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.

Update on vascular endothelial Ca2+signalling: A tale of ion channels, pumps and transporters

“…This, in turn, leads to the blockade of several key steps of the angiogenic process, such as proliferation and tubulogenesis [205,206,338] . These data are supported by the finding that, under resting conditions, the ER Ca 2+ sensor is homogeneously distributed throughout the cytosol, whereas emptying of the InsP3-sensitive Ca 2+ reservoir in the absence of extracellular Ca 2+ induces Stim1 homo-oligomerization and redistribution into sub-plasmalemmal puncta [206,218,339] . Both processes are recovered upon Ca 2+ restoration to the bathing solution, as Ca 2+ entry recharges the intracellular Ca 2+ pool and inactivates Stim1 [340] .…”

“…These triggering sites remain constant during repetitive cell stimulation [393,401,402] . Indeed, when the information embedded within the [Ca 2+ ]i increase must be conveyed over a long time, both the elementary events and the global Ca 2+ sweeps can adopt an oscillatory pattern [16,72,339,396] . However, the physiological outcome of local Ca 2+ spikes depends on the Ca 2+ -sensitive decoders located within a few nanometers from the channel pore [395] , and the frequency of repetitive Ca 2+ waves encodes for the selective engagement of specific downstream targets [403][404][405] .…”

“…Unlike the oscillatory Ca 2+ response to extracellular ligands [72,406,407] , the amplitude of the Ca 2+ spikes increases with the intensity of the mechanical stimulation [22][23][24][25] . Agonist-evoked Ca 2+ oscillations require PLC activation by either GPCRs or TKRs [72,339,404,405,408] . The following InsP3-dependent Ca 2+ mobilization may be amplified by adjacent RyRs via CICR [44,134] and requires Ca 2+ inflow to supply releasable Ca 2+ over time and maintain InsP3R sensitivity to the ligand [72,407,409,410] .…”

Vascular Endothelial Growth Factor Stimulates Endothelial Colony Forming Cells Proliferation and Tubulogenesis by Inducing Oscillations in Intracellular Ca2+ Concentration

“…Alternatively, the specific Ca 2þ signatures evoked by VEGF might depend on the different phases of the cell cycle in adjacent ECFCs, as observed in oligodendroglial cells [28]. Finally, the heterogeneity in VEGFinduced Ca 2þ oscillations in ECFCs might be ascribed to the different density or distribution of the underlying signaling pathways, such as InsP 3 Rs [25,26,55]. This hypothesis is corroborated by the stochastic nature of the Ca 2þ spikes.…”

Automated Analysis of Dynamic Ca²⁺ Signals in Image Sequences

“…Spatial and temporal control of Ca 2+ is thought to underlie physiological signal specificity, and therefore the analysis of Ca 2+ signal patterns is of considerable interest to investigators in multiple fields 5 . Ca 2+ indicator dyes such as Fluo-4 and Fura-2 are commonly employed to measure intracellular Ca 2+ signals with fluorescence microscopy [5][6][7][8][9][10][11][12] . Typically, temporal Ca 2+ signals are evaluated as time-dependent changes in mean fluorescence within a user-defined area, or region of interest (ROI) 5,6,[13][14][15][16] .…”