Role of ERK/MAPK in endothelin receptor signaling in human aortic smooth muscle cells

Background  

Endothelin-1 (ET-1) is a potent vasoactive peptide, which induces vasoconstriction and proliferation in vascular smooth muscle cells (VSMCs) through activation of endothelin type A (ET_A) and type B (ET_B) receptors. The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein kinases (MAPK) are involved in ET-1-induced VSMC contraction and proliferation. This study was designed to investigate the ET_A and ET_B receptor intracellular signaling in human VSMCs and used phosphorylation (activation) of ERK1/2 as a functional signal molecule for endothelin receptor activity.     

Endocardial endothelial celsl stimulate proliferation and collagen synthesis of cardiac fibroblasts

Given that vascular endothelial cells play an important role in the modulation of vascular structure and function, we hypothesized that endocardial endothelial cells (EECs) may have a modulator role in regulating the cardiac interstitial cells. Endocardial endothelial cells were isolated from freshly collected pig hearts and cardiac fibroblasts were isolated from 3- to 4-d-old Wistar rats. Fibroblasts were cultured in the presence or absence of conditioned medium from EECs. Proliferation of cardiac fibroblasts was measured by the incorporation of [³H]-Thymidine and collagen synthesis was assayed by the incorporation of [³H]-proline. To determine the involvement of signaling mediators, in separate experiments, cardiac fibroblasts were incubated with BQ123 (selective ET_A receptor antagonist), PD142893 (nonselective ET_A/ET_B receptor antagonist), Bis-indolylmaleimide (PKC inhibitor), PD 098059 (MEK inhibitor), or neutralizing anti-transforming growth factor (TGF)-β-antibody. Endocardial endothelium-derived factors endothelin (ET)-1, TGF-β, and Angiotensin (Ang)-II in the conditioned medium were assayed by enzyme-linked immunosorbent assay using commercially available kits. We report here evidence that suggest that endocardial endothelial cells stimulate both proliferation and collagen synthesis of cardiac fibroblasts. The response seems to be mediated by endothelin through its ET_A receptor. Our results also indicate that protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) pathways are essential for the EEC-induced proliferation of cardiac fibroblasts.     

TNF-alpha upregulates the A2B adenosine receptor gene: The role of NAD(P)H oxidase 4

Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-α) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A_2B adenosine receptor (A_2BAR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-α. Here, we show a regulatory loop by which TNF-α upregulates the A_2BAR in VSMC in vitro and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A_2BAR, and Nox inhibitors dampen the effect of TNF-α. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A_2BAR.     

Increased expression of vascular endothelin type B and angiotensin type 1 receptors in patients with ischemic heart disease

Background

Endothelin-1 and angiotensin II are strong vasoconstrictors. Patients with ischemic heart disease have elevated plasma levels of endothelin-1 and angiotensin II and show increased vascular tone. The aim of the present study was to examine the endothelin and angiotensin II receptor expression in subcutaneous arteries from patients with different degrees of ischemic heart disease.

Methods

Subcutaneous arteries were obtained, by biopsy from the abdomen, from patients undergoing coronary artery bypass graft (CABG) surgery because of ischemic heart disease (n = 15), patients with angina pectoris without established myocardial infarction (n = 15) and matched cardiovascular healthy controls (n = 15). Endothelin type A (ET_A) and type B (ET_B), and angiotensin type 1 (AT₁) and type 2 (AT₂) receptors expression and function were examined using immunohistochemistry, Western blot and in vitro pharmacology.

Results

ET_A and, to a lesser extent, ET_B receptor staining was observed in the healthy vascular smooth muscle cells. The level of ET_B receptor expression was higher in patients undergoing CABG surgery (250% ± 23%; P < 0.05) and in the patients with angina pectoris (199% ± 6%; P < 0.05), than in the healthy controls (100% ± 28%). The data was confirmed by Western blotting. Arteries from CABG patients showed increased vasoconstriction upon administration of the selective ET_B receptor agonist sarafotoxin S6c, compared to healthy controls (P < 0.05). No such difference was found for the ET_A receptors. AT₁ and, to a lesser extent, AT₂ receptor immunostaining was seen in the vascular smooth muscle cells. The level of AT₁ receptor expression was higher in both the angina pectoris (128% ± 25%; P < 0.05) and in the CABG patients (203% ± 41%; P < 0.05), as compared to the healthy controls (100% ± 25%). The increased AT₁ receptor expression was confirmed by Western blotting. Myograph experiment did however not show any change in vasoconstriction to angiotensin II in CABG patients compared to healthy controls (P = n.s).

