Sarafotoxin S6c Research Articles

Natriuretic response to renal medullary endothelin B receptor activation is impaired in Dahl-salt sensitive rats on a high-fat diet.

Renal medullary endothelin B receptors (ET(B)) mediate sodium excretion and blood pressure (BP) control. Several animal models of hypertension have impaired renal medullary ET(B) function. We found that 4-week high-caloric diet elevated systolic BP in Dahl salt-sensitive (Dahl S) rats (126+/-2 vs. 143+/-3 mm Hg, p<0.05). We hypothesized that renal medullary ET(B) function is dysfunctional in DS rats fed a high-caloric diet. We compared the diuretic and natriuretic response to intramedullary infusion of ET(B) agonist sarafotoxin 6c (S6c) in DS rats fed either a normal or high-caloric diet for 4 weeks. Urine was collected during intramedullary infusion of saline for baseline collection followed by intramedullary infusion of either saline or S6c. We first examined the ET(B) function in DS rats fed a normal diet. S6c increased urine flow (2.7+/-0.3 microl/min during baseline vs. 5.1+/-0.6 microl/min after S6c; p<0.05; n=5) and sodium excretion (0.28+/-0.05 vs. 0.81+/-0.17 micromol/min; p<0.05), suggesting that DS rats have renal medullary ET(B) function. However, DS rats fed a high-caloric diet displayed a significant increase in urine flow (2.7+/-0.4 vs. 4.2+/-0.4 microl/min, baseline vs. S6c infusion, respectively; p<0.05, n=6), but no significant change in sodium excretion in response to S6c (0.32+/-0.06 vs. 0.45+/-0.10 micromol/min). These data demonstrate that renal medullary ET(B) function is impaired in DS rats fed a high-caloric diet, which may be contributed to the elevation of blood pressure during high-caloric feeding in this model.

Expression of endothelin type B receptors (EDNRB) on smooth muscle cells is controlled by ternary complex factors and the actin cytoskeleton

Endothelin signaling plays an important role in physiology and disease and one of the endothelin receptors, the type B receptor (ETB or EDNRB), is known to be highly plastic, being upregulated in smooth muscle cells by arterial injury and following organ culture in vitro. Herein, we hypothesized that this transcriptional plasticity may arise in part because EDNRB is controlled by ternary complex factors and by the myocardin family coactivator MKL2. In line with this hypothesis we found significant correlations between the ternary complex factors ELK3, FLI1 and EDNRB, and between MKL2 and EDNRB at the mRNA level in human arteries. Overexpression of MKL2 in human coronary artery smooth muscle cells (HCASMC) led to a 30‐ fold induction of EDNRB, and this effect was antagonized by myocardin (MYOCD) which also correlated negatively with EDNRB at the tissue level. Overexpression of ELK3 and FLI1 induced expression of EDNRB, whereas the expression of classical smooth muscle markers was reduced. Depolymerization of actin using latrunculin B (LatB) increased EDNRB on both mRNA and protein levels, and increased calcium responses on stimulation with the ETB‐selective agonist sarafotoxin 6c (S6c) and endothelin 1 (ET‐1). Pretreatment with LatB in organ culture increased S6c and ET‐1‐induced contraction. We also found that ROCK inhibition using Y27632 induced EDNRB expression, and that the effect of LatB is antagonized by MAP‐kinase inhibition. Transcript‐specific primers and promoter reporter assays showed that the EDNRB_2 isoform is regulated by LatB in HCASMC. We finally demonstrate that the mRNA level of EDNRB is increased in rat carotid lesions and human carotid plaques, and that ELK3/FLI1 induction closely parallels EDNRB induction. In all, these studies uncover transcriptional control mechanisms for the endothelin type B receptor (EDNRB) that may explain its plasticity and could be targeted for therapy.Support or Funding InformationThis study was supported by grants from the Medical Faculty and The Royal Physiographic Society. KKK was supported by Marie Curie ITN Small Artery Remodelling of European Union.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Homocysteine up-regulates ETB receptors via suppression of autophagy in vascular smooth muscle cells

The change of autophagy is implicated in cardiovascular diseases (CVDs). Homocysteine (Hcy) up-regulates endothelin type B (ETB) receptors in vascular smooth muscle cells (VSMCs). However, it is unclear whether autophagy is involved in Hcy-induced-up-regulation of ETB receptors in VSMCs. The present study was designed to examine the hypothesis that Hcy up-regulates ETB receptors by inhibiting autophagy in VSMCs. Hcy treated the rat superior mesenteric artery (SMA) without endothelium in the presence and absence of AICAR, rapamycin or MHY1485 for 24 h. The contractile responses to sarafotoxin 6c (S6c) (an ETB receptor agonist) were studied using a sensitive myograph. Levels of protein expression were determined using Western blot analysis. Punctate staining of LC3B was exanimated by immunofluorescence using confocal microscopy. The results showed that Hcy inhibited AMPK, and activated mTOR, followed by impairing autophagy, and increased the levels of ETB receptor protein expression and the ETB receptor-mediated contractile responses to S6c in SMA without endothelium. However, these effects were reversed by AICAR or rapamycin. Additionally, MHY1485 up-regulated the AICAR-inhibited ETB receptor-mediated contractile response and the levels of ETB receptor protein expression in presence of Hcy. In conclusion, this suggested that Hcy up-regulated ETB receptors by inhibiting autophagy in VSMCs via AMPK/mTOR signaling pathway.

Facial hyperalgesia due to direct action of endothelin-1 in the trigeminal ganglion of mice.

This study assessed the ability of endothelin-1 (ET-1) to evoke heat hyperalgesia when injected directly into the trigeminal ganglia (TG) of mice and determined the receptors implicated in this effect. The effects of TG ETA and ETB receptor blockade on alleviation of heat hyperalgesia in a model of trigeminal neuropathic pain induced by infraorbital nerve constriction (CION) were also examined. Naive mice received an intraganglionar (i.g.) injection of ET-1 (0.3-3 pmol) or the selective ETB R agonist sarafotoxin S6c (3-30 pmol), and response latencies to ipsilateral heat stimulation were assessed before the treatment and at 1-h intervals up to 5 h after the treatment. Heat hyperalgesia induced by i.g. ET-1 or CION was assessed after i.g. injections of ETA R and ETB R antagonists (BQ-123 and BQ-788, respectively, each at 0.5 nmol). Intraganglionar ET-1 or sarafotoxin S6c injection induced heat hyperalgesia lasting 4 and 2 h, respectively. Heat hyperalgesia induced by ET-1 was attenuated by i.g. BQ-123 or BQ-788. On day 5 after CION, i.g. BQ-788 injection produced a more robust antihyperalgesic effect compared with BQ-123. ET-1 injection into the TG promotes ETA R/ETB R-mediated facial heat hyperalgesia, and both receptors are clearly implicated in CION-induced hyperalgesia in the murine TG system.

Up-regulation of contractile endothelin receptors by airborne fine particulate matter in rat mesenteric arteries via activation of MAPK pathway.

Fine particle matters (PM2.5) is a well-known risk factor for cardiovascular diseases. However, the underlying molecular mechanisms are largely unknown. Vascular hyper-reactivity plays an important roles in the pathogenesis of cardiovascular diseases. The present study was designed to investigate a hypothesis that PM2.5 up-regulated endothelin receptors in mesenteric artery and the potential underlying mechanisms. Rat mesenteric arteries were cultured with PM2.5. The artery contractile responses were recorded by a sensitive myograph. ETB and ETA receptor expressions of mRNA and protein were assessed by quantitative real-time PCR, Western blotting, and immunohistochemistry, respectively. Results showed that ETB receptor agonist, sarafotoxin 6c induced a negligible contraction in fresh artery segments, while ETA receptor agonist, ET-1 induced an obvious contraction. After organ culture, the contraction curve mediated by ETB and ETA receptors were shifted toward the left. PM2.5 1.0μg/ml cultured for 16h further enhanced ETB and ETA receptor-mediated contractile responses with a markedly increased maximal contraction. The organ culture enhanced ETB and ETA receptor mRNA and protein levels from fresh arteries, which were further increased by PM2.5. The U0126 (MEK/ERK1/2 inhibitor) and SB203580 (p38 inhibitor) significantly attenuated both organ cultured-induced and PM2.5-induced up-regulation of ETB receptor. U0126 also suppressed organ culture-increased and PM2.5-increased expressions of ETA receptor. SB203580 only suppressed PM2.5-induced enhanced expressions of ETA receptor In conclusion, airborne PM2.5 up-regulates ETB and ETA receptors of mesenteric artery via p38 MAPK and MEK/ERK1/2 MAPK pathways.

Distortion of KB estimates of endothelin-1 ETA and ETB receptor antagonists in pulmonary arteries: Possible role of an endothelin-1 clearance mechanism.

