Endothelial PtdIns(4,5)P2 Levels Underlie a Controlling Mechanism of TRPV4 Channel Activity in the Cerebral Circulation

Abstract

The transient receptor potential vanilloid 4 (TRPV4) channel in the mesenteric endothelium plays a pivotal role in mediating Ca2+ entry leading to membrane hyperpolarization. We previously showed that endothelial cells in the cerebral circulation express functional TRPV4 channels, which are less sensitive to the TRPV4 agonist GSK1016790A (GSK101) when compared to their peripheral counterparts. This suggests that TRPV4 channels are differentially regulated, depending on the vascular bed and/or cellular environment. Here, we employed patch clamp electrophysiology and endothelial cells freshly isolated from C57BL/6 mouse brain capillaries (cECs) and pial arteries to explore possible mechanisms underlying the disparate regulation of TRPV4 channels. While conventional whole‐cell recordings in cECs revealed a TRPV4 current comparable to that previously recorded in peripheral ECs, perforated patch clamp electrophysiology indicated reduced sensitivity to GSK101; an observation that suggested a role for an intracellular factor. Along this suggestion, inclusion of physiological levels of intracellular ATP, but not GTP or ATP‐γ‐S, rendered cECs less sensitive to TRPV4 agonists (GSK101 or 4αPDD). The latter finding proposed a potential role for ATP hydrolysis/kinase activity in the inhibitory regulation of TRPV4. Consistent with a hypothetical role for kinases, intracellular ATP was insufficient to suppress TRPV4 current in Mg2+‐free pipette solutions. Pharmacological suppression of protein kinases A, C or G failed to reverse the ATP‐mediated effect. Alternatively, inhibitors of the phosphatidylinositol 4‐kinase (PIK93, wortmannin, phenylarsine oxide and LY294002) significantly masked the inhibitory effect of ATP on TRPV4 currents; suggesting that ATP may act as a precursor for phosphoinositides that suppress TRPV4 activity. In line with this hypothesis, intracellular application of exogenous PtdIns(4,5)P2 (diC8‐PIP2), but not PtdIns(4)P, suppressed GSK101‐induced currents. Accumulation of endogenous PtdIns(4,5)P2 (U73122) similarly inhibited TRPV4 current. Furthermore, scavenging PtdIns(4,5)P2 using poly‐L‐lysine reversed ATP‐induced inhibition. This regulatory mechanism is not delimited to capillaries; importantly, a similar inhibitory effect of PtdIns(4,5)P2 or ATP was evident in arterial ECs isolated from pial cerebral arteries. In conclusion, TRPV4 channel activity is regulated in the cerebral circulation due to, at least in part, modulation by the phosphoinositide PtdIns(4,5)P2. The inhibitory role of PtdIns(4,5)P2 represents a mechanism underlying the tighter control over TRPV4 channel activity in cerebral arteries and capillaries. It may further allude to a possible crosstalk between G‐protein coupled receptors, that dynamically alter cellular PtdIns(4,5)P2 levels, and the TRPV4 channel. These regulatory cascades may have broad implications for TRPV4 channel control in diverse cell types beyond the endothelium.Support or Funding InformationSupported by: Totman Medical Research Trust, Fondation Leducq, and NIH (R01 HL121706, PO1 HL‐095488, R37‐DK‐053832).