Agonist and cholesterol modulate the alpha7 acetylcholine receptor in non-neural endothelial cells

AYALA PEÑA, V.B.; BONINI, I.C.; BARRANTES, F.J.

Puerto Madryn

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2010

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

The "neuronal" a7-type nicotinic acetylcholine receptor (a7AChR) is found in various non-neural tissues, including vascular endothelium, where its peculiar ionotropic (high Ca2+ permeability) and metabotropic (Ca2+-mediated intracellular cascades) properties may play important roles in angiogenesis, inflammation and atherogenesis. Molecular properties of the a7AChR, its response to nicotine stimulation, and its cellular distribution were studied here using a combination of pharmacological, biochemical and fluorescence microscopy tools. a7AChRs in rat arterial endothelial cells (RAEC) were found to undergo agonist (nicotine)-induced upregulation (from 53±16 to 385.2±46.8 fmol/mg protein with 50 mM nicotine), increasing their cell-surface exposure. a7AChRs occurred predominantly in the “non-raft" subcellular fractions, yet cholesterol depletion mediated by cyclodextrin treatment reduced the number of cell-surface a7AChRs. Nicotine was found to increase the affinity of the a7AChR for crystal violet, an openchannel blocker. Under basal conditions, a7AChRs in endothelial cells displayed a high-affinity, presumably desensitized conformation (Kd ~0.76 nM), and both nicotine and cyclodextrin affected their cell-surface expression.a7-type nicotinic acetylcholine receptor (a7AChR) is found in various non-neural tissues, including vascular endothelium, where its peculiar ionotropic (high Ca2+ permeability) and metabotropic (Ca2+-mediated intracellular cascades) properties may play important roles in angiogenesis, inflammation and atherogenesis. Molecular properties of the a7AChR, its response to nicotine stimulation, and its cellular distribution were studied here using a combination of pharmacological, biochemical and fluorescence microscopy tools. a7AChRs in rat arterial endothelial cells (RAEC) were found to undergo agonist (nicotine)-induced upregulation (from 53±16 to 385.2±46.8 fmol/mg protein with 50 mM nicotine), increasing their cell-surface exposure. a7AChRs occurred predominantly in the “non-raft" subcellular fractions, yet cholesterol depletion mediated by cyclodextrin treatment reduced the number of cell-surface a7AChRs. Nicotine was found to increase the affinity of the a7AChR for crystal violet, an openchannel blocker. Under basal conditions, a7AChRs in endothelial cells displayed a high-affinity, presumably desensitized conformation (Kd ~0.76 nM), and both nicotine and cyclodextrin affected their cell-surface expression.2+ permeability) and metabotropic (Ca2+-mediated intracellular cascades) properties may play important roles in angiogenesis, inflammation and atherogenesis. Molecular properties of the a7AChR, its response to nicotine stimulation, and its cellular distribution were studied here using a combination of pharmacological, biochemical and fluorescence microscopy tools. a7AChRs in rat arterial endothelial cells (RAEC) were found to undergo agonist (nicotine)-induced upregulation (from 53±16 to 385.2±46.8 fmol/mg protein with 50 mM nicotine), increasing their cell-surface exposure. a7AChRs occurred predominantly in the “non-raft" subcellular fractions, yet cholesterol depletion mediated by cyclodextrin treatment reduced the number of cell-surface a7AChRs. Nicotine was found to increase the affinity of the a7AChR for crystal violet, an openchannel blocker. Under basal conditions, a7AChRs in endothelial cells displayed a high-affinity, presumably desensitized conformation (Kd ~0.76 nM), and both nicotine and cyclodextrin affected their cell-surface expression.2+-mediated intracellular cascades) properties may play important roles in angiogenesis, inflammation and atherogenesis. Molecular properties of the a7AChR, its response to nicotine stimulation, and its cellular distribution were studied here using a combination of pharmacological, biochemical and fluorescence microscopy tools. a7AChRs in rat arterial endothelial cells (RAEC) were found to undergo agonist (nicotine)-induced upregulation (from 53±16 to 385.2±46.8 fmol/mg protein with 50 mM nicotine), increasing their cell-surface exposure. a7AChRs occurred predominantly in the “non-raft" subcellular fractions, yet cholesterol depletion mediated by cyclodextrin treatment reduced the number of cell-surface a7AChRs. Nicotine was found to increase the affinity of the a7AChR for crystal violet, an openchannel blocker. Under basal conditions, a7AChRs in endothelial cells displayed a high-affinity, presumably desensitized conformation (Kd ~0.76 nM), and both nicotine and cyclodextrin affected their cell-surface expression.a7AChR, its response to nicotine stimulation, and its cellular distribution were studied here using a combination of pharmacological, biochemical and fluorescence microscopy tools. a7AChRs in rat arterial endothelial cells (RAEC) were found to undergo agonist (nicotine)-induced upregulation (from 53±16 to 385.2±46.8 fmol/mg protein with 50 mM nicotine), increasing their cell-surface exposure. a7AChRs occurred predominantly in the “non-raft" subcellular fractions, yet cholesterol depletion mediated by cyclodextrin treatment reduced the number of cell-surface a7AChRs. Nicotine was found to increase the affinity of the a7AChR for crystal violet, an openchannel blocker. Under basal conditions, a7AChRs in endothelial cells displayed a high-affinity, presumably desensitized conformation (Kd ~0.76 nM), and both nicotine and cyclodextrin affected their cell-surface expression.a7AChRs in rat arterial endothelial cells (RAEC) were found to undergo agonist (nicotine)-induced upregulation (from 53±16 to 385.2±46.8 fmol/mg protein with 50 mM nicotine), increasing their cell-surface exposure. a7AChRs occurred predominantly in the “non-raft" subcellular fractions, yet cholesterol depletion mediated by cyclodextrin treatment reduced the number of cell-surface a7AChRs. Nicotine was found to increase the affinity of the a7AChR for crystal violet, an openchannel blocker. Under basal conditions, a7AChRs in endothelial cells displayed a high-affinity, presumably desensitized conformation (Kd ~0.76 nM), and both nicotine and cyclodextrin affected their cell-surface expression.mM nicotine), increasing their cell-surface exposure. a7AChRs occurred predominantly in the “non-raft" subcellular fractions, yet cholesterol depletion mediated by cyclodextrin treatment reduced the number of cell-surface a7AChRs. Nicotine was found to increase the affinity of the a7AChR for crystal violet, an openchannel blocker. Under basal conditions, a7AChRs in endothelial cells displayed a high-affinity, presumably desensitized conformation (Kd ~0.76 nM), and both nicotine and cyclodextrin affected their cell-surface expression.a7AChRs occurred predominantly in the “non-raft" subcellular fractions, yet cholesterol depletion mediated by cyclodextrin treatment reduced the number of cell-surface a7AChRs. Nicotine was found to increase the affinity of the a7AChR for crystal violet, an openchannel blocker. Under basal conditions, a7AChRs in endothelial cells displayed a high-affinity, presumably desensitized conformation (Kd ~0.76 nM), and both nicotine and cyclodextrin affected their cell-surface expression.a7AChRs. Nicotine was found to increase the affinity of the a7AChR for crystal violet, an openchannel blocker. Under basal conditions, a7AChRs in endothelial cells displayed a high-affinity, presumably desensitized conformation (Kd ~0.76 nM), and both nicotine and cyclodextrin affected their cell-surface expression.a7AChR for crystal violet, an openchannel blocker. Under basal conditions, a7AChRs in endothelial cells displayed a high-affinity, presumably desensitized conformation (Kd ~0.76 nM), and both nicotine and cyclodextrin affected their cell-surface expression.a7AChRs in endothelial cells displayed a high-affinity, presumably desensitized conformation (Kd ~0.76 nM), and both nicotine and cyclodextrin affected their cell-surface expression. Supported by grants from Philip Morris USA and Philip Morris International to FJB.