The Plasma Membrane Channel TRPV4 Evokes Endothelium‐Dependent Relaxation via a Novel Ca 2+ ‐Induced Ca 2+ Release Acting, at the Endothelial IP 3 Receptor

The vascular endothelium lines the the entire vascular network and is a critical regulator of vascular tone given its ability to sense the complex composition of vasoactive substances in the circulating blood, as well as hemodynamic stresses and mechanical forces acting on the blood vessel wall. TRPV4 is a Ca 2+ permeable cation channel which is expressed in the membrane of the endothelium and is activated by osmotic, mechanical and chemical stimuli, including the pharmacological agent GSK1016790A (GSK101). TRPV4 is an important contributor to endothelial regulation of vascular tone. Ca 2+ influx from outside cells is a key messenger in the signalling cascades which transduce activating stimuli sensed by the endothelium into a functional output in the underlying smooth muscle. Previous studies suggested that the TRPV4 activation evoked endothelium‐dependent relaxation solely as a result of Ca 2+ influx via TRPV4. However, recently, we found the endothelium‐dependent vasorelaxation evoked by TRPV4 activation was attenuated when Ca 2+ release from the internal store was prevented. Here, we investigated the link between TRPV4 activation and Ca 2+ release from the internal store. In mesenteric arteries from 8–12 week old Sprague Dawley rats, we visualised Ca 2+ signalling (Cal‐520/AM) in response to GSK101(20 nM) and used pharmacological agents to characterise the signalling pathway leading to vasorelaxation. In conjunction, we also measured the contractile response of the arteries in response to the vasoconstrictor phenylephrine (PE), in the absence and presence of GSK101, the endothelium and after Ca 2+ store depletion. All vessels were perfused with MOPS buffer in which all drugs were diluted. Images were captured using a high‐sensitivity CCD camera and analysed using custom analysis packages written in Python. The TRPV4 activator GSK101 (20 nM) stimulated a ‘slow’ continuous global rise in intracellular Ca 2+ on which ‘fast’ sporadic intracellular waves which propagated both within and between cells occurred. All Ca 2+ signals (slow and fast) were inhibited by the removal of extracellular Ca 2+ or by prior treatment with the TRPV inhibitors, Ruthenium Red and HC067047. 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 IP 3 ‐receptor with 2‐APB inhibited only the widespread propagating waves. These data suggest that TRPV4 activation caused Ca 2+ influx that induced IP 3 ‐evoked Ca 2+ release and propagating Ca 2+ waves. In PE pre‐constricted vessels, GSK101 (20 nM) induced a dilation that was entirely dependent upon an intact endothelium. Prior depletion of the intracellular store using CPA prevented the inhibitory effect of GSK on PE‐induced contractions indicating that vasodilation induced by the TRPV4 activator GSK101 is dependent upon IP 3 ‐stimulated Ca 2+ release from the endothelium's internal Ca 2+ store. These results suggest that Ca 2+ influx via the plasma membrane channel TPRV4, evokes a novel Ca 2+ ‐induced Ca 2+ release like mechanism acting at the IP 3 receptor in the intact endothelium to evoke vasodilation. Support or Funding Information BHF This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .