Gαq/11-mediated intracellular calcium responses to retrograde flow in endothelial cells

Abstract

Disturbed flow patterns, including reversal in flow direction, are key factors in the development of dysfunctional endothelial cells (ECs) and atherosclerotic lesions. An almost immediate response of ECs to fluid shear stress is the increase in cytosolic calcium concentration ([Ca(2+)](i)). Whether the source of [Ca(2+)](i) is extracellular, released from Ca(2+) intracellular stores, or both is still undefined, though it is likely dependent on the nature of forces involved. We have previously shown that a change in flow direction (retrograde flow) on a flow-adapted endothelial monolayer induces the remodeling of the cell-cell junction along with a dramatic [Ca(2+)](i) burst compared with cells exposed to unidirectional or orthograde flow. The heterotrimeric G protein-α q and 11 subunit (Gα(q/11)) is a likely candidate in effecting shear-induced increases in [Ca(2+)](i) since its expression is enriched at the junction and has been previously shown to be activated within seconds after onset of flow. In flow-adapted human ECs, we have investigated to what extent the Gα(q/11) pathway mediates calcium dynamics after reversal in flow direction. We observed that the elapsed time to peak [Ca(2+)](i) response to a 10 dyn/cm(2) retrograde shear stress was increased by 11 s in cells silenced with small interfering RNA directed against Gα(q/11). A similar lag in [Ca(2+)](i) transient was observed after cells were treated with the phospholipase C (PLC)-βγ inhibitor, U-73122, or the phosphatidylinositol-specific PLC inhibitor, edelfosine, compared with controls. Lower levels of inositol 1,4,5-trisphosphate accumulation seconds after the onset of flow correlated with the increased lag in [Ca(2+)](i) responses observed with the different treatments. In addition, inhibition of the inositol 1,4,5-trisphosphate receptor entirely abrogated flow-induced [Ca(2+)](i). Taken together, our results identify the Gα(q/11)-PLC pathway as the initial trigger for retrograde flow-induced endoplasmic reticulum calcium store release, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to the reversal of blood flow.