RELATIVE CONTRIBUTION OF PHOSPHOINOSITIDES AND PHOSPHATIDYLCHOLINE HYDROLYSIS TO THE ACTIONS OF CARBAMYLCHOLINE, THYROTROPIN, AND PHORBOL ESTERS ON DOG THYROID SLICES - REGULATION OF CYTIDINE MONOPHOSPHATE PHOSPHATIDIC-ACID ACCUMULATION AND PHOSPHOLIPASE-D ACTIVITY .2. ACTIONS OF PHORBOL ESTERS

The effects of phorbol dibutyrate (PDBu) on phosphatidylbutanol (PtdBu t) generation in [H-3]palmitate- or [H-3]myristate-prelabeled dog thyr oid slices were measured to assess the activity of phospholipase-D (PL D) in the presence or absence of the two inhibitors of protein kinase- C (PKC), staurosporine (STSP) and calphostin-C. The actions of the sam e agents on [H-3]cytidine monophosphate-phosphatidic acid accumulation were also determined to evaluate phosphatidic (PA) generation and ino sitol recycling to phosphatidylinositol. The effluxes of [H-3]choline and [H-3]ethanolamine induced by the phorbol ester from prelabeled sli ces were also evaluated. PDBu (5 x 10(-9) to 5 x 10(-6) M) potently st imulated PLD activity, with a concomitant increase in fatty acids inco rporation in phosphatidylcholine (PtdCho). However, under no condition did the phorbol ester result in cytidine monophosphate-phosphatidic a cid accumulation. It stimulated the efflux of choline and ethanolamine while decreasing choline and ethanolamine phosphates in the slices an d incubation medium. Calphostin-C, inhibiting PKC, decreased PtdBut an d PtdCho formation induced by the phorbol ester, as opposed to STSP (5 x 10(-6) M), which did not affect these actions of PDBu and, moreover , reproduced by itself the effects of the phorbol ester on choline eff lux and PtdBut generation despite efficient inhibition of other effect s of PKC. These data demonstrate the existence in thyroid tissue of a PLD-hydrolyzing PtdCho, which was stimulated by phorbol esters and STS P. They also suggest that the PA formed after PKC stimulation and subs equent PLD activation is channeled toward PtdCho resynthesis when intr acellular Ca2+ is not increased, whereas the PA accumulated with a con comitant increase in intracellular Ca2+ is diverted toward phosphatidy linositol synthesis. The physiological relevance of this Ca-independen t stimulation of a PKC-coupled PLD in thyroid metabolism could be rela ted to the growth-inducing and dedifferentiating effects of the phorbo l esters.