Tumor promoters as probes of protein kinase C in dog thyroid cell: Inhibition of the primary effects of carbamylocholine and reproduction of some distal effects

Abstract

The acute effects of phorbol esters, used as probes of protein kinase C activation, were studied on dog thyroid slices incubated in vitro. The derivatives used were: tetradecanoylphorbol acetate (TPA), phorbol-12,13, didecanoate (PDD), phorbol-12,13-diacetate (PDA), and phorbol dibutyrate (PDBu) and as inactive controls, phorbol itself, phorbol-12, myristate and phorbol-13, acetate, in concentrations ranging from 5.10 −8 to 5.10 −6 mol/L. The active phorbol esters had no effect on basal cyclic AMP concentrations; they inhibited cyclic AMP accumulation induced by prostaglandin E 1 but not that induced by thyrotropin (TSH) 1 mU/mL and forskolin 10 μmol/L. Phorbol esters like carbamylcholine acutely stimulated iodide organification and inhibited the stimulation of hormone secretion resulting from TSH, Cholera Toxin, forskolin, and Bu 2-cyclic AMP action. These metabolic effects did not require the presence of extracellular Ca ++, and could not be antagonized by Ca ++ depletion or manganese addition. The active phorbol esters abolished the cyclic AMP independent increased PI turnover induced by TSH 10 mU/mL or carbamylcholine (Cchol) 10 −6 mol/L but did not affect the basal incorporation of 32P into phosphatidylinositol. They reduced the 45Ca efflux from preloaded slices below basal levels and blocked the increased 45Ca release induced by TSH and Cchol. They also inhibited the increase in cyclic GMP concentrations resulting from Cchol action but not the effect of the ionophore A23187 (10 −5 mol/L) nor the basal levels of cyclic GMP. They relieved the inhibitions of TSH induced cyclic AMP accumulation by Cchol 10 −6 to 10 −5 mol/L and prostaglandin F 1α (2 μg/mL), but not the inhibitions caused by norepinephrine 100 μmol/L, adenosine (5 mmol/L), or potassium iodide (10 −4 mol/L) additions. None of the above described effects could be reproduced by the inactive phorbol esters. In conclusion, the data presented here show that active phorbol esters reproduce the effects of carbachol and prostaglandin F α on iodine metabolism in dog thyroid slices. On the other hand, they inhibit phosphatidylinositol turnover, calcium translocation, and the consequences of increased ionized free calcium in the slices, ie, cyclic GMP accumulation and inhibition of cyclic AMP accumulation. This suggests that diacylglycerol released by the hydrolysis of phosphatidylinositol in response to thyrotropin and acetylcholine acts in parallel with calcium as an intracellular signal controlling iodine metabolism but also as a feedback inhibition of the initial interaction of these agonists with this system.