Evidence for functionally active protease-activated receptor-4 (PAR-4) in human vascular smooth muscle cells.
1. This study investigates, whether in addition to the protease-activated receptor-1 (PAR-1), PAR-4 is present in vascular smooth muscle cells (SMC) of the human saphenous vein and whether this receptor is functionally active. PAR-1 and PAR-4 are stimulated by thrombin and by the synthetic peptides SFLLRN and GYPGQV, respectively. 2. mRNAs for both, PAR-1 and PAR-4, were detected in the SMC by using reverse transcriptase polymerase chain reaction (RT - PCR). 3. Treatment of the SMC with GYPGQV (200 microM) resulted in a transient increase in free intracellular calcium. This calcium signal was completely abolished after a preceding challenge with thrombin (10 nM), indicating homologous receptor desensitization. 4. Stimulation of the SMC with 10 nM thrombin or 200 microM SFLLRN caused a time-dependent activation of the extracellular signal-regulated kinases-1/2 (ERK-1/2) with a maximum at 5 min. In contrast, 100 nM thrombin as well as 200 microM of GYPGQV induced a prolonged phosphorylation of ERK-1/2 with a maximum at 60 min. These data suggest that PAR-1 and PAR-4 are activated by thrombin at distinct concentrations and with distinct kinetics. 5. GYPGQV stimulated [(3)H]-thymidine incorporation in SMC. At 500 microM, the peptide increased DNA synthesis 2.5 fold above controls. A comparable mitogenic effect was obtained after stimulation of the SMC by 10 nM thrombin or 100 microM SFLLRN, respectively. 6. These data indicate that a functionally active PAR-4 is present in SMC and, in addition to PAR-1, might contribute to thrombin-induced mitogenesis.
Friedrich-Schiller-Universität Jena, Zentrum für Vaskuläre Biologie und Medizin, Nordhäuser Str. 78, D-99089 Erfurt, Germany. bretschn@zmhk.cf.uni-jena.de
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Bretschneider
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Kaufmann
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Braun
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Nowak
E
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Glusa
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Schrör
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000187:drug effects / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018179:Receptors, Thrombin / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013917:Thrombin / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors
D009842:Oligopeptides; D012333:RNA, Messenger; D018179:Receptors, Thrombin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin; C112908:protease-activated receptor 4; D002118:Calcium; D013936:Thymidine