Busch G L, Wiesinger H, Gulbins E, Wagner H J, Hamprecht B, Lang F
Institutes of Physiology, University of Tubingen, Germany.
Biochim Biophys Acta. 1996 Dec 4;1285(2):212-8. doi: 10.1016/s0005-2736(96)00163-0.
A variety of pathological conditions lead to swelling of astrocytes, which in turn stimulates ion release by activation of ion channels at the plasma membrane. In the present study, acridine orange and fluorescein isothiocyanate coupled to dextran (FITC-dextran) have been used to examine the effect of cell swelling on pH in acidic compartments of cultured astroglial cells. Both NH4Cl (2 mM) and chloroquine (10 microM), known to alkalinize acidic cellular compartments, led to the expected increase in acridine orange fluorescence intensity. Similar, albeit smaller, effects were elicited by a reduction of extracellular osmolarity (-80 mOsm) and treatment of the cells with glutamate (l mM), manoeuvres which enhanced cell volume. Determination of changes in the FITC-dextran fluorescence ratio (485/440 nm) allowed quantification of the pH changes in lysosomal compartments. Treatment with NH4Cl, reduced extracellular osmolarity and glutamate increased lysosomal pH by 0.65 +/- 0.07, 0.85 +/- 0.14 and 0.25 +/- 0.07, respectively. Measurement of cytosolic pH using 2',7',-bis-(2-carboxyethyl)-5- (and -6) carboxyfluorescein (BCECF) demonstrated a pronounced acidification following cell swelling, observed with both reduced extracellular osmolarity (by 0.23 +/- 0.05 pH units) and 1 mM glutamate (by 0.26 +/- 0.02 pH units). In conclusion, pH within lysosomes and possibly other acidic cellular compartments of astrocytes is increased by cell swelling, which may have important consequences for astrocyte function.
多种病理状况会导致星形胶质细胞肿胀，进而通过激活质膜上的离子通道刺激离子释放。在本研究中，吖啶橙和异硫氰酸荧光素偶联葡聚糖（FITC-葡聚糖）已被用于检测细胞肿胀对培养的星形胶质细胞酸性区室中pH的影响。已知可使细胞酸性区室碱化的氯化铵（2 mM）和氯喹（10 μM）均导致吖啶橙荧光强度出现预期增加。细胞外渗透压降低（-80 mOsm）以及用谷氨酸（1 mM）处理细胞（这些操作会增加细胞体积）也引发了类似但较小的影响。通过测定FITC-葡聚糖荧光比率（485/440 nm）的变化，可以对溶酶体区室中的pH变化进行定量。用氯化铵处理、降低细胞外渗透压以及使用谷氨酸分别使溶酶体pH升高了0.65±0.07、0.85±0.14和0.25±0.07。使用2',7'-双（2-羧乙基）-5-（和-6）羧基荧光素（BCECF）测量胞质pH表明，在细胞肿胀后出现了明显的酸化，细胞外渗透压降低（降低0.23±0.05个pH单位）和使用1 mM谷氨酸（降低0.26±0.02个pH单位）时均观察到这种情况。总之，星形胶质细胞溶酶体以及可能其他酸性细胞区室中的pH会因细胞肿胀而升高，这可能对星形胶质细胞功能产生重要影响。
