M3 muscarinic cholinoceptors are linked to phosphoinositide metabolism in rat cerebellar granule cells.
Primary cultures of rat cerebellar granule cells are shown to possess a high density (283 +/- 48 fmol/mg of protein) of muscarinic receptor sites, defined using N-[3H]methylscopolamine [( 3H]NMS), with a KD of 0.18 +/- 0.01 nM measured after culture in vitro for 7 days. Displacement of specific [3H]NMS binding demonstrated a muscarinic receptor with low affinity for pirenzepine (Ki: 240 nM); further investigation using antagonists, AF-DX 116 and 4-DAMP to discriminate between M2 and M3 receptors respectively, revealed low M2 affinity (Ki: 600 nM) and high M3 affinity (Ki: 2.4 nM), indicative of the M3 receptor subtype. The robust muscarinic receptor stimulation of [3H]inositol phosphate formation, previously observed in these cells, was confirmed. Inhibition of this response followed a similar profile to the binding data, exhibiting weak inhibitory effects for pirenzepine (Ki: 710 nM) and AF-DX 116 (Ki: 5000 nM), but a potent action for 4-DAMP (Ki: 2.4 nM). The opposite profile seen for AF-DX 116 and 4-DAMP is indicative of a M3 receptor subtype expressed on these cells and linked to phosphoinositide hydrolysis. Further studies demonstrated that M3 receptor stimulation caused a rapid, transient increase in the second messenger inositol 1,4,5-trisphosphate, suggesting that potential Ca(2+)-homeostatic and neuromodulatory effects may be mediated by this response.
Whitham
E M
EM
Department of Pharmacology and Therapeutics, University of Leicester, U.K.
Challiss
R A
RA
Nahorski
S R
SR
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008297:Male; D010276:Parasympatholytics / Q000378:metabolism / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D010880:Piperidines / Q000494:pharmacology; D010890:Pirenzepine / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*
D007295:Inositol Phosphates; D010276:Parasympatholytics; D010716:Phosphatidylinositols; D010880:Piperidines; D011976:Receptors, Muscarinic; D010890:Pirenzepine; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; C048593:otenzepad