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 α-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 μM) 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 bredykinin, 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 ³H-labeled inositol phosphates (IP₂ and IP₃) in UMR 106-H5 cells, with increases in [³H]1,4,5-IP₃ detectable as early as 15 sec after the addition of thrombin. Bradykinin increased [³H]IP production to a lesser extent than thrombin, whereas PTH neither increased [³H]IP accumulation nor potentiated the [³H]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-IPa/Ca_i ²⁺. pathway.