Intrinsic hydrogen ion buffering in rat CNS neurones maintained in culture

Abstract

SUMMARYThe intrinsic proton buffering power (/1) 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,) was estimated to be 7.03 + 0.04 (n = 22) in Hepes-buffered media and /3i estimated from the addition and removal of weak bases was ca 10 mM (pH unit)-' at pHi values near to 7. Estimates of /#j made from butyric acid challenges were inconsistent with estimates made at the same pH, using NH4C1 withdrawal.However, estimating /?j with butyrate in the presence of the monocarboxylate ion transport inhibitor a-cyano-hydroxy-cinnamate (CHC) yielded /, values commensurate with those measured using NH4Cl. Application of CHC alone lead to a rapid fall in pH,, suggesting a significant contribution of the monocarboxylate transporter to pHi regulation. /3i was also estimated from a step increase in extracellular PCo2* This yielded a value of 11 mM at an average pH, of 7 1, which is similar to that of the other estimates reported here. /3j was found to increase with decreasing pH,: each unit drop in pH, increased buffering power by about 60 %. Blockade of pHi regulation did not significantly affect estimates of #3. The change in buffering power with pH could be closely modelled from the known concentrations of free amino acids and organic phosphates.

On the role of bicarbonate as a hydrogen ion buffer in rat CNS neurones

“…Mean total buffering power of four cells subjected to these two conditions was estimated to be 6*03 mm (pH unit)-1 at a mean pHi of 7-25 in Cl--containing media and 7.45 mM (pH unit)-1 at a mean pHi of 7 24 in Cl--free media. We have already reported that values of intrinsic buffering power measured from cells bathed in Na+-containing and Na+free solutions were not significantly different (see Amos & Richards, 1996) and estimates of AT under the same conditions also showed no significant difference (n = 5 cells). Estimates offT in the presence of acetazolamide It has been suggested (Thomas, 1976) that the presence of carbonic anhydrase (CA) is necessary for the CO2-HCO3system to work to its full potential.…”

“…(1) The Hender-son-Hasselbalch equation is not valid in this situation. Since application of the Hendersor I -lasselbalch equation gave sensible and consistent estimates of intrinsic buffering when exogenous weak acids and weak bases were added and removed or when P(, was varied from ca 18 to 36 mmHg (see Amos & Richards, 1996), there seems every reason to believe it to apply in this system. In any event, such an explanation would raise as maLny difficulties as it would resolve.…”

“…Szatkowski & Thomas, 1989; Vaughan-Jones & Wu, 1990; Amos & Richards, 1996). In rat CNS neurones /3A increases from about 8 to 14 mm (pH unit)-' as pHi falls from 7.7 to 6.7 (see Amos & Richards, 1996, their Fig. 6).…”

“…Some of these findings have been presented to The Physiological Society in a preliminary form (Amos, Pocock & Richards, 1993). METHODS Neonatal rats (3-4 days old) were anaesthetized with isoflurane and then decapitated.…”

“…Vaughan-Jones & Wu, 1990) or by takiing a reading of' pH, immediately after the addition of the base (e.g. Amos & Richards, 1996). The justificationi for the latter procedure is that, for rat CNS neurones, the maximum displacement of pHi occurs within 2 s of the addition of base while the half-time for the subsequent decline in pHi is of the or(der of' 1 min or more (i.e.…”