A NOVEL PHOSPHOLIPASE-C INHIBITOR AND PHORBOL ESTERS REVEAL SELECTIVEREGULATION OF THROMBIN-STIMULATED 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 pool s in UMR 106-H5 rat osteosarcoma cells. The present studies were desig ned to explore the molecular basis of this differential signaling. Max imally effective concentrations of both PTH (240 nM) and thrombin (10 U/ml) produced a rapid intracellular free calcium (Ca-i(++)) transient (a 2- to 3-fold increase) that was inhibited by pretreatment with the phospholipase C inhibitor 1-[6-[[17 5(10)-trien-17-yl]amino]hexyl]1H- pyrrole-2,5-dione (U73,122) in a dose-dependent manner (IC50 = 3 mu M) . Inhibition by U73,122 was not associated with a change in PTH-stimul ated adenylate cyclase activity, whereas inositol phosphate accumulati on, detected only in response to thrombin, was inhibited 23 to 45%. Pr ior exposure of cells for 5 min with the protein kinase C activators p horbol 12-myristate 13-acetate (8-80 nM) and phorbol 12,13-dibutyrate (80 nM) weakly inhibited (less than or equal to 30%) the peak Ca-i(++) increase in response to thrombin but completely blocked the Ca-i(++) response to PTH. In contrast, 12-myristate 13-acetate produced a 1.55- fold increase in the maximal stimulatory effect of PTH on adenylate cy clase activity. These data suggest that activation of phospholipase C is a prerequisite for both PTH- and thrombin-stimulated increases in C a-i(++) and that protein kinase C differentially regulates the ability of these agents to raise Ca-i(++). Collectively the results support t he notion that the IP3/calcium mobilizing pathways utilized by PTH and thrombin are compartmentalized. Further, the finding that phorbol est ers produce opposite effects on PTH-stimulated adenylate cyclase and C a-i(++) suggests that protein kinase C can alter the cellular response to PTH by regulating the relative strength of signaling via these pat hways.