Regulation of glycoprotein hormone free alpha-subunit secretion and intracellular alpha-subunit content in primary pituitary cells.
The effects and interactions of GnRH, TRH, a cAMP analog, a protein kinase-C (PKC) activator, a calcium ionophore, and a calcium channel blocker on pituitary glycoprotein hormone free alpha-subunit secretion and intracellular free alpha-subunit content were investigated. Treatment of dispersed rat pituitary cells with GnRH (100 nM) effected a time-dependent release of alpha-subunit, reaching a 4.5-fold increase (P < 0.05) at 24 h. Smaller effects were observed with TRH (10 nM). A rapid and progressive fall in intracellular alpha-subunit content was observed for 8 h after stimulation with GnRH (61% decrease; P < 0.05) or TRH (55% decrease; P < 0.05), which then remained constant at 24 h. The cAMP analogue 8-bromo-cAMP augmented a late release of alpha-subunit (4.5-fold increase at 24 h; P < 0.05) without affecting levels of alpha-subunit within the cells. Co-addition of 8-bromo-cAMP with GnRH or TRH arrested the marked fall in intracellular alpha-subunit seen with GnRH or TRH alone. These results suggest that although cAMP is capable of stimulating alpha-subunit secretion and maintaining cell content in the face of GnRH- and TRH-stimulated secretion, it does not mediate their effects on alpha-subunit. Like GnRH, the PKC activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) rapidly stimulated alpha-subunit secretion (1.7-fold increase at 4 h; P < 0.05) and progressively lowered cell content over 24h (73% decrease; P < 0.01). This similarity of action and the lack of demonstration of additive effects of TPA with GnRH or TRH imply a role for PKC as a mediator of GnRH and TRH action on alpha-subunit. Using verapamil (50 microM) to block L-type calcium channels had no effect on either basal or GnRH-stimulated alpha-secretion over 24 h. The calcium ionophore A23187 (3 microM) blocked the stimulatory effects of GnRH on alpha-subunit release and alone inhibited free alpha-subunit secretion (28% decrease at 24 h; P < 0.05). Our results suggest that neither cAMP nor an influx of extracellular calcium mediates the effects of GnRH or TRH on free alpha-subunit secretion. Accordingly, we postulate that PKC is involved in the actions of GnRH and TRH on alpha-subunit in rat pituitary cells, although further studies are required in PKC-depleted cells to confirm this hypothesis.
Holdstock
J G
JG
Department of Clinical Biochemistry, London Hospital Medical College, United Kingdom.
Burrin
J M
JM
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D015292:Glycoprotein Hormones, alpha Subunit / Q000096:biosynthesis* / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D007700:Kinetics; D007986:Luteinizing Hormone / Q000096:biosynthesis* / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*; D013997:Time Factors; D014700:Verapamil / Q000494:pharmacology
D015292:Glycoprotein Hormones, alpha Subunit; D015124:8-Bromo Cyclic Adenosine Monophosphate; D007987:Gonadotropin-Releasing Hormone; D000001:Calcimycin; D013973:Thyrotropin-Releasing Hormone; D007986:Luteinizing Hormone; D014700:Verapamil; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate