The mechanism of agonist induced Ca2+ signalling in intact endothelial cells studied confocally in in situ arteries

Abstract

In endothelial cells there remain uncertainties in the details of how Ca2+ signals are generated and maintained, especially in intact preparations. In particular the role of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), in contributing to the components of agonist-induced signals is unclear.The aim of this work was to increase understanding of the detailed mechanism of Ca2+ signalling in endothelial cells using real time confocal imaging of Fluo-4 loaded intact rat tail arteries in response to muscarinic stimulation. In particular we have focused on the role of SERCA, and its interplay with capacitative Ca2+ entry (CCE) and ER Ca2+ release and uptake. We have determined its contribution to the Ca2+ signal and how it varies with different physiological stimuli, including single and repeated carbachol applications and brief and prolonged exposures.In agreement with previous work, carbachol stimulated a rise in intracellular Ca2+ in the endothelial cells, consisting of a rapid initial phase, then a plateau upon which oscillations of Ca2+ were superimposed, followed by a decline to basal Ca2+ levels upon carbachol removal. Our data support the following conclusions: (i) the size (amplitude and duration) of the Ca2+ spike and early oscillations are limited by SERCA activity, thus both are increased if SERCA is inhibited. (ii) SERCA activity is such that brief applications of carbachol do not trigger CCE, presumably because the fall in luminal Ca2+ is not sufficient to trigger it. However, longer applications sufficient to deplete the ER or even partial SERCA inhibition stimulate CCE. (iii) Ca2+ entry occurs via STIM-mediated CCE and SERCA contributes to the cessation of CCE. In conclusion our data show how SERCA function is crucial to shaping endothelial cell Ca signals and its dynamic interplay with both CCE and ER Ca releases.