Activation of AMP-activated protein kinase by tributyltin induces neuronal cell death
Introduction
AMP-activated protein kinase (AMPK) is a well-conserved heterotrimeric protein kinase that senses energy state. The control of AMPK activity is complex and involves allosteric stimulation by AMP, as well as phosphorylation by AMPK kinase. Phosphorylation of Thr-172 in the α-subunit is required for AMPK activation (Young et al., 2005). Under normal conditions, the very low AMP/ATP ratio keeps AMPK in the inactive form, while an increase of the AMP/ATP ratio activates AMPK. In peripheral tissues, this activation can acutely regulate cellular metabolism and chronically regulate gene expression to restore ATP levels (Zhou et al., 2000, Woods et al., 2000). For example, AMPK enhances fatty acid oxidation and glucose uptake (Iglesias et al., 2004, Smith et al., 2005). AMPK is abundantly expressed in neurons, such as cultured cortical and hippocampal neurons, in addition to peripheral tissues (Culmsee et al., 2001, Landree et al., 2004), but little is known about its role in neurons. AMPK is involved in cell death, in addition to cell survival. For example, McCullough et al. (2005) reported that AMPK promotes neuronal injury in stroke, and Garcia-Gil et al. (2003) demonstrated that 5′-aminoimidazole-4-carboxamide riboside (AICAR), an AMPK activator, induces apoptosis in neuroblastoma cells.
Tributyltin chloride (TBT) has been widely used as a component of antifouling paint. Although the use of TBT in antifouling paints has been restricted recently (van Wezel and van Vlaardingen, 2004), TBT and its degradation products, dibutyltin and monobutyltin, will remain in marine sediments for some years. Human exposure to organotin compounds arises from drinking water that has been contaminated with industrial effluents and through leaching of the compounds from polyvinyl chloride water pipes (Snoeij et al., 1987). Tsuda et al. (1995) reported that the daily intake of TBT in Japan was 2.2 to 6.9 μg, and Whalen et al. (1999) reported the presence of butyltin compounds, including TBT, at concentrations between 50 nM and 400 nM in human blood.
It is known that one of the targets of TBT toxicity is neurons (O'Callaghan and Miller, 1988). We have recently reported that TBT induces cell death in cultured rat cortical neurons (Nakatsu et al., 2006). It increased extracellular glutamate concentration, and overactivation of glutamate receptors generated reactive oxygen species and phosphorylated extracellular-regulated kinase (ERK) (Nakatsu et al., 2006). The mechanism of TBT-induced glutamate release remains unknown, but TBT was reported to disrupt mitochondrial function by inhibiting ATP synthase, resulting in a decrease of ATP content (von Ballmoos et al., 2004). We considered that the ATP reduction might lead to glutamate release through a mediator such as AMPK.
Therefore, we investigated whether AMPK activation is involved in TBT-induced extracellular glutamate release and cell death in cultured rat cortical neurons. Our results suggest that AMPK is activated by TBT and this activation induces neuronal death through extracellular glutamate release.
Section snippets
Materials
Eagle's minimal essential salt medium (Eagle's MEM) was purchased from Nissui Pharmaceutical (Tokyo, Japan). Fetal calf serum (FCS) and horse serum (HS) were purchased from JRH Biosciences (Lenexa, KS). Tributyltin chloride, trypan blue, NaHCO3 and glucose were purchased from Wako (Osaka, Japan). Glutamine, arabinocylcytosine and protease inhibitor cocktail were purchased from Sigma (St. Louis, MO). Compound C were purchased from Calbiochem (Darmstadt, Germany). Primary antibodies and
Effect of TBT on intracellular ATP
Firstly, we examined the effect of TBT on the intracellular ATP levels in cultured cortical neurons, because it has been reported that TBT inhibits ATP synthase (Matsuno-Yagi and Hatefi, 1993, von Ballmoos et al., 2004). Exposure of cells to 500 nM TBT for 24 h significantly decreased intracellular ATP (Fig. 1A), although less than 500 nM TBT had no effect. Intracellular ATP levels declined rapidly within 30 min after 500 nM TBT exposure and the reduction was time-dependent (Fig. 1B).
Involvement of AMPK in TBT-induced neurotoxicity
Because
Discussion
In this study, we have demonstrated two new aspects of TBT neurotoxicity. One is that TBT-induced cell death is mediated via phosphorylation of AMPK. The other is that AMPK is involved in extracellular glutamate release caused by TBT.
We confirmed that most neurons in our culture system express AMPK by using total-AMPK antibody (Fig. 2A). This is in agreement with the finding by Landree et al. (2004) that AMPK is abundantly expressed in cortical neurons.
As shown in Fig. 1B, 500 nM TBT
Acknowledgments
We thank Ms. Naoko Takai for her technical assistance. We also thank the Analysis Center of Life Science and the Research Center for Molecular Medicine, Hiroshima University for the use of their facilities. This study was supported in part by a Grant-in-Aid for young scientists (B) 17790099 and 19790106 (to Y.K.) from the Ministry of Education, Science, Sports, and Culture, Japan, and by a Satake Research Grant (to Y.K.) from Hiroshima University Foundation.
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