Human Endothelin Research Articles

Crystal structure of human endothelin ETB receptor in complex with peptide inverse agonist IRL2500

Endothelin receptors (ETA and ETB) are G-protein-coupled receptors activated by endothelin-1 and are involved in blood pressure regulation. IRL2500 is a peptide-mimetic of the C-terminal tripeptide of endothelin-1, and has been characterized as a potent ETB-selective antagonist, which has preventive effects against brain edema. Here, we report the crystal structure of the human ETB receptor in complex with IRL2500 at 2.7 Å-resolution. The structure revealed the different binding modes between IRL2500 and endothelin-1, and provides structural insights into its ETB-selectivity. Notably, the biphenyl group of IRL2500 penetrates into the transmembrane core proximal to D2.50, thus stabilizing the inactive conformation. Using the newly-established constitutively active mutant, we clearly demonstrate that IRL2500 functions as an inverse agonist for the ETB receptor. The current findings will expand the chemical space of ETR antagonists and facilitate the design of inverse agonists for other class A GPCRs.

Abstract P404: Sex-specific Associations of Plasma Endothelin-1 With Incidence of Stroke in African Americans: the Jackson Heart Study

Background: Endothelin-1 (ET-1) is a vasoconstrictive peptide with cerebral action, also associated with the degree of atherosclerosis. Previous research found that estrogen has inhibitory effect on ET-1 level, which points to putative differences of ET-1 by sex. As stroke is the fifth leading cause of death in the U.S., killing approximately 133,000 people per year, more among African Americans (AA), we aimed to identify the sex differences of the association between ET-1 and incident stroke among AA. Methods: Our AA study participants (N= 4995), with ages 21-90 years and no prevalent stroke, were enrolled in the Jackson Heart Study in Jackson, Mississippi. Stroke events were ascertained through 2011 by reviewing medical records for hospitalizations of JHS participants. Plasma ET levels were measured at the baseline study visit (2000-2004) and expressed in picograms/ml by QuantiGlo Human Endothelin-1 Immunoassay (R&D Systems Inc). Hazard ratios (HR) and 95% confidence intervals (95% CI) were estimated using Cox proportional hazard regression models. Several interactions were assessed such as by sex, smoking and hypertension. Results: Baseline plasma ET-1 level (mean=1.35, median= 1.30) was significantly lower in women (mean =1.32) than in men (mean =1.40; P-value <0.0001). Sex was not an effect modifier of the association between ET-1 and time to stroke event (p for interaction =0.18). After adjusted for age, ET-1 was associated with reduced risk of stroke [HR (95% CI) for all gender: 0.61 (0.40, 0.94), P trend =0.015, among men: 0.32 (0.14, 0.72), P trend =0.003, but not among women: 0.96 (0.54, 1.68), P trend =0.68, for highest vs. lowest quartile, respectively]. After additional adjustment for hypertension and smoking status, the association was still significant among men [0.35 (0.15, 0.79), P trend =0.007, for highest vs. lowest quartile, respectively], but not among women. No statistically significant associations were found among both men and women after additional adjustment for BMI, cigarette smoking, anti-hypercholesterolemic medication status, diabetes and hypertension status, cholesterol levels and homocysteine. No interactions by smoking or hypertension were detected. Conclusion: Our study indicates a non-statistical significant lower risk of stroke with higher levels of endothelin-1, with variation by sex. Our findings warrant confirmation in other epidemiologic studies.

Activation of PERK in ET-1- and thrombin-induced pulmonary fibroblast differentiation: Inhibitory effects of curcumin.

In the present study, we investigated the role of PKR-like endoplasmic reticular kinase (PERK), an endoplasmic reticulum (ER) stress kinase, in endothelin 1 (ET-1)- and thrombin-induced pulmonary fibrosis (PF), and the preventive effects of curcumin (CUR). Using the human embryonic WI-38 lung fibroblast cell line, ET-1 and thrombin induced the expression of ER stress-related proteins (CCAAT-enhancer-binding protein homologous protein, PERK, and binding immunoglobulin protein), a profibrogenic factor (cellular communication network factor 2 [CCN2]), and differentiation markers including α-smooth muscle actin (α-SMA), collagen I (Col I), and Col IV. Knockdown of PERK expression via small interfering RNA (siRNA) significantly reduced the increases in CCN2, α-SMA, Col I, and Col IV proteins in WI-38 cells according to western blot analysis and immunohistochemistry (IHC). Activation of c-Jun N-terminal kinase (JNK) was observed in ET-1- and thrombin-treated WI-38 cells, and the addition of a JNK inhibitor (SP) suppressed the induction of the indicated proteins by ET-1 and thrombin. Thapsigargin (TG), an ER stress inducer, elevated expressions of PERK and ER stress-related proteins with increased differentiation of WI-38 cells. Knockdown of PERK by siRNA or the PERK inhibitor glycogen synthesis kinase reduced expressions of the differentiation markers, α-SMA and Col IV, in WI-38 cells. CUR concentration-dependently inhibited ET-1- or thrombin-induced CCN2, α-SMA, and vimentin proteins with decreased levels of phosphorylated mitogen-activated protein kinase and PERK in WI-38 cells. An in vivo bleomycin-induced PF study showed that an intraperitoneal injection of CUR (30 mg/kg) reduced expressions of α-SMA, CCN2, Col IV, and vimentin in lung tissues via IHC staining using specific antibodies. This study is the first to demonstrate that PERK activation contributes to pulmonary fibroblast differentiation elicited by ET-1 or thrombin, and the inhibitory activity of CUR against PF is demonstrated herein.

The X-ray crystal structure of human endothelin 1, a polypeptide hormone regulator of blood pressure.

Human endothelin is a 21-amino-acid polypeptide, constrained by two intra-chain disulfide bridges, that is made by endothelial cells. It is the most potent vasoconstrictor in the body and is crucially important in the regulation of blood pressure. It plays a major role in a host of medical conditions, including hypertension, diabetes, stroke and cancer. Endothelin was crystallized 28 years ago in the putative space group P6122, but the structure was never successfully solved by X-ray diffraction. Using X-ray diffraction data from 1992, the structure has now been solved. Assuming a unit cell belonging to space group P61 and a twin fraction of 0.28, a solution emerged with two, almost identical, closely associated molecules in the asymmetric unit. Although the data extended to beyond 1.8 Å resolution, a model containing 25 waters was refined to 1.85 Å resolution with an R of 0.216 and an Rfree of 0.284. The disulfide-constrained `core' of the molecule, amino-acid residues 1-15, has a main-chain conformation that is essentially the same as endothelin when bound to its receptor, but many side-chain rotamers are different. The carboxy-terminal `tail' comprising amino-acid residues 16-21 is extended as when receptor-bound, but it exhibits a different conformation with respect to the `core'. The dimer that comprises the asymmetric unit is maintained almost exclusively by hydrophobic interactions and may be stable in an aqueous medium.

Comparison of p38 MAPK, soluble endoglin and endothelin-1 level in severe preeclampsia and HELLP syndrome patients

Objective: To compare the levels of p38 mitogen-activated protein kinase (MAPK), soluble endoglin and endothelin-1 in the serum of patients with severe preeclampsia, hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome and normal pregnancies.Methods: This study was an observational analytic cross-sectional study performed at Wahidin Sudirohusodo Hospital, Makassar, Indonesia, in the period of 5th February 2016 to 20th January 2017. P38 MAPK, soluble endoglin and endothelin-1 levels of patients with normal pregnancies, severe preeclampsia and HELLP syndrome were measured by enzyme-linked immunoabsorbentassay technique, using kits of human soluble endoglin, endothelin-1 and p38 MAPK, Quantikine immunoassay: R&D System Inc.Results: Level of serum p38 MAPK in HELLP syndrome group was higher than in severe preeclampsia and normal pregnancy groups. Soluble endoglin and endothelin-1 levels in pregnancies with severe preeclampsia and HELLP syndrome were higher than normal pregnancy but there was no significant difference between these two groups (P>0.05). Levels of p38 MAPK, soluble endoglin and endothelin-1 also had a positive linear correlation with systolic and diastolic blood pressures (P<0.05).Conclusions: P38 MAPK in serum may be a marker for evidence of the severe hypoxia and its application may be considered for the diagnosis of HELLP syndrome.

Anti-hangover and anti-hypertensive effects in vitro of fermented persimmon juice

ABSTRACTThis study aimed to develop and characterize a fermented persimmon juice with potential anti-hangover and anti-hypertensive effects using gamma-aminobutyric acid (GABA)-producing lactic acid bacteria. Results showed the co-fermentation of Lactobacillus plantarum C17 and Lactobacillus pentosus Lp-B produced the highest yield of 2.102 mmol/L of GABA. After 48 h of fermentation, GABA was produced at the conversion rate of 69.32% from glutamate. The alcohol dehydrogenase activation rate, acetaldehyde dehydrogenase activation activity and hydroxyl inhibition rate of fermented persimmon juice were 38.91%, 34.72%, and 16.79%, respectively, which displayed higher anti-hangover activity compared to unfermented persimmon. Moreover, the angiotensin-converting enzyme inhibition ability of fermented persimmon juice was 42.34% and the expression level of human endothelin-1 was reduced to 34.64 μg/mL. These findings indicated GABA-enriched fermented persimmon juice had potential anti-hangover and anti-hypertensive properties.

Periodic Assessment Of Antenatal And Post Natal Serum Endothelin1 and Nitric Oxide Levels In Hypertensive Disorders Of Pregnancy

Introduction: Hypertensive Disorder of Pregnancy (HDP) is an independent risk factor of cardiovascular (CVS) disease with persistent endothelial dysfunction, that occurs after the initial insult during pregnancy and persists post partum postulated to be the pathophysiology. Endothelin-1 (ET-1), a potent vasoconstrictor, has been identified as a pivotal mediator in both essential hypertension and HDP. Disturbances in Nitric Oxide (NO) bioavailability found in endothelial dysfunction may increase susceptibility to cardiovascular diseases such as hypertension. Materials and Methods: Thirty six pregnant women at 30-36 weeks period of gestation from the following categories (i) pregnancy induced hypertension (PIH) (ii) chronic hypertension during pregnancy (CH) and (iii) normal pregnant women (Control). Blood pressure indices measurements and sample collection was done at antepartum (30-36 weeks), post partum (8 weeks and 12 weeks). Endothelin-1 was measured using the Human ET-1 (Endothelin-1) ELISA Kit and serum NO was measured using the Nitric Oxide (total) detection kit. Results: All blood pressure indices were significantly higher in HDP patients compared to control during antenatal and post partum periods. Serum ET-1 was significantly higher in patients with HDP compared to control during antenatal until 3 months post partum. This was accompanied by significantly lower levels of serum NO in HDP patients. Conclusion: ET-1 levels in patients with history of HDP irrespective of post partum blood pressure contributes to persistent endothelial dysfunction.

Abstract P151: Short- and Long-Term Induction of Human Endothelin-1 Overexpression in Mice Up-Regulated the Expression of Khdrbs3 in Mesenteric Arteries

