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 (pHo) decrease caused by a [HCO3 -]o decrease at constant [CO2]o-usually causes intracellular pH (pHi) to fall. Here we determine the extent to which the pHi decrease depends on the pHo 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 pHo (pure acidosis, pAc), or stabilize [CO2]o and pHo while decreasing [HCO3 -]o (pure metabolic/down, pMet↓). Using the fluorescent dye 2',7'-bis-2-carboxyethyl)-5(and-6)carboxyfluorescein (BCECF) to monitor pHi in rat hippocampal neurons in primary culture, we find that-in naïve neurons-the pHi 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 pHo 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 pHo 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 pHi response during challenge #2 (MAc2, pAc2, or pMet↓2).