Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.     

Regulated interaction of endothelin B receptor with caveolin-1.

The peptide hormone endothelin transmits various signals through G protein-coupled receptors, the endothelin type A (ETAR) and B (ETBR) receptors. Caveolae are specialized lipid rafts containing polymerized caveolins. We examined the interaction of ETBR with caveolin-1, expressed in Sf9, COS-1, and HEK293 cells, and its effects on the subcellular distribution and the signal transduction of ETBR. ETBR formed a complex with caveolin-1 in cells in which these two proteins were coexpressed and in the mixture after purification and reconstitution (as examined by immunoprecipitation) suggesting the direct binding of ETBR with caveolin-1. The complex formed efficiently only when the ETBR was ligand-free or bound to an antagonist, RES-701-1, whereas the addition of ET-1 or another antagonist, BQ788, dissociated the complex, suggesting the structural recognition of ETBR by caveolin-1. In contrast, the ETAR bound to caveolin-1 regardless of ligand binding. Caveolin-1 utilized its scaffolding domain (residues 82-101) and the C-terminal domain (residues 136-178) to bind to ETBR, as for other signalling molecules. Furthermore, the amount of ETBR localized in caveolae increased significantly with the expression of caveolin-1 and decreased with the addition of ET-1. The disruption of caveolae by filipin reduced the ET-1-derived phosphorylation of ERK1/2. These results suggest the possibility that the binding to caveolin-1 retains the ligand-free ETBR in caveolae and regulates the ET signal.     

A reversible radioligand specific for the ETB receptor: [125I]Tyr13-Suc-[Glu9,Ala11,15]-endothelin-1(8- 21), [125I]IRL 1620.

Suc-[Glu9,Ala11,15]-endothelin(ET)-1(8-21), IRL 1620, is a linear ET-analog specific for the ET-isopeptide-nonselective ETB receptor. The radio-iodinated analog, [125I]IRL 1620, showed a single class of saturable binding to the ETB receptors in porcine lung membranes with a Kd of 18 pM and a Bmax of 930 fmol/mg protein, which are almost comparable to the values obtained with [125I]ET-3 (6 pM and 900 fmol/mg protein). In competitive binding assays with [125I]IRL 1620, unlabeled ET-1, ET-3, IRL 1620 and [monoiodo-Tyr13]-IRL 1620 showed almost identical displacement curves with Ki of 8 to 16 pM. However, [125I]IRL 1620 was dissociated from the binding sites by addition of an excess amount (100 nM) of any of these unlabeled peptides, each with the same t1/2 of 100 min. This was in marked contrast to [125I]ET-3 which was hardly dissociated from the binding sites.     

Expression of endothelin 1 and endothelin A receptor in HPV-associated cervical carcinoma: new potential targets for anticancer therapy.

Human papillomaviruses (HPV) are associated with cervical cancer and interact with growth factors that may enhance malignant transformation of cervical carcinoma cells. Endothelin-1 (ET-1) is released from HPV transfected keratinocytes and induces increased growth response in these cell lines in comparison with normal cells. In the present study several cervical carcinoma cell lines have been analyzed to investigate the expression of ET-1 and its receptors as well as their involvement in tumor growth. All HPV-positive cancer cells secreted ET-1 and expressed mRNA for ET-1 and its receptors, whereas a HPV-negative carcinoma cell line expressed only the ETBR mRNA and didn't secrete ET-1. Binding studies showed that HPV-associated cells expressed an increased number of functional ETAR. ET-1 stimulated a marked dose-dependent increase in [3H]-thymidine incorporation with respect to the normal cells whereas ET-3 and ETBR agonists had no effect. In HPV-positive cancer cells, a specific antagonist of ETAR inhibited the proliferation induced by ET-1 and substantially reduced the basal growth rate of unstimulated cervical tumor cells, whereas the ETBR antagonist had no effect. These results demonstrate that ET-1 participates in the progression of neoplastic growth in HPV-associated carcinoma, in which ETAR are increased and could be targeted for antitumor therapy.     

