A PHOSPHOLIPASE-C INHIBITOR, U-73122, BLOCKS TSH-INDUCED INOSITOL TRISPHOSPHATE PRODUCTION, CA2+ INCREASE AND ARACHIDONIC-ACID RELEASE IN FRTL-5 THYROID-CELLS

To characterize the role of phospholipase C (FLC)-mediated intrathyroid signal transduction by thyrotropin, we studied the effect of U-73122, an aminosteroid inhibitor of PLC-dependent activity, on TSH-activated PLC-Ca2+ and arachidonic acid (AA) signalling systems in cultured FRTL-5 rat thyroid cells. In the presence of extracellular Ca2+, TSH (0.1 mu M) increased intracellular free calcium concentration ([Ca2+](i)) by 63 +/- 6% with a sustained plateau phase, and AA release by 160 +/- 16%. By deletion of extracellular Ca2+, TSH induced a similar maximal [Ca2+](i) increase, but the plateau phase and AA release were entirely suppressed. U-73122 (5 mu M) inhibited TSH stimulation of H-3-labelled inositol trisphosphates (IP3) production by 73 +/- 3% (P < 0.01) in one study, and completely in another. U-73122 concentration-dependently blocked the TSH-induced Ca2+ increase in either the presence or absence of external Ca2+. U-73122 also showed a similar concentration-response inhibition of TSH-induced AA release. These results provide direct evidence of PLC mediation of TSH-stimulated signal transduction in FRTL-5 thyroid cells. TSH-induced external Ca2+ entry, as well as intracellular Ca2+ mobilization, is probably a PLC-mediated process. From an IP3-sensitive intracellular pool, TSH induces intracellular Ca2+ mobilization. External Ca2+ entry seems to be a prerequisite for TSH-induced AA release. U-73122 inhibition of both cytosolic Ca2+ increase and AA release further confirms [Ca2+](i) dependence for TSH stimulation of AA release.