ppET-1 mRNA Research Articles

Elevated Endothelin-1 Expression in Dogs with Heartworm Disease

We explored the involvement of endothelin-1 (ET-1) in the pathophysiology of dog dirofilariasis (heartworm disease caused by Dirofilaria immitis) by analyzing mRNA levels of preproendothelin-1 (PPET-1), the precursor form of ET-1, in cardiopulmonary organs as well as ET-1 peptide levels in plasma. To determine the cDNA sequence and primary protein structure of dog PPET-1, we performed molecular cloning of the full-length cDNA. Based on the determined sequence information, comparative expression analysis of PPET-1 mRNA was carried out by real-time polymerase chain reaction on cardiopulmonary organs from healthy (n=5) and filarial (n=5) dogs. Filarial dogs showed a significantly (p<0.05) higher mRNA expression level in the heart (about one hundred times) and lung (about ten times) than healthy dogs. Analysis of plasma ET-1 levels in healthy (n=10) and filarial (n=10) dogs showed that filarial dogs (6.9+/-2.7 pg/ml) have significantly (p<0.01) increased plasma ET-1 levels compared with healthy dogs (1.4+/-0.3 pg/ml). To assess the pathophysiological significance of ET-1 in dirofilariasis relative to other cardiopulmonary disorders, plasma ET-1 levels determined in dogs diagnosed with mitral regurgitation (n=10), tricuspid regurgitation (n=5), ventricular septal defect (n=5), and patent ductus arteriosus (n=5) were compared to plasma ET-1 levels in filarial dogs. Filarial dogs, which commonly develop serious pulmonary hypertension, exhibited by far the highest ET-1 levels of the disease states examined. Based on the fact that ET-1 is a potent bioactive mediator that induces vasoconstriction and promotes vascular remodeling, these findings suggest that ET-1 plays an important role in the pathophysiology of dog dirofilariasis as an aggravating factor by inducing pulmonary hypertension.

Early activation of cardiac and renal endothelin systems in experimental heart failure.

We investigated the time course of the expression of cardiac and renal endothelin systems in tachycardia-induced heart failure in dogs. Eleven beagles underwent rapid pacing at a progressively increased rate over a period of 5 wk, with a weekly clinical examination, echocardiography, measurement of circulating and urinary endothelin-1 (ET-1), and myocardial and renal tissue biopsies. Real-time quantitative PCR was used for determinations of tissue prepro-ET-1 (ppET-1), ET-1-converting enzyme (ECE-1), and ETA and ETB receptor mRNA. Cardiac and renal tissue ET-1 contents were evaluated by immunostaining and measured by radioimmunoassay at autopsy. Rapid pacing caused a progressive increase in end-systolic and end-diastolic ventricular volumes (P < 0.05) from week 2 together with a decrease in ejection fraction and in mean velocity of circumferential shortening (P < 0.05) from week 1. These changes were tightly correlated to myocardial ppET-1 and renal ETA receptor mRNA and less so to myocardial ECE-1 mRNA, and they occurred before any increase in plasma and urinary ET-1 (P < 0.05 from week 4) and clinical signs of heart failure. Renal ppET-1 did not change. Both cardiac and renal ET-1 peptide contents were increased at autopsy. We conclude that tachycardia-induced heart failure in dogs is characterized by an early activation of the cardiac and renal tissue endothelin systems, which occurs before any changes in circulating and urinary ET-1 and is closely related to altered ventricular function.

Endothelin-1 synthesis and secretion in human retinal pigment epithelial cells (ARPE-19): differential regulation by cholinergics and TNF-alpha.

Endothelin (ET)-1 can produce nerve damage analogous to that in optic neuropathies such as glaucoma. The precise source of endothelin in the posterior segment of the eye remains unclear. The present study was conducted to determine whether the retinal pigment epithelium (RPE), which helps maintain the outer blood-retinal barrier, is a local source for ET-1 and whether the amount of ET-1 secreted by the RPE is be differentially regulated by cholinergics and the cytokine TNF-alpha. Human retinal pigment epithelial cells (ARPE-19) were cultured either to a mature state (mRPE) for 4 weeks, with well-defined tight junctions, or to a young state (yRPE) for 4 days, with incompletely formed tight junctions. ET-1-like immunoreactivity was determined by immunocytochemistry, and secreted ET-1 was quantified by radioimmunoassay in both cell types. Cells were stimulated with the cholinergic agonist carbachol or with the cytokine TNF-alpha for specific periods. The expression of muscarinic receptor subtypes M1 and M3 and the peripheral membrane protein ZO-1 were analyzed by immunoblot and immunocytochemistry, respectively. Expression of preproendothelin-1 (ppET-1) mRNA after application of different stimuli at specific time points was determined by real-time RT-PCR. Carbachol-mediated elevation in intracellular calcium ([Ca2+]i) was also measured in the presence or absence of a selective muscarinic receptor antagonist. Constitutive synthesis and secretion of ET-1 was greater in mRPE than in yRPE cells. TNF-alpha caused a significant increase in ppET-1 mRNA levels and ET-1 secretion in both phenotypes. The disruption and subsequent breakdown of the tight junction barrier was evident in either phenotype after treatments with TNF-alpha. There was a concentration-dependent increase in [Ca2+]i in both y- and mRPE cells in response to CCh. CCh at 1 microM significantly increased ET-1 secretion, a response observed in yRPE but not in mRPE cells. This effect may be mediated primarily by the M3 receptor subtype and is phospholipase C (PLC) dependent. Regulation of ET-1 release in ARPE-19 cells was differentially regulated by TNF-alpha and CCh and was dependent on the age of the culture. RPE may be a source for ET-1 in the retina, and its increased release may become more important during breakdown of the blood-retinal barrier, as seen after TNF-alpha treatment.

