THROMBIN AND PARATHYROID-HORMONE MOBILIZE INTRACELLULAR CALCIUM IN RAT OSTEOSARCOMA CELLS BY DISTINCT PATHWAYS

The mechanisms by which PTH and thrombin mobilize intracellular Ca2+ (Ca(i)2+) were examined in UMR 106-H5 rat osteosarcoma cells. Bovine PTH-(1-34) (24 pM to 240 nM) produced a dose-dependent increase in CA(i)2+ (EC50, 3 nM), which returned to baseline within 75 sec. Human alpha-thrombin produced an increase in Ca(i)2+ (EC(max), 10 U/ml) which was similar to that of PTH with respect to both magnitude and time course. Chelation of extracellular calcium with 5.0 mM EGTA did not alter the Ca(i)2+ response to either PTH or thrombin. When added together at maximally effective concentrations, PTH and thrombin produced additive effects on Ca(i)2+ in the presence and absence of EGTA. The additive effects of PTH and thrombin on Ca(i)2+ were confirmed at the single cell level, using laser-based image analysis. Bradykinin (1-mu-M) produced a significant increase in Ca(i)2+ in UMR 106-H5 cells which was of lesser magnitude than the peak 2- to 3-fold increase elicited by PTH or thrombin. Preexposure of cells to 10 U/ml thrombin for 2 min abolished the Ca(i)2+ response to bradykinin, whereas preexposure to 240 nM PTH had no effect on the Ca(i)2+ response to bradykinin. Thrombin elicited a rapid increase in the accumulation of H-3-labeled inositol phosphates (IP2 and IP3) in UMR 106-H5 cells, with increases in [H-3]1,4,5-IP3 detectable as early as 15 sec after the addition of thrombin. Bradykinin increased [H-3]IP production to a lesser extent than thrombin, whereas PTH neither increased [H-3]IP accumulation nor potentiated the [H-3]IP response to thrombin. The results suggest that thrombin and bradykinin mobilize Ca(i)2+ from a shared IP3-responsive calcium pool, whereas PTH may use signals in addition to 1,4,5-IP3 to mobilize calcium from a distinct cellular calcium pool. Alternatively, specific calcium compartmentalization exists, and there is differential coupling of these agonists to the 1,4,5-IP3/Ca(i)2+ pathway.