A phospholipase C inhibitor, U-73122, blocks TSH-induced inositol trisphosphate production, Ca2+ increase and arachidonic acid release in FRTL-5 thyroid cells

Abstract To characterize the role of phospholipase C (PLC)-mediated intrathyroid signal transduction by thyrotropin, we studied the effect of U-73122, an aminosteroid inhibitor of PLC-dependent activity, on TSH-activated PLC-Ca 2+ and arachidonic acid (AA) signalling systems in cultured FRTL-5 rat thyroid cells. In the presence of extracellular Ca 2+ , TSH (0.1 μM) increased intracellular free calcium concentration ([Ca 2+ ] i ) by 63 ± 6% with a sustained plateau phase, and AA release by 160 ± 16%. By deletion of extracellular Ca 2+ , TSH induced a similar maximal [Ca 2+ ] i increase, but the plateau phase and AA release were entirely suppressed. U-73122 (5 μM) inhibited TSH stimulation of 3 H-labelled inositol trisphosphates (IP 3 ) production by 73 ± 3% ( P 2+ increase in either the presence or absence of external Ca 2+ . 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 Ca 2+ entry, as well as intracellular Ca 2+ mobilization, is probably a PLC-mediated process. From an IP 3 -sensitive intracellular pool, TSH induces intracellular Ca 2+ mobilization. External Ca 2+ entry seems to be a prerequisite for TSH-induced AA release. U-73122 inhibition of both cytosolic Ca 2+ increase and AA release further confirms [Ca 2+ ] i dependence for TSH stimulation of AA release.