α7-type acetylcholine receptor localization and its modulation by nicotine and cholesterol in vascular endothelial cells

The neuronal-type 7 nicotinic acetylcholine receptor (7AChR) is also found in various non-neural tissues, including vascular endothelium, where its peculiar ionotropic properties (high Ca2+ permeability) and its supervening Ca2+-mediated intracellular cascades may play important roles in physiology (angiogenesis) and pathology (inflammation and atherogenesis). Changes in molecular (up-regulation, affinity and conformational states) and cellular (distribution, association with membranes) properties of the 7AChR related to angiogenesis (wound-repair cell migration) and atherogenesis (alterations in cholesterol content) were studied in living endothelial cells, with the aim of determining whether such changes constitute early markers of inflammatory response. The combination of pharmacological, biochemical and fluorescence microscopy tools showed that 7AChRs in rat arterial endothelial (RAEC) and human venous endothelial (HUVEC) cells occur at extremely low expression levels (~50 fmol/mg protein) but undergo agonist-induced up-regulation at relatively high nicotine concentrations (~300-fold with 50 M ligand), increasing their cell-surface exposure. When analyzed in terms of cold Triton X-100 solubility and subcellular distribution, 7AChRs occur in the "non-raft" subcellular membrane fractions.Acute cholesterol depletion reduced not only cholesterol levels but the number of cell-surface 7AChRs. Nicotine exposure markedly stimulated cell migration and accelerated wound repair, which drastically diminished in cells deprived of the sterol. The angiogenic effect of nicotine appears to be synergistic with cholesterol content. Finally, the apparent KD of 7AChRs for the open-channel blocker crystal violet was found to be ~600-fold lower in receptor-enriched membranes obtained from up-regulated HUVEC.Fil: Ayala Peña, Victoria Belen. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. UNESCO Chair of Biophysics & Molecular Neurobiology; ArgentinaFil: Bonini, Ida Clara. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. UNESCO Chair of Biophysics & Molecular Neurobiology; ArgentinaFil: Antollini, Silvia Susana. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. UNESCO Chair of Biophysics & Molecular Neurobiology; ArgentinaFil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. UNESCO Chair of Biophysics & Molecular Neurobiology; Argentin