A role for increased mRNA stability in the induction of endothelin-1 synthesis by lipopolysaccharide

An association exists between infection and cardiovascular diseases, including atherosclerosis, stroke and myocardial infarction. This may involve endothelin-1 (ET-1) which has been implicated in these and other vascular pathologies. ET-1 synthesis is controlled primarily by the level of its mRNA and numerous stimuli, including infection, lead to elevated ET-1 levels. Here, we have investigated the regulation of ET-1 release and preproET-1 (ppET-1) mRNA in bovine aortic endothelial cells by lipopolysaccharide (LPS). ET-1 release from bovine aortic endothelial cells was stimulated by LPS and reporter gene assays implicated LPS-induced ppET-1 transcription. However, changes in transcription were modest compared to increases in ET-1 synthesis. Therefore, ppET-1 mRNA levels were measured by real-time reverse transcription–polymerase chain reaction. The effect of LPS on ppET-1 mRNA levels was more marked than on transcription (1.2-fold increase in transcription vs. 5.5-fold increase in ppET-1 mRNA). Analysis of ppET-1 mRNA stability by real-time reverse transcription–polymerase chain reaction showed that LPS increased its half-life by approximately 2-fold. Thus, upregulated ppET-1 mRNA and hence increased ET-1 synthesis may be due to both increased transcription and reduced mRNA degradation. These effects of LPS on mRNA stability may be a key mechanism in vascular pathologies through which many proteins are induced in response to infection.

Role of Protein Kinase C on the Expression of Platelet-Derived Growth Factor and Endothelin-1 in the Retina of Diabetic Rats and Cultured Retinal Capillary Pericytes

Increased expression of endothelin-1 (ET-1) is associated with diabetic retinopathy and vasculopathy, although the molecular explanation has not been defined. The effects of high glucose and protein kinase C (PKC) activation on platelet-derived growth factor (PDGF)-BB and of ET-1 expression in the retina of streptozotocin (STZ)-induced diabetic rats and bovine retinal pericytes (BRPC) were examined. In 4-week diabetic rats, PDGF-B and prepro-ET-1 (ppET-1) mRNA levels increased significantly by 2.8- and 1.9-fold, respectively, as quantified by RT-PCR. Treatment with PKC-beta isoform-specific inhibitor (LY333531) or insulin normalized retinal ET-1 and PDGF-B expression. In BRPC, high glucose levels increased ppET-1 and PDGF-B mRNA expression by 1.7- and 1.9-fold, respectively. The addition of PDGF-BB but not PDGF-AA increased expression of ppET-1 and vascular endothelial growth factor mRNA by 1.6- and 2.1-fold, respectively, with both inhibited by AG1296, a selective PDGF receptor kinase inhibitor. A general PKC inhibitor, GF109203X, suppressed PDGF-BB's induction of ET-1 mRNA. Thus, increased ET-1 expression in diabetic retina could be due to increased expression of PDGF-BB, mediated via PDGF-beta receptors in part by PKC activation. The novel demonstration of elevated expression of PDGF-B and its induction by PKC activation identifies a potential new molecular step in the pathogenesis of diabetic retinopathy.

Endothelin-1 receptors and biosynthesis in the corpus luteum: Molecular and physiological implications

Endothelin-1 (ET-1), a 21-amino acid peptide was initially identified as a potent vasoconstrictor, ET-1 plays an important role in the female reproductive cycle: its quick ascent during luteal regression, ability to inhibit steroidogenesis in vitro and in vivo, combined with the observation that the luteolytic effects of prostaglandin F2α (PGF2α) were delayed by pretreatment with ET-1 receptors type A (ETA) antagonists suggest that this peptide functions as an important element of the luteolytic cascade. The observation that ETA receptor expression was inversely correlated with steroidogenesis in luteal cells; namely factors which stimulated steroidogenesis inhibited ETA receptor levels is also in accord with the inhibitory role of ET-1 in corpus luteum (CL) function. Contrary to the mature mid cycle CL, the CL of early cycle is refractory to PGF2α-induced luteolysis. PGF2α administered at early luteal phase (day 4 of the cycle) failed to increase luteal ET-1 gene expression or its ETA receptors. In contrast, both genes were markedly induced in mid cycle CL exposed to PGF2α. ET-1 gene is transcribed as prepro ET-1 (ppET-1) and the active form of peptide is derived from the inactive intermediate big ET-1, by endothelin-converting enzyme-1 (ECE-1), therefore alterations in mature ET-1 levels can be achieved by modulating the expression of ppET-1 and/or ECE-1. Analysis using in situ hybridization and enriched luteal cell subpopulations showed that both steroidogenic and endothelial cells of the CL expressed high levels of ECE-1 mRNA. The ppET-1 mRNA, on the other hand, was only expressed by resident endothelial cells, suggesting that luteal parenchymal and endothelial cells cooperate in the biosynthesis of mature bioactive ET-1. A significant, four-fold elevation in ECE-1 expression (mRNA and protein levels) occurred during the transition of the CL from early to mid luteal phase. This increase was accompanied by a significant rise in ET-1 peptide. Surprisingly however, ppET-1 mRNA levels remained similar during early and mid luteal phase. Collectively, these studies demonstrate that: (a) the various components of ET-1 system (ET-1/ECE-1/ETA) are dynamically and independently regulated during bovine luteal life span. (b) The CL becomes PGF2α-responsive only when both ppET-1 and ECE-1 genes are expressed at a level which enable an uninterrupted ET-1 biosynthesis.

Correction

HomeHypertensionVol. 39, No. 1Correction Free AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessCorrectionPDF/EPUBCorrection Originally published1 Apr 2018https://doi.org/10.1161/hyp.39.1.e9Hypertension. 2002;39:e9–e11This article corrects the followingLocalization of the Endothelin System in Aldosterone-Producing AdenomasIn the article “Localization of the Endothelium System in Aldosterone-Producing Adenomas” by Egidy et al (Hypertension 2001;38:1137–1142), the figures and legends were mismatched. Following is the correct match of the figures and legends.Download figureDownload PowerPointFigure 1. Components of the ET system in APAs. ETA and ETB receptors, ECE-1, PPET-1, and GAPDH mRNAs were detected by RT-PCR using the specific primers described previously22 and total RNA from the 20 frozen adrenal cortex samples. Only 5 samples are shown in Figure 2. Thirty cycles of amplification gave each gene at the expected size: 675 bp (ETA), 400 bp (ETB), 341 bp (PPET-1), 622 bp (ECE-1), and 649 bp (GAPDH). The molecular weight markers shown on the right are λ/HindIII, φX174 DNA/HaeIII.Download figureDownload PowerPointFigure 2. Localization of the ET system in APAs. ISH was performed on sections of adrenal cortex from the 20 patients with the antisense probes for ETA (a and b), ETB (c and d), ECE-1 (e and f), and PPET-1 (g and h). Photographs representative of the 20 samples studied are shown in dark-field illumination (left) and in bright-field illumination (right). ETA mRNA was found in compact cells of the zona glomerulosa (arrow, a) and ETB mRNA in vascular structures (arrow, c). ECE-1 mRNA labeling was ubiquitous and intense, and no specific signal for PPET-1 mRNA was detected. Scale bar 50 μm.Download figureDownload PowerPointFigure 3. Cellular distribution of ETA and ETB receptor mRNAs in APAs. ISH with the antisense probe for ETA (a) and ETB (b) receptors. Microphotographs representative of the 20 samples studied are shown in bright-field illumination. Immunohistochemistry was performed with anti-αSMA (c) (smooth muscle cell marker) and anti-CD31 (d) (endothelial cell marker) antibodies to identify the cells labeled with the ETA and ETB probes. ETA mRNA labeling was found in secretory cells (arrow) and smooth muscle cells (arrowhead), and ETB mRNA labeling was found mainly in endothelial cells (arrow) and smooth muscle cells (arrowhead). Scale bar 20 μm.Download figureDownload PowerPointFigure 4. ET-1 immunoreactivity. ET-1 immunohistochemistry was performed in paraffin-embedded specimens of stable transfected CHO/PPET-1/ECE-1 cells (a), CHO/ETB cells (b), CHO/ETB cells incubated with ET-1 for 30 minutes at 4°C (c), and untransfected CHO cells (d). CHO/PPET-1/ECE-1 cells contained ET-1 that was recognized by the antibody (arrow, a). No immunoreactivity was detected in CHO/ETB cells (b) or in untransfected CHO cells (d). In contrast, ET-1 incubated with CHO/ETB and bound to ETB receptors was recognized by the anti–ET-1 antibody (arrow, c).Download figureDownload PowerPointFigure 5. Cellular distribution of PPET-1 and ECE-1 mRNAs in APAs. ISH with the antisense probes for ECE-1 (a) and PPET-1 (c and e). Microphotographs representative of the 20 APA samples studied are shown in bright-field illumination. Immunohistochemistry was performed with the anti–ECE-1 (b) and anti–ET-1 (d and f) antibodies. There was intense ubiquitous labeling for ECE-1 mRNA and protein, whereas no specific labeling was detected for PPET-1 mRNA (c). However, strong ET-1 immunoreactivity was found in endothelial cells (arrow, d). Thickened precapillary arterioles were found in 6 of the 20 samples and contained PPET-1 mRNA (arrow, e) and ET-1 (arrow, f). Scale bar 20 μm. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesLocalization of the Endothelin System in Aldosterone-Producing AdenomasGiorgia Egidy, et al. Hypertension. 2001;38:1137-1142 January 2002Vol 39, Issue 1 Advertisement Article InformationMetrics https://doi.org/10.1161/hyp.39.1.e9 Originally publishedApril 1, 2018 PDF download Advertisement

Regional distribution of endothelin-1 and endothelin converting enzyme-1 in porcine endotoxemia.

Endothelin-1 (ET-1) levels are markedly increased in sepsis. Since ET-1 is primarily transcriptionally regulated, there should be a corresponding increase in pre-pro-endothelin-1 (ppET-1). Our objective was to determine whether ppET-1 is increased in pigs with a low systemic vascular resistance. We also examined the distribution of ET-1 and the regulation of endothelin-converting enzyme 1 (ECE-1), the rate limiting enzyme in ET-1 production. We anesthetized and ventilated 16 pigs. We measured arterial, pulmonary, and central venous pressures, as well as cardiac output. ET-1 was measured by radioimmunoassay in plasma and in multiple tissues. We infused 20 microg/kg of endotoxin over 2 h and then sacrificed the animals. ppET-1 and ECE-1 mRNA were assessed by Northern analysis. We performed immunohistochemistry for the assessment of tissue ET-1 and ECE-1. The systemic vascular resistance rose at 30 min, but fell by 120 min. Plasma ET-1 more than doubled by 2 h. However, there was no change in the concentration of ET-1 in any tissue except in the pulmonary artery. By immunohistochemistry, there was also no change in ET-1 in aorta, vena cava, heart, lung, liver, and kidney. Distribution of ECE-1 followed that of ET-1 on immunohistochemistry. There was a significant increase in ppET-1 mRNA in liver, kidney papillae, and vena cava, and a tendency for an increase in other tissues. This was paralleled by an increase in ECE-1 mRNA. In conclusion, the amount of ECE-1 mRNA and protein parallel those of ET-1. Endotoxemia is associated with a marked increase in plasma ET-1 and an increase in ppET-1 and ECE-1 mRNA in multiple tissues; however, there was no significant change in tissue ET-1 except in the pulmonary artery. The rise in plasma levels without a change in tissue levels suggests a greater release into the vasculature in sepsis than under normal conditions.

Myocardial expression of the endothelin system in endotoxin-treated rats.

Although circulating plasma levels of endothelin (ET)-1 are elevated in endotoxemia, little is known about the myocardial expression of the ET system in endotoxic shock. We assessed the temporal mRNA expression pattern of key components of the ET system (pre-pro ET (ppET) -1, -2, ET-converting enzyme-1, ET(A) and ET(B) receptors) by reverse transcription polymerase chain reaction in a rat model of early endotoxic shock. Lipopolysaccharide (5 mg/kg, i.p.) caused a transient increase (p < 0.05) in inducible nitric oxide synthase mRNA expression. ppET-1 mRNA expression was increased at 2 h (approximately 12-fold increase; p < 0.05) in the lipopolysaccharide compared with the saline group and ppET-2 mRNA expression was unaltered. ET-converting enzyme-1, ET(A), and ET(B) receptor mRNA expression was unaltered in the lipopolysaccharide compared with the saline group. While ppET-1 mRNA expression is selectively upregulated in ventricular myocardium of lipopolysaccharide-treated rats, an absence of alteration in ET-converting enzyme-1 mRNA expression suggests an excess capacity of ET-converting enzyme-1 to cope with the increased expression of ET-1. At the level of the receptor, endotoxic shock did not affect the expression of either ET(A) or ET(B) receptor mRNA. These data are consistent with the increased expression of myocardial ET-1 as an acute-phase response due to hemodynamic instability associated with the early stages of endotoxic shock.

