ENDOTHELIN ASSOCIATION WITH CULTURED RAT HEPATIC ENDOTHELIAL-CELLS - FUNCTIONAL-CHARACTERIZATION

Endothelin is a potent vasoactive peptide whose concentration increase s in a number of pathophysiological states. In the intact animal, the liver is known to sequester approximate to 12% of an injected bolus of [I-125]endothelin-1 ([I-125]ET-1). Endothelial cells (ECs) isolated f rom rat liver were maintained in culture in order to examine their rol e in ET sequestration. LECs were shown to express predominantly ETB re ceptors both by association assays and by Northern blot analysis. In t hese cells the reaction between [I-125]ET-1 and its receptor was essen tially irreversible. Ligand binding experiments performed at 4 degrees C showed that LECs in early culture (approximate to 3 h) had 4.3 +/- 0.8 fmol of ET receptors per 10(6) cells; this number fell progressive ly to less than or equal to 1 fmol/l0(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 f reshly isolated cells, mRNA for the ETB receptor decreased 4-fold in e arly culture, and recovered somewhat at 24 h. At 37 degrees C [I-125]E T-1 bound by the cells was rapidly internalized, with concomitant down -regulation of ET receptors. Recovery of down-regulated ET receptors w as sensitive to cycloheximide, making short-term receptor recycling un likely. Metabolism of [I-125]ET-1 was low at short (< 4 h) exposure ti mes, and at 24 h showed a concentration dependence similar to that of ligand association, suggesting that ET-1 metabolism primarily was intr acellular. ET stimulation of Kupffer cells and other hepatic cell type s is known to activate phosphoinositide signaling, but no such activat ion 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 cont ribute substantially to the liver's previously reported ability to seq uester systemically administered ET. (C) 1997 Elsevier Science B.V.