Boess F G, Balasubramanian M K, Brammer M J, Campbell I C
Department of Neuroscience, Institute of Psychiatry, DeCrespigny Park, London, England.
J Neurochem. 1990 Jul;55(1):230-6. doi: 10.1111/j.1471-4159.1990.tb08843.x.
In synaptosomes prepared from rat cerebral cortex, free cytosolic calcium concentration ([Ca2+]i) was measured using the fluorescent dye fura-2. Incubation of fura-2-loaded synaptosomes with carbachol increased [Ca2+]i in a dose-dependent manner (1-1,000 microM), with a maximum response of 22 +/- 2% at approximately 100 microM and an EC50 (calculated concentration producing 50% of the maximum response) of 30 microM. The effect of carbachol (100 microM) on [Ca2+]i was antagonised by atropine, but not by hexamethonium (10 microM). The calculated concentration of atropine needed for 50% inhibition (IC50) was 260 nM. The rise in [Ca2+]i produced by carbachol was reduced in the absence of extrasynaptosomal Ca2+ and effectively blocked by the L-type calcium channel blocker nifedipine (with an IC50 of 29 nM). The response to carbachol was reduced if the synaptosomes were preincubated with the protein kinase inhibitors H7 [1-(5-isoquinolinylsulfonyl)-2- methylpiperazine] (from 17% in the solvent control to 4%) and staurosporine (from 20% in the solvent control to 3%). These results show that stimulation of muscarinic acetylcholine receptors in synaptosomes increases [Ca2+]i by protein kinase-dependent activation of 1,4-dihydropyridine-sensitive calcium channels.
在从大鼠大脑皮层制备的突触体中，使用荧光染料fura - 2测量游离胞质钙浓度（[Ca2 +]i）。用卡巴胆碱孵育负载fura - 2的突触体，[Ca2 +]i以剂量依赖性方式增加（1 - 1000 microM），在约100 microM时最大反应为22±2%，半数有效浓度（产生最大反应50%的计算浓度）为30 microM。卡巴胆碱（100 microM）对[Ca2 +]i的作用被阿托品拮抗，但不被六甲铵（10 microM）拮抗。50%抑制所需的阿托品计算浓度（IC50）为260 nM。在没有突触体外Ca2 +的情况下，卡巴胆碱引起的[Ca2 +]i升高降低，并被L型钙通道阻滞剂硝苯地平有效阻断（IC50为29 nM）。如果突触体用蛋白激酶抑制剂H7 [1 - （5 - 异喹啉磺酰基） - 2 - 甲基哌嗪]（从溶剂对照中的17%降至4%）和星形孢菌素（从溶剂对照中的20%降至3%）预孵育，对卡巴胆碱的反应会降低。这些结果表明，突触体中毒蕈碱型乙酰胆碱受体的刺激通过蛋白激酶依赖性激活1,4 - 二氢吡啶敏感的钙通道增加[Ca2 +]i。
