Regulation of Cl--HCO3- exchangers by cAMP-dependent protein kinase in adult rat hippocampal CA1 neurons

Abstract

The contributions of HCO(3)(-)-dependent, DIDS-sensitive mechanisms to the maintenance of steady-state pH(i), 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 HCO(3)(-)/CO(2) to neurons with "low" (pH(i) < or = 7.20) and "high" (pH(i) > 7.20) initial pH(i) values under Hepes-buffered conditions, increased and decreased steady-state pH(i), respectively. Conversely, under HCO(3)(-)/CO(2)-buffered conditions, DIDS caused pH(i) to decrease and increase in neurons with low and high initial pH(i) values, respectively. In the presence, but not the absence, of HCO(3)(-), the PKA inhibitor Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS; 50 microM) evoked DIDS-sensitive increases and decreases in pH(i) in neurons with low and high initial pH(i) values, respectively. In contrast, in neurons with low initial pH(i) values, activation of PKA with the Sp isomer of cAMPS (Sp-cAMPS; 25 microM) elicited increases in pH(i) that were smaller in the presence than in the absence of HCO(3)(-), whereas in neurons with high initial pH(i) values, Sp-cAMPS-evoked rises in pH(i) were larger in the presence than in the absence of HCO(3)(-); the differences between the effects of Sp-cAMPS on pH(i) under the different buffering conditions were attenuated by DIDS. Consistent with the possibility that changes in the activities of HCO(3)(-)-dependent, DIDS-sensitive mechanisms contribute to the steady-state pH(i) changes evoked by the PKA modulators, in neurons with initial pH(i) values < or = 7.20, Rp-cAMPS concurrently inhibited Na(+)-independent Cl(-)-HCO(3)(-) exchange and stimulated Na(+)-dependent Cl(-)-HCO(3)(-) exchange; in contrast, Sp-cAMPS concurrently stimulated Na(+)-independent Cl(-)-HCO(3)(-) exchange and inhibited Na(+)-dependent Cl(-)-HCO(3)(-) exchange. Data from a limited number of neurons with initial pH(i) 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 pH(i) values. Taken together, the results indicate that the effects of modulating PKA activity on steady-state pH(i) in rat CA1 neurons under HCO(3)(-)/CO(2)-buffered conditions reflect not only changes in Na(+)-H(+) exchange activity but also changes in Na(+)-dependent and Na(+)-independent Cl(-)-HCO(3)(-) exchange activity that, in turn, may be dependent upon the initial pH(i).

Figure 1. Effects on pH_i of the transition from Hepes- to HCO₃⁻/CO₂-buffered medium, and the addition of DIDS to HCO₃⁻/CO₂-buffered medium

A, a neuron perfused with a Hepes-buffered medium (pH 7.35) had a low initial pH_i (continuous line). Upon exposure to HCO₃⁻/CO₂-buffered medium at a constant pH_o, pH_i decreased transiently and then increased to a new steady-state level. In contrast, a different neuron with a high initial pH_i in Hepes-buffered medium (○) exhibited an internal acidification upon exposure to HCO₃⁻/CO₂. B, the changes in pH_i (ΔpH_i) evoked by the addition of HCO₃⁻/CO₂ plotted against initial pH_i values in Hepes-buffered medium (n = 39). A linear least squares regression fit to the data points (r²= 0.84) had a negative slope and intersected the abscissa at pH_i 7.21. C, in a neuron with a low initial pH_i value under HCO₃⁻/CO₂-buffered conditions at pH_o 7.35 (continuous line), DIDS caused pH_i to decrease to a new steady-state level. In contrast, DIDS caused pH_i to rise in a different neuron with a high initial pH_i (○). D, ΔpH_i elicited by 200 μm DIDS applied under HCO₃⁻/CO₂-buffered conditions plotted against initial pH_i values (n = 44). A linear least squares regression fit to the data points (r²= 0.75) had a similar x-intercept (pH_i 7.17) but an opposite slope to the fit representing the pH_i response to HCO₃⁻/CO₂ application.