Dual endothelin ETA and ETB receptor antagonists are approved therapy for pulmonary artery hypertension (PAH). We hypothesized that ETB receptor‐mediated clearance of endothelin‐1 at specific vascular sites may compromise this targeted therapy. Concentration‐response curves (CRC) to endothelin‐1 or the ETB agonist sarafotoxin S6c were constructed, with endothelin receptor antagonists, in various rat and mouse isolated arteries using wire myography or in rat isolated trachea. In rat small mesenteric arteries, bosentan displaced endothelin‐1 CRC competitively indicative of ETA receptor antagonism. In rat small pulmonary arteries, bosentan 10 μmol L−1 left‐shifted the endothelin‐1 CRC, demonstrating potentiation consistent with antagonism of an ETB receptor‐mediated endothelin‐1 clearance mechanism. Removal of endothelium or L‐NAME did not alter the EC 50 or Emax of endothelin‐1 nor increase the antagonism by BQ788. In the presence of BQ788 and L‐NAME, bosentan displayed ETA receptor antagonism. In rat trachea (ETB), bosentan was a competitive ETB antagonist against endothelin‐1 or sarafotoxin S6c. Modeling showed the importance of dual receptor antagonism where the potency ratio of ETA to ETB antagonism is close to unity. In conclusion, the rat pulmonary artery is an example of a special vascular bed where the resistance to antagonism of endothelin‐1 constriction by ET dual antagonists, such as bosentan or the ETB antagonist BQ788, is possibly due to the competition of potentiation of endothelin‐1 by blockade of ETB‐mediated endothelin‐1 clearance located on smooth muscle and antagonism of ETA‐ and ETB‐mediated contraction. This conclusion may have direct application for the efficacy of endothelin‐1 antagonists for treating PAH.

Contractile Responses in Spontaneously Hypertensive Rats after Transient Middle Cerebral Artery Occlusion

Stroke is one of the leading causes of mortality and morbidity worldwide, and few therapeutic treatments have shown beneficial effect clinically. One reason for this could be the lack of risk factors incorporated into the preclinical stroke research. We have previously demonstrated phenotypic receptor changes to be one of the injurious mechanisms occurring after stroke but mostly in healthy rats. The aim of this study was to investigate if hypertension has an effect on vasoconstrictive receptor responses to endothelin 1, sarafotoxin 6c and angiotensin II after stroke by inducing transient middle cerebral artery occlusion in spontaneously hypertensive rats and Wistar-Kyoto rats using the wire-myograph. We demonstrated an increased contractile response to endothelin 1 and extracellular potassium as well as an increased carbachol-induced dilator response in the middle cerebral arteries from hypertensive rats after stroke. This study demonstrates the importance of including risk factors in experimental stroke research.

Intrarenal activation of endothelin type B receptors improves kidney oxygenation in type 1 diabetic rats

About one-third of patients with type 1 diabetes develops kidney disease. The mechanism is largely unknown, but intrarenal hypoxia has been proposed as a unifying mechanism for chronic kidney disease, including diabetic nephropathy. The endothelin system has recently been demonstrated to regulate oxygen availability in the diabetic kidney via a pathway involving endothelin type A receptors (ETA-R). These receptors mainly mediate vasoconstriction and tubular sodium retention, and inhibition of ETA-R improves intrarenal oxygenation in the diabetic kidney. Endothelin type B receptors (ETB-R) can induce vasodilation of the renal vasculature and also regulate tubular sodium handling. However, the role of ETB-R in kidney oxygen homeostasis is unknown. The effects of acute intrarenal ETB-R activation (sarafotoxin 6c for 30-40 min; 0.78 pmol/h directly into the renal artery) on kidney function and oxygen metabolism were investigated in normoglycemic controls and insulinopenic male Sprague-Dawley rats administered streptozotocin (55 mg/kg) 2 wk before the acute experiments. Intrarenal activation of ETB-R improved oxygenation in the hypoxic diabetic kidney. However, the effects on diabetes-induced increased kidney oxygen consumption could not explain the improved oxygenation. Rather, the improved kidney oxygenation was due to hemodynamic effects increasing oxygen delivery without increasing glomerular filtration or tubular sodium load. In conclusion, increased ETB-R signaling in the diabetic kidney improves intrarenal tissue oxygenation due to increased oxygen delivery secondary to increased renal blood flow.

