Regulation of Cl−−Hco3− exchangers by cAMP‐dependent protein kinase in adult rat hippocampal CA1 neurons

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Abstract
The contributions of HCO3−‐dependent, DIDS‐sensitive mechanisms to the maintenance of steady‐state pHi, and the regulation of their activities by cAMP‐dependent protein kinase (PKA), were investigated in CA1 neurons with the H+‐sensitive fluorophore, BCECF. The addition of HCO3−/CO2 to neurons with ‘low’ (pHi≤ 7.20) and ‘high’ (pHi > 7.20) initial pHi values under Hepes‐buffered conditions, increased and decreased steady‐state pHi, respectively. Conversely, under HCO3−/CO2‐buffered conditions, DIDS caused pHi to decrease and increase in neurons with low and high initial pHi values, respectively. In the presence, but not the absence, of HCO3−, the PKA inhibitor Rp‐adenosine‐3′,5′‐cyclic monophosphorothioate (Rp‐cAMPS; 50 μm) evoked DIDS‐sensitive increases and decreases in pHi in neurons with low and high initial pHi values, respectively. In contrast, in neurons with low initial pHi values, activation of PKA with the Sp isomer of cAMPS (Sp‐cAMPS; 25 μm) elicited increases in pHi that were smaller in the presence than in the absence of HCO3−, whereas in neurons with high initial pHi values, Sp‐cAMPS‐evoked rises in pHi were larger in the presence than in the absence of HCO3−; the differences between the effects of Sp‐cAMPS on pHi under the different buffering conditions were attenuated by DIDS. Consistent with the possibility that changes in the activities of HCO3−‐dependent, DIDS‐sensitive mechanisms contribute to the steady‐state pHi changes evoked by the PKA modulators, in neurons with initial pHi values ≤ 7.20, Rp‐cAMPS concurrently inhibited Na+‐independent Cl−‐HCO3− exchange and stimulated Na+‐dependent Cl−‐HCO3− exchange; in contrast, Sp‐cAMPS concurrently stimulated Na+‐independent Cl−‐HCO3− exchange and inhibited Na+‐dependent Cl−‐HCO3− exchange. Data from a limited number of neurons with initial pHi values > 7.20 suggested that the directions of the reciprocal changes in anion exchange activities (inhibition or stimulation) evoked by Rp‐ and Sp‐cAMPS may be opposite in cells with low vs. high resting pHi values. Taken together, the results indicate that the effects of modulating PKA activity on steady‐state pHi in rat CA1 neurons under HCO3−/CO2‐buffered conditions reflect not only changes in Na+‐H+ exchange activity but also changes in Na+‐dependent and Na+‐independent Cl−‐HCO3− exchange activity that, in turn, may be dependent upon the initial pHi.