title: Thrombin stimulates inositol phosphate production and intracellular free calcium by a pertussis toxin-insensitive mechanism in osteosarcoma cells.
abstract: Human alpha-thrombin is known to elicit bone resorption in vitro and has been proposed as a mediator of increased bone turnover in inflammatory diseases. We used UMR 106-H5 rat osteoblast-like osteosarcoma cells to explore the signal transduction mechanism utilized by thrombin in bone. Thrombin produced a dose-dependent increase in the accumulation of [3H]inositol phosphates (IPs) in UMR 106-H5 cells prelabeled with [3H]myo-inositol (EC50 15 U/ml). In saponin-permeabilized cells, GTP gamma S increased [3H]IP production, whereas GDP beta S inhibited the response to both GTP gamma S and thrombin, indicating involvement of a G-protein in thrombin action. Thrombin produced a dose-dependent increase in intracellular free calcium (Cai2+) in UMR 106-H5 cells (EC50 1 U/ml; maximal increase 4-fold), as well as a small (20%) increase in [3H]thymidine incorporation. Treatment of UMR 106-H5 membranes with pertussis toxin (PT) and [32P]NAD+ resulted in labeling of a 40-kDa protein. However, pretreatment of cells with a dose of PT sufficient to produce maximal endogenous labeling of this protein failed to influence thrombin action on IP accumulation, Cai2+, or [3H]thymidine incorporation. In contrast, PT treatment of CCL39 hamster lung fibroblasts significantly blunted thrombin-stimulated [3H]IP accumulation and [3H]thymidine incorporation. These results suggest that thrombin raises Cai2+ in UMR 106-H5 cells by activating polyphosphoinositide-specific phospholipase C. Whereas in fibroblasts and platelets, thrombin receptors appear to couple to both PT-sensitive and PT-insensitive G-proteins, only a PT-insensitive G-protein appears to mediate thrombin action in UMR 106-H5 cells. Either these cells lack the relevant PT-sensitive G-protein or they possess thrombin receptors that selectively couple to a pertussis toxin-insensitive G-protein.
authors: Endocrine Section, Veterans Administration Medical Center, San Francisco, California 94121.
authors: M
authors: M
authors: Babich
authors: K L
authors: KL
authors: King
authors: R A
authors: RA
authors: Nissenson
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007294:Inositol / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D012516:Osteosarcoma / Q000378:metabolism*; D037342:Pertussis Toxin; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D013873:Thionucleotides / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
chemical_list: D007295:Inositol Phosphates; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007294:Inositol; C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D013917:Thrombin; D002118:Calcium