Intracellular processes associated with vesicular transport from endoplasmic reticulum to Golgi and exocytosis: ethanol-induced changes in membrane biogenesis.
Membrane biogenesis, expressed in endoplasmic reticulum (ER) by formation of transport vesicles, was studied in the liver of ethanol-fed and pair-fed rats. In ER of ethanol-fed animals, the endogenous synthesis of phosphatidylcholine (PC) and its contribution to ER transport vesicles were reduced by 50%, as compared to that in pair-fed controls. Reduction of PC synthesis and of its presence in ER-transport vesicles was also observed in pair-fed controls when the native cytosol was replaced with that from ethanol-fed animals. In contrast, preincubation of ER membranes from ethanol-fed animals with cytosol from controls led to the stimulation of PC synthesis in ER and its contribution to ER-transport vesicles. Analysis of water soluble metabolites of [methyl-14C]choline phosphate revealed the accumulation of CDP-choline precursor in samples derived from ethanol-fed rats. Concomitantly, the endogenous synthesis of phosphatidylinositol (PI) in the ER of ethanol-fed animals was stimulated up to 400-500%, but declined when the cytosol from ethanol-fed rats was replaced with that from the controls. The restoration of PC synthesis, the normalization of PI synthase activity, and, similar to control, the contribution of PC to ER-transport vesicles in ethanol-fed animals was achieved when ER membranes were preincubated with diglycerides or the cytosol was treated with ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Conversely, addition of CaCl2-EGTA buffer containing 3 microM free Ca2+ to control samples, led to a reduction in PC synthesis. The studies on the effect of free Ca2+ on PI synthase and phosphatidic acid (PA) phosphatase activity established that in the presence of 1-3 microM free Ca2+, PI synthase activity remained constant, whereas that of PA phosphatase was reduced by 40% at 1 microM Ca2+, and no activity was detected when free Ca2+ was adjusted to 3 microM. The results suggest that modified membrane biogenesis in the liver of ethanol-fed rats is connected to the elevated free Ca2+ in the cytosol, which appears to regulate phosphatase activity. Accumulation of CDP-choline, decreased activity of PA phosphatase, and increased contribution of PI lipids to ER-transport vesicle membrane suggest that in ethanol-fed animals diglycerides are depleted and PA is utilized in a CDP-diacylglycerol pathway, thus leading to the generation of a different group of phospholipids and consequently modified ER-transport vesicle membrane.
Slomiany
A
A
Research Center, University of Medicine and Dentistry of New Jersey, Newark 07103-2400.
Grabska
M
M
Piotrowski
E
E
Sengupta
S
S
Morita
M
M
Kasinathan
C
C
Slomiany
B L
BL
D000428:Alcohol Drinking / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D002118:Calcium / Q000378:metabolism; D019916:Choline-Phosphate Cytidylyltransferase; D003566:Cytidine Diphosphate Choline / Q000378:metabolism; D003567:Cytidine Diphosphate Diglycerides / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D002798:Diacylglycerol Cholinephosphotransferase / Q000032:analysis; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D006056:Golgi Apparatus / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D008566:Membranes / Q000737:chemistry / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000032:analysis; D015388:Organelles / Q000737:chemistry / Q000378:metabolism*; D010711:Phosphatidate Phosphatase / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017855:Transferases (Other Substituted Phosphate Groups) / Q000032:analysis
D003567:Cytidine Diphosphate Diglycerides; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D003566:Cytidine Diphosphate Choline; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; D017855:Transferases (Other Substituted Phosphate Groups); D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D002798:Diacylglycerol Cholinephosphotransferase; D010711:Phosphatidate Phosphatase; D002118:Calcium