Intrinsic hydrogen ion buffering in rat CNS neurones maintained in culture

Abstract

The intrinsic proton buffering power (β1) of individual rat hippocampal and neocortical neurones maintained in culture has been investigated using the fluorescent dye 2',7'-bis(carboxymethyl)-5,6-carboxyfluorescein) (BCECF). The steady-state intracellular pH (pH(i)) was estimated to be 7.03 ± 0.04 (n = 22) in Hepes-buffered media and β1 estimated from the addition and removal of weak bases was ca 10 mM (pH unit)-1 at pH(i) values near to 7. Estimates of β1 made from butyric acid challenges were inconsistent with estimates made at the same pH(i) using NH4Cl withdrawal. However, estimating β1 with butyrate in the presence of the monocarboxylate ion transport inhibitor α-cyano-hydroxy-cinnamate (CHC) yielded β1 values commensurate with those measured using NH4Cl. Application of CHC alone lead to a rapid fall in pH(i), suggesting a significant contribution of the monocarboxylate transporter to pH(i) regulation. β1 was also estimated from a step increase in extracellular P(CO2). This yielded a value of 11 mM at an average pH(i) of 7.1, which is similar to that of the other estimates reported here. β1 was found to increase with decreasing pH(i): each unit drop in pH(i) increased buffering power by about 60%. Blockade of pH(i) regulation did not significantly affect estimates of β1. The change in buffering power with pH could be closely modelled from the known concentrations of free amino acids and organic phosphates.