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Pharmacological actions of nobiletin in the modulation of platelet function.

Vaiyapuri S, Roweth H, Ali MS, Unsworth AJ, Stainer AR, Flora GD, Crescente M, Jones CI, Moraes LA, Gibbins JM - Br. J. Pharmacol. (2015)

Bottom Line: The effect of nobiletin in vivo was assessed by measuring tail bleeding time using C57BL/6 mice.Nobiletin extended bleeding time in mice and reduced the phosphorylation of PKB (Akt) and PLCγ2 within the collagen receptor (glycoprotein VI)-stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of vasodilator-stimulated phosphoprotein, a protein whose activity is associated with inhibitory cyclic nucleotide signalling.Therefore, nobiletin may represent a potential antithrombotic agent of dietary origins.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK.

No MeSH data available.


Related in: MedlinePlus

Nobiletin inhibits phosphorylation of PLCγ2 and PKB (Akt). Washed human platelets were stimulated with CRP-XL (1 μg·mL−1) in the absence or presence of various concentrations of nobiletin before the analysis by immunoblotting using anti-phosphotyrosine antibody (A) and phospho-specific antibodies for proteins involved in the GPVI pathway such as Syk pY323, LAT pY200, PLCγ2 pY759 and Akt pS473 (B). Total level of 14-3-3ζ was measured on each sample as a loading control. The blots shown in the figure are representative of four separate experiments. R represents resting platelets. Data presented in (B) represent mean ± SD (n = 4) and P-values were calculated using non-parametric repeated measures anova (Friedman test; ****P < 0.0001).
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fig05: Nobiletin inhibits phosphorylation of PLCγ2 and PKB (Akt). Washed human platelets were stimulated with CRP-XL (1 μg·mL−1) in the absence or presence of various concentrations of nobiletin before the analysis by immunoblotting using anti-phosphotyrosine antibody (A) and phospho-specific antibodies for proteins involved in the GPVI pathway such as Syk pY323, LAT pY200, PLCγ2 pY759 and Akt pS473 (B). Total level of 14-3-3ζ was measured on each sample as a loading control. The blots shown in the figure are representative of four separate experiments. R represents resting platelets. Data presented in (B) represent mean ± SD (n = 4) and P-values were calculated using non-parametric repeated measures anova (Friedman test; ****P < 0.0001).

Mentions: Since collagen and CRP-XL-stimulated platelet activation was affected by nobiletin, the phosphorylation levels of different signalling proteins involved in the GPVI pathway were analysed. Washed human platelets were stimulated at 37°C in an aggregometer under conditions (in presence of 1 mM EGTA, 10 μM indomethacin and 2 U·mL−1 apyrase) that disfavour platelet aggregation with CRP-XL (1 μg·mL−1) in the presence of various concentrations of nobiletin (12.5, 25, 50, 100, 150 and 200 μM) or vehicle control, and lysates were prepared to analyse the phosphorylation of different signalling proteins. Total tyrosine phosphorylation (Figure 5A) and the phosphorylation of individual proteins such as Syk (pY323) and LAT (pY200; Figure 5B) were not affected by nobiletin. However, tyrosine phosphorylation of PLCγ2 (pY759) was diminished at all concentrations of nobiletin used (Figure 5B). Similarly, the serine phosphorylation of PKB (or Akt) (pS473) was also affected in a concentration-dependent manner by nobiletin (Figure 5B). Although the phosphorylation levels were quantified under non-aggregation conditions, the level of inhibition of phosphorylation of PLCγ2 and Akt were similar to the inhibitory effects observed on platelet aggregation at the same concentration of agonist (Figure 1G and 1H). For example, 12.5 μM of nobiletin inhibited around 20–30% of CRP-XL-induced platelet aggregation and phosphorylation of PLCγ2 and Akt. These data suggest that nobiletin may not influence the immediate effectors within the GPVI pathway (such as Syk and LAT) although it can modulate the later signalling events controlled by PLCγ2 and Akt, which are shared with activation mechanisms stimulated by other agonists. The inhibitory effects of nobiletin on PLCγ2 and Akt phosphorylation indicate its potential effect on PI3K-mediated platelet signalling.


Pharmacological actions of nobiletin in the modulation of platelet function.

Vaiyapuri S, Roweth H, Ali MS, Unsworth AJ, Stainer AR, Flora GD, Crescente M, Jones CI, Moraes LA, Gibbins JM - Br. J. Pharmacol. (2015)

Nobiletin inhibits phosphorylation of PLCγ2 and PKB (Akt). Washed human platelets were stimulated with CRP-XL (1 μg·mL−1) in the absence or presence of various concentrations of nobiletin before the analysis by immunoblotting using anti-phosphotyrosine antibody (A) and phospho-specific antibodies for proteins involved in the GPVI pathway such as Syk pY323, LAT pY200, PLCγ2 pY759 and Akt pS473 (B). Total level of 14-3-3ζ was measured on each sample as a loading control. The blots shown in the figure are representative of four separate experiments. R represents resting platelets. Data presented in (B) represent mean ± SD (n = 4) and P-values were calculated using non-parametric repeated measures anova (Friedman test; ****P < 0.0001).
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Related In: Results  -  Collection

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fig05: Nobiletin inhibits phosphorylation of PLCγ2 and PKB (Akt). Washed human platelets were stimulated with CRP-XL (1 μg·mL−1) in the absence or presence of various concentrations of nobiletin before the analysis by immunoblotting using anti-phosphotyrosine antibody (A) and phospho-specific antibodies for proteins involved in the GPVI pathway such as Syk pY323, LAT pY200, PLCγ2 pY759 and Akt pS473 (B). Total level of 14-3-3ζ was measured on each sample as a loading control. The blots shown in the figure are representative of four separate experiments. R represents resting platelets. Data presented in (B) represent mean ± SD (n = 4) and P-values were calculated using non-parametric repeated measures anova (Friedman test; ****P < 0.0001).
Mentions: Since collagen and CRP-XL-stimulated platelet activation was affected by nobiletin, the phosphorylation levels of different signalling proteins involved in the GPVI pathway were analysed. Washed human platelets were stimulated at 37°C in an aggregometer under conditions (in presence of 1 mM EGTA, 10 μM indomethacin and 2 U·mL−1 apyrase) that disfavour platelet aggregation with CRP-XL (1 μg·mL−1) in the presence of various concentrations of nobiletin (12.5, 25, 50, 100, 150 and 200 μM) or vehicle control, and lysates were prepared to analyse the phosphorylation of different signalling proteins. Total tyrosine phosphorylation (Figure 5A) and the phosphorylation of individual proteins such as Syk (pY323) and LAT (pY200; Figure 5B) were not affected by nobiletin. However, tyrosine phosphorylation of PLCγ2 (pY759) was diminished at all concentrations of nobiletin used (Figure 5B). Similarly, the serine phosphorylation of PKB (or Akt) (pS473) was also affected in a concentration-dependent manner by nobiletin (Figure 5B). Although the phosphorylation levels were quantified under non-aggregation conditions, the level of inhibition of phosphorylation of PLCγ2 and Akt were similar to the inhibitory effects observed on platelet aggregation at the same concentration of agonist (Figure 1G and 1H). For example, 12.5 μM of nobiletin inhibited around 20–30% of CRP-XL-induced platelet aggregation and phosphorylation of PLCγ2 and Akt. These data suggest that nobiletin may not influence the immediate effectors within the GPVI pathway (such as Syk and LAT) although it can modulate the later signalling events controlled by PLCγ2 and Akt, which are shared with activation mechanisms stimulated by other agonists. The inhibitory effects of nobiletin on PLCγ2 and Akt phosphorylation indicate its potential effect on PI3K-mediated platelet signalling.

Bottom Line: The effect of nobiletin in vivo was assessed by measuring tail bleeding time using C57BL/6 mice.Nobiletin extended bleeding time in mice and reduced the phosphorylation of PKB (Akt) and PLCγ2 within the collagen receptor (glycoprotein VI)-stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of vasodilator-stimulated phosphoprotein, a protein whose activity is associated with inhibitory cyclic nucleotide signalling.Therefore, nobiletin may represent a potential antithrombotic agent of dietary origins.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK.

No MeSH data available.


Related in: MedlinePlus