Taurine Efflux and Intracellular pH during Astrocyte Volume Regulation
Cytotoxic cerebral edema is characterized by enlarged astroglial cells. In tissue culture, osmotically swollen astrocytes return toward normal volume over a period of 15–30 min in a process termed regulatory volume decrease (RVD). RVD is due, in part, to net efflux of taurine and other amino acids. Our objective in these studies was to examine changes in astrocyte intracellular pH (pHi) which may be related to taurine loss during RVD. We hypothesized net efflux of anionic taurine abandons a proton inside the cell, thus lowering pHi. Primary cultures of cerebral astrocytes were prepared from neonatal rat pups and grown on glass coverslips. Confluent cells were loaded at 37°C with the fluorescent pH indicator BCECF. Fluorescence intensity ratios for excitation wavelengths of 440 nm and 494 nm (530 nm emission) were computed every 2 sec. Intensity ratios were calibrated to pHi at the end of each experiment using 140 mM KCl plus 8.6 μM nigericin at pH 7.4. pHi was measured in isoosmotic Hepes-buffered saline (290 mOsm) and then in hypoosmotic Hepes-buffered saline (200 mOsm) in the presence of 0.5 mM amiloride. Some solutions also contained 150 μM niflumic acid (NA). Cellular taurine content was determined in parallel studies using HPLC. Changes in pHi were compared between groups using Student’s t-test with Bonferroni correction. Significance was assumed if p<0.05. In isoosmotic saline, mean ± SEM pHi was 7.58 ± 0.04 and decreased to 7.35 ± 0.09 after adding amiloride. Hypoosmotic exposure caused a further drop in pHi of 0.29 ± 0.03 within 15 min. Recovery of pHi in isoosmotic saline was amiloride-sensitive. Subsequent hypoosmotic exposure after recovery in isoosmotic saline produced a change in pHi which was 81 ± 9% of the change measured during the initial hypoosmotic exposure. Taurine content decreased from 147 ± 6 nmol/(mg protein) to 116 ± 7 nmol/(mg protein) during the 15 min hypoosmotic exposure in 0.5 mM amiloride. NA significantly reduced the hypoosmotically induced change in pHi to 0.17 ± 0.02 while completely blocking taurine loss. Assuming an intracellular buffering power of 13 mM, the NA-sensitive intracellular acidification of cells during hypoosmotic exposure in the presence of 0.5 mM amiloride corresponds to 1.6 mequiv/l additional intracellular H+. This increase in intracellular H+ content is equivalent to approximately 32% of the NA-sensitive loss of taurine. The association of changes in pHi with taurine efflux is supported by these data; however, efflux of other weak acids may contribute to intracellular acidification during astrocyte RVD and a significant portion of taurine must leave the cell with a proton.
KeywordsRegulatory Volume Decrease Niflumic Acid Intracellular Acidification Fluorescence Intensity Ratio Taurine Content
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