TRPV4 Activation Evokes Coordinated, Multicellular Signalling via IP3‐Evoked Ca2+ Signalling in the Endothelium of Intact Blood Vessels

Abstract

The vascular endothelium is the innermost layer of cells lining blood vessels and is the key regulator of vascular tone. A critical feature of endothelial signalling, in the control of vascular function, is the endothelium's ability to coordinate function by communicating information between cells. Changes in the intracellular Ca2+ concentration is a major regulator of endothelial function. Central to an understanding of the control and coordination of endothelial function is an appreciation of the control of Ca2+ signalling across cells. While Ca2+ release from the internal store is of acknowledged significance in the control of endothelial function, the mechanism(s) permitting Ca2+ influx to coordinate activity across cells are less well understood. Ca2+ influx via the transient receptor potential vanilloid channel 4 (TRPV4), a non‐selective Ca2+ permeant cation channel, is reported to be an important contributor of endothelial signalling and vascular tone. TRPV4 is activated by thermal and mechanical stimuli, endogenous signalling molecules such as phorbol esters and the downstream metabolites of arachidonic acid, as well as pharmacological compounds including GSK1016790A (GSK101) and 4alphaPDD. However, how Ca2+ influx via one or few channels on the outside membrane of endothelial cells is communicated across multiple cells is not understood.Here in the endothelium of isolated mesenteric arteries (8–12 week old Sprague Dawley rats) intracellular Ca2+ signalling in response to TRPV4 activation was visualised using the Ca2+ indicator Cal 520/AM (80–100 cells per field of view). En face artery preparations were perfused with TRPV4 activators diluted in MOPS buffer, at a rate of 1.5 ml/min. Ca2+ images were captured using a high‐sensitivity CCD camera and analysed using a custom analysis package written in Python. The TRPV4 activators GSK101 (20 nM) and 4alphaPDD (5 μM) initially evoked sporadic, localized rises in Ca2+. The Ca2+ rises increased in frequency and amplitude and endothelial cells eventually exhibited a global increase in intracellular Ca2+ that propagated as widespread waves within and between cells. Both Ca2+ influx and widespread propagated waves were prevented by the removal of extracellular Ca2+ or pre‐incubation with the TRPV4 antagonists HC067047 or ruthenium red. On the other hand, prior depletion of the intracellular store with cyclopiazonic acid (CPA) or inhibition of phospholipase C (PLC) using U73122, or block of the IP3‐receptor with 2‐APB inhibited the widespread propagating waves. However, neither U73122 nor 2‐APB prevented the global rise in Ca2+ evoked by GSK101. These data suggest that TRPV4 activation causes Ca2+ influx that induces IP3 and store dependent propagating waves. Ca2+ influx and propagating waves may be an important contributor for vasorelaxation mediated by TRPV4.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.