title: Fluid shear stress activates Ca(2+) influx into human endothelial cells via P2X4 purinoceptors.
abstract: Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) influx. Human umbilical vein ECs loaded with the Ca(2+) indicator Indo-1/AM were exposed to laminar flow of Hanks' balanced salt solution at various concentrations of ATP, and changes in [Ca(2+)](i) were monitored with confocal laser scanning microscopy. A stepwise increase in shear stress elicited a corresponding stepwise increase in [Ca(2+)](i) at 250 nmol/L ATP. The shear stress-dependent increase in [Ca(2+)](i) was not affected by phospholipase C inhibitor (U-73122) but disappeared after the chelation of extracellular Ca(2+) with EGTA, indicating that the Ca(2+) increase was due to Ca(2+) influx. Antisense oligonucleotides designed to knockout P2X4 expression abolished the shear stress-dependent Ca(2+) influx seen at 250 nmol/L ATP in human umbilical vein ECs. Human embryonic kidney 293 cells showed no Ca(2+) response to flow at 2 micromol/L ATP, but when transfected with P2X4 cDNA, they began to express P2X4 purinoceptors and to show shear stress-dependent Ca(2+) 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 Ca(2+) signaling.
authors.affiliation: Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Yamamoto
authors.forename: R
authors.initials: R
authors.lastname: Korenaga
authors.forename: A
authors.initials: A
authors.lastname: Kamiya
authors.forename: J
authors.initials: J
authors.lastname: Ando
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D004963:Estrenes / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D007211:Indoles; D018613:Microscopy, Confocal; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D058848:Receptors, Purinergic P2X4; D012212:Rheology; D013314:Stress, Mechanical; D014162:Transfection; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D014471:Umbilical Veins
chemical_list: D004963:Estrenes; D005456:Fluorescent Dyes; D007211:Indoles; D016376:Oligonucleotides, Antisense; C548226:P2RX4 protein, human; D011760:Pyrrolidinones; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases; C048960:indo-1; D002118:Calcium