Tail vein injection of mmLDL upregulates mouse mesenteric artery ETB receptors via activation of the ERK1/2 pathway

Minimally modified low density lipoprotein (mmLDL) is a risk factor for cardiovascular disease. This study investigated the effect of mmLDL on mouse mesenteric artery endothelin type B (ETB) receptors and its molecular mechanism. Mice were injected with normal saline (NS group), mmLDL in the tail vein (mmLDL group), or with both mmLDL and an intraperitoneal injection of the ERK1/2 pathway-specific inhibitor U0126 (mmLDL+U0126 group). The dose-response curve of mesenteric artery contraction induced by sarafotoxin 6c (S6c), the ETB receptor agonist, was measured using a sensitive myograph system. ELISAs, RT-PCR and Western blot were used to determine the serum concentrations of mouse oxidized low density lipoprotein (oxLDL), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) as well as the expression of ETB receptors, ICAM-1, VCAM-1 and phosphorylated-extracellular signal-regulated kinase 1/2 (p-ERK1/2). The S6c-induced contraction dose-response curve was significantly enhanced by mmLDL treatment and showed a significantly higher Emax value than in the NS group (P<0.001), and the ETB receptor mRNA and protein expression in the vascular wall was significantly higher than in the NS group. The serum concentration and expression of ICAM-1 and VCAM-1 were also increased by mmLDL treatment, but intraperitoneal injection of U0126 inhibited these changes as well as the increase in p-ERK1/2 protein in the vessel wall caused by mmLDL. ICAM-1 and VCAM-1 serum concentrations were positively correlated with the S6c-induced maximum contraction of blood vessels. Increased in vivo levels of mmLDL increased the serum concentrations and expression of ICAM-1 and VCAM-1 by activating the ERK1/2 pathway, resulting in the expression of ETB receptors and the enhancement of contractile function in vascular smooth muscle. Understanding the effect of mmLDL on ETB receptors and its mechanism can provide ideas for cardiovascular disease prevention and treatment.

Crosstalk between the angiotensin and endothelin system in the cerebrovasculature after experimental induced subarachnoid hemorrhage.

Under physiologic conditions, losartan showed a dose-dependent antagonistic effect to the endothelin-1 (ET-1)-mediated vasoconstriction. This reduced vasoconstriction was abolished after preincubation with an endothelin B1 receptor (ET(B1)-receptor) antagonist. Also, an increased ET(B1)-receptor-dependent relaxation to sarafotoxin S6c (S6c; an ET(B1)-receptor agonist) was detected by preincubation with losartan. Investigations after experimental induced subarachnoid hemorrhage (SAH) are still missing. Therefore, we analyzed losartan in a further pathological setup. Cerebral vasospasm was induced by a modified double hemorrhage model. Rats were sacrificed on day 3 and isometric force of basilar artery ring segments was measured. Parallel to physiological conditions, after SAH, the ET-1-induced vasoconstriction was decreased by preincubation with losartan. This reduced contraction has been abolished after preincubation with BQ-788, an ET(B1)-receptor antagonist. In precontracted vessels, ET-1 induced a higher vasorelaxation under losartan and the endothelin A receptor (ET(A)-receptor) antagonist BQ-123. After SAH, losartan caused a modulatory effect on the ET(B1)-receptor-dependent vasorelaxation. It further induced an upregulation of the NO pathway. Under losartan, the formerly known loss of the ET(B1)-receptor vasomotor function was abolished and a significantly increased relaxation, accompanied with an enhanced sensitivity of the ET(B1)-receptor, has been detected. Also, the dose-dependent antagonistic effect to the ET-1-induced contraction can be effected by angiotensin II type 1 receptor (AT1-receptor) antagonism due to losartan directly via the ET(B1)-receptor.

Vitamin C Deficiency Reduces Muscarinic Receptor Coronary Artery Vasoconstriction and Plasma Tetrahydrobiopterin Concentration in Guinea Pigs.

Vitamin C (vitC) deficiency is associated with increased cardiovascular disease risk, but its specific interplay with arteriolar function is unclear. This study investigates the effect of vitC deficiency in guinea pigs on plasma biopterin status and the vasomotor responses in coronary arteries exposed to vasoconstrictor/-dilator agents. Dunkin Hartley female guinea pigs (n = 32) were randomized to high (1500 mg/kg diet) or low (0 to 50 mg/kg diet) vitC for 10–12 weeks. At euthanasia, coronary artery segments were dissected and mounted in a wire-myograph. Vasomotor responses to potassium, carbachol, sodium nitroprusside (SNP), U46619, sarafotoxin 6c (S6c) and endothelin-1 (ET-1) were recorded. Plasma vitC and tetrahydrobiopterin were measured by HPLC. Plasma vitC status reflected the diets with deficient animals displaying reduced tetrahydrobiopterin. Vasoconstrictor responses to carbachol were significantly decreased in vitC deficient coronary arteries independent of their general vasoconstrictor/vasodilator capacity (p < 0.001). Moreover, in vitC deficient animals, carbachol-induced vasodilator responses correlated with coronary artery diameter (p < 0.001). Inhibition of cyclooxygenases with indomethacin increased carbachol-induced vasoconstriction, suggesting an augmented carbachol-induced release of vasodilator prostanoids. Atropine abolished carbachol-induced vasomotion, supporting a specific muscarinic receptor effect. Arterial responses to SNP, potassium, S6c, U46619 and ET-1 were unaffected by vitC status. The study shows that vitC deficiency decreases tetrahydrobiopterin concentrations and muscarinic receptor mediated contraction in coronary arteries. This attenuated vasoconstrictor response may be linked to altered production of vasoactive arachidonic acid metabolites and reduced muscarinic receptor expression/signaling.

