Stimulation of adenylate cyclase activity in rat thymocytes in vitro by 3,3,5’ tri-iodothyronine

Abstract

In view of our previous demonstration that T3 promptly increases the cAMP concentration in freshly isolated rat thymocytes in vitro, we studied the effects of T3 on adenylate cyclase activity in a crude thymocyte plasma membrane preparation. In common with adenylate cyclase in other tissues, the enzyme in rat thymocytes was activated by NaF, GTP, 5'-guanylylimidodiphosphate, and beta-adrenergic agonists and was inhibited by high concentrations of calcium. In the presence of 1 microM Ca+2, T3 induced a time-dependent increase in adenylate cyclase activity that was statistically significant between 1 and 2 min and maximum between 2 and 5 min after hormone addition. As judged from observations made at 5 min, the effect of T3 was dose dependent over the range 1 nM to 1 microM. The stimulatory effect of T3 was calcium dependent, since it was abolished by EGTA at a concentration (0.5 mM) that did not alter basal enzyme activity, and the effect of T3 in the presence of EGTA was restored by the addition of either 0.1 or 1 mM Ca+2. As judged from the lack of hydrolysis of added cAMP, phosphodiesterase activity in the assay mixture was nil in both the presence and absence of T3. Both epinephrine and the specific beta-adrenergic agonist isoproterenol, but not the alpha-agonist phenylephrine, increased adenylate cyclase activity, and their effects appeared to be additive to that of T3. The beta-adrenergic antagonist L-alprenolol, in doses that did not influence basal adenylate cyclase activity, produced a dose-related inhibition of the stimulatory effect of T3 and of the effects of epinephrine and isoproterenol as well. Neither D-alprenolol nor the alpha-antagonist phentolamine had any effect. Various thyronine analogs displayed a rank order of potency in stimulating adenylate cyclase activity very similar to their relative potencies in increasing cAMP concentration in the intact thymocyte. These findings reveal that T3 stimulates adenylate cyclase activity in rat thymocyte plasma membrane preparations. With respect to calcium dependence, inhibition by alprenolol, and response to thyronine analogs, this effect has properties similar to those of the increase in cellular cAMP concentration induced by T3 in the intact thymocyte. It can be concluded, therefore, that the effect of T3 to increase 2-deoxyglucose uptake by the rat thymocyte in vitro, a response consequent to an increase in thymocyte cAMP concentration, derives from a stimulatory effect of the hormone on adenylate cyclase itself