A novel phospholipase C inhibitor and phorbol esters reveal selective regulation of thrombin- and parathyroid hormone-stimulated signaling pathways in rat osteosarcoma cells.
Previous studies have demonstrated that parathyroid hormone (PTH) and human alpha-thrombin mobilize intracellular calcium from distinct pools in UMR 106-H5 rat osteosarcoma cells. The present studies were designed to explore the molecular basis of this differential signaling. Maximally effective concentrations of both PTH (240 nM) and thrombin (10 U/ml) produced a rapid intracellular free calcium (Cai++) transient (a 2- to 3-fold increase) that was inhibited by pretreatment with the phospholipase C inhibitor 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]- 1H-pyrrole-2,5-dione (U73,122) in a dose-dependent manner (IC50 = 3 microM). Inhibition by U73,122 was not associated with a change in PTH-stimulated adenylate cyclase activity, whereas inositol phosphate accumulation, detected only in response to thrombin, was inhibited 23 to 45%. Prior exposure of cells for 5 min with the protein kinase C activators phorbol 12-myristate 13-acetate (8-80 nM) and phorbol 12,13-dibutyrate (80 nM) weakly inhibited (< or = 30%) the peak Cai++ increase in response to thrombin but completely blocked the Cai++ response to PTH. In contrast, 12-myristate 13-acetate produced a 1.55-fold increase in the maximal stimulatory effect of PTH on adenylate cyclase activity. These data suggest that activation of phospholipase C is a prerequisite for both PTH- and thrombin-stimulated increases in Cai++ and that protein kinase C differentially regulates the ability of these agents to raise Cai++. Collectively the results support the notion that the IP3/calcium mobilizing pathways utilized by PTH and thrombin are compartmentalized.(ABSTRACT TRUNCATED AT 250 WORDS)
Babich
M
M
Department of Biomedical Sciences, University of Illinois College of Medicine, Rockford.
Alford
G E
GE
Nissenson
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001859:Bone Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000503:physiopathology*; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation; D004963:Estrenes / Q000494:pharmacology*; D007295:Inositol Phosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D012516:Osteosarcoma / Q000201:enzymology / Q000378:metabolism / Q000503:physiopathology*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000502:physiology; D011760:Pyrrolidinones / Q000494:pharmacology*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology*; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*
D004963:Estrenes; D007295:Inositol Phosphates; D010281:Parathyroid Hormone; D010703:Phorbol Esters; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium