Interaction of organophosphorus compounds with muscarinic receptors in SH‐SY5Y human neuroblastoma cells

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Abstract
Human neuroblastoma cells (line SH‐SY5Y) were used to examine the interaction of single exposure to organophosphorus compounds (OPs) with muscarinic receptors. In this study, SH‐SY5Y cells were exposed for 30 min to concentrations of paraoxon, diisopropyl phos‐phorofluoridate (DFP), phenyl saligenin cyclic phosphate (PSP), and mipafox (N,N'‐diiso‐propyl phosphorodiamide fluoridate) that ranged between 10−9 M and 10−3 M (10−2 M for mipafox). Ability to interfere with muscarinic receptor binding was determined by change in the binding of the nonspecific antagonist [3H]‐N‐methylscopolamine (3H‐NMS). Concentrations of paraoxon >0.5 × 10−3 M and PSP 1 × 10−3 M significantly inhibited the binding of a saturating concentration of 3H‐NMS. Concentrations of >10−5 M paraoxon or PSP could significantly inhibit the binding of a half‐saturating concentration of 3H‐NMS. Studies using specific antagonists for muscarinic subtypes (pirenzepine for M1, AFDX‐116 for M2, and 4‐DAMP for M3) indicated that SH‐SY5Y cells have muscarinic receptors most sensitive to the specific antagonist for the M3 subtype (IC50 of 10−8 M for 4‐DAMP compared to 2.5 × 10−6 M and 2.7 × 10−5 M for pirenzepine and AFDX‐116, respectively). As M3 receptor stimulation results in formation of inositol phosphates from membrane phos‐phoinositides, the capability of OPs to alter levels of inositol phosphates and agonist‐stimulated increases in inositol phosphate formation was examined. Intact cells were prelabeled with [3H]myo‐inositol and then incubated for 15 min with the OPs before addition of 10−5 M to 10−3 M carbachol. Levels of inositol phosphates were determined as the amount of aqueous soluble radiolabeled product extracted from the reaction mixture. Paraoxon and PSP, but not mipafox or DFP, decreased basal levels of inositol phosphates in a concentration‐related manner. This could be overcome in cells stimulated with carbachol, a muscarinic agonist, and with sodium fluoride, which does not act at muscarinic receptors. These results indicate that certain OPs, upon acute exposure, interact with muscarinic receptors, but that they also have effects on levels of inositol phosphates that may be associated with another site of action in SH‐SY5Y cells.

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