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(Circulation Research. 2000;87:385.)
© 2000 American Heart Association, Inc.

Cellular Biology

Fluid Shear Stress Activates Ca²⁺ Influx Into Human Endothelial Cells via P2X4 Purinoceptors

Kimiko Yamamoto, Risa Korenaga, Akira Kamiya, Joji Ando

From the Department of Biomedical Engineering (K.Y., R.K., J.A.), Graduate School of Medicine, University of Tokyo, Tokyo, Japan; and Interdisciplinary Science Center (A.K.), Nihon University, Tokyo, Japan.

Correspondence to Dr Joji Ando, Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 Japan. E-mail joji{at}m.u-tokyo.ac.jp

Abstract—Ca²⁺ signaling plays an important role in endothelial cell (EC) responses to shear stress generated by blood flow. Our previous studies demonstrated that bovine fetal aortic ECs showed a shear stress–dependent Ca²⁺ influx when exposed to flow in the presence of extracellular ATP. However, the molecular mechanisms of this process, including the ion channels responsible for the Ca²⁺ response, have not been clarified. Here, we demonstrate that P2X4 purinoceptors, a subtype of ATP-operated cation channels, are involved in the shear stress–mediated Ca²⁺ influx. Human umbilical vein ECs loaded with the Ca²⁺ indicator Indo-1/AM were exposed to laminar flow of Hanks’ balanced salt solution at various concentrations of ATP, and changes in [Ca²⁺]_i were monitored with confocal laser scanning microscopy. A stepwise increase in shear stress elicited a corresponding stepwise increase in [Ca²⁺]_i at 250 nmol/L ATP. The shear stress–dependent increase in [Ca²⁺]_i was not affected by phospholipase C inhibitor (U-73122) but disappeared after the chelation of extracellular Ca²⁺ with EGTA, indicating that the Ca²⁺ increase was due to Ca²⁺ influx. Antisense oligonucleotides designed to knockout P2X4 expression abolished the shear stress–dependent Ca²⁺ influx seen at 250 nmol/L ATP in human umbilical vein ECs. Human embryonic kidney 293 cells showed no Ca²⁺ response to flow at 2 µmol/L ATP, but when transfected with P2X4 cDNA, they began to express P2X4 purinoceptors and to show shear stress–dependent Ca²⁺ influx. P2X4 purinoceptors may have a "shear-transducer" property through which shear stress is perceived directly or indirectly and transmitted into the cell interior via Ca²⁺ signaling.

Key Words: endothelium • shear stress • purinergic receptors/purinoceptors • Ca²⁺ • adenosine triphosphate

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