Expression and regulation of endothelin-1 and its receptors in human penile smooth muscle cells

We report for the first time that penile smooth muscle cells (SMC) not only respond to, but also synthesize, endothelin-1 (ET-1), one of the main regulators of SMC activity. Immunohistochemical studies indicated that, beside endothelial cells (EC), SMC of the human adult and fetal penis also express ET-1 and its converting enzyme, ECE-1. Accordingly, cultures of adult penile stromal cells express these genes. We also prepared and characterized penile SMC from human fetuses. These cells express SMC specific markers such as alpha smooth muscle actin and phosphodiesterase type 5A3 along with hallmarks of androgen-dependent cells (androgen receptor and 5alpha reductase type 2). Human fetal penile SMC (hfPSMC) are immunopositive for ET-1 and release ET-1. ET-1 expression in hfPSMC was strongly increased by several factors such as transforming growth factor-beta1 (TGF-beta1), interleukin-1alpha (IL-1alpha), ET-1 itself and prolonged (24 h) hypoxia. This latter condition not only affected ET-1 expression but also responsiveness. While at normal oxygen tension, hfPSMC responded to ET-1 with a decreased proliferation mediated by the endothelin-A receptors and TGF-beta1; however, during hypoxia, ET-1 stimulated cell growth. Accordingly, prolonged hypoxia up-regulated endothelin-B receptor mRNA expression. In conclusion, our results indicate that in penile tissues SMC produce ET-1 and that such production is modulated by factors involved in penile physiology and tissue remodelling. In addition, the hfPSMC we have characterized might be a useful model for studying biochemical aspects of the human erectile process in vitro.     

Signaling pathways involved in the A and B receptor-mediated cortisol secretagogue effect of endothelins in the human adrenal cortex

Endothelins (ETs) are a family of 21-amino acid hypertensive peptides, which together with their receptors ETA and ETB are expressed in human adrenal cortex. Evidence has been provided that ETs exert a potent secretagogue effect on human adrenocortical cells, acting through both ETA and ETB receptors. Therefore, it seemed worthwhile to study the signaling cascades mediating the cortisol secretagogue effect of the two receptor subtypes. Normal adrenal glands were obtained from consenting patients undergoing unilateral nephrectomy with ipsilateral adrenalectomy for renal cancer. Dispersed zona fasciculata-reticularis (ZF/R) cells were obtained by collagenase digestion and mechanical disaggregation. The selective activation of ETA and ETB receptors was obtained by exposing dispersed cells to ET-1 plus the ETB receptor antagonist BQ-788 and to the selective ETB receptor agonist BQ-3020, respectively. ETA and ETB receptors about equally contributed to the cortisol response of dispersed ZF/R cells to ETs. The phospholipase (PL) C inhibitor U-73122 abolished ETA-mediated secretory response, but only partially prevented the ETB-mediated one. The phosphatidylinositol 3-kinase inhibitor wortmannin and the protein kinase (PK) C inhibitor calphostin-C significantly blunted the secretory responses ensuing from the activation of both receptor subtypes, while the Ca(2+)-channel blocker nifedipine was ineffective. The ETB receptor-, but not the ETA receptor-mediated cortisol response was partially reversed by the cyclooxygenase (COX) inhibitor indomethacin, which when added together with U-73122 abolished it. The inhibitors of adenylate cyclase, PKA, tyrosine kinase and lipoxygenase did not affect the secretory response to the activation of either receptor subtype. ETA-receptor activation raised inositol triphosphate (IP3) production from dispersed ZF/R cells, while ETB-receptor stimulation enhanced both IP3 and prostaglandin-E(2) production. Collectively, our findings indicate that ETs stimulate cortisol secretion from human ZF/R cells, acting through ETA receptors exclusively coupled with PLC/PKC-dependent pathway and ETB receptors coupled with both PLC/PKC- and COX-dependent cascades.     

Localization and distribution of endothelin receptor subtypes in pulmonary vasculature of normal and hypoxia-exposed rats

To clarify the roles of two different endothelin (ET) receptors in the pulmonary vasculature, the localization and distribution of endothelin-A (ETA) and ETB receptors were investigated in rat lung under normal and hypoxic conditions by an immunohistochemical method. We also carried out in situ hybridization for ETB receptor. In normal rats, ETA receptor is localized in the media of the pulmonary artery and vein with predominant distribution in such proximal segments as elastic arteries and large muscular arteries. ETB receptor is expressed in the intima and media of pulmonary vessels. The distribution of ETB receptor in the media predominates in the distal segments of the pulmonary artery, whereas its distribution in the intima is greater in the proximal segments. Immunoreactivity for ETA receptor increases in the media of the distal segments of the pulmonary artery after exposure to hypobaric hypoxia. Semiquantitative evaluation showed immunoreactivity for ETA receptor in the pulmonary arteries accompanying the terminal bronchioles, respiratory bronchioles, and alveolar ducts to be increased by 2.5-, 5-, and 20-fold after 14 d exposure to hypoxia, respectively. The messenger RNA and immunoreactivity for ETB receptor increased significantly in the intima of the distal segments of pulmonary artery after 7 and 14 d exposure to hypoxia. These results suggest that the vasoconstrictive effects of ET-1 are exerted mainly through ETA receptor in the proximal segments of the pulmonary artery and vein, whereas its effects in the distal segments are mediated by ETA and ETB receptors in normal rats. ETA receptors that increase in resistance arteries after exposure to hypoxia appear to play an important role in the vascular remodeling associated with hypoxic pulmonary hypertension. Because ETB receptors in the endothelium mediate ET-1-induced vasodilatory effects, the increase in endothelial ETB receptors may counteract the development of hypoxic pulmonary hypertension.     

Expression of endothelin-1 and its receptors in the lungs of broiler chickens exposed to high-altitude hypoxia

To investigate the influence of exposure to high-altitude (HA) hypoxia on the expressions of endothelin-1 (ET-1), endothelin type A (ETA) and endothelin type B (ETB) receptors in broiler chickens, immunohistochemistry studies were performed in the lungs. Six hundred 1-day-old male broiler chickens were randomly divided into two groups: group A, birds maintained under rich oxygen conditions (oxygen content 21%); and group B, birds exposed to HA hypoxia (oxygen content 13%). Our data showed that exposure to altitude elevated ET-1 and ETA gene expressions at 21 and 28 days of age when compared with the rich oxygen group. Meanwhile, a marked decline in ETB expression was observed at 28 days of age in the course of HA, although there were no significant changes (P>0.05) at 7, 14 and 21 days of age. The increased response was accompanied by adverse effects on weekly body weight gain and ascites mortality. These observations suggested that ET-1, ETA and ETB genes are normally expressed in the lungs of birds. Increased levels of ET-1 and ETA and decreased ETB gene expression in the lungs are probably involved in the lung dysfunction of broiler chickens with developmental ascites.     

Chronic hypoxia enhances endothelin-1-induced intracellular calcium elevation in rat carotid body chemoreceptors and up-regulates ETA receptor expression

Endothelin-1 (ET-1) excites carotid body (CB) chemoreceptors and induces mitosis of the chemoreceptors in chronic hypoxia. The aim of the present study was to examine the hypothesis that up-regulation of both ETA receptor and endogenous ET-1 expression in CB chemoreceptors enhances the response of intracellular Ca2+ to ET-1 following adaptation to chronic hypoxia (10% inspired O2 for 3-4 weeks). Cytosolic free [Ca2+] ([Ca2+]i) in type-I (glomus) cells freshly dissociated from rat CBs was measured by spectrofluorometry. Application of exogenous ET-1 (1-100 nM) concentration-dependently elevated [Ca2+]i in the glomus cells. This response to ET-1 (100 nM) was 49% greater in the chronically hypoxic (CH) group. The ET-1 response was abolished completely by the ETA receptor antagonist BQ610 (1 microM), but not by the ETB antagonist BQ788 (1 microM). The transient [Ca2+]i elevation induced by caffeine (30 mM) in the normoxic group was similar to that in the CH group, suggesting no differences in the intracellular Ca2+ stores. In situ hybridization with a digoxigenin-labelled antisense ETA receptor mRNA oligonucleotide probe revealed very intense and ubiquitous specific expression of ETA receptors in the lobules of glomus cells in the CH group, whereas staining in normoxic controls was light. Immunohistochemical studies revealed intense cytoplasmic staining for ET-1-immunoreactivity in most of the cell clusters in glomera in the CBs of CH rats but was faint in normoxic CBs. These findings indicate increased expression of both the ETA receptor and ET-1 in CB chemoreceptors during chronic hypoxia. Taken together, our results suggest that the [Ca2+]i response to ET-1 in rat CB chemoreceptors is augmented by up-regulation of ETA receptors and ET-1 expression. The enhancement of the paracrine/autocrine effect of ET-1 on the chemoreceptors is consistent with an excitatory and mitogenic role of the ET-1 and ETA receptor in the CB during chronic hypoxia.     

Endothelin receptor expression in human lungs of newborns with congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a major cause of refractory respiratory failure in the neonatal period and is characterized by persistent pulmonary hypertension of the newborn (PPHN) and pulmonary hypoplasia. Endothelin-1 (ET-1) dysregulation may play a significant role in the pathophysiology of PPHN and ET-1 acts through binding to type A (ETA) and type B (ETB) receptors. Therefore, ETA and ETB receptor protein expression was studied using immunohistochemistry in 10 lung specimens obtained from newborns with CDH, and 4 normal lung specimens, in order to explore whether dysregulation of ETA and ETB expression contributes to PPHN. ETA and ETB mRNAs were then quantified using real-time RT-PCR in laser-microdissected pulmonary resistive arteries. In the lungs of newborns with CDH, immunohistochemistry of both ETA and ETB receptors demonstrated over-expression in the thickened media of pulmonary arteries. Using laser microdissection and real-time RT-PCR, higher levels of ETA and ETB mRNA were found in CDH pulmonary arteries than in controls: this increase was more pronounced for ETA mRNA. This study provides the first demonstration of ET-1 receptor dysregulation in association with structural alteration of pulmonary arteries in newborns with CDH and PPHN. This dysregulation preferentially affects the ETA receptor. These results suggest that dysregulation of ET-1 receptors may contribute to PPHN associated with CDH.     

Buffering action of endogenous nitric oxide on the adrenocortical secretagogue effect of endothelins in the rat

The secretagogue effect of endothelins (ETs) on the rat adrenal cortex is mediated by the ETB receptor. ETB receptors are coupled with nitric oxide (NO) synthase (NOS), and NO is known to inhibit steroid-hormone secretion from adrenal cortex. We investigated whether ETB-mediated NO production interferes with the stimulatory action of ETs on rat adrenal cortex. The selective agonist of ETB receptor BQ-3020 concentration-dependently increased aldosterone secretion from dispersed zona glomerulosa (ZG) cells and corticosterone secretion from dispersed zona fasciculata-reticularis (ZF/R) cells, and the NOS inhibitor NG-nitro-L-arginine methylester (L-NAME) potentiated the effect of BQ-3020 in a concentration-dependent manner. The guanylate cyclase inhibitor Ly-83583, at a concentration suppressing guanylin- and L-arginine-induced cyclic-GMP release from dispersed adrenocortical cells, did not affect the secretory response of ZG and ZF/R cells to BQ-3020. ET-1, an agonist of both ETA and ETB receptors, stimulated the release of both aldosterone and corticosterone by in situ perfused rat adrenal gland. This effect was potentiated by L-NAME and unaffected by Ly-83583. Collectively, our findings allow us to suggest that endogenous NO exerts in vivo and in vitro a cyclic-GMP-independent buffering action on the ETB receptor-mediated adrenocortical secretagogue action of ETs.     

