Stimulation of Muscarinic Acetylcholine Receptors Increases Synaptosomal Free Calcium Concentration by Protein Kinase‐Dependent Opening of L‐Type Calcium Channels

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Abstract
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 μM), with a maximum response of 22 × 2% at approximately 100 μM and an EC50 (calculated concentration producing 50% of the maximum response) of 30 μM The effect of carbachol (100 μM) on [Ca2+], was antagonised by atropine, but not by hexamethonium (10 μM). 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‐dihydropyridinesensitive calcium channels.