Figure 2. Effects of Rp- and Sp-cAMPS on pH_i

A, Rp-cAMPS, applied under HCO₃⁻/CO₂-buffered conditions (pH_o 7.35) for the period indicated by the bar above the traces, evoked increases and decreases in pH_i in two different neurons with low and high initial pH_i values, respectively, prior to the addition of the PKA inhibitor. B, the changes in pH_i (ΔpH_i) elicited by 50 μm Rp-cAMPS under HCO₃⁻/CO₂-buffered control conditions (•) or following 10-15 min pre-treatment with 200 μm DIDS (▵) were plotted against pH_i values measured prior to the addition of the PKA inhibitor and each set of data points was fitted by least squares regression (r²≥ 0.80). The changes in pH_i evoked by Rp-cAMPS were dependent upon the initial pH_i and, as illustrated in the inset, were attenuated by DIDS. C, four different neurons, two with similarly low initial pH_i values and two with similarly high initial pH_i values, were exposed to 25 μm Sp-cAMPS under HCO₃⁻/CO₂- (continuous line) or Hepes- (□) buffered conditions. In neurons with initial pH_i values ≤ 7.20, Sp-cAMPS evoked a greater increase in pH_i under Hepes- than under HCO₃⁻/CO₂-buffered conditions; in contrast, in neurons with initial pH_i values > 7.20, Sp-cAMPS evoked a smaller increase in pH_i under Hepes- than under HCO₃⁻/CO₂-buffered conditions. D, ΔpH_i elicited by 25 μm Sp-cAMPS in the nominal absence (□) or presence (•) of HCO₃⁻/CO₂ were plotted against initial values of pH_i and a regression line was fitted to each set of data points. The vertical dotted line represents the division between ‘low’ (initial pH_i≤ 7.20) and ‘high’ (initial pH_i > 7.20) pH_i neurons. E, the same experiment as that shown in A, but conducted in the absence of external Na⁺. Rp-cAMPS evoked an increase and a decrease in pH_i in two different neurons with low and high initial pH_i values, respectively. F, under Na⁺_o-free, HCO₃⁻-buffered conditions, the addition of 25 μm Sp-cAMPS caused pH_i to increase in a neuron with a high initial pH_i, and pH_i to decrease in a different neuron with a low initial pH_i.

Figure 3. Effects of Rp-cAMPS on pH_i recovery from base loading in the presence and absence of Na⁺_o

All data were obtained from neurons with initial pH_i values ≤ 7.20 under HCO₃⁻/CO₂-buffered conditions. Alkali loads were imposed by applying and withdrawing a 10 % CO₂/39 mm HCO₃⁻ solution, as indicated by the short bars above the traces in A and B. A, following an initial alkali load, pH_i was allowed to recover and a second alkali load was imposed in the presence of DIDS (applied for the period indicated by the long bar above the trace). Superimposed is a record (○) obtained from a different cell that was exposed to Cl⁻-free medium (applied for the period indicated by the short bar beneath the trace) at the peak of the second alkali load (the first part of the record from this experiment has been omitted for clarity). B, after recovery from an initial alkali load imposed under control conditions, the neuron was exposed to 50 μm Rp-cAMPS. Rp-cAMPS evoked a rise in pH_i (see Fig. 2A) and a second alkali load was then imposed; the rate of pH_i recovery was decreased in the presence of Rp-cAMPS. C, mean rates of pH_i recovery following alkali loads imposed in the absence (•) and presence (○) of 50 μm Rp-cAMPS plotted against absolute values of pH_i. Data points were obtained from 14 experiments of the type illustrated in B; error bars represent s.e.m.D, mean rates of pH_i recovery from alkali loads obtained in the absence (•) and presence (○) of 50 μm Rp-cAMPS under Na⁺_o-free conditions plotted against absolute values of pH_i. Data points were obtained from seven experiments of the type illustrated in B, except in the absence of external Na⁺; error bars represent s.e.m.

