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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 dephosphorylation of ERK. Lane 1, unstimulated platelets (base); Lane 2, thrombin (0.05 U/mL); Lane 3, thrombin (0.05 U/mL) + G-Ro (50 μM); Lane 4, thrombin (0.05 U/mL) + G-Ro (100 μM); Lane 5, thrombin (0.05 U/mL) + G-Ro (200 μM); Lane 6, thrombin (0.05 U/mL) + G-Ro (300 μM). Western blotting was performed 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.
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fig7: Effects of G-Ro on dephosphorylation of ERK. Lane 1, unstimulated platelets (base); Lane 2, thrombin (0.05 U/mL); Lane 3, thrombin (0.05 U/mL) + G-Ro (50 μM); Lane 4, thrombin (0.05 U/mL) + G-Ro (100 μM); Lane 5, thrombin (0.05 U/mL) + G-Ro (200 μM); Lane 6, thrombin (0.05 U/mL) + G-Ro (300 μM). Western blotting was performed 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.

Mentions: It is known that Ca2+ mobilized from DTS (ER) is involved in phosphorylation of PI3K and ERK to influx extracellular Ca2+ [13, 14, 17, 18]. Here, we investigated the effect of G-Ro on dephosphorylation of ERK (1/2). As shown in Figure 7, in unstimulated platelets, ERK 1 (44 kDa) phosphorylation only was observed (Figure 7, Lane 1). Thrombin potently phosphorylated both ERK 1 (44 kDa) and ERK 2 (42 kDa) (Figure 7, Lane 2). However, G-Ro (50 to 300 μM) dose-dependently inhibited thrombin-induced phosphorylation of ERK 2 (42 kDa), and the ratio of p-ERK 2 to ERK 2 (42 kDa) (Figure 7, Lanes 3 to 6).


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 dephosphorylation of ERK. Lane 1, unstimulated platelets (base); Lane 2, thrombin (0.05 U/mL); Lane 3, thrombin (0.05 U/mL) + G-Ro (50 μM); Lane 4, thrombin (0.05 U/mL) + G-Ro (100 μM); Lane 5, thrombin (0.05 U/mL) + G-Ro (200 μM); Lane 6, thrombin (0.05 U/mL) + G-Ro (300 μM). Western blotting was performed 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig7: Effects of G-Ro on dephosphorylation of ERK. Lane 1, unstimulated platelets (base); Lane 2, thrombin (0.05 U/mL); Lane 3, thrombin (0.05 U/mL) + G-Ro (50 μM); Lane 4, thrombin (0.05 U/mL) + G-Ro (100 μM); Lane 5, thrombin (0.05 U/mL) + G-Ro (200 μM); Lane 6, thrombin (0.05 U/mL) + G-Ro (300 μM). Western blotting was performed 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.
Mentions: It is known that Ca2+ mobilized from DTS (ER) is involved in phosphorylation of PI3K and ERK to influx extracellular Ca2+ [13, 14, 17, 18]. Here, we investigated the effect of G-Ro on dephosphorylation of ERK (1/2). As shown in Figure 7, in unstimulated platelets, ERK 1 (44 kDa) phosphorylation only was observed (Figure 7, Lane 1). Thrombin potently phosphorylated both ERK 1 (44 kDa) and ERK 2 (42 kDa) (Figure 7, Lane 2). However, G-Ro (50 to 300 μM) dose-dependently inhibited thrombin-induced phosphorylation of ERK 2 (42 kDa), and the ratio of p-ERK 2 to ERK 2 (42 kDa) (Figure 7, Lanes 3 to 6).

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