Calcium‐handling of Endothelin A and B Receptors in Coronary Artery upon Post Ischemic Reperfusion Injury in Rats

BackgroundIschemia‐reperfusion has been demonstrated to increase coronary artery resistance and increase the sensitivity toward the potent vasoconstrictor peptide endothelin‐1 (ET‐1). The increased sensitivity to ET‐1 is associated with an up‐regulation of vasoconstrictive ETB receptors in the vascular smooth muscle cells. This project aims to investigate any causal link between the ETB receptor‐upregulation and a shift in calcium sensitivity/handling in the vascular smooth muscle cells after experimental myocardial ischemia‐reperfusion injury.MethodsMale SD rats (~320 gram) were either subjected to experimental left anterior descending (LAD) coronary artery occlusion for 30 min followed by 24 hours of reperfusion, or sham operation. Contractile properties of artery segments distal to the ligature were measured with wire‐myography or along with simultaneous measures of the intracellular Ca2+ concentration ([Ca2+]i) using Fura‐2. The Ca2+ sensitivity of the arteries was determined during stimulation of the ETB‐R agonist Sarafotoxin 6c (S6c) followed by cumulative addition of extracellular Ca2+ (up to 2.5 mM). The experiments were subsequent repeated with ET‐1 as stimulating agent to access dual ETB‐R and ETA‐R mediated Ca2+ sensitization. Thapsigargin, 2‐APB and Felodipine were in addition used in order to identify Ca2+ handling mechanisms and their relative contribution for the ETB‐R and ETA‐R mediated contraction following ischemia‐reperfusion injury.ResultsPost ischemic reperfusion injury significantly increased vascular tone of the LAD coronary artery in response to [Ca2+]o. Prior incubation with either 125 mM of K+ or the ETB‐R antagonist BQ788 showed, that this difference in contractility was neither due to changes in membrane depolarization or a constitutive activity of the ETB‐R after ischemia and reperfusion injury. The evoked agonist‐responses of the Endothelin receptor subtypes, confirmed the enhanced ETB‐R mediated tone after ischemia‐reperfusion injury, whereas dual ETA‐R and ETB‐R mediated tone seems unaffected between surgery groups. The enhanced ETB‐R contractility develops an equal [Ca2+]i –tone relationship mainly from extracellular Ca2+ influx, through activation of voltage‐gated Ca2+ channels.ConclusionThe ETA‐R mediated contraction was unaffected between surgery groups and show equal dependence on intracellular store‐release and extracellular Ca2+ influx. The ET‐1 evoked [Ca2+]i –tone balance was leftward‐shifted opposed to the ETB‐R and suggest the ETA‐Rs to additionally stimulate downstream Ca2+ sensitivity mechanisms.Support or Funding InformationFaculty of Health and Medical Sciences, University of Copenhagen, Denmark. Lundbeck Foundation, Grant of Excellence, Denmark. R59‐AR‐5404.

Myocardial ischemia-reperfusion enhances transcriptional expression of endothelin-1 and vasoconstrictor ETB receptors via the protein kinase MEK-ERK1/2 signaling pathway in rat.

BackgroundCoronary artery remodelling and vasospasm is a complication of acute myocardial ischemia and reperfusion. The underlying mechanisms are complex, but the vasoconstrictor peptide endothelin-1 is suggested to have an important role. This study aimed to determine whether the expression of endothelin-1 and its receptors are regulated in the myocardium and in coronary arteries after experimental ischemia-reperfusion. Furthermore, we evaluated whether treatment with a specific MEK1/2 inhibitor, U0126, modified the expression and function of these proteins.Methods and findingsSprague-Dawley rats were randomly divided into three groups: sham-operated, ischemia-reperfusion with vehicle treatment and ischemia-reperfusion with U0126 treatment. Ischemia was induced by ligating the left anterior descending coronary artery for 30 minutes followed by reperfusion. U0126 was administered before ischemia and repeated 6 hours after start of reperfusion. The contractile properties of isolated coronary arteries to endothelin-1 and sarafotoxin 6c were evaluated using wire-myography. The gene expression of endothelin-1 and endothelin receptors were measured using qPCR. Distribution and localization of proteins (pERK1/2, prepro-endothelin-1, endothelin-1, and endothelin ETA and ETB receptors) were analysed by Western blot and immunohistochemistry. We found that pERK1/2 was significantly augmented in the ischemic area 3 hours after ischemia-reperfusion; this correlated with increased ETB receptor and ET-1 gene expressions in ischemic myocardium and in coronary arteries. ETB receptor-mediated vasoconstriction was observed to be increased in coronary arteries 24 hours after ischemia-reperfusion. Treatment with U0126 reduced pERK1/2, expression of ET-1 and ETB receptor, and ETB receptor-mediated vasoconstriction.ConclusionsThese findings suggest that the MEK-ERK1/2 signaling pathway is important for regulating endothelin-1 and ETB receptors in myocardium and coronary arteries after ischemia-reperfusion in the ischemic region. Inhibition of the MEK-ERK1/2 pathway may provide a novel target for reducing ischemia-reperfusion damage in the heart.

The sirt1/NF-kB signaling pathway is involved in regulation of endothelin type B receptors mediated by homocysteine in vascular smooth muscle cells.

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases (CVDs). Endothelin type B (ETB) receptors were involved in the pathogenesis of CVDs. However, Sirtuin 1(Sirt1) has potential protect roles for CVDs. The present study was designed to examine the hypothesis that homocysteine up-regulates ETB receptors through down-regulation of Sirt 1. In vitro experiments were performed in rat superior mesenteric artery (SMA). The rat SMA was cultured in serum free medium for 24h in the presence and absence of homocysteine (Hcy) with or without resveroral (Res) (a Sirt 1agonist). In vivo, the rats received subcutaneous injections of Hcy in the presence of or absence of Res for 3 weeks. The contractile responses to sarafotoxin 6c (S6c) (an ETB receptor agonist) were studied using a sensitive myograph. Levels of protein expression were determined using western blotting. The blood pressure of rat was measured via a noninvasive tail-cuff plethysmography method. We observed that Hcy increased the level of ETB receptor protein expression and the ETB receptor-mediated contractile responses induced by S6c, and decreased level of Sirt1 protein expression in SMA without endothelium in vitro. However, these effects were reversed by Res. Moreover, Res also blocked the up-regulation of acetylized p65 induced by Hcy. The in vivo study showed that HHcy down-regulated Sirt 1, and up-regulated acetylized p65 and ETB receptor protein expression, and elevated the blood pressure of rats. However, Res could block these effects. In conclusion, this suggested that Hcy regulated ETB receptor expression through sirt1/nuclear factor-κB signaling pathway.

Homocysteine regulates endothelin type B receptors in vascular smooth muscle cells

Vascular smooth muscle endothelin type B (ETB) receptor is involved in the pathogenesis of cardiovascular diseases (CVDs). Hyperhomocysteinemia is an independent risk factor for CVDs. The present study was designed to examine the hypothesis that homocysteine (Hcy) up-regulates vascular smooth muscle ETB receptors. In vitro experiments were performed in rat superior mesenteric artery (SMA) and vascular smooth muscle cells (VSMCs). The rat SMA or VSMCs were cultured in serum-free medium for 24h in the presence and absence of Hcy with or without specific inhibitors for the ERK1/2 signaling pathway and NF-κB. In vivo, the rats received subcutaneous injections of Hcy in the presence or absence of specific inhibitors for the ERK1/2 signaling pathway (U0126) for 3weeks. Levels of protein expression were determined using Western blot analysis. The contractile responses to sarafotoxin 6c (an ETB receptor agonist) were studied using a sensitive myograph. The blood pressure of the rats was measured via a noninvasive tail-cuff plethysmography method. The results from in vitro experiments showed that Hcy concentration-dependently increased the ETB receptor-mediated contractile responses, and up-regulated ETB receptor expression, in rat SMA. Blockage of the ERK1/2 signaling pathway and NF-κB using the MEK1/2 inhibitor (PD98059 and U0126) or IκB kinase inhibitor (wedelolactone) significantly abolished Hcy-induced up-regulation of ETB receptor. Finally, we used VSMCs as a cellular model to further validate our finding. In vivo study found that hyperhomocysteinemia up-regulated ETB receptor expression, and elevated the blood pressure of rats via the ERK1/2 signaling pathway. In conclusion, Hcy up-regulated vascular smooth muscle ETB receptor via activation of the ERK1/2 signaling pathway and NF-κB.

Effects of endothelin family on ANP secretion

The endothelins (ET) peptide family consists of ET-1, ET-2, ET-3, and sarafotoxin (s6C, a snake venom) and their actions appears to be different among isoforms. The aim of this study was to compare the secretagogue effect of ET-1 on atrial natriuretic peptide (ANP) secretion with ET-3 and evaluate its physiological meaning. Isolated nonbeating atria from male Sprague-Dawley rats were used to evaluate stretch-activated ANP secretion in response to ET-1, ET-2, ET-3, and s6C. Changes in mean blood pressure (MAP) were measured during acute injection of ET-1 and ET-3 with and without natriuretic peptide receptor-A antagonist (A71915) in anesthetized rats. Changes in atrial volume induced by increased atrial pressure from o to 1, 2, 4, or 6cm H2O caused proportional increases in mechanically-stimulated extracellular fluid (ECF) translocation and stretch-activated ANP secretion. ET-1 (10nM) augmented basal and stretch-activated ANP secretion in terms of ECF translocation, which was blocked by the pretreatment with ETA receptor antagonist (BQ123, 1μM) but not by ETB receptor antagonist (BQ788, 1μM). ETA receptor antagonist itself suppressed stretch-activated ANP secretion. As compared to ET-1- induced ANP secretion (3.2-fold by 10nM), the secretagogue effects of ANP secretion by ET-2 was similar (2.8-fold by 10nM) and ET-3 and s6C were less potent (1.7-fold and 1.5-fold by 100nM, respectively). Acute injection of ET-1 or ET-3 increased mean blood pressure (MAP), which was augmented in the presence of natriuretic peptide receptor-A antagonist. Therefore, we suggest that the order of secretagogue effect of ET family on ANP secretion was ET-1≥ET-2>>ET-3>s6C and ET-1-induced ANP secretion negatively regulates the pressor effect of ET-1.

Serial Measurements of Splanchnic Vein Diameters in Rats Using High-Frequency Ultrasound.

The purpose of this study was to investigate serial ultrasound imaging in rats as a fully non-invasive method to (1) quantify the diameters of splanchnic veins in real time as an indirect surrogate for the capacitance function of those veins, and (2) assess the effects of drugs on venous dimensions. A 21 MHz probe was used on anesthetized male Sprague–Dawley rats to collect images containing the portal vein (PV), superior mesenteric vein (SMV), abdominal inferior vena cava (IVC), and splenic vein (SpV; used as a landmark in timed studies) and the abdominal aorta (AA). Stable landmarks were established that allowed reproducible quantification of cross-sectional diameters within an animal. The average diameters of vessels measured every 5 min over 45 min remained within 0.75 ± 0.15% (PV), 0.2 ± 0.09% (SMV), 0.5 ± 0.12% (IVC), and 0.38 ± 0.06% (AA) of baseline (PV: 2.0 ± 0.12 mm; SMV: 1.7 ± 0.04 mm; IVC: 3.2 ± 0.1 mm; AA: 2.3 ± 0.14 mm). The maximal effects of the vasodilator sodium nitroprusside (SNP; 2 mg/kg, i.v. bolus) on venous diameters were determined 5 min post SNP bolus; the diameters of all noted veins were significantly increased by SNP, while mean arterial pressure (MAP) decreased 29 ± 4 mmHg. By contrast, administration of the venoconstrictor sarafotoxin (S6c; 5 ng/kg, i.v. bolus) significantly decreased PV and SpV, but not IVC, SMV, or AA, diameters 5 min post S6c bolus; MAP increased by 6 ± 2 mmHg. In order to determine if resting splanchnic vein diameters were stable over much longer periods of time, vessel diameters were measured every 2 weeks for 8 weeks. Measurements were found to be highly reproducible within animals over this time period. Finally, to evaluate the utility of vein imaging in a chronic condition, images were acquired from 4-week deoxycorticosterone acetate salt (DOCA-salt) hypertensive and normotensive (SHAM) control rats. All vessel diameters increased from baseline while MAP increased (67 ± 4 mmHg) in DOCA-salt rats compared to SHAM at 4 weeks after pellet implantation. Vessel diameters remained unchanged in SHAM animals. Together, these results support serial ultrasound imaging as a non-invasive, reliable technique able to measure acute and chronic changes in the diameter of splanchnic veins in intact rats.