Increased levels of endothelin-1 and differential endothelin type A and B receptor expression in scleroderma-associated fibrotic lung disease.

In addition to their vasoactive action, endothelins are potent peptides in the regulation of both cell proliferation and the turnover of extracellular matrix. Using immunohistochemical, autoradiographic, and molecular analyses, we have studied the localization and expression of endothelin-1 and endothelin A (ETA) and B (ETB) receptors in scleroderma-associated fibrotic lung disease. Increased ET-1 immunoreactivity was found in sclerotic tissue compared with control and was associated with the vasculature, pulmonary interstitium, and bronchial and alveolar epithelium. Microautoradiographic analysis after 125I-labeled ET-1 binding showed a two- to threefold increase in the expression of total ET-1 receptors in scleroderma lung tissue localized to the alveolar epithelium and the pulmonary interstitium which was composed of mainly fibroblastic cells with macrophages and some microvessels. RNAse protection assay revealed significantly reduced ETA receptor and slightly raised ETB message levels in systemic sclerosis lung. Surface expression of functional ET receptors was examined by targeted receptor blocking using mixed and receptor-subtype-selective ligands. A consistent decrease in ETA receptor binding sites was noted primarily within the interstitium and vasculature, in contrast to a slight increase in ETB receptors. Elevated ET-1 and the cell-specific pattern of endothelin receptor expression suggest that the endothelins may represent important mediators that influence the pathology of scleroderma-associated lung disease and other fibrotic conditions.     

Interleukin-1β attenuates endothelin B receptor-mediated airway contractions in a murine in vitro model of asthma: roles of endothelin converting enzyme and mitogen-activated protein kinase pathways

BACKGROUND: Asthma is a chronic airway disease, known to involve several inflammatory mediators. Little is known about how these mediators interact in order to produce or attenuate even basic features of the disease, like airway hyper-reactivity and remodelling. Endothelin-1 (ET-1) and IL-1beta are two mediators suggested to play important roles in the induction of airway inflammation. OBJECTIVE: To investigate the interactions between ET-1 and IL-1beta, using a novel in vitro model of asthma, focusing on airway smooth muscle contractility. METHODS: Isolated murine tracheal segments were cultured from 1 to 8 days in the absence and presence of IL-1beta. The subsequent contractile responses to sarafotoxin 6c (S6c) (selective agonist for ETB receptor) and sarafotoxin 6b (S6b) (ETA and ETB receptor agonist) were recorded by a myographs system. In all experiments, ETB receptors were desensitized before the contractile response to S6b was recorded. Thus, the response to S6b is only mediated by ETA receptors in the present study. The mRNA expressions for ET-1 and endothelin (ET) receptors were quantified by real-time PCR. RESULTS: Organ culture in the presence of IL-1beta attenuated the maximal contraction induced by S6c, but not S6b. This reduction was concentration-dependent and was significant after 2, 4 and 8 days of culture. To investigate the mechanisms behind this, inhibitors for endothelin converting enzyme (ECE) phosphoramidon, c-JUN N-terminal kinase (JNK) SP600125, extracellular-signal-regulated kinase 1/2(ERK 1/2) PD98059 and p38 pathway SB203580 were used. Individually, SP600125 and PD98059, but not SB203580, could partly reverse the reduction induced by IL-1beta. An additional effect was obtained when SP600125 and PD98059 were combined. The mRNA expressions for ET-1 and ETB receptor were up- and down-regulated, respectively, by IL-1beta. CONCLUSION: Presence of IL-1beta in the airways attenuate the contractile response mediated via ETB receptors, an effect dependent on ECE, JNK and ERK 1/2 pathways.     

