Growth Factors and Taurine Protect against Excitotoxicity by Stabilizing Calcium Homeostasis and Energy Metabolism

Calcium accumulation as a function of glutamate receptor activation. Cells were depolarized with various concentrations of glutamate, NMDA plus glycine (10 μm), and kainate plus MK-801 (10 μm) as indicated.⁴⁵Ca²⁺ accumulation was determined 15 min after depolarization. Each data point represents mean ± SEM of three experiments. ANOVA showed significant main effect of agonists (F_(2,30) = 85.30; p< 0.0001) and concentrations (F_(4,30)= 121.91; p < 0.0001). The interaction was also significant (F_(8,30) = 25.32;p < 0.0001). Post hoc tests indicated that 10, 100, and 1000 μm glutamate caused a significant increase in ⁴⁵Ca²⁺accumulation above control levels (p < 0.001), whereas 100 and 1000 μm NMDA were significantly different from controls (p < 0.001). Kainate in the presence of 10 μm MK-801 did not induce a significant increase above baseline. Ten micromolar the kainate receptor antagonist DNQX or 10 μm the NMDA receptor antagonist MK-801 completely blocked glutamate-induced⁴⁵Ca²⁺ accumulation.

Taurine and horse serum downregulate glutamate-induced calcium accumulation. Taurine modulation of glutamate-induced ⁴⁵Ca²⁺ accumulation was analyzed in early postnatal cerebellar granule cells cultured in serum-free medium and supplemented with different concentrations of HS (0, 2, and 10%, respectively). Cells were initially plated under serum conditions (10% HS plus 5% FCS). After 24 hr in vitro, cultures were switched to serum-free medium (15% N-2 supplement). At 3 days in vitro, 0.25 ml of culture medium was removed and replaced with 0.25 ml of fresh medium supplemented with HS to give the desired final concentration (0, 2, or 10%). Ten millimolar taurine was added at this time. Twenty-four hours later,⁴⁵Ca²⁺ accumulation was measured after depolarization with 1 mm glutamate for 30 min. Data represent mean ± SEM from at least three separate experiments. A two-way ANOVA showed a statistically significant interaction between HS concentrations and NAA treatment (F_(6,24) = 65.82; p< 0.0001). There was a significant main effect of HS concentrations (F_(2,24) = 200; p< 0.0001) and NAA treatment (F_(3,24) = 200; p < 0.0001). Post hoc tests showed that glutamate-induced ⁴⁵Ca²⁺accumulation was significantly higher than control (p < 0.001) under 0 and 2%, but not 10%, HS.

Time course accumulation of calcium under glutamate depolarization. A, Taurine was added simultaneously with glutamate. B, Cultures were pretreated with taurine (10 mm) for 24 hr before addition glutamate (1 mm). Each data point represents the mean ± SEM of three separate experiments. A two-way ANOVA showed a statistically significant main effect of taurine treatment (F_(2,48) = 33.88; p< 0.0001) and time of depolarization (F_(7,48) = 103.92;p < 0.0001). The interaction between taurine and time was also significant (F_(14,48) = 5.53; p < 0.0001). Post hoc tests indicated that glutamate caused a significant (p < 0.05) increase in⁴⁵Ca²⁺ accumulation after 5 min and thereafter (p < 0.0001). A, Only at 30 min did taurine significantly (p< 0.001) reduce ⁴⁵Ca²⁺ accumulation. InB, taurine induced a significant (p < 0.05) reduction 15 min after depolarization.

Interaction of GFs and taurine during calcium uptake modulation. Cells were pretreated with bFGF and BDNF (10 ng/ml) and with taurine (10 mm) or a combination thereof for 24 hr. Glutamate was added for 30 min, and⁴⁵Ca²⁺ and accumulation were measured as described. Data represent mean ± SEM from three separate experiments. The two-way ANOVA showed a statistically significant interaction between growth factors and NAAs (F_(9,32) = 25.10; p< 0.0001). There was also a significant main effect of growth factors (F_(3,32) = 32.09; p< 0.0001) and NAAs (F_(3,32) = 304.52;p < 0.0001). Post hoc tests indicated that glutamate significantly increased intracellular⁴⁵Ca²⁺ under all conditions examined (p < 0.005). bFGF or bFGF and BDNF caused a significant reduction in glutamate-induced⁴⁵Ca²⁺ accumulation (p < 0.001). Taurine alone or combined with GFs significantly reduced ⁴⁵Ca²⁺accumulation (p < 0.0005) in the presence of glutamate.

Regulation of [Ca²⁺]_i by taurine and bFGF. [Ca²⁺]_i was determined by confocal microscopy in cells attached to coverslips and loaded with 5 μm fluo-3. Images were acquired at 30 sec intervals.A–D show one set of data of one representative experiment. Each determination was repeated at least twice or more. Although the measurements vary from one experiment to another, the outcome was identical. Each point represents the mean ± SEM of [Ca²⁺]_i determined as indicated in Materials and Methods. A, Glutamate (1 mm) was applied directly to the recording chamber as indicated by thearrow and was present throughout the recording period (n = 73 cells). B, Cells were pretreated with 10 mm taurine for 24 hr before glutamate addition (n = 82). Taurine was also present during the recording. C, Cultures were pretreated for 24 hr with 10 ng/ml bFGF (n = 65). D, The extent of photobleaching of the signal intensities. The software and microscope settings were maintained the same as forA–C, but images were acquired at 20 sec intervals (n = 57).

Combination of taurine and bFGF counteracted the glutamate-induced decrease in MtECG. Cells were treated with BDNF (10 ng/ml), bFGF (10 ng/ml), or taurine (10 mm) as indicated. Twenty-four hours later, cells were depolarized with 100 μm glutamate for 30 min. Data represent mean ± SEM of three experiments. A two-way ANOVA involving the interactions between NAAs and GFs was significant (F_(6,24) = 2.65; p< 0.05). The main effect of NAAs was highly significant (F_(3,24) = 155.9; p< 0.0001). The effect of GFs was also significant (F_(2,24) = 30.35; p< 0.0001). Post hoc tests indicated that BDNF and bFGF induced significantly (p < 0.005) increased rhodamine uptake. Glutamate significantly (p< 0.01) reduced rhodamine uptake but not in cultures pretreated with taurine and bFGF compared with untreated controls.