Figure 4. Effects of Sp-cAMPS on pH_i recovery from base loading in the presence and absence of Na⁺_o

All data were obtained from neurons with initial pH_i values ≤ 7.20 under HCO₃⁻/CO₂-buffered conditions. A, after the recovery of pH_i from a control alkali load imposed under Na⁺_o-containing conditions, 25 μm Sp-cAMPS evoked a slow increase in steady-state pH_i (see Fig. 2C). A second alkali load was then imposed and pH_i recovered at a faster rate, and to a new steady-state level, in the continued presence of Sp-cAMPS. B, the pH_i dependence of pH_i recovery under Na⁺_o-containing conditions in the absence (•) and presence (○) of 25 μm Sp-cAMPS. Data points were obtained from nine experiments of the type illustrated in A; error bars represent s.e.m. The alkaline shift in the pH_i-dependence of pH_i recovery from alkali loads evoked by Sp-cAMPS reflects the increase in pH_i elicited by Sp-cAMPS in the presence of Na⁺_o in neurons with resting pH_i values ≤ 7.20. C, mean rates of pH_i recovery from base loading in the absence (•) and presence (○) of 25 μm Sp-cAMPS under Na⁺_o-free conditions plotted against absolute values of pH_i. Data points were obtained from five experiments of the type illustrated in A, except in the absence of external Na⁺. The acidic shift in the pH_i-dependence of pH_i recovery from alkali loads in the presence of Sp-cAMPS reflects the decrease in pH_i elicited by Sp-cAMPS in the absence of Na⁺_o in neurons with resting pH_i values ≤ 7.20 (see Fig. 2F).

Figure 5. Effects of Rp- and Sp-cAMPS on changes in pH_i evoked by removal of Cl⁻_o

All data were obtained from neurons with initial pH_i values ≤ 7.20 under HCO₃⁻/CO₂-buffered conditions. A, the acute removal of Cl⁻_o (for the period indicated by the short bar above the trace) evoked an increase in pH_i which recovered upon the reintroduction of the anion. Rp-cAMPS (50 μm) was then applied and, after pH_i increased to a new steady-state level (see Fig. 2A), the removal of Cl⁻_o evoked an increase in pH_i that was smaller than in the absence of the PKA inhibitor. Increases in pH_i evoked by the acute removal of Cl⁻_o in the presence of a PKA modulator were measured as the difference between the maximum pH_i observed in the absence of Cl⁻_o and the plateau pH_i value observed following the reintroduction of Cl⁻_o in the continued presence of the PKA modulator. B, a neuron with a low initial pH_i (continuous line) was exposed to Cl⁻-free medium, which caused an increase in pH_i. Sp-cAMPS (25 μm) was then applied; the PKA activator evoked an increase in pH_i (see Fig. 2C) and subsequent exposure to Cl⁻-free medium caused a large internal alkalinization. Superimposed is a record (□) obtained from a different low pH_i neuron in which 25 μm Sp-cAMPS was co-applied with 200 μm DIDS; the gap in the trace represents a 5.5 min break in the record. The rise in pH_i observed during exposure to Cl⁻-free medium in the presence of Sp-cAMPS was blocked by DIDS. C, the increases in pH_i evoked by transient exposure to Cl⁻-free medium are shown in three different neurons with similar initial pH_i values under control conditions (continuous line) and following pre-treatment with 25 μm Sp-cAMPS (▿) or 50 μm Rp-cAMPS (○).

