title: Active and peripheral anionic sites of acetylcholinesterase have differential modulation effects on cell proliferation, adhesion and neuritogenesis in the NG108-15 cell line.
abstract: The classical enzymatic role of acetylcholinesterase (AChE) is to terminate impulse transmission at cholinergic synapses through rapid hydrolysis of acetylcholine (ACh). Inactivation of this enzyme's catalytic site is the primary mechanism of acute toxicity of OP insecticides (e.g. parathion, chlorpyrifos). There is now sufficient evidence to suggest that AChE has a neurotrophic function that may be altered by organophosphate (OP) exposure, resulting in defects of neuronal growth and development, though the clarification of the mechanisms involved require further in vitro investigation. In the present study, the mouse neuroblastoma×rat glioma hybrid NG108-15 cell line was used to investigate the differential effects between inhibition of the catalytic site and peripheral anionic site (PAS) of acetylcholinesterase (AChE) on cell adhesion, proliferation and neuritogenesis, in the presence and absence of human red blood cell (hRBC) AChE (ED3.1.1.7). AChE active-site inhibitor paraoxon (PO; 0.1-1.0μM), when added to NG108-15 cells grown on AChE-coated plates, had no effect on cell proliferation, but exerted a significant reduction in strongly adherent viable cells accompanied by mostly short process formations, with 18% of cells considered to be neuritogenic, similar to that observed on uncoated plates. In contrast, PO had no significant effect on cell adhesion and proliferation of NG108-15 cells on uncoated plates. The PAS-ligand thioflavin-T (Th-T; 0.5-25μM), however, decreased cell adhesion and proliferation, on both uncoated and ACh-E coated plates, with less magnitude on AChE-coated plates. Taken together, these results suggest that strong cell adherence and neuritogenesis are sensitive to PO in this cell culture model, with no impact on proliferation, in the presence of membrane bound AChE-coating, while there is no sensitivity to PO on uncoated plates. On the other hand, binding of Th-T directly to the PAS affects both cell adherence and proliferation, with less magnitude in the presence of membrane-bound AChE. The current study indicates that PO is deleterious in neural development during critical periods of strong cell adhesion and differentiation, interfering with AChE trophic function.
authors.affiliation: Rutgers, New Jersey Medical School-Graduate School of Biomedical Sciences, 185 South Orange Avenue, MSB H609, Newark, NJ 07103, United States. Electronic address: hcampanha@ymail.com.
authors.forename: Helen M
authors.initials: HM
authors.lastname: Campanha
authors.affiliation: REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal.
authors.forename: Félix
authors.initials: F
authors.lastname: Carvalho
authors.affiliation: U.S. Environmental Protection Agency, National Center for Environmental Assessment, Washington, DC, United States.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Schlosser
mesh_terms: D000110:Acetylcholinesterase / Q000502:physiology*; D000818:Animals; D052160:Benzothiazoles; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects*; D006801:Humans; D051379:Mice; D016501:Neurites / Q000187:drug effects* / Q000502:physiology; D010261:Paraoxon / Q000633:toxicity*; D051381:Rats; D013844:Thiazoles / Q000633:toxicity*
chemical_list: D052160:Benzothiazoles; D013844:Thiazoles; C009462:thioflavin T; D000110:Acetylcholinesterase; D010261:Paraoxon
keywords: Acetylcholinesterase; Acylation site; Cell adhesion; Neuritogenesis; Peripheral anionic site; Proliferation