Sequential activation of phospholipase-C and -D in agonist-stimulated gonadotrophs.
The contributions of phospholipase-C and -D to diacylglycerol (DG) formation during agonist-induced cell signaling were investigated in rat pituitary cells and alpha T3-1 gonadotrophs. In both cell types, GnRH caused a biphasic increase in DG formation, with an initial spike within 60 sec, followed by a larger and sustained rise to reach a second peak after 15 min of stimulation. Both phases of DG production were temporally correlated with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation, consistent with the dependence of DG formation on phospholipase-C-mediated phosphoinositide hydrolysis. However, the ability of GnRH to stimulate phosphatidylethanol (PEt) in the presence of ethanol suggested that phospholipase-D may also participate in DG formation. Two inhibitors of phospholipase-C-dependent phosphoinositide hydrolysis, U73122 and neomycin sulfate, reduced the PEt as well as the Ins(1,4,5)P3 response to GnRH, indicating that phospholipase-D is activated during phospholipase-C-dependent signaling in pituitary gonadotrophs. The production of both DG and PEt was increased by treatment with the active phorbol ester phorbol 12-myristate 13-acetate (PMA), but not with inactive 4 alpha-phorbol 13-didecanoate, indicating that stimulation of protein kinase-C leads to activation of phospholipase-D. In accord with this, GnRH- and PMA-induced elevations of DG and PEt production were attenuated or abolished in protein kinase-C-depleted cells. In contrast, short and long term stimulation with PMA had no effect on basal inositol phosphate production. Also, GnRH-induced inositol phosphate production was not affected by protein kinase-C depletion. Finally, U73122 and neomycin sulfate did not inhibit PMA-induced PEt formation. These data indicate that GnRH activates a dual phospholipase pathway in a sequential and synchronized manner; phospholipase-C initiates the biphasic increase in Ins(1,4,5)P3 and DG formation, and protein kinase-C mediates the integration of phospholipase-D into the signaling response during the sustained phase of agonist stimulation.
Zheng
L
L
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Stojilkovic
S S
SS
Hunyady
L
L
Krsmanovic
L Z
LZ
Catt
K J
KJ
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D004963:Estrenes / Q000494:pharmacology; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*
D004075:Diglycerides; D004963:Estrenes; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D007987:Gonadotropin-Releasing Hormone; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium