Coupling of transfected muscarinic acetylcholine receptor subtypes to phospholipase D.
Muscarinic receptor-induced changes in the activities of phospholipase D (PLD) and of phosphoinositide-phospholipase C (PI-PLC) were investigated in human embryonic kidney (HEK) cells transfected with, and stably expressing, the human m1, m2, m3, and m4 mAChR subtypes, respectively. PLD and PI-PLC activities in these four transfected cell lines as well as in nontransfected cells were measured by the formation of [3H]phosphatidylethanol [( 3H]PEt) and [3H]inositol phosphates [( 3H]IP) after labeling cellular phospholipids with [3H]oleic acid and [3H]inositol. The muscarinic receptor agonist carbachol had no significant effects on [3H]PEt and [3H]IP formation in nontransfected HEK cells. In cells expressing the m1 or m3 receptors carbachol (1 mM; in the presence of 400 mM ethanol and 10 mM lithium chloride) caused the formation of [3H]PEt of about 12,000 cpm/mg protein (basal PEt formation was not measurable) and increased [3H]IP formation by 20,000-30,000 cpm/mg (a 7-10-fold increase over basal levels). The EC50 values (0.3-1.5 microM) were similar for both effects and both mAChR subtypes. In contrast, in cells expressing m2 or m4 receptor subtypes the magnitude of [3H]PEt (about 4,000 cpm/mg protein) or [3H]IP (3,000-4,000 cpm/mg) formation was much smaller and the EC50 values (20-40 microM) much higher than for the m1 and m3 receptors. Neomycin (1 mM) inhibited the m1 and m3 receptor-mediated production of IP by 50%, whereas the PEt formation was attenuated by 20% in the same cells. We conclude that activation of all of the four mAChR subtypes, although with different efficiencies, can stimulate PLD. The m1 and m3 receptor-mediated stimulation of the PLD may be at least partially independent of the PI-PLC stimulation.
Sandmann
J
J
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge 02139.
Peralta
E G
EG
Wurtman
R J
RJ
D000109:Acetylcholine / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D004789:Enzyme Activation; D020404:Glycerophospholipids; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D010712:Phosphatidic Acids / Q000096:biosynthesis; D010739:Phospholipase D / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D020404:Glycerophospholipids; D007295:Inositol Phosphates; D010712:Phosphatidic Acids; D011976:Receptors, Muscarinic; C051521:phosphatidylethanol; D002217:Carbachol; D010739:Phospholipase D; D000109:Acetylcholine