DOR activation inhibits anoxic/ischemic Na+ influx through Na+ channels via PKC mechanisms in the cortex

Abstract

Activation of delta-opioid receptors (DOR) is neuroprotective against hypoxic/ischemic injury in the cortex, which is at least partially related to its action against hypoxic/ischemic disruption of ionic homeostasis that triggers neuronal injury. Na(+) influx through TTX-sensitive voltage-gated Na(+) channels may be a main mechanism for hypoxia-induced disruption of K(+) homeostasis, with DOR activation attenuating the disruption of ionic homeostasis by targeting voltage-gated Na(+) channels. In the present study we examined the role of DOR in the regulation of Na(+) influx in anoxia and simulated ischemia (oxygen-glucose deprivation) as well as the effect of DOR activation on the Na(+) influx induced by a Na(+) channel opener without anoxic/ischemic stress and explored a potential PKC mechanism underlying the DOR action. We directly measured extracellular Na(+) activity in mouse cortical slices with Na(+) selective electrodes and found that (1) anoxia-induced Na(+) influx occurred mainly through TTX-sensitive Na(+) channels; (2) DOR activation inhibited the anoxia/ischemia-induced Na(+) influx; (3) veratridine, a Na(+) channel opener, enhanced the anoxia-induced Na(+) influx; this could be attenuated by DOR activation; (4) DOR activation did not reduce the anoxia-induced Na(+) influx in the presence of chelerythrine, a broad-spectrum PKC blocker; and (5) DOR effects were blocked by PKCβII peptide inhibitor, and PKCθ pseudosubstrate inhibitor, respectively. We conclude that DOR activation inhibits anoxia-induced Na(+) influx through Na(+) channels via PKC (especially PKCβII and PKCθ isoforms) dependent mechanisms in the cortex.

Fig. 1

Effect of Na⁺ channel blockade on anoxia-induced Na⁺ influx in mouse cortical slices. Anoxia induced a sudden large drop in [Na⁺]o (A), which could be completely abolished in most slices (7 out of 11) by voltage-gated Na⁺ channel blocker, TTX (B). OGD test showed these slices in B had good viability (C). Of the remaining 4 (36%) TTX-treated slices that still responded to anoxia, two showed a reduced drop in anoxia-induced [Na⁺]o (D) and two had no appreciable changes in anoxia-induced [Na⁺]o drop. These results suggest that the blockade of voltage-gated Na⁺ channels in the cortex largely blocked the anoxia-induced Na⁺ influx in the cortex.

Fig. 2

Effect of DOR activation with UFP 512 (1, 5 μM) on anoxia-induced Na⁺ influx in mouse cortical slices. Trace recordings of A: control (Cont), B: UFP 512 (1 μM), C: UFP 512 (5 μM). D–F are statistical results of each recording parameter. *p<0.05, **p<0.01 as compared with the controls. Note that UFP 512 at 1 and 5 μM significantly attenuated anoxic decrease in [Na⁺]o, suggesting that DOR activation attenuates anoxic Na⁺ influx.

Fig. 3

Effect of DOR activation with UFP 512 on ischemia-induced Na⁺ influx in mouse cortical slices. Trace recordings of A: anoxia; B: oxygen-glucose deprivation (OGD); C: OGD+UFP 512 (5 μM). D–F are statistical results of each recording parameter. **p<0.01, ***p<0.001 as compared with anoxia; ^##p<0.01, ^###p<0.001 as compared with OGD. Note that OGD induced a sudden drop in [Na⁺]o to a greater extent than anoxia in all the slices investigated, which could be attenuated by UFP 512 (5 μM).

