Amphoterin (HMGB1) is a 30-kD heparinbinding 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. Unactivated 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-␥ (IFN-␥)-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. IntroductionCirculating monocytes adhere to sites of vascular injury where they participate together with other cells in the regulation of blood clotting, inflammation, and wound healing. Adhesion to other cells and extracellular matrix components is a prerequisite for migration and tissue recruitment of monocytes. 1,2 The knowledge of molecules involved in monocyte transendothelial migration is rapidly increasing. However, the overall picture of the transendothelial migration mechanism is not completely understood. 2 Amphoterin is a 30-kD heparin-binding protein widely expressed in humans and other organisms, and it is abundantly expressed in the developing brain as well as in various immature and transformed cell lines. [3][4][5][6] It was isolated as an extracellular neurite outgrowth-promoting protein, but its amino acid sequence turned out to be identical to high-mobility groupϪ1 protein. 5,7 In a new nomenclature of high-mobility group proteins amphoterin and other proteins identical in the cDNA sequence are called as HMGB1 (high-mobility group B-1). 8 We have used the designation amphoterin for the protein occurring in the extracellular space and interacting with the cell surface. 5 Surface-bound amphoterin is adhesive for neural cells and platelets, and it induces extension of membrane processes in adherent cells. 3,9,10 Amphoterin binds to plasma membrane lipids, mainly to phosphatidylserine and sulfatide, and enhances and localizes plasminogen activation. 6,9,[11][12][13] In neurons, neurite outgrowth on amphoterin s...

1 SH-SY5Y cells express a7 and a3* subtypes of nicotinic acetylcholine receptors (AChR). Numbers of these receptors are upregulated by chronic treatment with nicotinic agonists or KCl. In this study we have examined the functional consequences of these drug treatments on nicotine-or KCl-evoked increases in [Ca 2+ ] i , in SH-SY5Y cells. 2 In untreated cells, nicotine increased [Ca 2+ ] i (EC 50 7.5 mM). Responses to 10 mM nicotine were abolished by the non-selective nicotinic antagonist mecamylamine and were partially blocked by a7-selective antagonists, the a3b2*-selective antagonist a-conotoxin-MII, and by cadmium and verapamil. 3 After treatment for 4 days with nicotinic agonists, nicotine-evoked increases in [Ca 2+ ] i were signi®cantly decreased by about 25%. Nicotine-evoked responses were paradoxically increased in the presence of acute methyllycaconitine (MLA; an a7-selective antagonist) although other a7-selective antagonists were without e ect, while a-conotoxin-MII gave a partial inhibition. The increase observed with MLA was abolished by mecamylamine but not by a-conotoxin-MII and was still observed 24 h after chronic nicotine treatment. 4 After treatment for 4 days with KCl, nicotine-evoked increases in [Ca 2+ ] i were also decreased by 25%, but acute MLA was without e ect. Responses to 20 mM KCl were unchanged by prior treatment with nicotine or KCl. Treatment for 4 days with 5 mM verapamil reduced responses to both nicotine and KCl by about 50%. 5 Multiple nicotinic AChR subtypes contribute to nicotine-evoked increases in [Ca 2+ ] i in SH-SY5Y cells. Responses to acute nicotine are reduced after chronic nicotine or KCl treatment, with loss of the component attributed to the a7 subtype. However, in nicotine-treated cells this e ect is reversed when nicotine stimulation is applied in the presence of acute MLA. The antagonist may assist in converting a non-functional a7 nicotinic AChR to a conducting state.

Nicotine releases dopamine in the brain by activating neuronal nicotinic acetylcholine receptors (nAChRs). Chronic nicotine treatment increases the number of nAChRs, which represents plasticity of the brain. Together these phenomena have been suggested to have a role in the development of nicotine addiction. In the brain nAChRs can be localized synaptically, extrasynaptically or intracellularly. The purpose of these studies was to clarify the effects of chronic nicotine treatment on the localization of beta2 and alpha7 nAChR subunits in brain areas involved in nicotine addiction. Nicotine was administered orally in drinking water to male NMRI mice for 7 weeks. At the end of chronic nicotine treatment the localization of the nAChR subunits was studied in the dorsal striatum and in the ventral tegmental area (VTA) by using electron microscopy. In the brain areas studied beta2 and alpha7 subunits were localized presynaptically and postsynaptically in axon endings and in dendrites. In both areas the majority of the beta2 and alpha7 subunits were localized at extrasynaptic sites. In response to chronic nicotine treatment the beta2 and alpha7 nAChR subunit labelling was increased at synaptic and extrasynaptic sites as well as intracellularly. This suggests that the trafficking of nAChR subunits is increased as a result of chronic nicotine treatment and nAChRs in all parts of neurons could have functional roles in the formation of nicotine addiction.

No abstract

(-)-Methadone acts as an agonist at opioid receptors. Both (+)-and (-)-enantiomers of methadone have been suggested to be potent non-competitive antagonists of a3b4 neuronal nicotinic acetylcholine receptors (nAChRs). In the present study, we have examined interactions of methadone with nAChRs by using receptor binding assays, patch-clamp recording and calcium fluorometry imaging with SH-SY5Y cells naturally expressing a7 and a3* nAChR subtypes and SH-EP1-ha7 cells heterologously expressing human a7 nAChRs. Methadone potently inhibited binding of [ 3 H]methyllycaconitine to a7 nAChRs and that of [ 3 H]epibatidine to a3* nAChRs. Methadone pretreatment induced up-regulation of epibatidine binding sites in SH-SY5Y cells. Using whole-cell patch-clamp recording, both isomers of methadone activated cation currents via mecamylamine-sensitive nAChRs in SH-SY5Y cells. Nicotine and both (+)-and (-)-methadone evoked increases in [Ca 2+ ] i in both fluo-3AM loaded cell lines, and these effects were blocked by mecamylamine and by the a7 selective antagonist methyllycaconitine, suggesting effects of methadone as a7-nAChR agonist. Sensitivity of sustained nicotine and methadone effects to blockade by CdCl 2 , ryanodine and xestospongin-c implicates voltage-operated Ca 2+ channels and intracellular Ca 2+ stores as downstream modulators of elevated [Ca 2+ ] i . Collectively, our results suggest that methadone engages in complex and potentially pharmacologically significant interactions with nAChRs.