Roles of Cl^- in cellular signal transduction

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1. Roles of intracellular Cl^- concentration ( [Cl^-] _i) in c-fos expression in the brain.Intracerebroventricular injection of ethacrynic acid that inhibits neuronal Cl^- extrusion and induces glutamate release, increased c-fos expression in hippocampal neurons in mice. Analyzes of the time courses by Northern blot and in situ hybridization showed two peaks of c-fos mRNA levels 60 min and 10-14 days after the injection of ethacrynic acid. In parallel with the time couses, levels of nerve growth factor mRNA also increased in the same brain regions.2. Roles of [Cl^-] _i in responses to beta-adrenoceptor stimulation.Adrenoceptor-mediated Cl^- transport in cultured rabbit corneal endothelium was examined using a Cl^--sensitive fluorescent dye. The corneal endothelial cells accumulated Cl^<> upon beta-adrenoceptor stimulation under the condition of low [Cl^-] _i (<30mM), but not under the conditions of [Cl^-] _i<greater than or equal>30mM,probably via acceleration of G-protein subunit dissociation followed by activation of cAMP-dependent proteinkinase and Na^+/K^+/Cl^- cotransporter.3. Role of [Cl^-] _i in responses to metabotropic glutamate receptor (mGluR) stimulation.When [Cl^-] _i was changed, stimulation of mGluRl alpha expressed in Chinese hamster ovary cells yielded different amounts of IP_3 with a peak at 20mM of [Cl^-] _i. Pertussis toxin (PTX) blocked the [Cl^-] _i-sensitive IP_3 formation suggesting that activation of mGluR-coupled PTX-sensitive G-protein is sensitive to [Cl^-] _i.4. A new [Cl^-] _i regulating system.Neuronal outwardly directed Cl^- transport system (Cl^- pump) was identified as 520 kDa protein that primarily transports Cl^- in an ATP-dependent manner to lower neuronal [Cl^-] _i when reconstituted in liposomes.