CALCIUM MOBILIZATION AND PROTEASE-ACTIVATED RECEPTOR CLEAVAGE AFTER THROMBIN STIMULATION IN MOTOR-NEURONS

Thrombin, the ultimate enzyme in the blood coagulation cascade, has pr ominent actions on various cells, including neurons. As in platelets, thrombin increases [Ca2+](i) mobilization in neurons, and also retract s neurites. Both these effects are mediated through a G protein-couple d, proteolytically activated receptor for thrombin (PAR-1). Prolonged exposure to thrombin kills neurons via apoptosis, that may also involv e PAR-1 activation. Increased [Ca2+](i) has been a unifying mechanism proposed for cell death in several neurodegenerative diseases. Thrombi n-elevated calcium levels may activate intracellular cascades in neuro ns 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 h ave on the neuronal response to thrombin. We studied calcium mobilizat ion by thrombin in a model motor neuronal cell line, NSC19, using fluo rescence image analysis. Confirming effects in other neuronal types, t hrombin caused dramatic increases in [Ca2+](i) levels, both transientl y 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 confirme d that PAR-1 protein and mRNA expression occurred in motor neurons. We found that cholera toxin inhibited thrombin-mediated Ca2+ influx, per tussis toxin did not significantly alter thrombin action, and lovastat in, a small 21-kDa Ras GTPase (Rho) modulator, showed a tendency to re duce 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.