Sinusoidal endothelial liver cells in vitro release endothelin. Augmentation by transforming growth factor beta and Kupffer cell-conditioned media
ABSTRACT Endothelin is the most potent vasoconstrictor peptide known today. Using a radioimmunoassay for endothelin, we measured immunoreactive endothelin in culture media of guinea pig sinusoidal endothelial liver cells and human umbilical vein endothelial cells. A time-dependent release of immunoreactive endothelin by confluent sinusoidal endothelial liver cells in culture was found. Sinusoidal endothelial liver cells produced similar amounts of immunoreactive endothelin as umbilical vein endothelial cells, about 900 pg/microgram DNA per 24 h. In the presence of transforming growth factor beta a dose-dependent increase of immunoreactive endothelin release was measured. The maximal increase of 50% was found at a concentration of 1 ng transforming growth factor per ml. To a similar extent Kupffer cell-conditioned media augmented the release of immunoreactive endothelin by sinusoidal endothelial liver cells, especially when Kupffer cells had been stimulated by endotoxin. Endotoxin itself did not alter the release of immunoreactive endothelin. Endothelin released by sinusoidal endothelial liver cells might influence the pericytes of the liver, i.e., the Ito-cells.
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ABSTRACT: Postcapillary endothelium at the sites of inflammation undergoes a series of changes collectively termed endothelial cell activation. Activated endothelium expresses immunologically relevant surface proteins that include MHC class II antigens (Ags) and adhesion proteins, as well as exhibits a number of functional changes. Endothelial activation has not been thoroughly studied in CNS endothelium. We have examined cytokine-mediated endothelial activation in isolated rat CNS microvessels. Freshly isolated rat CNS microvessels are viable in culture for at least 72 h. Untreated microvessels express no endothelial activation antigens, but do exhibit constitutive expression of the transferrin receptor (tfR). INFγ induces a dose-dependent increase in both MHC class II antigens and tfR measured by immunofluorescent staining and quantitated by laser cytometry. IFNγ-mediated endothelial cell activation could be inhibited with as little as 2 ng/mL TGF-β1, although 100% inhibition was seen with 10 ng/mL TGF-β1. Cytokinepreactivated endothelial expression of class II Ag and tfR could also be inhibited by TGF-β1. TGF-β1-treated microvessels become anergic to IFNγ stimulation. Results suggest that TGF-β1 may have a regulatory role in endothelial activation.Neurochemical Pathology 08/1994; 22(3):161-175. DOI:10.1007/BF03160103
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ABSTRACT: Endothelin, a potent vasoactive peptide originally isolated from the vascular endothelial cells, exerts glycogenolytic and vasoconstrictive actions in the perfused rat liver. In this paper we demonstrate high-affinity binding sites for endothelin-1 (ET-1) on rat hepatocytes. Upon incubation at 37 degrees C, association of ET-1 with hepatocytes occurred in a time-dependent manner, was maximal between 3 and 6 h, and subsequently declined; at this temperature ET-1 was rapidly internalized with the internalized ligand exceeding the surface-bound ligand at all time points. The rate of association of 125I-ET-1 with hepatocytes was much slower when the binding assay was performed at 4 degrees C; sequestration of ET-1 in hepatocytes was also substantially reduced at this temperature. ET-1 was extremely potent in stimulating phosphoinositide metabolism in hepatocytes, with significant activation of this signal transduction process occurring at ET-1 concentrations as low as 0.1 pM, with an EC50 of 1 pM. The effect of ET-1 was coupled via a pertussis toxin-sensitive G-protein. Cholera toxin did not affect ET-1-mediated phosphoinositide metabolism and neither toxin influenced the association of 125I-ET-1 with hepatocytes. PAGE of hepatocyte membranes following exposure of the cells to 125I-ET-1 and cross-linking revealed labelling of three major proteins with apparent molecular masses of 32, 49 and 72 kDa. 125I-ET-1 labelling of each of these proteins was inhibited by unlabelled ET-1, whereas unlabelled ET-3 inhibited the labelling of only the 32 and 49 kDa proteins. 125I-ET-3 labelled the 49 kDa protein and this labelling was inhibited by both unlabelled ET-1 and ET-3. Each of these receptors appears to be functional, since both ET-1 and ET-3 stimulated phosphoinositide metabolism in hepatocytes. Down-regulation of ET-1 association and desensitization of ET-1-induced phosphoinositide metabolism occurred upon incubation of hepatocytes with the homologous ligand. Following down-regulation, the ET-1 receptor was restored to the surface of the hepatocyte by prolonged incubation, although the ET-1-stimulated phosphoinositide response remained inhibited even after complete recovery of the ET-1 association capability. These results demonstrate the presence of multiple high-affinity receptors for ET-1 on hepatocytes and the direct action of this peptide on hepatic parenchymal cells via the phosphoinositide signal transduction pathway.Biochemical Journal 12/1992; 287 ( Pt 3)(3):897-904. DOI:10.1042/bj2870897 · 4.40 Impact Factor
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ABSTRACT: This study was designed to investigate the mechanism for ethanol-induced hepatic vasoconstriction in isolated perfused rat liver. Upon initiation of ethanol infusion into the portal vein at concentrations ranging from 25 to 100 mM, portal pressure began to increase in a concentration-dependent manner and reached maximal levels in 2-5 min (initial phase), followed by a gradual decrease over the period of ethanol infusion (escape phenomenon). Endothelin-1 antiserum significantly inhibited this ethanol-induced hepatic vasoconstriction by 45-80%. Cessation of infusion of endothelin-1 antiserum was followed by a subsequent increase in portal pressure. On the other hand, when a nitric oxide synthesis inhibitor, NG-monomethyl-L-arginine (L-NMMA), was infused into the portal vein simultaneously with ethanol, the initial phase of the response of portal pressure to ethanol was not altered and the peak values of portal pressure remained unchanged. However, after the peak increase in portal pressure, the rate of decrease was less than in the absence of L-NMMA. Thus, L-NMMA diminished the escape phenomenon and sustained the vasoconstriction. This study supports the hypothesis that two endothelium-derived vasoactive factors, endothelin-1 and nitric oxide, regulate hepatic vascular tone in the presence of ethanol.Journal of Clinical Investigation 05/1993; 91(4):1337-42. DOI:10.1172/JCI116334 · 13.22 Impact Factor