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

Abstract

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)P2 hydrolysis remains inconclusive. We have compared the effects of TSH and of ATP (acting via P2-purinergic receptors) on the inositol lipids and polyphosphates of [2-3H]inositol-labelled FRTL-5 rat thyroid cells. ATP initiated a rapid decrease in 3H-labelled PtdIns4P and PtdIns(4,5)P2, 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)P3, Ins(1,3,4,5)P4, Ins(1,3,4)P3 and Ins(1,4)P2, confirming activation of PtdIns(4,5)P2 hydrolysis. No concentration of TSH provoked detectable accumulation of Ins(1,4,5)P3 or Ins(1,4)P2 during the first few minutes of stimulation. However, an InsP3 [with the chromatographic properties of Ins(1,3,4)P3] and two InsP4 isomers [neither of which was Ins(1,3,4,5)P4] 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 P2-purinergic receptors and alpha 1-adrenergic receptors provokes PIC activation, an accumulation of Ins(1,4,5)P3 and its metabolites and rapid [Ca2+]i mobilization in FRTL-5 cells. By contrast, TSH provokes no rapid PIC-catalysed PtdIns(4,5)P2 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 InsP3 and two isomers of InsP4.

Differential effect of thyroid-stimulating hormone (TSH) on intracellular free calcium and cAMP in cells transfected with the human TSH receptor

“…While many studies have concluded or assumed that the effects of TSH on inositol phosphate turnover and calcium are the result of activation of phospholipase C, the kinetics of TSH-induced changes in membrane inositol phosphates do not correspond to those of a classical phospholipase C agonist (Singh et al 1996). Indeed, data from this laboratory show that, under conditions in which lithium significantly inhibited TSH-stimulated cAMP accumulation in cultured pig cells, there was no hydrolysis of phosphatidylinositol or accumulation of diacylglycerol .…”

“…Possibly, the accumulating cAMP was exerting some crosstalk, which ultimately led to an elevation in intracellular calcium concentration. This possibility has previously been explored by ourselves and other workers using forskolin to increase cAMP (Ye et al 1991, Singh et al 1996. Both studies concluded that forskolin was unable to mimic the actions of TSH on intracellular calcium (Ye et al 1991) or inositol phosphate turnover (Singh et al 1996).…”

“…This possibility has previously been explored by ourselves and other workers using forskolin to increase cAMP (Ye et al 1991, Singh et al 1996. Both studies concluded that forskolin was unable to mimic the actions of TSH on intracellular calcium (Ye et al 1991) or inositol phosphate turnover (Singh et al 1996). In the current study, cells were incubated (in the absence of 3-isobutyl-1-methylxanthine) with either a single addition of TSH (10 mU/ml) or increasing additions of TSH (0·01, 0·1, 1 and 10 mU/ml at 5 min intervals).…”

“…The agonist ATP was used as a positive control for activation of phospholipase C via P 2 -purinergic receptors (Sato et al 1992, Singh et al 1996. Cells responded to a single addition of ATP with a rapid increase in calcium in the majority (79%) of cells challenged.…”

“…dog (Rani et al 1985), rat FRTL-5 cells (Corda et al 1985, Mac Neil et al 1994, Singh et al 1996, pig (Takasu et al 1986, Ye et al 1991, CHO cells transfected with the human TSH receptor (Van Sande et al 1990, Mac Neil et al 1994 and human thyroid cells (Mac Neil et al 1994, Singh et al 1996). A recent study shows that the effects of TSH on inositol phosphate and calcium do not fit the classical pattern of agonists activating phospholipase C (Singh et al 1996), leaving unresolved the nature of this TSH-induced signalling.…”