Obesity regulates renal endothelin and endothelin ETA receptor expression in vivo. Differential effects of chronic ETA receptor blockade

ETA receptors have been implicated in obesity-associated hypertension (Hypertension 1999; 33: 1169). We characterized the renal endothelin system in diet-induced obesity and determined the effects of chronic treatment with the ETA antagonist darusentan. C57BL/6J mice were fed a standard diet (control) or a high-fat diet (Harlan TD88137) with or without darusentan (50 mg/kg/d, 30 wk). Total RNA was extracted from whole kidneys and mRNA expression of preproendothelin-1 (ppET-1), ETA receptors, and β-actin were determined by RT-PCR using mouse-specific primers. PCR-products were normalized vs. β-actin or 18S rRNA. Renal ET-1 protein was measured by RIA/HPLC. High fat diet increased body weight by 257% compared to 54% (control diet). Darusentan had no effect on body weight in obese mice (263%) and treatments had no effect on systolic blood pressure. Obesity was associated with upregulation of renal ETA receptors (144±5% vs 100±7%, p<0.05 vs. control) and to a lesser extent, preproendothelin-1 (113±5% vs.100±2%, p<0.05 vs. control). In obese mice chronic darusentan treatment in part prevented the ETA receptor upregulation (126% vs. 144±5%, p<0.05) but had no significant effect on ppET-1 mRNA expression (101±9 vs. 100±2%, n.s.). Renal ET-1 protein increased in obese animals (from 190±18 to 267±19 pg/g tissue, p<0.05 vs. control). This increase was not affected by concomitant darusentan treatment (n.s.). These data for the first time demonstrate that obesity in normotensive rats is associated with upregulation of renal ETA receptor expression suggesting that body weight per se affects ET receptor expression in the kidney. Our data further indicate that in this model ETA receptors control expression of the ETA receptor but not the ppET-1 gene, suggesting autocrine regulation in vivo. These mechanisms might contribute to the pathogenesis of obesity-associated diseases affecting the kidney and/or blood pressure

Early sequence of cardiac adaptations and growth factor formation in pressure- and volume-overload hypertrophy.

To investigate the time sequence of cardiac growth factor formation, echocardiographic and hemodynamic measurements were performed at scheduled times, and mRNAs for angiotensinogen, prepro-endothelin-1 (ppET-1), and insulin-like growth factor I (IGF-I) were quantified with RT-PCR and localized with in situ hybridization in pigs (fluothane anesthesia) by use of pressure or volume overload (aortic banding and aorta-cava fistula, respectively). Relative peptide formation was also measured by radioimmunoassay. In pressure overload, angiotensinogen and ppET-1 mRNA overexpression on myocytes (13 times vs. sham at 3 h and 112 times at 6 h, respectively) was followed by recovery (12 h) of initially decreased (0.5-6 h) myocardial contractility. In volume overload, contractility was not decreased, the angiotensinogen gene was slightly upregulated at 6 h (6.7 times), and ppET-1 was not overexpressed. IGF-I mRNA was overexpressed on myocytes (at 24 h) in both volume and pressure overload (14 times and 37 times, respectively). In the latter setting, a second ppET-1 overexpression was detectable on myocytes at 7 days. In conclusion, acute cardiac adaptation responses involve different growth factor activation over time in pressure versus volume overload; growth factors initially support myocardial contractility and thereafter induce myocardial hypertrophy.

Regulation of the endothelin system by shear stress in human endothelial cells.

In this study, the effect of shear stress on the expression of genes of the human endothelin-1 system was examined. Primary cultures of human umbilical vein endothelial cells (HUVEC) were exposed to laminar shear stress of 1, 15 or 30 dyn cm-2 (i.e. 0.1, 1.5 or 3 N m-2) (venous and two different arterial levels of shear stress) in a cone-and-plate viscometer. Laminar shear stress transiently upregulates preproendothelin-1 (ppET-1) mRNA, reaching its maximum after 30 min (approx 1.7-fold increase). In contrast, long-term application of shear stress (24 h) causes downregulation of ppET-1 mRNA in a dose-dependent manner. Arterial levels of shear stress result in downregulation of endothelin-converting enzyme-1 isoform ECE-1a (predominating in HUVEC) to 36.2 +/- 8.5 %, and isoform ECE-1b mRNA to 72.3 +/- 1.9 % of static control level. The endothelin-1 (ET-1) release is downregulated by laminar shear stress in a dose-dependent manner. This downregulation of ppET-1 mRNA and ET-1 release is not affected by inhibition of protein kinase C (PKC), or tyrosine kinase. Inhibition of endothelial NO synthase (L-NAME, 500 microm) prevents downregulation of ppET-1 mRNA by shear stress. In contrast, increasing degrees of long-term shear stress upregulate endothelin receptor type B (ETB) mRNA by a NO- and PKC-, but not tyrosine kinase-dependent mechanism. In conclusion, our data suggest the downregulation of human endothelin synthesis, and an upregulation of the ETB receptor by long-term arterial laminar shear stress. These effects might contribute to the vasoprotective and anti-arteriosclerotic potential of arterial laminar shear stress.

