Thrombin and parathyroid hormone mobilize intracellular calcium in rat osteosarcoma cells by distinct pathways.
The mechanisms by which PTH and thrombin mobilize intracellular Ca2+ (Cai2+) were examined in UMR 106-H5 rat osteosarcoma cells. Bovine PTH-(1-34) (24 pM to 240 nM) produced a dose-dependent increase in Cai2+ (EC50, 3 nM), which returned to baseline within 75 sec. Human alpha-thrombin produced an increase in Cai2+ (ECmax, 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 Cai2+ response to either PTH or thrombin. When added together at maximally effective concentrations, PTH and thrombin produced additive effects on Cai2+ in the presence and absence of EGTA. The additive effects of PTH and thrombin on Cai2+ were confirmed at the single cell level, using laser-based image analysis. Bradykinin (1 microM) produced a significant increase in Cai2+ 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 Cai2+ response to bradykinin, whereas preexposure to 240 nM PTH had no effect on the Cai2+ 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 Cai2+ 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/Cai2+ pathway.
Babich
M
M
Endocrine Section, Veterans Affairs Medical Center, San Francisco, California 94121.
Choi
H
H
Johnson
R M
RM
King
K L
KL
Alford
G E
GE
Nissenson
R A
RA
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D007295:Inositol Phosphates / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D012516:Osteosarcoma; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D051381:Rats; D019379:Teriparatide; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors
D007295:Inositol Phosphates; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D019379:Teriparatide; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D002118:Calcium