Cell-polymer interactions of fluorescent polystyrene latex particles coated with thermosensitive poly(N-isopropylacrylamide) and poly(N-vinylcaprolactam) or grafted with poly(ethylene oxide)-macromonomer.

Cell-polymer interactions of thermosensitive poly(N-isopropylacrylamide) (PNIPAM) or poly(N-vinylcaprolactam) (PVCL) coated particles with RAW264.7 macrophages and intestinal Caco-2 cells were evaluated. Nanosized particles were prepared by modifying the surface of fluorescent polystyrene (FPS) particles with the thermosensitive polymer gels or with poly(ethylene oxide) (PEO)-macromonomer grafts. The particles were characterized by IR-spectroscopy for functional groups, light scattering for size distribution and zeta-potential for surface charge. Effects of temperature and polymer coating/grafting on the cellular interactions were evaluated by cell association/uptake and visualized by confocal scanning microscope. PEO and PNIPAM inhibited the polymer-cell contact by steric repulsion, evidenced by weak attachment of the particles. PVCL-coated FPS was adsorbed on the cells more strongly, especially at 37 degrees C, because of more hydrophobic nature at higher temperatures. The results suggest feasibility of the PNIPAM and PVCL for biotechnological/pharmaceutical applications, as the cell-particle interactions may be modified by size, surface charge, hydrophobicity, steric repulsion and temperature.

Effect of chronic nicotine treatment on localization of neuronal nicotinic acetylcholine receptors at cellular level.

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.

Methadone increases intracellular calcium in SH-SY5Y and SH-EP1-halpha7 cells by activating neuronal nicotinic acetylcholine receptors.

(-)-Methadone acts as an agonist at opioid receptors. Both (+)- and (-)-enantiomers of methadone have been suggested to be potent non-competitive antagonists of alpha3beta4 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 alpha7 and alpha3* nAChR subtypes and SH-EP1-halpha7 cells heterologously expressing human alpha7 nAChRs. Methadone potently inhibited binding of [3H]methyllycaconitine to alpha7 nAChRs and that of [3H]epibatidine to alpha3* 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 [Ca2+]i in both fluo-3AM loaded cell lines, and these effects were blocked by mecamylamine and by the alpha7 selective antagonist methyllycaconitine, suggesting effects of methadone as alpha7-nAChR agonist. Sensitivity of sustained nicotine and methadone effects to blockade by CdCl2, ryanodine and xestospongin-c implicates voltage-operated Ca2+ channels and intracellular Ca2+ stores as downstream modulators of elevated [Ca2+]i. Collectively, our results suggest that methadone engages in complex and potentially pharmacologically significant interactions with nAChRs.

Up-regulation of beta2 and alpha7 subunit containing nicotinic acetylcholine receptors in mouse striatum at cellular level.

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.