Conclusion

The results demonstrate, for the first time, upregulation of ET_B and AT₁ receptors in vascular smooth muscle cells in ischemic heart disease. These receptors may play a role in the pathophysiology of ischemic heart disease and could provide important targets for pharmaceutical interventions.     

Discovery of a series of pyrrolidine-based endothelin receptor antagonists with enhanced ETA receptor selectivity

Endothelins, ET-1, ET-2, and ET-3 are potent vasoconstricting and mitogenic 21-amino acid bicyclic peptides, which exert their effects upon binding to the ET_A and ET_B receptors. The ET_A receptor mediates vasoconstriction and smooth muscle cell proliferation, and the ET_B receptor mediates different effects in different tissues, including nitric oxide release from endothelial cells, and vasoconstriction in certain vascular cell types. Selective antagonists of endothelin receptor subtypes may prove useful in determining the role of endothelin in various tissue types and disease states, and hence as therapeutic agents for such diseases. The pyrrolidine carboxylic acid A-127722 has been disclosed as a potent and ET_A-selective antagonist, and is currently undergoing clinical trials. In our efforts to find antagonists with altered selectivity (ET_A-selective, ET_B-selective, or nonselective), we investigated the SAR of the 2-substituent on the pyrrolidine. Compounds with alkyl groups at the 2-position possessed ET_A selectivity improved over A-127722 (1400-fold selective), with the best of these compounds showing nearly 19,000-fold selectivity.     

An endothelin type A receptor-expressing cell to characterize endothelin-1 binding and screen antagonist

Endothelin-1 (ET-1) induces contraction of vascular smooth muscle through binding to endothelin type A receptor (ET_AR). COS-7 cells stably expressing high levels of the ET_AR were established (designated COS-7(ET_AR)). The COS-7(ET_AR) cell bound [¹²⁵I]ET-1 with a K_d of 932 ± 161 pM and a B_max of 74 ± 13 fmol/2 × 10⁵ cells. [¹²⁵I]ET-1 binding was inhibited by ET-1 and the ET_AR antagonist BQ-610, but not by the endothelin type B receptor (ET_BR) antagonist BQ-788. In clones expressing two ET_AR mutants containing D46N or R53Q substitutions in the first extracellular domain of the receptor, [¹²⁵I]ET-1 binding activity was dramatically reduced. This suggests that these single amino acid substitutions alter the three-dimensional structure of the ligand-binding domain of the ET_AR. Using COS-7(ET_AR) cell, we showed that Ca²⁺ or Mg²⁺ was essential for ET-1 binding to the ET_AR and that ET-1 treatment induced postreceptor signaling, that is, intracellular accumulation of cyclic AMP (cAMP) and Ca²⁺ mobilization. The COS-7(ET_AR) established in this study will be a useful tool for screening ET-1 antagonists for treating hypertension.     

Endothelin receptor dimers evaluated by FRET, ligand binding, and calcium mobilization

Endothelin-1 (ET-1) mediates physiological responses via endothelin A (ET_A) and B (ET_B) receptors, which may form homo- and heterodimers with unknown function. Here, we investigated ET-receptor dimerization using fluorescence resonance energy transfer (FRET) between receptors tagged with CFP (donor) and receptors tagged with tetracysteine-FlAsH (fluorescein arsenical hairpin) (acceptor) expressed in HEK293 cells. FRET efficiencies were 15%, 22%, and 27% for ET_A/ET_A, ET_B/ET_B and ET_A/ET_B, respectively, and dimerization was further supported by coimmunoprecipitation. For all dimer pairs, the natural but nonselective ligand ET-1 rapidly (≤30 s) reduced FRET by >50%, but did not detectably reduce coimmunoprecipitation. ET-1 stimulated a transient increase in intracellular Ca²⁺ ([Ca²⁺]_i) lasting 1-2 min for both homodimer pairs, and these ET-1 actions on FRET and [Ca²⁺]_i elevation were blocked by the appropriate subtype-selective antagonist. In contrast, ET_A/ET_B heterodimers mediated a sustained [Ca²⁺]_i increase lasting >10 min, and required a combination of ET_A and ET_B antagonists to block the observed FRET and [Ca²⁺]_i responses. The sensitive CFP/FlAsH FRET assay used here provides new insights into endothelin-receptor dimer function, and represents a unique approach to characterize G-protein-coupled receptor oligomers, including their pharmacology.     

