The influence of calcium transients on intracellular pH in cortical neurons in primary culture

The objective of this study was to assess the influence of Ca²⁺ influx on intracellular pH (pH_i) of neocortical neurons in primary culture. Neurons were exposed to glutamate (100–500 μM) or KCl (50 mM), and pH_i was recorded with microspectroflurometric techniques. Additional experiments were carried out in which calcium influx was triggered by ionomycin (2 μM) or the calcium ionophore 4-Br-A23187 (2 μM). Glutamate exposure either caused no, or only a small decrease in pH_i (ΔpH ≈ 0.06 units). When a decrease was observed, a rebound rise in pH_i above control was observed upon termination of glutamate exposure. In about 20% of the cells, the acidification was more pronounced (ΔpH ≈ 0.20 units), but all these cells had high control pH_i values, and showed gradual acidification. Exposure of cells to 50 mM KCl consistently increased pH_i. Since this increase was similar in the presence and nominal absence of HCO₃⁻, it probably did not reflect influx of HCO₃⁻ via a Na⁺-HCO₃⁻ symporter. Furthermore, since it occurred in the absence of external Ca²⁺ (or a measurable rise in Ca_i²⁺) it seemed independent of Ca²⁺ influx. It is tentatively concluded that the rise in pH_i was due to reduced passive influx of H⁺ along the electrochemical gradient, which is reduced by depolarization. In Ca²⁺-containing solutions, depolarization led to a rebound increase in pH_i above control. This, and the rebound found after glutamate transients, may reflect Ca²⁺-triggered phosphorylation and upregulation of the Na⁺/H⁺ antiporter which extrudes H⁺ from the cell. Ionomycin and 4-Br-A23187 gave rise to a large rise in Ca_i²⁺ and to alkalinization of the cell (ΔpH ≈ 0.5). Since amiloride or removal of Na⁺ from the external solution did not alter the rise in pH_i, it was probably not due to accelerated H⁺ extrusion. However, removal of Ca²⁺ from extracellular fluid prevented the rise, suggesting that it was secondary to Ca²⁺/2H⁺ exchange across plasma membranes.