Thyrotropin-releasing hormone-stimulated [3H]inositol metabolism in GH3 pituitary tumor cells. Studies with lithium.

GH3 pituitary tumor cells were labeled to isotopic equilibrium with [3H]inositol. Thyrotropin-releasing hormone (TRH), which has been shown to stimulate 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 two metabolites accumulated in a multiphasic manner with an initial peak 5-10 sec 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 calcium ionophore A23187. Incubation of GH3 cells with TRH in the presence of lithium led to an enhanced accumulation of [3H]inositol 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 lithium was added 30 min after TRH, [3H]inositol monophosphate accumulated at the same rate as was found when TRH and lithium were added together, indicating that the TRH-induced phospholipid response in GH3 cells does not desensitize. Under normal conditions, approximately equal amounts of the three [3H]inositol phosphates were formed within 5 sec of TRH addition. However, when TRH was added to cells grown chronically in lithium-containing medium, or to cells incubating at a subphysiological temperature (25 degrees), greater than 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-bisphosphate and, perhaps also, of phosphatidylinositol 4-phosphate.