Evidence that an increase in cytoplasmic calcium is the initiating event in certain plasma membrane-mediated responses to 3,5,3'-triiodothyronine in rat thymocytes.

The present studies were undertaken to explore further the mechanism by which T3 increases adenylate cyclase activity and the uptake of the sugar analog 2-deoxyglucose (2-DG) in freshly isolated rat thymocytes. In studies of cells preloaded with the fluorescent probe quin-2, whose fluorescence intensity increases linearly with increases in cytoplasmic free calcium concentration [( Ca2+]i), we have now demonstrated that T3 increases [Ca2+]i in thymocytes suspended in buffer containing 1 mM Ca2+. This effect was extremely prompt, becoming evident much less than 1 min after the addition of T3 and reaching maximal values in about 5-8 min. The subsequent time course of the T3 effect was obscured by an increase in fluorescence intensity in control thymocytes not exposed to T3, beginning after about 8 min of incubation. However, the T3 effect, after reaching its peak, appeared to remain stable for about 5 min and then to decline, abating completely in 18-30 min. No effect of T3 on [Ca2+]i was observed when thymocytes were suspended in Ca2+-free medium. The effect of T3 on [Ca2+]i was concentration dependent, and as with its actions on thymocyte adenylate cyclase activity, cAMP concentration and 2-DG uptake, the lowest effective concentration of T3 was 1 nM. Among several thyronine analogs studied, L-T3 was the most potent, followed in decreasing order of potency by L-T4, D-T3, 3,5-diiodo-3'-isopropyl-L-thyronine, and D-T4. rT3, 3,5-diiodo-L-thyronine, and D,L-thyronine were without effect. l-Alprenolol alone (10 microM) produced a modest increase in thymocyte [Ca2+]i, but, as it does with the effect of T3 on cellular cAMP concentration and 2-DG uptake, it markedly inhibited or abolished the stimulatory effect of T3 on [Ca2+]i. From these observations we conclude that T3 initiates the increase in thymocyte [Ca2+]i by enhancing the influx of extracellular calcium, though the possibility that it also releases calcium from an intracellular calcium pool cannot be excluded. Since the effects of T3 on thymocyte adenylate cyclase activity, cAMP concentration, and 2-DG uptake occur subsequent to these effects on calcium metabolism and require the presence of Ca2+ in the extracellular fluid, we suggest that an increase in [Ca2+]i, due at least partly to an influx of extracellular calcium, is the initiating event in these plasma membrane-mediated responses of the rat thymocyte to T3.