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Volume regulated anion channel currents of rat hippocampal neurons and their contribution to oxygen-and-glucose deprivation induced neuronal death.

Zhang H, Cao HJ, Kimelberg HK, Zhou M - PLoS ONE (2011)

Bottom Line: The OGD induced VRAC currents were significantly inhibited by inhibitors for glutamate AMPA (30 µM NBQX) and NMDA (40 µM AP-5) receptors in the OGD solution, supporting the view that induction of AD requires an excessive Na(+)-loading via these receptors that in turn to activate neuronal VRAC.In the presence of NPPB and DCPIB in the post-OGD reperfusion solution, the OGD induced CA1 pyramidal neuron death, as measured by TO-PRO-3-I staining, was significantly reduced, although DCPIB did not appear to be an effective neuronal VRAC blocker.Altogether, we show that rat hippocampal pyramidal neurons express functional VRAC, and ischemic conditions can initial neuronal VRAC activation that may contribute to ischemic neuronal damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China. zhanghq_04@yahoo.com

ABSTRACT
Volume-regulated anion channels (VRAC) are widely expressed chloride channels that are critical for the cell volume regulation. In the mammalian central nervous system, the physiological expression of neuronal VRAC and its role in cerebral ischemia are issues largely unknown. We show that hypoosmotic medium induce an outwardly rectifying chloride conductance in CA1 pyramidal neurons in rat hippocampal slices. The induced chloride conductance was sensitive to some of the VRAC inhibitors, namely, IAA-94 (300 µM) and NPPB (100 µM), but not to tamoxifen (10 µM). Using oxygen-and-glucose deprivation (OGD) to simulate ischemic conditions in slices, VRAC activation appeared after OGD induced anoxic depolarization (AD) that showed a progressive increase in current amplitude over the period of post-OGD reperfusion. The OGD induced VRAC currents were significantly inhibited by inhibitors for glutamate AMPA (30 µM NBQX) and NMDA (40 µM AP-5) receptors in the OGD solution, supporting the view that induction of AD requires an excessive Na(+)-loading via these receptors that in turn to activate neuronal VRAC. In the presence of NPPB and DCPIB in the post-OGD reperfusion solution, the OGD induced CA1 pyramidal neuron death, as measured by TO-PRO-3-I staining, was significantly reduced, although DCPIB did not appear to be an effective neuronal VRAC blocker. Altogether, we show that rat hippocampal pyramidal neurons express functional VRAC, and ischemic conditions can initial neuronal VRAC activation that may contribute to ischemic neuronal damage.

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OGD induced VRAC activation requires Na- +loading through glutamate receptors.A. In the presence of 40 µM AP-5 and 30 µM NBQX in the OGD solution to inhibit ionotropic glutamate receptors, the 25 min OGD induced neuronal electrophysiological changes seen in the Fig. 3A were largely attenuated. Also, the activation of VRAC in the reperfusion stage was significantly inhibited. B. Shows the differences in the outward current amplitudes between 6 min and 20 min post-OGD under the following conditions: 1) control; 2) in the presence of DNDS+ bicuculline +furosemide in the OGD and 3) in the presence of AP-5+NBQX in the OGD solution. * Indicates that the difference between the control and AP-5+NBQX groups was statistically significant at p<0.05.
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pone-0016803-g005: OGD induced VRAC activation requires Na- +loading through glutamate receptors.A. In the presence of 40 µM AP-5 and 30 µM NBQX in the OGD solution to inhibit ionotropic glutamate receptors, the 25 min OGD induced neuronal electrophysiological changes seen in the Fig. 3A were largely attenuated. Also, the activation of VRAC in the reperfusion stage was significantly inhibited. B. Shows the differences in the outward current amplitudes between 6 min and 20 min post-OGD under the following conditions: 1) control; 2) in the presence of DNDS+ bicuculline +furosemide in the OGD and 3) in the presence of AP-5+NBQX in the OGD solution. * Indicates that the difference between the control and AP-5+NBQX groups was statistically significant at p<0.05.

Mentions: Excessive Na+-loading through ionotropic glutamate receptors has been showed to initiate an impaired regulatory volume regulation that eventually lead to the necrotic cell death [12], [36]. We next asked whether OGD-induced glutamate receptor activation is required for the neuronal VRAC activation in the reperfusion stage. To address this question, neuronal NMDA inhibitor, 40 µM AP-5, and 30 µM of AMPA receptor inhibitors NBQX, were added together in the OGD solution. We found that not only was the OGD activated membrane currents decreased, but the AD that always occurred at the end of 25 min OGD treatment (Fig. 3), was also prevented. Noticeably, in the presence of AP-5 and NBQX in the OGD solution, the post-OGD anion conductance was significantly reduced by 53%. Specifically, the values of I20 min –I6 min were 444.5±75.1 pA in the OGD control group and 235.3±38.1 pA in the OGD+AP-5+NBQX group (n = 4 for each group, p<0.01,) (Fig. 5A–B). In contrast, addition of a cocktail containing DNDS/bicuculline/furosemide in OGD solution did not significantly affect the post-OGD VRAC currents (396.1±92.4 pA, n = 5, p>0.05, Fig. 5B). Student's t test was used for above data analyses. These results demonstrate that OGD induced stimulation of ionotropic glutamate receptors plays a crucial role in mediating neuronal VRAC activation.