Effect of angiotensin II receptor blocker on angiotensin II stimulated dna synthesis of cultured human aortic smooth muscle cells

To examine the role of the renin-angiotensin system on human vascular smooth muscle cell (VSMC) replication, we studied the effect of DUP753, an angiotensin II (ANG II) type 1 receptor antagonist, on ANG II stimulated tritiated-thymidine (³H-Tdr) incorporation into cultured human aortic VSMC. ANG II stimulated DNA synthesis of VSMC in a dose-dependent manner as estimated by ³H-Tdr incorporation (control; 2993 ± 486 cpm, 10⁻⁸M; 3360 ± 350 cpm, 10⁻⁷M; 3474 ± 516 cpm, 10⁻⁶M; 4889 ± 320 cpm, P < 0. 01). The effects of ANG II were clearly inhibited by 10⁻⁶M DUP 753 (ANG II 10⁻⁸M; 3360 ± 350 vs 509 ± 39 cpm, 10⁻⁷M; 3474 ± 516 vs 661 ± 36 cpm, 10⁻⁶M; 4889 ± 320 vs 806 ± 76 cpm, each P < 0. 01). This receptor antagonist decreased the basal ³H-Tdr incorporation of VSMC from 2933 ± 486 to 411 ±78 cpm (P < 0. 01). Furthermore, DUP 753 decreased 10⁻⁷M ANG II-stimulated ³H-Tdr incorporation of VSMC in a dose-dependent manner (control; 2627 ± 256 cpm, 10⁻⁹M; 2145 ± 143 cpm, 10⁻⁸M; 1047 ± 543 cpm, 10⁻⁷M; 639 ± 169 cpm, 10⁻⁶M; 642 ± 59 cpm, P < 0. 01). These observations suggest that, in human VSMC, ANG II type 1 receptors are important for the regulation of both stimulated and basal cell proliferation. It may therefore be worth while to examine the clinical usefulness of DUP 753 for preventing abnormal VSMC growth.     

Characterization and localization of the endothelin receptors in human end-stage heart failure: ETA/ETB receptor system—Role in the failing human heart

Endothelin (ET) is a potent vasoconstrictor peptide implicated in numerous human diseases, including ischemic cardiomyopathy (ICM). ET binds to receptors ET_A and ET_B. Specific ET receptors were characterized in left atria of patients with end-stage heart failure due to ischemic cardiomyopathy (ICM) (n = 9) and healthy controls (n = 9). Saturation assays revealed a K_d and B_max of 28 ± 8 pM and 87 ± 22 fmol mg^?1 of protein, respectively, from healthy atria and 50 ± 9 pM and 162 ± 19 fmol mg^?1 of protein, respectively, from diseased atria (p < 0.05). For competition studies, we obtained a percentage of ET_A receptors using BQ123, 55% ± 5 and 66% ± 4 in healthy and diseased atria, respectively (p < 0.05). The percentage of ET_B using BQ3020 was 55% ± 14 in healthy and 59% ± 10 in diseased atria. Microautoradiography studies showed a greater number of ET receptors, predominately ET_A, existed in the myocardial layer of diseased atria compared with healthy atria. The percentage of occupied area was greater in the endocardium than the epicardium in diseased atria. These results showed an increase in the ET_A/ET_B receptor system in the failing human heart compared with the non-failing human heart.     

Endothelin-1 binding to endothelin receptors in the rat anterior pituitary gland: possible formation of an ETA-ETB receptor heterodimer

1. Interaction in the recognition of endothelin-1 (ET-1), a typical bivalent ET receptor-ligand, between ET_A and ET_B receptors was investigated in the rat anterior pituitary gland, using our quantitative receptor autoradiographic method with tissue sections preserving the cell-membrane structure and ET receptor-related compounds.2. In saturation binding studies with increasing concentrations (0.77–200 pM) of ¹²⁵I-ET-1 (nonselective bivalent radioligand), ¹²⁵I-ET-1 binding to the rat anterior pituitary gland was saturable and single with a K _D of 71 pM and a B _max of 120 fmol mg^–1. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, binding parameters were 8.3 pM of K _D and 8.0 fmol mg^–1 of B _max, whereas 10 nM sarafotoxin S6c (ET_B agonist) exerted little change in these binding parameters (K _D, 72 pM; B _max, 110 fmol mg^–1).3. Competition binding studies with a fixed amount (3.8 pM) of ¹²⁵I-ET-1 revealed that when 1.0 M BQ-123 was present in the incubation buffer, ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620 (ET_B agonist), and BQ-788 (ET_B antagonist) competitively inhibited ¹²⁵I-ET-1 binding with K _is of 140, 18, 350 pM, and 14 nM, respectively, however, these compounds were not significant competitors for ¹²⁵I-ET-1 binding in the case of absence of BQ-123.4. In cold-ligand saturation studies with a fixed amount (390 pM) of ¹²⁵I-IRL 1620 (ET_B radioligand), IRL1620 bound to a single population of the ET_B receptor, and no change was observed in binding characteristics in the presence of 1.0 M BQ-123. ¹²⁵I-IRL1620 binding was competitively inhibited by ET-1 and ET-3 in the absence of BQ-123, with K _is of 20 and 29 pM, respectively, the affinities being much the same as those of 29 nM, in the presence of 1.0 M BQ-123.5. Two nonbivalent ET_A antagonists, BQ-123 and PD151242, were highly sensitive and full competitors for ¹²⁵I-ET-1 binding (5.0 pM), in the presence of 10 nM sarafotoxin S6c.6. Taken together with the present finding that mRNAs encoding the rat ET_A and the ET_B receptors are expressed in the anterior pituitary gland, we tentatively conclude that although there are ET_A and ET_B receptors with a functional binding capability for ET receptor-ligands, the ET_B receptor does not independently recognize ET-1 without the aid of the ET_A receptor. If this thesis is tenable, then ET-1 can bridge between the two receptors to form an ET_A–ET_B receptor heterodimer.     

Selective and non-selective et antagonists reveal an ETA/ETB receptor mediated ET-1-induced antinociceptive effect in PAG area of mice

The injection of endothelin-1 (ET-1) (2 pmol) into the dorsolateral periaqueductal gray area (PAG) of mice produces antinociceptive effect as underscored by increases in the latency time for the reaction to a hot plate. Pretreatment of the PAG area with bosentan (10 nmol) (a mixed ET_A/ET_B receptor antagonist), FR 139317 (5 nmol) (ET_A receptor selective antagonist) or BQ-788 (5 nmol) (ET_B receptor selective antagonist) greatly reduced the antinociceptive effect induced by ET-1. Therefore, ET-1 induces antinociceptive effects via both ET_A/ET_B receptors. In addition, since ET-antagonists lowered per se the control reaction time of the mice when administered alone to the PAG area, we would suggest that endogenous ET-1 acting within the PAG area contributes to the suppression of pain.     

Design,synthesis and evaluation of 1,3,6-trisubstituted-4-oxo-1,4-dihydroquinoline-2-carboxylic acid derivatives as ETA receptor selective antagonists using FRET assay

The endothelin axis and in particular the two receptor subtypes, ET_A and ET_B, are under investigation for the treatment of various diseases such as pulmonary arterial hypertension, fibrosis, renal failure and cancer. Previous work in our lab has shown that 1,3,6-trisubstituted-4-oxo-1,4-dihydroquinoline-2-carboxylic acid derivatives exhibit noteworthy endothelin receptor antagonist activity. A series of analogues with modifications centered around position 6 of the heterocyclic quinolone core and replacement of the aryl carboxylic acid group with an isosteric tetrazole ring was designed and synthesized to further optimize the structure activity relationship. The endothelin receptor antagonist activity was determined by in vitro Förster resonance energy transfer (FRET) using GeneBLAzer® assay technology. The most potent member of this series exhibited ET_A receptor antagonist activity in the subnanomolar range with an IC₅₀ value of 0.8 nM, and was 1000-fold selective for the ET_A receptor compared to the ET_B receptor. Its activity and selectivity profile resembles that of the most recently approved drug, macitentan.     

