CHOLINERGIC STIMULATION INCREASES THROMBIN ACTIVITY AND GENE-EXPRESSION IN CULTURED MOUSE MUSCLE

Activity-dependent synapse reduction is a major determinant of neuromu scular innervation. Previous research has shown that nanomolar concent rations of hirudin, a specific thrombin antagonist, significantly atte nuates this reduction, and protease nexin 1 (PN1), an endogenous throm bin inhibitor closely localized to the neuromuscular synapse, can inhi bit synapse reduction at similar concentrations. Protease inhibitors w hich do not inhibit thrombin, including cystatin and aprotinin, had no effect on synapse reduction. We present a series of experiments exami ning whether prothrombin and/or PN1 gene expression, as well as thromb in activity, are regulated in muscle cultures by acetylcholine (ACh) r eceptor activation. We also studied the effect of exogenous thrombin o n synapse elimination in co-cultures of muscle and cholinergic neurons . Cultured muscle cells were electrically blocked with tetrodotoxin (T TX), or co-treated with ACh in order to isolate ACh receptor activatio n. Electrical blockade resulted in a decrease in thrombin release to a bout two-thirds of control values. The application of ACh to electrica lly blocked muscle cultures resulted in a 2.5-fold increase in thrombi n activity released into the medium and a 2-fold increase in prothromb in gene expression. In contrast, ACh treatment in the presence of TTX had no effect on PN1 gene expression compared to treatment with TTX al one. In addition, exogenous thrombin significantly increased synapse e limination in unstimulated muscle/cholinergic neuron co-cultures. Thes e results suggest that thrombin or a thrombin-like molecule released f rom muscle is required for activity-dependent synapse elimination and is regulated by neuromuscular activity. (C) 1997 Elsevier Science B.V.