Role of protein kinase A in the regulation of cytosolic free calcium in human osteoblast-like SaOS-2 cells.

We have used wild-type and adenosine 3',5'-cyclic monophosphate (cAMP)-resistant mutant osteoblast-like SaOS-2 cells to investigate the role of protein kinase A (PKA) in the regulation of cytosolic free Ca2+ concentration ([Ca2+]i). Basal levels of [Ca2+]i were the same in wild-type (127 +/- 6.1 nM) and transfected (117 +/- 6.8 nM) SaOS-2 cells, although 45Ca2+ efflux was slower in the transfected cells. In wild-type cells, thapsigargin (TG, > or = 200 nM), an inhibitor of the Ca(2+)-ATPase activity of the endoplasmic reticulum, acutely increased [Ca2+]i (by up to 2-fold), which then returned promptly to basal [Ca2+]i. In cAMP-resistant cells, TG elicited a significantly greater acute rise in [Ca2+]i, which then decayed to an elevated plateau level. In mutant cells, high concentrations of dibutyryladenosine 3',5'-cyclic monophosphate, which overcome the PKA blockade, restored the changes in [Ca2+]i to the wild-type pattern. In cAMP-resistant, TG-blocked cells, ionomycin (or alpha-thrombin) induced a further elevation in [Ca2+]i, which then declined rapidly to the original basal level. We conclude that basal PKA activity is involved actively in regulation of [Ca2+]i in SaOS-2 cells by promoting Ca2+ efflux from the cell and, possibly, by inhibiting Ca2+ release from or stimulating net Ca2+ sequestration into the ER. We have also obtained evidence for an alternate Ca(2+)-triggered Ca2+ reuptake mechanism in SaOS-2 cells that is not dependent on either Ca(2+)-ATPase or PKA.