Polyamine Uptake in Cultured Astrocytes

Abstract

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...

The Role of Glia in Stress

“…A solution to their origins comes from the idea that PAs are probably taken up from external sources by glial cells. 114, 126, 194 It has been hypothesized that PAs are taken up by glia from the blood circulation, cerebral spinal fluid (CSF) or from macrophages penetrating the blood-brain barrier. One of the possible pathway is the organic cation transporter (OCT) system which is expressed in glia.…”

“…9, 78, 80, 239–244 It was suggested that PAs could be taken up from external sources by glial cells via hypothetical transporters which would bring PAs into the cells 114, 126, 194,195 by transporters localized at the endfeet that are attached to blood vessels and brain ventricles. According to this view, the blood circulation and CSF would be the major sources of PA uptake.…”

“…Recent data show that SPD may be taken up by transporters such as polyspecific electrogenic organic cation transporters SLC22A subfamily 196 that were suggested as a pathway for monoamines such as dopamine, tetraethylammonium and others, 126, 127, 194, 195 that are indeed also well suited for PAs. 196 Such transporters may function in a reverse mode releasing PAs by exchanging with other OCT substrates 245 , or, as it was shown for glutamate reverse transport 246 or dopamine reverse transport 247, 248 with similar and different mechanisms.…”

Neuronal and Glial Responses to Polyamines in the Ischemic Brain

“…However, little is known about mechanisms underlying transport, uptake and release of polyamines in neurons and glia. Polyamines are taken up in cerebral cortex slices , cultured cerebellar astrocytes (Dot et al, 2000) and synaptosomes (Gilad and Gilad, 1991). In a recent research paper, Masuko et al, characterized polyamine transport systems in synaptic vesicles, synaptosomes and glial cells, as well as the release of spermine from hippocampal slices.…”

“…Polyamines are taken up with Km values in a micromolar or high nanomolar range in many cases, whereas polyamine transport systems have relatively low affinities for polyamines (Km: several hundreds micromolar range) in synaptosomes and glial cells (Dot et al, 2000;Masuko et al, 2003). However, Vmax values of polyamine transporters are greater in synaptosomes and glial cells than those in bovine lymphocytes (Kakinuma et al, 1988).…”