Elsevier

Biochemical Pharmacology

Volume 56, Issue 11, 1 December 1998, Pages 1481-1484
Biochemical Pharmacology

Molecular and Cellular Pharmacology
Evidence for separate effects of U73122 on phospholipase C and calcium channels in human platelets

https://doi.org/10.1016/S0006-2952(98)00146-4Get rights and content

Abstract

U73122 ({1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)exyl]-1H-pyrrole-2,5-dione}) is generally used as a selective inhibitor of phospholipase C (PLC) and the related rise in cytosolic Ca2+. Recently, by using hepatocytes, it was suggested that its action sites are different for PLC activation and increase in Ca2+ concentration. To verify whether U73122 has different sites for inhibiting PLC activation and calcium responses in human platelets, aggregation, Mn2+ influx, cytosolic Ca2+ increase and PLC activation were studied in response to thrombin and the synthetic agonist of the thromboxane receptor U46619 (9,11-dideoxy-9α,11α-methanoepoxyprostaglandin F). With both agonists, U73122 inhibited aggregation, Mn2+ influx and the enhancement of cytosolic calcium at concentrations of 2 μM or lower, while 10 μM was necessary to inhibit PLC activation. Our results suggested that U73122 is much more active in antagonizing Ca2+ channels, both the intracellular ones, which are activated by formation of inositol 1,4,5 P3 and those present on plasma membrane, than in reducing the activation of PLC.

Section snippets

Platelet preparation

Platelet rich plasma was obtained after centrifugation (180 g for 15 min) of blood samples taken by venipuncture from healthy volunteers into acid/citrate/dextrose (ACD) [11]. Platelet-rich plasma was recentrifuged at 800 g for 20 min and the pellet was resuspended in 0.5 vol of autologous platelet poor plasma. The concentrated platelets were incubated with aspirin (1 mM) (Sigma) for at least 15 min at 37° and with either the fluorescent indicator Fura-2-AM (4 μM) (Molecular Probes) for 30 min

Results

FIG. 1, FIG. 2 show that U73122 (1–10 μM) dose dependently inhibited the maximum platelet aggregation, Mn2+ influx, changes in intracellular calcium concentration and PLC activation induced by 1 U/mL of thrombin (Fig. 1) and 1 μM U46619 (Fig. 2). Platelet aggregation and manganese influx induced by both agonists were the only responses significantly reduced after preincubation with 1 μM U73122. Increases in intracellular calcium in response to thrombin or U46619 showed a significant reduction

Discussion

Our results show that U73122 is more active in antagonizing Ca2+ channels, both the intracellular ones which are activated by IP3 and those present on plasma membrane, than in inhibiting PLC. In fact, this drug significantly reduced platelet aggregation, the influx of Mn2+, and Ca2+ mobilization at a concentration of 1 to 2 μM, while the production of the stable metabolite of phosphatidylinositol 4,5 P2, Ins 1,3,4 P3, was significantly reduced only by using U73122 at the concentration of 10 μM.

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