Molecular and Cellular PharmacologyEvidence for separate effects of U73122 on phospholipase C and calcium channels in human platelets
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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Cited by (39)
Recent advances in understanding the molecular role of phosphoinositide-specific phospholipase C gamma 1 as an emerging onco-driver and novel therapeutic target in human carcinogenesis
2021, Biochimica et Biophysica Acta - Reviews on CancerCitation Excerpt :Earlier, focusing on PIP2-based small molecule inhibitors, several fluorogenic substrate analogues such as substrate 2, DDPB and lysoDDPB against PLC isoforms were developed [175–177]. Moreover, several other compounds especially aminosteroid U73122 was most frequently reported to be used as a potent PLC inhibitor; although, its off-target effect on several other unrelated targets such as calcium pumps and high cytotoxicity were reported [178–180]. Therefore, in an urgent need to develop novel small molecule inhibitors against PLC isoforms including PLCγ1, Reynisson et al. identified several potent PLCγ inhibitors such as CCT196700, CCT196714 and CCT196789 which exerted high selectivity and low cytotoxicity in several biochemical assays such as 3H-PIP2 flash-plate biochemical assay and Calcium release assay [181].
Enhanced Ca<sup>2+</sup> handling in thioglycolate-elicited peritoneal macrophages
2021, Cell CalciumCitation Excerpt :Therefore, the TRP family channels contributing to constitutive Ca2+ influx seemed to be largely PLC-dependently activated in TGPMs. On the other hand, it has been reported that U73122 weakly attenuates SOCE responses in several cell types, suggesting a functional link between the STIM-Orai system and PI turnover [24–26]. U73122 obviously inhibited SOCE in TGPMs (Fig. 5D), and this inhibition degree (−48.7 ± 1.7 % in Δ F340/F380) was roughly similar to those of GSK7975A (−53.2 ± 1.4 %) and SKF96365 (−56.4 ± 2.3 %).
Phospholipase C families: Common themes and versatility in physiology and pathology
2020, Progress in Lipid ResearchCitation Excerpt :These include the most frequently used aminosteroid U73122 [1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione] and edelfosine or ET-18-OCH3 (1-0-octadecyl-2-0-methyl-glycerol-3-phosphocholine) [424,425]. However, mechanisms of action remain unclear and a number of reports, in particular for the most widely used U73122, suggest other targets; these include calcium pumps, ion channels and unrelated enzymes regulating lipid metabolism [426–436]. Initial assessments in vitro have suggested that the inhibitory effects of U73122 could be demonstrated only when using specific lipid composition and further studies have shown that treatment of purified PLC isoforms with this compound had surprisingly diverse effects on activity, including increased activity of PLCγ [437,438].
Phosphatidylinositol-3,4,5-trisphosphate stimulates Ca<sup>2+</sup> elevation and Akt phosphorylation to constitute a major mechanism of thromboxane A<inf>2</inf> formation in human platelets
2015, Cellular SignallingCitation Excerpt :The presence of 1 U/ml apyrase reduced the aggregation to a similar extent. As PIP3 is implicated in the activation of PLCγ isoforms [22], we tested the PLC inhibitor U73122 [with caution as it has been reported to inhibit platelet aggregation and Ca2 + entry at lower doses than for PLC [23]]. At 1 μM, U73122 inhibited DiC8-PIP3 induced platelet aggregation by 93% (P < 0.0001).
Dysfunction of phospholipase Cγ in immune disorders and cancer
2014, Trends in Biochemical SciencesSmall molecule inhibitors of phospholipase C from a novel high-throughput screen
2013, Journal of Biological Chemistry