Analysis of immunostaining and western blotting of endothelin 1 and its receptors in mitral stenosis.

INTRODUCTION: Rheumatic Fever represents a serious public health problem in developing countries, with thousands of new cases each year. It is an autoimmune disease, which occurs in response to infection by streptococcus A. OBJECTIVE: The aim of this study was to evaluate the immunolabeling and protein expression for endothelin-1 and 3 (ET-1, ET-3) and its receptors (ETA, ETB) in rheumatic mitral valves. METHODS: Immunohistochemistry was used to identify ET-1/ET-3 and ETA/ETB receptors in rheumatic and control mitral valves. Quantitative analysis of immunostaining for ET-1/ET-3 and ETA/ETB receptors was performed. In addition, western blot analysis was carried out to assess protein levels in tissue samples. RESULTS: ET-1 and ETA receptor immunostaining predominated in stenotic valves, mainly associated with fibrotic regions, inflammatory areas and neovascularization. Quantitative analysis showed that the average area with positive expression of ET-1 was 18.21 ± 14.96%. For ETA and ETB, the mean expressed areas were respectively 15.06 ± 13.13% and 9.20 ± 11.09%. ET-3 did not have a significant expression. The correlation between the expression of both endothelin receptors were strongly positive (R = 0.74, P = 0.02), but the correlation between ET-1 and its receptor were negative for both ETA (R = -0.37, P = 0.25), and ETB (R = -0.14, P = 0.39). This data was supported by western blot analysis. CONCLUSION: The strong correlation between ET-1 and its receptors suggests that both play a role in the pathophysiology of rheumatic mitral valve stenosis and may potentially act as biomarkers of this disease.

The comparison of traditional and modified harvesting techniques of left internal mammary artery regarding endothelin-1/2/3 expression and free flow capacity.

AIM: We have conducted this study to seek and observe visual clues through immunohistochemical staining for differences in Et-1/2/3 expression and the free-flow capacity measuring the blood flow through grafts, in the left internal mammary artery grafts prepared either with clipped or nonclipped techniques. METHODS: A total of 40 consecutive patients with a diagnosis of coronary artery disease who would benefit from elective coronary artery bypass graft surgery were randomised into two groups consisting 20 patients each. Left internal mammary artery was harvested by a traditional clipped (control group) and a modified nonclipped (study group) technique in each of the groups. All harvested arterial segments were evaluated for luminal endothelial integrity through hematoxylin&eosin and immunohistochemical staining. RESULTS: The free-flow capacity of left internal mammary artery grafts were significantly higher in nonclipped arteries when compared with that of clipped ones (P=0.001). The arterial lumen of the nonclipped segments were visibly more dilated than the clipped ones. Nonclipped segments presented a lighter immunostaining for Et-1/2/3 when compared with the clipped ones (P<0.001). CONCLUSION: We believe that lesser endothelial damage caused by the lower intraluminal pressure in modifiedly harvested left internal mammary artery segments has positive implications on intraoperative and postoperative cardiac events related to graft vasospasm, especially related with endothelins. We recommend modified left internal mammary artery harvesting in patients going under coronary artery bypass graft operation.

Expression levels of endothelin-1, endothelin-2, and endothelin-3 vary during the initial, lag, and late phase of orthodontic tooth movement in rats.

Endothelins (ET)-1, ET-2, and ET-3 are one group of cytokines likely to be released during orthodontic tooth movement (OTM). Therefore, the expression of ET levels was investigated to determine the importance and involvement of isopeptides during the several phases of OTM. Thirty-two male Wistar rats (12-13 weeks old) were divided into four groups of eight: control, 14, 28, and 42 day groups. Tooth movement was induced by a closed-coil spring inserted between the upper left first molar and the upper incisors. The distance between the teeth was measured on days 0, 2, 7, 14, 21, 28, 35, and 42 using a digital calliper. The rate of tooth movement was calculated. The animals were sacrificed on days 14, 28, and 42 and gene expression levels of all three ET were determined using reverse transcription polymerase chain reaction. Statistical analysis was performed using two-way analysis of variance, Bonferroni's correction, and paired t-tests. The distance between the teeth decreased in all appliance groups (P < 0.001). The rate of tooth movement was 0.20 +/- 0.02, 0.03 +/- 0.01, and 0.06 +/- 0.02 mm/day between days 0-2, 3-21, and 22-42, respectively. On day 14, gene expression levels for ET-1 (P < 0.05) and ET-3 (P < 0.001) increased compared with day 0. On day 28, a downregulation of ET-3 was observed when compared with day 0 (P < 0.001). On day 42, ET-1 (P < 0.001) and ET-3 (P < 0.01) gene expression levels were strongly upregulated, while ET-2 gene expression level was downregulated (P < 0.01) when compared with day 0. ET-1 and ET-3, but not ET-2, are involved in all three phases of OTM, and ET-1 seems to be the predominant form in the late phase of OTM.

Expression of endothelin system in neuroblastic tumors: close association of endothelin-1 and endothelin B receptor expression with differentiation of tumor cells.

Although a critical role of the endothelin (ET) system in differentiation of neural crest cells has been reported, implication of the ET system in human neuroblastic tumors has not been fully elucidated. We immunohistochemically examined for localization of ET-1, ET-3, ET-A receptor (ET-A), and ET-B receptor (ET-B) in 24 ganglioneuromas, 8 ganglioneuroblastomas, 37 neuroblastomas, 14 normal sympathetic ganglia, and 10 fetal adrenal glands with regard to neuroblastic cell differentiation. Neuroblasts in fetal adrenal glands expressed ET-B (100%) alone. Immature ganglionic cells in sympathetic ganglia of fetus frequently expressed ET-1 (86%) and ET-B (100%), while ET-A was occasionally detected (28%). Ganglionic cells of mature adult ganglia consistently harbored ET-1 (100%) and, infrequently, ET-3 (21%) or ET-B (29%). Expression of ET-1 and ET-B was closely associated with tumor cell differentiation: the expression frequency of ET-1 or ET-B was 16% or 46% in neuroblastomas; 100% or 88% in ganglioneuroblastomas; and 96% or 92% in ganglioneuromas. In contrast, ET-3 and ET-A showed no association with tumor cell differentiation: the expression frequency of ET-3 or ET-A was 26% or 14% in neuroblastomas; 63% or 13% in ganglioneuroblastomas; and 29% or 21% in ganglioneuromas. In conclusion, ET-1 and ET-B are expressed with differentiation of neuroblastic tumors.

Salivary immunoreactive endothelin in patients with upper gastrointestinal diseases.

Endothelins have been implicated in gastric mucosal damage in a variety of animal models. Furthermore, clinical reports also show elevated gastric mucosal endothelin-1 levels in patients suffering from peptic ulcer diseases. We have demonstrated, first, the presence of immunoreactive endothelin (IR-ET) in human saliva. We also show that endothelins are rather stable in human saliva. The present study was undertaken to determine whether patients with endoscopically proven upper gastrointestinal diseases have a salivary excess of IR-ET, compared with patients with a normal esophagogastroduodenoscopy. Saliva was collected from fasting subjects prior to esophagogastroduodenoscopy. The levels of IR-ET were measured by the radioimmunoassay method. The salivary concentrations of IR-ET in the studied subjects were as follows: 8.9 +/- 1.0 fmol/mL (mean +/- standard error of the mean) for patients with gastric ulcers (n = 18); 7.3 +/- 1.0 fmol/mL for patients with duodenal ulcers (n = 22); and 6.8 +/- 0.6 fmol/mL for patients with gastritis (n = 28). These values are all higher than that of normal subjects (4.4 +/- 0.5 fmol/mL, n = 20; P < 0.001, P < 0.01, and P < 0.05, respectively). No significant differences in salivary IR-ET were noted between patients with a normal esophagogastroduodenoscopy and patients with esophagitis (3.8 +/- 0.7 fmol/mL, n = 4) or gastric cancer (5.3 +/- 1.4 fmol/mL, n = 4). There were no significant differences in the salivary IR-ET levels between males and females. However, the salivary IR-ET levels in the smokers (8.0 +/- 0.6 fmol/mL, n = 38) were significantly higher (P < 0.01) than those of the non-smokers (6.0 +/- 0.4 fmol/mL, n = 58). There was no correlation of IR-ET levels with age. Our findings suggest that salivary endothelin may have a contributing role in certain gastroduodenal diseases.

Immunoreactive endothelin in Taiwanese thyroid cystic fluid.

Endothelin-1 is a major vasoconstrictor peptide, first found in endothelial cells and later in many other tissues, including the thyroid gland. It is well known that endothelins can act as autocrine and/or paracrine regulators of thyroid homeostasis and growth. Previously we have demonstrated that immunoreactive endothelins (IR-ET) are present in various human body fluids, and IR-ET has also been detected in pathologic breast and thyroid cystic fluids. In this study, the IR-ET in Taiwanese thyroid cystic fluid was measured by radioimmunoassay and characterized by chromatography. Human thyroid cystic fluid was obtained by fine needle aspiration, was centrifuged, and the supernatant was stored at -20 degrees C until IR-ET assay. IR-ET has been detected in 25 of 33 samples of thyroid cystic fluid [25 cases, 4.11 +/- 0.31 fmol/mL (mean +/- standard error of the mean); other eight cases, undetectable]. Gel permeation chromatography of the extract of pooled cystic fluid showed only one major peak at the elution position of human endothelin-1 standard. No difference in cystic IR-ET levels was found in our patients with cystic nodules in relation to differences in thyroid function. It is probable that endothelin-1 is produced by the epithelial cells lining the thyroid cysts, and the increased levels of IR-ET in cystic fluid found in our patients could either be secondary to cystic nodule development or have a role in goiter formation.

Localization and endothelin-3 dependence of stem cells of the enteric nervous system in the embryonic colon.

BACKGROUND/PURPOSE: The aganglionosis in a variable length of the distal gut found in Hirschsprung's disease results from the abnormal prenatal development of neural crest-derived stem cells of the enteric nervous system. The cytokine endothelin-3 is necessary for successful colonization of the distal gut, but the location of this interaction with neural crest-derived stem cells remains to be established. The hypothesis tested here is that the stem cells of the enteric nervous system (ENS) in the colon are located at the leading edge of the migrating wave of neural crest-derived stem cells and that these cells require colonic endothelin-3 for complete colonization of the gut. METHODS: Explants of 11.5-day-old embryonic intact mouse gut and isolated colon were cultured for 72 hours in the presence and absence of the endothelin-B receptor antagonist, BQ788. Specimens then were sectioned and stained by immunohistochemistry to assess enteric nervous system development. RESULTS: Isolated colon contained a very low number (mean, 73 cells; range, 37 to 106; n = 8) of neural crest-derived stem cells, which had just entered its proximal end at the leading edge of neural crest cell migration. After 72 hours of culture, progeny of these few neural crest-derived stem cells had colonized the colon at an equivalent ganglionic density to those in intact gut. Furthermore, neuronal differentiation, as shown by the appearance of nitric oxide synthase positive neurons, also was equivalent to intact gut. Blockade of the endothelin-B receptor produced terminal aganglionosis in both isolated colons and intact gut. CONCLUSIONS: The very small number of cells that first enter the proximal colon at the leading edge of neural crest cell migration have the ability to colonize the entire colon normally in an ET-3-dependent manner. These cells therefore have the functional characteristics expected of the stem cells of the colonic enteric nervous system. Furthermore, the normal development of these cells is dependent on the endothelin-3 expressed by the mesenchymal cells of the colon itself.

Endothelin-1 production by prostate cancer cell lines is up-regulated by factors involved in cancer progression and down-regulated by androgens.

BACKGROUND: Recent data demonstrate that endothelin-1 (ET-1) concentration increases in plasma of men with advanced, hormone-refractory prostate adenocarcinoma. In addition, ET-1 is involved in osteblastic remodelling and new bone formation, suggesting a role for this vasoactive peptide in the metastatic progression of prostate cancer to the bone. METHODS: We investigated the regulation of ET-1 expression in androgen-sensitive and insensitive prostate cancer cell lines by androgens and several factors involved in progression of prostate cancer (EGF) and bone remodelling (TGFbeta-1, IL1-alpha and IGF-1). RESULTS: Northern analysis and radio immunoassay demonstrated that all the ET-1 pathways are tuned off in the androgen-sensitive LNCaP cell line when compared to the androgen-insensitive PC-3 and DU145. In PC-3 cells transfected with a full-length androgen receptor expression vector (PC-3-AR), treatment with androgens reduced gene expression and secretion of ET-1 without affecting the gene expression of ET-3. Collectively, these data support a role for androgens in the regulation of ET-1 production by prostate adenocarcinoma cells. In PC-3 and DU145 cells, ET-1 gene expression and secretion were up-regulated by TGFbeta-1, EGF and IL1-alpha, whereas IGF-1 was ineffective. Conversely, none of the treatments affected ECE-1 or ET-3 gene expression. CONCLUSIONS: In conclusion, ET-1 production by prostate adenocarcinoma cells is down-regulated by androgens and up-regulated by factors involved in tumour progression indicating a role for this peptide in the biology of prostate cancer. In view of the role exerted by ET-1 in the process of bone metastasis, our data suggest the use of ET-1 receptor antagonists in the treatment of advanced prostate cancer.

