THE ROLE OF THE CALPAIN-CALPASTATIN SYSTEM IN THYROTROPIN-RELEASING HORMONE-INDUCED SELECTIVE DOWN-REGULATION OF A PROTEIN-KINASE-C ISOZYME, NPKC-EPSILON, IN RAT PITUITARY GH(4)C(1) CELLS

We have examined the mechanism for the selective down-regulation of pr otein kinase C epsilon (nPKC epsilon) in rat pituitary GH(4)C(1) cells responding to thyrotropin-releasing hormone (TRH) stimulation. Among various low molecular weight protease inhibitors examined, only a cyst eine protease inhibitor (calpain inhibitor I, N-acetyl-Leu-Leu-norleuc inal) blocked the down-regulation of nPKC epsilon. Furthermore, the in troduction of a synthetic calpastatin peptide, an exclusively specific inhibitor of calpain, into the cells also reduced the down-regulation , suggesting the involvement of calpain among all the intracellular cy steine proteases in this process. In accordance, we observed TRH-induc ed translocation of m-calpain from the cytosol to the membrane and the concomitant up regulation of calpastatin isoforms; presumably, the fo rmer represents activation of the protease initiating the kinase degra dation, while the latter constitutes a negative feedback system protec ting the cells from activated calpain. These results suggest that in G H(4)C(1) cells, TRH mobilizes both protease (m-calpain) and inhibitor (calpastatin) as a strictly regulating system for the nPKC epsilon pat hway mediating TRH signals.