Background: Transgenic mouse with tamoxifen-inducible endothelium-restricted human ET-1 overexpression (ieET-1) exhibited blood pressure (BP) elevation three weeks (short-term) and three months after induction (long-term). Vascular injury was observed only after long-term exposure to endothelial ET-1 overexpression. It is unknown whether short or long-term exposure to ET-1 overexpression results in gene dysregulation. We aimed to identify differentially expressed (DE) microRNAs (miRs) and genes in mesenteric arteries of ieET-1 mice after short- and long-term induction of human ET-1 overexpression. Methods and Results: Ten to 12-week old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase under the control of endothelium-specific Tie2 promoter, were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and sacrificed 16 days or 3 month later. RNA was extracted from mesenteric arteries of ieCre and ieET-1 mice and used for small and total RNA-sequencing using Illumina HiSeq-2500. EdgeR was used for differential expression analysis (false discovery rate &lt;0.05, fold change ≤ or ≥ 1.5). No DE miRs were identified. DE genes were identified in ieET-1 compared to ieCre mice after short-term induction (mRNAs: 1 up and 6 down; non-coding [nc]RNAs: 3 up) and after long-term induction (mRNA: 1 up). Khdrbs3 (KH domain containing, RNA binding, signal transduction associated 3), which was up-regulated after both short- and long-term exposure to endothelial ET-1 overexpression, was validated by reverse transcription-quantitative PCR (RT-qPCR). We demonstrated a correlation between RNA-sequencing and RT-qPCR data for short- and long-term groups (r=0.8, P &lt;0.0005). The mRNA expression of Khdrbs3 was 2 times more in fibroblasts than in smooth muscle cells and endothelial cells (2.37±0.28 vs. 1.11±0.17 and 1.12±0.12, P &lt;0.01). Conclusions: The results showed that short- and long-term exposure to endothelial ET-1 overexpression up-regulated Khdrbs3 in mesenteric arteries. In vitro study revealed that this gene was expressed to a greater level in fibroblasts than other vascular cells. However, the role of Khdrbs3 in ET-1-induced vascular injury remains to be determined.

Antibodies targeting human endothelin-1 receptors reveal different conformational states in cancer cells.

The endothelin axis (endothelins and their receptors) is strongly involved in physiological and pathological processes. ET-1 plays a crucial role in particular in tumor diseases. Endothelin-1 receptors (ET(A) and ET(B)) are deregulated and overexpressed in several tumors such as melanoma and glioma. We studied the binding of 24 monoclonal antibodies directed against human ET(B) receptors (hET(B)) to different melanoma cell lines. Few of these mAbs bound to all the melanoma cell lines. One of them, rendomab B49, bound to ET(B) receptors expressed at the surface of human glioma stem cells. More recently, we produced new antibodies directed against human ET(A) receptor (hET(A)). Several antibodies have been isolated and have been screened on different tumoral cells lines. As for the mAbs directed against the hET(B) receptor only some of new antibodies directed against ET(A) receptor are capable to bind the human tumoral cell lines. Rendomab A63 directed against hET(A) is one of them. We report the specificity and binding properties of these mAbs and consider their potential use in diagnosis by an in vivo imaging approach.

Clinical observation of recombinant human endostatin combined with cisplatin in the treatment of pleural effusion of lung cancer

Objective To investigate the clinical effect of recombinant human endostatin combined with cisplatin in the treatment of malignant pleural effusion of lung cancer. Methods 100 lung cancer patients with malignant pleural effusion were selected and divided into observation group and control group according to the random number table method, with 50 cases in each group.The control group was treated with cisplatin 40mg/m2 single-dose pleural perfusion.The observation group was treated with 40mg/m2 of cisplatin on the basis of 45mg of recombinant human endostatin.All the patients were treated twice a week, treatment for 3 weeks.The clinical efficacy, adverse reactions, the immune function indicators before and after treatment, quality of life improvement were compared between the two groups. Results (1)In the control group, 10 cases were cured, markedly effective in 24 cases, effective in 12 cases, the total effective rate was 92%.In the observation group, 4 cases were cured, markedly effective in 14 cases, effective in 18 cases, the total effective rate was 72%, the difference between the two groups was statistically significant (χ2=9.15, P 0.05), but the number of adverse reactions in the observation group was less than that in the control group, which indicated that the safety of the observation group was high.(3) Before treatment, the CD4+, CD8+ and CD4+/CD8+ between the two groups had no statistically significant differences (t=0.03, 0.26, 0.67, all P>0.05). After treatment, the percentage of CD4+ and CD4+/CD8+ in the observation group were higher than those in the control group, the percentage of CD8+in the observation group was lower than that in the control group, the differences were statistically significant (t=11.58, 12.36, 10.84, all P<0.05). (4) In the observation group, improved in 31 cases, stable in 10 cases, decreased in 9 cases.In the control group, improved in 25 cases, stable in 12 cases, decreased in 13 cases.There was in statistically significant difference between the two groups (χ2=2.30, P<0.05). Conclusion Recombinant human endostatin combined with cisplatin in the treatment of pleural effusion of lung cancer is significant, which can enhance the immune function of patients, improve the quality of life of patients, and with good safety, less adverse reactions. Key words: Recombinant human vascular endothelin; Cisplatin; lung cancer; Pleural effusion; Clinical efficacy

Systematic optimization of cell-free synthesized human endothelin B receptor folding.

