title: Calcium mobilization and protease-activated receptor cleavage after thrombin stimulation in motor neurons.
abstract: Thrombin, the ultimate enzyme in the blood coagulation cascade, has prominent actions on various cells, including neurons. As in platelets, thrombin increases [Ca2+]i mobilization in neurons, and also retracts neurites. Both these effects are mediated through a G protein-coupled, proteolytically activated receptor for thrombin (PAR-1). Prolonged exposure to thrombin kills neurons via apoptosis, that may also involve PAR-1 activation. Increased [Ca2+]i has been a unifying mechanism proposed for cell death in several neurodegenerative diseases. Thrombin-elevated calcium levels may activate intracellular cascades in neurons leading to cell death. Since thrombin mediates its diverse effects on cells through both heterotrimeric and monomeric G proteins, we also explored what effect altering differential G protein coupling would have on the neuronal response to thrombin. We studied calcium mobilization by thrombin in a model motor neuronal cell line, NSC19, using fluorescence image analysis. Confirming effects in other neuronal types, thrombin caused dramatic increases in [Ca2+]i levels, both transiently and after prolonged exposure, which involved activation and cleavage of the PAR-1 receptor. Using enzyme linked immunosorbent assay (ELISA) and dot-blot analysis, we found that the N-terminal fragment of PAR-1 was released into the medium after exposure to thrombin. We confirmed that PAR-1 protein and mRNA expression occurred in motor neurons. We found that cholera toxin inhibited thrombin-mediated Ca2+ influx, pertussis toxin did not significantly alter thrombin action, and lovastatin, a small 21-kDa Ras GTPase (Rho) modulator, showed a tendency to reduce the thrombin effect. These data indicate that thrombin-increased [Ca2+]i, sufficient to trigger cell death in motor neurons, might be approached in vivo by modulating thrombin signaling through PAR-1.
authors.affiliation: Fluorescence Imaging Laboratories Department of Veterans Affairs Medical Center, Kansas City, MO 64128, USA.
authors.forename: I V
authors.initials: IV
authors.lastname: Smirnova
authors.forename: S
authors.initials: S
authors.lastname: Vamos
authors.forename: T
authors.initials: T
authors.lastname: Wiegmann
authors.forename: B A
authors.initials: BA
authors.lastname: Citron
authors.forename: P M
authors.initials: PM
authors.lastname: Arnold
authors.forename: B W
authors.initials: BW
authors.lastname: Festoff
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006822:Hybrid Cells; D007399:Interphase / Q000187:drug effects; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D008148:Lovastatin / Q000494:pharmacology; D051379:Mice; D009046:Motor Neurons / Q000187:drug effects* / Q000378:metabolism*; D037342:Pertussis Toxin; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000187:drug effects / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D010566:Virulence Factors, Bordetella; D002772:Cholera Toxin; D008148:Lovastatin; D037342:Pertussis Toxin; D013917:Thrombin; D019204:GTP-Binding Proteins; D002118:Calcium