Activation of cyclic nucleotide formation in murine neuroblastoma N1E-115 cells by modified human thrombins

The activity of alpha-thrombin and chemically modified derivatives of this enzyme in stimulating cGMP formation in murine neuroblastoma clone N1E-115 cells is reported. The rank order potency of the compounds falls into three classes: 1) alpha-thrombin is the most potent and effective; 2) the catalytically active enzymes gamma-thrombin, trypsin, and nitro-alpha-thrombin are approximately 50-fold less potent than alpha-thrombin; and 3) active site blocked derivatives of alpha-thrombin are 100 to 1000-fold less potent than alpha-thrombin. Native alpha-thrombin consistently produces the most effective response, usually 1.5 to 3-fold greater than any of the other compounds tested. Preincubation of cells with quinacrine, an inhibitor of phospholipase A2, or with the lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid prior to thrombin challenge resulted in a concentration-dependent attenuation of the response. Indomethacin was without effect in modifying the response. These results suggest that thrombin stimulation of neuroblastoma cells involves the release of arachidonic acid and its metabolism by lipoxygenase. These results clearly demonstrate the activity of alpha-thrombin in stimulating a receptor-mediated response in cultured nerve cells. The results are discussed in relation to the interaction of endogenous alpha-thrombin with nerve cells following invasive trauma and to the possible presence of endogenous proteinases with a neurotransmitter-like function.