Mechanisms and receptor subtypes involved in the stimulatory action of endothelin-1 on rat adrenal zona glomerulosa

Endothelin (ET)-1 is the prototype of a family of 21-amino acid residue hypertensive peptides, acting through two subtypes of receptors, named ETA and ETB. ETs and their receptors are expressed in the adrenal cortex and medulla, and ET-1 enhances both corticosteroid and catecholamine release. ET-1 concentration-dependently (from 10(-11) to 10(-8) M) increased aldosterone secretion of both dispersed rat zona glomerulosa (ZG) cells and adrenal slices containing a core of medullary chromaffin tissue, but the response of the latter preparations was significantly more intense than that of the formers. The stimulatory effect of 10(-8) M ET-1 on dispersed ZG cells was blocked by the ETB-receptor antagonist BQ-788 (10(-7) M), but not by the ETA-receptor antagonist BQ-123 (10(-7) M); conversely, both ET-receptors antagonists counteracted aldosterone response of adrenal slices to ET-1. The -adrenoceptor antagonist l-alprenolol (10(-6) M) did not affect aldosterone response of dispersed ZG cells to ET-1 (10(-8) M), but it significantly lowered that of adrenal slices. l-Alprenolol also counteracted the aldosterone response of adrenal slices to the pure activation of ETB or ETA receptors, as obtained by using the selective ETB-receptor agonist BQ-3020 (10(-8) M) or ET-1 (10(-8) M) plus BQ-788 (10(-7) M). ET-1 concentration-dependently (from 10(-9) to 10(-8)/10(-7) M) stimulated catecholamine release by adrenal slices, and the effect was counteracted by both BQ-123 and BQ-788 (10(-7) M). Collectively, our findings suggest that, when the integrity of adrenal tissue is preserved, a two-fold mechanism underlies the aldosterone secretagogue action of ET-1 in the rat: i) a direct mechanism mediated by ETB receptors located on ZG cells; and ii) an indirect mechanism involving the ETA and ETB receptor-mediated local release of catecholamines, which in turn stimulate ZG cells in a paracrine manner.     

Modulation of human colon tumor-stromal interactions by the endothelin system

Tumor neovascularization is considered to be a critical step in the development of a malignant tumor. Endothelin (ET)-1 is a powerful vasoconstrictor and mitogenic peptide that is produced by many cancer cell lines. The cellular distribution of the ET components was evaluated in human colon tumors and compared to normal colon. There was more of the ET components (preproET-1, endothelin-converting enzyme-1, and ETA and ETB receptors) in adenomas and adenocarcinomas than in the normal colon. There was overproduction of preproET-1 and endothelin-converting enzyme-1 in carcinoma cells and stromal vessels, suggesting that they are a local source of ET-1. ETA receptors were present in stromal myofibroblasts of neoplastic tissue, and there were large amounts of ETB receptors in the endothelium and myofibroblasts. There was also a redistribution of alpha-smooth muscle actin-positive cells in the vascular structures of tumors. An experimental rat model of induced colon cancer treated for 30 days with bosentan, a mixed antagonist of both ET receptors, confirmed the morphological changes observed during the tumor vascularization. Our data suggest that ET-1 and its receptor play a role in colon cancer progression, with ET-1 functioning as a negative modulator of the stromal response.     

Effects of endothelin-1 on epithelial ion transport in human airways

Endothelin-1 (ET-1) exerts many biological effects in airways, including bronchoconstriction, airway mucus secretion, cell proliferation, and inflammation. We investigated the effect of ET-1 on Na absorption and Cl secretion in human bronchial epithelial cells. Addition of 10(-7) M ET-1 had no effect on the inhibition of the short circuit current (Isc) induced by amiloride, a Na channel blocker. Addition of 10(-7) M ET-1 to the apical bath in the presence of amiloride increased Isc in cultured human bronchial epithelial cells studied in Ussing chambers. No effect was observed when ET-1 was added to basolateral bath, indicating that the involved ET-1 receptors are likely present only in the apical membrane of the cells. Use of Cl-free solutions and bumetanide reduced the ET-1-induced increases in Isc, indicating that ET-1 stimulates Cl secretion. The ET-1-induced increase in Isc was prevented by exposure to the ETB receptor antagonist BQ-788 but not to the ETA receptor antagonist BQ-123. ET-1 did not raise intracellular Ca levels, but increased the intracellular concentration of cAMP. These findings indicate that ET-1 is a Cl secretagogue in human airways and acts presumably through apically located ETB receptors and activation of the cAMP pathway.     

