A transfected m5 muscarinic acetylcholine receptor stimulates phospholipase A2 by inducing both calcium influx and activation of protein kinase C.
Receptor-mediated arachidonic acid release and its relationship to phospholipase A2 and phospholipase C activation were investigated in Chinese hamster ovary cells transfected with and expressing the m5 muscarinic receptor. Carbachol, a muscarinic receptor agonist, stimulated the release of arachidonic acid and inositol phosphates with similar potencies. In addition, carbachol and the phorbol ester, phorbol-12-myristate, 13-acetate (PMA), stimulated protein kinase C (PKC) activity. PMA potentiated the carbachol-stimulated release of arachidonic acid, but had no effect on release of inositol phosphates. Long-term preincubation with PMA or carbachol inhibited PKC activity and prevented carbachol-stimulated release of arachidonic acid, but not inositol phosphates, suggesting that release of arachidonic acid, but not release of inositol phosphates, required activation of PKC. Carbachol stimulated the release of [3H]lysophosphatidylcholine from [3H]choline prelabeled cells, suggesting that phospholipase A2 was involved in the release of arachidonic acid. The role of calcium in carbachol-stimulated release of arachidonic acid was also investigated. Carbachol stimulated a transient followed by a sustained increase in intracellular calcium. In the absence of extracellular calcium, the transient rise in intracellular calcium was maintained but the sustained increase in intracellular calcium and the release of arachidonic acid were abolished. Carbachol stimulated a sustained influx of 45Ca++. We conclude that the combined effect of PKC activation and sustained elevation of intracellular calcium, from an extracellular source, is essential for m5 muscarinic receptor activation of phospholipase A2.
Felder
C C
CC
Section on Pharmacology, National Institute of Mental Health, Bethesda, Maryland.
Dieter
P
P
Kinsella
J
J
Tamura
K
K
Kanterman
R Y
RY
Axelrod
J
J
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D004789:Enzyme Activation; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011493:Protein Kinase C / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000235:genetics / Q000502:physiology*; D013997:Time Factors; D014162:Transfection
D001095:Arachidonic Acids; D007295:Inositol Phosphates; D011976:Receptors, Muscarinic; D016718:Arachidonic Acid; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium