Expression pattern of endothelin system components and localization of smooth muscle cells in the human pre-ovulatory follicle.

BACKGROUND: Whether ovarian follicular rupture involves contractile activity or not has been debated for decades. Recently, study in the rodents has indicated that an endogenously produced potent vasoconstrictive peptide, endothelin-2 (EDN2), may induce follicular constriction immediately prior to ovulation. This study was aimed to systematically characterize the human ovarian endothelin system and localize smooth muscle cells to assess the possible involvement of contractile activity in human ovulation. METHODS: This is a prospective experimental study. Study subjects were 20 women aged 20-38 years who underwent IVF owing to tubal or male factors. Expression patterns of messenger RNAs (mRNAs) for EDN1, EDN2, EDN3, endothelin-converting enzyme-1 (ECE1 and ECE2), endothelin receptor A (ET(A)) and ET(B) in the granulosa cells (GCs) and cumulus cells and endothelin peptide concentration in the pre-ovulatory follicles were measured at 36 h after hCG injection. In addition, localization of ovarian smooth muscle cells and endothelin receptor expression were determined in normal (non-IVF patient) ovaries. RESULTS: Pre-ovulatory follicles express mRNA for EDN1 and EDN2, ECE1, ECE2, ET(A) and ET(B), but not EDN3, contain highly concentrated endothelin peptides (105.9 pg/ml) and are surrounded by theca externa that are made mostly of multicell layer non-vascular smooth muscle cells. ET(A) expression is localized in the smooth muscle cells of theca externa, theca interna and GC, whereas ET(B) expression is confined to theca interna. CONCLUSIONS: Pre-ovulatory follicles contain highly concentrated endothelins and are surrounded by non-vascular smooth muscle cells that express endothelin receptor, suggesting involvement of endothelin-induced contractile action in ovulation in the human ovary.

Vascular remodeling in the internal mammary artery graft and association with in situ endothelin-1 and receptor expression.

BACKGROUND: The vasoconstricting peptide endothelin-1 (ET-1) has been associated with atherosclerotic cardiovascular disease, vascular smooth muscle cell (VSMC) growth stimulation, and intimal thickening. ET-1 binds 2 receptor subtypes, endothelin A and B, and the ETA receptor mediates vasoconstriction and VSMC growth. This study aims to quantitatively assess arterial remodeling variables and compare them with changes in ET-1, ETA, and ETB expression in the internal mammary artery (IMA). METHODS AND RESULTS: Specimens from 55 coronary artery disease (CAD) patients (45 men, 10 women; mean age 65 years) and 14 control IMA specimens (from 7 men and 7 women; mean age 45 years) were collected. IMA cross sections were assessed by histochemical and immunohistochemical staining methods to quantify the levels of medionecrosis, fibrosis, VSMC growth, ET-1, ETA, ETB, and macrophage infiltration. The percentage area of medionecrosis in the patients was almost double that in the controls (31.85+/-14.52% versus 17.10+/-9.96%, P=0.0006). Total and type 1 collagen was significantly increased compared with controls (65.8+/-18.3% versus 33.7+/-13.7%, P=0.07, and 14.2+/-10.0% versus 4.8+/-2.8%, P=0.01, respectively). Despite ACE and/or statin therapy, ET-1 expression and cell cycling were significantly elevated in the patient IMAs relative to the controls (46.27+/-18.46 versus 8.56+/-8.42, P=0.0001, and 37.29+/-12.88 versus 11.06+/-8.18, P=0.0001, respectively). ETA and ETB staining was elevated in the patient vessels (46.88+/-11.52% versus 18.58+/-7.65%, P=0.0001, and 42.98+/-7.08% versus 34.73+/-5.20%, P=0.0067, respectively). A mild presence of macrophages was noted in all sections. CONCLUSIONS: Elevated distribution of collagen indicative of fibrosis coupled with increased cell cycling and high levels of ET-1 and ETA expression in the absence of chronic inflammation suggests altered IMA VSMC regulation is fundamental to the remodeling process.

Gene expression, localization, and characterization of endothelin A and B receptors in the human adrenal cortex.

