REGULATION AND METABOLIC ROLE OF PHOSPHOLIPASE-D ACTIVITY IN HUMAN THYROID AND CULTURED DOG THYROCYTES

The actions of TSH, ATP, the ionophore A23187, the endoplasmic reticul um Ca2+-ATPase inhibitor thapsigargin, and phorbol dibutyrate (PDBu) o n H-3-cytidine-monophosphate phosphatidic acid (H-3-CMP-PA) accumulati on were studied in human thyroid slices to evaluate PA generation and inositol recycling towards phosphatidylinositol synthesis. The effects of the same agonists also were measured on phosphatidylbutanol (PtdBu t) generation in H-3-palmitate or H-3-myristate prelabeled slices to a ssess the activity of phospholipase D (PLD). The phospholipid target o f this PLD was determined on H-3-choline prelabeled human thyroid slic es by measuring H-3-choline release in incubation medium and slices an d H-3-choline incorporation in phospholipids. TSH(10 U/L) stimulated H -3-CMP-PA accumulation in an LiCl- and propranolol-insensitive way, as well as H-3-fatty acids incorporation into PA, diacylglycerol, and ph osphatidylcholine (PtdCho) with no evidence of dose-dependent effects and had no detectable action on PLD activity. The effects of TSH were not reproduced by Bu(2)cAMP or forskolin. Thapsigargin and A23187 both increased CMP-PA accumulation and PtdBut generation, whereas ATP only stimulated PLD activity. The phorbol ester PDBu (5 x 10(-7) mol/L) in creased PtdBut formation and H-3-fatty acid incorporation into PtdCho, but had no effect on CMP-PA generation. Staurosporine (STSP) (5 x 10( -6) mol/L), a nonspecific inhibitor of protein kinase C, unexpectedly reproduced the effects of PDBu. The increase of H-3-choline in slices' supernatant and the decrease of H-3-choline-labeled PtdCho induced by PDBu, ATP, thapsigargin, and STSP indicate that the activated PLD hyd rolyzed PtdCho. We suggest that the PA generation induced by PLD stimu lation could contribute to the stimulated H2O2 formation and iodide or ganification observed with the agonists inducing PtdBut accumulation. Indeed, Bu(2)cAMP and forskolin, known to decrease iodide organificati on in human thyroid, inhibited the PLD stimulation induced by ATP and PDBu. In cultured dog thyrocytes, phorbol esters, and STSP induced DNA synthesis and dedifferentiation, whereas thapsigargin inhibited TSH-i nduced growth and killed phorbol esters stimulated cells, suggesting a positive role of PLD stimulation towards dedifferentiated growth and of simultaneously raised [Ca2+](i) and stimulated protein kinase C-PLD towards growth arrest and cellular death.