Yan G M, Lin S Z, Irwin R P, Paul S M
Section on Molecular Pharmacology, National Institute of Mental Health, Bethesda, Maryland 20892.
Mol Pharmacol. 1995 Feb;47(2):248-57.
We have recently reported that the majority of cultured rat cerebellar granule neurons undergo apoptosis when maintained in the presence of physiological concentrations of K+ (nondepolarizing conditions). We now report that exposure of cultured cerebellar granule neurons, maintained under nondepolarizing conditions, to the muscarinic cholinergic receptor (mAchR) agonists carbachol and muscarine results in a concentration- and time-dependent inhibition of apoptosis. The nicotinic cholinergic receptor agonist (-)-nicotine fails to mimic, and the nicotinic cholinergic receptor antagonist dihydro-beta-erythroidine fails to antagonize, the survival-promoting effects of carbachol. In contrast, relatively low concentrations of atropine completely prevent the effects of carbachol in blocking apoptotic death of cultured granule neurons. Although the m1- and m2-preferring mAchR antagonists pirenzepine and gallamine, respectively, fail to reverse the effects of carbachol, the m3-preferring antagonist 4-diphenylacetoxyl-N- methylpiperidine methiodide completely blocks the survival-promoting effects of carbachol. These data demonstrate that activation of the mAchR (possibly of the m3 subtype) blocks apoptosis of cultured cerebellar granule neurons. The antiapoptotic effects of mAchR agonists are not indirectly mediated via glutamate release from granule neurons, because antagonists of either N-methyl-D-aspartate or non-N-methyl-D-aspartate glutamate receptors fail to affect the antiapoptotic effects of carbachol or muscarine. Moreover, exposure of cultured cerebellar granule neurons to antiapoptotic concentrations of carbachol, in contrast to high concentrations of K+ or glutamate receptor agonists, results in only a small and transient elevation of the intracellular Ca2+ concentration, as measured by fura-2 microfluorimetry. Slow neurotransmitters such as acetylcholine, acting via their cognate G protein-coupled receptors, may prevent neuronal apoptosis in the developing (and perhaps adult) central nervous system.
我们最近报道，当培养的大鼠小脑颗粒神经元在生理浓度的K⁺存在下（非去极化条件）维持培养时，大多数神经元会发生凋亡。我们现在报告，在非去极化条件下培养的小脑颗粒神经元暴露于毒蕈碱型胆碱能受体（mAchR）激动剂卡巴胆碱和毒蕈碱，会导致凋亡受到浓度和时间依赖性的抑制。烟碱型胆碱能受体激动剂（-）-尼古丁不能模拟卡巴胆碱的促存活作用，烟碱型胆碱能受体拮抗剂二氢-β-刺桐碱也不能拮抗卡巴胆碱的促存活作用。相反，相对低浓度的阿托品能完全阻止卡巴胆碱对培养的颗粒神经元凋亡死亡的阻断作用。虽然分别偏好m1和m2的mAchR拮抗剂哌仑西平和加拉明不能逆转卡巴胆碱的作用，但偏好m3的拮抗剂4-二苯基乙酰氧基-N-甲基哌啶甲碘化物能完全阻断卡巴胆碱的促存活作用。这些数据表明，mAchR（可能是m3亚型）的激活可阻断培养的小脑颗粒神经元的凋亡。mAchR激动剂的抗凋亡作用不是通过颗粒神经元释放谷氨酸间接介导的，因为N-甲基-D-天冬氨酸或非N-甲基-D-天冬氨酸谷氨酸受体的拮抗剂都不能影响卡巴胆碱或毒蕈碱的抗凋亡作用。此外，与高浓度的K⁺或谷氨酸受体激动剂相比，培养的小脑颗粒神经元暴露于抗凋亡浓度的卡巴胆碱时，用fura-2显微荧光测定法测量，细胞内Ca²⁺浓度仅出现小幅短暂升高。像乙酰胆碱这样的慢递质，通过其同源的G蛋白偶联受体起作用，可能在发育中的（也许还有成年的）中枢神经系统中预防神经元凋亡。
