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Here we present an exciting finding that activation of delta-opioid receptor (DOR), which is highly expressed in the cortex, reduced anoxic Na+ influx and K+ leakage in the cortex by restricting Na+ influx through voltage-gated Na+ channels. |
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Furthermore, we show for the first time with direct evidence that DOR expression/activation indeed plays an inhibitory role in Na+ channel regulation by decreasing the amplitude of sodium currents and increasing activation threshold of Na+ channels. |
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DOR activation reduced anoxic Na+ influx in the cortex |
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To obtain the first evidence as to whether DOR activation reduces Na+ influx in anoxia, we measured the changes in extracellular [Na+] induced by anoxia in mouse cortical slices. |
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These results suggested that DOR activation inhibits anoxia-induced Na+ influx in the cortex. |
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DOR activation attenuated Na+-triggered K+ leakage in the cortex |
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Overloaded intracellular Na+ may trigger substantial K+ efflux that plays a key role in neuronal injury In our previous work, we have demonstrated that DOR-mediated inhibition of Na+ entry constitutes a major mechanism underlying the DOR-protection against anoxic K+ derangement in the cortex. |
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Also as shown in Table 1, the similar treatment as in Figure 1 (i.e., DOR activation with UFP-512) decreased K+ leakage by 39.06+-9.32% (p<0.001) and prolonged the anoxic response by 2.4+-0.3 times (p<0.001, n=14), accompanying with the reduction of anoxic Na+ influx. |
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Since the major route of Na+ influx is voltage-gated Na+ channels that are highly expressed in the cortex, we asked whether DOR targets at the Na+ influx through voltage-gated Na+ channels, thus attenuating K+ leakage. |
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Therefore, we applied TTX, a potent and specific voltage-gated Na+ channel blocker, to the cortical slices and tested its effect on DOR protection from anoxic K+ leakage. |
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These results suggest that anoxic K+ leakage was greatly dependent on Na+ influx via the voltage-gated Na+ channels and DOR signal may target at the voltage-gated Na+ channels to restrict Na+ influx and thus K+ leakage in the cortex. |
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DOR expression and activation down-regulated Na+ channel function |
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Since the major subtype of the voltage-gated Na+ channels in the cortex is Na1.2 channels, we further asked whether DOR truly plays an inhibitory role in the regulation of Na1.2 channels. |
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Because there is no specific activator/blocker for individual subtype of Na+ channels, we expressed Na1.2 channels in Xenopus oocytes and then tested the effects of DOR expression and activation on Na1.2 channel function. |
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3.3 Na+ currents were inhibited by DOR activation in oocytes with co-expression of DOR, but not in those without DOR expression |
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To determine the role of DOR in Na+ channel regulation, we co-expressed Na1.2 channels and DOR in the oocytes and then recorded sodium currents. |
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Because DOR density critically affects neuronal responses to hypoxia, we expressed DOR at different levels by injecting various amounts of DOR cRNA into the oocytes and then tested their effects on sodium currents. |
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To maximize effects of DOR expression, we extended the culture time to 72 hours in this set of the experiments since the protein expression increases with time after the injection of cRNA as shown above. |
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In the group with the injection of 1 ng DOR cRNA, activation of DOR with 5 microM of UFP-512 had little effect on sodium currents (n=5, data not shown). |
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When the injected DOR cRNA was increased to 10 ng, DOR activation with the same amount of UFP-512 significantly reduced the sodium current by 27.4% (p=0.01285, n=4) (Figure 5A). |
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When the injected DOR cRNA was increased to 20 ng, 5 microM of UFP-512 decreased sodium current amplitude by 35.2 % (p=0.03042, n=9; Figure 5C). |
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In the oocytes with a large amount of DOR cRNA (10 and 20 ng), UFP-512 significantly inhibited sodium currents within a broad range of depolarization potential (-30 to +40 mV) (Figure 5A, 5C). |
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Interestingly, our data suggest that DOR expression alone (without DOR activation) could reduce the size of sodium currents. |
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As shown in Figure 6, the size of sodium currents was much smaller in oocytes with injection of 20 ng DOR cRNA than in those with 10 ng DOR cRNA. |
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To further characterize the DOR-induced inhibition of sodium currents, we analyzed the conductance/amplitude-voltage relationship by fitting with a Boltzmann equation in the oocytes with injection of 10 and 20 ng of DOR cRNA. |
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Our results showed that DOR activation right shifted the curves (Figure 5B and 5D), suggesting that DOR activation not only reduced the size of sodium currents but also increased the threshold of sodium channel activation. |
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With DOR co-expression, the evoked peak currents appeared at different depolarization potentials, typically 4 different types of recordings, i.e., 20 mV, 0 mV, -10 mV, and -30 mV. |
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Despite of this difference, DOR activation could further inhibit sodium currents in all these oocytes with DOR expression (Figure 7). |
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3.5 DOR activation reduced Na+ channel currents in a dose-response manner |
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We performed a dose-response study to further clarify the DOR activation-induced inhibition of sodium currents. |
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In the oocytes with co-expression of Na1.2 channels (5 ng cRNA) and DOR (10 ng cRNA) under the same experimental conditions, we applied UFP-512 at concentrations of 0, 0.1, 0.5, 2.5 and 5 microM and compared their effects on the sodium currents (Figure 8). |
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The response of extracellular [Na+] to anoxia decreased when UFP-512 (1-5 microM), a specific and potent DOR agonist, was applied to the slices starting 20 min. |
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When the concentration was increased to 5 microM, the DOR-mediated inhibition of sodium currents could not be further strengthened (Figure 8E, 8F), suggesting a saturable dose-response manner in terms of the DOR agonist induced inhibition on the sodium currents. |
