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- (greater than or equal to 24 h) and concentration- (greater than or equal to 2 mM) dependently, resulting in a depolarizing shift of the equilibrium potential of the GABA, receptor-Cl- channel opening (E-GABA). 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 mu M 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 mu M calphostin C and 10 mu M 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine dihydrochloride (H-7), and of transcription and de novo protein synthesis, 1 mu M actinomycin D and 0.5 mu g/ml cycloheximide, while a PKC activator, 0.1 mu M 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. (C) 1998 Elsevier Science B.V. All rights reserved.