Key role for the epsilon isoform of protein kinase C in painful alcoholic neuropathy in the rat

Abstract

Chronic alcohol consumption produces a painful peripheral neuropathy for which there is no reliably successful therapy, attributable to, in great part, a lack of understanding of the underlying mechanisms. We tested the hypothesis that neuropathic pain associated with chronic alcohol consumption is a result of abnormal peripheral nociceptor function. In rats maintained on a diet to simulate chronic alcohol consumption in humans, mechanical hyperalgesia was present by the fourth week and maximal at 10 weeks. Thermal hyperalgesia and mechanical allodynia were also present. Mechanical threshold of C-fibers in ethanol fed rats was lowered, and the number of action potentials during sustained stimulation increased. The hyperalgesia was acutely attenuated by intradermal injection of nonselective protein kinase C (PKC) or selective PKCepsilon inhibitors injected at the site of nociceptive testing. Western immunoblot analysis indicated a higher level of PKCepsilon in dorsal root ganglia from alcohol-fed rats, supporting a role for enhanced PKCepsilon second-messenger signaling in nociceptors contributing to alcohol-induced hyperalgesia.

Fig. 1.

A, Effect of long-term ethanol feeding on body weight using the Lieber–DeCarli liquid diet pair-feeding technique. The average weight of ethanol-fed (filled squares; n = 17) and isocalorically fed control (open triangles;n = 12) rats is plotted against the duration of administration of the liquid diet. Each point represents mean ± SEM weight (in grams) (errorbars). B, Decrease in paw-withdrawal threshold to mechanical stimulation of the dorsum of the rat hindpaw induced by chronic administration of ethanol (alcohol-induced mechanical hyperalgesia). The paw-withdrawal threshold (in grams) of ethanol-fed (filled squares;n = 34 paws) and control diet-fed (open triangles; n = 24 paws) rats plotted against the duration (in weeks) of chronic exposure to ethanol administered by the Lieber–DeCarli liquid diet technique and pair-fed isocaloric diet controls. From the fourth week of ethanol diet feeding, *p < 0.0001 (repeated measures ANOVA).

Fig. 2.

A, Effect of WIPTIDE (WIP; 1 μg; n = 12 paws), BIMM (1 μg; n = 12 paws), and PKCε-I (1 μg; n = 12 paws) on the baseline (BL) paw threshold (grams ± SEM) of ethanol-fed (filled bars; n = 12 paws) and isocalorically fed control (hatched bars;n = 12 paws) rats. *p < 0.0001 (ANOVA, followed by Fisher's PLSD post hoc test).NS, Not statistically significant. B, Reduction in lowered paw-withdrawal threshold (hyperalgesia) produced by WIPTIDE (WIP; 1 μg; n = 12 paws), BIMM (1 μg; n = 12 paws), and PKCε-I (1 μg; n = 12 paws) plotted as percentage change in nociceptive threshold in ethanol-fed (filled bars; n = 12 paws) and isocalorically fed control (hatched bars; n = 12 paws) rats. *p < 0.0001 (one-way ANOVA and Fisher's PLSD post hoc test). NS, Not statistically significant.

Fig. 3.

A, Effect of chronic alcohol consumption on response to stimulation by calibrated 1.32, 3.63, 10.0, and 27.5 mN (represented on the abscissa by 1,2, 3, and 4) VFH measured in ethanol-fed (filled squares;n = 24 paws) and control-diet fed (open triangles; n = 12 paws) rats. *p < 0.05 (repeated measures ANOVA).NS, Not statistically significant. B, Effect on sensitivity, as measured by the paw-withdrawal latency, to thermal stimulation from a 50 W radiant heat stimulus, in ethanol-fed (filled bar; n = 12 paws) and control (hatched bar; n = 12 paws) rats. *p < 0.03 (Student's unpairedt test).

Fig. 4.

Effect of ethanol withdrawal (initiation of the CD in a group of rats fed ED in the preceding 12 weeks) on the paw threshold (in grams) of rats fed for 12 weeks on ED (12ED, filledbar;n = 34 paws) and at 2 (12ED+2CD,hatched bar; n = 6 paws) and 5 (12ED+5CD, cross-hatched bar;n = 6 paws) weeks after initiation of CD. *p < 0.0002. NS, Not significant (p > 0.05). Comparisons were by one-way ANOVA and Fisher's post hoc test.

Fig. 5.

Conduction velocity and mechanical threshold of C-fibers in CD and ED rats. A, There was no difference in conduction velocity between C-fibers from ED (filled bar; n = 10) and CD (hatched bar; n = 14) rats. Unpairedt test, p > 0.05. B, The mechanical threshold of C-fibers from ED rats (filled bar; n = 10) was significantly lower than that of C-fibers from control rats (hatched bar;n = 14) using nonparametric Mann–WhitneyU test; * p < 0.05.C, The number of action potentials evoked by sustained (60 sec) threshold stimulation was significantly greater in C-fibers in ED rats (filled bar; n = 10) compared with controls (hatched bar;n = 14). D, The number of action potentials evoked by sustained suprathreshold (10 gm) stimulation was significantly greater in C-fibers in ethanol-fed rats (filled bar; n = 10) compared with controls (hatched bar; n = 14). Unpaired (one-tailed) t test; *p < 0.05 (Student's unpaired t test).

Fig. 6.

PKCε levels in DRG from CD and ED rats.A, Representative blot of PKC immunoreactivity in DRG samples from CD and ED rats (after 10 weeks of ethanol administration). Proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with PKCε-specific antibody. B, Mean ± SEM for data showed a statistically significant difference between control diet-fed (hatched bar;n = 10) and ethanol-fed (filled bar; n = 11) rats. *p< 0.01 (Student's t test).