Journal of Neuroscience, Vol 6, 3739-3748, Copyright © 1986 by Society for Neuroscience
Biochemical and physical analyses of newly synthesized muscarinic acetylcholine receptors in cultured embryonic chicken cardiac cells
DD Hunter and NM Nathanson
Exposure of cultured embryonic chicken cardiac cells to the muscarinic
agonist carbachol results in a 70-80% decrease in the number of muscarinic
acetylcholine receptors (mAChR) expressed on the surface of the cells.
Removal of the agonist results in a gradual increase in mAChR number
because of the accumulation of newly synthesized receptors, reaching the
control level in 14 hr. Measurements of increases in K+ permeability
elicited by carbachol show that even after the complete recovery of
receptor number, the sensitivity to agonist is reduced. The EC50 for
carbachol is 13-fold higher in cells that have been exposed to carbachol
and allowed to recover for 18 hr than in control cells, but is not
significantly different from the EC50 for control cells 24 hr after agonist
removal. The sensitivity of the mAChR- mediated inhibition of adenylate
cyclase is also decreased at 18 hr, and recovers by 24 hr. These increases
in sensitivity of mAChR-mediated responses are not blocked by
administration of cycloheximide, and thus do not require de novo protein
synthesis. The number of surface mAChR available for ligand binding can be
reduced by 85-100% by treatment with the affinity-alkylating antagonist
propylbenzilylcholine mustard. Newly synthesized mAChR that appear
following affinity alkylation are also poorly coupled to mAChR-mediated
increases in K+ permeability, indicating that decreased physiological
sensitivity is not due to a nonspecific effect of long-term agonist
exposure on general cellular function, but reflects, rather, an intrinsic
property of newly synthesized mAChR. The decrease in sensitivity of the
mAChR-mediated responses is due neither to a lack of expression of mAChR on
the surface nor to reduced agonist affinity of the mAChR. Cells exhibiting
decreased responsiveness contain GTP-binding proteins, which function
normally in the inhibition of adenylate cyclase and appear to be identical
to pertussis toxin substrates from control cells using gel electrophoresis;
therefore, the decreased sensitivity does not appear to be the result of an
alteration in coupling proteins. These cells also contain mAChR that do not
differ from those in control cells either by molecular weight or
isoelectric point. Thus, the diminished sensitivity observed in cells
containing newly synthesized receptors is either caused by a small change
in mAChR not detected by these electrophoretic techniques or by a change in
an as-yet-undefined component of mAChR transduction system in the heart.
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