Endothelin-1, endothelin-3 and their receptors (endothelin(A) and endothelin(B)) in chronic renal transplant rejection in rats.

Endothelins (ET) are a family of vasoactive peptides that play an important role in several disorders affecting kidneys. In this study we investigated the expressions of ET-1, ET-3, and their receptors, ET(A) and ET(B), in a rat chronic renal transplant rejection model. Renal allografts were performed (F344 --> Lewis) with bilateral nephrectomy in recipients. For isograft control, lewis --> lewis transplantations were performed. All recipients were sacrificed 140 days after transplantation and the grafts were analyzed histologically. ET-1 and ET-3 protein expression in grafts was measured by immunohistochemistry and ELISA. Semiquantitative RT-PCR methods were used for mRNA levels of ET-1, ET-3, ET(A) and ET(B). No evidence of chronical rejection was manifested in isografts. The allografted rats showed proteinuria and increased serum creatinine levels. Histologically, renal allografts showed atrophy and sclerosis of the glomeruli, cortical scarring and vascular intimal thickening. Immunohistochemically, ET-1 and ET-3 were localized in the convoluted tubules, collecting ducts, endothelium and smooth muscle cells of the large blood vessels. Significantly increased staining for ET-1 and ET-3 were found in allografts compared to isografts. Simultaneously, ELISA for ET-1 and ET-3 showed elevated protein concentrations in allografts compared to isografts. Allografts showed significantly increased ET-1- and ET-3 mRNA compared to isografts. On the other hand, a significant down regulation of the ET(A) mRNA was noted, and the ET(B) mRNA remained unchanged. The data from the present study suggest that alteration of ET system may be of importance in the pathogenesis of chronic renal transplant rejection.

ET(B) receptor blockade potentiates the pressor response to big endothelin-1 but not big endothelin-2 in the anesthetized rabbit.

The precursor of endothelin-1, big endothelin-1, is considered to be a more reliable marker of systemic production of vasoactive peptide. However, it is largely unclear whether ET(B) receptor-dependent clearance and endothelium-derived relaxing factors affect the precursor in a similar manner to mature ET-1. These ET(B)-dependent modulations of big ET-1 and big ET-2 pressor properties were therefore studied in the anesthetized rabbit. When injected into the left cardiac ventricle, ET-1 and ET-2 (0.01 to 1 nmol/kg) each induced biphasic responses (a depressor followed by a pressor response), whereas big ET-1 and big ET-2 (0.1 to 3 nmol/kg) caused only protracted pressor responses. The highest dose of big ET-1 caused significantly greater responses than ET-1, ET-2, or big ET-2. A selective ET(A) receptor antagonist, BQ-123 (1 mg/kg), markedly reduced pressor responses to all 4 peptides, whereas blockade of ET(B) receptors with BQ-788 (0.25 mg/kg) sharply potentiated the responses to ET-1, ET-2, and big ET-1, but not to big ET-2. Indomethacin (10 mg/kg) sharply potentiated the pressor response to ET-1 (1 nmol/kg), but not big ET-1, at all time points. In control animals, ET-1, but not big ET-1, also triggered an indomethacin-sensitive increase in circulating prostacyclin. Finally, systemically administered big ET-1, but not big ET-2, induced a phosphoramidon-sensitive increase in plasma IR-ET. Our results suggest a significant limiting role of ET(B) receptors on pressor responses to big ET-1. In contrast, the same receptor entities do not modulate the hemodynamic properties of the ET-2 precursor, given that, unlike big ET-1, it is poorly converted in the pulmonary or systemic circulation in anesthetized rabbits.

