ETA Receptor mRNA Research Articles

Endothelins-1/3 and endothelin-A/B receptors expressing glial cells with special reference to activated microglia in experimentally induced cerebral ischemia in the adult rats

We reported previously that amoeboid microglial cells (AMC) in the developing brain exhibited endothelins (ETs) expression which diminished with advancing age and was undetected in microglia in the more mature brain. This study sought to explore if microglia in the adult would be induced to express ETs in altered conditions. By immunofluorescence microscopy, ETs and endothelin (ET)-B receptor were undetected in microglial cells in sham-operated and normal control rats. However, in adult rats subjected to middle cerebral artery occlusion (MCAO), lectin labeled activated microglia which occurred in large numbers in the marginal zones in the ischemic cortex at 3 days and 1 week intensely expressed ETs specifically endothelin (ET)-1 and ET-B receptor; ET-3 and ET-A receptor were absent in these cells. By RT-PCR and ELISA, ET-1 and -3 mRNA and protein expression level was progressively increased in the ischemic cerebral cortex after MCAO compared with the controls. ET-A and ET-B receptor mRNA and protein levels were concomitantly up-regulated. It is suggested that increased release of ET-1 following MCAO by massive activated microglia can exert an immediate constriction of local blood vessels bearing ET-A receptor. ET-1 may also interact with activated microglia endowed with ET-B receptor via an autocrine manner that may be linked to chemokines/cytokines production. ET-1, ET-3 and ET-B receptor were also localized in reactive astrocytes along with some oligodendrocytes. We conclude that activated microglia together with other glial cells in the marginal zone after MCAO are the main cellular source of ETs that may be involved in regulation of vascular constriction and glial chemokines/cytokines production. However, dissecting the role of individual component of the endothelin system in the various glial cells, notably activated microglia, would be vital in designing of an effective therapeutic strategy for clinical treatment of stroke in which microglial cells have been implicated.

Proliferative stimulation of the vascular endothelin-1 axis in vitro and ex vivo by infection with Chlamydia pneumoniae

Endothelin-1 (ET-1) is a vasoactive peptide that modifies vascular function via the G-protein coupled transmembrane receptors, Endothelin-A receptor (ETAR) and Endothelin-B receptor (ETBR). Dysregulation of the ET-1 axis plays a role in atherosclerotic development as it triggers cell proliferation, inflammation, and vasoconstriction. The respiratory pathogen Chlamydia pneumoniae (Cp) has been recovered from atherosclerotic lesions, and related to atherogenesis, via activation of vascular small GTPases and leukocyte recruitment. Cp effectively reprograms host cell signalling and is able to enter an intracellular persistent state in vascular cells that is refractory to antibiotics. Upon chlamydial infection, vascular smooth muscle cells, which do not produce significant ET-1 under physiological conditions were switched into a fundamental source of ET-1 mRNA and protein in a p38-MAP-kinase-dependent pathway. Endothelial cells did not overproduce ET-1 but showed upregulation of mitogenic ETAR mRNA and protein while the counterbalancing ETBR, which regulates ET-1 clearance, remained unaffected. This disruption of the ET-1 axis was confirmed in an ex vivo mouse aortic ring model, and resulted in endothelial cell proliferation that could be abrogated by ETAR-siRNA and the selective ETAR-antagonist BQ-123. Chronic chlamydial infection of the vascular wall might represent a permanent noxious stimulus linked to the endothelial cell proliferation characteristic of early atherosclerosis. Suppression of this deleterious paracrine loop by ETAR antagonism opens up a new option of preventing possible vascular sequelae of otherwise untreatable chronic chlamydial infection. In conclusion, this is the first study to demonstrate infection to dysregulate the ET-1 axis towards inducing a proatherogenic proliferative phenotype.

Reactive oxygen species-dependent endothelin signaling is required for augmented hypoxic sensory response of the neonatal carotid body by intermittent hypoxia

We previously reported that intermittent hypoxia (IH) augments hypoxic sensory response (HSR) and increases the number of glomus cells in neonatal carotid bodies. In the present study, we tested the hypothesis that recruitment of endothelin-1 (ET-1) signaling by reactive oxygen species (ROS) plays a critical role in IH-evoked changes in neonatal carotid bodies. Experiments were performed on neonatal rats exposed either to 10 days of IH (P0-P10; 8 h/day) or to normoxia. IH augmented HSR of the carotid bodies ex vivo and resulted in hyperplasia of glomus cells. The effects of IH were associated with enhanced basal release of ET-1 under normoxia, sensitization of carotid body response to exogenous ET-1, and upregulation of ET(A) but not an ET(B) receptor mRNA without altering the ET-1 content. An ET(A) but not ET(B) receptor antagonist prevented augmented HSR by IH. ROS levels were elevated in carotid bodies from IH-treated rat pups as evidenced by increased levels of malondialdehyde. Systemic administration of manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 5 mg/kg ip), a scavenger of O(2)(*-), prevented IH-induced elevation of ROS, basal release of ET-1, upregulation of ET(A) mRNA, and augmented HSR. In striking contrast, MnTMPyP treatment had no significant effect on IH-induced hyperplasia of glomus cells. These results demonstrate that IH-evoked increase in HSR involve a ROS-mediated increase in basal ET-1 release and upregulation of ET(A) receptor mRNA.

Up-regulation of endothelin type B receptors in the human internal mammary artery in culture is dependent on protein kinase C and mitogen-activated kinase signaling pathways

BackgroundUp-regulation of vascular endothelin type B (ETB) receptors is implicated in the pathogenesis of cardiovascular disease. Culture of intact arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for, ex vivo, in detail delineation of the regulation of endothelin receptors. We hypothesize that mitogen-activated kinases (MAPK) and protein kinase C (PKC) are involved in the regulation of endothelin ETB receptors in human internal mammary arteries.MethodsHuman internal mammary arteries were obtained during coronary artery bypass graft surgery and were studied before and after 24 hours of organ culture, using in vitro pharmacology, real time PCR and Western blot techniques. Sarafotoxin 6c and endothelin-1 were used to examine the endothelin ETA and ETB receptor effects, respectively. The involvement of PKC and MAPK in the endothelin receptor regulation was examined by culture in the presence of antagonists.ResultsThe endohtelin-1-induced contraction (after endothelin ETB receptor desensitization) and the endothelin ETA receptor mRNA expression levels were not altered by culture. The sarafotoxin 6c contraction, endothelin ETB receptor protein and mRNA expression levels were increased after organ culture. This increase was antagonized by; (1) PKC inhibitors (10 μM bisindolylmaleimide I and 10 μM Ro-32-0432), and (2) inhibitors of the p38, extracellular signal related kinases 1 and 2 (ERK1/2) and C-jun terminal kinase (JNK) MAPK pathways (10 μM SB203580, 10 μM PD98059 and 10 μM SP600125, respectively).ConclusionIn conclusion, PKC and MAPK seem to be involved in the up-regulation of endothelin ETB receptor expression in human internal mammary arteries. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the development of vascular endothelin ETB receptor changes in cardiovascular disease.

Endothelin type A receptor antagonist normalizes blood pressure in rats exposed to eucapnic intermittent hypoxia

We have reported that eucapnic intermittent hypoxia (E-IH) causes systemic hypertension, elevates plasma endothelin 1 (ET-1) levels, and augments vascular reactivity to ET-1 and that a nonspecific ET-1 receptor antagonist acutely lowers blood pressure in E-IH-exposed rats. However, the effect of chronic ET-1 receptor inhibition has not been evaluated, and the ET receptor subtype mediating the vascular effects has not been established. We hypothesized that E-IH causes systemic hypertension through the increased ET-1 activation of vascular ET type A (ET(A)) receptors. We found that mean arterial pressure (MAP) increased after 14 days of 7 h/day E-IH exposure (109 +/- 2 to 137 +/- 4 mmHg; P < 0.005) but did not change in sham-exposed rats. The ET(A) receptor antagonist BQ-123 (10 to 1,000 nmol/kg iv) acutely decreased MAP dose dependently in conscious E-IH but not sham rats, and continuous infusion of BQ-123 (100 nmol.kg(-1).day(-1) sc for 14 days) prevented E-IH-induced increases in MAP. ET-1-induced constriction was augmented in small mesenteric arteries from rats exposed 14 days to E-IH compared with those from sham rats. Constriction was blocked by the ET(A) receptor antagonist BQ-123 (10 microM) but not by the ET type B (ET(B)) receptor antagonist BQ-788 (100 microM). ET(A) receptor mRNA content was greater in renal medulla and coronary arteries from E-IH rats. ET(B) receptor mRNA was not different in any tissues examined, whereas ET-1 mRNA was increased in the heart and in the renal medulla. Thus augmented ET-1-dependent vasoconstriction via vascular ET(A) receptors appears to elevate blood pressure in E-IH-exposed rats.