Early increase in pulmonary vascular reactivity with overexpression of endothelin-1 and vascular endothelial growth factor in canine experimental heart failure

Heart failure is a cause of pulmonary vasoconstriction and remodelling, leading to pulmonary hypertension (PH) and decreased survival. The pathobiology of PH in heart failure remains incompletely understood. We investigated pulmonary vascular function and signalling molecules in early stage PH secondary to experimental heart failure. Eight beagle dogs with overpacing-induced heart failure underwent haemodynamic assessment and postmortem pulmonary arterial reactivity, morphometry and quantification of genes encoding for factors involved in vascular reactivity and remodelling: endothelin-1 (ET-1), ETA and ETB receptors, vascular endothelial growth factor (VEGF), VEGF receptors 1 and 2 (VEGFR1 and VEGFR2), endothelial nitric oxide synthase, angiopoietin-1, bone morphogenetic protein receptors (BMPR1A and BMPR2), serotonin transporter (5-HTT) and the 5-HT(2B) receptor. Overpacing was associated with a decrease in cardiac output and an increase in pulmonary vascular pressures. However, there were no changes in pulmonary vascular resistance or in arteriolar medial thickness. There were increased expressions of genes encoding for ET-1, ETB, VEGF and VEGFR2, while expression of the other genes analysed remained unchanged. In vitro, pulmonary arteries showed decreased relaxation and increased reactivity, while systemic mammary arteries were unaffected. Early PH in heart failure is characterized by altered vasoreactivity and increased ET-1/ETB and VEGF/VEGFR2 signalling.     

Cloprostenol, a prostaglandin F(2alpha) analog, induces hypoxia in rat placenta: BOLD contrast MRI

Blood oxygen level dependent (BOLD) contrast was used to monitor hypoxia induced by cloprostenol, a prostaglandin F(2alpha) (PGF(2alpha)) analog, in the rat embryo-placental unit (EPU). It is shown that administration of cloprostenol (0.025 mg/rat) at mid-gestation (day 16) reduced EPU oxygenation, as detected by BOLD contrast MRI, in correlation with induction of vascular endothelial growth factor (VEGF) gene (Vegfa) expression in the corresponding placenta (r = 0.56, p = 0.03). Elevated VEGF mRNA expression in response to cloprostenol treatment was also observed at early gestation (day 9) in the forming placenta (p = 0.04) and uterus (p = 0.03). Cloprostenol increased the expression levels of endothelin-1 (ET-1) gene (Edn1) (p = 0.03) and its corresponding peptide (p = 0.02) in the forming placenta, as well as the expression of the endothelin receptor type A (ETA) gene (Ednra) in both the forming placenta (p = 0.009) and the uterus (p = 0.01). The levels of the endothelin receptor type B (ETB) gene (Ednrb) were not affected in response to cloprostenol, but a significant elevation in the expression level of this receptor was observed in the uterus at mid- and late gestation (day 22) (p = 0.04 and 0.01 respectively), suggesting a role for ETB in the vasodilatory status of the pregnant uterus. It is suggested that PGF(2alpha) induces uteroplacental vasoconstriction in the rat, and that ET-1 may take part in mediating this effect, probably via activation of ETA receptor. The uteroplacental vasoconstriction induces hypoxia, as manifested by significant changes in BOLD MRI and by upregulation of VEGF.     

Effects of endothelin-1 on the membrane potential and slow waves in circular smooth muscle of rat gastric antrum.

