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 mum ATP to t-BBEC 117 concentration-dependently increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in two phases: an initial transient phase and a later and smaller sustained one. These two phases of [Ca(2+)](i) rise were mainly due to Ca(2+) release and sustained Ca(2+) influx, respectively. The pretreatment with apamin, a selective blocker of small conductance Ca(2+)-activated K(+) channels (SK), significantly reduced both the [Ca(2+)](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 Ca(2+)-activated K(+) channel expressed in t-BBEC 117, by siRNA significantly reduced both the sustained phase of the [Ca(2+)](i) rise and the K(+) current induced by ATP. Cell proliferation was increased significantly by the presence of the stable ATP analogue ATPgammaS. This effect was blunted by UCL1684, a synthesized SK blocker. In conclusion, in brain endothelial cells ATP-induced [Ca(2+)](i) rise activates SK2 current, and the subsequent membrane hyperpolarization enhances Ca(2+) entry presumably through transient receptor potential channels. This positive feedback mechanism can account for the augmented cell proliferation by ATP.