Transforming growth factor-β regulates endothelin-1 signaling in the newborn mouse lung during hypoxia exposure

We have previously shown that inhibition of transforming growth factor-β (TGF-β) signaling attenuates hypoxia-induced inhibition of alveolar development and abnormal pulmonary vascular remodeling in the newborn mice and that endothelin-A receptor (ETAR) antagonists prevent and reverse the vascular remodeling. The current study tested the hypothesis that inhibition of TGF-β signaling attenuates endothelin-1 (ET-1) expression and thereby reduces effects of hypoxia on the newborn lung. C57BL/6 mice were exposed from birth to 2 wk of age to either air or hypoxia (12% O(2)) while being given either BQ610 (ETAR antagonist), BQ788 (ETBR antagonist), 1D11 (TGF-β neutralizing antibody), or vehicle. Lung function and development and TGF-β and ET-1 synthesis were assessed. Hypoxia inhibited alveolar development, decreased lung compliance, and increased lung resistance. These effects were associated with increased TGF-β synthesis and signaling and increased ET-1 synthesis. BQ610 (but not BQ788) improved lung function, without altering alveolar development or increased TGF-β signaling in hypoxia-exposed animals. Inhibition of TGF-β signaling reduced ET-1 in vivo, which was confirmed in vitro in mouse pulmonary endothelial, fibroblast, and epithelial cells. ETAR blockade improves function but not development of the hypoxic newborn lung. Reduction of ET-1 via inhibition of TGF-β signaling indicates that TGF-β is upstream of ET-1 during hypoxia-induced signaling in the newborn lung.     

Endothelin Signaling Pathways in Rat Adrenal Medulla

1. We further characterized the effect of endothelins (ETs) on receptor-mediated phosphoinositide (PI) turnover, nitric oxide synthase (NOS) activation, and cGMP formation in whole rat adrenal medulla.2. The PI hydrolysis was assessed as accumulation of inositol monophosphates (InsP₁) in the presence of 10 mM LiCl in whole tissue and the analysis of inositol-1-phosphate by Dowex anion exchange chromatography. NOS activity was assayed by monitoring the conversion of radiolabeled L-arginine to L-citrulline. Cyclic GMP formation was assessed as accumulation of cGMP in whole tissue in the presence of phosphodiesterase inhibition, and the amount of cGMP formed was determined by radioimmuno-antibody procedure.3. ET-1 and ET-3 increased PI turnover by 30% in whole adrenal medulla prelabeled with [³H] myoinositol. Both ETs isoforms, at equimolar doses, increased NOS activity and cGMP levels in similar degree. The selective ET_B receptor agonist, IRL-1620, also increased cGMP formation, mimicking the effects of ETs, while IRL-1620 did not alter the PI metabolism. ETs-induced InsP₁ accumulation and cGMP was dependent on extracellular calcium. The effect of ETs on PI turnover was inhibited by neomycin. The L-arginine analogue, N-nitro-L-arginine (L-NAME), and two inhibitors of soluble guanylyl cyclase, methylene blue and ODQ, significantly inhibited the increase in cGMP production induced by ETs or IRL-1620. The selective ET_A receptor antagonist, BQ 123, inhibited the ETs-induced increase in PI turnover, while the selective ET_B receptor antagonist, BQ 788, was ineffective. Likewise, BQ 788, significantly inhibited ET-1- or ET-3-induced NOS activation and cGMP generation but not ETs-induced InsP₁ accumulation.4. Our data indicate that stimulation of PI turnover and NO-induced cGMP generation constitutes ETs signaling pathways in rat adrenal medulla. The former action is mediated through activation of ET_A receptor, while the latter through the activation of ET_B receptor. These results support the role of endothelins in the regulation of adrenal medulla function.     

A novel radioligand [125I]BQ-3020 selective for endothelin (ETB) receptors.

A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeu-AspIleIleTrp (BQ-3020), is highly selective for ETB receptors. BQ-3020 displaces [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC50: 0.2nM) at a concentration 4,700 times lower than that to ETA receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ETA antagonist BQ-123 failed to inhibit this BQ-3020-induced vasoconstriction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ETB agonistic activity. The radioligand [125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd = 34.4pM), whereas it scarcely binds to VSMC. [125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC50: 0.07-0.17nM). However, the binding of [125I]BQ-3020 was insensitive to the ETA antagonist BQ-123 and other bioactive peptides. Both [125I]ET-1 and [125I]BQ-3020 show slow onset and offset binding kinetics to ETB receptors. These data indicate that the radioligand [125I]BQ-3020 selectively labels ETB receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu6 to Trp21, but not on the structure formed by its two disulfide bridges.     

Localization of endothelin-A and -B receptors during the postnatal development of rat cerebellum

Intense expression of mRNA of endothelin-B receptor (ETBR) has been detected in the Bergmann glia of cerebellum by in situ hybridization, but the intracellular localization has not been reported because of the absence of a useful antibody for immunohistochemical investigations. We made polyclonal antibodies against the carboxyl terminus of human ETBR (420-442) and ETAR (403-427), and performed light- and electron-microscopic immunohistochemistry of the wild-type and ETBR-deficient (sl/sl) rat cerebella. Localization of ETBR during postnatal development was examined by double-staining immunofluorescence using antibodies against ETBR and S-100 beta. In the wild-type rats, ETBR immunoreactivity appeared from postnatal day 5 (P5) and was distributed diffusely in the processes and cell bodies of S-100 beta-positive glial cells. By P14, ETBR immunoreactivity was concentrated in the Golgi apparatus of Bergmann glial cell soma and the plasma membrane of its processes. The ETBR-positive astrocytes in the granular layer decreased in number during P7-14 and had disappeared by week 3. At 3 weeks, ETBR immunoreactivity was restricted to the Golgi apparatus of Bergmann glia. In the sl/sl rats, ETBR immunoreactivity was not observed at all. In contrast to ETBR, ETAR immunoreactivity appeared transiently in the cytoplasm of all astrocytes (Bergmann glia and astrocytes in the granular layer) in the 9- to 14-day-old wild rats and 7- to 14-day-old sl/sl rats, and disappeared within 3 weeks in both. Granule cells did not express immunoreactivity for ETBR and ETAR from the neonatal stage to adulthood. Changes in the intracellular localization of ETBR and transient expression of ETAR may be correlated with the changes of glial functions and proliferation during postnatal development of rat cerebellum.     

The potassium channel opener BRL 38227 inhibits binding of [125I]-labelled endothelin-1 to rat cardiac membranes.

Binding of [125I]-labelled endothelin-1 (ET-1) to rat cardiac membranes and the effects of endothelin-1 (ET-1), endothelin-3 (ET-3), the calcium channel antagonist nifedipine, and both enantiomers of the potassium channel opener cromakalim (BRL 34915) on binding have been examined. Specific binding of [125I]-ET-1 was inhibited in a concentration dependent manner by both unlabelled ET-1 (10(-12)-10(-7) M) and ET-3 (10(-12)-10(-6) M). Nifedipine (10(-11)-10(-5) M) did not affect [125I]-ET-1 binding. However, BRL 38227 (10(-11)-10(-5) M), the biologically active isomer of cromakalim, significantly inhibited [125I]-ET-1 binding. The inactive isomer, BRL 38226 (10(-11)-10(-5) M) had no effect. These findings provide the first evidence for a stereospecific interaction between BRL 38227 and an ET-1 binding site in rat cardiac membranes.     

