Rapid flow-induced activation of Gα q/11 is independent of Piezo1 activation.

Endothelial cell (EC) mechanochemical transduction is the process by which mechanical stimuli are sensed by ECs and transduced into biochemical signals and ultimately into physiological responses. Identifying the mechanosensor/mechanochemical transducer(s) and describing the mechanism(s) by which they receive and transmit the signals has remained a central focus within the field. The heterotrimeric G protein, Gα _q/11 , is proposed to be part of a macromolecular complex together with PECAM-1 at EC junctions and may constitute the mechanochemical transducer as it is rapidly activated within seconds of flow onset. The mechanically activated cation channel Piezo1 has recently been implicated due to its involvement in mediating early responses, such as calcium and ATP release. Here, we investigate the role of Piezo1 in rapid shear stress-induced Gα _q/11 activation. We show that flow-induced dissociation of Gα _q/11 from PECAM-1 in ECs at 15 s is abrogated by BIM-46187, a selective inhibitor of Gα _q/11 activation, suggesting that Gα _q/11 activation is required for PECAM-1/Gα _q/11 dissociation. Although siRNA knockdown of Piezo1 caused a dramatic decrease in PECAM-1/Gα _q/11 association in the basal condition, it had no effect on flow-induced dissociation. Interestingly, siRNA knockdown of Piezo1 caused a marked decrease in PECAM-1 expression. Additionally, selective blockade of Piezo1 with ion channel inhibitors had no effect on flow-induced PECAM-1/Gα _q/11 dissociations. Lastly, flow onset caused increased association of Gβ ₁ with Piezo1 as well as with the p101 subunit of phosphoinositide 3-kinase, which were both blocked by the Gβγ inhibitor gallein. Together, our results indicate that flow-induced activation of Piezo1 is not upstream of G protein activation.