BIIE 0246 australia The fact that Yoda can activate Piezo in
The fact that Yoda1 can activate Piezo1 in the absence of other cellular components other than a cell membrane , suggests that it may directly interact with and activate Piezo1. However, this does not preclude Yoda1 from interacting and activating non-Piezo channels, particularly in endothelial cells. It is possible that Yoda1 activates Akt through Piezo1-independent mechanisms and that both gadolinium and ruthenium red can block Yoda1-induced phosphorylation of Akt because they are broader spectrum inhibitors than GsMTx4. For example, Gd3+ has been demonstrated to block other stretch-activated ion channels  as well as transient receptor potential (TRP) channels, such as the TRPML3 channel . Ruthenium red is also known to block TRP channels and is a well described antagonist of ryanodine receptors, a class of intracellular Ca2+ release channel expressed by skeletal and BIIE 0246 australia . All except two of the 28 identified TRP channels allow Ca2+ influx and at least 19 isoforms are expressed in vascular endothelial cells . Therefore, it is possible that in addition to activating Piezo1 in ECs, Yoda1 also activates one or more TRP channels, which leads to increased intracellular Ca2+, which in turn contributes to the activation of both Akt and ERK1/2. To our knowledge, this is the first study to show that Yoda1 stimulation increases ERK1/2 phosphorylation in ECs or in any cell type for that matter. However, the lack of a substantial blocking effect of three widely-used antagonists against Piezo1 (i.e. ruthenium red, gadolinium, and GsMTx4) suggests that Yoda1-induced ERK1/2 phosphorylation is not mediated specifically through this channel. Our findings differ from that of two recent studies. First, it has been demonstrated that ruthenium red significantly inhibits ERK1/2 activation in dental stem cells by low-intensity pulsed ultrasound (LIPUS) and therefore concluded that it is regulated by Piezo1 . Secondly, it has been reported that stretch induces calcium-dependent activation of ERK1/2 in epithelial cells that is Piezo1-mediated, since it can be blocked by gadolinium addition . One likely explanation is that Yoda1 differs in its ability to activate Piezo1 and potentially other channels than either LIPUS or cell stretching. Our data suggests that Yoda1 has the ability to stimulate ERK1/2 activation in ECs through other calcium channels and/or mechanisms. A previous study showed that at a concentration of 1.5 μM, which was the concentration used in the present study, Yoda1 does not trigger a Ca2+ response in human embryonic kidney (HEK) 293T cells transfected with human Piezo1 . In fact, there was no appreciable increase in Ca2+ influx in human Piezo1-transfected HEK293T cells until they were stimulated with Yoda1 concentrations at and above 6 μM, which implies that activation of Piezo1 by Yoda1 is concentration-dependent. In another study, Yoda1 at a lower concentration (1 μM) was shown to induce a robust Ca2+ response in human umbilical endothelial cells (HUVECs) . This discrepancy may be explained by the possibility that Piezo1 was not activated by Yoda1 in the latter study, and that there are inherent differences in the endogenous expression of other ion channels that control Ca2+ influx in ECs compared to HEK293T cells, thereby allowing Yoda1 to still cause a Ca2+ response. TRPV4 is one such Ca2+ -permeable channel that is highly expressed by ECs, but not expressed by HEK293T [21,22]. Interestingly, TRPV4 has been shown to be activated by shear stress and to play a regulatory role in vascular tone [23,24]. TRPM7 is another example of a Ca2+ -permeable channel that is expressed in ECs , but not reported to be expressed by HEK293T cells. Additionally, it has been shown to rapidly accumulate at the plasma membrane in response to shear stress . Despite ECs having low levels of endogenous TRPM7-like current that is not increased by shear stress , TRPM7 channels have been implicated in regulating cell survival [27,28] and cell proliferation via the ERK signaling pathway [25,29].