Thyroid-stimulating hormone rapidly stimulates inositol polyphosphate formation in FRTL-5 thyrocytes without activating phosphoinositidase C

The thyroid-stimulating hormone (TSH) receptor is widely regarded as one of a limited number of G-protein-coupled receptors that activate both adenylate cyclase and phosphoinositidase C (PIC) via G-proteins, but the existing experimental evidence for TSH-stimulated PtdIns(4,5)P-2 hydrolysis remains inconclusive. We have compared the effects of TSH and of ATP (acting via P-2-purinergic receptors) on the inositol lipids and polyphosphates of [2-H-3]inositol-labelled FRTL-5 rat thyroid cells. ATP initiated a rapid decrease in H-3-labelled PtdIns4P and PtdIns(4,5)P-2, whereas TSH did not. Stimulation with ATP and, less consistently, with noradrenaline (acting via alpha-adrenergic receptors) provoked rapid formation of Ins(1,4,5)P-3, Ins(1,3,4,5)P-4, Ins(1,3,4)P-3 and Ins(1,4)P-2, confirming activation of PtdIns(4,5)P-2 hydrolysis. No concentration of TSH provoked detectable accumulation of Ins(1,4,5)P-3 or Ins(1,4)P-2 during the first few minutes of stimulation. However, an InsP(3) [with the chromatographic properties of Ins(1,3,4)P-3] and two InsP(4) isomers [neither of which was Ins( 1,3,4,5)P-4] accumulated quickly in TSH-stimulated cells. ATP immediately provoked a large increase in intracellular calcium concentration ([Ca2+](i)) in Indo 1-AM-loaded cells. TSH provoked a small and delayed [Ca2+](i) elevation in only some experiments. We therefore confirm that activation of P-2-purinergic receptors and alpha(1)-adrenergic receptors provokes PIC activation, an accumulation of Ins(1,4,5)P-3 and its metabolites and rapid [Ca2+](i) mobilization in FRTL-5 cells. By contrast, TSH provokes no rapid PIC-catalysed PtdIns(4,5)P-2 hydrolysis or immediate [Ca2+](i) mobilization. These results fail to support the widespread view that the TSH receptor of FRTL-5 cells signals, in part, through PIC activation. Our results suggest that TSH activates another, still undefined, mechanism that causes accumulation of an InsP(3) and two isomers of InsP(4).