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)