Endothelin in human plasma and culture medium of aortic endothelial cells—Detection and characterization with radioimmunoassay using monoclonal antibody
Abstract
We have developed monoclonal (KY-ET-1-I) and polyclonal (ET-F5) antibodies against endothelin-1 (ET-1) and established sensitive radioimmunoassays (RIAs) with different specificities. The RIA with KY-ET-1-I detected ET-1, ET-2 and ET-3, while the RIA with ET-F5 recognized ET-3 very weakly. Using these RIAs, we have investigated the concentration and molecular forms of ET-1-like immunoreactivity (-LI) in culture medium of bovine aortic endothelial cells and human plasma. Culture medium of endothelial cells contained two major components compatible with big ET and ET-1. ET-1-LI was also detected in human plasma. ET-1-LI in human plasma consisted of apparent two components, the small molecular form emerging at the position of ET-1 and the large form with the peak eluting at the preceding fraction of the elution position of big ET. The concentration of the small form of ET in human plasma was about 5 pg/ml.
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Cited by (86)
Endothelins: Vasoactive modulators of renal function in health and disease
2001, Pharmacology and TherapeuticsVasoactive autocoids with directly opposing actions on the renal vasculature, glomerular function, and in salt and water homeostasis have been demonstrated in the kidney. In the renal cortex, endothelin (ET)-1 and angiotensin-II cause vasoconstriction, decreasing renal blood flow, and glomerular filtration rate, whereas bradykinin and atrial natriuretic peptide cause vasodilation and increase glomerular capillary permeability. ET-1 causes vasoconstriction of the afferent and efferent arteries and outer medullary descending vasa recta, thereby decreasing vasa recta and papillary blood flow, while bradykinin has the opposite effect. ET-1 stimulates cell proliferation, increasing the expression of several genes, including collagenase, prostaglandin endoperoxidase synthase, and platelet-derived growth factor. ET-1 promotes natriuresis via the ET-B receptor, causing down-regulation of the epithelial Na+ channel in the renal tubule. Thus, ETs affect three major aspects of renal physiology: vascular and mesangial tone, Na+ and water excretion, and cell proliferation and matrix formation.
Endotoxin markedly elevates plasma concentration and gene transcription of adrenomedullin in rat
1995, Biochemical and Biophysical Research CommunicationsA marked increase in adrenomedullin (AM) concentration was observed in rat plasma collected after intravenous infusion of lipopolysaccharide (LPS). Plasma immunoreactive (ir-) AM concentration increased already after 1 h exposure to 5 mg/kg of LPS and was elevated to 49.7±2.3 fmol/ml (mean±SEM) at 3 h after injection in contrast to 2.7±0.3 fmol/ml in saline-injected control rats. Plasma ir-AM level increased in a dose-dependent manner in a range of 0.008 to 5.0 mg/kg of LPS. AM gene transcription in LPS-injected rats was augmented more than 3-fold in ileum, liver, lung, aorta, and 2.4-fold even in skeletal muscle, in which AM was not thought to be produced in myocytes. These results, along with our recent data that AM production in cultured vascular smooth muscle cells (VSMCs) is stimulated with LPS, indicate that AM production is highly augmented in blood vessel, lung and intestine by administration of LPS. Since VSMCs express AM-specific receptors, secreted AM is deduced to actually exert a vasorelaxant effect especially in the endotoxin shock.
Endothelin-1 levels in plasma and cerebrospinal fluidfollowing subarachnoid haemorrhage
1995, Journal of Clinical NeuroscienceA serial measurement of endothelin-1(ET-1) levels in plasma, cisternal and ventricular cerebrospinal fluid(CSF) was performed in 16 patients with subarachnoid haemorrhage (SAH). The patients were classified as grade III or IV according to the clinical grade of Hunt and Hess, and computerised tomography(CT) was classified as Fisher's CT group 3. Cisternal and ventricular CSF and plasma were obtained from the patients on the day of operation days 0–3, days 5–8 and days 14–18 after SAH. ET-I concentration in each sample was quantified by sandwich-enzyme immunoassay. ET-I levels in plasma and CSF were the highest between days 0–3 and then decreased. The ET-I levels in the cisternal CSF were significantly higher during days 0–3(p<0. 01) and days 5–8(p<0. 01) than those in the ventricular CSF It is suggested that ET-I could play an important role in the early stages of the cerebral vasospasm.
Endothelin-1 inhibits the adipose differentiation of cultured human adipocyte precursor cells
1994, MetabolismWe studied the effect of endothelin-1 (ET-1) on the differentiation of adipocyte precusor cells obtained from human adipose tissue and cultured in a serum-free hormone-supplemented medium. ET-1 was found to inhibit in a dose-dependent manner the accumulation of lipid droplets and the expression of glycerol-3-phosphate dehydrogenase (GPDH), a marker of adipose differentiation. The half-maximal inhibitory effect was observed in the range of 8.5 × 10−10 mol/L. Full inhibition required the continuous exposure of the cells to ET-1. The prevention of adipose conversion was not associated with a stimulation of mitogenesis. The presence of staurosporine, an inhibitor of the protein kinase C signaling pathway, completely prevented the inhibitory effect of ET-1 on adipose differentiation. Addition of ET-1 to newly developed fat cells also caused a suppression of GPDH activity without changing adipocyte morphology. Again, the magnitude of this effect was dependent on the exposure time. These findings suggest that ET-1 is a potent modulator of fat cell formation in man, which may act through activation of protein kinase C. Because of the close spatial relationship between fat cell precursors and blood vessels, ET-1 may exert its action in a paracrine manner.
Endothelin family peptides in human plasma and urine: Their molecular forms and concentrations
1994, PeptidesMolecular forms and concentrations of endothelin (ET) family peptides in normal human plasma and urine were investigated using five different sandwich-type enzyme immunoassays (sandwich-EIAs) and reverse-phase high performance liquid chromatography (RP-HPLC). We found that immunoreactive (IR-) big ET-3 and IR-big ET-2 were the major species in both plasma and urine from normal humans. Big ET-2(1–38) was found to be the major form of IR-big ET-2 in plasma. Urinary IR-big ET-2 and plasma and urinary IR-big ET-3 were heterogeneous, and the IR-big ET-3s were larger () than authentic big ET-3. IR-big ET-1, IR-ET-1, and IR-ET-3 were found at relatively low concentrations in human plasma and urine, and their major forms were identical to authentic big ET-1, ET-1, and ET-3, respectively. Although Et-1 and other ET family peptides have often been measured using RIAs with board-spectrum antibodies, the presence of high concentrations of IR-big ET-2 and IR-big ET-3, but not IR-ET-1, in plasma and urine indicates that the clinical significance of ET family peptides should be investigated with assay methods specific to each ET and big ET.
Inhibition of the Pharmacological Effects of Endothelin
1994, Progress in Medicinal ChemistryThis chapter focuses on the inhibition of the pharmacological effects of endothelin. Endothelin is one of the most potent vasoconstrictors known and its effects on the vasculature are characterized by an unusually long duration of action. Endothelin is also a potent cellular mitogen and a constrictor of non-vascular smooth muscle. With such profound biological actions, it is of little surprise that a vast amount of effort has been directed toward establishing the physiological or pathological role of endothelin and toward inhibiting its actions. The pharmacological effects of endothelin can be prevented either by inhibiting the formation of endothelin or by blocking the action of endothelin at its receptors. Endothelin also has effects on isolated cardiac preparations, although these are less marked than on vascular tissues. The positive inotropic response varies considerably between species. Endothelin has many other biological actions, but among the most important of these are the effects on the airways, the gastro-intestinal tract, the kidney and the central nervous system. In addition to its effects on the pulmonary circulation, endothelin has marked effects on non-vascular airway smooth muscle.