Octyl Gallate Inhibits ATP-induced Intracellular Calcium Increase in PC12 Cells by Inhibiting Multiple Pathways

Yujie Guo; Yi Jae Hong; Hyun-Jong Jang; Myung-Jun Kim; Duck-Joo Rhie; Yang-Hyeok Jo; Sang June Hahn; Shin Hee Yoon

doi:10.4196/kjpp.2010.14.1.21

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Guo, Hong, Jang, Kim, Rhie, Jo, Hahn, and Yoon: Octyl Gallate Inhibits ATP-induced Intracellular Calcium Increase in PC12 Cells by Inhibiting Multiple Pathways

Original Article

Korean J Physiol Pharmacol 2010;14(1):21-28.

Published online: 18 February 2010

DOI: https://doi.org/10.4196/kjpp.2010.14.1.21

Octyl Gallate Inhibits ATP-induced Intracellular Calcium Increase in PC12 Cells by Inhibiting Multiple Pathways

Yujie Guo, Yi Jae Hong, Hyun-Jong Jang, Myung-Jun Kim, Duck-Joo Rhie, Yang-Hyeok Jo, Sang June Hahn, Shin Hee Yoon

Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea

Corresponding to: Shin Hee Yoon, Department of Physiology, College of Medicine, The Catholic University of Korea, 505, Banpo-dong, Seocho-gu, Seoul 137-701, Korea. (Tel) 82-2-2258-7276, (Fax) 82-2-532-9575, (E-mail) s-hyoon@catholic.ac.kr

Received 17 December 2009 Revised 18 January 2010 Accepted 26 January 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Phenolic compounds affect intracellular free Ca²⁺ concentration ([Ca²⁺]_i) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca²⁺ signaling in PC12 cells using fura-2-based digital Ca²⁺ imaging and whole-cell patch clamping. Treatment with ATP (100 μM) for 90 s induced increases in [Ca²⁺]_i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20 μM) for 10 min inhibited the ATP-induced [Ca²⁺]_i response in a concentration-dependent manner (IC₅₀=2.84 μM). Treatment with octyl gallate (3 μM) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca²⁺ with nominally Ca²⁺-free HEPES HBSS or depletion of intracellular Ca²⁺ stores with thapsigargin (1 μM). Treatment for 10 min with the L-type Ca²⁺ channel antagonist nimodipine (1 μM) significantly inhibited the ATP-induced [Ca²⁺]_i increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca²⁺]_i increase induced by 50 mM KCl. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein (50 μM) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced [Ca²⁺]_i increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca²⁺ from extracellular space and P2Y receptor-induced release of Ca²⁺ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca²⁺ responses by inhibiting the secondary activation of voltage-gated Ca²⁺ channels.

Keywords: Ca²⁺, Flavonoid, Octyl gallate, PC12 cells, Phenolic compound, Purinergic receptor, Voltage-gated Ca²⁺ channels

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Fig. 1.

Concentration-dependent inhibitory effects of octyl gallate on the ATP-induced [Ca²⁺]_i increase in PC12 cells. (A∼G) After pretreating cells with various concentration of octyl gallate, subsequent ATP-induced [Ca²⁺]_i response was observed. Image pairs were collected at 3∼60 s intervals. ATP and octyl gallate were applied as indicated by the horizontal bars. (H) Summary of concentration-response data. The ATP-induced response amplitude is presented as a percentage of the initial control (relative to peak 1) (n=99, 35, 26, 30, 39, 21, 15 at 0, 100 nM, 300 nM, 1 μM, 3 μM, 10 μM, 20 μM, respectively). A non-linear least-squares fit by the prism software 5.0 to the concentration-response data yielded an IC₅₀ of 2.84 μM for octyl gallate. Data represent mean±SEM.

Fig. 2.

