptor Expression in Rat Cerebral Artery

Background: The present study was designed to examine the influence of inositol 1,4,5-trisphosphate receptor (IP3R) and calcium calmodulin-dependent protein kinase (CaMK) on endothelin receptor regulation during organ culture in rat middle cerebral arteries (MCA). Methods: MCA segments were incubated with or without xestospongin C (XeC), an IP3R inhibitor, or KN93, a CaMKII blocker. The mRNA levels of the ETA and ETB receptors, nuclear factor of activated T cells activating protein (NFam1), CaMKII, IP3R and Downstream regulatory element antagonist modulator (DREAM) protein were determined by real-time PCR or immunohistochemistry. Contractile responses to endothelin-1 (ET-1) and sarafotoxin 6c (S6c) were studied by a sensitive myograph and the intracellular calcium levels [Ca2+]i were evaluated by the FURA-2AM. Results: The contractile responses to ET-1 and S6c, the ETB receptors mRNA level and the baseline level of intracellular calcium [Ca2+]i were all increased after 24 h organ culture. Incubation with XeC or KN93 attenuated ETB receptor expression and increase in [Ca2+]i induced by ET-1 or S6c. XeC decreased the NFam1 mRNA levels while KN93 decreased DREAM protein levels. Conclusions: The study suggests that CaMKII and calcium release via IP3R are involved in the mechanisms regulate ETB receptor putatively via DREAM and NFam1, respectively.

Abstract 258: Angiotensin 1-7 Attenuates Endothelin-1-Induced Endothelial Cell Inflammation and Growth Through Nitric Oxide Production and Activation of Mas and EndothelinB Receptors

In pulmonary hypertension, where the endothelin system plays a major role, the vasoprotective axis of the rennin angiotensin system (ACE2-Ang (1-7)-Mas) seems to be protective. However, the exact mechanisms are still elusive and whether Ang 1-7 counterbalancing effects are beyond interactions with Ang II system is unknown. In this study, we assessed whether Ang 1-7 influences/interacts with the ET-1 system in endothelial cells. Cultured human microvascular endothelial cells (HMEC) were studied. HMEC were stimulated with ET-1 (10-7 mol/L) in the absence and presence of Ang 1-7 (10-7 mol/L), BQ788 (an ETBR antagonist), BQ 123 (an ETAR antagonist) and A779 (Mas receptor inhibitor) (10-6 mol/L). Expression of pro-inflammatory mediator (VCAM-1), cell growth marker (PCNA), Mas, ETBR expression and eNOS activation was determined by immunoblotting. ET-1 significantly increased expression of VCAM-1 (138.90% vs control, p&lt;0.05) and PCNA (125% vs control, p&lt;0.05). Ang 1-7 alone did not modulate pro-inflammatory and growth mediators, but significantly inhibited the effects of ET-1 on VCAM-1 (95.55%) and PCNA (103.83%) expression, an effect mediated by Mas receptor activation (after A779: VCAM-1: 226.15%; PCNA: 120% vs control, p&lt;0.05). Ang 1-7 increased NO production (Ctl: 7.5 vs Ang 1-7: 20 RFU/ug of protein, by microfluorescence). Inhibition of Ang 1-7-induced NO production by L-NAME, inhibited Ang 1-7-mediated effects on ET-1-induced VCAM-1 (160%) and PCNA (125%), p&lt;0.05. Ang 1-7 significantly increased expression of ETB receptors (175.63% vs control, p&lt;0.05), an effect attenuated by A779. Ang 1-7 (166.94% vs control, p&lt;0.05) and ET-1 (146.04% vs control, p&lt;0.05) increased eNOS phosphorylation in HMEC. Blockade of Mas and ETB receptor inhibited Ang 1-7 and ET-1 effects on eNOS activation. BQ123, but not BQ788, blocked ET-1-stimulated inflammation/growth in HMEC (VCAM-1: 75%, PCNA: 100%, p&lt;0.05). In conclusion, Ang 1-7 negatively modulates proinflammatory and mitogenic actions of ET-1, through crosstalk between Mas and ETB receptors, and increase in NO production. These data highlight some molecular mechanisms whereby Ang 1-7 may exert beneficial effects in pulmonary hypertension and suggests a novel mechanism for Ang 1-7 signalling in HMEC.

Age-associated alterations in retinal arteriole reactivity to endothelin-1 differ between the sexes

Endothelin-1 (ET-1) is a vasoconstrictor implicated in age-related retinal pathologies. This study determined whether responses to ET-1 differed in retinal arterioles isolated from adult (2–3 months) and aged (>20 months) Fischer 344 rats of both sexes. Risk factors for retinal disease (retinal perfusion pressure, intraocular pressure, blood glucose) were not affected by age. However, sensitivity to ET-1 declined with age, especially in females. Vasoconstrictor responses to 50mM KCl and Ca2+ release by caffeine (10mM) were similar in all groups. Retinal ETA and ETB receptor expression also was similar in young and aged rats, regardless of sex. Contractions elicited by 10nM ET-1 were inhibited by the ETA antagonist BQ-123 (1μM) in all groups. In contrast, the ETB antagonist BQ-788 (1μM) restored ET-1-induced contractions in aged female vessels, but had no effect in any other group. Removal of the endothelium also restored contractions in vessels from aged females but not males. Thus, responsiveness to ET-1 declines with age in retinal microvasculature. In males, this is likely mediated by age-related changes in the ETA receptor signaling pathway. By contrast, effects of ET-1 on endothelial ETB receptors attenuate vasoconstrictor responses in aged females.

Loss of renal medullary endothelin B receptor function during salt deprivation is regulated by angiotensin II

We have recently demonstrated that chronic infusion of exogenous ANG II, which induces blood pressure elevation, attenuates renal medullary endothelin B (ET(B)) receptor function in rats. Moreover, this was associated with a reduction of ET(B) receptor expression in the renal inner medulla. The aim of this present work was to investigate the effect of a physiological increase in endogenous ANG II (low-salt diet) on the renal ET system, including ET(B) receptor function. We hypothesized that endogenous ANG II reduces renal medullary ET(B) receptor function during low-salt intake. Rats were placed on a low-salt diet (0.01-0.02% NaCl) for 2 wk to allow an increase in endogenous ANG II. In rats on normal-salt chow, the stimulation of renal medullary ET(B) receptor by ET(B) receptor agonist sarafotoxin 6c (S6c) causes an increase in water (3.6 ± 0.4 from baseline vs. 10.5 ± 1.3 μl/min following S6c infusion; P < 0.05) and sodium excretion (0.38 ± 0.06 vs. 1.23 ± 0.17 μmol/min; P < 0.05). The low-salt diet reduced the ET(B)-dependent diuresis (4.5 ± 0.5 vs. 6.1 ± 0.9 μl/min) and natriuresis (0.40 ± 0.11 vs. 0.46 ± 0.12 μmol/min) in response to acute intramedullary infusion of S6c. Chronic treatment with candesartan restored renal medullary ET(B) receptor function; urine flow was 7.1 ± 0.9 vs. 15.9 ± 1.7 μl/min (P < 0.05), and sodium excretion was 0.4 ± 0.1 vs. 1.1 ± 0.1 μmol/min (P < 0.05) before and after intramedullary S6c infusion, respectively. Receptor binding assays determined that the sodium-depleted diet resulted in a similar level of ET(B) receptor binding in renal inner medulla compared with rats on a normal-salt diet. Candesartan reduced renal inner medullary ET(B) receptor binding (1,414 ± 95 vs. 862 ± 50 fmol/mg; P < 0.05). We conclude that endogenous ANG II attenuates renal medullary ET(B) receptor function to conserve sodium during salt deprivation independently of receptor expression.

Sa1487 Gene Expression Profiling and Endothelin in Acute Experimental Pancreatitis

AIM:To analyze gene expression profiles in an experimental pancreatitis and provide functional reversal of hypersensitivity with candidate gene endothelin-1 antagonists.METHODS:Dibutyltin dichloride(DBTC) is a chemical used as a polyvinyl carbonate stabilizer/catalyzer,biocide in agriculture,antifouling agent in paint and fabric.DBTC induces an acute pancreatitis flare through generation of reactive oxygen species.Lewis-inbred rats received a single i.v.injection with either DBTC or vehicle.Spinal cord and dorsal root ganglia(DRG) were taken at the peak of inflammation and processed for transcriptional profiling with a cDNA microarray biased for rat brain-specific genes.In a second study,groups of animals with DBTC-induced pancreatitis were treated with endothelin(ET) receptor antagonists [ET-A(BQ123) and ET-B BQ788)].Spontaneous pain related mechanical and thermal hypersensitivity were measured.Immunohistochemical analysis was performed using anti-ET-A and ET-B antibodies on sections from pancreatic tissues and DRG of the T10-12 spinal segments.RESULTS:Animals developed acute pancreatic inflammation persisting 7-10 d as confirmed by pathological studies(edema in parenchyma,loss of pancreatic architecture and islets,infiltration of inflammatory cells,neutrophil and mononuclear cells,degeneration,vacuolization and necrosis of acinar cells) and the painrelated behaviors(cutaneous secondary mechanical and thermal hypersensitivity).Gene expression profile was different in the spinal cord from animals with pancreatitis compared to the vehicle control group.Over 260 up-regulated and 60 down-regulated unique genes could be classified into 8 functional gene families:circulatory/acute phase/immunomodulatory;extracellular matrix;structural;channel/receptor/transporter;signaling transduction;transcription/translation-related;antioxidants/chaperones/heat shock;pancreatic and other enzymes.ET-1 was among the 52 candidate genes upregulated greater than 2-fold in animals with pancreatic inflammation and visceral pain-related behavior.Treatments with the ET-A(BQ123) and ET-B(BQ-788) antagonists revealed significant protection against inflammatory pain related mechanical and thermal hypersensitivity behaviors in animals with pancreatitis(P 0.05).Open field spontaneous behavioral activity(at baseline,day 6 and 30 min after drug treatments(BQ123,BQ788) showed overall stable activity levels indicating that the drugs produced no undesirable effects on normal exploratory behaviors,except for a trend toward reduction of the active time and increase in resting time at the highest dose(300 μmol/L).Immunocytochemical localization revealed that expression of ET-A and ET-B receptors increased in DRG from animals with pancreatitis.Endothelin receptor localization was combined in dual staining with neuronal marker NeuN,and glia marker,glial fibrillary acidic protein.ET-A was expressed in the cell bodies and occasional nuclei of DRG neurons in na ve animals.However,phenotypic expression of ET-A receptor was greatly increased in neurons of all sizes in animals with pancreatitis.Similarly,ET-B receptor was localized in neurons and in the satellite glia,as well as in the Schwann cell glial myelin sheaths surrounding the axons passing through the DRG.CONCLUSION:Endothelin-receptor antagonists protect against inflammatory pain responses without interfering with normal exploratory behaviors.Candidate genes can serve as future biomarkers for diagnosis and/or targeted gene therapy.

Endothelins induce choleresis through ETB receptors coupled to nitric oxide release and vago‐vagal reflexes

We previously showed that brain applied ET‐1 and ET‐3 in rats induce choleresis or cholestasis depending on the dose. In this study we evaluated if endothelins (ETs) regulated bile secretion when peripherally infused. Animals were prepared with jugular vein and bile duct cannulation to infuse drugs and collect bile samples. At the doses used ETs did not affect hepatic blood flow or portal venous pressure. ETs induced significant choleresis, being ET‐1 more potent than ET‐3. ETs increased bile salts, electrolytes and glutathione excretion supporting enhanced bile acid dependent and independent flows. ETs‐induced choleresis was mediated by ETB receptors coupled to nitric oxide and abolished by truncal vagotomy or capsaicin perivagal application. RT‐PCR and western blot analysis showed ETA and ETB receptor expression in the vagus nerve. ETs through ETB stimulated sodium taurocholate co transporter polypeptide (Ntcp) and aquaporin 8 (AQP8) translocation to the plasma membrane and augmented Ntcp, AQP8, bile salt export pump (Bsep) and multidrug resistance protein 2 (Mrp2) in microsomal membranes. Real time PCR studies showed that exposure to ETs increased the mRNAs of these transporters. ETs induced choleresis through ETB receptors coupled to nitric oxide and vago‐vagal reflexes without involving hemodynamic changes. These findings suggest that ETs may play a beneficial role when bile secretion is impaired.(CONICET PIP 0370/ ANPCyT‐PICT2008‐0716)