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The modulation of the activity of intracellular pH regulating mechanisms by second messenger systems in rat hippocampal CA1 neurons Brett, Christopher L.

Abstract

The effects of modulating the activities of the cAMP/cAMP-activated protein kinase (PICA) and calmodulin (CaM)/Ca²⁺-CaM activated protein kinase II (CaMK-II) second messenger systems on steady-state (SS) intracellular pH (pH[sub i]) and on rates of pH[sub i] recovery following imposed acid and/or alkaline loads were investigated in acutely dissociated adult rat hippocampal CA1 neurons loaded with the fluorescent hydrogen ion indicator 2',7'-bis-(2- carboxyethyl)-5(6)-carboxyfluorescein. Alkali extrusion under HC03⁻/C0₂-buffered conditions was dependent on the activity of a 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS)-sensitive, Na⁺-independent HC0₃⁻/Ci⁻ exchanger (NIAE). Acting in concert with an acid-extruding Na⁺-dependent HC0₃⁻/Ci⁻ exchanger (NDAE) and a Na⁺/H⁺-exchanger (NHE), the NIAE functioned to maintain SS pH[sub i] near the external pH value. Under HC03⁻-free conditions, stimulation of cAMP/PKA increased both SS pH[sub i] and rates of pH[sub i] recovery from imposed acid loads. The increases in SS pH[sub i] were enhanced by protein phosphatase inhibition, unaffected by changes in intracellular free calcium ([Ca²⁺][sub i]), and dependent on the presence of external Na⁺ . These results indicate that stimulating cAMP/PKA activates the NHE in rat hippocampal neurons. Conversely, inhibition of cAMP/PKA failed to affect NHE activity. However, in the presence of HC03⁻/C0₂ , both stimulation and inhibition of cAMP/PKA evoked changes in SS pH[sub i] and rates of pH[sub i] recovery from internal acid (and alkaline) loads. Further study indicated that modulation of the activity of the cAMP/PKA pathway changes the activities of the NIAE and NDAE in a manner depending on resting pH[sub i]. Inhibitors of CaM evoked rises in SS pHj that were dependent on the initial resting pHj and external Na+ , and increased rates of pH[sub i] recovery from acid loads imposed in either the presence or absence of HC03⁻, indicating that CaM inhibitors activate the NHE. CaM inhibitorevoked increases in SS pH[sub i] were minimally affected by changes in [Ca²⁺][sub i], but were not mediated by changes in the activities of the cAMP/PKA second messenger pathway, calcineurin or CaMKII. The results indicate that the activity of the NHE can be modulated by more than one intracellular second messenger system. Furthermore, the activities of the three pH[sub i] regulating mechanisms can be altered concomitantly by a single second messenger system. Ultimately, the described second messenger control of the activities of pH[sub i] regulating mechanisms present in rat hippocampal CA1 neurons may contribute to some of the effects of neurotransmitters on neuronal excitability and function.

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