Figure 6. Effects of Sp-cAMPS on pH_i recovery from acid loads at room temperature

A, two consecutive acid loads, the second in the presence of 25 μm Sp-cAMPS, were imposed on a neuron with a low initial pH_i under Hepes-buffered conditions. Rates of pH_i recovery from both acid loads were similar and, in contrast to the effects of Sp-cAMPS under Hepes-buffered conditions at 37 °C (see Smith et al. 1998), pH_i failed to recover to a higher steady-state level in the presence of Sp-cAMPS. B, rates of pH_i recovery from acid loads performed in the absence (•) and presence (○) of 25 μm Sp-cAMPS plotted against absolute values of pH_i; data points were obtained from nine experiments of the type shown in A (error bars represent s.e.m.). Sp-cAMPS failed to significantly affect the rate of pH_i recovery at any absolute value of pH_i. C, the pH_i dependence of pH_i recovery under HCO₃⁻/CO₂-buffered conditions at RT in the absence (•) and presence (○) of 25 μm Sp-cAMPS; data points were obtained from six experiments of the type shown in A, except in the presence of HCO₃⁻/CO₂. Rates of pH_i recovery were significantly reduced in the presence of Sp-cAMPS at all absolute values of pH_i. D, rates of pH_i recovery from acid loads imposed on high pH_i neurons (n = 6) under HCO₃⁻/CO₂-buffered conditions at RT in the absence (•) and presence (○) of 25 μm Sp-cAMPS plotted against absolute values of pH_i. In contrast to its effect in low pH_i neurons, Sp-cAMPS increased rates of pH_i recovery in neurons with high initial pH_i values.

Figure 7. Effects of Rp-cAMPS on pH_i recovery from acid loads in low pH_i neurons

A, an acid load was imposed on a low pH_i neuron under HCO₃⁻/CO₂-buffered conditions at RT and, after recovery of pH_i, 50 μm Rp-cAMPS was applied. Rp-cAMPS increased both steady-state pH_i and the rate of pH_i recovery from the second acid load. B, the pH_i dependence of the rate of pH_i recovery from acid loads imposed under HCO₃⁻/CO₂-buffered conditions at RT in the absence (•) and presence (○) of 50 μm Rp-cAMPS; data points were obtained from 12 experiments of the type shown in A. C, the same experiment as shown in A, but conducted at 37 °C. D, the pH_i dependence of the rate of pH_i recovery from acid loads performed at 37 °C in the absence (•) and presence (○) of 50 μm Rp-cAMPS; data points were obtained from eight experiments of the type shown in C. In contrast to observations made in the nominal absence of HCO₃⁻/CO₂, Rp-cAMPS applied to low pH_i neurons in the presence of HCO₃⁻/CO₂ increased rates of pH_i recovery at all absolute values of pH_i and shifted the pH_i dependence of the rate of pH_i recovery in an alkaline direction.

Figure 8. Effects Rp- and Sp-cAMPS on alkalinizations evoked by HCO₃⁻/CO₂ under Cl⁻_o-free conditions

A, under Cl⁻_o-free conditions, repeated exposures to HCO₃⁻/CO₂ caused pH_i to transiently decrease and then increase to a new steady-state level; the magnitude of the HCO₃⁻/CO₂-evoked increase in pH_i declined with each successive application, resulting in a progressive reduction in the rate at which pH_i reached each new steady-state level (see C, Control). B, the effects on pH_i of exposing two different low pH_i neurons to HCO₃⁻/CO₂ under Cl⁻_o-free conditions in the presence of 50 μm Rp-cAMPS (left-hand side) or 25 μm Sp-cAMPS (right-hand side). Under each condition, filled circles identify the record obtained during the first exposure to HCO₃⁻/CO₂; traces identified by open circles represent the changes in pH_i observed during the second (Rp-cAMPS) or fourth (Sp-cAMPS) exposures to HCO₃⁻/CO₂ in the respective series. C, rates of alkalinization observed during consecutive applications of HCO₃⁻/CO₂ in the continuous absence of Cl⁻_o were calculated at an absolute pH_i value of 7.00 and normalized to the mean rate of alkalinization observed during the first application of HCO₃⁻/CO₂ under each experimental condition. The resulting normalized rates of alkalinization are presented as percentage values (± s.e.m.) under control conditions and in the presence of 50 μm Rp-cAMPS and 25 μm Sp-cAMPS. (Number of neurons examined under each experimental condition). *P < 0.05 for the difference between the first normalized rate of alkalinization under a given experimental condition.