Sarafotoxin S6c Research Articles

Abstract P1170: Changes In Endothelin Signaling In A Long-term Rat Coronary Culture Resembles Chronic Coronary Syndrome

Background: Coronary artery disease generates changes in the structure and function of the blood vessels. 48hrs organ culture (mimicking the acute loss of blood flow) of coronary arteries induces the upregulation of contractile endothelin type B (ETB) receptors, resembling the pattern seen in ischemic heart disease. MEK inhibitors (U0126 and Trametinib), can acutely inhibit the upregulation of these receptors, illustrating the importance of this pathway. The 48hrs organ culture model has been proven successful for translation in acute ischemic disease, but arterial changes have not been studied in a long-term chronic model which could be important for chronic coronary syndrome. Purpose: 1) Investigate the changes of ETB receptors after incubation in a long-term optimized medium for organ culture, mimicking the reduction of blood flow in chronic coronary syndrome and 2) determine whether Trametinib, would have treatment effect on the upregulation of ETB receptors in both long term and short term culture. Methods: Rat coronary arteries were incubated in organ culture for 48hrs in DMEM ± trametinib (“acute”) or in a long-term optimized medium for organ culture for 14 days (“chronic”). After the incubation, arteries were mounted stainless steel wires (40 μm) on a Mulvany-Halpern arterial wire myograph and cumulative concentration response curves were made to measure arterial contractility to the ETB receptor specific agonist Sarafotoxin (S6c). Trametinib was added at time 0 (for both the acute and chronic organ culture) or 7 days delayed. Results: For the “acute” culture, we observed a high contractile effect in response to S6c (Emax=136.5±18%). The presence of Trametinib greatly reduced this contractile effect (Emax=10±1.9%) and arteries were comparable to fresh (Emax=0.8±0.7%). The rat coronary arteries in the “chronic” culture maintained their ability to contract in response to potassium, and S6c had a strong contractile effect (Emax=74.64±17%). Interestingly, neither trametinib addition at time 0 nor at day 7 could inhibit the functional upregulation of ETB in the chronic model. Conclusion: Chronic coronary syndrome may require new inhibitors of ETB receptor upregulation. This could be investigated with our new long-term rat coronary artery model.

The Effect of Losartan on Neuroinflammation as Well as on Endothelin-1- and Serotonin-Induced Vasoconstriction in a Double-Haemorrhage Rat Model.

Poor patient outcome after aneurysmal subarachnoid haemorrhage (SAH) is due to a multifactorial process. Delayed cerebral vasospasm, ischemic neurological deficits, and infarction are the most feared acute sequelae triggered by enhanced synthesis of serotonin and endothelin-1 (ET-1). During the past decades, multiple drugs have been analysed for protective effects without resounding success. Therefore, the authors wanted to analyse the potential beneficial role of Losartan (LOS). Male Sprague Dawley rats were randomised into either a group receiving two injections of blood into the cisterna magna (SAH group) or a group receiving two injections of isotonic sodium chloride (sham group). The animals were culled on day five and basilar artery ring segments were used for in vitro tension studies. Sarafotoxin S6c caused a dose-dependent vasorelaxation in sham and SAH segments, which was more pronounced in sham segments. LOS, applied in a concentration of 10-3 M, was able to significantly reduce serotonin- (p < 0.01) and ET-1- (p < 0.05, p < 0.01) mediated vasoconstriction in sham segments. These findings, along with the well-known beneficial effects of LOS on restoring the impaired endothelin-B1-receptor function after SAH, as well as on the neuroprotectional and antiepileptogenic aspects, might be implemented in advancing tailored concepts to sufficiently ameliorate patients' functional outcome after SAH.

Tetrahydroxystilbene glucoside attenuated homocysteine-upregulated endothelin receptors in vascular smooth muscle cells via the ERK1 /2 /NF-κB signaling pathway.

