UBC Theses and Dissertations
Regulation of intracellular pH in cultured foetal rat hippocampal pyramidal neurones Baxter, Keith Allen
The mechanisms regulating intracellular pH (pH[sub i]) were investigated in cultured foetal rat hippocampal pyramidal neurones loaded with the pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(or 6)-carboxyfluorescein. At room temperature (~20°C), steady-state pH[sub i] was 6.85 in the nominal absence of external HCO₃⁻, and increased to 7.15 in the presence of HCO₃⁻. In HCO₃⁻-free medium at 37°C, steady-state pH[sub i] rested at the substantially higher level of 7.23, whereas in HCO₃⁻ -containing solutions at 37°C, pH[sub i] was reduced to 7.13. Regardless of temperature and in the absence of HCO₃⁻, the removal of extracellular Na caused an immediate and sustained intracellular acidification, suggesting the dominance of a Na⁺-dependent mechanism(s) maintaining steady-state pH[sub i]. In HCO₃⁻/CO₂- buffered medium at room temperature, a moderate intracellular acidification was observed during the application of the anion exchanger inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) or after the removal of HCO₃⁻, indicating the contribution of HCO₃⁻/CI- exchange to the maintenance of baseline pH[sub i]. Moreover, this anion exchanger could participate in pH[sub i] regulation even in the absence of extracellular Na⁺. At 37°C, however, DIDS did not alter steady-state pH[sub i] in the presence of HCO3-. Though extremely sensitive to the removal of extracellular Na⁺ at both temperatures, neither steady-state pH[sub i] nor the rate of pH[sub i], restoration from an imposed acid load were influenced by the application of ethylisopropylamiloride, a potent inhibitor of Na⁺/H+ exchange. Following an NH4+ -induced intracellular acidification, the rate of pH[sub i] recovery to baseline levels was faster at 37°C than at room temperature. Furthermore, in contrast to experiments performed at room temperature, the addition of HCO₃⁻ to the perfusate did not increase the rate of pH[sub i] recovery at 37°C. The results of this study suggest that at 37°C, the dominant regulator of pH[sub i] in hippocampal neurones is a Na⁺-dependent, HCO₃⁻-independent acid extrusion mechanism (probably an amiloride insensitive variant of the Na⁺/H+ exchanger). At room temperature, this Na⁺-dependent acid extrusion mechanism remains active, but the regulation of pH[sub i] appears to be supplemented by the activity of a Na⁺-independent HCO₃⁻/CI- exchanger.
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