Modulation of Adenylate Cyclase/Cyclic AMP Response by Thyrotropin and Prostaglandin E2 in Cultured Thyroid Cells. 1. Negative Regulation

Abstract

Isolated porcine thyroid cells, cultured in the presence of thyrotropin ( 2 0.25 mU/ml) or prostaglandin EZ ( 2 0.1 pM), showed decreased adenosine 3': 5'-monophosphate (cyclic AMP) response to further thyrotropin or prostaglandin EZ stimulation, respectively.Kinetics of the refractory process to thyrotropin and prostaglandin EZ are different : ( a) maximal refractoriness to prostaglandin EZ was attained after 2 -6 h exposure to prostaglandin EZ while refractoriness to thyrotropin was maximal only after 12-24 h ; (b) the degree of refractoriness to prostaglandin Ez was much greater than that to thyrotropin.Refractoriness to thyrotropin or prostaglandin EZ is characterized : by specificity for each thyroid stimulator; by dependence upon the dose of thyrotropin or prostaglandin E2 in culture, e.g. induction of high degree of refractoriness with 0.5 mU/ml thyrotropin (or 1 pM prostaglandin Ez), which elicits only a small cyclic AMP increase; by time requirement for induction; by partial effect; by changes of maximum activation of cyclic AMP response; by reversibility.This refractoriness of the cyclic AMP response was not induced by dibutyryl adenosine 3': 5'-monophosphate. It was not attributed to increased cyclic AMP-phosphodiesterase activity, but to alterations in the receptor-adenylate cyclase system. Prevention of refractoriness to thyrotropin or prostaglandin EZ by incubation of cells in the presence of actinomycin D, puromycin and cycloheximide suggests that new RNA and protein syntheses are required for the development of the refractory state.

Increases of prostacyclin and PGE2 levels after acute thyrotropin stimulation in cultured porcine thyroid cells

“…not induce refractoriness to further stimulation [6][7][8][9]. After 6 days' incubation, the cells were washed and then incubated with 50 mU TSH/ml, 10/~M (35/.tg/ ml) PGE2 or 10 btM (3.7/~g/ml) PGI2 in the presence of 10 mM theophylline in room air to get the time courses of cAMP synthesis of control and TSH-supplemented cells after acute stimulation with TSH, PGE2 or PGI2 ( fig.3).…”

“…Thyroid cells were obtained as in [6]. Freshly isolated cells were suspended (3 X 106 cells/ml) in Eagle minimum essential medium supplemented with 10% newborn calf serum (Flow Labs) and antibiotics (penicillin, 200 units/ml; streptomycin, 50/ag/ml).…”

Specific inhibition by Na+ of TSH‐stimulated thyroid cell adenylate cyclase

“…Cells were incubated for 4 days as unstirred suspension in untreated polystyrene dishes (10 ml cell suspension/100 mm dishes) in 95% air/5% CO2 water-saturated atmosphere at 37°C. Under these conditions cells reorganized into follicles and displayed maximal responsiveness to acute TSH stimulation [1,11].…”

“…Thyrotropin (TSH), isoproterenol and prostaglandin E: (PGE2) increase acutely intracellular cAMP levels in cultured porcine thyroid cells [1,2] whereas muscarinic agonists decrease this stimulation [3].…”

“…Responsiveness to the various stimulators can be modified by culture conditions and by the chronic treatment by the agonists themselves [2,4].…”