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J. Biol. Chem., Vol. 281, Issue 50, 38430-38439, December 15, 2006

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 281/50/38430    most recent M603917200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yamazaki, D. Articles by Imaizumi, Y. Search for Related Content PubMed PubMed Citation Articles by Yamazaki, D. Articles by Imaizumi, Y. Social Bookmarking                      What's this?

Novel Functions of Small Conductance Ca²⁺-activated K⁺ Channel in Enhanced Cell Proliferation by ATP in Brain Endothelial Cells^*

Daiju Yamazaki, Mineyoshi Aoyama, Susumu Ohya, Katsuhiko Muraki^¶, Kiyofumi Asai, and Yuji Imaizumi¹

From the Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuho-ku, Nagoya 467-8603, the Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-ku, Nagoya 467-8601, and the ^¶Cell Signaling & Ion Channel Research Group, Cellular Pharmacology, School of Pharmacy, Aichi Gakvin University, Chikusaku Nagoya 464-8650, Japan

Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB), which is essential for maintaining homeostasis of the brain. Net cellular turnover, which results from the balance between cell death and proliferation, is important in maintaining BBB homeostasis. Here we report a novel mechanism that underlies ATP-induced cell proliferation in t-BBEC 117, a cell line derived from bovine brain endothelial cells. Application of 0.1-30 µM ATP to t-BBEC 117 concentration-dependently increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) in two phases: an initial transient phase and a later and smaller sustained one. These two phases of [Ca²⁺]_i rise were mainly due to Ca²⁺ release and sustained Ca²⁺ influx, respectively. The pretreatment with apamin, a selective blocker of small conductance Ca²⁺-activated K⁺ channels (SK), significantly reduced both the [Ca²⁺]_i increase and K⁺ current induced by ATP. Transcripts corresponding to P2Yx, SK2, and transient receptor potential channels were detected in t-BBEC 117. Knock down of SK2 protein, which was the predominant Ca²⁺-activated K⁺ channel expressed in t-BBEC 117, by siRNA significantly reduced both the sustained phase of the [Ca²⁺]_i rise and the K⁺ current induced by ATP. Cell proliferation was increased significantly by the presence of the stable ATP analogue ATPS. This effect was blunted by UCL1684, a synthesized SK blocker. In conclusion, in brain endothelial cells ATP-induced [Ca²⁺]_i rise activates SK2 current, and the subsequent membrane hyperpolarization enhances Ca²⁺ entry presumably through transient receptor potential channels. This positive feedback mechanism can account for the augmented cell proliferation by ATP.

Received for publication, April 24, 2006 , and in revised form, September 11, 2006.

The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB114474 [GenBank] .

^* This work was supported by a grant-in-aid for Scientific Research (B) from the Japan Society for the Promotion of Science and by a grant-in-aid for Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation (to Y. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Tables S1 and S2.

¹ To whom correspondence should be addressed. Tel./Fax: 81-52-836-3431; E-mail: yimaizum{at}phar.nagoya-cu.ac.jp.

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