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

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 .mu.M), with a maximum response of 22 .+-. 2% at approximately 100 .mu.M and an EC50 (calculated concentration producing 50% of the maximum response) of 30 .mu.M. The effect of carbachol (100 .mu.M) on [Ca2+]i was antagonised by atropine, but not by hexamethonium (10 .mu.M). 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 (withan 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.