Thrombin modulates and reverses neuroblastoma neurite outgrowth.
Previous studies have shown that neuroblastoma cells and several types of primary neuronal cells in culture rapidly extend neurites when switched from serum-containing to serum-free medium. The present studies on cloned neuroblastoma cells show that thrombin blocked this spontaneous differentiation at 2 nM with a half-maximal potency of 50 pM. This required the catalytic activity of thrombin and was reversed upon thrombin removal. Thrombin also caused cells in serum-free medium to retract their neurites at equally low concentrations. Two other serine proteases, urokinase and plasmin, did not block or reverse neurite extension even at 100-fold higher concentrations. A specific assay for thrombin indicated that thrombin detected in serum-containing medium from neuroblastoma cultures was derived from serum and that it was likely responsible for much of the known capacity of serum to maintain neuroblastoma cells in a nondifferentiated state. This was supported by the finding that heparin addition reduced the thrombin concentration in serum-containing medium and stimulated neurite outgrowth from neuroblastoma cells in serum-containing medium. Studies on the ability of thrombin to modulate neurite outgrowth by other agents showed that it blocked and reversed the neurite outgrowth activity of two thrombin inhibitors: protease nexin-1 (which is identical to glial-derived neurite-promoting factor) and hirudin. Thrombin, however, did not block the neurite-promoting activity of dibutyryl cAMP or prostaglandin E1. These results suggest a specific role for thrombin in control of neurite outgrowth.
Gurwitz
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Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717.
Cunningham
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DD
D000527:Alprostadil / Q000494:pharmacology; D000818:Animals; D001369:Axons / Q000187:drug effects / Q000648:ultrastructure; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D002999:Clone Cells; D003470:Culture Media; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D051379:Mice; D009447:Neuroblastoma / Q000473:pathology*; D013917:Thrombin / Q000494:pharmacology*
D003470:Culture Media; D006629:Hirudins; D003994:Bucladesine; D013917:Thrombin; D000527:Alprostadil