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.

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.

Endothelin expression and responsiveness in human ovarian carcinoma cell lines.

To elucidate the potential role of endothelins (ETs) as growth regulators in ovarian carcinoma cells in culture, expression of endothelins and their receptors were measured in two ovarian cancer cell lines (PEO4 and PEO14), together with the effect of the exogenous addition of endothelins on the growth of these cell lines in vitro. RT-PCR analysis of mRNA prepared from PEO4 and PEO14 indicated the presence of ET-1 and ET-3 mRNA. Immunoreactive ET-1-like peptide was found in media from cultures of both PEO4 (1.7 +/- 0.4 fmol/10(6) cells/72 h) and PEO14 (20.2 +/- 6.8 fmol/10(6) cells/72 h) cell lines. Radioligand binding studies using 125I-ET-1 and membrane fractions were consistent with PEO4 cells having two receptor sites of either high affinity (Kd = 0.065 nM, Bmax = 0.047 pmol/mg protein) or lower affinity sites (Kd = 0.49 nM, Bmax = 0.23 pmol/mg protein). Studies using membrane fractions of PEO14 cells indicated that this cell line has only a single lower affinity binding site (Kd = 0.56 nM, Bmax = 0.31 pmol/mg protein). However, RT-PCR analysis indicated the presence of mRNA from both ETA and ETB receptors in PEO4 and PEO14 cell lines. Exogenous addition of ETs to PEO4 and PEO14 cells at concentrations of 10(-10)-10(-7)M resulted in specific dose-dependent increases in cell number for ET-1 (with maximum effects at 10(-10) and 10(-9)M for PEO4 and PEO14, respectively) and ET-2 (maximum effects at 10(-8) and 10(-9)M for PEO4 and PEO14, respectively) but not for ET-3. Experiments on the growth of PEO14 cells using BQ123 (ETA-R) antagonist and "antisense" oligonucleotide against the ETA-R, in the absence of exogenous ETs, suggested that immunoreactive ET-1-like material secreted by PEO14 cells can affect their growth in an autocrine manner. These results would be consistent with ET-1 acting as a possible autocrine growth regulator in human ovarian carcinoma cells.

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.

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.

The characterization, localization and regulation of endothelin in ovine pars intermedia.

The pituitary intermediate lobe (IL) contains a single population of cells and has recently been shown to express endothelin (ET)-like peptides. The IL thus provides an excellent in vivo model to study regulation, function and processing of ET in an endocrine cell. The primary aims of the present study were to locate and characterize the precise molecular forms of ET in the ovine IL and determine if levels and/or processing of ET is under dopaminergic or other influences. We have developed a radioimmunoassay (RIA) that detects each form of ET and, when combined with reverse phase-HPLC (RP-HPLC), shows the ovine IL to contain predominantly the ET-1 isoform. In addition, using a specific anti-endothelin antiserum for immunohistochemistry (IHC), we localized ET-1 with alpha-melanocyte stimulating hormone (alpha-MSH) within the melanotroph. The effects of dopamine agonists, antagonists and hypothalamo-pituitary disconnection (HPD) on both tissue levels and processing of ET in the ovine IL were also examined. Normal sheep were treated chronically with haloperidol or bromocriptine to investigate the possibility of dopaminergic regulation of ET in the IL. In the haloperidol-treated group, plasma prolactin levels did not vary significantly from day 0 to day 8, but the bromocriptine treatment reduced prolactin levels (t = 9.4 P < 0.01). Neither bromocriptine nor haloperidol, however, affected tissue ET peptide levels or forms. After HPD, the HPLC profile of pooled IL showed that ET-1 levels in the IL are slightly increased with no change in molecular forms.

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.

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.

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.

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.

Localization of endothelin-1 and endothelin-3 in the cochlea.

The distribution of endothelin-1 (ET-1) and endothelin-3 (ET-3) was studied by indirect immunostaining of decalcified guinea pig and rat cochleae. No species differences were observed. Perikarya and processes of spiral ganglion cells were highly reactive for both ET-1 and ET-3. The epithelial lining of the cochlear duct stained for ET-1 and ET-3, but reactivity for ET-1 was higher in the lining cells of the inner sulcus, Claudius', and Hensen's cells, while the tympanic covering layer of the basilar membrane stained stronger for ET-3 compared to ET-1. In the stria vascularis, all cell types stained for ET-3, while marginal cells were more reactive for ET-1. Spiral ligament fibroblasts were reactive for ET-1, but not for ET-3. Connective tissue cells of the spiral limbus stained for both endothelins. The region of synapses on outer hair cells reacted for ET-1 and ET-3 but sensory cells remained unstained. Endothelins are discussed to act as modulatory peptides, possibly interfering with nitric oxide, prostaglandins, and atrial natriuretic peptide in the lateral cochlear wall (lateral cochlear wall, i.e. stria vascularis and spiral ligament). The occurrence of endothelins in cochlear neurons suggest their potential role as neurotransmitters.

[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.

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.

Endothelins in canine genitourinary tissues.

PURPOSE: To characterize the functional effect of endothelin-1 (ET1) and endothelin-3 (ET3), immunohistochemically localize ET1-like immunoreactivity, and measure the tissue levels of immunoreactive endothelin (irET) in canine genitourinary (GU) tissues. MATERIALS AND METHODS: Canine GU tissues were characterized by measuring ET1 levels using a RIA, immunohistochemical staining of ET1 and isometric tension studies. RESULTS: Immunoreactive endothelin was present, to varying degrees, in the vas deferens, ureter, prostate, bladder and urethra. Functionally, ET1 demonstrated the typical concentration response characteristics in the canine bladder base, bladder body, and prostate. The maximal tension (Emax) measured following ET1 challenge was approximately 20-fold greater in the bladder body (0.67 +/- 0.21 g/mm.2) and bladder base (0.48 +/- 0.18 g/mm.2) as compared to the prostate 0.04 +/- 0.001 g/mm.2 The Emax of ET3 in the bladder body (0.31 +/- 0.12 g/mm.2) and bladder base (0.19 +/- 0.08 g/mm.2) was significantly lower than the corresponding Emax of ET1. No measurable contractile response was elicited by ET3 in the canine prostate. Immunohistochemical staining localized the ET-like immunoreactivity to the glandular epithelium of the prostate and the transitional epithelium of the bladder. CONCLUSIONS: Endothelins are ubiquitous in the canine lower GU tract with predominant localization to the epithelial elements. Endothelins are also functionally active in canine GU tissues, but the specific role of endothelins in the physiology and pathophysiology of GU tissues requires further investigation.

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, 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.

Localization of endothelin peptides in human kidney.

We investigated the synthesis and localization of endothelin isoforms in the human kidney using the reverse-transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. PCR products corresponding to the expected size for mRNA encoding ET-1, ET-2 and ET-3 were found in homogenates of renal medulla, cortex and vessels from each of five individuals. Using four rabbit polyclonal antibodies to assess the distribution of mature ET, Big ET-1, Big ET-2 and Big ET-3 immunoreactivity in the human kidney, mature IR ET localized to the cytoplasm of endothelial cells lining intra-renal blood vessels including interlobular and arcuate arteries, arterioles and adjacent arcuate veins, all of which showed strongly positive staining. IR Big ET-1 co-localized with the mature peptide. No specific staining was detected within these anatomical regions when pre-immune sera were substituted or primary antibody omitted. Mature IR ET also localized to the cytoplasm of endothelial cells within the glomerulus. Other capillary endothelial cells did not stain, and other structures stained only faintly by comparison. IR Big ET-2 and Big ET-3 could not be detected. These results show that human kidney contains mRNA encoding all three peptide isoforms, but only mature ET and Big ET-1 peptides could be detected by immunocytochemical staining. This provides further evidence that ET-1 may function as a renal peptide in humans, as it is locally synthesized within the kidney.

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.

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).