Abstract
TRPM7, a divalent cation channel, plays an important role in neurons damaged from cerebral ischemia due to permitting intracellular calcium overload. This study aimed to explore whether magnesium was transported via a TRPM7 channel into the intracellular space of rat hippocampal neurons after 1 h of oxygen-glucose deprivation (OGD) and acute chemical ischemia (CI) by using methods of the Mg(2+) fluorescent probe Mag-Fura-2 to detect intracellular magnesium concentration ([Mg(2+)](i)) and flame atomic absorption spectrometry to measure extracellular magnesium concentration ([Mg(2+)](o)). The results showed that the neuronal [Mg(2+)](i) was 1.51-fold higher after 1 h of OGD at a basal level, and the increase of neuronal [Mg(2+)](i) reached a peak after 1 h of OGD and was kept for 60 min with re-oxygenation. Meanwhile, the [Mg(2+)](o) decreased after 1 h of OGD and recovered to the pre-ischemic level within 15 min after re-oxygenation. In the case of CI, the [Mg(2+)](i) peak immediately appeared in hippocampal neurons. This increase of [Mg(2+)](i) declined by removing extracellular magnesium in OGD or CI. Furthermore, by using Gd(3+) or 2-aminoethoxydiphenyl borate to inhibit TRPM7 channels, the [Mg(2+)](i) increase, which was induced by OGD or CI, was attenuated without altering the basal level of [Mg(2+)](i). By silencing TRPM7 with shRNA in hippocampal neurons, it was found that not only was the increase of [Mg(2+)](i) induced by OGD or CI but also the basal levels of [Mg(2+)](i) were attenuated. In contrast, overexpression of TRPM7 in HEK293 cells exaggerated both the basal levels and increased [Mg(2+)](i) after 1 h of OGD/CI. These results suggest that anoxia induced the increase of [Mg(2+)](i) via TRPM7 channels in rat hippocampal neurons.
Highlights
China Grant 30870793. □S The on-line version of this article contains supplemental “Experimental Procedures,” “Results,” and Figs. 1–5. 1 Both authors contributed to this work. 2 To whom correspondence should be addressed
oxygen-glucose deprivation (OGD) or chemical ischemia (CI) Induces the Increase of Mag-Fura-2 Fluorescence Intensity That May Reflect [Mg2ϩ]i in Rat Hippocampal Neurons—We first examined whether OGD/reperfusion or CI, which mimics the condition of brain ischemia [12, 27], affects the intracellular magnesium in cultured rat hippocampal neurons using a magnesium indicator Mag-Fura-2
When neurons were reperfused for 30 min following 1 h OGD, the [Mg2ϩ]i increase was on the decline but still above the basal level
Summary
China Grant 30870793. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental “Experimental Procedures,” “Results,” and Figs. 1–5. 1 Both authors contributed to this work. 2 To whom correspondence should be addressed. Previous results from our laboratory are consistent with others that have shown the TRPM7 channel contributes to neuronal death after ischemia [14]. This indicates that TRPM7 is an essential mediator of ischemic death. Whether neuronal [Mg2ϩ]i3 changes during the anoxia conditions and whether TRPM7 channels play a role in neuronal magnesium dynamic movement in physiological or ischemic levels have never been checked. Using the classic models of cerebral ischemia injury in vitro, we investigated the effects of oxygen-glucose deprivation and acute chemical ischemia on the magnesium dynamic movement in rat hippocampal neurons and HEK293 cells and explored the possible pathway-TRPM7 channel
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