Inhibitory effects of octyl gallate on the ATP-induced release of Ca²⁺ from intracellular stores and Ca²⁺ influx from the extracellular space. (A) Reproducible [Ca²⁺]_i increases were elicited by super fusion with 100 μM ATP for 90 s at 30 min intervals. ATP-induced [Ca²⁺]_i were recorded after treatment with nominally Ca²⁺-free HEPES-HBSS for 2 min (B) or with thapsigargin (1 μM) for 15 min (C); then 20 min later the ATP-induced responses were recorded in the presence of octyl gallate (3 μM) for 10 min. (D) Summary of the effects of octyl gallate on ATP-induced [Ca²⁺]_i increase. The amplitude of the ATP-induced response is presented as a percentage of the initial control (relative to peak 1) for the control (n=99), nominally Ca²⁺-free HEPES-HBSS-treated (0 Ca²⁺, n=33), 0 Ca²⁺ plus octyl gallate-treated (n=33), thapsigargin-treated (n=59), and thapsigargin plus octyl gallate-treated (n=59) cells. Data represent mean±SEM. ^∗p<0.05 relative to respective control (paired Student's t-test). ⁺p<0.05 relative to respective non-octyl gallate-treated cells (paired Student's t-test).

Fig. 3.

Inhibitory effects of octyl gallate on the ATP-induced secondary Ca²⁺ influx through voltage-gated Ca²⁺ channels. Effects of nimodipine and octyl gallate on ATP (100 μM)-induced [Ca²⁺]_i increase in untreated cells (A1) and thapsigargin-treated cells (A2). Cells were pretreated with thapsigargin (1 μM) for 45 min during the fura-2 loading period, since thapsigargin-induced increase in [Ca²⁺]_i returns to near basal levels after a 15 min exposure to thapsigargin. The amplitude of the ATP-induced response after treatment of vehicle (control) (n=25, n=24), nimodipine (n=25, n=24), octyl gallate (n=25, n=24), nimodipine plus octyl gallate (n=25, n=24) is presented as a percentage of the initial control in untreated and thapsigargin-treated cells, respectively. (B) Effects of octyl gallate on KCl (50 mM K⁺ HEPES-HBSS)-induced [Ca²⁺]_i increase. The amplitude of the KCl-induced response after treatment of vehicle (n=31) or octyl gallate (n=26) is presented as a percentage of the initial control. Data represent mean±SEM. ^∗p<0.05 relative to respective control or KCl-treated cell; p<0.05 relative to respective nomodipine or octyl gallate-treated cells (one way ANOVA followed by Bonferroni's test and paired Student's t-test).

Fig. 4.

ATP-induced [Ca²⁺]_i increase were not inhibited by treatment with PKC inhibitors staurosporin, GF10923X or the tyrosine kinase inhibitor genistein. ATP-induced responses were induced in the presence of vehicle (control, n=59), octyl gallate (3 μM, n=39), staurosprin (100 nM, n=30), staurosprin (100 nM) plus octyl gallate (3 μM) (n=29), GF 109203X (300 nM, n=32), GF 109203X (300 nM) plus octyl gallate (3 μM) (n=29), genistein (50 μM, n=35), genistein (50 μM) plus octyl gallate (3 μM) (n=34) for 10 min following 90 s exposure to ATP and a 20 min wash. The amplitude of the ATP-induced response is presented as a percentage of the initial control responses. Data are expressed as mean±SEM. ^∗p<0.05 relative to respective non-octyl gallate-treated cells (one way ANOVA followed by Bonferroni's test).

Fig. 5.

Inhibitory effects of octyl gallate on ATP-induced inward currents in PC12 cell. (A) Application of ATP (100 μM, 10 s) evoked inward currents (control). In the same cells, a second application of ATP induced inward current after 10 min washout (vehicle) (n=9). (B) Pretreatment with 3 μM octyl gallate for 10 min inhibited the ATP-induced inward current (octyl gallate, n=6). (C) Summary of the effect of octyl gallate on ATP-induced inward currents. The amplitude of second ATP-induced response (I_p2) is presented as a percentage of the initial control (I_p1) (I_p2/I_p1). Data are expressed as mean±SEM. ^∗p<0.05 relative to control (non-paired student's t-test).

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