Elevated perfusion pressure upregulates endothelin-1 and endothelin B receptor expression in the rabbit carotid artery.

To investigate the hypothesis that high blood pressure activates the endothelin system in the vessel wall, isolated segments of the rabbit carotid artery were subjected to different levels of perfusion pressure. Both preproendothelin-1 (ppET-1) mRNA abundance and intravascular ET-1 peptide content were strongly upregulated on raising the intraluminal pressure from 90 to 160 mm Hg for 3 to 12 hours, and this increase in ppET-1 mRNA occurred predominantly in the endothelial cells. Endothelin-converting enzyme-1 and endothelin A receptor (ET(A)-R) expression were pressure-insensitive, whereas that of the ET(B)-R in the smooth muscle cells was also significantly enhanced. Both the pressure-induced increase in ppET-1 and ET(B)-R expression required RNA synthesis because they were abolished by actinomycin D. The nuclear signaling mechanisms involved therein, however, appeared to be different. Thus, the pressure-induced expression of ppET-1 and activation of CCAAT-enhancer binding proteins beta and delta were blocked by the tyrosine kinase inhibitor herbimycin A, whereas ET(B)-R expression and the nuclear translocation of activator protein-1 were abolished by the protein kinase C inhibitor Ro 31-8220. One consequence of these presumably deformation-induced changes in gene expression was an increased rate of apoptosis of the smooth muscle cells in the media that if transferable to the situation in human blood vessels may contribute to hypertension-induced arterial remodeling.

Regulation of the myocardial endothelin system by angiotensin-II and losartan.

Evidence for interactions between the endothelin (ET) and renin-angiotensin systems is plentiful in vitro, but few studies have investigated these interactions in vivo. In the study reported here, we have investigated the influence of chronic angiotensin-II (A-II) infusion in vivo on expression of preproendothelin-1 (PPET-1) and endothelin-A- (ET(A)) and endothelin-B- (ET(B)) receptor mRNA in the heart. The role of the angiotensin type 1 (AT1)-receptor in mediating the actions of A-II was studied using losartan, the selective AT1-receptor antagonist. Male rats received an infusion of A-II (200 ng/kg/min) or vehicle for 14 days via mini-osmotic pumps; losartan (10 mg/kg/day) was administered in the drinking water. PPET-1 and ET(A)- and ET(B)-receptor mRNA were detected in heart sections using nonradioactive antisense in situ hybridization. Independent treatments with either A-II or losartan had no significant effect on PPET-1, ET(A)- or ET(B)-receptor expression. Combined treatment resulted in an increase in PPET-1 mRNA (p < 0.001) and ET(B)-receptor mRNA expression (p < 0.01), while ET(A)-receptor mRNA expression was decreased (p < 0.001). These results suggest that selective AT1-receptor blockade, in the presence of an elevated plasma A-II concentration, causes upregulation of ET-1 synthesis in the myocardium as well as modification of ET receptor expression. These effects may be mediated via angiotensin type 2 (AT2)-receptors.

Regulation of the Myocardial Endothelin System by Angiotensin-II and Losartan

Summary: Evidence for interactions between the endothelin (ET) and renin-angiotensin systems is plentiful in vitro, but few studies have investigated these interactions in vivo. In the study reported here, we have investigated the influence of chronic angiotensin-II (A-II) infusion in vivo on expression of preproendothelin-1 (PPET-1) and endothelin-A-(ETA) and endothelin-B- (ETB) receptor mRNA in the heart. The role of the angiotensin type 1 (AT1)-receptor in mediating the actions of A-II was studied using losartan, the selective AT1-receptor antagonist. Male rats received an infusion of A-II (200 ng/kg/min) or vehicle for 14 days via mini-osmotic pumps; losartan (10 mg/kg/day) was administered in the drinking water. PPET-1 and ETA- and ETB-receptor mRNA were detected in heart sections using nonradioactive antisense in situ hybridization. Independent treatments with either A-II or losartan had no significant effect on PPET-1, ETA- or ETB-receptor expression. Combined treatment resulted in an increase in PPET-1 mRNA (p <0.001) and ETB-receptor mRNA expression (p <0.01), while ETA-receptor mRNA expression was decreased (p < 0.001). These results suggest that selective AT1-receptor blockade, in the presence of an elevated plasma A-II concentration, causes upregulation of ET-1 synthesis in the myocardium as well as modification of ET receptor expression. These effects may be mediated via angiotensin type 2 (AT2)-receptors.

Cold Ischemia Induces Endothelin Gene Upregulation in the Preserved Kidney

Background. Prolonged cold ischemia time (CIT) can lead to posttransplant renal dysfunction; however, the pathophysiology remains unclear. Endothelin (ET), a potent vasoconstrictive peptide, may play a role in this injury. The purpose of this study was to determine if cold ischemia could induce renal ET-1 gene upregulation and to localize ET-1 peptide expression in the hypothermic kidney.Materials and methods. Kidneys from Lewis rats were perfused with Viaspan, harvested, and stored at 4°C for varying periods of CIT: 0, 6, 24, and 48 h. Preproendothelin-1 (ppET-1) gene upregulation was measured using a reverse-transcription polymerase-chain reaction. ET-1 peptide expression was localized using immunohistochemistry.Results. Control kidneys (0 h CIT) had 0.56 ± 0.22 DU of ppET-1 mRNA. After 6 h of CIT, a 2.3-fold increase in this level was noted. Following 24 h of CIT, ppET-1 mRNA was significantly upregulated to 1.96 ± 0.38 DU (P < 0.05). Immunohistochemistry revealed typical vascular ET-1 staining in control kidneys. At 6 h of CIT, a significant increase in the expression of ET-1 was noted in the peritubular capillaries and vasa recta. After 24 h, intense staining for ET-1 was seen in the medullary collecting ducts. After 48 h of CIT, early cellular necrosis was present along with global decreases in ET-1 expression and ppET-1 mRNA levels.Conclusions. This study demonstrates that 24 h of cold preservation can induce significant upregulation of the renal ET-1 gene and increase expression of the ET-1 peptide localized to both vascular endothelial and tubular epithelial surfaces of the kidney. Consequently, prolonged cold ischemia prior to transplantation may lead to delayed renal function following revascularization via endothelin-induced vasoconstriction and/or tubular impairment.

Endothelin up-regulation and localization following renal ischemia and reperfusion

Endothelin (ET), a potent vasoconstrictor, is known to play a role in ischemic acute renal failure. Although preproET-1 (ppET-1) mRNA is known to be up-regulated following ischemia/reperfusion injury, it has not been determined which component of the injury (ischemia or reperfusion) leads to initial gene up-regulation. Likewise, although ET-1 peptide expression has been localized in the normal kidney, its expression pattern in the ischemic kidney has not been determined. Therefore, the purpose of this study was twofold: (a) to determine whether ischemia alone or ischemia plus reperfusion is required for the up-regulation of ppET-1 mRNA to occur, and (b) to localize ET-1 peptide expression following ischemia in the rat kidney to clarify better the role of ET in the pathophysiology of ischemia-induced acute renal failure. Male Lewis rats underwent clamping of the right renal vascular pedicle for either 30 minutes of ischemia (group 1), 60 minutes of ischemia (group 2), 30 minutes of ischemia followed by 30 minutes of reperfusion (group 3), or 60 minutes of ischemia followed by three hours of reperfusion (group 4). The contralateral kidney acted as a control. ppET-1 mRNA up-regulation and ET-1 peptide expression were examined using the reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. Reverse transcription-polymerase chain reaction yielded a control (nonischemic) value of 0.6 +/- 0.2 densitometric units (DU) of ppET-1 mRNA in the kidney. Group 1 levels (30 min of ischemia alone) were 1.8 +/- 0.4 DU, a threefold increase (P < 0.05). Group 2 levels (60 min of ischemia alone) increased almost six times above baseline, 3.5 +/- 0.2 DU (P < 0.01), whereas both group 3 and group 4 (ischemia plus reperfusion) did not experience any further significant increases in mRNA levels (1.9 +/- 0.4 DU and 2.8 +/- 0.6 DU, respectively) beyond levels in group 1 or 2 animals subjected to similar ischemic periods. ET-1 peptide expression in the ischemic kidneys was significantly increased over controls and was clearly localized to the endothelium of the peritubular capillary network of the kidney. Initial ET-1 gene up-regulation in the kidney occurs secondary to ischemia, but reperfusion most likely contributes to sustaining this up-regulation. The marked increase of ET-1 in the peritubular capillary network suggests that ET-induced vasoconstriction may have a pathophysiological role in ischemic acute tubular necrosis.

Chronic Intrauterine Pulmonary Hypertension Increases Preproendothelin-1 and Decreases Endothelin B Receptor mRNA Expression in the Ovine Fetal Lung

Endothelin-1 (ET-1), a potent vasoconstrictor and smooth muscle mitogen, is produced from its precursor, preproendothelin-1 (ppET-1), by ET-converting enzyme (ECE-1) activity. 1 Ivy DD Le Cras TD Horan MP et al. Increased lung preproET-1 and decreased ETB-receptor gene expression in fetal pulmonary hypertension. Am J Physiol. 1998; 274: L535-L541 PubMed Google Scholar Past studies have suggested that ET-1 contributes to high pulmonary vascular resistance (PVR) in the normal fetal lung and that the hemodynamic effects of ET-1 are mediated through stimulation of ETa and ETb receptors, which cause vasoconstriction and vasodilation, respectively. Increased ET-1 production may also contribute to high PVR in an experimental model of persistent pulmonary hypertension of the newborn, induced by intrauterine ligation of the ductus arteriosus (DA). In this model, closure of the DA in the late gestation fetal lamb causes progressive elevation of PVR in utero, marked right ventricular hypertrophy, hypertensive pulmonary vascular remodeling, and sustained elevation of PVR despite mechanical ventilation with high fraction of inspired oxygen after delivery. Based on past physiologic studies from our laboratory, we hypothesized that increased ET-1 production and altered ETa and ETb receptor activities contribute to the pathophysiology of this experimental model of persistent pulmonary hypertension of the newborn. To define mechanisms of altered ET-1 activity and to study further the role of ET-1 in perinatal pulmonary hypertension, we studied lung mRNA expression of ppET-1, ECE-1, and ETa and ETb receptors in normal and hypertensive fetal lambs. Total RNA was isolated from whole lung tissue in normal late gestation fetuses (135±3 days; term=147 days) and from animals with pulmonary hypertension after DA ligation for 8 days (134±4 days). Northern blot analysis was performed with cDNA probes and was normalized to the signal for 18s rRNA. We found a 71±24% increase in steady state ppET-1 mRNA (p<0.05) and a 62±5% decrease in ETb mRNA expression after DA ligation (p<0.05). ECE-1 and ETa receptor mRNA expression did not change. We conclude that chronic intrauterine pulmonary hypertension caused by DA ligation increases steady state ppET-1 mRNA and decreases ETb receptor mRNA without changing ECE-1 mRNA or ETa receptor mRNA expression. These findings suggest that both increased ET-1 production and decreased ETb receptor expression favor increased vasoconstrictor tone and contribute to high PVR in experimental neonatal pulmonary hypertension.

Transient, isopeptide-specific induction of myocardial endothelin-1 mRNA in congestive heart failure in rats.

Increased myocardial expression of preproendothelin-1 (ppET-1) mRNA has been associated with congestive heart failure (CHF) in rats. However, the time course and isoform pattern of ppET mRNA induction and the cellular localization of ET in failing hearts are unknown. Thus our aim was to investigate myocardial ppET mRNA expression in CHF rats during the first 6 wk after induction of myocardial infarction. Furthermore, performing immunohistochemical analysis, we also investigated the origin and localization of immunoreactive endothelin (ET) in different regions of the failing heart. Ribonuclease protection assays revealed a marked increase in ppET-1 mRNA levels in rat myocardial tissues during CHF. The induction of ppET-1 mRNA was isopeptide specific and transient. The most substantial upregulation was observed in the infarcted area, where maximal expression of ppET-1 mRNA was observed after 7 days (25-fold increase, P < 0.05). However, a marked and statistically significant induction of ppET-1 mRNA was also observed in the nonischemic myocardium. Immunohistochemical analysis revealed ET-1-like immunoreactivity in cardiomyocytes, vascular endothelial cells, macrophages, and proliferating fibroblasts. Thus immunohistochemistry revealed the structural basis for the dramatic upregulation of the myocardial ET system in the infarcted region, suggesting a role for ET in the healing process after myocardial infarction. However, the global upregulation of ppET-1 mRNA in the heart also suggests an autocrine/paracrine regulatory mechanism in the nonischemic myocardium during CHF.

IL-1 beta inhibits ET-1 production by ATII cells in vitro: evidence for involvement of cyclooxygenase 2 pathway.

The aim of this study was to characterize the effect of alveolar macrophage (AM) secretory products on endothelin (ET)-1 production by rat alveolar type II (ATII) cells in primary culture. We quantified preproendothelin (ppET)-1 mRNA by Northern blot and ET-1 concentrations in cell supernatants by enzyme-linked immunosorbent assay. Conditioned medium (CM) from rat adherent AM decreased the ppET-1 mRNA levels in ATII cells and reduced ET-1 concentrations in cell culture supernatants. This effect was mediated by interleukin (IL)-1 beta as shown by pretreatment of CM with an anti-IL-1 beta neutralizing antiserum. IL-1 beta effect was dependent on protein synthesis, was partially prevented with indomethacin, and was totally prevented with dexamethasone. Specific inhibition of cyclooxygenase 2 activity completely reversed the effect of IL-1 beta. We conclude that rat AM inhibit ET-1 production by rat ATII cells in vitro through IL-1 beta secretion. The IL-1 beta-mediated inhibition is dependent on the cyclooxygenase 2 pathway. Downregulation of ET-1 production by activated AM could limit the intra-alveolar burden of this profibrogenic peptide and thus could prevent fibrosis development.