2,3,5,4'-Tetrahydrostilbene-2-o-β-d-glucoside (TSG) is the main active component of Polygonum multiflorum Thunb. It has effects on hypertension. However, the mechanism is unclear. Current research is devoted to exploring the mechanism of TSG improving HHcy-induced hypertension. The mice received a subcutaneous injection of Hcy in the presence or absence of TSG for 4 weeks. Blood pressure (BP) was measured using a noninvasive tail-cuff plethysmography method. Levels of plasma Hcy and endothelin-1 were measured using ELISA. Rat SMA without endothelium was cultured in a serum-free medium in the presence or absence of TSG with or without Hcy. The contractile response to sarafotoxin 6c or endothein-1 was studied using a sensitive myography. The levels of protein were detected using Western blotting. The results showed that TSG lowered HHcy-elevated BP and decreased levels of plasma Hcy and endothelin-1 in mice. Furthermore, the results showed that TSG inhibited Hcy-upregulated ET receptor expression and ET receptor-mediated contractile responses as well as the levels of p-ERK1/2 and p-p65 in SMA. In vivo results further validate the in vitro results. In conclusion, TSG can decrease the levels of plasma Hcy and ET-1 and downregulate Hcy-upregulated ET receptors in VSMCs by inhibiting the ERK1/2 /NF-κB/ETB2 pathway to lower the BP.

Inhibitors of the MAPK/ NF-κB pathway attenuate the upregulation of the ETB receptor mediated by high glucose in vascular smooth muscle cells

BackgroundIncreased vascular smooth muscle cell (VSMC) endothelin type B (ETB) receptor expression is involved in cardiovascular diseases. High glucose (HG) in diabetes is closely related to cardiovascular complications. Although diabetes upregulates VSMC endothelin subtype B (ETB) receptors, its mechanism is still unclear. Our aim is to investigate the mechanism of HG-induced ETB receptors in VSMCs. MethodsRat superior mesenteric arteries (SMAs) without endothelium were cultured in medium without serum for 24 h. HG with or without mitogen-activated protein kinase (MAPK) signaling pathway inhibitors and downstream nuclear factor-kappaB (NF-κB) inhibitors was coincubated with SMAs. A sensitive myograph detected the contractile responses to sarafotoxin 6c. Western blotting and immunofluorescence staining were used to determine protein expression. ResultsHG promoted the expression of VSMC ETB receptors in rat SMAs and enhanced the ETB receptor-induced contractile response. The results showed that HG increased vascular smooth muscle cell (VSMC) ETB receptor expression and ETB receptor-induced contractile responses in rat SMAs. Both extracellular signal-related kinase 1 and 2 (ERK1/2) inhibitors (U0126) and P38 inhibitors (SB203580) significantly inhibited HG-increased VSMC ETB receptors. However, a C-jun terminal kinase (p-JNK) inhibitor (SP600125) did not affect HG- upregulated VSMC ETB receptors. Further study showed that NF-κB using an IκB kinase inhibitor (wedelolactone) also significantly inhibited HG-increased VSMC ETB receptors. ConclusionIn conclusion, HG upregulated the VSMC ETB receptor by activating the ERK1/2- or P38- NF-κB signaling pathway.

Ovariectomy Reduces Vasocontractile Responses of Rat Middle Cerebral Arteries After Focal Cerebral Ischemia.

Effects of sex hormones on stroke outcome are not fully understood. A deleterious consequence of cerebral ischemia is upregulation of vasoconstrictor receptors in cerebral arteries that exacerbate stroke injury. Here, we tested the hypothesis that female sex hormones alter vasocontractile responses after experimental stroke in vivo or after organ culture in vitro, a model of vasocontractile receptor upregulation. Female rats with intact ovaries and ovariectomized (OVX) females treated with 17β-estradiol, progesterone, or placebo were subjected to transient, unilateral middle cerebral artery occlusion followed by reperfusion (I/R). The maximum contractile response, measured my wire myography, in response to the endothelin B receptor agonist sarafotoxin 6c was increased in female arteries after I/R, but the maximum response was significantly lower in arteries from OVX females. Maximum contraction mediated by the serotonin agonist 5-carboxamidotryptamine was diminished after I/R, with arteries from OVX females showing a greater decrease in maximum contractile response. Contraction elicited by angiotensin II was similar in all arteries. Neither estrogen nor progesterone treatment of OVX females affected I/R-induced changes in endothelin B- and 5-carboxamidotryptamine-induced vasocontraction. These findings suggest that sex hormones do not directly influence vasocontractile alterations that occur after ischemic stroke; however, loss of ovarian function does impact this process.

