The sustained second phase of hormone-stimulated diacylglycerol accumulation does not activate protein kinase C in GH3 cells.
Numerous hormones activate cells through receptor-regulated hydrolysis of phosphoinositides resulting in elevated cellular diacylglycerol (DAG), an activator of protein kinase C (PKC). Our previous studies showed that thyrotropin-releasing hormone (TRH) treatment of GH3 cells stimulated a rapid (less than 10 s) but transient (less than 60 s) association of cytosolic PKC with the membrane. In this study, we investigated the roles of hormone-stimulated Ca2+ and DAG levels in initiating and terminating the membrane association of PKC. The initial effects of TRH were not mimicked by elevating CA2+ levels, however, inhibiting TRH-stimulated Ca2+ increases blocked hormone-stimulated PKC translocation. Hence, the TRH stimulation of both Ca2+ and DAG levels were essential for the initial PKC translocation. The termination of PKC membrane association could not be attributed to proteolysis of PKC nor to limiting Ca2+ levels. Treatment of cells with phorbol diesters potentiated and prolonged the effects of TRH on PKC translocation, suggesting that DAG levels limited the membrane association of PKC. Since TRH stimulated a sustained increase in DAG levels, DAG composition was analyzed. There was a marked shift in DAG from tetraenoic (at 15 s) to more saturated DAGs at longer times. In addition, increases in plasma membrane DAG in response to TRH were transient rather than sustained. We propose that the TRH stimulation of PKC translocation is short-lived due to the metabolism of plasma membrane DAGs which are effective in promoting PKC activation. In contrast, DAGs which accumulate in intracellular membranes during the sustained phase of TRH treatment appear to be ineffective as activators of PKC.
Martin
T F
TF
Department of Zoology, University of Wisconsin, Madison 53706.
Hsieh
K P
KP
Porter
B W
BW
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D001692:Biological Transport / Q000187:drug effects; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002645:Chickens / Q000276:immunology; D004075:Diglycerides / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D005989:Glycerides / Q000378:metabolism*; D015151:Immunoblotting; D010911:Pituitary Neoplasms; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*
D000906:Antibodies; D004075:Diglycerides; D005989:Glycerides; D013973:Thyrotropin-Releasing Hormone; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium