Intracellular Ca²⁺ mobilization by thyrotropin, carbachol, and adenosine triphosphate in dog thyroid cells

The effect of TSH, carbachol (CC), and ATP on intracellular calcium concentration ([Ca²⁺]i) in primary cultures of dog thyroid cells was examined using the fluorescent Ca²⁺ indicator fura-2. TSH caused an increase in [Ca²⁺]i at 37 C, but not 22 C, while it increased cAMP formation in these cells at both 22 and 37 C. CC and ATP increased [Ca²⁺]i at both 22 and 37 C. The CC-induced increase in [Ca²⁺]i was under muscarinic receptor control, and it was biphasic, with an initial spike followed by a sustained increase at a lower level. TSH and ATP were weaker agonists compared to CC, since maximal doses of TSH (100−500 mU/ml) and ATP (100−500 μM) increased [Ca²⁺]i by 40−70% over basal levels, compared to a 2-to 4-fold increase in [Ca²⁺] induced by maximal doses of CC (10−50 μM). The TSH-induced increase in [Ca²⁺]i was transient, returning to basal levels within 1−2 min after application of the agonist. All three agents were able to transiently increase [Ca²⁺]i in Ca²⁺-free/EGTA buffer, indicating the source of this [Ca²⁺]i to be internal stores. In the presence of the inorganic Ca²⁺ channel blockers La³⁺, Ni²⁺, and Co²⁺, the peak [Ca²⁺]i change was little affected, while the persistent response to CC and ATP was blocked, indicating dependence of this phase on influx of Ca²⁺. Paradoxically, these channel blockers abolished the effect of TSH on [Ca²⁺]i. TSH stimulation of cAMP formation was also inhibited 80−90% by these blockers, but not in Ca²⁺-free/EGTA buffer. These results suggest that the Ca²⁺ channel blockers may have actions in addition to inhibition of Ca²⁺ entry in these cells. TMB-8 [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate HCl] specifically blocked both the initial and sustained increase induced by CC, while having no effect on ATP or TSH-induced [Ca²⁺];, suggesting that TMB-8 may not be a general antagonist of Ca²⁺ mobilization. Activators of protein kinase-C, such as phorbol esters or an analog of diacylglycerol, inhibited the [Ca²⁺]i rise induced by all the three agonists used, indicating a regulatory role of protein kinase-C activation on [Ca²⁺]j in these cells. In FRTL-5 cells, [Ca²⁺]i was also increased by TSH and ATP, but not by CC. ATP, however, was a more effective agonist than in dog thyroid cells, while TSH increased [Ca²⁺]i by a similar magnitude in both cell types. The results of the present study demonstrate that TSH, albeit of lesser potency than CC, increases [Ca²⁺]; by causing intracellular Ca²⁺ mobilization in cultured dog thyroid cells. The failure to detect such an increase previously could have been due to the small magnitude of this [Ca²⁺]i change and the prior use of quin²⁺ as the Ca²⁺ indicator.