Localization Of Endothelin Research Articles

EXPRESSION FEATURES OF CALCIUM-DEPENDENT RECEPTORS IN THE LUNGS IN EXPERIMENTAL COMPENSATED TRAUMATIC BRAIN INJURY

Purpose of the study: to assess the histochemical features of the localization of calciumdependent receptors of the left lung cell membrane on the 8th day of experimental compensated traumatic brain injury. Materials and methods: The object of the study was male albino outbred rats, three months old, weight 270-300 grams. Created two groups of animals: 1 - control: intact rats; 2- experimental: rats with TBI. To create a head injury, a model of a falling weight was used. Animals were withdrawn from the experiment on the 8th day of head injury. We studied paraffin sections of the left lung of rats. Using the Abcam kits (USA), an immunohistochemical study of the localization of endothelin -1 on sections of the left lung was carried out, the histochemical detection of calcium cations was performed using alizarin red C. Results. The morphofunctional characteristics of the lungs of rats with experimental traumatic brain injury were investigated. It was found that when the brain of experimental animals is damaged, lipid-associated lymphoid clusters appear in the root of the left lung of rats, and the content of endothelin-1 in cardiomyocytes of the pulmonary vein and epithelium of terminal bronchioles increases. A change in the histochemical characteristics of the localization of calcium cations in the cells and tissues of the left lung is revealed, which indicates a tendency towards the development of obstruction of terminal bronchioles and migration of macrophages into the parenchyma of the alveoli. Conclusion: Thus, with TBI, the mechanisms of humoral regulation of the respiratory system function significantly change. Histochemical study of endothelin 1 revealed that the expression in the lung tissues is calcium-dependent. receptor and mediator systems of cell membranes. These data are confirmed by staining the lungs with alizarin red C.

06 Localization of endothelin a receptor in the transverse tubules of cardiac myocytes

06 Localization of endothelin a receptor in the transverse tubules of cardiac myocytes

Endothelin immunoreactivity and mRNA expression in sensory and sympathetic neurones following selective denervation

The localization of endothelin (ET) in perivascular nerve varicosities supports pharmacological evidence that ET is a neurotransmitter in the autonomic nervous system. To examine the potential source of ET previously localized in cerebrovascular nerves, ganglia which send projections to these vessels were immunolabelled for ET and examined at the ultrastructural level. The trigeminal (TG) and superior cervical ganglia (SCG) were examined in control rats and following either sensory denervation or sympathectomy. In control TG, ET immunolabelling was detected throughout the cytoplasm of a subpopulation of neurones whereas in the SCG only the occasional ET-positive neurone was seen. Following sensory denervation with capsaicin, very few ET-immunoreactive nerve cell bodies or nerve fibres were detected in the TG compared with control ganglia, suggesting that ET is predominantly localized in primary afferent neurones, although some remaining myelinated nerve fibres stained positively. ET labelling of neurones in the SCG was unaffected by sensory denervation. Following selective damage to sympathetic nerves with 6-hydroxydopamine, there was a marked increase in intensity of ET-labelling of nerve fibres in the TG, probably due to increased availability of nerve growth factor for sensory nerves. There was no effect on ET immunoreactivity in the nerve cell bodies and nerve fibres within the SCG. However, in situ hybridization techniques demonstrated that 6-hydroxydopamine sympathectomy resulted in a marked increase in ET-1 mRNA expression in the SCG neurones. In conclusion, sensory nerves projecting from the TG are a more likely source of ET-positive perivascular nerves in cerebral arteries than sympathetic nerves from the SCG. Damaged sympathetic neurones markedly increase ET mRNA expression. In view of the neuroprotective properties of ET, this may represent a compensatory mechanism to promote repair.

Localization of endothelin A receptors in the rat pituitary TSH cells: light- and electron-microscopic immunohistochemical studies.

Endothelins modulate hormonal secretion in the pituitary gland. Intense signaling of endothelin A receptors (ET(A)R) has been detected by in situ hybridization, binding assay and receptor autoradiography. We used light- and electron-microscopic immunohistochemistry of ET(A)R with polyclonal antibody against a synthetic peptide corresponding to the carboxyl terminus (403-427) of human ET(A)R. Immunoreactivity was observed in 6-8% of anterior pituitary cells, which were rather large polygonal or stellate cells. These cells were often clustered. Double-staining immunofluorescence showed that the ET(A)R-positive cells immunoreacted with antibody against the beta-subunit of thyroid-stimulating hormone (TSH), but not adrenocorticotropic hormone (ACTH) or lutenizing hormone beta (LHbeta). Pre- and postembedding electron-microscopic immunohistochemistry showed that ET(A)R-positive cells had vacuolated or parallel-lined rough endoplasmic reticulum (rER) and numerous round granules in their periphery and the elongated processes. By pre-embedding immunohistochemistry, diaminobenzidine tetrahydrochloride (DAB) products were shown to be mostly located around the granules and occasionally underneath the plasma membrane. By postembedding immunohistochemistry, granules in the ET(A)R-positive cells were 90-150 nm in diameter, and colloidal gold particles due to ET(A)R were associated with about 10% of these granules. These results indicate that ET(A) receptors are associated mostly with the secretory granules of TSH cells.

Endothelin Receptor Subtype Distribution Predisposes Coronary Arteries to Damage

Several vasoactive drugs that lower blood pressure and increase heart rate induce regional cardiotoxicity in the dog, most frequently of right coronary arteries and right atrium. The basis for this selective damage is thought to result from local changes in vascular tone and blood flow. Administration of an endothelin receptor antagonist (ETRA, SB 209670) to dogs induced damage most frequent and severe in the right coronary artery and right atrium. Because site predisposition may correlate with distribution of vasoactive receptors, the objectives of this study were to map endothelin (ET) receptor distribution and density within regions of dog heart using both gene (mRNA) and protein expression endpoints for dog ET(A) and ET(B) receptors, and, additionally, correlate ET receptor subtype density with regional cardiac blood flow. A 10- to 15-mmHg reduction in mean arterial pressure with a concomitant increase in heart rate (10-20%), a six- and twofold increase in regional blood flow to the right and left atrium, respectively, and acute hemorrhage, medial necrosis, and inflammation were observed in the right coronary arteries and arteries of the right atrium after ETRA infusion for 5 days. Radioligand protein binding to quantify both ET receptors in normal dog heart indicated a twofold greater density of ET receptors in atrial regions versus ventricular regions. Importantly, ET receptor density in coronary arteries was markedly (about five- to sixfold) increased above that in atrial or ventricular tissues. ET receptor subtype characterization indicated ET(B) receptors were three times more prevalent in right coronary arteries compared to left coronary arteries and in situ hybridization confirmed localization of ET(B) in vascular smooth muscle. ET(A) receptor density was comparable in right and left coronary arteries. Quantitative real-time polymerase chain reaction for ET(A) and ET(B) receptor mRNA transcripts supported the site prevalence for message distribution. Consequently, the composite of protein and message expression profiles for ET(A) and ET(B) receptors indicated a disproportionate distribution of ET(B) receptors within right coronary artery of dog and this, along with functional measures of blood flow after ETRA infusion indicated a predisposition for exaggerated pharmacological responses and subsequent damage to right coronary arteries by ET and/or ETRAs.

The significance of endothelin for generation of endocochlear potential.

The role of intermediate cells (ICs) in the stria vascularis (SV) of the cochlear ducts in the generation of endocochlear potential (EP) is clear because certain mutants cannot generate EP. Recent reports have shown that endothelin (ET) stimulates or inhibits the function of Na+, K(+)-ATPase in various organs. This study was designed to examine the immunocytochemical localization of ET and its receptor in the SV. The cochlear ducts of WBB6F1 (+/+) mice and mutants (W/Wv) devoid of the ICs were used for light and electron microscopic immunocytochemistry using rabbit anti-ET-1, ET-3, and ETA receptor antisera. The location of Na+, K(+)-ATPase using rabbit anti-rat Na+, K(+)-ATPase was also examined. Immunoreactivity to Na+, K(+)-ATPase was seen in the marginal cells (MCs) in both species. Immunoreactivity to ET-1 and ET-3 was preferentially localized in the rough endoplasmic reticulum and cytoplasmic vesicles of the ICs and along the plasma membrane of the ICs and in the MCs apposed to the ICs. Immunoreactive sites for ETA were almost identical to those of ET-1 and ET-3. Because Na+, K(+)-ATPase activities also exist in the MCs of the mutants, it appears likely that ET, synthesized and released by the ICs, may participate in generation of EP by regulating the function of Na+, K(+)-ATPase for production of the endolymph of the MCs.

Transient, isopeptide-specific induction of myocardial endothelin-1 mRNA in congestive heart failure in rats.

Increased myocardial expression of preproendothelin-1 (ppET-1) mRNA has been associated with congestive heart failure (CHF) in rats. However, the time course and isoform pattern of ppET mRNA induction and the cellular localization of ET in failing hearts are unknown. Thus our aim was to investigate myocardial ppET mRNA expression in CHF rats during the first 6 wk after induction of myocardial infarction. Furthermore, performing immunohistochemical analysis, we also investigated the origin and localization of immunoreactive endothelin (ET) in different regions of the failing heart. Ribonuclease protection assays revealed a marked increase in ppET-1 mRNA levels in rat myocardial tissues during CHF. The induction of ppET-1 mRNA was isopeptide specific and transient. The most substantial upregulation was observed in the infarcted area, where maximal expression of ppET-1 mRNA was observed after 7 days (25-fold increase, P < 0.05). However, a marked and statistically significant induction of ppET-1 mRNA was also observed in the nonischemic myocardium. Immunohistochemical analysis revealed ET-1-like immunoreactivity in cardiomyocytes, vascular endothelial cells, macrophages, and proliferating fibroblasts. Thus immunohistochemistry revealed the structural basis for the dramatic upregulation of the myocardial ET system in the infarcted region, suggesting a role for ET in the healing process after myocardial infarction. However, the global upregulation of ppET-1 mRNA in the heart also suggests an autocrine/paracrine regulatory mechanism in the nonischemic myocardium during CHF.

Endothelin production and effects of endothelin antagonism during experimental airway inflammation.

Endothelin (ET) has strong bronchoconstrictor properties, stimulate mucus secretion and mucosal edema, and may also exert proinflammatory effects. Therefore, ET may play a pathogenic role in inflammatory airway diseases such as bronchial asthma. The production and localization of ET and the effect of blocking ET receptors was investigated in rats during airway inflammation induced by intratracheal instillation of dextran (Sephadex). We observed a considerable increase in the concentration of ET in bronchoalveolar lavage fluid (BALF) during the early phase of inflammation, with an increase from 2.2 +/- 0.6 pg/ml in controls to 40.0 +/- 6.7 pg/ml at Day 1, declining to 5.3 +/- 1.1 pg/ml at Day 14. Correlated with the ET response in BALF was a considerable increase in total cell count (r = 0.61), eosinophils (r = 0.83), and neutrophils (r = 0.81). Plasma ET was not elevated. Immunohistochemical analyses revealed ET-like staining in the bronchial epithelium. Treatment with the ET receptor antagonist bosentan inhibited the increase in eosinophils in BALF and reduced the inflammatory reaction in the lung tissue. In summary, a considerable increase in the ET concentration in BALF was demonstrated during the acute phase of an experimental eosinophilic airway inflammation, coinciding with an increased ET-like immunostaining in the bronchial epithelium. Treatment with an ET receptor antagonist inhibited the inflammatory response in BALF and in the tissue.

Regular immunohistochemical localization of endothelin-1 and endothelin-B receptor in normal, hyperplastic and neoplastic human adrenocortical cells.

The localization of endothelin (ET)-1/big ET-1, ET-3/big ET-3, ET-A and ET-B receptor was immunohistochemically examined in human adrenal glands composed of 36 normal cases, nine hyperplasia, 70 adenomas and seven carcinomas of cortical cells. In normal adrenals, ET-1/big ET-1 and ET-B receptor were regularly detected in the cortical cells, especially in the zona fasciculata for ET-1 and zona glomerulosa for ET-B receptor but not in the medulla, while ET-A receptor localized occasionally in endothelial cells or rarely in cortical cells and ET-3/big ET-3 was very limited in the cortical cells. In hyperplasia, adenoma and carcinoma, ET-1/big ET-1 and ET-B receptor showed frequent localization, although focal distribution of the ET-B receptor was rather predominant in these groups. ET-A receptor and ET-3/big ET-3 were very infrequently expressed. Functioning versus non-functioning and hypertensive versus normotensive cases revealed no significant differences in the frequency of positive cells for ET-1/big ET-1, ET-3/big ET-3, ET-A receptor or ET-B receptor. Alternatively, the frequency of immunoreactivity to ET-1/big ET-1 or ET-B receptor significantly decreased in hyperplasia, adenoma and carcinoma, when compared with that of normal adrenal cortex. The present study, therefore, indicates that ET-1/big ET-1 and ET-B receptor are a prevalent ligand-receptor system in normal and hyperplastic/neoplastic adrenocortical cells, even with a malignant profile, and may contribute in maintaining adrenocortical cell function or cell viability but not cell growth or systemic hypertension.

Immunohistochemical and in situ Hybridization Studies on Endothelin in the Rat Eye.

