Essential fatty acid deficiency in cultured SK-N-SH human neuroblastoma cells.
SK-N-SH neuroblastoma cells grown under standard culture conditions contain significant amounts of Mead acid (20:3 omega 9) in phospholipids, indicating essential fatty acid (EFA) deficiency. The amount of esterified 20:3 omega 9 was augmented by growth in a chemically defined EFA-free medium, whereas its presence could be virtually eliminated by supplementation of the culture medium with either arachidonic (20:4 omega 6; AA), eicosapentaenoic (20:5 omega 3; EPA), or linolenic (18:3 omega 3) acids. Substitution of Mead acid for omega 6 fatty acids, particularly evident in phosphatidylinositol (PI), indicates a compensatory replacement of omega 9 for omega 6 fatty acids during EFA deficiency. Studies evaluating [3H]scopolamine binding to the M3 muscarinic acetylcholine receptors (mAChRs) present in these neurotumor cells as well as effects of carbachol on phosphoinositide turnover and intracellular Ca2+ mobilization, indicate that the biosubstitution of 20:4 omega 6 with 20:3 omega 9 does not detectably impair these measures of signal transduction. Stimulation of mAChRs with carbachol increased the cellular mass of diacylglycerol (DAG) approximately 60%. On the basis of distinctive fatty acid "signatures" of each of the phospholipid classes, it is concluded that the DAG initially released following muscarinic stimulation is derived from phosphoinositide breakdown. After several minutes, however, a significant amount of DAG comes from phosphatidylcholine (PC) as well. In contrast to DAG, the composition of phosphatidate (PA) following receptor stimulation closely resembles that of the phosphoinositides, even at the later time points examined. These results support a selective phosphorylation of DAG arising from the stimulated breakdown of phosphoinositides, favoring the conservation of the 1-stearoyl, 2-arachidonoyl (or 20:3 omega 9) moiety.
Stubbs
E B
EB
Department of Psychiatry, University of Michigan, Ann Arbor 48104-1687.
Carlson
R O
RO
Lee
C
C
Fisher
S K
SK
Hajra
A K
AK
Agranoff
B W
BW
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000032:analysis; D016718:Arachidonic Acid / Q000032:analysis; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D005228:Fatty Acids, Essential / Q000172:deficiency*; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism; D011982:Receptors, Prostaglandin / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D004075:Diglycerides; D005228:Fatty Acids, Essential; D010743:Phospholipids; D011950:Receptors, Cholinergic; D011982:Receptors, Prostaglandin; D016718:Arachidonic Acid; D002217:Carbachol; D015126:8,11,14-Eicosatrienoic Acid; C010944:mead acid