Identification of a binding domain of the endothelin-B receptor using a selective IRL-1620-derived photoprobe

On the basis of the structure of IRL-1620, a specific agonist of the endothelin-B receptor subtype (ET(B)), a few photosensitive analogues were developed to investigate the binding domain of the receptor. Among those, a derivative containing the photoreactive amino acid, p-benzoyl-l-phenylalanine in position 5 showed, as assessed with endothelin-A (ET(A)) and ET(B) receptor paradigms, pharmacological properties very similar to those of IRL-1620. The binding capacity of the probe was also evaluated on transfected Chinese hamster ovary (CHO) cells overexpressing the human ET(B) receptor. Data showed that binding of the radiolabeled peptide was inhibited by ET-1 and IRL-1620. Therefore, this photolabile probe was used to label the ET(B) receptor found in CHO cells. Photolabeling produced a ligand-protein complex appearing on SDS-PAGE at around 49 kDa. An excess of ET-1 or IRL-1620 completely abolished the formation of the complex, showing the selectivity of the photoprobe. Digestions of the [Bpa(5),Tyr((125)I)(6)]IRL-1620-ET(B) complex were carried out, and receptor fragments were analyzed to define the region of the receptor where the ligand interacts. Results showed that Endo Lys-C digestion gave a 3.8-kDa fragment corresponding to the Asp(274)-Lys(303) segment, whereas migration after V8 digestion revealed a fragment of 4.6 kDa. Because the fragments of these two digestions must overlap, the latter would be the Trp(275)-Asp(313) stretch. A cleavage with CNBr confirmed the identity of the binding domain by giving a fragment of 3.6 kDa, corresponding to Gln(267)-Met(296). Thus, the combined cleavage data strongly suggested that the agonist binding domain of ET(B) includes a portion of the fifth transmembrane domain, between residues Trp(275) and Met(296).     

Nonpeptidic antagonists of ETA and ETB receptors reverse the ET-1-induced sustained increase of cytosolic and nuclear calcium in human aortic vascular smooth muscle cells

Our previous work showed that ET-1 induced a concentration-dependent increase of cytosolic Ca2+ ([Ca]c) and nuclear Ca2+ ([Ca]n) in human aortic vascular smooth muscle cells (hVSMCs). In the present study, using hVSMCs and 3-dimensional confocal microscopy coupled to the Ca2+ fluorescent probe Fluo-3, we showed that peptidic antagonists of ETA and ETB receptors (BQ-123 (10(-6) mol/L) and BQ-788 (10(-7) mol/L), respectively) prevented, but did not reverse, ET-1-induced sustained increase of [Ca]c and [Ca]n. In contrast, nonpeptidic antagonists of ETA and ETB (respectively, BMS-182874 (10(-8)-10(-6) mol/L) and A-192621 (10(-7) mol/L)) both prevented and reversed ET-1-induced sustained increase of [Ca]c and [Ca]n. Furthermore, activation of the ETB receptor alone using the specific agonist IRL-1620 (10(-9) mol/L) induced sustained increases of [Ca]c and [Ca]n, and subsequent administration of ET-1 (10(-7) mol/L) further increased nuclear Ca2+. ET-1-induced increase of [Ca]c and [Ca]n was completely blocked by extracellular application of the Ca2+ chelator EGTA. Pretreatment with the G protein inhibitors pertussis toxin (PTX) and cholera toxin (CTX) also prevented the ET-1 response; however, strong membrane depolarization with KCl (30 mmol/L) subsequently induced sustained increase of [Ca]c and [Ca]n. Pretreatment of hVSMCs with either the PKC activator phorbol-12,13-dibutyrate or the PKC inhibitor bisindolylmaleimide did not affect ET-1-induced sustained increase of intracellular Ca2+. These results suggest that both ETA- and ETB-receptor activation contribute to ET-1-induced sustained increase of [Ca]c and [Ca]n in hVSMCs. Moreover, in contrast to the peptidic antagonists of ET-1 receptors, the nonpeptidic ETA-receptor antagonist BMS-182874 and the nonpeptidic ETB-receptor antagonist A-192621 were able to reverse the effect of ET-1. Nonpeptidic ETA- and ETB-receptor antagonists may therefore be better pharmacological tools for blocking ET-1-induced sustained increase of intracellular Ca2+ in hVSMCs. Our results also suggest that the ET-1-induced sustained increase of [Ca]c and [Ca]n is not mediated via activation of PKC, but via a PTX- and CTX-sensitive G protein calcium influx through the R-type Ca2+ channel.     

