Calcium mobilization and protease-activated receptor cleavage after thrombin stimulation in motor neurons.
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.
Smirnova
I V
IV
Fluorescence Imaging Laboratories Department of Veterans Affairs Medical Center, Kansas City, MO 64128, USA.
Vamos
S
S
Wiegmann
T
T
Citron
B A
BA
Arnold
P M
PM
Festoff
B W
BW
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
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