THYROTROPIN DOES NOT ACTIVATE THE PHOSPHATIDYLINOSITOL BISPHOSPHATE HYDROLYZING PHOSPHOLIPASE-C IN THE DOG THYROID

The effects of thyrotropin (TSH) and carbamylcholine (Cchol) on labeling by [H-3]inositol of inositol lipids (i.e. total phosphoinositides (PI)) and inositol phosphates (IP) and on diacylglycerol (DAG) generation was studied in dog thyroid slices. Both agents (TSH 1-250 mU/ml, Cchol 10(-6) to 10(-4) M) increased the incorporation of [H-3]inositol into PI and IP during 4 h labeling experiments; but the [H-3]IP/[H-3]PI ratio as compared to the control one was not modified by TSH (10 mU/ml: 1.03 +/- 0.24) while it was increased by Cchol (10(-5) M: 6.14 +/- 1.81). Slices prelabeled in the absence of agonists were then incubated in the presence or absence of 10 mM LiCl +/- 10(-4) M inositol. With LiCl alone, Cchol increased [H-3]IP generation, while no such effect of TSH could be detected. However, in the absence of LiCl or in the presence of both LiCl and 10(-4) M inositol, TSH and Cchol both increased [H-3]PI and [H-3]IP, but IP and PI labeling remained strictly proportional with TSH (10 mU/ml: [H-3]IP/[H-3]PI ratio = 1.03 +/- 0.06 vs. control), while Cchol increased this ratio (10(-5) M = 2.44 +/- 0.24) with a preferential accumulation of IP. Both agonists stimulated DAG formation with similar kinetics and maximal effects (400% of control at 60 min). These findings (1) confirm that Cchol activates the classical pathway of the phosphatidylinositol bisphosphate hydrolyzing phospholipase C (PIP2-PLC); (2) suggest by contrast that the IP-PI effect of TSH does not proceed through PIP2 hydrolysis since it was blocked by LiCl and since TSH did not modify the IP/PI ratio of the tissue in any condition. TSH could stimulate the hydrolysis of other PI substrates, since molar DAG generation was similar with both agonists.