Ca2+ ionophores affect phosphoinositide metabolism differently than thyrotropin-releasing hormone in GH3 pituitary cells
Thyrotropin-releasing hormone (TRH) stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) by a phospholipase C (or phosphodiesterase) and elevates cytoplasmic-free Ca2+ concentration ([Ca2+]i) in GH3 pituitary cells. To explore whether hydrolysis of PtdIns-4,5-P2 is secondary to the elevation of [Ca2+]i, we studied the effects of Ca2+ ionophores, A23187 and ionomycin. In cells prelabeled with [3H]myoinositol, A23187 caused a rapid decrease in the levels of [3H]PtdIns-4,5-P2, [3H]PtdIns-4-P, and [3H]PtdIns to 88 +/- 2%, 88 +/- 4%, and 86 +/- 1% of control, respectively, and increased [3H]inositol bisphosphate to 200 +/- 20% at 0.5 min. There was no increase in [3H] Ins-P3; the lack of a measurable increase in [3H]Ins-P3 was not due to its rapid dephosphorylation. In cells prelabeled with [14C]stearic acid, A23187 increased [14C]diacylglycerol and [14C]phosphatidic acid to 166 +/- 20% and 174 +/- 17% of control, respectively. In cells prelabeled with [3H]arachidonic acid, A23187, but not TRH, increased unesterified [3H]arachidonic acid to 166 +/- 8% of control. Similar effects were observed with ionomycin. Hence, Ca2+ ionophores stimulate phosphodiesteratic hydrolysis of PtdIns-4-P but not of PtdIns-4,5-P2 and elevate the level of unesterified arachidonic acid in GH3 cells. These data demonstrate that Ca2+ ionophores affect phosphoinositide metabolism differently than TRH and suggest that TRH stimulation of PtdIns-4,5-P2 hydrolysis is not secondary to the elevation of [Ca2+]i.