Stimulation of intrarenal endothelin B receptors with Sarafotoxin 6c improves kidney oxygenation in rats with diabetic nephropathy by augmented oxygen availability

Diabetic nephropathy is the leading cause of end stage renal disease. The damage progression has recently been suggested to involve decreased kidney oxygenation as a result of increased oxygen consumption. Furthermore, endothelin A receptors (ETA) are highly related to kidney damage meanwhile endothelin B receptors (ETB) seem to be more protective towards diabetic nephropathy. Therefore, in this study we examined how acute in vivo intrarenal stimulation of ETB affects the progression of kidney damage in rats with diabetic nephropathy.Two weeks post induction of hyperglycemia (Streptozotocin 55 mg/kg) rats werestudied for kidney function and oxygen metabolism during acute in vivo procedures. Parameters were collected during a baseline period with intrarenal saline infusion and during an experimental period with Sarafotoxin 6c (S6c) infusion (0.78125 pmol/h).Hyperglycemic rats had lower kidney oxygenation which increased during S6c infusion. Meanwhile the increased oxygen consumption and glomerular filtration rate was unaffected by the treatment. Renal blood flow, delivered oxygen, urinary sodium excretion and oxidative stress levels increased in both groups during treatment compared to baseline.Conclusively, stimulating ETB with S6c seems to improve the diminished kidney oxygenation in diabetic nephropathy in the same manner as inhibiting ETA, by improving oxygen availability, although not to the same extent.Support or Funding InformationAll experiments were conducted according to FASEB Statement of Principles. This study was supported by the Swedish Research Council, the Swedish Diabetes Foundation and the Swedish Heart Lung Foundation.

Reduced Mechanical Stretch Induces Enhanced Endothelin B Receptor-Mediated Contractility via Activation of Focal Adhesion Kinase and Extracellular Regulated Kinase 1/2 in Cerebral Arteries from Rat.

Cerebral ischaemia results in enhanced endothelin B (ETB ) receptor-mediated contraction and receptor protein expression in the affected cerebrovascular smooth muscle cells (SMC). Organ culture of cerebral arteries is a method to induce similar alterations in ETB receptor expression. We suggest that rapid and sustained reduction in wall tension/stretch is a possible trigger mechanism for this vascular remodelling. Isolated rat middle cerebral artery (MCA) segments were incubated in a wire myograph with or without mechanical stretch, prior to assessment of their contractile response to the selective ETB receptor agonist sarafotoxin 6c. The involvement of extracellular regulated kinase (ERK) 1/2 and focal adhesion kinase (FAK) was studied by their specific inhibitors U0126 and PF-228, respectively. Compared with their stretched counterparts, unstretched MCA segments showed a significantly increased ETB receptor-mediated contractile response after 12 hr of incubation, which was attenuated by either U0126 or PF-228. The functionally increased ETB -mediated contractility could be attributed to two different mechanisms: (i) a difference in ETB receptor localization from primarily endothelial expression to SMC expression and (ii) an increased calcium sensitivity of the SMCs due to an increased expression of the calcium channel transient receptor potential canonical 1. Collectively, our results present a possible mechanism linking lack of vessel wall stretch/tension to changes in ETB receptor-mediated contractility via triggering of an early mechanosensitive signalling pathway involving ERK1/2 and FAK signalling. A mechanism likely to be an initiating factor for the increased ETB receptor-mediated contractility found after cerebral ischaemia.

High salt intake increases endothelin B receptor function in the renal medulla of rats

AimsEndothelin (ET)-1 promotes natriuresis via the endothelin B receptor (ETB) within the renal medulla. In male rats, direct interstitial infusion of ET-1 into the renal medulla has no effect on renal sodium and water excretion but is associated with endothelin A receptor (ETA)-dependent reductions in medullary blood flow. Loss of ETB function leads to salt-sensitive hypertension. We hypothesized that HS intake would increase the natriuretic and diuretic response to renal medullary infusion of ET peptides. Main methodsMale Sprague–Dawley (SD) rats were fed a normal (NS) or high (HS) salt diet for 7days. Rats were anesthetized and a catheter implanted in the renal medulla for interstitial infusion along with a ureteral catheter for urine collection. Medullary infusion of a low dose of ETB receptor agonist, sarafotoxin 6c (S6c; 0.15μg/kg/h), or ET-1 (0.45μg/kg/h) was used to determine changes in sodium excretion (UNaV). Key findingsIn HS fed rats, intramedullary infusion of a low dose of S6c induced a significant increase in UNaV, roughly 2-fold over baseline, compared to no response to this low dose in NS fed rats. In HS fed rats, intramedullary infusion of ET-1 induced a significantly greater increase in UNaV compared to NS fed rats, although this increase was not different from the HS time control studies. SignificanceWe conclude that high salt intake enhances the diuretic and natriuretic effects of ETB receptor activation in vivo consistent with a role for the ETB receptor in maintaining fluid-electrolyte homeostasis.