Chloride concentration in cultured hippocampal neurons increases during long-term exposure to ammonia through enhanced expression of an anion exchanger.
The effects of long-term exposure to ammonia on [Cl-]i in cultured hippocampal neurons were examined. Ammonia increased the [Cl-]i time- (>/=24 h) and concentration- (>/=2 mM) dependently, resulting in a depolarizing shift of the equilibrium potential of the GABAA receptor-Cl- channel opening (EGABA). Such an effect of ammonia was diminished by the inhibitors of Cl-/HCO3- exchangers, 0.1 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 0.1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and a carbonic anhydrase inhibitor, 2 mM acetazolamide, but not by a Na+/K+/2Cl-cotransport inhibitor, 50 microM bumetanide, suggesting an enhanced Cl-/HCO3- exchange activity by ammonia. The ammonia-induced increase in [Cl-]i was also abolished by the inhibitors of protein kinase C (PKC), 0.1 microM calphostin C and 10 microM 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine dihydrochloride (H-7), and of transcription and de novo protein synthesis, 1 microM actinomycin D and 0.5 microg/ml cycloheximide, while a PKC activator, 0.1 h microM phorbor 12-myristate 13-acetate (PMA), increased the [Cl-]i. The mRNA level of the AE3 Cl-/HCO3- exchanger was increased by ammonia in a calphostin C- and H-7-sensitive manner. The AE3-like immunoreactivity was also increased by ammonia. These findings suggest that long-term exposure to ammonia increases the expression of AE3 through the activation of PKC, resulting in an increase in [Cl-]i in neurons and a reduction of inhibitory postsynaptic potentials.
Irie
T
T
Department of Pharmacology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka, 570-8506, Japan.
Hara
M
M
Yasukura
T
T
Minamino
M
M
Omori
K
K
Matsuda
H
H
Inoue
K
K
Inagaki
C
C
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000641:Ammonia / Q000494:pharmacology*; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D017920:Antiporters / Q000378:metabolism*; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D004594:Electrophysiology; D005456:Fluorescent Dyes; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009994:Osmolar Concentration; D011806:Quinolinium Compounds; D051381:Rats / Q000196:embryology*; D011963:Receptors, GABA-A / Q000502:physiology; D013997:Time Factors
D017920:Antiporters; D002712:Chlorides; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; D011963:Receptors, GABA-A; D000643:Ammonium Chloride; C085493:N-(ethoxycarbonylmethyl)-6-methoxyquinolinium; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000641:Ammonia; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid