Increased Na, K-ATPase alpha2 isoform gene expression by ammonia in astrocytes and in brain in vivo.
In mouse astrocyte cultures identical to those used in the present study ammonia increases the production of ouabain-like compounds and Na, K-ATPase activity (Kala et al., 2000). Increased activity of Na, K-ATPase could be the result of enhanced production of ouabain-like compounds, since cultured rat astrocytes react to prolonged exposure to a high concentration of ouabain with an upregulation of the Na, K-ATPase alpha(1) isoform (Hosoi et al., 1997). However, unlike astrocytes in brain in vivo and mouse primary cultures, cultured rat astrocytes do not express the astrocyte-specific alpha(2) isoform, which shows a higher affinity for ouabain (EC(50) approximately 0.1 microM) than the alpha(1) isoform (EC(50) approximately 10 microM). In the present study we have investigated (i) effects of ammonia on mRNA and protein expression of alpha(1) and alpha(2) isoforms in primary cultures of mouse astrocytes; (ii) effects of hyperammonia obtained by urease injection on mRNA and protein expression of alpha(1) and alpha(2) isoforms in the brain in vivo; and (iii) effect on observed upregulation of gene expression of AG1478, an inhibitor of the EGF receptor-tyrosine kinase, PP1, an inhibitor of Src, and GM6001, an inhibitor of Zn(2+)-dependent metalloproteinases in the cultured cells. It was established that alpha(2) mRNA and protein expression, but not alpha(1) expression, was upregulated in cultured astrocytes by 1-4 days of exposure to 3 or 5 mM ammonia and that similar upregulation, contrasted by a downregulation of the neuronal alpha(3) subunit occurred in the hyperammonemic brain. Based on the effects of the inhibitors and literature data it is concluded that ammonia activates formation of an endogenous ouabain-like compound, which binds to the Na, K-ATPase, activating Src, which in turn stimulates the receptor-tyrosine kinase of the EGF receptor, leading to activation of the Ras, Raf, MEK pathway and phosphorylation of ERK(1/2), which eventually causes upregulation of alpha(2) gene expression.
Xue
Zhanxia
Z
Department of Clinical Pharmacology, China Medical University, Shenyang, PR China.
Li
Baoman
B
Gu
Li
L
Hu
Xiaoling
X
Li
Min
M
Butterworth
Roger F
RF
Peng
Liang
L
D000641:Ammonia / Q000494:pharmacology*; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology*; D015153:Blotting, Western; D001921:Brain / Q000187:drug effects / Q000201:enzymology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D016391:Genes, src / Q000502:physiology; D022124:Hyperammonemia / Q000097:blood; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D011480:Protease Inhibitors / Q000494:pharmacology; D011799:Quinazolines; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics*; D020032:Tyrphostins / Q000494:pharmacology; D014510:Urease / Q000494:pharmacology
D004151:Dipeptides; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D011480:Protease Inhibitors; D011799:Quinazolines; D012333:RNA, Messenger; D020032:Tyrphostins; C101044:RTKI cpd; D004815:Epidermal Growth Factor; D000641:Ammonia; D066246:ErbB Receptors; D014510:Urease; C401838:Atp1a2 protein, mouse; D000254:Sodium-Potassium-Exchanging ATPase