Compelling evidence indicates that the endothelium-derived potent vasoconstrictor endothelin-1 (ET-1) stimulates aldosterone secretion by interacting with specific receptors. Although two different ET-1 receptors have been identified and cloned, the receptor subtype involved in mediating aldosterone secretion is still unknown. Accordingly, we wished to investigate whether the genes of ET-1 and of its receptors A and B are expressed in the normal human adrenal cortex. We designed specific primers for ET-1 and the ETA and ETB receptors genes and developed a reverse transcription polymerase chain reaction (RT-PCR) with chemiluminescent quantitation of the cDNA. In addition, we carried out 125I ET-1 displacement studies with cold ET-1, ET-3 and the specific ETA and ETB ligands BQ123 and sarafotoxin 6C. Localization of each receptor subtype was also investigated by autoradiography. Binding experiments were first individually analyzed by Scatchard and Hofstee plot and then coanalyzed by the nonlinear iterative curve fitting program Ligand. Histologically normal adrenal cortex tissue, obtained from kidney cancer patients (n = 7), and an aldosterone-producing adenoma (APA), which is histogenetically derived from the zona glomerulosa (ZG) cells, were studied. Results showed that the ET-1, ETA and ETB mRNA can be detected by RT-PCR in all adrenal cortices as well as in the APA. The best fitting of the 125I ET-1 displacement binding data was consistently provided by a two-site model both in the normal adrenal cortex (F = 22.1, P < 0.0001) and in the APA (F = 18.4, P < 0.0001). In the former the density (Bmax) of the ETA and ETB subtype was 2.6 +/- 0.5 pmol/mg protein (m +/- SEM) and 1.19 +/- 0.6, respectively. The dissociation constant (Kd) of ET-1, ET-3, S6C, and BQ-123 for each receptor subtype resulted to be within the range reported for human tissue for the ETA and ETB receptors. In the APA tissue the Bmax tended to be lower (1.33 and 0.8 pmol/mg protein, for the ETA and ETB, respectively) but the Kd were similar. Autoradiographic studies confirmed the presence of both receptor subtypes on the ZG as well as on APA cells. Thus, the genes of ET-1 and both its receptor subtypes ETA and ETB are actively transcribed in the human adrenal cortex. Furthermore, both receptor subtypes are translated into proteins in ZG and APA cells.

Osmolar regulation of endothelin signaling in rat renal medullary interstitial cells.

We tested the hypothesis that endothelin (ET) responsiveness in the renal medulla is modulated by ambient osmolarity. Cultured renal medullary interstitial cells (RMICs) were incubated from 3 to 24 h in isosmolar culture medium (300 mOsm/kg H2O) or media rendered hyperosmolar (600 mOsm/kg H2O) by the addition of urea. Under hyperosmolar conditions, the peak of ET-evoked Ca2+ transient was blunted by 45-58% (P < 0.02) and PGE2 accumulation decreased from 16- to 2-fold above basal values (P < 0.001). To explore whether hyperosmolar conditions blunt intracellular signaling via modulation of receptor number or expression, kinetics of ET binding and Northern blot analysis of ETA receptor mRNA was performed. Under hyperosmolar conditions, ETA receptor density was reduced by 84% versus isosmolar conditions (238 +/- 12 vs. 1450 +/- 184 fmol/mg) (P < 0.01). In contrast to the ligand binding studies, ETA receptor mRNA was increased by 58% (P < 0.05) in cells grown under hyperosmolar versus isosmolar media. These observations indicate that in the hyperosmolar setting, ET-evoked intracellular signaling is blunted in RMICs due to ET receptor downregulation. Since ETA receptor mRNA is increased under hyperosmolar conditions, we conclude that ET receptor downregulation is the consequence of either decreased translation of message, increased degradation of receptor peptide, or increased internalization of specific receptor sites.

Expression and localization of endothelin-1 and endothelin receptors in human meningiomas. Evidence for a role in tumoral growth.

