Gene expression of the transporters and biosynthetic enzymes of the osmolytes in astrocyte primary cultures exposed to hyperosmotic conditions.
Sorbitol, myo-inositol, betaine, and taurine are held as organic osmolytes. When cells are exposed to a hyperosmotic medium, they accumulate these organic compounds and thus achieve osmotic equilibrium with the medium while maintaining their volume. In astrocyte primary cultures adapted to a chemically defined medium and then exposed to a medium made 30% hyperosmotic by adding sodium chloride or raffinose, we have comparatively investigated the expression of the genes encoding the proteins that control the cellular accumulation of these osmolytes, namely sorbitol biosynthetic enzyme, aldose reductase (AR), taurine biosynthetic enzymes, cysteine dioxygenase (CDO), and cysteine sulfinic acid decarboxylase (CSD), and the transporters of taurine (TauT), myo-inositol (SMIT), and betaine (BGT1) by assaying the corresponding mRNA levels through relative quantitative RT-PCR. When exposed to the hyperosmotic medium the astrocytes shrank rapidly and then slowly regained their initial volume after several hours. CDO- and CSD-mRNA remained unchanged, whereas AR-mRNA appeared increased only with the medium made hyperosmotic with sodium chloride. The mRNA levels of the transporters only showed significant and comparable increases in both hyperosmotic conditions. They were all significantly higher after 4-h exposure and back or close to normal values after 24-h exposure. The maximum level occurred at around 4 h (SMIT), 8 h (BGT1), and 12 h (TauT). The amplitude of BGT1-mRNA increase was much larger. When taurine was added to the hyperosmotic medium the cell volume recovery was greatly accelerated and the osmo-induced overexpression of TauT-, SMIT-, and BGT1-mRNA was fully prevented. The activation of the genes encoding the osmolyte transporters appears to be triggered when the cell shrinks below a certain volume threshold and prolonged once the cell volume has regained this threshold value most likely as a result of a marked inertia of the transducing pathway. Since the upregulation pattern of the transporters of the different osmolytes notably differs, we speculate that the activation threshold varies from one gene to another.
Bitoun
M
M
Institut National de la Santé et de la Recherche Médicale, Unité INSERM 433, Faculté de Médecine RTH Laennec, Lyon, France.
Tappaz
M
M
D000449:Aldehyde Reductase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D050558:Cysteine Dioxygenase; D049308:Dioxygenases; D004798:Enzymes / Q000235:genetics*; D050485:GABA Plasma Membrane Transport Proteins; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009994:Osmolar Concentration; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D027981:Symporters; D013654:Taurine / Q000494:pharmacology; D013997:Time Factors; D014882:Water-Electrolyte Balance / Q000187:drug effects / Q000502:physiology*; D000099247:Taurine Transporters
D000449:Aldehyde Reductase; D002262:Carboxy-Lyases; D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D050558:Cysteine Dioxygenase; D049308:Dioxygenases; D004798:Enzymes; D050485:GABA Plasma Membrane Transport Proteins; D006360:Heat-Shock Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010105:Oxygenases; D012333:RNA, Messenger; D027981:Symporters; D013654:Taurine; D000099247:Taurine Transporters; C072207:betaine plasma membrane transport proteins; C073884:SLC5A3 protein, human; C024981:cysteine decarboxylase