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

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

Endothelins and their receptors in cirrhotic and neoplastic livers of Canadian and Chinese populations.

BACKGROUND: Endothelins (ETs) are 21 amino acid peptides with widespread tissue distribution and functions. In this study, we retrospectively investigated immunoreactive ET-1, ET-3 as well as ET receptors by ligand binding and autoradiography in hepatic cirrhosis and neoplasms. MATERIALS AND METHODS: Formalin fixed paraffin embedded tissues from 30 hepatocellular carcinomas (HCC), 4 fibrolamellar carcinomas (FLC), and 7 liver metastatic adenocarcinomas (Ad) from colon were collected from the Pathology Department of London Health Science Centre. Adjacent cirrhotic livers were obtained from 17 cases and adjacent normal liver was present in 12 cases. In addition, 15 HCCs, 6 cirrhotic and 8 normal livers were obtained from Normal Bethune University for Medical Sciences in China. The slides were stained for ET-1 and ET-3 with a polyclonal antibody and scored. Autoradiographic localization of ET-receptors with 125I-ET-1 was carried out in some of the cases. RESULTS: In the normal liver, hepatocytes, biliary epithelium, vascular endothelium and smooth muscle cells were positive for both ET-1 and ET-3. Higher immnunoreactivity for ET-1 and ET-3 was seen in cirrhosis. HCCs showed variation in immunoreactivity, with overall scoring not different from normal livers. FLCs showed consistent higher immunoreactivity for both ET-1 and ET-3, while in Ads the immunoreactivity was decreased. Increased ET-receptors, representing both ETA and ETB subtypes were seen in both cirrhosis and in HCC. CONCLUSION: Alterations in both ETs and their receptors were found in cirrhosis and neoplastic liver diseases.

Ultrastructural localization of nitric oxide synthase and endothelin in the renal and mesenteric arteries of the golden hamster: differences during and after arousal from hibernation.

This is a study of the electron-immunocytochemical localization of nitric oxide synthase (type III) and endothelin in renal and mesenteric artery endothelial cells of normal (active) and hibernating hamsters, as well as hamsters exposed to the cold but not hibernating, and hamsters aroused for 2h following hibernation. In the renal artery of hibernating hamsters and cold-exposed hamsters, a subpopulation of nitric oxide synthase-positive endothelial cells displayed immunoprecipitate predominantly in the vicinity of the Golgi complex indicating intracellular translocation from the cytoplasm to the Golgi complex. In hibernating animals, the percentages of both nitric oxide synthase-positive and endothelin-positive endothelial cells were notably lower than those observed either in active, cold-exposed or aroused animals. These changes may reflect a reduced endothelial contribution to the maintenance of vascular tone in these vessels during hibernation and an upregulation of expression of nitric oxide synthase and endothelin in the endothelium early on during arousal from hibernation.

Specific sandwich-type enzyme immunoassays for smooth muscle constricting novel 31-amino acid endothelins.

We established highly sensitive and specific sandwich-enzyme immunoassays (EIAs) for three newly discovered bioactive 31-amino acid endothelins [ETs(1-31)], which can detect as little as 0.16 pg/well of ET-1(1-31), 0.39 pg/well of ET-2(1-31), and 0.16 pg/well of ET-3(1-31). The EIAs showed no crossreactivity with 21-amino acid endothelins [ETs(1-21)] or big ETs at the usual assay concentrations below 1-5 ng/ml. In reversed-phase HPLC, immunoreactive ETs(1-31) in the granulocytes of normal human subjects eluted at the exact positions of authentic ETs(1-31), except for the presence of one additional unknown immunoreactive ET-1(1-31). The results also indicate that ETs(1-31) exist in the granulocytes at levels higher than or similar to those of ETs(1-21). This study is the first to establish EIAs for novel bioactive ETs(1-31). These assays can be utilized to assess the pathophysiological roles of ETs(1-31).

Quantitative peptide bioanalysis using column-switching nano liquid chromatography/mass spectrometry.

Many endogenous peptides are circulating in bodily fluids at the low pmol l-1 range, placing high demands on the bioanalytical procedure. In order to analyze these minute concentrations in complex matrices, a miniaturized liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) bioanalysis method was developed using custom-made nanoLC columns (75 microns i.d.) and a micro-electrospray interface (micro ESI). To be able to analyze large sample volumes in order to cope with low biological analyte concentrations, the nanoLC/ESI-MS method was coupled to an on-line preconcentration (PC) system based on a strong anion-exchange material. This method was used to analyze endothelin peptides (ETs) in complex matrices, which are potent vasoconstrictors of M(r) approximately 2500 Da. The ET isoforms could be simultaneously analyzed with detection limits down to 30 pmol l-1 in cell supernatants (1.5 fmol on column). The method was linear from 50 to 2000 pmol l-1 with correlation coefficients of 0.99 for two of the three endothelin isoforms. Several other parameters, such as matrix effects and recovery, were also investigated.

[Immunocytochemical studies on the endothelin peptides and their receptors in mast cells of the rat lung and gastrointestinal tract].

The distribution of endothelin (ET)-containing mast cells was immunohistochemically investigated in the rat lung and gastrointestinal tract using antibodies against Big ET-1, Big ET-2, Big ET-3, mature ETs and their receptors of ET-A, and ET-B. In the lung, numerous mature ETs-containing mast cells were present in connective tissue around the bronchus, bronchioles and in the interalveolar septa. The number of Big ET-2-containing mast cells was almost the same as that of Big ET-3-containing mast cells, while Big ET-1-positive mast cells were fewer than that of the other isopeptides. In all the regions of the gastrointestinal tract, immunoreactivity for mature ETs was found mainly in mast cells of the lamina propria, the number of Big ET-2 and Big ET-3-containing cells was almost the same similar to that found in the lung, while Big ET-1-containing cells were very few. Moreover, mast cells in not only lung but also gastrointestinal tract contain both of ET-A and ET-B receptors. Electron-microscopically, ET-immunoreaction products were mainly precipitated in the mast cell granules. Hence, we presume that ETs are synthesized in and secreted from mast cells in the rat lung and gastrointestinal tract; they act in an autocrine/paracrine fashion; and their main isopeptides are ET-2 and ET-3.

Immunocytochemical studies on endothelin in mast cells and macrophages in the rat gastrointestinal tract.

To reveal the distribution of endothelin (ET)-containing stromal cells (mast cells and macrophages), we investigated the rat gastrointestinal tract immunohistochemically using antibodies to Big ET-1, Big ET-2, Big ET-3, and mature ETs. In all the regions of the gastrointestinal tract, immunoreactivity for all the antibodies used was found in stromal cells that were located mainly in the lamina propria (not in the submucosa). The number of these cells was largest in the small intestine and smallest in the colon. Moreover, Big ET-2, which was originally identified in the gastrointestinal tract, was also found in many stromal cells, but Big ET-3-containing cells, unexpectedly, were found in almost the same number as Big ET-2-containing cells, while Big ET-1-containing cells were few. These immunopositive stromal cells seemed to be mast cells and macrophages from their histological features. Double-immunohistochemical staining revealed that 92% of the mature ETs-positive cells were mast cells; the rest were macrophages. Furthermore, we confirmed that mature ETs coexisted with ET-A or ET-B receptors in identical cells. Hence, we presume that ETs are synthesized in and secreted from stromal cells in the rat gastrointestinal tract, that their main isotypes are not only ET-2 but also ET-3, and that ETs may act in an autocrine/paracrine fashion.

Endothelin-1 production and agonist activities in cultured prostate-derived cells: implications for regulation of endothelin bioactivity and bioavailability in prostatic hyperplasia.

BACKGROUND: Endothelin-1 (ET-1) interacts with specific G-protein-coupled receptors to initiate short-term (contraction) and long-term (mitogenesis) events in target cells. ET-1 is an abundant prostate secretory protein that, in its biologically active form, elicits prostatic smooth muscle contraction. The present study was designed to determine the effects of ET-1 on prostate cell growth and to examine the regulation of endogenous ET-1 activity and bioavailability. METHODS: Primary cultures of prostate secretory epithelial (PE) and prostate fibromuscular stromal (PS) cells were established from benign human prostate tissue. RESULTS: In culture, PE cells secrete immunoreactive ET-1 (38.5 +/- 1.6 pg/ml/10(6) cells/24 hr) into the conditioned medium. Levels of immunoreactive ET-1 produced by PS cells were more than 10-fold lower. Endothelin-converting enzyme-1 (ECE-1) mRNA was detected in PE cells and not in PS cells; however, big ET-1 was the predominant immunoreactive ET-1 secretory product of PE cells. The ET(B) endothelin receptor was the predominant subtype in both PE and PS cells. In PS cells, but not PE cells, ET-1 induced significant inositol phosphate accumulation and [3H]-thymidine uptake. Agonist activity was inhibited by the ET(B) receptor selective antagonist, BQ 788. Intact PE cell monolayers secrete ET-1 through the apical surface, consistent with secretion of ET-1 into the glandular lumen in vivo. CONCLUSIONS: On the basis of these findings, regulation of ET-1 activity and bioavailability appears to be tightly regulated. Such findings have important implications in the pathophysiology of prostate disease.

Immunohistochemical localization of endothelin-1, endothelin-3 and endothelin receptors in human pheochromocytoma and paraganglioma.

Endothelin (ET) and its receptor system have been shown to exert various biological effects on different types of cells in addition to their well-known vasoconstrictor activity. Recently ET-1, ET-3 and the ETB receptor have been shown to play an important role in the development of neural crest-derived cells and, in this context, pheochromocytomas have been reported to harbor ET-1. Endothelin-3 or ET receptor subtypes, however, have not been examined in pheochromocytoma and paraganglioma so far. In the present study the immunohistochemical localization of ET-1/big ET-1, ET-3/big ET-3 and the ETA and ETB receptors were investigated to clarify the biological characteristics of these two tumors using 32 pheochromocytomas and 11 extra-adrenal paragangliomas. Endothelin-1/big ET-1 was detected in 19 pheochromocytomas (59%) and eight paragangliomas (72%), while ET-3/big ET-3 was detected in 10 pheochromocytomas (31%) and three paragangliomas (27%). The ETA receptor was found in 21 pheochromocytomas (66%) and in eight paragangliomas (73%), while the ETB receptor was found in 25 pheochromocytomas (78%) and in eight paragangliomas (73%). Normal adrenomedullary cells lacked each antigen examined. Endothelin-immunoreactive tumor cells were distributed focally or in a manner scattered, while receptor-immunostained tumor cells were distributed with a focal pattern for the ETA receptor and with a focal or diffuse pattern for the ETB receptor. Endothelin and its receptor coexisted in the same tumor in 21 of 28 ET-positive pheochromocytomas and in eight of 10 ET-positive paragangliomas. In addition, seven pheochromocytomas and two paragangliomas revealed positivity of the receptor(s) irrespective of the absence of ET-immunoreactivity. In conclusion, ET and its receptor are frequently and concomitantly expressed in the pheochromocytoma and paraganglioma. From the highly frequent expression of this system or the receptor(s), ET-receptor-mediated signal transduction of these tumors concerning growth and/or cell survival is expected, although definite biological significance of this ligand-receptor system in these tumors awaits further investigation.

Endothelin-1 and endothelin-3-like immunoreactivity in the eyes of diabetic and non-diabetic BB/W rats.

Endothelins (ETs) are a family of vasoactive peptides implicated in several disorders of the microvasculature. In the present study, the distribution of immunoreactive ET-1 and ET-3 was investigated in eyes from 8 month spontaneously diabetic BB/W rats and in age matched control animals. Both peptides showed similar immunoreactivity. In non-diabetic animals, corneal epithelium and endothelium, ciliary epithelium, lens epithelium, iris and the microvasculature of the sclera and choroid showed positive immunoreactivity. In the retina, photoreceptor inner segments showed positivity. In the inner nuclear layer, cells of both neuronal and glial origins showed positive immunoreactivity. Both the nuclei and the cytoplasm of the ganglion cells were positively stained. Retinal pigment epithelium showed patchy but consistent immunoreactivity. Capillary endothelial cells showed inconsistent positive staining. The pericytes were uniformly negative. In diabetic animals although overall intensity was increased, retinal pigment epithelium and ciliary epithelium showed no immunoreactivity. The corneal epithelium showed increased but patchy immunoreactivity. The altered intensity and distribution of ETs in diabetes suggest that ETs may be of importance in the pathogenesis of chronic occular complications in the diabetic BB/W rat.