In addition to its well-known homoeostatic actions in the cardiovascular system, ET-1 has been shown to constitute a potent growth regulatory peptide in various tissues. We have studied the expression of ET-1 and its receptors (ET-Ar and ET-Br) in human meningiomas (n = 35) as well as their involvement in cellular growth. By PCR of reverse-transcribed RNA we detected ET-1 mRNA in 91% (32 of 35), ET-Ar mRNA in 82% (29 of 35), and ET-Br mRNA in 42% (15 of 35) of human meningiomas examined. The localization of ET-1 mRNA, ET-Ar mRNA, and ET-1 peptide in tumoral cells was observed by in situ hybridization and immunohistochemistry, whereas ET-Br mRNA was expressed at low level only in cells belonging to blood vessels. In addition, we found that ET-1 stimulated [3H] thymidine incorporation in primary cell cultures of 20 meningiomas and that this effect could be blocked by BQ-123, a specific antagonist for ET-Ar. In contrast, RES-701-3, an antagonist of ET-Br, did not block the proliferative effect of ET-1. In conclusion, our data provide evidence that ET-1 constitutes an important growth factor for meningiomas acting via ET-Ar. We can hypothesize that ET-1, acting in concert with other growth factors and cytokines, is involved in the meningioma tumorigenesis.

Antagonism of endothelin action normalizes altered levels of VEGF and its signaling in the brain of stroke-prone spontaneously hypertensive rat.

Stroke-prone spontaneously hypertensive rats (SHRSP) often suffer from spontaneous stroke, in part, due to abnormalities in the cerebrovasculature. Here, we investigate the profile of key angiogenic factors and their basic signaling molecules in the brain of SHRSP during the age-dependent stages of hypertension. The profile of VEGF and its receptor, Flk-1, was dependent on age and stage of hypertension (i.e., down regulated at pre-hypertensive and malignant hypertensive stages, but up regulated at typical hypertensive stage), while that of its downstream components, pAkt and eNOS, were down regulated in a time-dependent manner in the frontal cortex of SHRSP compared to age-matched genetic control, normotensive WKY rats. On the other hand, the expression of endothelin-1 and its type A receptor (endothelin ETA receptor) were up regulated, depending on age and stage of hypertension. In contrast, levels of endothelin type B receptor were down regulated. The regional cerebral blood flow decreased during the development of malignant hypertension. Thus, subsequent experiments were designed to investigate whether endothelin-1 receptor antagonism, using endothelin-A/-B dual receptor antagonist SB209670, could normalize the molecular profile of these factors in SHRSP brain. Interestingly, blockage of endothelin-1 receptor restored to normal, levels of cerebral endothelin-1, endothelin ETA receptor and endothelin ETB receptor; VEGF and Flk-1; endothelial nitric oxide synthase (eNOS) and pAkt, in SHRSP, compared to age-matched WKY. Endothelin receptor blocker might be important to prevent the progression in the defect in VEGF and its angiogenic signaling cascade in the pathogenesis of hypertension-induced vascular remodeling in frontal cortex of SHRSP rats.

Endothelin-1 and its receptors on haemorrhoidal tissue: a potential site for therapeutic intervention.

BACKGROUND AND PURPOSE: Haemorrhoids is a common anorectal condition affecting millions worldwide. We have studied the effect of endothelin-1 (ET-1) and the role of endothelin ETA and ETB receptors in haemorrhoid tissue. EXPERIMENTAL APPROACH: Protein expression of ET-1, ETA and ETB receptors were compared between haemorrhoids and normal rectal submucosa using Western blot analysis, with the localization of proteins determined by autoradiography and immunohistochemistry. Effects of ET-1 and sarafotoxin 6a on human colonic and rectal arteries and veins was assessed by wire myography and the involvement of receptor subtypes established by selective antagonists. KEY RESULTS: Dense binding of [125 I]-ET-1 to haemorrhoidal sections was reduced by selective receptor antagonists. A higher density of ETB than ETA receptors was found in haemorrhoidal, than in control rectal tissue and confirmed by Western blot analysis. ETA and ETB receptors were localized to smooth muscle of haemorrhoidal arteries and veins, with ETB receptors on the endothelium. Human colonic and rectal arteries and veins were similarly sensitive to ET-1 and affected by the ETA selective antagonist, but sarafotoxin S6a-induced contractions were more pronounced in veins and antagonized by a selective ETB receptor antagonist. CONCLUSIONS AND IMPLICATIONS: ETA and ETB receptors are present in human haemorrhoids with ETB receptors predominating. ETA receptors are activated by ET-1 to mediate a contraction in arteries and veins, but the latter are selectively activated by sarafotoxin S6a - a response that involves ETB receptors at low concentrations. Selective ETB agonists may have therapeutic potential to reduce congestion of the haemorrhoidal venous sinusoids.

Emergence of smooth muscle cell endothelin B-mediated vasoconstriction in lambs with experimental congenital heart disease and increased pulmonary blood flow.

BACKGROUND: Endothelin-1 (ET-1) has been implicated in the pathophysiology of pulmonary hypertension. In 1-month-old lambs with increased pulmonary blood flow, we have demonstrated early alterations in the ET-1 cascade. The objective of this study was to investigate the role of potential later alterations of the ET cascade in the pathophysiology of pulmonary hypertension secondary to increased pulmonary blood flow. METHODS AND RESULTS: Eighteen fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt) and were studied 8 weeks after spontaneous delivery. Compared with age-matched control lambs, lung tissue ET-1 levels were increased in shunt lambs (317.2+/-113.8 versus 209.8+/-61.8 pg/g, P<0.05). In shunt lambs (n=9), exogenous ET-1 induced potent pulmonary vasoconstriction, which was blocked by the ETA receptor antagonist PD 156707 (n=3). This pulmonary vasoconstriction was mimicked by exogenous Ala1,3,11,15 ET-1 (4 Ala ET-1), the ETB receptor agonist, and was blocked by the ETB receptor antagonist BQ 788 (n=3). However, in control lambs (n=7), ET-1 and 4 Ala ET-1 did not change pulmonary vascular tone. In contrast to 4-week-old shunt lambs, immunohistochemistry revealed the emergence of ETB receptors on smooth muscle cells in the vasculature of 8-week-old shunt lambs. CONCLUSIONS: Over time, increased pulmonary blood flow and/or pressure results in the emergence of ETB-mediated vasoconstriction, which coincides with the emergence of ETB receptors on smooth muscle cells. These data suggest an important role for ETB receptors in the pathophysiology of pulmonary hypertension in this animal model of increased pulmonary blood flow.

Signalling and regulation of collagen I synthesis by ET-1 and TGF-beta1.

Endothelin-1 (ET-1) plays an important role in tissue remodelling and fibrogenesis by inducing synthesis of collagen I via protein kinase C (PKC). ET-1 signals are transduced by two receptor subtypes, the ETA- and ETB-receptors which activate different Galpha proteins. Here, we investigated the expression of both ET-receptor subtypes in human primary dermal fibroblasts and demonstrated that the ETA-receptor is the major ET-receptor subtype expressed. To determine further signalling intermediates, we inhibited Galphai and three phospholipases. Pharmacologic inhibition of Galphai, phosphatidylcholine-phospholipase C (PC-PLC) and phospholipase D (PLD), but not of phospholipase Cbeta, abolished the increase in collagen I by ET-1. Inhibition of all phospholipases revealed similar effects on TGF-beta1 induced collagen I synthesis, demonstrating involvement of PC-PLC and PLD in the signalling pathways elicited by ET-1 and TGF-beta1. ET-1 and TGF-beta1 each stimulated collagen I production and in an additive manner. ET-1 further induced connective tissue growth factor (CTGF), as did TGF-beta1, however, to lower levels. While rapid and sustained CTGF induction was seen following TGF-beta1 treatment, ET-1 increased CTGF in a biphasic manner with lower induction at 3 h and a delayed and higher induction after 5 days of permanent ET-1 treatment. Coincidentally at 5 days of permanent ET-1 stimulation, a switch in ET-receptor subtype expression to the ETB-receptor was observed. We conclude that the signalling pathways induced by ET-1 and TGF-beta1 leading to augmented collagen I production by fibroblasts converge on a similar signalling pathway. Thereby, long-time stimulation by ET-1 resulted in a changed ET-receptor subtype ratio and in a biphasic CTGF induction.

Endothelins and sarafotoxins: physiological regulation, receptor subtypes and transmembrane signaling.

The endothelins and sarafotoxins are two structurally related families of potent vasoactive peptides. Although the physiological functions of these peptides are not entirely clear, the endothelins are probably involved in pathophysiological conditions such as hypertension and heart failure. This review summarizes the state of the art in some areas of this intensively studied subject, including: (1) structure-function relationships of ET/SRTX, (2) ET concentrations in plasma, (3) ET/SRTX receptor subtypes and (4) signaling events mediated by the activation of ET/SRTX receptors.

Altered paracrine effect of endothelin in blood vessels of the hyperinsulinemic, insulin resistant obese Zucker rat.

OBJECTIVE: Earlier, we reported that high insulin incubation in vitro leads to increased ETA receptor expression in cultured rat aortic smooth muscle cells (Diabetes 1998, 47: 934-944). Our later observation of enhanced endothelin-1 evoked vasoconstriction in aorta from the hyperinsulinemic obese Zucker rat indicated that this interaction might also be relevant in vivo. To further examine the relationship between insulinemia and endothelin, we characterized endothelin receptor expression and endothelin-1 peptide levels in vascular tissues and plasma from young and old obese Zucker rats. METHODS: 12 and 40-week-old Zucker obese and lean rats were used. Plasma endothelin-1 levels and endothelin-1 peptide content in the mesenteric artery and in the thoracic aorta were examined by radioimmunoassay. Messenger RNA levels of endothelin-1 peptide and ETA and ETB receptors were examined in the aortic and mesenteric vessels using RT-PCR. RESULTS: Obese rats from both age groups had significantly higher plasma levels of insulin (4-10 fold), total cholesterol (2-3 fold), triglycerides (10-fold), and glucose (approximately 1.5 fold) than their lean counterparts. There was a trend toward worsening lipoproteinemia and glycemia, but improved insulinemia with age in the obese rats. In association with these changes, obese rats exhibited attenuated endothelin-1 peptide and preproET-1 mRNA levels, but conversely elevated ETA and ETB receptor mRNA levels in both aortic and mesenteric vessels. CONCLUSION: These data suggest that vascular tissue from the metabolically dysregulated obese Zucker rat exhibits attenuated endothelin-1 peptide production and elevated endothelin receptor levels. Since elevated insulin levels have been linked to increased endothelin receptor expression, it is plausible that hyperinsulinemia upregulates endothelin receptors contributing to elevated vasoconstrictor responses to endothelin-1 in this model of obesity and hypertension.

Activation of the cardiac endothelin system in left ventricular hypertrophy before onset of heart failure in TG(mREN2)27 rats.

OBJECTIVE: To characterize the cardiac angiotensin and endothelin (ET) system in compensated left ventricular hypertrophy due to long standing arterial hypertension and to assess the role of angiotensin and ET converting enzymes in mediating the observed changes of angiotensin and ET levels, respectively. METHODS: We studied the left ventricular renin-angiotensin system (RAS) and ET system in 20-week-old male transgenic hypertensive TG(mREN2)27 rats, a model of the monogenic renin-dependent form of severe hypertension. Age-matched Sprague-Dawley rats served as controls. RESULTS: TG(mREN2)27 rats exhibited left ventricular hypertrophy without signs of congestion. Transgene overexpression led to an activation of the tissue RAS with increased angiotensin II levels in spite of unchanged angiotensin converting enzyme (ACE) activity and ACE mRNA levels. ET-1 production was markedly increased in TG(mREN2)27 rats indicating that the ET-system was activated. Cardiac ET-1 in TG(mREN2)27 originated most likely from increased preproET-1 production because preproET-1 mRNA levels were increased but ET converting enzyme gene expression and activity were unchanged. Furthermore, ET-1 binding sites were significantly increased in TG(mREN2)27 rats without changes in K(D) values and ET(A)/ET(B) receptor ratios. ET(A) receptor gene expression was not altered whereas ET(B) receptor mRNA levels were up-regulated twofold in TG(mREN2)27 rats suggesting that ET(A) and ET(B) receptor expression may be regulated differentially. CONCLUSIONS: Cardiac ET and angiotensin systems are co-activated in compensated cardiac hypertrophy before onset of heart failure, and thus may be involved in the mechanism by which cardiac remodelling and progression of left ventricular dysfunction occur in TG(mREN2)27 rats.

Endothelin-1 and endothelin B receptor expression in pancreatic adenocarcinoma.

BACKGROUND: Endothelin-1 (ET-1) acting through endothelin A and B receptors (ETAR and ETBR) has been implicated in the development of cancer. The endothelin axis has not previously been characterised in human pancreatic adenocarcinoma (PAC). METHODS: Expression of ET-1, ETAR, ETBR, vascular endothelial growth factor and microvessel density (MVD) was determined by immunohistochemistry in 45 surgically resected human PACs and 15 non-cancer human pancreas samples. RESULTS: PAC had the highest staining intensity for ET-1 and ETBR: 38% PAC samples scored 2+ or more compared with 7% non-cancer sample in ET-1; 58% PAC samples scored 2+ compared with 0% non-cancer samples in ETBR. MVD was significantly lower in PAC compared with non-cancer tissue (p<0.0001). CONCLUSIONS: PAC was characterised by greater expression of ET-1 and ETBR compared with normal pancreas.

Effect of endothelin-1 on corticosteroid secretion by the frog adrenal gland is mediated by an endothelinA receptor.

We have previously reported that endothelin-1 (ET-1) stimulates the in vitro secretion of corticosterone and aldosterone from the adrenal gland of the frog Rana ridibunda. The aim of the present study was to investigate the pharmacological profile of the endothelin receptor subtype involved in the corticotropic effect of ET-1. The mixed ET(A)/ET(B) receptor antagonist Ro 47-0203 (10(-5) M) totally blocked the stimulatory effect of ET-1 (5 x 10(-9) M) on corticosterone and aldosterone secretion. The action of ET-1 was also inhibited by the selective ET(A) receptor antagonist BQ-485 (10(-7) M). In contrast, the selective ET(B) receptor antagonist IRL 1038 (10(-6) M) did not affect the response of the frog adrenal gland to ET-1. In addition, the selective ET(B) receptor agonist IRL 1620 (10(-6) M) did not mimic the stimulatory effect of ET-1. The high affinity ET(C) receptor agonist endothelin-3 (ET-3) stimulated corticosteroid secretion, but was 400 times less potent than ET-1. Moreover, the action of ET-3 was also blocked by BQ-485 (10(-7) M). These data indicate that the stimulatory effects of ET-1 and ET-3 on corticosteroid secretion by the frog adrenal gland are mediated by an ET(A) receptor subtype.

Endothelin activates large-conductance K+ channels in rat lactotrophs: reversal by long-term exposure to dopamine agonist.

Endothelin-1 (ET-1) inhibits PRL secretion from cultured rat lactotrophs. However, ET-1 stimulates PRL secretion after cultured lactotrophs have been exposed for 48 h to dopamine or D2 dopamine agonists. In the present study, we have used cell-attached and inside-out patch recordings to establish an ionic basis for these effects. Bath application of 20 nM ET-1 to untreated lactotrophs evoked a robust and persistent activation of large-conductance K+ channels in cell-attached patches. This effect of ET-1 had a long latency to onset, was maintained for as long as ET-1 was present, and required at least 10 min of washing in control saline before complete recovery was achieved. The stimulatory effect of 20 nM ET-1 on these channels was markedly attenuated in the presence of the selective ET(A) receptor antagonist BQ-610 (200 nM), or after pertussis toxin (200 ng/ml, 16 h) pretreatment. The unitary slope conductance of the ET-1 activated channels in cell attached patches was 165 and 95 pS when the recording electrodes contained 150 and 5.4 mM KCl, respectively. These channels were voltage-sensitive and their activity increased upon patch depolarization. Previously activated channels in cell-attached patches became quiescent immediately upon patch excision into Ca2+-free bath saline. Exposure of the intracellular surface to 0.1 microM Ca2+ restored the activity of these channels similar to the level seen before patch excision. In addition, preincubating the cells with the membrane-permeable Ca2+-chelator BAPTA-AM, or using Ca2+-free solution in the recording pipettes, prevented the activation of these channels by ET-1. The ET-1 activated large-conductance Ca2+-dependent K+ (BK(Ca)) channels were blocked by 20 mM tetraethylammonium but were insensitive to the K+ channel blockers apamin (1 microM), charybdotoxin (200 nM), or iberiotoxin (200 nM). Acute application of 10 microM dopamine and 20 nM ET-1 caused activation of BK(Ca) channels with indistinguishable kinetic properties, although the effect of dopamine occurred with shorter latency. After 48-h exposure to the specific D2 dopamine receptor agonist (+/-)-2-(N-phenyl-N-propyl) amino-5-hydroxytetralin hydrochloride (PPHT, 500 nM), bath application of 20 nM ET-1 resulted in inhibition of spontaneously active BK(Ca) channels. These data suggest that both the stimulatory and inhibitory effects of ET-1 on PRL secretion are mediated, at least in part, by actions on BK(Ca) channels, and that long term exposure to dopamine or D2 agonists alters the signaling pathways from the ET(A) receptor to BK(Ca) channels.

