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-[[17β-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-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrolidine-2,5-dione), were determined. Intracellular calcium transient 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 μM) manner with an IC 50 of 1.5–1.8 μM. A concentration of 6–8 μM was required for complete iinhibition 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 μM by the addition of EGTA, ET-1 signals were completely inhibited by 4–6 μM U-73122 and the IC 50 was 0.8 μM. In the low external calcium medium, the PTH response was abolished by 2 μM U-73122 ( IC 50 = 0.5 μ M ). U-73122, 8 μM, significantly ( P < 0.01) inhibited the effect of ET-1 on inositol triphosphate 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 processes in osteoblastic cells.