title: U-73122, a phospholipase C antagonist, inhibits effects of endothelin-1 and parathyroid hormone on signal transduction in UMR-106 osteoblastic cells.
abstract: Endothelin-1 (ET-1) and parathyroid hormone (PTH) increase calcium transients in rodent osteoblastic cells. To investigate the role of phospholipase C (PLC) in these hormone-stimulated calcium signals, the effects of U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)- trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), a reported PLC inhibitor, and its inactive analog, U-73343 (1-[6[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]- 1H-pyrrolidine-2,5-dione), were determined. Intracellular calcium transients were measured in UMR-106 cells with the fluorescent indicator fluo-3. In normal calcium containing medium, prior exposure (3 min) to U-73122 inhibited ET-1 and PTH stimulated calcium transients in a dose-dependent (0.2-10 microM) manner with an IC50 of 1.5-1.8 microM. A concentration of 6-8 microM was required for complete inhibition of responses to 100 nM ET-1 or PTH. U-73343 elicited no effects over this concentration range. In cells in which external calcium was reduced to less than 1 microM by the addition of EGTA, ET-1 signals were completely inhibited by 4-6 microM U-73122 and the IC50 was 0.8 microM. In the low external calcium medium, the PTH response was abolished by 2 microM U-73122 (IC50 = 0.5 microM). U-73122, 8 microM, significantly (P < 0.01) inhibited the effect of ET-1 on inositol trisphosphate production at 3 min whereas U-73343 did not. Pertussis toxin (100 ng/ml) likewise significantly inhibited the effect of ET-1 on phosphoinositol turnover as well as on intracellular calcium concentration. In conclusion, the results support the hypothesis that PLC plays a role in the calcium transients elicited by ET-1 and PTH, and that ET-1 transmits its signal in part via a pertussis toxin sensitive G-protein coupled receptor. Furthermore they suggest that U-73122 is useful for investigating PLC-mediated process in osteoblastic cells.
authors: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, IL 60611.
authors: A
authors: A
authors: Tatrai
authors: S K
authors: SK
authors: Lee
authors: P H
authors: PH
authors: Stern
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D016232:Endothelins / Q000494:pharmacology*; D004963:Estrenes / Q000494:pharmacology*; D007295:Inositol Phosphates / Q000096:biosynthesis; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology*; D011760:Pyrrolidinones / Q000494:pharmacology*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*
chemical_list: D016232:Endothelins; D004963:Estrenes; D007295:Inositol Phosphates; D010281:Parathyroid Hormone; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D010738:Type C Phospholipases; D002118:Calcium