Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus

Abstract

We studied the regulation of intracellular pH (pHi) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pHi. Intrinsic buffering power (βI) was 10.5 mM (pH unit)−1 at pHi 7.0, and decreased linearly with pHi; the best-fit line to the data had a slope of −10.0 mM (pH unit)−2. In the absence of HCO3 −, pHi recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pHi 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pHi. Acid extrusion was inhibited 68% (pHi 6.23) by substituting Li+ for Na+ in the bath solution. Switching from a CO2/HCO3 −-free to a CO2/HCO3 −-containing bath solution caused mean steady state pHi to increase from 6.82 to 6.90, due to a Na+-driven HCO3 − transporter. The HCO3 −-induced pHi increase was unaffected by amiloride, but was inhibited 75% (pHi 6.85) by 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and 65% (pHi 6.55–6.75) by pretreating astrocytes for up to ∼6.3 h with 400 μM 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). The CO2/HCO3 −-induced pHi increase was blocked when external Na+ was replaced with N-methyl-d-glucammonium (NMDG+). In the presence of HCO3 −, the Na+-driven HCO3 − transporter contributed to the pHi recovery from an acid load. For example, HCO3 − shifted the plot of acid-extrusion rate vs. pHi by 0.15–0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO3 − increased acid extrusion 3.8-fold (pHi 6.20). When astrocytes were acid loaded in amiloride, with Li+ as the major cation, HCO3 − failed to elicit a substantial increase in pHi. Thus, Li+ does not appear to substitute well for Na+ on the HCO3 − transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na+-driven HCO3 − transporter are the predominant acid extruders in astrocytes.

An acidic environment changes cyclin D1 localization and alters colony forming ability in gliomas

“…A pHi of 6.9-7.0 has been reported for normal astrocytes [6,37] and other normal brain cell types. Therefore the effect of the extracellular acidification observed in T98G and U87 glioma cells is not the effect of a non-physiological pHi, rather it appears to be a cell's response to their environment.…”

Chloride/anion channels in glial cell membranes

“…Given the relatively negative membrane potential, a passive distribution would result in an intracellular chloride concentration of ϳ7 mM. The intracellular chloride concentration of cultured astrocytes has been found to be Յ40 mM, using intracellular microelectrodes (Kettenmann, 1987); 43 mM and 45 mM, using radioactive chloride studies (Kimelberg, 1981;Walz and Mukerji, 1988, respectively); 36 mM with chloride-sensitive dyes (Bevensee et al, 1997); and 29 mM, using muscimol reversal potential in perforated patch measurements in which [Cl Ϫ ] i is left undisturbed (Bekar and Walz, 2002). Furthermore, in vivo estimates of cortical glial chloride concentration give values of 36 -46 mM (Smith et al, 1981).…”

Sleep and Epilepsy: A Key Role for Nitric Oxide?

“…The particularities of these impairments still remain to be further clarified. It may include local changes in pH because NO and NO releasers alter brain pH (33 It is also probable that the antiepileptic substances, like VPA and ESM, capable of triggering a release of NO, may counteract the SWD occurrence in balancing their cortical expression and in facilitating PS production within specific pontine structures. Brain glucose utilization may also be a restrictive factor for the alternative expression of PS or SWD in GAERS rats (34,35).…”