Continuous delta-opioid receptor activation reduces neuronal voltage-gated sodium channel (NaV1.7) levels through activation of protein kinase C in painful diabetic neuropathy

Abstract

The Na(V)1.7 tetrodotoxin-sensitive voltage-gated sodium channel isoform plays a critical role in nociception. In rodent models of diabetic neuropathy, increased Na(V)1.7 in dorsal root ganglia (DRG) neurons correlates with the emergence of pain-related behaviors characteristic of painful diabetic neuropathy (PDN). We examined the effect of transgene-mediated expression of enkephalin on pain-related behaviors and their biochemical correlates in DRG neurons. Transfection of DRG neurons by subcutaneous inoculation of a herpes simplex virus-based vector expressing proenkephalin reversed nocisponsive behavioral responses to heat, cold, and mechanical pressure characteristic of PDN. Vector-mediated enkephalin production in vivo prevented the increase in DRG Na(V)1.7 observed in PDN, an effect that correlated with inhibition of phosphorylation of p38 MAPK (mitogen-activated protein kinase) and protein kinase C (PKC). Primary DRG neurons in vitro exposed to 45 mm glucose for 18 h also demonstrated an increase in Na(V)1.7 and increased phosphorylation of p38 and PKC; these changes were prevented by transfection in vitro with the enkephalin-expressing vector. The effect of hyperglycemia on Na(V)1.7 production in vitro was mimicked by exposure to PMA and blocked by the myristolated PKC inhibitor 20-28 or the p38 inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole]; the effect of vector-mediated enkephalin on Na(V)1.7 levels was prevented by naltrindole. The results of these studies suggest that activation of the presynaptic delta-opioid receptor by enkephalin prevents the increase in neuronal Na(V)1.7 in DRG through inhibition of PKC and p38. These results establish a novel interaction between the delta-opioid receptor and voltage-gated sodium channels.

Figure 1.

Enkephalin expression resulting from inoculation of vE reverses nocisponsive behaviors in PDN. a, Thermal withdrawal latency (Hargreave's test) in seconds. b, Cold allodynia in seconds. c, Mechanical hyperalgesia (Randall–Selitto test) threshold in grams. C, Naive control; D, diabetic; DvE, diabetic inoculated with vE; DvZ, diabetic inoculated with vZ. All data are presented as mean ± SEM. n = 6–8 per group. ^#p < 0.001 or **p < 0.01 versus untreated diabetic animals or DvZ. d, Representative photomicrographs of enkephalin immunoreactivity 7 d after the vector inoculation. Scale bar, 20 μm.

Figure 2.

Enkephalin expression resulting from inoculation of vE reverses increase in Na_V1.7 in DRG characteristic of PDN. a, Amount of Na_V1.7 determined by Western blot, normalized to D or DvZ. C, Control; D, diabetic; DvE, diabetic inoculated with vE; DvZ, diabetic inoculated with vZ. ^#p < 0.001; n = 5 animals per group. b, c, Representative Western blots showing two independent samples from each group.

Figure 3.

Inoculation of vE prevents phosphorylation of p38 and PKC in diabetic DRG in vivo. a, Amount of p-p38 determined by Western blot, normalized to D or DvZ. ***p < 0.005; n = 5 animals per group. b, c, Representative Western blots showing two independent samples from each group. d, Amount of p-PKC determined by Western blot, normalized to D or DvZ. ^#p < 0.001; n = 5 animals per group. e, f, Representative Western blots showing two independent samples from each group. C, Control; D, diabetic; DvE, diabetic inoculated with vE; DvZ, diabetic inoculated with vZ.

Figure 4.

Transgene-mediated expression of enkephalin prevents the increase in Na_V1.7 resulting from exposure primary DRG neurons to hyperglycemic conditions in vitro. a, Amount of Na_V1.7 determined by Western blot, normalized to G or GvZ. ***p < 0.005; n = 3 wells per group. C, Control; G, hyperglycemia; GvE, hyperglycemia, transfected with vE; GvZ, hyperglycemia, transfected with vZ. b–d, Representative Western blots showing two independent samples from each group. d, The effect of vE is blocked by naltrindole (GvE+N). e, The amount of Na_V1.7 determined by Western blot, normalized to GvE. ^#p < 0.001; n = 3 wells per group.

Figure 5.

Transduction with vE prevents phosphorylation of p38 and PKC in DRG exposed to hyperglycemia in vitro. a, Amount of p-p38 determined by Western blot, normalized to G or GvZ. *p < 0.05; ***p < 0.005; n = 3 wells per group. b, c, Representative Western blots showing two independent samples from each group. d, Amount of p-PKC determined by Western blot, normalized to G or GvZ. *p < 0.05; **p < 0.01; n = 3 wells per group. e, f, Representative Western blots showing two independent samples from each group. C, Control; G, hyperglycemia; GvE, hyperglycemia, transfected with vE; GvZ, hyperglycemia, transfected with vZ.

Figure 6.

Naltrindole blocks the effect of vE on p38 and PKC in DRG exposed to hyperglycemia in vitro. a, b, Representative Western blots showing amount of p-p38 (a) and p-PKC (b) in primary DRG neurons exposed to hyperglycemia and transfected with vE in the absence (GvE) or presence (GvE+N) of 10 pm naltrindole. The difference between GvE and GvE+N was statistically significant for both p-p38 (p < 0.001) and p-PKC (p < 0.005).

Figure 7.

The amount of Na_V1.7 in DRG is regulated by PKC acting through p38. a, The amount of Na_V1.7 in control (C) DRG neurons in vitro and in DRG neurons exposed to PMA in 25 mm glucose-containing medium (P) or to 45 mm glucose (G). ^#p < 0.001; ***p < 0.005; n = 3 wells per group. b, Representative Western blots. c, The amount of Na_V1.7 in DRG neurons exposed to 45 mm glucose in vitro treated with the myristolated PKC inhibitor 20-28 (G+I) normalized to the amount of Na_V1.7 in cells exposed to 45 mm glucose (G). ^#p < 0.001. d, Representative Western blots. e, The amount of Na_V1.7 in DRG neurons exposed to 45 mm glucose in vitro treated with the p38 inhibitor SB202190 (G+I) normalized to the amount of Na_V1.7 in cells exposed to 45 mm glucose (G). ^#p < 0.001. f, Representative Western blots. g, The amount of phosphorylated p38 in DRG neurons exposed to 45 mm glucose in vitro treated with the myristolated PKC inhibitor 20-28 (G+I) normalized to the amount of phosphorylated p38 in cells exposed to 45 mm glucose (G). ***p < 0.005; n = 3 wells per group. h, Representative Western blots.