Tanshinone IIA inhibits endothelin-1 production in TNF-?-induced brain microvascular endothelial cells through suppression of endothelin-converting enzyme-1 synthesis

To investigate the effects of tanshinone IIA (Tan IIA) on the regulation of the production of endothelin (ET)-1 (including large ET-1), mRNA levels of ET-1, endothelin-converting enzyme-1 (ECE-1), endothelin-A receptor (ETA) and endothelin-B receptor (ETB) induced by TNF-alpha in rat brain microvascular endothelial cells (BMVEC). The ET-1 release (including large ET-1) into the culture medium was determined by enzyme immunoassay. The levels of ET-1, ECE-1, ETA, and ETB mRNA were measured by RT-PCR. Endothelin receptor binding was also tested. The induction of ET-1 release by TNF-alpha from cultured BMVEC was dose-dependently reduced by Tan IIA, but large ET-1 levels progressively increased in response to Tan IIA; the mRNA expression of ET-1 was unaffected. Tan IIA also caused a decrease in ETA receptor mRNA and ECE-1 expression in a dose-dependent manner. Endothelin receptor binding was unaltered in BMVEC stimulated with TNF-alpha alone or a combination of TNF-alpha and Tan IIA. These findings suggest that Tan IIA may inhibit ET-1 production in TNF-alpha-induced BMVEC through the suppression of ECE-1 synthesis.

ACTIVATED PROTEIN C REDUCES HEPATIC ET-1 TRANSCRIPT LEVELS AFTER TRAUMA AND SEPSIS

Introduction: Activated protein C (aPC) confers survival benefit in patients with sepsis; yet its protective mechanisms remain unclear. Recently, we found that aPC ameliorates ET-1 induced alterations in the hepatic microcirculation after femur fracture (FFx) and cecal ligation and puncture (CLP). Here, we hypothesize that the protective effects of aPC are mediated by a reduction in the vasoconstrictor ET-1 and its receptor, ETA. Methods: Male Sprague Dawley rats underwent control, sham, FFx, CLP, or FFx followed 24 hours by CLP. Subsequently, animals received either aPC (1 mg/kg) or saline intravenously, twice daily for four days. Twenty four hours after the final aPC or saline treatment, liver tissue was harvested for RT-PCR. Results: CLP and FFx + CLP resulted in a significant upregulation in ET-1 and ETA receptor transcripts when compared to control and sham. Treatment with aPC resulted in a reduction in ET-1 and ETA receptor mRNA levels after CLP and FFx+CLP (see Figure); no effect was seen in control, sham, and FFx animals.FigureConclusion: Treatment with aPC reduced the vasoconstrictor ET-1 and ETA receptor mRNA levels in CLP and FFx+CLP animals, which plays an important role in the protective effects of aPC on the observed hepatic microcirculation in response to ET-1. Figure: Saline treated vs aPC treated RT-PCR bands for ET-1 and ETA receptor. 1 = CLP and 2 = FFx + CLP

Involvement of Protein Kinases on the Upregulation of Endothelin Receptors in Rat Basilar and Mesenteric Arteries

Endothelin(B) (ET(B)) receptors are upregulated in experimental stroke or after 24 hrs of organ culture. This upregulation is manifested both as stronger contraction and as an increase in ET(B) receptor messenger RNA (mRNA) levels. The present study was designed to evaluate the importance of protein kinases (c-Jun N-terminal kinase [JNK], protein kinase C [PKC], and extracellular signal-regulated kinase [ERK1/2]) in ET(B) receptor upregulation after organ culture. Rat basilar and mesenteric arteries were incubated for 24 hrs in Dulbecco's modified Eagle's medium (DMEM) with or without the PKC inhibitor, RO-31-7549; the ERK1/2 inhibitor, SB386023; or the JNK inhibitor, SP600125, added 3, 6, or 12 hrs after initiation of incubation. Subsequently, vessel segments were mounted in myographs and the contractile responses to ET-1 and sarafotoxin 6c were studied. The ET(B) and ET(A) receptor mRNA levels were determined with a real-time polymerase chain reaction (PCR). The cellular localization and protein level of ET(B) receptors were evaluated by immunohistochemistry. The PKC and ERK1/2 inhibitors attenuated the contraction induced by S6c in the basilar arteries more than in the mesenteric arteries. The efficiency of the inhibitors was proportional to the incubation time. Real-time PCR showed a decrease in the ET(B) receptor mRNA levels in arteries treated with PKC or ERK inhibitors. The JNK inhibitor had a significant inhibitory effect on ET(B) receptor upregulation in the basilar arteries. Immunohistochemistry revealed that the ET(B) receptor upregulation occured in the smooth-muscle cells and that it had the same pattern as in the quantitative PCR. Our results show that the PKC, ERK1/2, and JNK are more important for the upregulation of contractile ET(B) receptors in cerebral arteries compared with mesenteric arteries. ERK1/2 seems to be more important for the ET(B) receptor upregulation, as compared with PKC and JNK. The evaluation of the time dependency suggests that the phenomenon can be reversed even after its initiation.

Smoking particles enhance endothelin A and endothelin B receptor-mediated contractions by enhancing translation in rat bronchi

BackgroundSmoking is known to cause chronic inflammatory changes in the bronchi and to contribute to airway hyper-reactivity, such as in bronchial asthma. To study the effect of smoking on the endothelin system in rat airways, bronchial segments were exposed to DMSO-soluble smoking particles (DSP) from cigarette smoke, to nicotine and to DMSO, respectively.MethodsIsolated rat bronchial segments were cultured for 24 hours in the presence or absence of DSP, nicotine or DMSO alone. Contractile responses to sarafotoxin 6c (a selective agonist for ETB receptors) and endothelin-1 (an ETA and ETB receptor agonist) were studied by use of a sensitive myograph. Before ET-1 was introduced, the ETB receptors were desensitized by use of S6c. The remaining contractility observed was considered to be the result of selective activation of the ETA receptors. ETA and ETB receptor mRNA expression was analyzed using real-time quantitative PCR. The location and concentration of ETA and ETB receptors were studied by means of immunohistochemistry together with confocal microscopy after overnight incubation with selective antibodies.ResultsAfter being cultured together with DSP for 24 hours the bronchial segments showed an increased contractility mediated by ETA and ETB receptors, whereas culturing them together with nicotine did not affect their contractility. The up-regulation of their contractility was blunted by cycloheximide treatment, a translational inhibitor. No significant change in the expression of ETA and ETB receptor mRNA through exposure to DMSO or to nicotine exposure alone occurred, although immunohistochemistry revealed a clear increase in ETA and ETB receptors in the smooth muscle after incubation in the presence of DSP. Taken as a whole, this is seen as the presence of a translation mechanism.ConclusionThe increased contractility of rat bronchi when exposed to DSP appears to be due to a translation mechanism.

Subarachnoid hemorrhage induced receptor upregulation in rat cerebral arteries.

