Phosphatidylinositol depletion in GH3 rat pituitary cells inhibits sustained responses to thyrotropin-releasing hormone. Reversal with myo-inositol.
TRH stimulation of rat pituitary (GH3) cells causes biphasic changes in cytoplasmic free Ca2+ concentration [( Ca2+]i) and PRL secretion. It has been proposed, based primarily on indirect evidence, that the first phase effects are mediated by inositol 1,4,5-trisphosphate, which releases Ca2+ from cellular stores, and the sustained effects are mediated by 1,2-diacylglycerol, which activates protein kinase C. To determine more directly if inositol lipid hydrolysis leading to protein kinase C activation is involved in the sustained effects of TRH, GH3 cells were depleted of phosphatidylinositol (PtdIns) by prestimulation and incubation in myo-inositol-free, Li(+)-containing medium. Cells depleted of PtdIns (to 53 +/- 3.2% of control) had unchanged PtdIns 4,5-bisphosphate content, and responded to TRH with a rapid elevation of inositol trisphosphate, and a first phase (or burst) elevation of [Ca2+]i and PRL secretion that was not different from that found in control cells. In contrast, in PtdIns-depleted cells, the prolonged generation of inositol phosphates, which are produced in equimolar amounts with 1,2-diacylglycerol, caused by TRH was virtually abolished, and the second phase (or sustained) elevation of [Ca2+]i and PRL secretion were inhibited by 50% and 40%, respectively. The inhibition of both sustained effects was reversed by adding 100 mM myo-inositol to the medium, which allowed for synthesis of PtdIns. Last, in cells in which protein kinase C was down-regulated by pretreatment with a phorbol ester, the sustained effects of TRH were inhibited also.(ABSTRACT TRUNCATED AT 250 WORDS)
Rodriguez
R
R
Department of Medicine, Cornell University Medical College, New York, New York 10021.
Imai
A
A
Gershengorn
M C
MC
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects; D007294:Inositol / Q000494:pharmacology*; D050356:Lipid Metabolism; D010716:Phosphatidylinositols / Q000502:physiology*; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011388:Prolactin / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*
D010716:Phosphatidylinositols; D007294:Inositol; D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium