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Inhibitory Effects of Cytosolic Ca(2+) Concentration by Ginsenoside Ro Are Dependent on Phosphorylation of IP3RI and Dephosphorylation of ERK in Human Platelets.

Kwon HW, Shin JH, Lee DH, Park HJ - Evid Based Complement Alternat Med (2015)

Bottom Line: This study was carried out to understand the Ca(2+)-antagonistic effect of ginsenoside Ro (G-Ro), an oleanane-type saponin in Panax ginseng.Phosphorylation of IP3RI (Ser(1756)) by G-Ro was decreased by PKA inhibitor Rp-8-Br-cAMPS.We demonstrate that G-Ro upregulates cAMP-dependent IP3RI (Ser(1756)) phosphorylation and downregulates phosphorylation of ERK 2 (42 kDa) to decrease thrombin-elevated [Ca(2+)] i , which contributes to inhibition of ATP and serotonin release, and p-selectin expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Laboratory Science, College of Biomedical Science and Engineering, Inje University, 197 Inje-ro, Gimhae, Gyungnam 621-749, Republic of Korea.

ABSTRACT
Intracellular Ca(2+) ([Ca(2+)] i ) is platelet aggregation-inducing molecule and is involved in activation of aggregation associated molecules. This study was carried out to understand the Ca(2+)-antagonistic effect of ginsenoside Ro (G-Ro), an oleanane-type saponin in Panax ginseng. G-Ro, without affecting leakage of lactate dehydrogenase, dose-dependently inhibited thrombin-induced platelet aggregation, and the half maximal inhibitory concentration was approximately 155 μM. G-Ro inhibited strongly thrombin-elevated [Ca(2+)] i , which was strongly increased by A-kinase inhibitor Rp-8-Br-cAMPS compared to G-kinase inhibitor Rp-8-Br-cGMPS. G-Ro increased the level of cAMP and subsequently elevated the phosphorylation of inositol 1, 4, 5-triphosphate receptor I (IP3RI) (Ser(1756)) to inhibit [Ca(2+)] i mobilization in thrombin-induced platelet aggregation. Phosphorylation of IP3RI (Ser(1756)) by G-Ro was decreased by PKA inhibitor Rp-8-Br-cAMPS. In addition, G-Ro inhibited thrombin-induced phosphorylation of ERK 2 (42 kDa), indicating inhibition of Ca(2+) influx across plasma membrane. We demonstrate that G-Ro upregulates cAMP-dependent IP3RI (Ser(1756)) phosphorylation and downregulates phosphorylation of ERK 2 (42 kDa) to decrease thrombin-elevated [Ca(2+)] i , which contributes to inhibition of ATP and serotonin release, and p-selectin expression. These results indicate that G-Ro in Panax ginseng is a beneficial novel Ca(2+)-antagonistic compound and may prevent platelet aggregation-mediated thrombotic disease.

No MeSH data available.


Related in: MedlinePlus

Effects of G-Ro on p-selectin expression. (a) The flow cytometry histograms on p-selectin expression. (A) Intact platelets (base); (B) thrombin (0.05 U/mL); (C) thrombin (0.05 U/mL) + G-Ro (50 μM); (D) thrombin (0.05 U/mL) + G-Ro (100 μM); (E) thrombin (0.05 U/mL) + G-Ro (200 μM); (F) thrombin (0.05 U/mL) + G-Ro (300 μM). (b) Effects of G-Ro on thrombin-induced p-selectin expression (%). (c) The flow cytometry histograms on p-selectin expression in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) and G-kinase inhibitor (Rp-8-Br-cGMPS). (A) Thrombin (0.05 U/mL) + G-Ro (300 μM) + Rp-8-Br-cAMPS (250 μM); (B) thrombin (0.05 U/mL) + G-Ro (300 μM) + Rp-8-Br-cGMPS (250 μM). (d) Effects of G-Ro on thrombin-induced p-selectin expression in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) and G-kinase inhibitor (Rp-8-Br-cGMPS) (%). Determination of p-selectin expression was carried out as described in “Section 2.” The data are expressed as the mean ± standard deviation (n = 4). ∗p < 0.05 versus the thrombin-stimulated human platelets; †p < 0.05 versus the thrombin-stimulated human platelets in the presence of G-Ro (300 μM).
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fig9: Effects of G-Ro on p-selectin expression. (a) The flow cytometry histograms on p-selectin expression. (A) Intact platelets (base); (B) thrombin (0.05 U/mL); (C) thrombin (0.05 U/mL) + G-Ro (50 μM); (D) thrombin (0.05 U/mL) + G-Ro (100 μM); (E) thrombin (0.05 U/mL) + G-Ro (200 μM); (F) thrombin (0.05 U/mL) + G-Ro (300 μM). (b) Effects of G-Ro on thrombin-induced p-selectin expression (%). (c) The flow cytometry histograms on p-selectin expression in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) and G-kinase inhibitor (Rp-8-Br-cGMPS). (A) Thrombin (0.05 U/mL) + G-Ro (300 μM) + Rp-8-Br-cAMPS (250 μM); (B) thrombin (0.05 U/mL) + G-Ro (300 μM) + Rp-8-Br-cGMPS (250 μM). (d) Effects of G-Ro on thrombin-induced p-selectin expression in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) and G-kinase inhibitor (Rp-8-Br-cGMPS) (%). Determination of p-selectin expression was carried out as described in “Section 2.” The data are expressed as the mean ± standard deviation (n = 4). ∗p < 0.05 versus the thrombin-stimulated human platelets; †p < 0.05 versus the thrombin-stimulated human platelets in the presence of G-Ro (300 μM).

Mentions: Compounds in α-granule of platelets are known to be involved in inflammation, coagulation, and angiogenesis [36]; these are also Ca2+-dependently released by various platelet agonists. In particular, p-selectin is released from α-granule and is reexpressed to the platelet surface [37] and subsequently is involved in inflammation by binding to the p-selectin glycoprotein ligand-1 receptor on monocyte [38]. Therefore, we investigated the effect of G-Ro on p-selectin expression from α-granule. Thrombin stimulated the expression of p-selectin (Figure 9(a)-(B)) as compared with that by unstimulated platelets (Figure 9(a)-(A)). However, G-Ro (50 to 300 μM) dose-dependently inhibited thrombin-induced expression of p-selectin (Figures 9(a)-(C)~(F) and 9(b)). PKA inhibitor Rp-8-Br-cAMPS increased G-Ro-reduced (300 μM) p-selectin expression (Figures 9(c)-(A) and 9(d)), but PKG inhibitor Rp-8-Br-cGMPS did not increase as compared with that by Rp-8-Br-cAMPS (Figures 9(c)-(B) and 9(d)).


Inhibitory Effects of Cytosolic Ca(2+) Concentration by Ginsenoside Ro Are Dependent on Phosphorylation of IP3RI and Dephosphorylation of ERK in Human Platelets.

Kwon HW, Shin JH, Lee DH, Park HJ - Evid Based Complement Alternat Med (2015)

Effects of G-Ro on p-selectin expression. (a) The flow cytometry histograms on p-selectin expression. (A) Intact platelets (base); (B) thrombin (0.05 U/mL); (C) thrombin (0.05 U/mL) + G-Ro (50 μM); (D) thrombin (0.05 U/mL) + G-Ro (100 μM); (E) thrombin (0.05 U/mL) + G-Ro (200 μM); (F) thrombin (0.05 U/mL) + G-Ro (300 μM). (b) Effects of G-Ro on thrombin-induced p-selectin expression (%). (c) The flow cytometry histograms on p-selectin expression in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) and G-kinase inhibitor (Rp-8-Br-cGMPS). (A) Thrombin (0.05 U/mL) + G-Ro (300 μM) + Rp-8-Br-cAMPS (250 μM); (B) thrombin (0.05 U/mL) + G-Ro (300 μM) + Rp-8-Br-cGMPS (250 μM). (d) Effects of G-Ro on thrombin-induced p-selectin expression in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) and G-kinase inhibitor (Rp-8-Br-cGMPS) (%). Determination of p-selectin expression was carried out as described in “Section 2.” The data are expressed as the mean ± standard deviation (n = 4). ∗p < 0.05 versus the thrombin-stimulated human platelets; †p < 0.05 versus the thrombin-stimulated human platelets in the presence of G-Ro (300 μM).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig9: Effects of G-Ro on p-selectin expression. (a) The flow cytometry histograms on p-selectin expression. (A) Intact platelets (base); (B) thrombin (0.05 U/mL); (C) thrombin (0.05 U/mL) + G-Ro (50 μM); (D) thrombin (0.05 U/mL) + G-Ro (100 μM); (E) thrombin (0.05 U/mL) + G-Ro (200 μM); (F) thrombin (0.05 U/mL) + G-Ro (300 μM). (b) Effects of G-Ro on thrombin-induced p-selectin expression (%). (c) The flow cytometry histograms on p-selectin expression in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) and G-kinase inhibitor (Rp-8-Br-cGMPS). (A) Thrombin (0.05 U/mL) + G-Ro (300 μM) + Rp-8-Br-cAMPS (250 μM); (B) thrombin (0.05 U/mL) + G-Ro (300 μM) + Rp-8-Br-cGMPS (250 μM). (d) Effects of G-Ro on thrombin-induced p-selectin expression in the presence of A-kinase inhibitor (Rp-8-Br-cAMPS) and G-kinase inhibitor (Rp-8-Br-cGMPS) (%). Determination of p-selectin expression was carried out as described in “Section 2.” The data are expressed as the mean ± standard deviation (n = 4). ∗p < 0.05 versus the thrombin-stimulated human platelets; †p < 0.05 versus the thrombin-stimulated human platelets in the presence of G-Ro (300 μM).
Mentions: Compounds in α-granule of platelets are known to be involved in inflammation, coagulation, and angiogenesis [36]; these are also Ca2+-dependently released by various platelet agonists. In particular, p-selectin is released from α-granule and is reexpressed to the platelet surface [37] and subsequently is involved in inflammation by binding to the p-selectin glycoprotein ligand-1 receptor on monocyte [38]. Therefore, we investigated the effect of G-Ro on p-selectin expression from α-granule. Thrombin stimulated the expression of p-selectin (Figure 9(a)-(B)) as compared with that by unstimulated platelets (Figure 9(a)-(A)). However, G-Ro (50 to 300 μM) dose-dependently inhibited thrombin-induced expression of p-selectin (Figures 9(a)-(C)~(F) and 9(b)). PKA inhibitor Rp-8-Br-cAMPS increased G-Ro-reduced (300 μM) p-selectin expression (Figures 9(c)-(A) and 9(d)), but PKG inhibitor Rp-8-Br-cGMPS did not increase as compared with that by Rp-8-Br-cAMPS (Figures 9(c)-(B) and 9(d)).

Bottom Line: This study was carried out to understand the Ca(2+)-antagonistic effect of ginsenoside Ro (G-Ro), an oleanane-type saponin in Panax ginseng.Phosphorylation of IP3RI (Ser(1756)) by G-Ro was decreased by PKA inhibitor Rp-8-Br-cAMPS.We demonstrate that G-Ro upregulates cAMP-dependent IP3RI (Ser(1756)) phosphorylation and downregulates phosphorylation of ERK 2 (42 kDa) to decrease thrombin-elevated [Ca(2+)] i , which contributes to inhibition of ATP and serotonin release, and p-selectin expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Laboratory Science, College of Biomedical Science and Engineering, Inje University, 197 Inje-ro, Gimhae, Gyungnam 621-749, Republic of Korea.

ABSTRACT
Intracellular Ca(2+) ([Ca(2+)] i ) is platelet aggregation-inducing molecule and is involved in activation of aggregation associated molecules. This study was carried out to understand the Ca(2+)-antagonistic effect of ginsenoside Ro (G-Ro), an oleanane-type saponin in Panax ginseng. G-Ro, without affecting leakage of lactate dehydrogenase, dose-dependently inhibited thrombin-induced platelet aggregation, and the half maximal inhibitory concentration was approximately 155 μM. G-Ro inhibited strongly thrombin-elevated [Ca(2+)] i , which was strongly increased by A-kinase inhibitor Rp-8-Br-cAMPS compared to G-kinase inhibitor Rp-8-Br-cGMPS. G-Ro increased the level of cAMP and subsequently elevated the phosphorylation of inositol 1, 4, 5-triphosphate receptor I (IP3RI) (Ser(1756)) to inhibit [Ca(2+)] i mobilization in thrombin-induced platelet aggregation. Phosphorylation of IP3RI (Ser(1756)) by G-Ro was decreased by PKA inhibitor Rp-8-Br-cAMPS. In addition, G-Ro inhibited thrombin-induced phosphorylation of ERK 2 (42 kDa), indicating inhibition of Ca(2+) influx across plasma membrane. We demonstrate that G-Ro upregulates cAMP-dependent IP3RI (Ser(1756)) phosphorylation and downregulates phosphorylation of ERK 2 (42 kDa) to decrease thrombin-elevated [Ca(2+)] i , which contributes to inhibition of ATP and serotonin release, and p-selectin expression. These results indicate that G-Ro in Panax ginseng is a beneficial novel Ca(2+)-antagonistic compound and may prevent platelet aggregation-mediated thrombotic disease.

No MeSH data available.


Related in: MedlinePlus