ET-B receptors involvement in peripheral opioid analgesia induced by light-emitting diode photobiomodulation in male and female mice

Currently, photobiomodulation therapy (PBMT) is gaining space in the scientific and clinical environment. To help elucidate the importance of irradiance, this study evaluated the effect of two different PBMT irradiances (3.5 and 90 mW/cm2), given a fixed wavelength of 630 nm and a dose of 2 J/cm2, on mechanical hyperalgesia following Complete Freund's Adjuvant (CFA) intraplantar (i.pl.) injection in mice. Additionally, we investigated the role of peripheral opioid and endothelin-B receptors (ETB-R), as well as sex differences in treatment outcome. Different groups of male or female mice were evaluated 6 and 96 h after CFA. Mechanical hyperalgesia was evaluated 30 min after treatments. Naloxone or Bq-788 administration, fifteen minutes before PBMT or Sarafotoxin S6c, helped determine the involvement of peripheral opioid and ETB-Rs on PBMT. Lastly, ETB-Rs skin immunocontent in both sexes was quantified after PBMT consecutive daily treatments. PBMT at an irradiance of 90 mW/cm2, was more effective than 3.5 mW/cm2. Bq-788 and naloxone administration prevented the effects of PBMT and SRTX S6c; however, PBMT did not influence peripheral ETB-Rs immunocontent. The results suggest that irradiance influences PMBT effect; and that activation of ETB-R play a role in peripheral PBMT opioid induced analgesia. Lastly, PMBT effects do not appear to be sex-dependent.

Endothelin-1 decreases the expression of Ephrin-A and B subtypes in cultured rat astrocytes through ETB receptors

Ephrin family proteins are cell surface molecules that regulate several cellular functions through cell-cell interactions. During nervous tissue repair after injury, the expression of ephrin subtypes in astrocytes is altered, affecting the axonal elongation and migration of neuronal precursors. However, the mechanism regulating the expression of ephrin subtypes in astrocytes has not been investigated. Herein, we studied the effects of endothelin-1 (ET-1) on the expression of ephrin subtypes in cultured rat astrocytes. Our results showed that ET-1 (100 nM) treatment for 1−24 h reduced the expression of ephrin-A2, -A4, -B2, and -B3 mRNA and protein in astrocytes, whereas the expression of ephrin-A1, -A3, -A5, and -B1 mRNA were not affected. Sarafotoxin S6c, a selective ETB receptor agonist, decreased the expression of ephrin-A2, -A4, -B2, and -B3 in cultured astrocytes. The decrease in ephrin-A2, -A4, -B2, and -B3 expression by ET-1 treatment was reduced in the presence of BQ788, an ETB receptor antagonist, while FR139317, an ETA receptor antagonist, had no effects. These results suggest that ET-1 is a signaling molecule that downregulates ephrin-A2, -A4, -B2, and -B3 expression in astrocytes.

Endothelin receptor B controls the production of fibroblast growth factor 23.

Endothelin-1 (ET-1) is a member of the endothelin family of peptide hormones first discovered as endothelium-derived mediators regulating vascular tone. ET-1 also regulates the proliferation and differentiation of bone cells that synthesize fibroblast growth factor 23 (FGF23). FGF23 is a hormone controlling renal phosphate and vitamin D metabolism. Here, we studied the role of ET-1 and endothelin receptor B (ETB) for FGF23 production. Fgf23 gene expression was studied in IDG-SW3 bone cells by quantitative RT-PCR. ETB-expressing (etb+/+ ) and rescued ETB-deficient mice (etb-/- ) were studied in metabolic cages. Their serum FGF23, PTH, and 1,25(OH)2 D3 concentrations were determined by ELISA, serum and urinary phosphate and Ca2+ by photometric methods. ET-1 and ETB agonist sarafotoxin 6c suppressed Fgf23 mRNA in IDG-SW3 cells. Serum C-terminal and intact FGF23 as well as bone Fgf23 mRNA levels were significantly higher in etb-/- mice than in etb+/+ mice. Renal phosphate excretion was significantly higher in etb-/- mice despite lower phosphate levels. In addition, etb-/- animals exhibited calciuria and a significantly higher serum 1,25(OH)2 D3 concentration compared to etb+/+ mice. In conclusion, ETB-dependent ET-1 signaling is a potent suppressor of FGF23 formation. This effect is likely to be of clinical relevance given the use of endothelin receptor antagonists in various diseases.

Metformin inhibited homocysteine-induced upregulation of endothelin receptors through the Sirt1/NF-κB signaling pathway in vascular smooth muscle cells

Metformin (Met) can improve atherosclerosis (As). Abnormal endothelin receptors [including endothelin type A (ETA) or type B (ETB) receptor] in vascular smooth muscle cells (VSMCs) are involved in As. Hyperhomocysteinemia (HHcy) is an independent risk factor for As. The present study was designed to test our hypothesis that Met inhibits the upregulation of endothelin receptors induced by homocysteine (Hcy) in VSMCs. Rat superior mesenteric artery (SMA) without endothelium, as an in vitro model, was cultured in serum-free medium for 24 h in the presence of Hcy with or without Met or nicotinamide (Nic). In vivo, rats received subcutaneous injections of Hcy in the presence or absence of Met or Nic for 3 weeks. Levels of protein expression were determined by Western blotting. The contractile responses to sarafotoxin 6c (an ETB receptor agonist) or ET-1 (an ETA/ ETB receptor agonist) were studied using a sensitive myograph. The blood pressure of rats was measured using a noninvasive tail-cuff plethysmography method. The results showed that Met could significantly inhibit the Hcy-induced upregulation of endothelin receptors (including ETA and ETB receptor) protein expression and endothelin receptor-mediated vasoconstriction, and it recovered the Hcy-induced decrease in silent information regulator 1 (Sirt1) in a dosage-dependent manner in SMA. However, Nic (a Sirt1 inhibitor) recovered the levels of Met-inhibited endothelin receptors and acetylated p65. Furthermore, the in vivo results showed that Met not only significantly the inhibited HHcy-induced upregulation of endothelin receptors and acetylated p65 but also recovered the HHcy-induced decrease in Sirt1 in a dosage-dependent manner in SMA. In addition, Met significantly inhibited the HHcy-induced blood pressure elevation. However, these effects were reverted by Nic. In conclusion, these data demonstrated that Met inhibited the Hcy-induced increase in endothelin receptor expression by activating Sirt1 and then inhibiting NF-κB in VSMCs. These findings may provide insights into the mechanism underlying of Met-treated cardiovascular diseases induced by Hcy.

Effects of a new magnesium-rich artificial cerebrospinal fluid on contractile 5-hydroxytryptamine and endothelin receptors in rat cerebral arteries

ABSTRACTObjectives Cerebral vasospasm after subarachnoid haemorrhage (SAH) is associated with cerebrovascular contractile receptor upregulation resulted from haemolysis in the subarachnoid space. This study developed a new magnesium-rich artificial cerebrospinal fluid (MACSF) formula and investigated its effects on receptor-mediated contraction in rat basilar arteries.Methods Clear and haemorrhagic cerebrospinal fluid (CSF) were collected from patients with hydrocephalus or SAH. MACSF was freshly prepared using clinical intravenous injections. Rat basilar arteries were segmented and incubated with clear CSF, haemorrhagic CSF or MACSF. The contractile responses were studied by myograph. The messenger ribonucleic acid (mRNA) and protein expression of 5-hydroxytryptamine 1B (5-HT1B), endothelin subtype B (ETB) and endothelin subtype A (ETA) receptors were evaluated by real-time polymerase chain reaction (PCR) and Western blot analyses.Results Haemorrhagic CSF exposure shifted the contractile curves induced by 5-hydroxytryptamine (5-HT), sarafotoxins 6c (S6c) and endothelin-1 (ET-1) leftward with increased maximal contraction values. Furthermore, mRNA and protein expression were markedly elevated for 5-HT1B, ETB and ETA receptors on arteries exposed to haemorrhagic CSF. However, the contractile responses to 5-HT, S6c or ET-1 and expression of 5-HT1B, ETB and ETA receptors in rat cerebral arteries exposed to MACSF remained unaffected compared to those exposed to clear CSF. Besides, unlike normal saline which can inactive in-vitro vessels, MACSF can maintain their physiological activity.Conclusion Haemorrhagic CSF induces upregulation of 5-HT1B, ETB and ETA receptors in rat cerebral arteries. However, MACSF can maintain in-vitro rat basilar arteries in good physiological activity and normal expression of contractile 5-HT and ET receptors.

Endothelin ETA- and ETB-Receptor-Mediated Inhibition of Noradrenaline Release From Isolated Rat Stomach

We examined the effect of endothelin-1, endothelin-3 and sarafotoxin S6c on the release of noradrenaline from gastric sympathetic nerve terminals using an isolated, vascularly perfused rat stomach. The release of noradrenaline evoked by electrical stimulation of the gastric postganglionic sympathetic nerves (at 2.5 Hz for 1 min) was inhibited by endothelin-1 (10-10 – 10-8 M), endothelin-3 (10-9 – 10-8 M) and sarafotoxin S6c (a highly selective agonist of endothelin ETB receptors) (10-9 – 10-8 M) in a concentration-dependent manner; the inhibitory potencies were as follows: endothelin-1 > endothelin-3 > sarafotoxin S6c. The inhibitory effect of endothelin-1 (3 × 10-9 M) on noradrenaline release was abolished by BQ-123 (a selective antagonist of endothelin ETA receptors) in a dose-dependent manner (10-7 and 10-6 M), but not influenced by BQ-788 (a selective antagonist of endothelin ETB receptors) (10-7 and 10-6 M). The endothelin-1-induced inhibition of noradrenaline release was attenuated by pretreatment with pertussis toxin (10 μg/animal, i.v., 4 days before experiments), but not influenced by indomethacin (3 × 10-6 M). These results indicate that endothelin ETA and ETB receptors located on the sympathetic nerve terminals play a role in the inhibition of noradrenaline release from the rat stomach: endothelin ETA receptor-mediated inhibition is carried out by pertussis toxin-sensitive and indomethacin-insensitive mechanisms.

Role of endothelin-1 clearance in the haemodynamic responses to endothelin-1 in the pulmonary and hindquarter vasculature of anaesthetised rats.

In the pulmonary vasculature there is clearance of endothelin-1 from the circulation mediated by endothelin ETB receptors. This study explored the haemodynamic effects of endothelin-1 and its clearance in the pulmonary and hindquarter vasculature in anaesthetised rats. Carotid and pulmonary artery pressures and pulmonary and hindquarter blood flows were measured. In each rat, a single endothelin-1 or sarafotoxin S6C cumulative dose-response curve was generated with or without antagonist pretreatment (i.v.). Endothelin-1 caused an acute fall in MAP and rise in hindquarter vascular conductance (HVC) followed by a marked increase in MAP at 5 min with falls in HVC and pulmonary vascular conductance (PVC). Bosentan (10, 20 & 30 mg/kg) pretreatment caused dose-dependent inhibition of the MAP increase as well as PVC and HVC decreases to endothelin-1. Similarly, macitentan (30 mg/kg) or ambrisentan (10 mg/kg) caused significant block of responses to endothelin-1. Sarafotoxin S6C caused acute falls in MAP and increases in HVC and then small falls in PVC and HVC, all prevented by pretreatment with ETB antagonist BQ788 (1 mg/kg). Pretreatment with BQ788 enhanced endothelin-1 potency by 2.5-fold in PVC and 2.4-fold in HVC. With BQ788 and bosentan, the fall in HVC response was completely blocked, but there were residual MAP rises and PVC falls at the highest endothelin-1 dose. Our work confirms the role of ETB receptors in the pulmonary vasculature that decrease the circulating levels of endothelin-1. This has important consequences in selecting an appropriate ETA and ETB dual receptor antagonist to effectively block endothelin-1-mediated pulmonary vasoconstriction.

