Activation of cathepsin B involved in enkephalin production by bradykinin and its cleavage products in cultured fibroblasts of the rat dental pulp.

Mechanisms of cathepsin B activation involved in methionine-enkephalin (ME) production induced by bradykinin (BK), des-Arg9-BK or L-arginine (L-Arg) were studied using cultured fibroblasts of the rat dental pulp, especially from a viewpoint of intracellular signal transduction. BK, des-Arg9-BK, L-Arg or cysteine enhanced the release of ME-like peptides from the cells, and the release of ME-like peptides induced by des-Arg9-BK was inhibited by des-Arg9-[Leu8]-BK (BK B1-receptor antagonist) and E-64 (a specific inhibitor of cysteine proteinases). The activation of cathepsin B by BK or des-Arg9-BK was inhibited by des-Arg9-[Leu8]-BK or islet-activating protein (IAP), and the activation of cathepsin B by L-Arg was inhibited by Leu-Arg (kyotorphin-receptor antagonist) or Botulinum C3-enzyme. The activation of cathepsin B by those stimulants was dependent on calcium ion. These results suggest that the ME production by BK or des-Arg9-BK may be mediated by Ca(2+)-dependent cathepsin B activation through B1-receptors and IAP-sensitive G-proteins, whereas the production by L-Arg may be mediated by Ca(2+)-dependent cathepsin B activation through kyotorphin-receptor and Botulinum C3-enzyme-sensitive G-proteins. On the other hand, the activation of cathepsin B was inhibited by neomycin B (phospholipase C inhibitor) and various serine/threonine kinase inhibitors. These results indicate that phospholipase C and serine/threonine kinases are involved in the activation of cathepsin B by BK, des-Arg9-BK or L-Arg. Genistein inhibited the activation of cathepsin B by des-Arg9-BK or L-Arg in a different fashion, suggesting that tyrosine kinase(s) is also involved in the activation. Cathepsin B activation by BK or L-Arg but not des-Arg9-BK was inhibited by L-NMMA (inhibitor of NO synthesis), and the activation by L-Arg was enhanced by beta-glycerophosphate (beta-GP: inhibitor of phosphatases), while the activation by BK or des-Arg9-BK was inhibited by beta-GP. These results suggest that BK-induced cathepsin B activation in the fibroblasts may be due to a combined effect of des-Arg9-BK and L-Arg.