Phosphatase inhibition reveals a calcium entry pathway dependent on protein kinase A in thyroid FRTL-5 cells: comparison with store-operated calcium entry.

Calcium entry through store-operated calcium channels is an important entry mechanism. In the present report we have described a novel calcium entry pathway that is independent of depletion of intracellular calcium stores. Treatment of the cells with the phosphatase inhibitor calyculin A (caly A), which blocked thapsigargin-evoked store-operated calcium entry (SOCE), induced a potent concentration-dependent calcium entry. In a calcium-free buffer, acute addition of caly A evoked a very modest increase in cytosolic free calcium ([Ca(2+)](i)). This increase was not from the agonist-mobilizable calcium stores, as the thapsigargin-evoked increase in [Ca(2+)](i) was unaltered in caly A-treated cells. The caly A-evoked calcium entry was not blocked by Gd(3+) or 2-APB, whereas SOCE was. Caly A enhanced the entry of barium, indicating that the increase in intracellular calcium was not the result of a decreased extrusion of calcium from the cytosol. Jasplakinolide and cytochalasin D had only marginal effects on calcium entry. The protein kinase A (PKA) inhibitor H-89 and an inhibitory peptide for PKA abolished the caly A-evoked entry of both calcium and barium. The SOCE was, however, enhanced in cells treated with H-89. In cells grown in the absence of thyrotropin (TSH), the caly A-evoked entry of calcium was smaller compared with cells grown in TSH-containing buffer. Stimulation of cells grown without TSH with forskolin or TSH restored the calyculin A-evoked calcium entry to that seen in cells grown in TSH-containing buffer. SOCE was decreased in these cells. Our results thus suggest that TSH, through the production of cAMP and activation of PKA, regulates a calcium entry pathway in thyroid cells. The pathway is distinctly different from the SOCE. As TSH is the main regulator of thyroid cells, we suggest that the novel calcium entry pathway participates in the regulation of basal calcium levels in thyroid cells.