Cell-free production of G-protein coupled receptors is becoming attractive for biochemical characterization, ligand screening or even structural purposes. However, despite high production levels within the range of mg/mL, the fraction of functionally folded receptor is frequently below 1%. In synthetic cell-free reactions, numerous factors that affect the efficient folding and stability of translated membrane proteins can be addressed by the appropriate design of the synthetic expression environment. We demonstrate the systematic quality optimization of the cell-free synthesized human endothelin B receptor by a combined approach of lipid screening, redox optimization, and molecular engineering. Key parameters for receptor folding are the implementation of nanodiscs, the selection of suitable lipid environments for co-translational solubilization, as well as providing an optimized redox system for essential disulfide bridge formation. In addition, enrichment with chaperones as well as receptor engineering by thermostabilization further supported the folding into ligand binding conformation. In summary, we provide evidence that the initial co-translational folding process rather than long-term stability of the receptor is limiting. The folding efficiency could be improved by more than 103-fold and under optimized conditions, up to 1.6 nmol or ∼100 µg of ligand binding competent receptor could be produced per mL of reaction mixture in a timescale of less than 24 h. The identified parameters affect rather common characteristics of G-protein receptors and are thus likely to improve the folding of similar targets as well. The optimized process provides full-length receptors embedded in defined membrane environments and in quantities and quality sufficient for throughput screening applications.

Correlation of single-nucleotide polymorphisms of the 1C3435T and ECE-1 genes with the efficacy of irbesartan in primary hypertension

Objective: To investigate the correlation of single-nucleotide polymorphisms of the Multidrug Resistance Gene (MDR) 1C3435T and the human Endothelin Converting Enzyme (ECE-1) gene with the efficacy of irbesartan in primary hypertension. Methods: A total of 186 patients with primary hypertension in our hospital from May 2015 to December 2016 received irbesartan oral medication. The 1C3435T and ECE-1 gene polymorphic sites were detected. Meanwhile, the plasma concentration was measured every hour after taking medicine for 8 h, and the plasma concentration peak was recorded. The systolic and diastolic blood pressures of the patients were recorded before and after treatment. The relationships between the different genotypes and plasma concentrations, as well as the drop of blood pressure, were compared. The antihypertensive effects of different genotypes were also investigated. Results: The distribution frequencies of the 1C3435T CT, TT, and CC genotypes were 37.63%, 34.95%, and 27.42%, respectively, whereas those of the C-338A CA, AA, and CC genotypes were 32.80%, 30.65%, and 36.56%, respectively. The plasma concentration peak, drop of blood pressure, and total effective rate of the 1C3435T TT genotype were significantly higher than those of CT and CC (P<0.05). The corresponding data of CT were significantly also higher than those of CC (P<0.05). Moreover, the plasma drug concentration, drop of blood pressure, and total effective rate of the C-338A CC genotype were significantly higher than those of CA and AA (P<0.05), and the corresponding data of CA were significantly higher than those of AA (P<0.05). Conclusion: During the oral treatment of primary hypertension by irbesartan tablets, the MDR1 1C3435T TT and ECE-1 C-338A CC genotypes can significantly increase the plasma concentration peak and significantly improve the curative effect.

Three-Month Endothelial Human Endothelin-1 Overexpression Causes Blood Pressure Elevation and Vascular and Kidney Injury.

