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Confocal microscope analysis of [Ca2+]i and45Ca2+ uptake studies showed that bFGF reduced the magnitude of glutamate-induced calcium uptake with no apparent regulation thereafter

Confocal microscope analysis of [Ca2+]i and45Ca2+ uptake studies showed that bFGF reduced the magnitude of glutamate-induced calcium uptake with no apparent regulation thereafter. WS 12 other hand, did not affect the level of WS 12 calcium uptake induced by glutamate but rather the duration of the maximal response; this maximal response was transient and returned to basal WS 12 levels 10 min after glutamate receptor stimulation. We conclude from these data that bFGF and taurine prevent glutamate excitotoxicity through regulation of [Ca2+]i and mitochondrial energy metabolism. Furthermore, the neuroprotective role of taurine and bFGF was enhanced by their collaboration. (Trenkner and Dykens, 1986;Trenkner, 1990), suggesting that the neuroprotective effect of taurine, as well as GFs, may be mediated primarily through modulation of intracellular calcium homeostasis. Energy metabolism is recognized as one of the fundamental processes necessary for the maintenance of neuronal structures and functions (Mattson et al., 1993; Beal, 1995). The activity of the mitochondrial electrochemical gradient (MtECG) and the amount of energy it produces are calcium-regulated (Hertz et al., 1988; White and Reynolds, 1995;Budd and Nicholls, 1996). Furthermore, mitochondria may be involved in glutamate toxicity (Schinder et al., 1996; White and Reynolds, 1996). Because the mitochondria have large capacity for calcium uptake (Nicholls and Akerman, 1982; Gunter et al., 1994), they might have a neuroprotective role by removing calcium from the cytoplasm (Budd and Nicholls, 1996; Stout et al., 1998). Mitochondria also have been found to be WS 12 essential in controlling certain apoptotic pathways (Green and Reed, 1998) through the release of caspase activators, such as cytochrome c (Liu et al., 1996) and apoptosis-inducing factors (Susin et al., 1996). We and others have demonstrated that depletion WS 12 of cellular energy levels increased the vulnerability toward excitotoxins, leading to cell death (Budd and Nicholls, 1995; Trenkner et al., 1996;Schinder et al., 1996). This study shows that a sustained rise in intracellular calcium levels and a decrease in Rabbit polyclonal to APEH MtECG were primarily responsible for the degenerative actions of glutamate, which can be controlled through different mechanisms by taurine and basic fibroblast growth factor (bFGF). MATERIALS AND METHODS Brain derived neurotrophic factor (BDNF) and bFGF were obtained from Promega (Madison, WI). Minimum essential medium (MEM), horse serum (HS), fetal calf serum (FCS), penicillin, streptomycin, and N-2 supplement were purchased from Life Technologies (Grand Island, NY). Culture dishes were obtained from Falcon (Lincoln Park, NJ). Trypsin and DNase were purchased from Worthington (Freehold, NJ). NMDA, kainate, and MK-801 came from Tocris Cookson (Bristol, UK). Fluo-3, fluorescein diacetate (FDA), and propidium iodide (PI), were obtained from Molecular Probes (Eugene, OR). Percoll, Triton X-100, cytosine arabinoside (Ara C), poly-d-lysine (PDL), rhodamine 123, taurine, and glutamate were obtained from Sigma (St. Louis, MO). 45CaCl2was received from Amersham Pharmacia Biotech (Arlington Heights, IL). All other chemicals were of high-quality cell culture grade. The development of cerebellar granule neurons depends on growth conditions. To characterize the role of glial cells and exogenously added growth factors on the survival and function of granule neurons, we used four culture conditions, as follows. (1) Mixed cultures: Cerebellar granule cells were prepared from 7-d-old mice as described previously (Trenkner and Sidman, 1977; Trenkner, 1991). Briefly, the entire cerebellum was removed, and single cell suspensions were prepared by trypsinization and trituration in 1% trypsin in Ca2+CMg2+-free isotonic phosphate buffer (CMF-PBS). Cells were washed in CMF-PBS and resuspended in culture medium (MEM), supplemented with 0.25% glucose, 2 mm glutamine, 10% HS, 5% FCS, and 25 U/ml both penicillin and streptomycin. Cells were seeded into PDL-coated dishes and incubated at 37C in a moist chamber under 5% CO2. (2) Enriched neuronal cultures: Mixed cultures were prepared as described above, but after 24 hr the medium was.