Endothelin association with cultured rat hepatic endothelial cells: functional characterization.
Endothelin is a potent vasoactive peptide whose concentration increases in a number of pathophysiological states. In the intact animal, the liver is known to sequester approximately 12% of an injected bolus of [125I]endothelin-1 ([125I]ET-1). Endothelial cells (ECs) isolated from rat liver were maintained in culture in order to examine their role in ET sequestration. LECs were shown to express predominantly ET(B) receptors both by association assays and by Northern blot analysis. In these cells the reaction between [125I]ET-1 and its receptor was essentially irreversible. Ligand binding experiments performed at 4 degrees C showed that LECs in early culture (approximately 3 h) had 4.3 +/- 0.8 fmol of ET receptors per 10(6) cells; this number fell progressively to < or = 1 fmol/10(6) cells during 24 h of culture. The decrease in receptor numbers could be blocked by maintaining the cells at 4 degrees C. Northern blot analysis showed that relative to freshly isolated cells, mRNA for the ET(B) receptor decreased 4-fold in early culture, and recovered somewhat at 24 h. At 37 degrees C [125I]ET-1 bound by the cells was rapidly internalized, with concomitant down-regulation of ET receptors. Recovery of down-regulated ET receptors was sensitive to cycloheximide, making short-term receptor recycling unlikely. Metabolism of [125I]ET-1 was low at short (< 4 h) exposure times, and at 24 h showed a concentration dependence similar to that of ligand association, suggesting that ET-1 metabolism primarily was intracellular. ET stimulation of Kupffer cells and other hepatic cell types is known to activate phosphoinositide signaling, but no such activation was seen in LECs. Moreover, ET did not appear to stimulate protein tyrosine kinase activity in LECs. While hepatic LECs may lack some of the ET-dependent responses seen in other cell types, they likely contribute substantially to the liver's previously reported ability to sequester systemically administered ET.
Eakes
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Department of Biochemistry, The University of Texas Health Science Center at San Antonio, 78284-7760, USA.
Harvey
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SA
Olson
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D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D015536:Down-Regulation / Q000502:physiology; D019332:Endothelin-1 / Q000378:metabolism* / Q000494:pharmacology; D004727:Endothelium / Q000166:cytology / Q000378:metabolism; D007457:Iodine Radioisotopes; D007728:Kupffer Cells / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D010716:Phosphatidylinositols / Q000378:metabolism; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013997:Time Factors
D019332:Endothelin-1; D007457:Iodine Radioisotopes; D010716:Phosphatidylinositols; D012333:RNA, Messenger; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D003513:Cycloheximide; D011505:Protein-Tyrosine Kinases