Endothelium-derived endothelin (ET)-1 has been implicated in the development of hypertension and end-organ damage, but its exact role remains unclear. We have shown that tamoxifen-inducible endothelium-restricted human ET-1 overexpressing (ieET-1) mice exhibited blood pressure rise after a 3-week induction in an ET type A (ETA) receptor-dependent manner, in absence of vascular and renal injury. It is unknown whether long-term ET-1 overexpression results in sustained blood pressure elevation and vascular and renal injury. Adult male ieET-1 and control tamoxifen-inducible endothelium-restricted Cre recombinase (ieCre) mice were induced with tamoxifen and 2.5 months later, were treated with or without the ETA receptor blocker atrasentan for 2 weeks. Three-month induction of endothelial human ET-1 overexpression increased blood pressure (P<0.01), reduced renal artery flow (P<0.001), and caused mesenteric small artery stiffening (P<0.05) and endothelial dysfunction (P<0.01). These changes were accompanied by enhanced mesenteric small artery Col1A1 and Col3A1 expression, and perivascular adipose tissue oxidative stress (P<0.05) and monocyte/macrophage infiltration (P<0.05). Early renal injury was demonstrated by increased kidney injury molecule-1 expression in renal cortex tubules (P<0.05), with, however, undetectable lesions using histochemistry staining and unchanged urinary albumin. There was associated increased myeloid (CD11b+) and myeloid-derived suppressive cell (CD11b+Gr-1+) renal infiltration (P<0.01) and greater frequency of myeloid and renal cells expressing the proinflammatory marker CD36 (P<0.05). Atrasentan reversed or reduced all of the above changes (P<0.05) except the endothelial dysfunction and collagen expression and reduced renal artery flow. These results demonstrate that long-term exposure to endothelial human ET-1 overexpression causes sustained blood pressure elevation and vascular and renal injury via ETA receptors.

X-ray structures of endothelin ETB receptor bound to clinical antagonist bosentan and its analog.

Endothelin receptors (ETRs) have crucial roles in vascular control and are targets for drugs designed to treat circulatory-system diseases and cancer progression. The nonpeptide dual-ETR antagonist bosentan is the first oral drug approved to treat pulmonary arterial hypertension. Here we report crystal structures of human endothelin ETB receptor bound to bosentan and to the ETB-selective analog K-8794, at 3.6-Å and 2.2-Å resolution, respectively. The K-8794-bound structure reveals the detailed water-mediated hydrogen-bonding network at the transmembrane core, which could account for the weak negative allosteric modulation of ETB by Na+ ions. The bosentan-bound structure reveals detailed interactions with ETB, which are probably conserved in the ETA receptor. A comparison of the two structures shows unexpected similarity between antagonist and agonist binding. Despite this similarity, bosentan sterically prevents the inward movement of transmembrane helix 6 (TM6), and thus exerts its antagonistic activity. These structural insights will facilitate the rational design of new ETR-targeting drugs.

Activation mechanism of endothelin ETB receptor by endothelin-1.

Endothelin, a 21-amino-acid peptide, participates in various physiological processes, such as regulation of vascular tone, humoral homeostasis, neural crest cell development and neurotransmission. Endothelin and its G-protein-coupled receptor are involved in the development of various diseases, such as pulmonary arterial hypertension, and thus are important therapeutic targets. Here we report crystal structures of human endothelin type B receptor in the ligand-free form and in complex with the endogenous agonist endothelin-1. The structures and mutation analysis reveal the mechanism for the isopeptide selectivity between endothelin-1 and -3. Transmembrane helices 1, 2, 6 and 7 move and envelop the entire endothelin peptide, in a virtually irreversible manner. The agonist-induced conformational changes are propagated to the receptor core and the cytoplasmic G-protein coupling interface, and probably induce conformational flexibility in TM6. A comparison with the M2 muscarinic receptor suggests a shared mechanism for signal transduction in class A G-protein-coupled receptors.

Expression and purification of an engineered human endothelin receptor B in a monomeric form.

In humans, two endothelin receptors, ETa and ETb, are activated by three endogenous 21-mer cyclic peptides, ET-1, ET-2, and ET-3, which control various physiological processes, including vasoconstriction, vasodilation, and stimulation of cell proliferation. The first stage of this study it to produce a stable solubilized and purified receptor in a monodisperse state. This article is focused on the engineering, expression, purification, and characterization of the endothelin receptor B for subsequent structural and functional studies.

Clueless with fewer cues from endothelin. Commentary: Venous endothelin guides sympathetic innervation of the developing mouse heart.