Fig. 4

DOR antagonist blocked the DOR attenuation of anoxic Na⁺ influx. Trace recordings of A: control (Cont), B: UFP 512 (5 μM), C: Naltrindole (NTI) (1 μM) and D: NTI+UFP 512. E–G are statistical results of each recording parameter. *p<0.05, **p<0.01, ***p<0.001 as compared with the controls; ^###p<0.001 vs. UFP 512. Note that NTI (1 μM) alone significantly increased anoxia-induced drop of [Na⁺]o, with a significant shortened Tmax and prolonged recovery from anoxic Na⁺ drop, suggesting a tonic activity of endogenous DOR. In the presence of NTI (1 μM), UFP 512 (5 μM) could not inhibit anoxic Na⁺ influx anymore.

Fig. 5

Effect of DOR activation on veratridine-enhanced anoxic drop in [Na⁺]o. Trace recordings of A: control (Cont), B: UFP 512 (5 μM), C: veratridine (1 μM) and D: veratridine+UFP 512. E–G are statistical results of each recording parameter. *p<0.05, **p<0.01 as compared with the controls; ^###p<0.001 vs. UFP 512; ^&&p<0.01, ^&&&p<0.001 as compared with veratridine. Note that perfusion of 1 μM veratridine itself for 20 min could not produce any obvious changes in [Na⁺]o in normoxia, but greatly enhanced anoxia-induced Na⁺ influx, which could be greatly attenuated by DOR activation with 5 μM UFP 512.

Fig. 6

Effect of DOR activation on Na⁺ influx induced by Na⁺ channel opener veratridine in normoxia. Trace recordings of A: veratridine (5 μM) (ver-5), B: UFP 512 (5 μM), C: veratridine (10 μM) (ver-10) and D: UFP 512 (5 μM). E–G are statistical results of each recording parameter. *p<0.05, **p<0.01 as compared with the controls; ^###p<0.001 vs. ver-5. Perfusion of 5 and 10 μM of veratridine induced a drop in [Na⁺]o to the same degree under normoxia; but a very short period of perfusion with 10 μM veratridine was sufficient to induce a major decrease in [Na⁺]o. Activation of DOR with UFP 512 (5 μM) significantly attenuated Na⁺ influx induced by 5 but not 10 of veratridine under normoxia.

Fig. 7

Involvement of PKC in DOR-mediated inhibition of anoxia-induced Na⁺ influx. Trace recordings of A: control (Cont), B: UFP 512 (5 μM), C: chelerythrine (CHEL) (10 μM) and D: CHEL+UFP 512. E–G are statistical results of each recording parameter. *p<0.05, **p<0.01, ***p<0.001 as compared with the controls; ^##p<0.01, ^###p<0.001 vs. UFP 512. Note that DOR-mediated inhibition of anoxia-induced Na⁺ influx was blocked by broad-spectrum PKC blocker, chelerythrine.

Fig. 8

Involvement of PKCβII in DOR-mediated inhibition of anoxia-induced Na⁺ influx. Trace recordings of A: control (Cont), B: UFP 512 (5 μM), C: PKCβII peptide inhibitor (PKCbII PI) (0.1 μM) and D: PKCbII PI+UFP 512. E–G are statistical results of each recording parameter. *p<0.05, **p<0.01 as compared with the controls; ^#p<0.05, ^##p<0.01 vs. UFP 512. Note that DOR-mediated inhibition of anoxia-induced Na⁺ influx was blocked by PKC βII peptide inhibitor.

Fig. 9

Involvement of PKCθ in DOR-mediated inhibition of anoxia-induced Na⁺ influx. Trace recordings of A: control (Cont), B: UFP 512 (5 μM), C: PKCθ pseudosubstrate inhibitor (PKCq PI) (0.1 μM) and D: PKCq PI+UFP 512. E–G are statistical results of each recording parameter. *p<0.05, **p<0.01, ***p<0.001 as compared with the controls; ^#p<0.05, ^###p<0.001 vs. UFP 512; ^&p<0.05 as compared with PKCq PI. Note that DOR-mediated inhibition of anoxia-induced Na⁺ influx was blocked by PKCθ pseudosubstrate inhibitor.