Ehrich M, Intropido L, Costa L G
Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg 24061-0442.
J Toxicol Environ Health. 1994 Sep;43(1):51-63. doi: 10.1080/15287399409531903.
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 phosphorofluoridate (DFP), phenyl saligenin cyclic phosphate (PSP), and mipafox (N,N'-diisopropyl 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 x 10(-3) M and PSP 1 x 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 x 10(-6) M and 2.7 x 10(-5) M for pirenzepine and AFDX-116, respectively). As M3 receptor stimulation results in formation of inositol phosphates from membrane phosphoinositides 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.
人类神经母细胞瘤细胞（SH-SY5Y细胞系）被用于研究单次暴露于有机磷化合物（OPs）与毒蕈碱受体之间的相互作用。在本研究中，将SH-SY5Y细胞暴露于浓度范围为10⁻⁹ M至10⁻³ M（米帕明为10⁻² M）的对氧磷、二异丙基氟磷酸酯（DFP）、苯基水杨苷环磷酸酯（PSP）和米帕明（N,N'-二异丙基磷酰胺氟化物）30分钟。通过非特异性拮抗剂[³H]-N-甲基东莨菪碱（³H-NMS）结合的变化来确定干扰毒蕈碱受体结合的能力。对氧磷浓度>0.5×10⁻³ M和PSP 1×10⁻³ M显著抑制饱和浓度的³H-NMS的结合。对氧磷或PSP浓度>10⁻⁵ M可显著抑制半饱和浓度的³H-NMS的结合。使用毒蕈碱亚型特异性拮抗剂（哌仑西平用于M1，AFDX-116用于M2，4-DAMP用于M3）的研究表明，SH-SY5Y细胞具有对M3亚型特异性拮抗剂最敏感的毒蕈碱受体（4-DAMP的IC50为10⁻⁸ M，而哌仑西平和AFDX-116分别为2.5×10⁻⁶ M和2.7×10⁻⁵ M）。由于M3受体刺激导致膜磷脂酰肌醇形成肌醇磷酸，因此研究了OPs改变肌醇磷酸水平以及激动剂刺激的肌醇磷酸形成增加的能力。完整细胞先用[³H]肌醇预标记，然后在加入10⁻⁵ M至10⁻³ M卡巴胆碱之前与OPs孵育15分钟。肌醇磷酸水平通过从反应混合物中提取的水溶性放射性标记产物的量来确定。对氧磷和PSP，但不是米帕明或DFP，以浓度相关的方式降低肌醇磷酸的基础水平。在用毒蕈碱激动剂卡巴胆碱和不作用于毒蕈碱受体的氟化钠刺激的细胞中，这种情况可以被克服。这些结果表明，某些OPs在急性暴露时与毒蕈碱受体相互作用，但它们也对肌醇磷酸水平有影响，这可能与SH-SY5Y细胞中的另一个作用位点有关。