Upregulation of endothelin-1 binding in tissues of salt-loaded stroke-prone spontaneously hypertensive rats

Upon maintained on a 1% NaCl drinking solution beginning at 7 weeks of age, the stroke-prone spontaneously hypertensive rat (SHRsp) developed severe hypertension and stroke; most died by 16 weeks. The mechanism by which these diseases evolve remains unclear. Endothelin-1 (ET-1) is a potent, peptidic vasoconstrictor and is implicated in the pathogenesis of various cardiovascular, renal, and central nervous system diseases. The purpose of the present study was to compare the binding of [125I]ET-1 to the brain, heart, kidney, liver, and spleen membrane preparations of 16-week-old SHRsp and age-matched normotensive Wistar-Kyoto rats (WKY). The KD values for [125I]ET-1 binding to the corresponding tissues of the two strains were not significantly different, except in the brain (SHRsp: 17 +/- 1 pM; WKY: 24 +/- 1 pM). In contrast, the Bmax values measured in the brain, heart, kidney, and liver of SHRsp were 1.5- to 2.1-fold greater than those of their WKY counterparts. Competition of [125I]ET-1 binding to the membrane preparations by the specific ETA receptor antagonist BQ-123 or the specific ETB receptor agonist sarafotoxin S6c revealed a similar proportion of ETA and ETB receptor subtypes in the corresponding tissues of the two rat strains. These results indicate that ET-1 binding is upregulated in SHRsp and suggest that ET-1 may play a pathophysiological role in this animal model of genetic hypertension.     

The inhibitory effect of [D-Arg1,D-Phe,D-Try7,9,Leu11] substance P on endothelin-1 binding sites in rat cardiac membranes.

The specific binding of [125I]ET-1 to rat cardiac membrane fragments was inhibited by [D-Arg1,D-Phe, D-Try7,9,Leu11] substance P [substance P(D)], a potent bombesin antagonist. This inhibitory effect required high concentrations (greater than 3X10(-6)M) of substance P(D) and was accompanied by a steep increase in non-specific binding, and not a decrease in total binding. Such results indicate that substance P(D) does not competitively inhibit the specific binding of [125I]ET-1 to rat cardiac membrane fragments.     

High affinity interaction of endothelin-3 with recombinant ETA receptors

Pharmacological evidence has suggested that endothelin-3 (ET-3) may act via a novel form of ET receptor that is shared by ETA receptor antagonists but not by ETB receptor selective agonists. This study analyses the properties of interaction of ET-3 with recombinant bovine ETA receptor. Apparent Kd(ET-3) values as low as 50 nM were defined from [125I]ET-1 binding experiments performed at low (5 microg/ml) protein concentrations in the assays. Larger (up to 1 microM) values were artefactually obtained in experiments performed at larger protein concentrations. The three monoiodo ET-3 derivatives were synthetized. ([125I]Y14)ET-3 did not recognize ETA receptors. ([125I]Y6)ET-3 labelled 18% of [125I]ET-1 binding sites with a Kd value of 320 pM. ([125I]Y13)ET-3 labelled 44% of [125I]ET-1 binding sites with a Kd value of 130 pM. High affinity ([125I]Y6)ET-3 and ([125I]Y13)ET-3 bindings were prevented by ET-1 (Kd = 5-7 pM), ET-3 (Kd = 70-250 pM), BQ-123 (Kd = 2 nM) and FR139317 (Kd = 2 nM) but not by low concentrations of 4-AlaET-1, sarafotoxin S6c or IRL1620. The three monoiodo ET-3 derivatives bound to recombinant rat ETB receptors with a pM affinity. The results suggest that ET-3, ([125I]Y6)ET-3 and ([125I]Y13)ET-3 should not be considered as ETB receptor specific ligands.     

