title: 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 Ca(2+) signals are generated and maintained, especially in intact preparations. In particular the role of the sarco-endoplasmic reticulum Ca(2+)-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 Ca(2+) 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 Ca(2+) entry (CCE) and ER Ca(2+) release and uptake. We have determined its contribution to the Ca(2+) 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 Ca(2+) in the endothelial cells, consisting of a rapid initial phase, then a plateau upon which oscillations of Ca(2+) were superimposed, followed by a decline to basal Ca(2+) levels upon carbachol removal. Our data support the following conclusions: (i) the size (amplitude and duration) of the Ca(2+) 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 Ca(2+) is not sufficient to trigger it. However, longer applications sufficient to deplete the ER or even partial SERCA inhibition stimulate CCE. (iii) Ca(2+) 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.
authors.affiliation: Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, UK.
authors.forename: S
authors.initials: S
authors.lastname: Mumtaz
authors.forename: G
authors.initials: G
authors.lastname: Burdyga
authors.forename: L
authors.initials: L
authors.lastname: Borisova
authors.forename: Susan
authors.initials: S
authors.lastname: Wray
authors.forename: T
authors.initials: T
authors.lastname: Burdyga
mesh_terms: D000818:Animals; D001158:Arteries / Q000166:cytology*; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000819:agonists* / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology; D048430:Cell Shape / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004963:Estrenes / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D007811:Lanthanum / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism; D018613:Microscopy, Confocal; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D012433:Ryanodine / Q000494:pharmacology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071737:Stromal Interaction Molecule 1; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D001896:Boron Compounds; D004963:Estrenes; D007211:Indoles; D008562:Membrane Glycoproteins; D011760:Pyrrolidinones; C518857:Stim1 protein, rat; D000071737:Stromal Interaction Molecule 1; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D012433:Ryanodine; D007811:Lanthanum; D002217:Carbachol; C109986:2-aminoethoxydiphenyl borate; D010738:Type C Phospholipases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid