Steady-state intracellular pH and recovery from NH3/NH4+-induced intracellular acid loads in primary cultures of rat hippocampal neurons and astrocytes

Abstract

Intracellular pH (pH i ) homeostasis is of vital importance because pH i fluctuations alter the net charge on intracellular weak acids and bases (e.g., amino-acid residues of proteins, metabolic intermediates, solutes like inorganic phosphate), and can result in cellular dysfunction. The rate of pH i change (dpH i /dt) is determined by the balance between acid loading (e.g., metabolic production of H + , influx of H + , efflux of HCO 3 − via transmembrane proteins of the SLC4 family) and acid extrusion (e.g., efflux of H + via Na-H exchangers in the SLC9 family, influx of HCO 3 − by members of the SLC4 family). A powerful approach for studying pH i regulation is to impose an acute intracellular acid load using the NH 4 + /NH 3 prepulse technique, and then assess dpH i /dt during the subsequent pH i recovery. Using two such prepulses and recoveries (twin pulses), one can compare 2 experimental conditions. The goal of the present study is to develop a data-analysis approach that facilitates a meaningful presentation of all twin-pulse experiments in a data set. Using primary cultures (~2 weeks) of embryonic (E18–E20) rat hippocampal neurons and astrocytes on coverslips, we loaded cells with the ratiometric pH-sensitive fluorescent dye BCECF, subjected the cells to twin NH 4 + /NH 3 pulses, and calibrated BCECF at the end of each experiment with nigericin at pH=7.0. A standard 12-point nigericin calibration curve allowed us to compute pH i vs. time. Because we studied cells in 21% O 2 but the nominal absence of CO 2 /HCO 3 − , the pH i recoveries mainly reflect Na-H exchange. We fitted the pH i -recovery time course with a double exponential (DExp) to reduce bias. Nevertheless, in both neurons and astrocytes plots of dpH i /dt vs. pH i appeared rather linear, indicating that the pH i recoveries were “quasi” single exponential (SExp). To exploit the near-linearity, for each pH i recovery, we fitted the plot of dpH i /dt vs pH i with a line, the slope of which is the quasi-single exponential rate constant (kqSExp), and the x-intercept of which is the asymptotic pH i of the recovery. We summarize the twin pH i recoveries from individual cells as miniature plots (“thumbnails”) in which we display, for each recovery, both the dpH i /dt values (from the DExp fit of pH i vs. time) vs. pH i and the line of best fit for these dpH i /dt values vs. pH i . A single figure can replicate such twin-pulse experiments from many dozens of cells, so that a reader, at a glance, can surmise the essentials of each pH i recovery. The present work provides background data for additional work in which we studied pH i recoveries at a reduced [O 2 ], with or without CO 2 /HCO 3 − . R01 NS018400, R01 HL160857, R01 DK128315 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.