Time course of recovery of endothelial cell surface thrombin receptor (PAR-1) expression.
We studied dynamics of cell surface expression of proteolytically activated thrombin receptor (PAR-1) in human pulmonary artery endothelial cells (HPAEC). PAR-1 activation was measured by changes in cytosolic calcium concentration ([Ca2+]i) and HPAEC retraction response (determined by real-time transendothelial monolayer electrical resistance). [Ca2+]i increase in response to thrombin was abolished by preexposure to 25 nM thrombin for >60 min, indicating PAR-1 desensitization, but preexposure to 25 nM thrombin for only 30 min or to 10 nM thrombin for up to 2 h did not desensitize PAR-1. Exposure to 10 or 25 nM thrombin decreased monolayer electrical resistance 40-60%. Cells preexposed to 10 nM thrombin, but not those preexposed to 25 nM thrombin, remained responsive to thrombin 3 h later. Loss of cell retractility was coupled to decreased cell surface PAR-1 expression as determined by immunofluorescence. Cell surface PAR-1 disappeared upon short-term (30 min) thrombin exposure but reappeared within 90 min after incubation in thrombin-free medium. Exposure to 25 nM thrombin for >60 min prevented rapid cycloheximide-insensitive PAR-1 reappearance. Cycloheximide-sensitive recovery of cell surface PAR-1 expression required 18 h. Therefore, both duration and concentration of thrombin exposure regulate the time course of recovery of HPAEC surface PAR-1 expression. The results support the hypothesis that initial recovery of PAR-1 surface expression in endothelial cells results from a rapidly mobilizable PAR-1 pool, whereas delayed recovery results from de novo PAR-1 synthesis. We conclude that thrombin itself regulates endothelial cell surface PAR-1 expression and that decreased surface expression interferes with thrombin-induced endothelial cell activation responses.
Ellis
C A
CA
Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612-7343, USA.
Tiruppathi
C
C
Sandoval
R
R
Niles
W D
WD
Malik
A B
AB
D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D048429:Cell Size / Q000502:physiology; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D009994:Osmolar Concentration; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D018179:Receptors, Thrombin / Q000187:drug effects / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors
D011500:Protein Synthesis Inhibitors; D018179:Receptors, Thrombin; D003513:Cycloheximide; D013917:Thrombin; D002118:Calcium