ETB-mediated regulation of extracellular levels of endothelin-1 in cultured human endothelial cells.
Our previous report suggests the important role that ETB receptors play in the clearance of circulating endothelin (ET)-1. The present study confirmed this finding by measuring the extracellular levels of immunoreactive (ir) ET-1 and intracellular contents of prepro (pp) ET-1 mRNA in the presence and absence of ETA- and ETB-selective antagonists (i.e., BQ-123 and BQ-788, respectively) in cultured human umbilical vein endothelial cells (HUVECs). ET-1 was secreted into the culture medium of HUVECs in a time-dependent manner with a plateau reached after incubation for more than 36 hr. In the presence of 10 microM BQ-788, the irET-1 level was enhanced significantly (i.e., up to 180% of control at 48 hr) whereas BQ-123 had no such effect. Specific binding of [125I]ET-1 to HUVECs was inhibited strongly by BQ-788 (IC50 = 2.4 nM) but very weakly by BQ-123 (IC50 = 1.4 microM), indicating that BQ-788 has a potent affinity for ETB receptors in HUVECs. The expression of ppET-1 mRNA was not changed by BQ-788. Extracellular levels of ET-1 decreased gradually after cellular treatment with cycloheximide. This decrease was significantly inhibited by BQ-788 but not by BQ-123 and was non-existent when the cells were incubated at 4 degrees C (where internalization of the receptor protein is not likely). In conclusion, HUVECs secrete ET-1 which, in turn, is internalized after binding to ETB receptors on HUVECs.
Ozaki
S
S
Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Japan.
Ohwaki
K
K
Ihara
M
M
Fukuroda
T
T
Ishikawa
K
K
Yano
M
M
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D065128:Endothelin Receptor Antagonists; D016232:Endothelins / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006020:Glycopeptides / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008168:Lung / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D017466:Receptors, Endothelin / Q000502:physiology*
D065128:Endothelin Receptor Antagonists; D016232:Endothelins; D006020:Glycopeptides; D009842:Oligopeptides; D010456:Peptides, Cyclic; D010880:Piperidines; D012333:RNA, Messenger; D017466:Receptors, Endothelin; C086539:BQ 788; D003513:Cycloheximide; C072247:cyclo(Trp-Asp-Pro-Val-Leu); C008890:phosphoramidon