Shear stress induced ionic current and FM1-43 influx via P2X4 ATP-receptor as a mechano-transduction pathway

Abstract

Endothelial cells (ECs) sense hemodynamic forces (pressure and shear stress) from the flowing blood. P2X4 is constitutively expressed at high levels in ECs. Yamamoto et al. found a shear stress-dependent Ca²⁺ influx mediated by the purinergic P2X4 receptor as confirmed by sense- and anti-sense oligonucleotide technique. Human embryonic kidney (HEK293) cells that had no Ca²⁺ response to flow, were able to show a cytosolic Ca²⁺ increase when transfected with P2X4. In order to determine if P2X4 can sense the shear force, we investigated whether direct mechanical stimulation of P2X4-containing outside-out patches were activated by flow. Patch-clamp recordings from HEK293 stably expressing of P2X4 showed single channel activity following flow stimulation with Hank's solution. The permeation property of the styryl dye FM1-43 through mechanotransduction channels was also assessed in our case. Epifluorescence and confocal microscopy showed FM1-43(5μM) uptake in HEK293-P2X4 both after ATP (10μM) or flow stimulation. These results indicate functional activity of P2X4 and suggest that P2X4 could likely be a primary shear-stress sensor in endothelial cells. [Jpn J Physiol 55 Suppl:S83 (2005)]