Korean J Physiol Pharmacol.  2010 Feb;14(1):21-28. 10.4196/kjpp.2010.14.1.21.

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

• Guo Y
• Hong YJ
• Jang HJ
• Kim MJ
• Rhie DJ
• Jo YH
• Hahn SJ
• Yoon SH

• KMID: 1457697
• DOI: http://doi.org/10.4196/kjpp.2010.14.1.21

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 micrometer) for 90 s induced increases in [Ca2+]i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20 micrometer) for 10 min inhibited the ATP-induced [Ca2+]i response in a concentration-dependent manner (IC50=2.84 micrometer). Treatment with octyl gallate (3 micrometer) 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 micrometer). Treatment for 10 min with the L-type Ca2+ channel antagonist nimodipine (1 micrometer) 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 micrometer) 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.

Keyword

Ca2+; Flavonoid; Octyl gallate; PC12 cells; Phenolic compound; Purinergic receptor; Voltage-gated Ca2+ channels

MeSH Terms

Adenosine Triphosphate
Animals
Calcium
Constriction
Extracellular Space
Gallic Acid
Genistein
HEPES
Indoles
Maleimides
Nimodipine
PC12 Cells
Phenol
Protein Kinase C
Protein-Tyrosine Kinases
Thapsigargin
Adenosine Triphosphate
Calcium
Gallic Acid
Genistein
HEPES
Indoles
Maleimides
Nimodipine
Phenol
Protein Kinase C
Protein-Tyrosine Kinases
Thapsigargin

Figure

• 

  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).

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  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 (Ip2) is presented as a percentage of the initial control (Ip1) (Ip2/Ip1). Data are expressed as mean±SEM. ∗p<0.05 relative to control (non-paired student's t-test).

Reference

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