THYROTROPIN-RELEASING HORMONE-STIMULATED [INOSITOL-H-3 METABOLISM IN GH3 PITUITARY-TUMOR CELLS - STUDIES WITH LITHIUM

Abstract

GH3 pituitary tumor cells were labeled to isotopic equilibrium with [3H]inositol. Rat pituitary tumor TRH which stimulates inositol phospholipid metabolism in these cells, enhanced the accumulation of [3H]inositol-derived radioactivity in the cell''s acid-soluble fraction. Separation of the [3H]inositol metabolites by ion-exchange chromatography revealed that TRH induced a rapid rise in the cellular content of [3H]inositol mono-, bis-, and trisphosphate. The latter 2 metabolites accumulated in a multiphasic manner with an initial peak 5-10 s after TRH addition. This was followed by a short-lived decline and a secondary rise which left the metabolite levels elevated for at least 50 min. The GH3 cell [3H]inositol monophosphate and [3H]inositol content also rose in response to TRH, but the latter accumulated with a considerably slower time course than the phosphorylated derivatives. None of these responses could be mimicked by the Ca ionophore A23187 [calcimycin]. Incubation of GH3 cells with TRH in the presence of Li led to an enhanced accumulation of [3H]ionositol monophosphate and, to a lesser extent, of [3H]inositol bis- and trisphosphate. This accumulation rose in a linear fashion with time for at least 20 min, by which point 50% of the [3H]inositol-containing phospholipids had been depleted. When Li was added 30 min after TRH, [3H]inositol monophosphate accumulated at the same rate as was found when TRH and Li were added together, indicating that the TRH-induced phospholipid response in GH3 cells does not desensitize. Under normal conditions, approximately equal amounts of the 3 [3H]inositol phosphates were formed within 5 s of TRH addition. However, when TRH was added to cells grown chronically in Li-containing medium, or to cells incubating at a subphysiological temperature (25.degree.), > 90% of the metabolites formed were the bis- or trisphosphates. This indicates that the primary event stimulated by TRH is the breakdown by phospholipase C of phosphatidylinositol 4,5-bisphosphatate and, perhaps also, of phosphatidylinositol 4-phosphate.