Journal List > Korean J Physiol Pharmacol > v.13(3) > 1025598

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

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.

REFERENCES

Acuna-Castillo C., Coddou C., Bull P., Brito J., Huidobro-Toro JP. Differential role of extracellular histidines in copper, zinc, magnesium and proton modulation of the P2×7 purinergic receptor. J Neurochem. 101:17–26. 2007.
Coleman ML., Sahai EA., Yeo M., Bosch M., Dewar A., Olson MF. Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat Cell Biol. 3:339–345. 2001.
crossref
Croft DR., Coleman ML., Li S., Robertson D., Sullivan T., Stewart CL., Olson MF. Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration. J Cell Biol. 168:245–255. 2005.
crossref
Delaleu N., Jonsson R., Koller MM. Sjogren's syndrome. Eur J Oral Sci. 113:101–113. 2005.
crossref
Franke H., Grosche J., Schadlich H., Krugel U., Allgaier C., Illes P. P2X receptor expression on astrocytes in the nucleus accumbens of rats. Neuroscience. 108:421–429. 2001.
crossref
Leverrier Y., Ridley AJ. Apoptosis: caspases orchestrate the ROCK ‘n’ bleb. Nat Cell Biol. 3:E91–93. 2001.
crossref
Minambres R., Guasch RM., Perez-Arago A., Guerri C. The RhoA/ROCK-I/MLC pathway is involved in the ethanol-induced apoptosis by anoikis in astrocytes. J Cell Sci. 119:271–282. 2006.
Morelli A., Chiozzi P., Chiesa A., Ferrari D., Sanz JM., Falzoni S., Pinton P., Rizzuto R., Olson MF., Di Virgilio F. Extracellular ATP causes ROCK I-dependent bleb formation in P2×7-transfected HEK293 cells. Mol Biol Cell. 14:2655–2664. 2003.
Nobile M., Monaldi I., Alloisio S., Cugnoli C., Ferroni S. ATP-induced, sustained calcium signalling in cultured rat cortical astrocytes: evidence for a non-capacitative, P2×7-like-mediated calcium entry. FEBS Lett. 538:71–76. 2003.
Novak I. ATP as a signaling molecule: the exocrine focus. News Physiol Sci. 18:12–17. 2003.
crossref
Pfeiffer ZA., Aga M., Prabhu U., Watters JJ., Hall DJ., Bertics PJ. The nucleotide receptor P2×7 mediates actin reorganization and membrane blebbing in RAW 264.7 macrophages via p38 MAP kinase and Rho. J Leukoc Biol. 75:1173–1182. 2004.
Sebbagh M., Renvoize C., Hamelin J., Riche N., Bertoglio J., Breard J. Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat Cell Biol. 3:346–352. 2001.
crossref
Sisto M., Lisi S., Castellana D., Scagliusi P., D'Amore M., Caprio S., Scagliusi A., Acquafredda A., Panaro MA., Mitolo V. Autoanti-bodies from Sjogren's syndrome induce activation of both the intrinsic and extrinsic apoptotic pathways in human salivary gland cell line A-253. J Autoimmun. 27:38–49. 2006.
Surprenant A., Rassendren F., Kawashima E., North RA., Buell G. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2×7). Science. 272:735–738. 1996.
Zhang X., Zhang M., Laties AM., Mitchell CH. Stimulation of P2×7 receptors elevates Ca2+ and kills retinal ganglion cells. Invest Ophthalmol Vis Sci. 46:2183–2191. 2005.

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.
kjpp-13-175f1.tif
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.
kjpp-13-175f2.tif
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.
kjpp-13-175f3.tif
TOOLS
Similar articles