Volume regulated anion channel currents of rat hippocampal neurons and their contribution to oxygen-and-glucose deprivation induced neuronal death.

Zhang H, Cao HJ, Kimelberg HK, Zhou M - PLoS ONE (2011)

OGD induced VRAC activation requires Na- +loading through glutamate receptors.A. In the presence of 40 µM AP-5 and 30 µM NBQX in the OGD solution to inhibit ionotropic glutamate receptors, the 25 min OGD induced neuronal electrophysiological changes seen in the Fig. 3A were largely attenuated. Also, the activation of VRAC in the reperfusion stage was significantly inhibited. B. Shows the differences in the outward current amplitudes between 6 min and 20 min post-OGD under the following conditions: 1) control; 2) in the presence of DNDS+ bicuculline +furosemide in the OGD and 3) in the presence of AP-5+NBQX in the OGD solution. * Indicates that the difference between the control and AP-5+NBQX groups was statistically significant at p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037944&req=5

pone-0016803-g005: OGD induced VRAC activation requires Na- +loading through glutamate receptors.A. In the presence of 40 µM AP-5 and 30 µM NBQX in the OGD solution to inhibit ionotropic glutamate receptors, the 25 min OGD induced neuronal electrophysiological changes seen in the Fig. 3A were largely attenuated. Also, the activation of VRAC in the reperfusion stage was significantly inhibited. B. Shows the differences in the outward current amplitudes between 6 min and 20 min post-OGD under the following conditions: 1) control; 2) in the presence of DNDS+ bicuculline +furosemide in the OGD and 3) in the presence of AP-5+NBQX in the OGD solution. * Indicates that the difference between the control and AP-5+NBQX groups was statistically significant at p<0.05.
Mentions: Excessive Na+-loading through ionotropic glutamate receptors has been showed to initiate an impaired regulatory volume regulation that eventually lead to the necrotic cell death [12], [36]. We next asked whether OGD-induced glutamate receptor activation is required for the neuronal VRAC activation in the reperfusion stage. To address this question, neuronal NMDA inhibitor, 40 µM AP-5, and 30 µM of AMPA receptor inhibitors NBQX, were added together in the OGD solution. We found that not only was the OGD activated membrane currents decreased, but the AD that always occurred at the end of 25 min OGD treatment (Fig. 3), was also prevented. Noticeably, in the presence of AP-5 and NBQX in the OGD solution, the post-OGD anion conductance was significantly reduced by 53%. Specifically, the values of I20 min –I6 min were 444.5±75.1 pA in the OGD control group and 235.3±38.1 pA in the OGD+AP-5+NBQX group (n = 4 for each group, p<0.01,) (Fig. 5A–B). In contrast, addition of a cocktail containing DNDS/bicuculline/furosemide in OGD solution did not significantly affect the post-OGD VRAC currents (396.1±92.4 pA, n = 5, p>0.05, Fig. 5B). Student's t test was used for above data analyses. These results demonstrate that OGD induced stimulation of ionotropic glutamate receptors plays a crucial role in mediating neuronal VRAC activation.

Bottom Line: The OGD induced VRAC currents were significantly inhibited by inhibitors for glutamate AMPA (30 µM NBQX) and NMDA (40 µM AP-5) receptors in the OGD solution, supporting the view that induction of AD requires an excessive Na(+)-loading via these receptors that in turn to activate neuronal VRAC.In the presence of NPPB and DCPIB in the post-OGD reperfusion solution, the OGD induced CA1 pyramidal neuron death, as measured by TO-PRO-3-I staining, was significantly reduced, although DCPIB did not appear to be an effective neuronal VRAC blocker.Altogether, we show that rat hippocampal pyramidal neurons express functional VRAC, and ischemic conditions can initial neuronal VRAC activation that may contribute to ischemic neuronal damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China. zhanghq_04@yahoo.com

ABSTRACT
Volume-regulated anion channels (VRAC) are widely expressed chloride channels that are critical for the cell volume regulation. In the mammalian central nervous system, the physiological expression of neuronal VRAC and its role in cerebral ischemia are issues largely unknown. We show that hypoosmotic medium induce an outwardly rectifying chloride conductance in CA1 pyramidal neurons in rat hippocampal slices. The induced chloride conductance was sensitive to some of the VRAC inhibitors, namely, IAA-94 (300 µM) and NPPB (100 µM), but not to tamoxifen (10 µM). Using oxygen-and-glucose deprivation (OGD) to simulate ischemic conditions in slices, VRAC activation appeared after OGD induced anoxic depolarization (AD) that showed a progressive increase in current amplitude over the period of post-OGD reperfusion. The OGD induced VRAC currents were significantly inhibited by inhibitors for glutamate AMPA (30 µM NBQX) and NMDA (40 µM AP-5) receptors in the OGD solution, supporting the view that induction of AD requires an excessive Na(+)-loading via these receptors that in turn to activate neuronal VRAC. In the presence of NPPB and DCPIB in the post-OGD reperfusion solution, the OGD induced CA1 pyramidal neuron death, as measured by TO-PRO-3-I staining, was significantly reduced, although DCPIB did not appear to be an effective neuronal VRAC blocker. Altogether, we show that rat hippocampal pyramidal neurons express functional VRAC, and ischemic conditions can initial neuronal VRAC activation that may contribute to ischemic neuronal damage.

Show MeSH
Related in: MedlinePlus