To demonstrate the distribution of endothelin (ET) in ocular tissues, we immunohistochemically elucidated the localization of ETs (ET-1, ET-2, ET-3) and their precursors (Big ETs) using specific antibodies that can distinguish precursor and mature types of each isopeptide. ET-A receptor and ET-1 and ET-3 mRNAs were also examined using immunohistochemical and in situ hybridization methods, respectively. Coincidentally, the positive reactivity for ET-1 and ET-3 mRNAs, Big ET-1, Big ET-2, Big ET-3, mature ETs and ET-A receptor was found in almost the same element of the rat ocular tissues, e. g., the retinal neural cells (ganglion, amacrine, bipolar and horizontal cells), the vessel walls in the retina and choroid, the non-pigment epithelium in the ciliary body and iris, the corneal epithelium (anterior surface) and the endothelium (posterior surface), and the epithelium of the lens. These observations indicated that the various ocular tissues biosynthesize and secrete all the isopeptides of ET which act in an autocrine/paracrine fashion, regulating the neural transmission, intraocular pressure, iris smooth muscle tone and ocular blood vessel tone.

Decreased contractile effect of endothelin-1 on hyperplastic prostate

1. 1. The contractile activity, binding activity and localization of endothelin (ET)-1 were evaluated in human nonhyperplastic (control) and hyperplastic prostates. 2. 2. ET-1 caused contraction of both prostates in a dose-dependent manner. However, this contraction was markedly decreased in hyperplastic prostates. 3. 3. B max and K d values of hyperplastic prostates were greater than those of the control. 4. 4. The muscle and proliferative epithelium of hyperplastic prostates showed strong staining for the anti-ET-1 antibody. However, the glandular epithelium of control prostates was weakly stained. 5. 5. These findings indicate that responsiveness to ET-1 is decreased, though the ET-1 and ET-1 receptors increase in the hyperplastic prostate. Namely, the increase in ET-1 receptors is not effective in regulating the contractile response of the prostate, because its expression is rather dominant in proliferated gland. 6. 6. These suggest that ET-1 may not have an important role in the release of the obstructive symptoms of benign prostatic hypertrophy.

Two subtypes of endothelin receptors and endothelin peptides are expressed in differential cell types of the rat placenta: in vitro receptor autoradiographic and in situ hybridization studies.

We studied the localization of endothelin (ET) receptors and ET peptides in the rat placenta. In vitro receptor autoradiographic and in situ hybridization studies revealed the differential and cell-specific distribution of ET receptor subtypes, suggesting that each ET receptor plays a different role in the function of the placenta. The expression of the ETB receptor was concentrated to cytotrophoblasts and trophoblastic giant cells of the basal zone, in which fetal cells directly face maternal cells. The ETA receptor was confined to the decidual tissue and vascular wall. Both ET receptors coexisted in the labyrinth in an approximately 50:50 ratio. Prepro-ET-1 messenger RNA (mRNA) was detected in cytotrophoblasts and trophoblastic giant cells of the basal zone and endothelial cells of vessels, whereas ET-1-like immunoreactivity was present not only in trophoblasts and endothelial cells, but also in the decidual tissue and vascular wall. ET-3 mRNA was localized in migrating cells. We also found changes in the expression levels of ET receptors by means of a cold ligand saturation study. The number of specific [125I]ET-1-binding sites was increased in the basal zone and labyrinth with gestation, but not in the decidual tissue. The enhancement of ETA receptor, ETB receptor, and prepro-ET-1 mRNA levels was also supposed, based on data obtained by RT-PCR Southern hybridization. On the other hand, ET-3 mRNA levels were reduced with gestation. These findings support the idea that ETs, through interaction with ETA and ETB receptors, play an important role in the regulation of placental growth and fetoplacental circulation through autocrine and paracrine mechanisms.

Localization of Endothelin-Like Immunoreactivity and Endothelin Binding Sites in Rabbit Eyes

Immunohistochemistry and in vitro autoradiographic techniques were used to investigate the localization of endothelin (ET)-like immunoreactivity and [125I]ET-1 binding sites in sections of ocular tissues of normal rabbits. ET-like immunoreactivity was localized to the corneal endothelium and the pre-epithelial layer of the cornea, which was specifically inhibited by addition of exogenous ET-1. Immunoreactivity for ET was also observed in the retinal ganglion layer and periocular fat tissues. These, however, were not inhibited in the presence of ET-1. Autoradiography showed binding sites for ET-1 in the iris and ciliary body. Emulsion-dipped slides of sections of rabbit eyes incubated with [125I]ET-1 showed specific labeling for binding sites over structures that were identified from serial sections stained with hematoxylineosin. These were the anterior stroma of the iris root, the walls of capillaries, the circular arteries, and the veins of the ciliary body and extraocular muscles. No binding sites were found in the cornea or conjunctiva. The difference in regional distributions of ET storage and binding sites in rabbit ocular tissue suggests that the peptide may have specific physiologic functions.

A novel ligand, [ 125I]BQ-3020, reveals the localization of endothelin ET B receptors

The precise localization of an endothelin (ET) receptor subtype, the ET B receptor, in porcine lung was elucidated by in vitro microautoradiography using a novel ET B-selective radioligand, [ 125I]BQ-3020 ([ 125I-Tyr]-N-acetyl-Leu-Met-Asp-Lys-Glu-Ala-Val- Tyr-Phe-Ala-His-Leu-Asp-Ile-Ile-Trp). Of the labeled native ET isopeptides, [ 125I]ET-3 is selective for ET B receptors. However, [ 125I]ET-3 was not suitable for autoradiography due to its high degree of non-specific binding. On the other hand, [ 125I]BQ-3020 showed extremely low non-specific binding on autoradiography. The distribution of [ 125I]BQ-3020 binding in porcine lung was clearly different from that of [ 125I]ET-1, which showed more widespread binding than [ 125I]BQ-3020 due to a high affinity to both ET A and ET B receptors. [ 125I]BQ-3020 was found to bind to parenchyma, parasympathetic ganglia, pulmonary and submucosal plexuses, but bound only slightly to circular smooth muscle layers and the epithelium of airway tracts. Although [ 125I]ET-1 bound to the smooth muscle layer of all blood vessels, the binding of [ 125I]BQ-3020 differed among blood vessels. [ 125I]BQ-3020 binding in blood vessels paralleled acetylcholinesterase activity, suggesting that ET B receptors in blood vessels are located on parasympathetic nerves. Thus, the radioligand [ 125I]BQ-3020 is very useful for studying the precise localization of ET B receptors.

Steroid modulation of oxytocin/vasopressin receptors in the uterus

Oxytocin (OT) and V1 vasopressin (VP) receptors are present simultaneously in several tissues, including the uterus. In myometrium these receptors mediate contractility, while in endometrium they mediate the release of other uterotonic substances as endothelin (ET). In rabbit myometrium, estrogens increase, while progesterone blunts neurohypophysial hormone receptors. However, the action of sex steroids on OT and V1 VP receptors differs in terms of the ED 50 and maximal effect. Therefore, at parturition, only OT receptors show a dramatic rise, while V1 VP receptors do not change, suggesting a major role for OT in labor. ET is a potent stimulator of uterine activity acting through specific receptors present on myometrial cells. These receptors as well as the endometrial localization of ET are modulated by sex steroids, indicating that ET might represent a paracrine regulator of uterine activity. In humans, OT but not V1 VP receptors increase as pregnancy progresses, confirming the primary relevance of OT in timing delivery.

Endothelin 1, an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root ganglia.

The localization of endothelin 1 mRNA and endothelin-like immunoreactivity was investigated in samples of neurologically normal nervous system tissue from 10 adults by using in situ hybridization and immunocytochemistry. Tissue sections of spinal cord and dorsal root ganglia were hybridized with an 35S-radiolabeled endothelin 1 complementary RNA probe. Autoradiograms showed labeled neurons in the spinal cord (laminae IV-VI and many motoneurons) and numerous small and large neurons in the dorsal root ganglia. Endothelin 1 transcripts were also found in association with the endothelial layer of some blood vessels in the white matter of the spinal cord. A similar distribution of immunoreactivity was seen using three antisera to endothelin 1, but fewer cells were immunostained than were labeled after the hybridization. Two other endothelin 1 antisera immunostained the endothelial lining of blood vessels in the spinal cord white matter but not neurons. In the ganglia, endothelin 1 transcripts were localized to all cells expressing beta-preprotachykinin and most expressing calcitonin gene-related peptide mRNAs; in the majority of the motoneurons, coexistence of endothelin 1 and calcitonin gene-related peptide mRNAs was noted. There was a similar pattern of coexistence for respective peptide immunoreactivities in the ganglia and spinal cord. The expression of endothelin 1 mRNA in distinct neuronal cell types suggests that this peptide plays a part in neural transmission/modulation and adds a further dimension to the known vascular-related actions of endothelin 1.