Endothelin receptors in human parathyroid gland.

We studied whether specific receptors for endothelins (ETs) exist in human parathyroid tissues and whether ETs may have any effect on secretion of PTH from parathyroid cells. Binding studies using [125I]ET-1 to the parathyroid membranes obtained from patients with hyperparathyroidism (2 adenomas, 2 hyperplasias) revealed that ET-1 competitively inhibited the binding of [125I]ET-1 to the membranes (the apparent Kd: 62 +/- 18 pM), whereas ET-3 showed biphasic and less steep inhibition curve than ET-1 in all tissue membranes examined. Northern blot analysis of poly(A)+ RNA from the parathyroid adenoma clearly demonstrated gene expression of both ETA and ETB receptors as well as preproET-1. ET-1 inhibited basal PTH secretion from dispersed adenoma cells more potently than ET-3. The present study clearly demonstrates the presence of both ETA and ETB receptor subtypes in human parathyroid tissues through which ETs may modulate PTH secretion in an autocrine and/or paracrine manner.     

Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor.

We describe novel potent endothelin (ET) antagonists that are highly potent and selective for the ETA receptor (selective to ET-1). Of the synthetic analogs based on ETA antagonist BE-18257A isolated from Streptomyces misakiensis (IC50 value for ETA receptor on porcine aortic smooth muscle cells (VSMCs); 1.4 microM), the compounds BQ-123 and BQ-153 greatly improved the binding affinity of [125I]ET-1 for ETA receptors on VSMCs (IC50; 7.3 and 8.6 nM, respectively), whereas they barely inhibited [125I]ET-1 binding to ETB receptors (nonselective with respect to isopeptides of ET family) in the cerebellar membranes (IC50; 18 and 54 microM, respectively). Associated with the increased affinity for ETA receptors, these peptides antagonized ET-1-induced constriction of isolated porcine coronary artery. However, there was a small amount of ET-1-induced vasoconstriction resistant to these antagonists, which paralleled the incomplete inhibition of [125I]ET-1 binding in the membrane of the aortic smooth muscle layer. These data suggest that the artery has both ETA and ETB receptors responsible for ET-1-induced vasoconstriction. The antagonists shifted the concentration-response curve to the right for ET-1 in the coronary artery, and increased the apparent dissociation constant in the Scatchard analysis of [125I]ET-1 binding on the VSMCs without affecting the binding capacity, indicative of the competitive antagonism for ETA receptor. In conscious rats, pretreatment with the antagonists markedly antagonized ET-1-induced sustained pressor responses in dose-dependent fashion without affecting ET-1-induced transient depressor action, suggesting that the pressor action is mediated by ETA receptors, while the depressor action is mediated by ETB receptors. In addition, pretreatment with the potent antagonists prevented ET-1-induced sudden death in mice. Thus, these potent ETA antagonists should provide a powerful tool for exploring the therapeutic uses of ETA antagonists in putative ET-1-related disorders.     

Endothelin-I induces gene expression through stimulation of endothelin type a receptors in normal rat kidney cells

RNA blots of total cellular RNA isolated from quiescent and endothelin (ET-1)-stimulated normal rat kidney (NRK) cells demonstrated that ET-1 induced the expression of c-jun, jun B, and c-fos mRNA in a time-dependent manner with maximal expression of mRNA by 1 hr after the addition of ET-1. Five hundred picomolal ET-1 was sufficient to induce maximal mRNA expression. These data agreed with saturation experiments which demonstrated that maximal binding of [¹²⁵I]ET-1 was achieved at concentrations greater than 100 pM. The K_d and B_max values for [¹²⁵I]ET-1 binding to NRK membranes were 20.5 pM and 22.2 fmol/mg protein, respectively. Competition experiments for the binding of [¹²⁵I]ET-1 to NRK membranes demonstrated that ET-1 was a more potent inhibitor (K_i = 0.047 nM) than ET-3 (K_i = 10.8 nM). No specific binding of [¹²⁵I]ET-3 (40 or 500 pM) to NRK membranes could be observed. The expression of c-jun, jun B, and c-fos mRNA was inhibited by the endothelin type A receptor (ET)-selective antagonist, BQ-123. Thus, these data demonstrate that ET-1 mediates the expression of immediate response gene mRNA in NRK cells via the ET_A receptor. ET-1 stimulation of NRK cells also upregulated EGF receptors, providing a possible mechanism for ET-1 complementation of epidermal growth factor (EGF) mitogenicity in NRK cells. © 1995 Wiley-Liss, Inc.     

Mechanical stretch induced interleukin-18 (IL-18) expression through Angiotensin subtype 1 receptor (AT1R) and endothelin-1 in cardiomyocytes

Interleukin-18 (IL-18) is a proinflammatory cytokine with multiple biological functions. We and others have demonstrated that an increased level of circulating IL-18 is one of the risk factors for cardiovascular diseases. Endothelin-1 (ET-1) has been reported to be a potent hypertrophy-promoting factor through RhoA and Rho-Kinase. Mechanical stretch induces a hypertrophic response, partly through the production of ET-1 through Endothelin A receptor (ETAR). Moreover, it has also been reported that mechanical stretch induces cardiac hypertrophy through Angiotensin subtype 1 receptor (AT1R). However, the mechanism by which the IL-18 gene expression is regulated in cardiomyocytes has not yet been fully understood. This study was designed to elucidate the functional significance of IL-18 gene expression in response to mechanical stretch. Neonatal rat cardiomyocytes cultured on silicone dishes were subjected to stretch. The moderate 20% mechanical stretch resulted in the elevation of IL-18 expression in a time-dependent manner with the maximal level achieved 36 hours after the stretch. Olmesartan, AT1R antagonist inhibited stretch-induced IL-18 expression. ETAR blockade BQ123 inhibited stretch-induced IL-18 expression. However, the Endothelin B receptor (ETBR) receptor blockade BQ788 did not inhibit this reaction. ET-1 induced IL-18 expression, with a peak induction after 4 hours of incubation. These results might suggest that stretch stimulation of cardiomyocytes induced ET-1 and, subsequently, ET-1 up-regulated the IL-18 expression. Furthermore, Fasudil, a Rho-Kinase inhibitor, and Simvastatin, a HMG-CoA reductase inhibitor, led to a significant reduction in mechanical stretch-induced IL-18 expression. These results indicated, for the first time, that IL-18 expression is induced by mechanical stretch in cardiomyocytes via the ETAR, AT1R, and the Rho/Rho-K pathways. The induction of IL-18 from cardiomyocytes by mechanical stress might cause the deterioration of cardiac functions in autocrine and paracrine fashion. The inhibition of IL-18 expression induced by mechanical stress might be one of the mechanisms that account for the beneficial cardiovascular effects of AT1R antagonist, ETAR blockade, Statin, and Rho-Kinase inhibitor.     

Endothelin-induced intracellular Ca2+ mobilization through its specific receptors in murine peritoneal macrophages.

We studied the presence of specific binding sites for endothelin (ET) and the effect of ET on cytosolic free Ca2+ concentration ([Ca2+]i) in murine thioglycolate-activated peritoneal macrophages. Scatchard analysis for binding experiments using [125I]ET-1 or [125I]ET-3 revealed the existence of a single class of binding sites. The binding parameters (Kd and Bmax) for [125I]ET-1 were almost identical to those for [125I]ET-3. In addition, unlabeled 3 ET isopeptides (ET-1, ET-2 and ET-3) inhibited the specific binding of both ET-1 and ET-3 with similar inhibitory potencies. All 3 ET isopeptides caused an increase in [Ca2+]i in the same dose-dependent manner (0.01-100 nM). These results demonstrate the existence of an ET receptor with the same affinity for all isoforms that mediates the ET-induced intracellular Ca2+ mobilization in murine peritoneal macrophages.