Objective The cerebral ischemia that occurs after a subarachnoid hemorrhage (SAH) often results in death or severe disability. Our hypothesis suggests that cerebral ischemia leads to pathophysiological endothelin (ET), 5-hydroxytryptamine (5-HT) and angiotensin (AngII) receptor changes in the vascular smooth muscle cells. Presently we wanted to study the time course for the upregulation of the receptors after SAH in cerebral arteries. Methods SAH was induced by injecting 250 μl blood into the prechiasmatic cistern. One, 3, 6, 12, 24 and 48 hrs after the SAH the basilar arteries (BA) and middle cerebral arteries (MCA) were harvested and contractile response to endothelin-1 (ET-1), 5-carboxamidotryptamine (5-CT) and AngII were investigated with a myograph. ETA, ETB, 5-HT1B, AT1 and AT2 receptor mRNA levels were analyzed by quantitative real-time PCR. Results SAH resulted in enhanced contraction to ET-1 and 5-CT compared to fresh or sham rats. Ang II (via AT1 recpetors) induced increased contractile response (in the presence of the AT2 receptor antagonist PD123319). The contractile responses to ET-1, 5-CT and Ang II were successively increased with time, with a maximum at 48 hrs for 5-CT and ET-1 and 24 hrs for Ang II. In parallel the ETB, 5-HT1B, AT1 and AT2 receptor mRNA levels were elevated by time. Conclusion The results demonstrate that SAH induces upregulation of ETB, 5-HT1B, AT1 receptors in a time-dependent manner both at functional and mRNA levels. The study was supported by the Swedish Heart-Lung Foundation and the Lundbeck Foundation.

Characterization of pulmonary endothelin receptors in healthy and recurrent airway obstruction (RAO)‐affected horses

Equine recurrent airway obstruction (RAO) is characterized by airway hyperreactivity. Endothelin-1 (ET-1), which is an inflammatory mediator has been implicated in the pathogenesis of various airway inflammatory diseases. Our objective was to determine and compare the expression of ETA and ETB receptors in the lungs of healthy and RAO horses. Seven of each healthy and RAO horses were used. Horses were euthanatized, lung samples were collected and either stored at −80°C or fixed in zinc-formalin. RT-PCR was performed by using appropriate primers. To detect ET receptor protein expression, Western blotting (WB) and immunohistochemistry (IHC) were performed. The WB and IHC techniques showed that ETA and ETB receptor expression is significantly greater in the lungs of RAO horses than in healthy. The RT-PCR showed greater ETB receptor mRNA expression in the lungs of RAO horses than those of healthy; however, no difference was observed in the ETA receptor mRNA expression between the two groups. This study demonstrated the over-expression of ET receptors, particularly ET-B, in the lungs of RAO horses. The study was supported by grants from NRI-USDA (#2001-35204-10809) and EHSP of SVM-LSU.

β2-Adrenoceptor Agonist Modulates Endothelin-1 Receptors in Human Isolated Bronchi

Chronic exposure of human isolated bronchi to beta(2)-adrenergic agonists, especially fenoterol, potentiates smooth muscle contraction in response to endothelin-1 (ET-1), a peptide implicated in chronic inflammatory airway diseases. Our objective was to determine whether ET-1 receptors ETA and ETB are involved in fenoterol enhancement. Twenty-two human bronchi were sensitized to ET-1 by prolonged incubation with 0.1 microM fenoterol (15 h, 21 degrees C). Removing the epithelium after fenoterol incubation limited the maximal contraction (0.10+/-0.36 g without epithelium versus 1.18+/-0.22 with, n=8, P=0.04). After 15 h incubation, 14 and 8 paired rings were fixed, respectively, for immunolabeling of bronchial ETA and ETB receptors, and to determine the mRNA expression levels using real-time quantitative reverse transcription polymerase chain reaction. ETA and ETB receptor mRNA expressions were 1.27- +/- 0.14-fold (not significant) and 2.24- +/- 0.28-fold (P<0.01) higher, respectively, in fenoterol-treated bronchi than in paired controls. Fenoterol incubation significantly increased epithelial ETA and ETB receptor labeling intensity scores (P=0.001 and P=0.002, respectively, versus controls), and enhanced the diffuse localization of ETA receptors on the epithelial cells (P=0.002 versus controls), but did not change the ETB-receptor immunolabeling intensity on airway smooth muscle. We conclude that fenoterol-induced sensitization of human isolated bronchi involves epithelial ETA and ETB receptors, which suggests perturbation of the epithelial regulation of airway smooth muscle contraction in response to ET-1.

The effect of endothelin receptor in androgen-independent prostate cancer

To study the expression of ET receptor and the apoptosis after intervened with ET receptor antagonist in androgen-independent prostate cancer. PC3, an androgen-independent prostate cancer cell line, was used. The expression of ETA and ETB receptor in PC3 was measured through RT-PCR. After intervened with selective ETA and ETB receptor antagonist, the apoptosis in PC3 was studied through flow cytometry and electron microscope. Clear signal was obtained in PC3 for ETA receptor mRNA transcript, while the signal for ETB receptor mRNA transcript was very weak. The expression of ETA receptor mRNA was obviously reduced and the apoptosis of PC3 cell was observed after intervened with selective ETA receptor antagonist. There was no change after intervened with selective ETB receptor antagonist. ET-1 exerts its effects through the ETA receptor subtype and ETB receptor is silenced in PC3. The expression of ETA was reduced and the apoptosis was observed in PC3 when ETA receptor was blocked. It was dose-dependent.

Developmental expression of endothelin receptors in postnatal swine mesenteric artery.

Studies were conducted to determine whether endothelin (ET) ETA and ETB receptor protein and mRNA expression is developmentally regulated in the postnatal swine mesenteric circulation. To this end, Western blotting and real-time reverse PCR were performed on protein and total RNA isolated from the mesenteric artery harvested from 3-, 10-, and 30-d-old swine. Western blot analysis revealed that ETA and ETB receptor protein expression in the swine mesenteric artery decreased over the age range studied; thus, ETA and ETB receptor protein expression was significantly greater in the 3-d-old group then progressively declined over the first postnatal month. Similar to the Western data, real-time PCR analysis revealed that ETA and ETB receptor mRNA expression also decreased over the age range studied; thus, ETA and ETB receptor mRNA expression was significantly greater in the 3-d-old group then progressively declined over the first postnatal month. Immunohistochemistry localized the ETA receptor to the vascular smooth muscle and the ETB receptor to the endothelial cell layer. Additionally, we report a partial cDNA sequence for the swine ETB receptor. We conclude that ETA and ETB receptor protein and mRNA expression is developmentally regulated in the postnatal swine mesenteric artery, being expressed to a greater degree in younger animals.

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.

Influence of Morphine and Naloxone on Endothelin and its Receptors in Newborn Piglet Brain Vascular Endothelial Cells: Clinical Implications in Neonatal Care

The present study examines the hypothesis that morphine exposure alters newborn brain vascular endothelial cell production of endothelin (ET)-1, as well as the mRNA expression of its receptors. Newborn piglet vascular endothelial cells were treated with morphine (100 ng/mL media), naloxone (100 ng/mL media), or drug-free media (control) for 6, 24, 48, and 96 h. Media was analyzed for ET-1 and big ET-1 levels and the cells were assessed for ETA and ETB receptor mRNA expression. Morphine exposure progressively increased ET-1 production from 6 to 96 h with concurrent reductions in big ET-1 levels starting at 24 h to almost undetectable levels by 96 h. Whereas ETA receptor mRNA expression increased 2-fold at 6 h and 4-fold at 96 h, ETB receptor mRNA expression remained unchanged. Naloxone exposure caused significant decreases in ET-1 levels, whereas an opposite effect was noted in big ET-1 levels, which increased from 6 through 96 h. Naloxone caused a progressive decrease in ETA receptor mRNA expression at 6 h through 96 h and a 2-fold increase in ETB receptor mRNA expression at 48 and 96 h. Increased ET-1 and its receptors in response to morphine may suggest altered cerebrovascular perfusion and brain metabolism in the immature piglet brain.

Role of endothelin in diabetic retinopathy.

