Characterization of phospholipase D activation by muscarinic receptors in human neuroblastoma SH-SY5Y cells.
The cholinergic regulation of phospholipase D activity was studied in SH-SY5Y human neuroblastoma cells with phosphatidylethanol formation as a specific marker for the enzyme activity. The muscarinic antagonists, hexahydrosiladifenidol and pirenzepine, inhibited carbachol-induced phosphatidylethanol formation in a concentration-dependent manner and the inhibitory constants indicated that muscarinic M1 receptors are responsible for the major part of the phospholipase D activation. The mechanism of receptor-mediated phospholipase D activation varies between different cell types and receptors. In SH-SY5Y cells, the carbachol-induced phospholipase D activity was inhibited by protein kinase C inhibitors. Since both phospholipases D and C are activated by muscarinic stimulation in SH-SY5Y cells, most of the phospholipase D activation is probably secondary to the protein kinase C activation that follows phospholipase C-mediated increase in diacylglycerols. Other kinases may be involved in the regulation since also a tyrosine kinase inhibitor decreased the phosphatidylethanol formation. Stimulation of G-protein(s) and increase in the intracellular Ca2+ concentration activated phospholipase D and may be additional mechanisms for the muscarinic regulation of phospholipase D in SH-SY5Y cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, increased the carbachol-induced formation of phosphatidic acid at the expense of 1,2-diacylglycerol. This indicates that phospholipase D contributes to the formation of 1,2-diacylglycerol after carbachol stimulation in SH-SY5Y cells.
Boyano-Adánez
M C
MC
Department of Medical Neurochemistry, Institute of Laboratory Medicine, Lund University Hospital, Sweden.
Lundqvist
C
C
Larsson
C
C
Gustavsson
L
L
D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000187:drug effects*
D011976:Receptors, Muscarinic; D002217:Carbachol; D010739:Phospholipase D