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 down arrow). 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 na & iuml;ve neurons-the pH(i) decrease caused by MAc is virtually the sum of those caused by pAc (similar to 70%) + pMet down arrow (similar to 30%). However, if we impose a first challenge (MAc1, pAc(1), or pMet down arrow(1)), allow the neurons to recover, and then impose a second challenge (MAc2, pAc(2), or pMet down arrow(2)), we find that pAc/pMet down arrow 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, pAc(1), or pMet down arrow(1))-presumably as detected by one or more acid-base sensors-has a major impact on the pH(i) response during challenge #2 (MAc2, pAc(2), or pMet down arrow(2)).