Clueless with fewer cues from endothelin. Commentary: Venous endothelin guides sympathetic innervation of the developing mouse heart.

Inducible human endothelin-1 overexpression in endothelium raises blood pressure via endothelin type A receptors.

The mechanisms of blood pressure regulation by endothelin-1 produced by endothelial cells are complex and still unclear. Transgenic mice with endothelium-restricted human endothelin-1 (EDN1) overexpression presented vascular damage but no significant change in blood pressure, which could be because of adaptation to life-long exposure to elevated endothelin-1 levels. We now generated a tamoxifen-inducible endothelium-restricted EDN1 overexpressing transgenic mouse (ieET-1) using Cre/loxP technology. Sixteen days after tamoxifen treatment, ieET-1 mice presented ≥10-fold increase in plasma endothelin-1 (P<0.01) and ≥20 mm Hg elevation in systolic blood pressure (P<0.01), which could be reversed by atrasentan (P<0.05). Endothelin-1 overexpression did not cause vascular or kidney injury or changes in kidney perfusion or function. However, endothelin type A and B receptor expression was differentially regulated in the mesenteric arteries and the kidney. Our results demonstrate using this ieET-1 mouse model that 21 days of induction of endothelin-1 overexpression caused endothelin-1-dependent elevated blood pressure mediated by endothelin type A receptors.

Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing.

G protein-coupled receptors, like many other membrane proteins, are notoriously difficult to synthesize in conventional cellular systems. Although expression in insect cells is considered the preferred technique for structural characterizations in particular, inefficient membrane translocation, instability, toxic effects and low yields still pose clear limitations for their production in living cells. Recent studies started to explore alternative strategies for the in vitro production of problematic membrane proteins in cell-free lysates in combination with supplied membranes. We provide a detailed study on the production efficiencies and quality of G protein-coupled receptors, Fab fragments and other proteins synthesized in insect cell lysates containing endogenous microsomes. Effects of different reaction kinetics, redox conditions and sample preparations on the specific activities of synthesized proteins have been analyzed. The extent of glycosylation, membrane translocation and percentages of ligand binding active fractions of synthesized protein samples have been determined. We provide strong evidence that membrane insertion of integral membrane proteins can represent a prime limiting factor for their preparative scale in vitro production. Improved expression protocols resulting into higher production rates yielded more active protein in case of Fab fragments, but not in case of the human endothelin B receptor.

Circadian Regulation of lncRNAs Connects Per1, Endothelin‐1, and GR

The circadian clock regulates expression of nearly half of all mammalian genes. The mechanism of this regulation involves four core clock proteins: Bmal1, CLOCK, Cry (homologs 1-2) and Per (homologs 1-3). We have demonstrated that Per1 is an aldosterone and glucocorticoid target gene in the kidney. Long, non-coding (lnc)RNAs regulate gene expression via diverse mechanisms and can either activate or silence genes. Growth arrest specific 5 (Gas5) is a lncRNA implicated in inhibition of glucocorticoid receptor (GR) action. We have shown that Gas5 mRNA is acutely upregulated in response to 4 hour aldosterone treatment in a renal collecting duct cell line. Gas5 expression is also affected by Per1 in vivo in the liver and the renal cortex of Per1 KO mice compared to wild type. Potential circadian regulation of Gas5 is further supported by the observation that Gas5 expression oscillates in synchronized human renal proximal tubule cells (HK2). Endothelin-1 (ET-1) production is inhibited by Per1 in the kidney. ET-1 is a peptide hormone that contributes to blood pressure control. We have identified a novel lncRNA transcribed from the human ET-1 gene (EDN1) locus. This antisense lncRNA (EDN1-AS) is expressed in HK2 cells, human umbilical vein endothelial (HUVEC) cells, human bronchoepithelial (S9) cells, human mammary (HMEC) cells and whole human kidney. The EDN1-AS lncRNA was also shown to oscillate over 24 hours in synchronized HK2 cells. Both GR and Per1 were detected at the EDN1-AS promoter. Thus, we have identified two lncRNAs as novel Per1 targets which may have implications for the regulation of ET-1 and GR action by the clock.