Stimulation of muscarinic acetylcholine receptors increases synaptosomal free calcium concentration by protein kinase-dependent opening of L-type calcium channels.
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.
Boess
F G
FG
Department of Neuroscience, Institute of Psychiatry, DeCrespigny Park, London, England.
Balasubramanian
M K
MK
Brammer
M J
MJ
Campbell
I C
IC
D000818:Animals; D001572:Benzofurans; D001696:Biomechanical Phenomena; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D008297:Male; D009994:Osmolar Concentration; D011494:Protein Kinases / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000502:physiology*; D013574:Synaptosomes / Q000378:metabolism*
D001572:Benzofurans; D015220:Calcium Channels; D005456:Fluorescent Dyes; D011976:Receptors, Muscarinic; D002217:Carbachol; D011494:Protein Kinases; D002118:Calcium; D016257:Fura-2