ABSTRACT
Chronic nicotine treatment increases the number of neuronal nicotinic acetylcholine receptors (nAChRs). Localization of nAChRs at a cellular level determines their functional role. However, changes in the localization of nAChRs caused by chronic nicotine treatment are not well known. In this study, we have examined the effects of chronic nicotine treatment on alpha7 and beta2 nAChR subunits in vitro in cell lines and in vivo in mouse striatum. In vitro, two different cell lines were used, SH-SY5Y cells endogenously expressing several nAChR subtypes and SH-EP1-halpha7 cells, transfected with the human alpha7 nAChR subunit gene. Effects of chronic nicotine treatment (10 microM, 3 days) were studied in vitro by using confocal and electron microscopy and calcium fluorometry. In vitro in SH-SY5Y cells, alpha7 and beta2 subunits formed groups, unlike alpha7 subunits in SH-EP1-halpha7 cells, which were partially localized on endoplastic reticulum. Chronic nicotine treatment did not change the localization of nAChRs in endosomes, but caused clustering of alpha7 subunits in SH-EP1-halpha7 cells. In vivo, nicotine was given to mice in their drinking water for 7 weeks. Results showed that alpha7 and beta2 subunits formed groups, and that chronic nicotine treatment increased the size of the clusters. As a conclusion, our data show that there are large intracellular pools of nAChR subunits, which are partially localized on endoplastic reticulum. Chronic nicotine treatment does not change endocytotic trafficking of nAChRs. Chronic nicotine treatment increased clustering of nAChRs, which could have a role in the release of dopamine (DA) evoked by nicotine.
Subject(s)
Corpus Striatum/metabolism , Neurons/metabolism , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Receptors, Nicotinic/drug effects , Animals , Cell Line , Corpus Striatum/drug effects , Endoplasmic Reticulum/metabolism , Humans , Image Processing, Computer-Assisted , In Vitro Techniques , Mice , Microscopy, Confocal , Microscopy, Electron, Transmission , Neurons/drug effects , Neurons/ultrastructure , Protein Transport/drug effects , Receptors, Nicotinic/metabolism , TransfectionABSTRACT
Amphoterin (HMGB1) is a 30-kD heparin-binding protein involved in process extension and migration of cells by a mechanism involving the receptor for advanced glycation end products (RAGE). High levels of amphoterin are released to serum during septic shock. We have studied the expression of amphoterin in monocytes and the role of amphoterin and RAGE in monocyte transendothelial migration. Un-activated monocytes in suspension did not reveal amphoterin on their surface, but adherent monocytes exported amphoterin to the cell surface. Immunohistochemical staining of arterial thrombi in vivo revealed amphoterin in mononuclear cells and in surrounding extracellular matrix. Amphoterin was secreted from phorbol ester and interferon-gamma (IFN-gamma)-activated macrophages, and the secretion was inhibited by blocking the adenosine 5'-triphosphate (ATP)-binding cassette transporter-1, a member of the multidrug resistance protein family. Amphoterin was specifically adhesive for monocytes in peripheral blood leukocyte adhesion assay. Adhesion caused an extensive spreading of cells, which was inhibited by the dominant-negative RAGE receptor (soluble ectodomain of RAGE), and adhesion up-regulated chromogranin expression in monocytes, also suggesting a RAGE-dependent interaction. Monocyte transendothelial migration was efficiently inhibited by anti-amphoterin and anti-RAGE antibodies and by the soluble RAGE. We suggest that amphoterin is an autocrine/paracrine regulator of monocyte invasion through the endothelium.
