Cellular responses to stimulation of the M5 muscarinic acetylcholine receptor as seen in murine L cells.
The membrane signaling properties of the neuronal type-5 muscarinic acetylcholine receptor (M5 AChR) as expressed in murine L cells were studied. Recipient Ltk- cells responded to ATP acting through a P2-purinergic receptor by increasing phosphoinositide hydrolysis 2-fold but were unresponsive to 17 receptor agonists that are stimulatory in other cells. L cells expressing the M5 AChR responded to carbachol (CCh) with an approximately 20-fold increase in phospholipase C activity, mobilization of Ca2+ from endogenous stores, causing a transient peak increase in the intracellular concentration of Ca2+ ([Ca2+]i), influx of extracellular Ca2+, causing a sustained increase in [Ca2+]i dependent on extracellular Ca2+, and release of [3H]arachidonic acid from prelabeled cells, without altering resting or prostaglandin E1-elevated intracellular cAMP levels. None of the effects of the M5 AChR were inhibited by pertussis toxin. The regulation of L cell [Ca2+]i was studied further. ATP had the same effects as CCh and the two agonists acted on a shared intracellular pool of Ca2+. The peak and sustained [Ca2+]i increases were reduced by cholera toxin and forskolin, neither of which altered significantly phosphoinositide hydrolysis. This is consistent with interference with the action of inositol 1,4,5-trisphosphate (IP3) through cAMP-mediated phosphorylation and suggests a continued involvement of IP3 during the sustained phase of [Ca+]i increases. The temporal pattern of the sustained [Ca2+]i increase differed whether elicited by CCh or ATP, and was enhanced in pertussis toxin-treated cells. This is consistent with existence of a kinetic control of the sustained [Ca2+]i change by a receptor-G protein-dependent mechanism independent of the IP3 effector site(s) (e.g. pulsatile activation of phospholipase C and/or pulsatile activation of a receptor/G protein-operated plasma membrane Ca2+ channel). Thus, the non-excitable L cell may be a good model for studying [Ca2+]i regulations, as may occur in other nonexcitable cells of which established cell lines do not exist, and for studying of receptors that as yet cannot be studied in their natural environment.
Liao
C F
CF
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Schilling
W P
WP
Birnbaumer
M
M
Birnbaumer
L
L
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000527:Alprostadil / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002772:Cholera Toxin / Q000494:pharmacology; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007739:L Cells; D051379:Mice; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D011976:Receptors, Muscarinic / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D001095:Arachidonic Acids; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D016718:Arachidonic Acid; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D002217:Carbachol; D002772:Cholera Toxin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D000527:Alprostadil; D002118:Calcium