Nuclear protein phosphorylation in rat cerebral cells following acute exposure to ethanol.
The sequelae of acute ethanol toxicity encompass a broad spectrum of metabolic and cellular derangements, including the induction of stress proteins in cells exposed to high levels of ethanol. In this investigation, the effects of ethanol exposure on nuclear protein synthesis and phosphorylation were compared by two-dimensional gel electrophoresis in glial-enriched cultures, adult rat cerebrum and regenerated liver. Cellular exposure to ethanol was at clinically relevant levels and tissue was analyzed at 1 h and 48 h after exposure. Cell nuclei were stained with propidium iodide, a DNA specific fluorochrome and flow cytometrically sorted to obtain cell cycle phase populations of nuclei for analysis. Ethanol treatment of intact rats and glial-enriched cultures induced phosphorylation of specific nuclear proteins, which were detected by two-dimensional electrophoresis and autoradiography. The autoradiographs of [32P]phosphate and [3H]leucine labeled proteins from glial-enriched cultures and from the G0/G1 phase of the regenerating liver tissue exhibited intense labeling indicative of active protein synthesis and phosphorylation. In contrast, the autoradiographs of proteins from adult rat cerebra showed substantial phosphorylation, but weak protein synthesis. Ethanol treatment was associated with phosphorylation of a 50,000 Mr protein in G0/G1 phase cells of the cultures and in predominantly G0 cells of the adult rat cerebra. A protein with similar characteristics was not found in ethanol exposed regenerating rat liver tissue and has not been observed in other 'heat shock' or 'stress' protein systems which we have previously studied.
Hinson
W G
WG
Department of Health and Human Services, Food and Drug Administration, National Center for Toxicological Research, Jefferson, Arkansas 72205-9502.
Pipkin
J L
JL
Anson
J F
JF
Davies
D L
DL
Burns
E R
ER
Casciano
D A
DA
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D000431:Ethanol / Q000494:pharmacology / Q000633:toxicity*; D005260:Female; D007202:Indicators and Reagents; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008115:Liver Regeneration; D008970:Molecular Weight; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism*; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D014316:Tritium
D007202:Indicators and Reagents; D009687:Nuclear Proteins; D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D014316:Tritium; D000431:Ethanol; D007930:Leucine