Electrophysiological effects of endothelin-1 (ET-1) on circular smooth muscle of rat gastric antrum were investigated by using intracellular membrane potential recording techniques. ET-1 (10 nM) caused an initial hyperpolarization of the membrane which was followed by a sustained depolarization. ET-1 also increased the frequency but not the amplitude of slow waves. In the presence of the endothelin type A (ETA) receptor antagonist, BQ123 (1 microM), ET-1 (10 nM) depolarized the membrane and increased the frequency of slow waves, but without the initial hyperpolarization. The selective endothelin type B (ETB) receptor agonist, sarafotoxin S6c (10 nM), also depolarized the membrane and increased the frequency of slow waves. In the presence of the ETB receptor antagonist, BQ788 (1 microM), ET-1 (10 nM) hyperpolarized the membrane. However, in the presence of BQ788, ET-1 caused neither the depolarization nor the increase in the frequency of the slow waves. The ET-1-induced hyperpolarization was completely abolished by apamin (0.1 microM). In the presence of apamin, ET-1 depolarized the membrane and increased the frequency of slow waves. The ET-1-induced depolarization was significantly attenuated by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.3 mM). The increase of the frequency by ET-1 was observed both in the presence and absence of DIDS. These results suggest that, ET-1 hyperpolarizes the membrane by the activation of Ca2+-activated K+ channels via ETA receptors, and depolarizes the membrane by the activation of Ca2+-activated Cl- channels via ETB receptors. ET-1 also appears to increase the frequency of slow waves via ETB receptors, however this mechanism would seem to be independent of membrane depolarization.     

Role of endothelin-1 in regulating proliferation of cultured human uterine smooth muscle cells

Endothelin-1 (ET-1) exhibits vasoconstricting and growth-promoting properties in vascular smooth muscle. Whether ET-1 has mitogenic properties in uterine smooth muscle cells, and which ET receptor subtype mediates this response, is unknown. The present study was undertaken to examine the proliferative potential of the ET family on human myometrial cells in culture. ET-1 stimulated DNA synthesis and proliferation of myometrial cells. The absence of a stimulating effect of endothelin-3 (ET-3) or the ETB agonist sarafotoxin 6c (S6c) was observed. The proliferative effect of 100nM ET-1 was blocked by the two ETA antagonists (BQ 123 and FR 139317), whereas the ETB antagonist IRL 1038 was ineffective. These data indicated that ET-1-induced DNA synthesis was mediated only by the ETA receptor subtype. Pertussis toxin (PTX) pretreatment completely abolished this effect, indicating that this pathway was coupled to the ETA receptor via the Gi protein family. PTX treatment partially decreased serum-induced DNA synthesis. This suggests that some factors from serum may operate via the G- protein in initiation of mitogenesis. Insulin-like growth factors (IGFs), epidermal growth factor (EGF) and insulin were found to be mitogens in the absence of serum, and they had no potentiating effect on ET-1-induced DNA synthesis. In the presence of 0.5% serum, EGF alone caused a weak increase in DNA synthesis, while all the growth factors were able to reduce the proliferative effect of ET-1. These findings on human myometrial cells in culture raise the possibility that, under certain conditions, ET-1 may function as a positive or as a negative modulator of smooth muscle proliferation.      

The role of endothelin receptor A during myelination of developing oligodendrocytes

Endothelin (ET)-1 and its receptors (ETA and ETB receptor) are present in the central nervous system. ET exerts biological effects on gliogenesis and glial cell functions. In order to define a possible mechanism of ETA receptor signaling, the distribution of the ETA receptor in developing oligodendrocytes and the effects of ET-1 on the myelination of oligodendrocytes were examined. ETA receptor immunoreactivity was confined to the perivascular elements of the blood vessels during early postnatal development. However later in development, ETA receptor immunoreactivity was no longer observed in the vessels but became localized to the myelinating oligodendrocytes of the primitive corpus callosum of the white matter, apart from the vessels. ET-1 induced myelin basic protein (MBP) in primary oligodendrocyte precursor cell culture though the ETA receptor and was blocked by an ETA receptor antagonist. In addition, ET-1 evoked the release of Ca(2+) which is a central regulator of oligodendrocyte differentiation. Our results provide a link between ET-1 and its ETA receptor and myelination during oligodendrocyte differentiation.     

Endothelin synthesis and receptors in porcine kidney

As insufficient information on the endothelin (ET) system in the porcine kidney is available at present, we investigated renal ET-1 synthesis and ET receptors in this species. Because ET specifically affects renal and glomerular haemodynamics and distal tubular reabsorption, we studied ET-1 synthesis in isolated glomeruli and in inner medullary collecting duct (IMCD) cells and preproET-1 mRNA in renal cortex, isolated glomeruli and papillary tissue. In addition, we characterized density and properties of ET receptors in membranes from isolated glomeruli and papillary tissue. In contrast to isolated IMCD cells, which synthesized 120 +/- 11 fmol h(-1) mg-1 protein of ET-1, no such synthesis was found with isolated glomeruli in our assay system. Nevertheless, with RT-PCR preproET(-1) mRNA was clearly present in renal cortex and glomeruli as well as in papillary tissue. Glomerular membranes were found to have ET receptors with Bmax of 1.6 +/- 0.2 pmol mg-1 protein and Kd of 311 +/- 33 pmol L(-1). Using BQ-123 (10-5 M), a specific blocker of ETA receptors, we found that 58% of total receptors are ETA receptors. Thus, presumably 42% are ETB receptors (Bmax 0.7 +/- 0.1 pmol mg-1 protein; Kd 429 +/- 110 pmol L(-1)). Bosentan (10-5 M), an ETA- and ETB-receptor antagonist, blocked all ET receptors in glomerular membranes. Papillary membranes showed ET receptors with Bmax of 2.1 +/- 0.2 pmol mg-1 protein and Kd of 137 +/- 11 pmol L(-1). In the presence of BQ-123 (10-5 M) we found that all receptors are ETB receptors (Bmax 2.3 +/- 0.4 pmol mg-1 protein; Kd 162 +/- 25 pmol L(-1)). Bosentan (10-5 M) again blocked all ET receptors in papillary membranes, thus confirming our previous finding that IMCD cells possess high-affinity ETB receptors mediating the diuretic effects of ET. Thus, in the porcine kidney the ET system may act in an autocrine/paracrine manner at the glomerular as well as at the IMCD level.     

Augmented expression of endothelin-1, endothelin-3 and the endothelin-B receptor in breast carcinoma

AIMS: Endothelins (ETs) are peptides expressed in many tumours which may stimulate angiogenesis and desmoplasia. Because ETs have not been extensively studied mammary neoplasia, we assessed ET protein and mRNA expression and receptor mRNA expression in normal and neoplastic breast tissues. METHODS AND RESULTS: Tissues from five normal breasts, six fibroadenomas, seven ductal carcinomas in situ (DCIS) and 25 invasive carcinomas were stained with anti-ET-1 and anti-ET-3 antibodies and analysed using a grading system. ET-1, ET-3, ETA and ETB mRNA expression was assessed by quantitative RT-PCR from eight carcinomas and five normals. Weak staining for ET-1 and ET-3 was detected in all normals. Moderate to strong staining was seen in 72% and 64% of carcinomas for ET-1 and ET-3, respectively. Most fibroadenomas showed weak positivity for ET-1 (83%) and ET-3 (67%). ET-1 and ET-3 mRNA levels were upregulated in carcinomas compared with normal breast. No ETA mRNA was not detected in any tissue. ETB mRNA was detected in normal breast and was increased in carcinomas. CONCLUSION: These results suggest that the ET system is altered in breast carcinomas and this may be of importance in the progression from in-situ to invasive carcinoma.