Phospholipid base exchange activity in rat liver plasma membranes. Evidence for regulation by G-protein and P2y-purinergic receptor.
Phospholipid base exchange activity using choline as substrate was detected in plasma membranes (PM) and other subcellular fractions of rat liver, with microsomes (MS) showing the highest specific activity. In contrast, phospholipase D activity was only detected in PM. In PM, choline exchanged for phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), whereas ethanolamine exchanged for PE and PS, and serine exchanged for PS. Ca2+ (10 microM or higher) stimulated choline incorporation into PC in MS and PM, whereas Mg2+ (10 microM or higher) stimulated it only in PM. Ethanolamine and serine incorporation into PM phospholipids was also stimulated by Ca2+, and inositol incorporation by Mn2+. Phospholipase D activity was substantial in the presence of EGTA and was slightly stimulated by Ca2+ concentrations less than 500 microM. It was undetectable without Mg2+. Low concentrations of oleate (1 mM or less) stimulated phospholipase D activity. These concentrations inhibited choline base exchange activity, whereas higher concentrations (3-8 mM) were stimulatory. Comparison of the subcellular distribution and Ca2+, Mg2+, and oleate effects on choline base exchange and phospholipase D activities supports the view that they are catalyzed by different enzymes. The incorporation of choline, but not ethanolamine or serine, into the phospholipids of PM, but not MS, was stimulated by micromolar concentrations of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) and other slowly hydrolyzable analogues of GTP. GDP, GMP, and other nucleoside triphosphates and their analogues were ineffective. GTP gamma S stimulation of base exchange activity was dependent upon Mg2+ and was inhibited by high concentrations of guanosine 5'-O-2-(thio)diphosphate. In the presence of low concentrations of GTP gamma S, ATP and its slowly hydrolyzable analogues stimulated base exchange activity. Dose-response curves for these nucleotides revealed a potency order consistent with mediation by purinergic receptors of the P2Y type. Base exchange activity stimulated by ATP plus GTP gamma S or GTP gamma S alone was not altered by treatment with pertussis or cholera toxins. These results suggest that the choline base exchange activity of liver PM is regulated by a pertussis toxin-insensitive G-protein linked to P2Y purinergic receptors.
Siddiqui
R A
RA
Howard Hughes Medical Institute Laboratory, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295.
Exton
J H
JH
D015720:5'-Nucleotidase / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002794:Choline / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005952:Glucose-6-Phosphatase / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006706:Homeostasis; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D019884:Nitrogenous Group Transferases; D010739:Phospholipase D / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D051381:Rats; D011983:Receptors, Purinergic / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014166:Transferases / Q000378:metabolism*
D006150:Guanine Nucleotides; D010743:Phospholipids; D011983:Receptors, Purinergic; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014166:Transferases; D019884:Nitrogenous Group Transferases; C022370:phospholipid serine base exchange enzyme; D015720:5'-Nucleotidase; D005952:Glucose-6-Phosphatase; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D002794:Choline; D002118:Calcium