Endothelin-1 (ET-1) is a 21 amino acid peptide originally purified from conditioned medium of cultures of porcine aortic endothelial cells. It is now known that there are three endothelin genes in the human genome (ET-1, ET-2, and ET-3 genes). ET-1 and ET-2 are both strong vasoconstrictors, whereas ET-3 is a potentially weaker vasoconstrictor compared to the other two isoforms. Besides being a very potent vasoconstrictor, ET-1 also acts as a mitogen on the vascular smooth muscle and thus it may play a role in the development of vascular diseases. There is evidence that impaired auto-regulation of blood flow is involved in the pathogenesis of diabetic microangiopathy. It is known that the ability of the diabetic's circulation to distribute blood is affected, especially during increased blood flow. In most tissues this causes no serious burden, but three tissues are usually susceptible to disturbance. They are the retina, renal cortex, and peripheral nerves. Retinal vascular auto-regulation is defined as the ability of the blood vessels to keep blood flow constant under varying perfusion pressure in order to match it to tissue oxygen and metabolic requirements. The failure of auto-regulation is an important and often early feature of diabetic retinopathy. Since human retina vessels lack extrinsic innervation, retinal vessel calibre and local blood flow are normally regulated by non-nervous mechanisms intrinsic to the retina. There is now a considerable body of evidence suggesting that retinal pericytes are the main regulators of vascular tone in the retinal capillaries because they contain components of contractile proteins similar to vascular smooth muscle cells and because they also possess ET-1 receptors. Furthermore. ET-1 has been shown to cause vasoconstriction of retinal vessels as well as to have mitogenic effects on retinal pericytes. Hence, alterations in the pericyte-ET interaction may have a role causing early hemodynamic and histopathological abnormalities found in diabetic retinopathy. On the contrary, Chakrabarti et al. demonstrate that retinas from the chronic diabetic BB/W rats (6 months) show an increase in ET-1, ET-3, ET(A) receptor and ET(B) receptor mRNA expressions when compared to those from control rats. Similar results are noted by them using immunohistochemical methods. Finally, an increased ocular, and retina tissue levels of ET-1 in diabetic rats have also been reported by Chakravarthy et al., as well as by Takagi et al. All of these findings suggest that endothelins may also be involved in the pathogenesis of more advanced diabetic retinopathy, such as capillary occlusion and subsequent neovascularization. This review summarizes the reported literature on the role of ET-1 in the development of diabetic retinopathy.

Fetal pulmonary hypertension prolongs the endothelin-1 induced increase in pulmonary artery smooth muscle cell cytosolic calcium concentration

Introduction: At birth, pulmonary artery (PA) blood flow increases 8–10 fold and PA pressure falls by 50% within 24 hours. The mechanisms responsible for postnatal adaptation of the pulmonary circulation remain incompletely understood. High serum endothelin-1 (ET-1) levels are found in infants with persistent pulmonary hypertension of the newborn. We hypothesized that chronic intrauterine pulmonary hypertension (PHT) potentiates agonist induced increase of cytosolic calcium concentration ([Ca2+]i) in fetal PA smooth muscle cells(SMC). Methods: Chronic intrauterine pulmonary hypertension was induced by intrauterine ligation of the ductus arteriosus (DA) in late-gestation fetal lambs. 7–10 days following DA ligation, animals were euthanized. Distal PAs (>4th generation) were removed from the lung and PA SMC were isolated and maintained in normoxic (95%O2) primary culture. Age-matched non-PHT controls were sacrificed at ∼132 days gestation and cells were cultured in the same manner. Fluorescence microscopy using fura-2 was used to determine the effect of ET-1 (10−6 -10−8 M) on PA SMC [Ca2+]i from PHT and control animals. To determine the effect of PHT on endothelin A and B (ETA and ETB) receptor mRNA expression, quantitative, internally controlled RT-PCR was performed on distal PAs and SMC in culture. Results: ET-1 (10−8 M) caused a monophasic increase in [Ca2+]i (206 +/- 47%, p < 0.01 vs baseline) above baseline in controls (n = 25), while PHT SMC [Ca2+]i did not increase significantly. When PHT SMC were treated with 10−6M ET-1, in those cells that responded, [Ca2+]i increased 174 +/- 81% above baseline(p < 0.01 vs baseline). Only 3/33 (9%) of PHT SMC responded to ET-1 compared to 90% of controls. Additionally, the duration of the increase in [Ca2+]i was longer in the PHT SMC: PHT SMC [Ca2+]i fell to baseline after 328 +/- 16 sec, where control [Ca2+]i fell to baseline after 117 sec. ETA and ETB receptor mRNA expression are both increased in PHT. Conclusions: PHT decreases PA SMC sensitivity to ET-1 by ∼100 fold, however, PHT increases the duration of the ET-1 induced increase in [Ca2+]i. We conclude that PHT prolongs the response of PA SMC to ET-1. This prolongation may lead to significant increases in PA SMC [Ca2+]i that results in attenuated postnatal adaptation of the pulmonary circulation.

