Effects of extracellular metabolic acidosis and out-of-equilibrium CO2/HCO3− solutions on intracellular pH in cultured rat hippocampal neurons

Metabolic acidosis (MAc)-an extracellular pH (pH_o) decrease caused by a [HCO₃ ^-]_o decrease at constant [CO₂]_o-usually causes intracellular pH (pH_i) to fall. Here we determine the extent to which the pH_i decrease depends on the pH_o decrease vs the concomitant [HCO₃ ^-]_o decrease. We use rapid-mixing to generate out-of-equilibrium CO₂/HCO₃ ^- solutions in which we stabilize [CO₂]_o and [HCO₃ ^-]_o while decreasing pH_o (pure acidosis, pAc), or stabilize [CO₂]_o and pH_o while decreasing [HCO₃ ^-]_o (pure metabolic/down, pMet↓). Using the fluorescent dye 2',7'-bis-2-carboxyethyl)-5(and-6)carboxyfluorescein (BCECF) to monitor pH_i in rat hippocampal neurons in primary culture, we find that-in naïve neurons-the pH_i decrease caused by MAc is virtually the sum of those caused by pAc (∼70%) + pMet↓ (∼30%). However, if we impose a first challenge (MAc₁, pAc₁, or pMet↓₁), allow the neurons to recover, and then impose a second challenge (MAc₂, pAc₂, or pMet↓₂), we find that pAc/pMet↓ additivity breaks down. In a twin-challenge protocol in which challenge #2 is MAc, the pH_o and [HCO₃ ^-]_o decreases during challenge #1 must be coincident in order to mimic the effects of MAc₁ on MAc₂. Conversely, if challenge #1 is MAc, then the pH_o and [HCO₃ ^-]_o decreases during challenge #2 must be coincident in order for MAc₁ to produce its physiological effects during the challenge #2 period. We conclude that the history of challenge #1 (MAc₁, pAc₁, or pMet↓₁)-presumably as detected by one or more acid-base sensors-has a major impact on the pH_i response during challenge #2 (MAc₂, pAc₂, or pMet↓₂).