Enhanced levels of endogenous endothelin-1 contribute to the over expression of Giα protein in vascular smooth muscle cells from SHR: Role of growth factor receptor activation

We earlier showed that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit increased expression of Gi proteins. Since the levels of endothelin-1 (ET-1) are enhanced in VSMC from SHR, we undertook the present study to examine the implication of endogenous ET-1 and the underlying mechanisms in the enhanced expression of Giα proteins in VSMC from SHR. The enhanced expression of Giα-2 and Giα-3 proteins in VSMC from SHR was inhibited by ET_A and ET_B receptor antagonists, BQ123 and BQ788 respectively. In addition, these antagonists also attenuated the enhanced inhibition of forskolin-stimulated adenylyl cyclase activity by low concentrations of GTPγS and by inhibitory hormones in VSMC from SHR compared to WKY. Furthermore, AG1295, AG1024 and PP2, inhibitors of platelet derived growth factor receptor (PDGFR), insulin-like growth factor 1 receptor (IGF-1R) and c-Src respectively, inhibited the enhanced expression of Giα protein and the enhanced phosphorylation of PDGFR and IGF-1R in VSMC from SHR to WKY levels. In addition, NAD(P)H oxidase inhibitor DPI and N-acetylcysteine (NAC), a scavenger of superoxide anion (O₂⁻) also inhibited the enhanced phosphorylation of PDGFR and IGF-1R and c-Src in VSMC from SHR to control levels. Furthermore, the augmented phosphorylation of ERK1/2 in VSMC from SHR was attenuated by BQ123 and BQ788, growth factor receptors inhibitors and PP2. These results suggest that the enhanced levels of endogenous ET-1 in VSMC from SHR increase oxidative stress, which through c-Src-mediated activation of growth factor receptors and associated MAP kinase signaling, contribute to the enhanced expression of Giα proteins.     

The Endothelin ETA Receptor Exists in the Caudal Solitary Tract Nucleus of the Rat Brain

1. The receptor autoradiographic method done on the rat lower brain stem and cerebellum plus ¹²⁵I-endothelin-1, BQ-123, an antagonist for the endothelin ET_A receptor, and sarafotoxin S6c, an agonist for the ET_B receptor, revealed minute amounts of the ET_A receptor coexisting with the ET_B receptor in the caudal solitary tract nucleus of the rat lower brain stem.2. The ET_B receptor is present predominantly in other parts of the lower brain stem.3. Knowledge of the heterogeneous distribution of the central endothelin receptor subtypes aids in understanding the neurophysiology of endothelins.     

Endothelin Receptors in Rat Pituitary Gland

1. We used the quantitative receptor autoradiographic method plus ¹²⁵I-endothelin-1 (¹²⁵I-ET-1), BQ-123, a specific antagonist for the endothelin ET_A receptor, and sarafotoxin S6c, a selective agonist for the ET_B receptor to investigate the ET receptor in the rat pituitary gland.2. The method revealed that the BQ-123-sensitive ET_A receptor was present predominantly in the anterior lobe and Rathke's pouch.3. The posterior lobe contained BQ-123-sensitive ET_A and sarafotoxin S6c-sensitive ET_B receptors, in almost the same proportion. There was no significant ¹²⁵I-ET-1 binding to the intermediate lobe.4. Knowledge of the heterogeneous distribution of ET receptor subtypes in the pituitary gland supplies information that will be pertinent to physiological investigations of the gland.     

Cigarette smoke upregulates rat coronary artery endothelin receptors in vivo

Background

Cigarette smoking is a strong cardiovascular risk factor and endothelin (ET) receptors are related to coronary artery diseases. The present study established an in vivo secondhand smoke (SHS) exposure model and investigated the hypothesis that cigarette smoke induces ET receptor upregulation in rat coronary arteries and its possible underlying mechanisms.

Methodology/Principal Findings

Rats were exposed to SHS for 200 min daily for 8 weeks. The coronary arteries were isolated and examined. The vasoconstriction was studied by a sensitive myograph. The expression of mRNA and protein for receptors was examined by real-time PCR, Western blot and immunofluorescence. Compared to fresh air exposure, SHS increased contractile responses mediated by endothelin type A (ET_A) and type B (ET_B) receptors in coronary arteries. In parallel, the expression of mRNA and protein for ET_A and ET_B receptors of smoke exposed rats were higher than that of animals exposed to fresh air, suggesting that SHS upregulates ET_A and ET_B receptors in coronary arteries in vivo. Immunofluorescence staining showed that the enhanced receptor expression was localized to the smooth muscle cells of coronary arteries. The protein levels of phosphorylated (p)-Raf-1 and p-ERK1/2 in smoke exposed rats were significantly higher than in control rats, demonstrating that SHS induces the activation of the Raf/ERK/MAPK pathway. Treatment with Raf-1 inhibitor GW5074 suppressed SHS-induced enhanced contraction mediated by ET_A receptors, and inhibited the elevated mRNA and protein levels of ET_A and ET_B receptors caused by SHS. The results of correlation and regression analysis showed that phosphorylation of Raf and ERK1/2 were independent determinants to affect protein expression of ET_B and ET_A receptors.

Conclusions/Significance

Cigarette smoke upregulates ET_B and ET_A receptors in rat coronary artery, which is associated with the activation of the Raf/ERK/MAPK pathway.     

Jab1 regulates levels of endothelin type A and B receptors by promoting ubiquitination and degradation

Endothelin type A receptor (ET_AR) plays an important role in some cardiovascular disorders where ET_AR levels are increased. However, regulatory mechanisms for ET_AR levels are unknown. Here, we identified Jun activation domain-binding protein 1 (Jab1) as an ET_AR-interacting protein by yeast two-hybrid screening of human heart cDNA library using carboxyl terminal tail (C-tail) of ET_AR as a bait. The interaction was confirmed by glutathione S-transferase pull-down assay, co-immunoprecipitation in HEK293T cells expressing ET_AR-myc and FLAG-Jab1, and confocal microscopy. Jab1 knockdown increased whole cell and cell surface levels of ET_AR and ET-1-induced ERK1/2 phosphorylation in HEK293T cells expressing ET_AR, whereas Jab1 overexpression decreased them. Jab1 overexpression accelerated disappearance rate of ET_AR after protein synthesis inhibition as an index of a degradation rate. ET_AR was constitutively ubiquitinated, and the level of ubiquitination was enhanced by Jab1 overexpression. Long-term ET-1 stimulation markedly accelerated the rate of ET_AR degradation and increased the amount of Jab1 bound to ET_AR with a maximal level of 500% at 3 h. In the absence of ET-1 stimulation, the level of ET_BR was lower than that of ET_AR and the degradation rate of ET_BR was markedly faster than that of ET_AR. Notably, the amount of Jab1 bound to ET_BR and ubiquitination level of ET_BR were markedly higher than those for ET_AR. Taken together, these results suggest that the amount of Jab1 bound to ETR regulates the degradation rate of ET_AR and ET_BR by modulating ubiquitination of these receptors, leading to changes in ET_AR and ET_BR levels.     

Endothelin causes transactivation of the EGFR and HER2 in non-small cell lung cancer cells

Endothelin (ET)-1 is an important peptide in cancer progression stimulating cellular proliferation, tumor angiogenesis and metastasis. ET-1 binds with high affinity to the ET_A receptor (R) and ET_BR on cancer cells. High levels of tumor ET-1 and ET_AR are associated with poor survival of lung cancer patients. Here the effects of ET-1 on epidermal growth factor (EGF)R and HER2 transactivation were investigated using non-small cell lung cancer (NSCLC) cells. ET_AR mRNA was present in all 10 NSCLC cell lines examined. Addition of ET-1 to NCI-H838 or H1975 cells increased EGFR, HER2 and ERK tyrosine phosphorylation within 2 min. The increase in EGFR and HER2 transactivation caused by ET-1 addition to NSCLC cells was inhibited by lapatinib (EGFR and HER2 tyrosine kinase inhibitor (TKI)), gefitinib (EGFR TKI), ZD4054 or BQ-123 (ET_AR antagonist), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor) or Tiron (superoxide scavenger). ET-1 addition to NSCLC cells increased cytosolic Ca²⁺ and reactive oxygen species. ET-1 increased NSCLC clonal growth, whereas BQ123, ZD4054, lapatinib or gefitinib inhibited proliferation. The results indicate that ET-1 may regulate NSCLC cellular proliferation in an EGFR- and HER2-dependent manner.     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.