α7-Type Acetylcholine Receptor Localization and its Modulation by Nicotine and Cholesterol in Vascular Endothelial Cells

The neuronal-type alpha 7 nicotinic acetylcholine receptor (alpha 7AChR) is also found in various non-neural tissues, including vascular endothelium, where its peculiar ionotropic properties (high Ca(2+) permeability) and its supervening Ca(2+)-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 alpha 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 alpha 7AChRs in rat arterial endothelial (RAEC) and human venous endothelial (HUVEC) cells occur at extremely low expression levels (similar to 50 fmol/mg protein) but undergo agonist-induced up-regulation at relatively high nicotine concentrations (similar to 300-fold with 50 mu M ligand), increasing their cell-surface exposure. When analyzed in terms of cold Triton X-100 solubility and subcellular distribution, alpha 7AChRs occur in the "non-raft" subcellular membrane fractions. Acute cholesterol depletion reduced not only cholesterol levels but also the number of cell-surface alpha 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 K(D) of alpha 7AChRs for the open-channel blocker crystal violet was found to be similar to 600-fold lower in receptor-enriched membranes obtained from up-regulated HUVEC. J. Cell. Biochem. 112: 3276-3288, 2011. (C) 2011 Wiley Periodicals, Inc.