THYROTROPIN-RELEASING-HORMONE STIMULATES RAPID BREAKDOWN OF PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE AND PHOSPHATIDYLINOSITOL 4-PHOSPHATE IN GH3 PITUITARY-TUMOR CELLS

Abstract

TRH induced a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5P2) and phosphatidylinositol 4-phosphate (PtdIns 4P) in [rat pituitary tumor] GH3 cells labeled to isotopic equilibrium with [3H]inositol. Within 10 s of the addition of TRH (1 .mu.M), there was a maximal 60% decrease in PtdIns 4,5P2 and 40% decrease in PtdIns 4P. Breakdown of phosphatidylinositol (PtdIns) occurred only after a lag of 30 s. While the reduced levels of the polyphosphoinositides had almost returned to control values by 5 min, the GH3 cell PtdIns content remained at .apprx. 85% of controls for at least 2 h. Both phosphatidic acid (PA) and 1,2-diacylglycerol levels increased in response to TRH in [32P]PO4- and [3H]glycerol-labeled GH3 cells. 1,2-Diacylglycerol accumulated in a biphasic manner with an early peak 10 s after addition of the peptide. This early rise in 1,2-diacylglycerol levels coincided in time and was equivalent in lipid mass with the decrease in the polyphosphoinositide content, suggesting the involvement of a phospholipase C-type enzyme. 1,2-Diacylglycerol levels subsequently fell toward control values and, after 3 min of treatment with TRH, rose again to levels 50% above normal. PA levels reached a peak value .apprx. 2-fold above normal 1 min after the addition of TRH. At all times after TRH addition, the bulk of the inositol phospholipid lost was recovered as 1,2-diacylglycerol. TRH possibly stimulates a cycle of events in which the breakdown of the polyphosphoinositides, PtdIns 4,5P2 and, perhaps, PtdIns 4P by a phsopholipase C enzyme could be the initiating event.