| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1994: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
We have studied on the role of protein kinase C (PKC) isozymes in hormonal secretion in rat pituitary GH4C1 cells. The cells express six PKC transcripts for cPKCalpha, cPKCbetaII,nPKCdelta, nPKCepsilon, nPKCeta, and aPKCzeta, but not for cPKCUPSILON or nPKCrheta. nPKCepsilon mRNA is the most abundant. Additional overexpression of nPKCepsilon, but not an inactive point mutant (nPKCepsilon436K->R) of nPKCepsilon, increases the basal and stimulated prolactin (PRL) secretion by 12-O-tetradecanoyl phorbol 13-acetate (TPA) or thyrotropin-releasing hormone (TRH). Immunocytofluorescence and immunoblot experiments indicated that TRH causes the transient translocation and subsequent down-regulation of overexpressed PKCepsilon together with endogenous one. Both the basal and TRH-stimulated PRL secretion are clearly correlated with the expression level of nPKCepsilon without changing the TRH receptor density or dose dependence. On the other hand, the overproduction of other endogenous PKC isozymes, cPKCalpha, cPKCbetaII,and nPKCdelta, does not affect any secretory responses. These findings clearly demonstrate that the expression level of nPKCepsilon in GH4C1 cells is rate-limiting for resting and TRH-stimulated PRL secretion, and provide the first direct evidence that nPKCepsilon plays a key role in hormonal secretory processes.
Moreover, we have investigated the mechanism for the selective down-regulation of nPKCepsilon responding to TRH stimulation, and indicated that TRH mobilizes both protease (m-calpain) and inhibitor (calpastatin) as a strictly regulating system for the nPKCepsilon pathway mediating TRH signals.
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