Signal Transduction in Astrocytes during Chronic or Acute Treatment with Drugs (SSRIs, Antibipolar Drugs, GABA-ergic Drugs, and Benzodiazepines) Ameliorating Mood Disorders
Figure 13
Glycogenolytic effect of GABA and/or addition of 5 mM K+, indicated as reduction of glycogen content, in astrocytes. Cultured astrocytes were incubated for 20 min in DMEM (containing 7.5 mM glucose) without any addition (control), addition of 5 mM K+ to a final extracellular concentration of 10 mM (+5 K+), of γ-aminobutyric acid (GABA) to a final concentration of 100 μM (GABA), or of GABA and K+ (GABA plus +5 K+). After the incubation, the astrocytes were washed three times with ice-cold phosphate-buffered saline (PBS) and sonicated in 30 mM HCl. The suspension was used to measure nonhydrolyzed glycosyl units of glycogen. Three 50 μL aliquots were sampled. In the first aliquot, 150 μL of acetate buffer (0.1 M, pH 4.65) was added. In the second, 150 μL of a solution containing 1% amyloglucosidase (10 mg/mL) in the acetate buffer was added in order to degrade remaining glycogen to glucose, and the mixture was incubated at room temperature for 30 min. Subsequently, the two aliquots were treated identically. Two mL of Tris-HCl buffer (0.1 M, pH 8.1) containing 3.3 mM MgCl2, 0.2 mM ATP, 25 μg/mL NADP, 4 μg/mL hexokinase, and 2 μg/mL glucose-6-phosphate dehydrogenase was added to each, and the mixture was incubated at room temperature for 30 min. The fluorescence of the NADPH formed in amounts equivalent to glucose metabolized by hexokinase was then read (excitation 340 nm; emission 450 nm). The first aliquot measures the sum of glucose and glucose-6-phosphate in the tissue, whereas the second aliquot in addition to those also measures the glycosyl units from glycogen remaining in the tissue. Determination of the difference between these two aliquots provides a measurement of the amount of the latter. The third aliquot was used to measure the protein content by the Lowry method to normalize the glycogen contents (nmol) per mg protein. Average glycogen contents were indicated as percentages of those under control conditions. All values were expressed as means ± S.E.M indicated by vertical bars and were from three-five individual cultures. *Statistically significant () difference from control. Results are unpublished experiments by J. Xu, D. Song, L. Hertz, and L. Peng.
