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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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The pharmacological characteristics of neuronal HAC.The left panel recordings are HACs of 5 different pyramidal neurons. After 25 min hypo exposure and HAC induction, a given VRAC inhibitor was added in the perfusion solutions as indicated, and the drug treatments were lasted for 30 min. The anion currents were induced by the alternate voltage pulses (see description in Fig. 1A). At the time points indicated as “a (control in iso)”, “b (25 min in hypo)” and “c” (30 min after addition of a putative VRAC inhibitor), the voltage step protocol described were delivered as in Fig. 1A and the resulting I-V curves are shown on the right hand panel next to the recording. According to the results presented in Fig. 1A, the amplitude of HAC should increase over a total of 55 min in hypo medium. Thus, should a putative VRAC inhibitor be effective, the HAC would be inhibited. Otherwise, HAC would further increase in the following 30 min of hypo stimulation. Accordingly, 30 µM IAA-94 (A1) and 100 µM NPPB (B1) were identified as effective inhibitors, while 10 µM tamoxifen (TAM, C1), 10 µM DCPIB (D1) and 500 µM DNDS (E1) were ineffective. I-V curves represent the mean ±SEM (vertical bars) for 4–6 recordings. **. Indicates a statistical significance of difference at p≤0.01 in the +40 mV step induced current amplitudes between the time points of “b” and “c”. The difference in the current amplitudes induced by voltage steps more positive than +40 mV also reached the same p value.
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pone-0016803-g002: The pharmacological characteristics of neuronal HAC.The left panel recordings are HACs of 5 different pyramidal neurons. After 25 min hypo exposure and HAC induction, a given VRAC inhibitor was added in the perfusion solutions as indicated, and the drug treatments were lasted for 30 min. The anion currents were induced by the alternate voltage pulses (see description in Fig. 1A). At the time points indicated as “a (control in iso)”, “b (25 min in hypo)” and “c” (30 min after addition of a putative VRAC inhibitor), the voltage step protocol described were delivered as in Fig. 1A and the resulting I-V curves are shown on the right hand panel next to the recording. According to the results presented in Fig. 1A, the amplitude of HAC should increase over a total of 55 min in hypo medium. Thus, should a putative VRAC inhibitor be effective, the HAC would be inhibited. Otherwise, HAC would further increase in the following 30 min of hypo stimulation. Accordingly, 30 µM IAA-94 (A1) and 100 µM NPPB (B1) were identified as effective inhibitors, while 10 µM tamoxifen (TAM, C1), 10 µM DCPIB (D1) and 500 µM DNDS (E1) were ineffective. I-V curves represent the mean ±SEM (vertical bars) for 4–6 recordings. **. Indicates a statistical significance of difference at p≤0.01 in the +40 mV step induced current amplitudes between the time points of “b” and “c”. The difference in the current amplitudes induced by voltage steps more positive than +40 mV also reached the same p value.

Mentions: VRAC currents can be inhibited by several pharmacological agents [3], [27], [28], however, the VRAC currents recorded from different cell types varied in their sensitivities to these inhibitors, e.g., the VRAC of the cultured cortical neurons were sensitive to IAA-94, NPPB, phloretin, SITS and DIDS, but not to tamoxifen [11], [29]. Because the HAC induced by a 25 min hypo could always be reversed to the control level after hypo withdrawal, we used 25 min hypo exposure to establish the pharmacological profile of HAC induced from CA1 pyramidal neurons. The effect of a given inhibitor was analyzed by comparing the difference of the voltage steps induced currents prior and at the end of drug treatment as indicated by the arrows in Fig. 2 (the steps induced current traces are not shown). The percentage inhibition of HAC by a given inhibitor was measured at the +100 mV step. All the I-V curves presented in Fig 2 were the averaged current amplitudes from a group of cells (n = 4–6). At +100 mV, the HAC were almost completely inhibited by 100 µM NPPB (97.6±5.0%, n = 4) and by 300 µM IAA-94 (88.5±5.0%, n = 5%) (Figs. 2, A1-2, B1-2). In contrast, 10 µM tamoxifen, 10 µM DCPIB and 400 µM DNDS increased the HAC by 30.2±11.0% (n = 4), 46.8±9.0% (n = 6) and 20.9±7.0% (n = 4), respectively (Fig. 2C1-2, D1-2, E1-2). This pharmacological profile is in good agreement with the VRAC reported from barrel cortex neurons in brain slices [12], [29]. Although test of type I Eisenman anion permeability sequence would provide additional evidence of VRAC involvement in HAC [30], however, the difficulty in achieving a full substitution of anions in the ambient of patched neurons with the use NaHCO3-based aCSF has limited us to pursue these data in slice recording.


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)

The pharmacological characteristics of neuronal HAC.The left panel recordings are HACs of 5 different pyramidal neurons. After 25 min hypo exposure and HAC induction, a given VRAC inhibitor was added in the perfusion solutions as indicated, and the drug treatments were lasted for 30 min. The anion currents were induced by the alternate voltage pulses (see description in Fig. 1A). At the time points indicated as “a (control in iso)”, “b (25 min in hypo)” and “c” (30 min after addition of a putative VRAC inhibitor), the voltage step protocol described were delivered as in Fig. 1A and the resulting I-V curves are shown on the right hand panel next to the recording. According to the results presented in Fig. 1A, the amplitude of HAC should increase over a total of 55 min in hypo medium. Thus, should a putative VRAC inhibitor be effective, the HAC would be inhibited. Otherwise, HAC would further increase in the following 30 min of hypo stimulation. Accordingly, 30 µM IAA-94 (A1) and 100 µM NPPB (B1) were identified as effective inhibitors, while 10 µM tamoxifen (TAM, C1), 10 µM DCPIB (D1) and 500 µM DNDS (E1) were ineffective. I-V curves represent the mean ±SEM (vertical bars) for 4–6 recordings. **. Indicates a statistical significance of difference at p≤0.01 in the +40 mV step induced current amplitudes between the time points of “b” and “c”. The difference in the current amplitudes induced by voltage steps more positive than +40 mV also reached the same p value.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037944&req=5

pone-0016803-g002: The pharmacological characteristics of neuronal HAC.The left panel recordings are HACs of 5 different pyramidal neurons. After 25 min hypo exposure and HAC induction, a given VRAC inhibitor was added in the perfusion solutions as indicated, and the drug treatments were lasted for 30 min. The anion currents were induced by the alternate voltage pulses (see description in Fig. 1A). At the time points indicated as “a (control in iso)”, “b (25 min in hypo)” and “c” (30 min after addition of a putative VRAC inhibitor), the voltage step protocol described were delivered as in Fig. 1A and the resulting I-V curves are shown on the right hand panel next to the recording. According to the results presented in Fig. 1A, the amplitude of HAC should increase over a total of 55 min in hypo medium. Thus, should a putative VRAC inhibitor be effective, the HAC would be inhibited. Otherwise, HAC would further increase in the following 30 min of hypo stimulation. Accordingly, 30 µM IAA-94 (A1) and 100 µM NPPB (B1) were identified as effective inhibitors, while 10 µM tamoxifen (TAM, C1), 10 µM DCPIB (D1) and 500 µM DNDS (E1) were ineffective. I-V curves represent the mean ±SEM (vertical bars) for 4–6 recordings. **. Indicates a statistical significance of difference at p≤0.01 in the +40 mV step induced current amplitudes between the time points of “b” and “c”. The difference in the current amplitudes induced by voltage steps more positive than +40 mV also reached the same p value.
Mentions: VRAC currents can be inhibited by several pharmacological agents [3], [27], [28], however, the VRAC currents recorded from different cell types varied in their sensitivities to these inhibitors, e.g., the VRAC of the cultured cortical neurons were sensitive to IAA-94, NPPB, phloretin, SITS and DIDS, but not to tamoxifen [11], [29]. Because the HAC induced by a 25 min hypo could always be reversed to the control level after hypo withdrawal, we used 25 min hypo exposure to establish the pharmacological profile of HAC induced from CA1 pyramidal neurons. The effect of a given inhibitor was analyzed by comparing the difference of the voltage steps induced currents prior and at the end of drug treatment as indicated by the arrows in Fig. 2 (the steps induced current traces are not shown). The percentage inhibition of HAC by a given inhibitor was measured at the +100 mV step. All the I-V curves presented in Fig 2 were the averaged current amplitudes from a group of cells (n = 4–6). At +100 mV, the HAC were almost completely inhibited by 100 µM NPPB (97.6±5.0%, n = 4) and by 300 µM IAA-94 (88.5±5.0%, n = 5%) (Figs. 2, A1-2, B1-2). In contrast, 10 µM tamoxifen, 10 µM DCPIB and 400 µM DNDS increased the HAC by 30.2±11.0% (n = 4), 46.8±9.0% (n = 6) and 20.9±7.0% (n = 4), respectively (Fig. 2C1-2, D1-2, E1-2). This pharmacological profile is in good agreement with the VRAC reported from barrel cortex neurons in brain slices [12], [29]. Although test of type I Eisenman anion permeability sequence would provide additional evidence of VRAC involvement in HAC [30], however, the difficulty in achieving a full substitution of anions in the ambient of patched neurons with the use NaHCO3-based aCSF has limited us to pursue these data in slice recording.

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