Bupropion Binds in the Middle (M2-9) of the Torpedo Nicotinic Acetylcholine Receptor Ion Channel

Abstract

Cigarette smoking is a significant risk factor for atherosclerosis, which involves the invasion of vascular smooth muscle cells (VSMCs) from the media to intima. Many invasive cells remodel the actin cytoskeleton to form podosomes, which regulate metalloproteinase (MMP) release for extracellular matrix (ECM) degradation. We tested the hypothesis that cigarette smoke extract (CSE) modulates the structure and function of podosomes in VSMCs. We found that, in response to PKC activation by phorbol dibutyrate (PDBu), untreated A7r5 VSMCs formed podosomes, whereas CSE, nicotine, and carbachol-treated cells formed actin-rich rings. The nicotinic acetylcholine receptor (nAChR) antagonist a-bungarotoxin abolished the effects of nicotine and carbachol on actin-rich ring formation. Immunofluorescence labeling experiments localized MMP-2 and nAChRs at the actin-rich rings. Nicotineinduced actin-rich ring formation required 6-12 hr exposure, and was sensitive to the protein synthesis inhibitor cycloheximide. Conditioned media collected from cell culture of nicotine-treated cells induced podosome remodeling in untreated cells. When cells were cultured on DQ-gelatin, untreated cells exhibited a fibrillar network of fluorescence from DQ-gelatin degradation, which, upon PDBu stimulation, reorganized into peripheral dots and migrated towards the perinuclear region. In contrast, nicotine-treated cells, upon PDBu stimulation, reorganized the fluorescence into perinuclear fibrils, which dispersed into small dots and disappeared rapidly over time. Results from this study suggest that nicotine, by activating nAChRs and inducing phenotypic modulation, may prime VSMCs to become hyperresponsive to PKC activation with an enhanced ability to remodel the actin cytoskeleton and release MMP for invasion of the ECM. This study was supported by NIH grant HL-52714. 5 Duquesne University, Pittsburgh, PA, USA. Bupropion is clinically prescribed for the treatment of depression (Wellbutrin) and for smoking cessation (Zyban). While there is consensus that the primary mechanism of action involves increased synaptic concentrations of dopamine and norepinephrine via inhibition of the respective reuptake transporters, DAT and NET, there is growing evidence that some of the therapeutic benefits of bupropion may result from non-competitive antagonism of neuronal nicotinic acetylcholine receptors (nAChRs), in particular a4b2 and a3b4 subtypes. To aid in elucidating the mechanism of bupropion action and the development of new therapeutic agents for smoking cessation and depression, our goal is to determine the specific molecular interactions between bupropion and several nAChR subtypes (Torpedo, a4b2, a3b4 ]-SADU-3-72 labeling within dM2 (dLeu265/dM2-9) and bM2 (bLeu257/ bM2-9). In the desensitized state, TCP-inhibitable (specific) labeling was identified within dM2 (dLeu265, dM2-9 with minor labeling of dSer258, dM2-2). Our results establish that bupropion binds in the middle (M2-9) of the Torpedo nAChR channel, with a slightly broader binding locus in the ...