Abstract:The properties and regulation of the polyamine transport system in brain are still poorly understood. The present study shows, for the first time, the existence of a polyamine transport system in cerebellar astrocytes and suggests that polyamine uptake is mediated by a single and saturable high-affinity transport system for putrescine, spermine, and spermidine (K m ϭ 3.2, 1.2, and 1.8 M, respectively). Although substitution of NaCl by choline chloride produced a decrease in the putrescine, spermine, and spermidine uptake, it seems that polyamine transport in cerebellar astrocytes is not mediated by an Na ϩ cotransport as in the presence of Na ϩ and cholinium, polyamine uptake was much lower than when measured in a sucrose-based medium. On the other hand, ouabain, gramicidin (a Na ϩ ionophore), and ionomycin (a Ca 2ϩ ionophore) produced a strong inhibition of polyamine uptake, suggesting that membrane potential could have an important role in the functioning of the astroglial polyamine uptake system. Moreover, protein kinase C inhibition produced an enhancement of polyamine uptake, whereas stimulation of protein kinase C with phorbol esters inhibited polyamine uptake. Alternatively, the tyrosine kinase inhibitor genistein caused a marked reduction in the uptake. No effects on polyamine uptake were observed with inhibitors and activators of cyclic AMP-dependent protein kinase or when Ca 2ϩ /calmodulin-dependent protein kinase II was inhibited with KN-62. These results suggest that the polyamine uptake system in cerebellar astrocytes could be modulated by protein kinase C and tyrosine kinase activities. Key Words: Uptake-Polyamines-Astrocytes-Protein kinases-Spermine-Spermidine-Putrescine. J. Neurochem. 75, 1917Neurochem. 75, -1926Neurochem. 75, (2000.Spermine, spermidine, and putrescine are ubiquitous polyamines that are synthesized from ornithine in a pathway that is controlled mainly by the rate of ornithine decarboxylase activity (Tabor and Tabor, 1984;Pegg and McCann, 1988). In the CNS, polyamines have been associated with normal growth and differentiation, and the changes in ornithine decarboxylase activity appear to closely follow the region-specific pattern of cell proliferation (Slotkin and Bartolome, 1986). Furthermore, polyamines have been reported to play a significant role in nerve growth and nerve regeneration (Ingoglia et al., 1982;Gilad and Gilad, 1983).In addition, intracellular polyamines have been described to have other roles in the CNS as second messengers (Koenig et al., 1983;Iqbal and Koenig, 1985) or as modulators of neuronal ion channels (Bowie and Mayer, 1995;Donevan and Ragawski, 1995). In particular, activity-dependent relief of the blocking effect of polyamines on ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors has been recently suggested to contribute to short-term plasticity in local cortical circuits (Rozov and Burnashev, 1999).Brain polyamines like putrescine, spermine, and spermidine can be released, in a Ca 2ϩ -dependent manner, by depolarizing co...

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Ceramide is able to induce the apoptotic death of cerebellar granule cells (CGC) in culture. However, previous reports did not agree on whether ceramide-induced apoptosis of CGC requires caspase activation. Here we have shown that addition of C2-ceramide is able to produce extensive death of cultured CGC, which is associated with chromatin condensation, ladder-like DNA fragmentation, and activation of caspases. Our results show that C2-ceramide activates caspases-3, -9, and -2 but not caspases-1 and -8. Caspase-9 activation was associated with cytochrome c release from mitochondria toward the cytosol and was followed by activation of caspases-2 and -3. PARP proteolysis was also observed after caspase-3 and -2 activation. The involvement of caspases-9, -3, and -2 in ceramide-mediated apoptotic death of CGC was further supported by the use of specific inhibitors.

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