Subarachnoid Hemorrhage Enhances Endothelin Receptor Expression and Function in Rat Cerebral Arteries

Inspired by organ culture-induced changes in the vascular endothelin (ET) receptor population, we investigated whether such changes occur in cerebral arteries in a rat subarachnoid hemorrhage (SAH) model. SAH was induced with injection of 250 microl of blood into the prechiasmatic cistern. After 2 days, the middle cerebral artery, basilar artery, and posterior communicating artery were harvested. Pharmacological studies were performed in vitro, and levels of messenger ribonucleic acid (mRNA) were quantified in real-time reverse transcriptase-polymerase chain reaction assays. In the middle cerebral artery and basilar artery from rats with induced SAH, enhanced biphasic responses to ET-1 were observed. The -log(50% effective concentration) value for the high-affinity phase was approximately 12, compared with approximately 8.5 for sham-operated animals. At a concentration of ET-1 of 10(-9) mmol/L (approximately equal to the physiological concentration of ET-1 in the plasma), submaximal contractions of 50 to 75% of the contraction obtained through stimulation with 60 mmol/L K(+) were now observed. Quantitative mRNA studies with the same arteries demonstrated significant increases in the number of copies of ET(B) receptor mRNA but not ET(A) receptor mRNA. Evidence of functional ET(B) receptors was provided in antagonist studies. The posterior communicating artery did not exhibit significant changes. The altered receptor profile observed may represent the final stage in the series of events leading from SAH to actual spasm of the artery. The pharmacological data for the ET(B) receptor suggest complex interactions between normally present ET(A) receptors and up-regulated ET(B) receptors.

HMG-CoA reductase inhibitor improves endothelial dysfunction in mineralocorticoid hypertension by inhibition of RhoA

Statins reduce cardiovascular morbidity and mortality. These beneficial effects are not fully explained by their lipid-lowering action. As such, we investigated the impact of a new statin, rosuvastatin, on endothelial function, the key event in early atherogenesis, in an experimental model of normocholesterolemic hypertension. Hypertension was induced in Wistar-Kyoto rats by inhibition of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) with Glycyrrhizic acid (GA). 11β-HSD2 provides mineralocorticoid receptor specificity for aldosterone by metabolising glucocorticoids to their receptor inactive 11-dehydro derivatives. GA was added to the drinking water (3 g/L) for 21 days. From days 8 to 21 rosuvastatin (20 mg/kg/d) or placebo were added to chow. Endothelium-dependent and -independent relaxation of isolated aortic rings to acetylcholine (ACh, 10-10-10-5 mol/L) and sodium nitroprusside (SNP, 10-10-10-5 mol/L) was measured. In addition, vascular reactivity to endothelin-1 (ET-1; 10-10-10-7 mol/L) was investigated. ETA and ETB receptor mRNA expression was determined by RT-PCR and RhoA activity by a pull-down assay. Systolic blood pressure increased in rats treated with GA (175 vs 153 mmHg in controls; p<0.01). Endothelium-dependent relaxations to acetylcholine were blunted after GA treatment (p≤0.005 vs control), while the responses to SNP remained unchanged. Rosuvastatin normalized NO-mediated endothelium-dependent relaxation in hypertensive animals (p≤0.01 vs placebo), although blood pressure and cholesterol levels were not affected by the statin. Vascular reactivity to ET-1 was increased by GA (p<0.01 vs control), but not affected by rosuvastatin. ETB receptor mRNA decreased in the GA group (p<0.05 vs control) and was upregulated by rosuvastatin (p<0.005; GA+rosuvastatin vs GA), whereas ETA receptor mRNA upregulation in the GA group (p<0.01 vs control) was partially prevented by the statin. In addition, GA increased Rho-GTP binding (p≤0.05 vs control) which was prevented in both groups by rosuvastatin treatment (p≤0.01 control+rosuvastatin vs control and GA+rosuvastatin vs GA). These data for the first time show that HMG-CoA inhibition improves endothelial dysfunction in normocholesterolemic mineralocorticoid hypertension without affecting blood pressure or cholesterol levels by correction of a stimulated endothelin system.