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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 elevates cGMP levels and increases VASP (S239) phosphorylation. The levels of cGMP (A) and cAMP (B) were measured in platelets (PRP) upon stimulation with CRP-XL (1 μg·mL−1) using elisa kits in the presence or absence of various concentrations of nobiletin. The concentration of cGMP and cAMP was calculated based on standard curves. R, resting; C, CRP-XL-treated platelets. Data represent mean ± SD (n = 4) and P-values were calculated using parametric repeated measures anova (****P < 0.0001). Platelet aggregation was measured using CRP-XL (0.5 μg·mL−1) together in the presence and absence of 20 μM ODQ, a soluble guanylate cyclase inhibitor (C and D) or 30 μM PKG inhibitor (Rp-8-Br-PET-cGMPS) (E and F) and 50 μM nobiletin for 5 min. C, O, P and N represent CRP-XL, ODQ, PKG inhibitor and nobiletin respectively. The level of aggregation obtained with CRP-XL at 5 min was taken as 100% to calculate the percentage of inhibition. The data from C + N were compared with C and C + O/P + N were compared with C + N. Data represent mean ± SD (n = 3) and P-values were calculated using Student's t-test as shown (**P < 0.01 and ***P < 0.001). The effect of various concentrations of nobiletin on phosphorylation of VASP [at positions pS239 (G) and pS157 (H)], a substrate for cyclic nucleotide-dependent PKs, was analysed in washed platelets upon stimulation with CRP-XL (1 μg·mL−1) by immunoblotting. The blots are representative of four separate experiments. Data 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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fig06: Nobiletin elevates cGMP levels and increases VASP (S239) phosphorylation. The levels of cGMP (A) and cAMP (B) were measured in platelets (PRP) upon stimulation with CRP-XL (1 μg·mL−1) using elisa kits in the presence or absence of various concentrations of nobiletin. The concentration of cGMP and cAMP was calculated based on standard curves. R, resting; C, CRP-XL-treated platelets. Data represent mean ± SD (n = 4) and P-values were calculated using parametric repeated measures anova (****P < 0.0001). Platelet aggregation was measured using CRP-XL (0.5 μg·mL−1) together in the presence and absence of 20 μM ODQ, a soluble guanylate cyclase inhibitor (C and D) or 30 μM PKG inhibitor (Rp-8-Br-PET-cGMPS) (E and F) and 50 μM nobiletin for 5 min. C, O, P and N represent CRP-XL, ODQ, PKG inhibitor and nobiletin respectively. The level of aggregation obtained with CRP-XL at 5 min was taken as 100% to calculate the percentage of inhibition. The data from C + N were compared with C and C + O/P + N were compared with C + N. Data represent mean ± SD (n = 3) and P-values were calculated using Student's t-test as shown (**P < 0.01 and ***P < 0.001). The effect of various concentrations of nobiletin on phosphorylation of VASP [at positions pS239 (G) and pS157 (H)], a substrate for cyclic nucleotide-dependent PKs, was analysed in washed platelets upon stimulation with CRP-XL (1 μg·mL−1) by immunoblotting. The blots are representative of four separate experiments. Data represent mean ± SD (n = 4) and P-values were calculated using non-parametric repeated measures anova (Friedman test; ****P < 0.0001).

Mentions: Under physiological conditions, endothelium-derived nitric oxide (NO) and PGI2 inhibit platelet function by elevating platelet cGMP and cAMP levels respectively. Since plant flavonoids such as tangeretin have been shown to act on cyclic nucleotide signalling (Ok et al., 2012), the effects of nobiletin on the levels of cGMP and cAMP in platelets were analysed in the presence of vehicle control or different concentrations (50, 100, 150 and 200 μM) of nobiletin. The levels of cGMP (Figure 6A) and cAMP (Figure 6B) were unaltered upon stimulation with CRP-XL (1 μg·mL−1), but the addition of nobiletin elevated cGMP levels significantly. Lower concentrations (less than 50 μM) of nobiletin did not increase the cGMP levels significantly (data not shown). cAMP levels, however, remained unaltered in the presence of all the concentrations of nobiletin used. To explore whether the elevation of cGMP levels by nobiletin is dependent on activation by CRP-XL, the level of cGMP was measured in resting platelets incubated with nobiletin (200 μM) for 5 min. Platelet cGMP levels were increased in platelets treated with nobiletin alone (Figure 6A) indicating that cGMP elevation is not dependent on agonist stimulation or platelet activation. These data suggest that nobiletin may inhibit platelet function through the elevation of cGMP and not through cAMP.


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 elevates cGMP levels and increases VASP (S239) phosphorylation. The levels of cGMP (A) and cAMP (B) were measured in platelets (PRP) upon stimulation with CRP-XL (1 μg·mL−1) using elisa kits in the presence or absence of various concentrations of nobiletin. The concentration of cGMP and cAMP was calculated based on standard curves. R, resting; C, CRP-XL-treated platelets. Data represent mean ± SD (n = 4) and P-values were calculated using parametric repeated measures anova (****P < 0.0001). Platelet aggregation was measured using CRP-XL (0.5 μg·mL−1) together in the presence and absence of 20 μM ODQ, a soluble guanylate cyclase inhibitor (C and D) or 30 μM PKG inhibitor (Rp-8-Br-PET-cGMPS) (E and F) and 50 μM nobiletin for 5 min. C, O, P and N represent CRP-XL, ODQ, PKG inhibitor and nobiletin respectively. The level of aggregation obtained with CRP-XL at 5 min was taken as 100% to calculate the percentage of inhibition. The data from C + N were compared with C and C + O/P + N were compared with C + N. Data represent mean ± SD (n = 3) and P-values were calculated using Student's t-test as shown (**P < 0.01 and ***P < 0.001). The effect of various concentrations of nobiletin on phosphorylation of VASP [at positions pS239 (G) and pS157 (H)], a substrate for cyclic nucleotide-dependent PKs, was analysed in washed platelets upon stimulation with CRP-XL (1 μg·mL−1) by immunoblotting. The blots are representative of four separate experiments. Data represent mean ± SD (n = 4) and P-values were calculated using non-parametric repeated measures anova (Friedman test; ****P < 0.0001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig06: Nobiletin elevates cGMP levels and increases VASP (S239) phosphorylation. The levels of cGMP (A) and cAMP (B) were measured in platelets (PRP) upon stimulation with CRP-XL (1 μg·mL−1) using elisa kits in the presence or absence of various concentrations of nobiletin. The concentration of cGMP and cAMP was calculated based on standard curves. R, resting; C, CRP-XL-treated platelets. Data represent mean ± SD (n = 4) and P-values were calculated using parametric repeated measures anova (****P < 0.0001). Platelet aggregation was measured using CRP-XL (0.5 μg·mL−1) together in the presence and absence of 20 μM ODQ, a soluble guanylate cyclase inhibitor (C and D) or 30 μM PKG inhibitor (Rp-8-Br-PET-cGMPS) (E and F) and 50 μM nobiletin for 5 min. C, O, P and N represent CRP-XL, ODQ, PKG inhibitor and nobiletin respectively. The level of aggregation obtained with CRP-XL at 5 min was taken as 100% to calculate the percentage of inhibition. The data from C + N were compared with C and C + O/P + N were compared with C + N. Data represent mean ± SD (n = 3) and P-values were calculated using Student's t-test as shown (**P < 0.01 and ***P < 0.001). The effect of various concentrations of nobiletin on phosphorylation of VASP [at positions pS239 (G) and pS157 (H)], a substrate for cyclic nucleotide-dependent PKs, was analysed in washed platelets upon stimulation with CRP-XL (1 μg·mL−1) by immunoblotting. The blots are representative of four separate experiments. Data represent mean ± SD (n = 4) and P-values were calculated using non-parametric repeated measures anova (Friedman test; ****P < 0.0001).
Mentions: Under physiological conditions, endothelium-derived nitric oxide (NO) and PGI2 inhibit platelet function by elevating platelet cGMP and cAMP levels respectively. Since plant flavonoids such as tangeretin have been shown to act on cyclic nucleotide signalling (Ok et al., 2012), the effects of nobiletin on the levels of cGMP and cAMP in platelets were analysed in the presence of vehicle control or different concentrations (50, 100, 150 and 200 μM) of nobiletin. The levels of cGMP (Figure 6A) and cAMP (Figure 6B) were unaltered upon stimulation with CRP-XL (1 μg·mL−1), but the addition of nobiletin elevated cGMP levels significantly. Lower concentrations (less than 50 μM) of nobiletin did not increase the cGMP levels significantly (data not shown). cAMP levels, however, remained unaltered in the presence of all the concentrations of nobiletin used. To explore whether the elevation of cGMP levels by nobiletin is dependent on activation by CRP-XL, the level of cGMP was measured in resting platelets incubated with nobiletin (200 μM) for 5 min. Platelet cGMP levels were increased in platelets treated with nobiletin alone (Figure 6A) indicating that cGMP elevation is not dependent on agonist stimulation or platelet activation. These data suggest that nobiletin may inhibit platelet function through the elevation of cGMP and not through cAMP.

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