Endocrinology, Vol 135, 2488-2496, Copyright © 1994 by Endocrine Society

ARTICLES

Relative contribution of phosphoinositides and phosphatidylcholine hydrolysis to the actions of carbamylcholine, thyrotropin (TSH), and phorbol esters on dog thyroid slices: regulation of cytidine monophosphate-phosphatidic acid accumulation and phospholipase-D activity. I. Actions of carbamylcholine, calcium ionophores, and TSH

C Lejeune, J Mockel and JE Dumont
Institute of Interdisciplinary Research, Hospital Erasme, Universite Libre de Bruxelles School of Medicine, Belgium.

The actions of carbamylcholine (Cchol), the ionophores A23187 and thapsigargin, and TSH on [3H]cytidine monophosphate-phosphatidic acid ([3H]CAMP-PA) accumulation were studied in prelabeled dog thyroid slices to evaluate phosphatidic acid (PA) generation and inositol recycling by phosphatidylinositol (PtdIns) synthesis. The effects of the same agonists were also measured on phosphatidylbutanol generation in [3H]palmitate- or [3H]myristate-prelabeled slices to assess the activity of phospholipase-D (PLD) and on the effluxes of myo- [3H]inositol and [3H]choline induced by these agents from prelabeled slices. Cchol (10(-6)-10(-4) M) increased inositol phosphate (InsP) generation, with no change in inositol efflux, and contracted the intracellular inositol pool. This suggests a stimulation of PtdIns synthesis as well as hydrolysis. The muscarinic agonist provoked a dramatic accumulation of CMP-PA in the presence of lithium chloride (10 mM), which suggests that when InsP hydrolysis is inhibited, inositol limits the rate of CMP-PA incorporation into PtdIns. Cchol also increased phosphatidylbutanol formation. The latter two actions of Cchol were reproduced by A23187 (10(-5) M) and thapsigargin (2 x 10(-6) M) and were inhibited by calphostin-C, an inhibitor of the regulatory site of protein kinase-C. Cchol also induced increased free choline efflux, with a decreased choline phosphate relative content of the medium. TSH (10 mU/ml) stimulated free inositol efflux and induced a slight and proportional increase in [3H]inositol incorporation in phosphoinositides and InsP. The hormone also increased PA and CMP-PA accumulation exclusively in the presence of the PA phosphatase inhibitor propranolol (10(-4) M), but had no detectable action on PLD activity. None of these effects of TSH was reproduced by forskolin or potentiated by lithium chloride (10 mM). The data demonstrate the existence in thyroid tissue of a PLD-hydrolyzing phosphatidylcholine that was stimulated by Cchol and increased intracellular Ca2+, but not by TSH. The results obtained, besides confirming that TSH does not stimulate PtdInsP2-PLC or affect phosphatidylcholine hydrolysis, suggest that the hormone, instead, stimulates de novo PtdIns synthesis and/or inositol transport. The physiological relevance of these actions of Cchol, increased intracellular Ca2+, and TSH in thyroid metabolism could be related to their divergent effects on thyroid cell metabolism.

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