Expression of endothelin type B receptors in uterine artery smooth muscle cells from pregnant Guinea pigs

IntroductionIt is well established that upregulation of endothelin type B (ETB) receptors in vascular smooth muscle cells plays a role in pathophysiological artery remodeling as response to ischemia and atherosclerosis. This study aimed to investigate the ETB receptors function and localization under normal physiological remodeling. Specifically, in the guinea pig uterine arteries during pregnancy. MethodsUterine artery contractility was assessed with sarafotoxin 6c and endothelin-1 in wire-myography in uterine arteries from non-pregnant and pregnant guinea pigs at gestational day 37 ± 5. Localization of ETB receptors, proliferation marker Ki-67, and SMC differentiation marker SM22α in uterine arteries were investigated with immunohistochemistry. ResultsUterine arteries from pregnant guinea pigs showed significantly increased ETB receptor-mediated vasoconstriction compared to uterine arteries from non-pregnant and to coronary arteries from pregnant guinea pigs (p < 0.001), suggesting that ETB-receptor upregulation in uterine artery SMCs is a normal physiological mechanism taking place during remodeling. Furthermore, uterine arteries from pregnant guinea pigs showed enhanced expression of ETB receptors, high density of Ki-67 positive SMCs and sparse SM22α staining in SMCs localized in the outer layer of the vessel wall. DiscussionOur results suggest that ETB receptors are expressed in dedifferentiated proliferating SMCs of uterine arteries in pregnant guinea pigs. This study provides novel insight into the function and expression of ETB receptors in uterine vascular remodeling during pregnancy.

Minimally modified low-density lipoprotein upregulates the ETB and α1 receptors in mouse mesenteric arteries in vivo by activating the PI3K/Akt pathway

The current study aimed to explore whether minimally modified low-density lipoprotein (mmLDL) via tail vein injection upregulates the ETB and α1 receptors in mouse mesenteric arteries by activating the PI3K/Akt pathway. The contraction curves of the mesenteric arteries caused by sarafotoxin 6c (S6c, ETB receptor agonist) and phenylephrine (PE, α1 receptor agonist) were measured by a myograph system. Serum oxLDL was detected using enzyme-linked immunosorbent assays. The levels of the ETB receptor, the α1 receptor, PI3K, p-PI3K and p-Akt were detected using real-time polymerase chain reaction and Western blot analyses. Minimally modified low-density lipoprotein noticeably enhanced the contraction effect curves of S6c and PE, with significantly increased Emax values (P<0.01), compared to those of the control group. This treatment significantly increased the mRNA expression and protein levels of the ETB and α1 receptors and the protein levels of p-PI3K and p-Akt in the vessel wall (P<0.01). LY294002 inhibited the effect of mmLDL. An increase in mmLDL activated the PI3K/Akt pathway, which upregulated the expression of the ETB and α1 receptors and enhanced the ETB and α1- receptor-mediated contractile function.

Afferent arteriole responsiveness to endothelin receptor activation: does sex matter?

BackgroundThe pathogenesis of hypertension is distinct between men and women. Endothelin-1 (ET-1) is a potential contributor to sex differences in the pathophysiology of hypertension. ET-1 participates in blood pressure regulation through activation of endothelin A (ETA) and endothelin B (ETB) receptors including those in the vasculature. Previous studies demonstrated that sex and sex hormones evoke discrepancies in ET-1-mediated control of vascular tone in different vascular beds. However, little is known about sex- and sex hormone-related differences in ET-1-dependent renal microvascular reactivity. Accordingly, we hypothesized that loss of sex hormones impairs afferent arteriole reactivity to ET-1.MethodsMale and female Sprague Dawley rats were subjected to gonadectomy or sham surgery (n = 6/group). After 3 weeks, kidneys from those rats were prepared for assessment of renal microvascular responses to ET-1 (ETA and ETB agonist, 10−12 to 10−8 M) and sarafotoxin 6c (S6c, ETB agonist, 10−12 to 10−8 M) using the blood-perfused juxtamedullary nephron preparation.ResultsControl afferent arteriole diameters at 100 mmHg were similar between sham male and female rats averaging 14.6 ± 0.3 and 15.3 ± 0.3 μm, respectively. Gonadectomy had no significant effect on control arteriole diameter. In sham males, ET-1 produced significant concentration-dependent decreases in afferent arteriole diameter, with 10−8 M ET-1 decreasing diameter by 84 ± 1%. ET-1 induced similar concentration-dependent vasoconstrictor responses in sham female rats, with 10−8 M ET-1 decreasing the diameter by 82 ± 1%. The afferent arteriolar vasoconstrictor responses to ET-1 were unchanged by ovariectomy or orchiectomy. Selective ETB receptor activation by S6c induced a concentration-dependent decline in afferent arteriole diameter, with 10−8 M S6c decreasing diameter by 77 ± 3 and 76 ± 3% in sham male and female rats, respectively. Notably, ovariectomy augmented the vasoconstrictor response to S6c (10−12 to 10−9 M), whereas orchiectomy had no significant impact on the responsiveness to ETB receptor activation.ConclusionThese data demonstrate that sex does not significantly influence afferent arteriole reactivity to ET receptor activation. Gonadectomy potentiated the responsiveness of the afferent arteriole to ETB-induced vasoconstriction in females, but not males, suggesting that female sex hormones influence ETB-mediated vasoconstriction in the renal microcirculation.

Constriction of Retinal Venules to Endothelin-1: Obligatory Roles of ETA Receptors, Extracellular Calcium Entry, and Rho Kinase.

PurposeEndothelin-1 (ET-1) is a potent vasoconstrictor peptide implicated in retinal venous pathologies such as diabetic retinopathy and retinal vein occlusion. However, underlying mechanisms contributing to venular constriction remain unknown. Thus, we examined the roles of ET-1 receptors, extracellular calcium (Ca2+), L-type voltage-operated calcium channels (L-VOCCs), Rho kinase (ROCK), and protein kinase C (PKC) in ET-1-induced constriction of retinal venules.MethodsPorcine retinal venules were isolated and pressurized for vasoreactivity study using videomicroscopic techniques. Protein and mRNA were analyzed using molecular tools.ResultsRetinal venules developed basal tone and constricted concentration-dependently to ET-1. The ETA receptor (ETAR) antagonist BQ123 abolished venular constriction to ET-1, but ETB receptor (ETBR) antagonist BQ788 had no effect on vasoconstriction. The ETBR agonist sarafotoxin S6c did not elicit vasomotor activity. In the absence of extracellular Ca2+, venules lost basal tone and ET-1–induced constriction was nearly abolished. Although L-VOCC inhibitor nifedipine also reduced basal tone and blocked vasoconstriction to L-VOCC activator Bay K8644, constriction of venules to ET-1 remained. The ROCK inhibitor H-1152 but not PKC inhibitor Gö 6983 prevented ET-1-induced vasoconstriction. Protein and mRNA expressions of ETARs and ETBRs, along with ROCK1 and ROCK2 isoforms, were detected in retinal venules.ConclusionsExtracellular Ca2+ entry via L-VOCCs is essential for developing and maintaining basal tone of porcine retinal venules. ET-1 causes significant constriction of retinal venules by activating ETARs and extracellular Ca2+ entry independent of L-VOCCs. Activation of ROCK signaling, without involvement of PKC, appears to mediate venular constriction to ET-1 in the porcine retina.

Molecular Determinants for Ligand Selectivity of the Cell-Free Synthesized Human Endothelin B Receptor

Extracellular domains of G-protein-coupled receptors act as initial molecular selectivity filters for subtype specific ligands and drugs. Chimeras of the human endothelin-B receptor containing structural units from the extracellular domains of the endothelin-A receptor were analyzed after their co-translational insertion into preformed nanodiscs. A short β-strand and a linker region in the second extracellular loop as well as parts of the extracellular N-terminal domain were identified as molecular discrimination sites for the endothelin-B receptor-selective agonists IRL1620, sarafotoxin 6c, 4Ala-ET-1 and ET-3, but not for the non-selective agonist ET-1 recognized by both endothelin receptors. A proposed second disulfide bridge in the endothelin-B receptor tethering the N-terminal domain with the third extracellular loop was not essential for ET-1 recognition and binding, but increased the receptor thermostability. We further demonstrate an experimental approach with cell-free synthesized engineered agonists to analyze the differential discrimination of peptide ligand topologies by the two endothelin receptors. The study is based on the engineering and cell-free insertion of G-protein-coupled receptors into defined membranes and may become interesting also for other targets as an alternative platform to reveal molecular details of ligand selectivity and ligand binding mechanisms.

Resveratrol down-regulates endothelin type B receptors in vascular smooth muscle cells via Sirt1/ERK1/2/NF-кB signaling pathways

Silent information regulator family protein 1 (Sirt1) has gained attention for protective effects against cardiovasc diseases. Vascular smooth muscle endothelin type B (ETB) receptors are related to the pathogenesis of cardiovascular diseases. Elevated oxidized low-density lipoprotein (ox-LDL) is associated with atherosclerosis. This study will investigate whether resveratrol (a Sirt1 activator, Res) is involved in oxidized low density lipoprotein (ox-LDL)-mediated- regulation of ETB receptors in rat superior mesenteric arteries (SMA). The rat SMA segments were cultured in the presence and absence of ox-LDL with or without Res and specific inhibitor (U0126) for the extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) for 24 h. After organ culture, the contractile responses to sarafotoxin 6c (S6c) were studied using a sensitive myograph, and the ETB receptor protein expression was detected using Western blotting. The results showed that Res concentration-dependently suppressed the ox-LDL -induced up-regulation of ETB receptors expression and receptor-mediated vasoconstriction. In addition, these effects could be inhibited by U0126. Furthermore, activity of ERK1/2 phosphorylation and P65 acetylation induced by ox-LDL were blocked by Res. In conclusion, Res down-regulated ETB receptors through up-regulating Sirt1 and followed by ERK1/2/NF-кB signaling pathways in the organ culture SMA.

Vasomotor dysfunction in human subcutaneous arteries exposed ex vivo to food-grade titanium dioxide

Animal studies have shown that titanium dioxide (TiO2) exposure affects arterial vasomotor function, whereas little is known about the effects in arteries from humans. This study investigated vasomotor responses after direct exposure of human subcutaneous arteries to food-grade TiO2 (E171) (14 or 140 μg/ml) for 30 min and 18 h. Vasomotor responses to bradykinin, 5-hydroxytryptamine (5-HT), sarafotoxin 6c (S6c) and nitroglycerin were recorded in wire-myographs. Vasoconstrictor responses to 5-HT were increased in arteries exposed to E171 for 18 h (P < 0.05). Furthermore, an increase in S6c responses was seen in low concentration E171 exposed arteries (30 min exposure; P < 0.05). The vasorelaxation response to nitroglycerin was increased in low concentration E171 exposed arteries (30 min exposure; P < 0.05). Vasorelaxation responses to bradykinin were unaffected after treatment with E171. There was no difference in gene expression levels of intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, 5-hydroxytryptamine receptor 1B, 5-hydroxytryptamine receptor 2A, endothelin receptor A and endothelin receptor B in E171 exposed arteries after exposure to TiO2 for 30 min or 18 h. In conclusion, this study shows that the same type of vasomotor dysfunction is found in artery segments of rats and humans following ex vivo exposure to E171.

High Glucose Upregulated Vascular Smooth Muscle Endothelin Subtype B Receptors via Inhibition of Autophagy in Rat Superior Mesenteric Arteries

Autophagy plays an important role in cardiovascular diseases. High glucose (HG) upregulates endothelin subtype B (ETB) receptors in vascular smooth muscle cells (VSMCs). However, it is unclear as to whether autophagy is involved in HG-induced upregulation of ETB receptors in VSMCs. The present study was designed to examine the hypothesis that HG upregulates ETB receptors by inhibiting autophagy in VSMCs. We studied HG-treated rat superior mesenteric artery (SMA) without endothelium in the presence and absence of 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), rapamycin, or MHY1485 for 24hr. We measured contractile responses to sarafotoxin 6c (S6c) (an ETB receptor agonist) using a sensitive myograph. Levels of protein expression were determined using Western blotting. HG impaired autophagy and increased the levels of ETB receptor protein expression and ETB receptor-mediated contractile responses to S6c in SMA. However, these effects were reversed by AICAR (an agonist of adenosine monophosphate [AMP]-activated protein kinase [AMPK]) and rapamycin (an inhibitor of mammalian target of rapamycin [mTOR]). However, MHY1485 (an agonist of mTOR) did not upregulate the AICAR-inhibited ETB receptor-mediated contractile responses or ETB receptor protein expression in the presence of HG. These data suggest that HG upregulated ETB receptors by inhibiting autophagy in VSMCs via AMPK and mTOR signaling pathways.