Autocrine-paracrine role of endothelin-1 in the regulation of aldosterone synthase expression and intracellular Ca2+ in human adrenocortical carcinoma NCI-H295 cells.

The role played by endothelin (ET-1) and its receptor subtypes A and B (ET(A) and ET(B)) in the functional regulation of human NCI-H295 adrenocortical carcinoma cells has been investigated. Reverse transcription-PCR with primers specific for prepro-ET-1, human ET-1 converting enzyme-1, ET(A), and ET(B) complementary DNAs consistently demonstrated the expression of all genes in NCI-H295 cells. The presence of mature ET-1 and both its receptor subtypes was confirmed by immunocytochemistry and autoradiography, respectively. Aldosterone synthase (AS) messenger RNA was also detected in NCI-H295 cells, and AS gene expression was enhanced by both ET-1 and the specific ET(B) agonist IRL-1620; this effect was not inhibited by either the ET(A) antagonist BQ-123 or the ET(B) antagonist BQ-788. A clear-cut increase in the intracellular Ca2+ concentration in NCI-H295 cells in response to ET(B), but not ET(A), activation was observed. In light of these findings, the following conclusions can be drawn: 1) NCI-H295 cells possess an active ET-1 biosynthetic pathway and are provided with ET(A) and ET(B) receptors; 2) ET-1 regulates in an autocrine/paracrine fashion the secretion of aldosterone by NCI-H295 cells by enhancing both AS transcription and raising the intracellular Ca2+ concentration; and 3) the former effect of ET-1 probably involves the activation of both receptor subtypes, whereas calcium response is exclusively mediated by the ET(B) receptor.

Endothelin-1-selective receptor in the arterial intima of patients with hypertension.

Endothelin-1 is a potent vasoconstrictor produced by vascular endothelial cells. A recently cloned endothelin-1-selective receptor, the endothelin-A receptor, mediates the vasoconstrictive action of endothelin-1. Because endothelin-1 also possesses mitogenic properties, it may play a role in regulating the proliferation of intimal smooth muscle cells. In this study, we analyzed the expression of endothelin-A receptor gene in the thickened arterial intima of patients with hypertension. Internal mammary artery specimens obtained from 12 patients undergoing cardiovascular surgery were subjected to in situ hybridization using a digoxigenin-labeled cRNA probe. High, homogeneous signals of endothelin-A receptor mRNA were observed in the medial smooth muscle cells of all vessels examined but not in the endothelial cells. Patients with hypertension displayed more severe intimal thickening than those without hypertension. Immunohistochemical analysis suggested that almost all of the intimal proliferative cells originated from smooth muscle cells. In contrast to media, endothelin-A receptor mRNA signals in intimal smooth muscle cells were low and heterogeneous. In the thickened arterial intima of hypertensive patients, the signals were detected just beneath the luminal endothelium but not deep in the intimal smooth muscle cell layer. By contrast, staining with anti-alpha-smooth muscle actin antibody was more intense in the deep layer than in the subendothelium. These findings suggest that the modulation of endothelin-A receptor gene expression in smooth muscle cells differs between the intima and media. Its regulated expression in intimal smooth muscle cells might affect the proliferative activity of these cells in patients with hypertension.

The effect of type 1 diabetes mellitus on vascular responses to endothelin-1 in pregnant women.

Diabetes is associated with vascular dysfunction, which may be due in part to altered vascular responses to endogenous peptides such as endothelin-1. These altered responses may also contribute to the decreased maternal peripheral resistance in pregnancy. The aim of this study was to examine the effect of diabetes on the vasoconstrictor response to endothelin-1 in pregnant women. Small arteries were isolated from nine healthy pregnant, seven type 1 diabetic pregnant women, and five healthy nonpregnant women. Contraction curves were performed on a wire myograph for noradrenaline (1 nM to 30 microM) and endothelin-1 (1 pM to 0.3 microM). Maximum responses and sensitivity were compared by t test. No differences in maximum response to noradrenaline or potassium were seen among the three groups. The maximum response to endothelin-1 was significantly increased in pregnancy (P < 0.05), whereas endothelin-1 sensitivity was reduced in the diabetic compared with the nondiabetic pregnant women (P < 0.05). Pregnant women have an increased maximum vasoconstriction response to endothelin-1 compared with nonpregnant women, whereas diabetic pregnant women demonstrate reduced sensitivity to endothelin-1. These observations suggest that endothelin-1 may play a role in maintaining peripheral vascular tone in normal pregnancy, and the decreased sensitivity seen in pregnant women with diabetes may reflect abnormal vascular reactivity.

Endothelin-1 and its receptor in hypertrophic cardiomyopathy.

Endothelin-1, a potent vasoconstrictor produced by vascular endothelial cells, activates the hypertrophic program in cultured heart muscle cells. However, the role of endothelin-1 in cardiac hypertrophy in humans is unknown. Therefore, we studied hypertrophic cardiomyopathy patients with normal pulmonary arterial pressure, in whom cardiac hypertrophy is a specific feature of the disease. Radioimmunoassay with a monoclonal antibody to human endothelin-1 showed that the plasma level of immunoreactive endothelin was more than twofold higher in hypertrophic cardiomyopathy patients than in control subjects (P < .005). In situ hybridization analysis of endomyocardial biopsy specimens showed positive signals of endothelin-1 type A receptor mRNA in ventricular myocytes of all specimens. The receptor expression in ventricular myocytes was similar between hypertrophic cardiomyopathy patients and control subjects. We propose that endothelin-1 might represent an important factor involved in hypertrophic cardiomyopathy. Whether endothelin-1 plays a causal role in cardiac hypertrophy or is a marker of its occurrence needs to be clarified.

Biochemical characterization and autoradiographic localization of [125I]endothelin-1 binding sites on trophoblast and blood vessels of human placenta.

The presence of endothelin binding sites in the human placenta raises the question of the precise localization of these receptors on well defined placental constituents. In order to find an answer to this problem various approaches were used. Specific binding sites for [125I] endothelin-1 (ET-1) were identified on human term placenta, not only on membranes of smooth muscles stem villi vessels, but also on trophoblastic plasma membranes prepared from trophoblast in culture. Scatchard analysis of binding data revealed a single class of high affinity binding sites with Kd values of 26 +/- 4 pmol/L for stem villi vessels and 126 +/- 4 pmol/L for trophoblast in culture, with maximum binding capacities of 681 +/- 61 and 224 +/- 53 fmol/mg protein, respectively. The anatomical localization of these binding sites was determined by in vitro autoradiography. Autoradiograms obtained from placental sections incubated with [125I]ET-1 indicate that [125I]ET-1 high affinity binding sites exist on placental stem villi vessels and on the trophoblastic layer of the villi. The latter localization was also found on autoradiograms of trophoblast in culture. The human placental syncytiotrophoblast is a polarized epithelium with the microvillous membrane, facing maternal blood space and the basal plasma membrane, facing fetal circulation. [125I]ET-1 high affinity binding sites are present on both membranes but the number of binding sites is higher on the basal plasma membrane. These findings lead to the suggestion that ET-1 may be involved in the regulation of the feto-placental circulation and may subserve specific trophoblastic functions.