Sung-Min Hwang; Na-Youn Koo; Se-Young Choi; Gae-Sig Chun; Joong-Soo Kim; Kyungpyo Park

doi:10.4196/kjpp.2009.13.3.175

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Hwang, Koo, Choi, Chun, Kim, and Park: P2×7 Receptor-mediated Membrane Blebbing in Salivary Epithelial Cells

Original Article

Korean J Physiol Pharmacol 2009;13(3):175-179.

Published online: 17 June 2009

DOI: https://doi.org/10.4196/kjpp.2009.13.3.175

P2×7 Receptor-mediated Membrane Blebbing in Salivary Epithelial Cells

Sung-Min Hwang¹, Na-Youn Koo¹, Se-Young Choi¹, Gae-Sig Chun², Joong-Soo Kim¹, Kyungpyo Park¹

¹Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul 110-749, Korea

²Department of Oral Physiology, School of Dentistry, Dankook University, Cheonan 330-714, Korea

Corresponding to: Kyungpyo Park, Department of Physiology, School of Dentistry, Seoul National University, 28, Yeongeon-dong, Chongnogu, Seoul 110-749, Korea. (Tel) 82-2-740-8658, (Fax) 82-2-762-5107, (E-mail) kppark@snu.ac.kr

Received 16 April 20096 May 2009 Accepted 18 May 2009

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

High concentrations of ATP induce membrane blebbing. However, the underlying mechanism involved in epithelial cells remains unclear. In this study, we investigated the role of the P2×7 receptor (P2×7R) in membrane blebbing using Par C5 cells. We stimulated the cells with 5 mM of ATP for 1 ~ 2 hrs and found the characteristics of membrane blebbing, a hallmark of apoptotic cell death. In addition, 500 μM Bz-ATP, a specific P2×7R agonist, induced membrane blebbing. However, 300 μM of Ox-ATP, a P2×7R antagonist, inhibited ATP-induced membrane blebbing, suggesting that ATP-induced membrane blebbing is mediated by P2×7R. We found that ATP-induced membrane blebbing was mediated by ROCK I activation and MLC phosphorylation, but not by caspase-3. Five mM of ATP evoked a biphasic [Ca²⁺]_i response; a transient [Ca²⁺]_i peak and sustained [Ca²⁺]_i increase secondary to ATP-stimulated Ca²⁺ influx. These results suggest that P2×7R plays a role in membrane blebbing of the salivary gland epithelial cells.

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Keywords: P2×7 receptor, Membrane blebbing, ROCKI, Ca²⁺ influx

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Fig. 1.

Membrane blebbing induced by ATP. (A) Par C5 cells were treated with 5 mM ATP, 5 mM ATP with 300 μM ox-ATP for 2 hrs, and 5 mM ATP in Ca²⁺-free solution. The cells were stained with Texas Red-X phalloidin. In addition, 5 mM of ATP for 2 hrs induced several membrane blebs per cell (arrows in the second panel). Membrane blebbing was reduced following pretreatment with 300 μM ox-ATP or 5 mM ATP in a Ca²⁺-free bath solution (the third and fourth panels). (B) The histogram summarizes the results of the percent of cells demonstrating membrane blebbing following treatment with 1 mM Bz-ATP, 5 mM ATP, 1 mM ADP, or 1 mM UTP. Membrane blebbing was also quantified following ATP or Bz-ATP stimulation following 2 hrs of incubation with 300 μM ox-ATP or in a Ca²⁺-free solution. The data are the means of five separate experiments. ∗p<0.05 is derived from comparisons with untreated control cells.

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Fig. 2.

ATP induced ROCK I activation and MLC phosphorylation in Par C5 cells. (A) ROCK I cleavage and phosphorylated-MLC (pMLC) from whole cell lysates (50 μg) were resolved by SDS-PAGE and blotted with specific antibodies. The blots were probed with antibodies against ROCK I, pMLC, and MLC (Control). The cells were treated with ATP for up to 2 hrs. (B) ATP-induced ROCK I cleavage and pMLC after pretreatment with 300 μM ox-ATP or in Ca²⁺-free solution. (C) ATP-induced ROCK I cleavage and pMLC after pretreatment with 300 μM caspase-3 inhibitor (DEVD-fmk) or 10 μM ROCK I inhibitor (Y-27632). (D) The effects of Y-27632 and DEVD-fmk on the percent of cells demonstrating membrane blebbing following 5 mM ATP stimulation. The number of cells containing membrane blebs was determined from a total of 500 cells. The data are means from five separate experiments ∗p<0.05 compared to untreated control cells.

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Fig. 3.

Measurement of cytoplasm free Ca²⁺ concentrations ([Ca²⁺]_i) mediated by P2×7R activation in fura-2 loaded Par C5 cells. (A) In the presence of 1 mM Mg²⁺, 5 mM ATP induced a transient and sustained elevation in [Ca²⁺]_i. In addition, 20 mM Mg²⁺ completely blocked the sustained increased in [Ca²⁺]. (B) In a Ca²⁺-free bath solution, the transient peak increase in [Ca²⁺]_i was measured following stimulation with ATP (5 mM) or UTP (1 mM). The sustained increase was absent in the Ca²⁺-free media. (C) Two hours following preincubation with 300 μM ox-ATP, a peak increase in [Ca²⁺]_i stimulated by 5 mM of ATP was present without evidence of a sustained increase. Each tracing is a typical representative sample of at least 10 separate experiments using different cell preparations.

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