Cell Calcium 41 (2007) 261–269 Cytosolic calcium microdomains by arachidonic acid and nitric oxide in endothelial cells Cristiana Tomatis a,b,1 , Alessandra Fiorio Pla a,b,1 , Luca Munaron a,b,∗ a Department of Animal and Human Biology, University of Torino, Italy b Nanostructured Interfaces and Surfaces Centre of Excellence (NIS), University of Torino, Italy Received 12 April 2006; received in revised form 5 July 2006; accepted 11 July 2006 Available online 22 August 2006 Abstract Intracellular calcium signals activated by growth factors in endothelial cells during angiogenesis regulate cytosolic and nuclear events involved in survival, proliferation and motility. Among the intracellular messengers released after proangiogenic stimulation (bFGF, VEGF), arachidonic acid (AA), nitric oxide (NO) and their metabolites play a key role and their effects are strictly related to calcium homeostasis. Recently, we showed that AA and NO are able to stimulate the opening of store-independent calcium-permeable channels in the plas- mamembrane of bovine aortic endothelial cells (BAECs). Here, we studied the intracellular spatiotemporal dynamics of AA- and NO-induced calcium increases following store-independent calcium entry from extracellular medium. Using confocal calcium imaging, we show that calcium entry is preferentially restricted to peripheral cytosolic microdomains and does not necessarily invade the nuclear region. These results support the existence of local mitogen-activated calcium signals. Several factors could account for this spatial restriction, including the geometry of the cells and the clusterization of calcium channels and other signalling molecules. Intracellular calcium fingerprints could contribute to the specificity of endothelial cell responses to angiogenic factors. © 2006 Elsevier Ltd. All rights reserved. Keywords: Confocal microscopy; Arachidonic acid; Nitric oxide; Calcium entry; Endothelial cells; Microdomains 1. Introduction Angiogenic process is a highly complex sequence of events in which endothelial cells (ECs) play a key role through their movement, proliferation and reorganization in new blood vessels [1–3]. Abbreviations: AA, arachidonic acid; CCE, capacitative calcium entry; Ca i 2+ , intracellular calcium; ECs, endothelial cells; NCCE, non-capacitative calcium entry; NO, nitric oxide ∗ Corresponding author at: Department of Animal and Human Biology, University of Torino, Via Accademia Albertina 13, 10123 Torino, Italy. Tel.: +39 11 6704667; fax: +39 11 6704508. E-mail address: luca.munaron@unito.it (L. Munaron). 1 Contributed equally to this work. Angiogenic factors binding to tyrosine kinase receptors, such as basic fibroblast growth factor (bFGF) and vascu- lar endothelial growth factors (VEGFs), trigger the release of several intracellular signalling cascades: among them, AA and NO pathways have been well described in ECs [4–9]. In particular, we showed that bFGF is able to induce an increase of intracellular calcium concentration (Ca i 2+ ) through the opening of store-independent calcium channels in the plasmamembrane: this event, leading to the so-called non-capacitative calcium entry (NCCE), is critically involved in the control of ECs proliferation [10–13]. Other types of extracellular stimula, such as the vasoactive compounds His- tamine, Bradikinin and ATP, trigger Ca i 2+ signals in ECs as well, mainly acting through G-protein-coupled receptors, IP 3 -dependent calcium release from intracellular stores and 0143-4160/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2006.07.003