Selective translocation of non-conventional protein kinase C isoenzymes by gonadotropin-releasing hormone (GnRH) in the gonadotrope-derived αT3-1 cell line

• Published in: Molecular and cellular endocrinology 1996-04, Vol.118 (1), p.103-111
• Main Authors: Kratzmeier, M., Poch, A., Mukhopadhyay, A.K., McArdle, C.A.
• Format: Article
• Language: English
• Subjects: Biological Transport - drug effects; Blotting, Western; Cell Line; Down regulation; Gonadotropin-releasing hormone (Gonadotropes); Gonadotropin-Releasing Hormone - pharmacology; Humans; Isoenzymes - metabolism; Phorbol Esters - pharmacology; Protein Kinase C - metabolism; Protein kinase C isoenzymes; Protein Kinase C-alpha; Protein Kinase C-epsilon; Translocation; αT3-1 cells
• ISSN: 0303-7207; 1872-8057
• DOI: 10.1016/0303-7207(96)03788-4

Gonadotropin-releasing hormone acts via G-protein coupled receptors to stimulate polyphosphoinositide-specific phospholipase C (PIC) with consequent elevation of cytosolic Ca 2+ and activation of protein kinase C (PKC). Whereas Ca 2+ is known to mediate stimulation of exocytotic gonadotropin release by GnRH, the identity of the PKC isoenzymes activated by GnRH and their physiological role in gonadotropes are poorly understood. In many systems translocation of PKC (from cytosolic to particulate fractions of cellular homogenates) has been taken as evidence of hormonal activation of PKC and down regulation of PKC (by prolonged treatment with PKC-activating phorbol esters) has been used extensively to investigate the role of PKC in hormone action. Here we have assessed the influence of GnRH and phorbol esters on translocation and down regulation of PKC isoenzymes identified by Western blotting with isoenzyme-specific antibodies in αT3-1 cells (a gonadotrope-derived cell line). These cells were found to possess PKCs α, ϵ and ζ but not β,δ (present in rat pituitaries) or γ (present in rat brains). In short-term stimulations (10 min), the PKC-activating phorbol esters, PMA and PDBu, caused concentration-dependent increases in the proportion of PKCα and PKCϵ recovered from the particulate fraction of αT3-1 cells, but did not induce measurable translocation of PKCζ. The inactive phorbol ester 4α PDBu did not cause translocation of any of these isoenzymes. GnRH treatment induced a concentration-dependent increase in the proportion of particulate PKCϵ and PKCζ but had no measurable effect on PKCα translocation. In longer incubations (6–48 h) GnRH failed to cause measurable down-regulation of these isoenzymes whereas PMA treatment led to a clear down regulation of PKCs α and ϵ (albeit with different kinetics). The data demonstrate the differential activation and down regulation of PKC isoenzymes by GnRH versus PMA, which are clearly pertinent to the design of experiments intended to address the role of such isoenzymes in GnRH action. Moreover, they provide the first demonstration of hormonal regulation of an atypical PKC isoenzyme (PKCζ) in pituitary cells.