Journal: Pain

Article Title: Pharmacologically enabling the degradation of Na V 1.8 channels to reduce neuropathic pain

doi: 10.1097/j.pain.0000000000003470

Figure Lengend Snippet: PY(A) peptide decreased Na V 1.8 expression. (A) PY(A) peptide sequence. (B) Proposed mechanism of lipidated peptides partitioning in the membrane and affecting Magi-1. (C) Cultured rat DRG neurons treated with 10 µM PY(A) peptide or 10 µM scrambled peptide control. Na V 1.8 immunofluorescence progressively decreases over 6 and 24 hours after incubation with PY(A) peptide. There is also an internalization of Na V 1.8 seen after 6 hours and continuing until 24 hours. (D) Quantification of Na V 1.8 immunofluorescence in cultured DRGs. Significance was determined using a one-way ANOVA with * P < 0.05; ** P < 0.01; *** P < 0.001. (E) Cultured rat DRG neurons treated with PY(A) peptide + vehicle, PY(A) peptide + bafilomycin (100 nM), or scrambled peptide + vehicle. Na V 1.8 immunofluorescence is decreased at 6 and 24 hours; however, the addition of Bafilomycin prevents the loss of Na V 1.8 immunofluorescence. (F) Quantification of Na V 1.8 immunofluorescence in cultured DRG neurons. Significance was determined using a one-way ANOVA with * P < 0.05; ** P < 0.01; *** P < 0.001. (G) Representative action potential firing from cultured neurons after 24 incubation with scrambled (above) PY(A) peptide (below). (H) Representative rheobase determination in cultured DRG neurons. PY(A) peptide increased threshold of firing. Red trace indicates selected trace to measure rheobase. For scrambled −140 pA, and for PY(A) peptide −440 pA. (I) Threshold of firing as a function of PY(A) peptide dose. The threshold for action potentials increased as the concentration of the peptide increased. Estimated IC 50 = 928 nM. ANOVA, analysis of variance; DRG, dorsal root ganglion.

Article Snippet: Cultured human DRG neuronal suspensions were purchased from Anabios (San Diego, CA).

Techniques: Expressing, Sequencing, Membrane, Cell Culture, Control, Immunofluorescence, Incubation, Concentration Assay

Journal: Pain

Article Title: Pharmacologically enabling the degradation of Na V 1.8 channels to reduce neuropathic pain

doi: 10.1097/j.pain.0000000000003470

Figure Lengend Snippet: PY(A)-H peptide decreased Na V 1.8 expression in human DRG neurons. (A) Human PY peptide (PY(A)-H) sequence. (B) Incubation of peptide alters Na V 1.8 expression in a first human donor. A similar Na V 1.8 immunofluorescence decrease is seen within 6 hours and continues to decrease at 24 hours of 10 µM PY(A)-H incubation. The scrambled peptide had no effect on Na V 1.8 expression. (C) Quantification of fluorescence in scrambled and PY(A)-H peptide–treated neurons over time. Significance was determined using one-way ANOVA ** P < 0.01; *** P < 0.001. (D) Incubation to peptide alters Na V 1.8 expression in a second human donor. Na V 1.8 immunofluorescence decreased within 6 hours of 10 µM PY(A)-H incubation and continued to decrease at 24 hours. The scrambled peptide did not affect Na V 1.8 expression. (E) Quantification of fluorescence in scrambled and PY(A)-H peptide–treated neurons over time. Significance was determined using one-way ANOVA ** P < 0.01; *** P < 0.001. (F) Immunofluorescence images of human DRG neurons taken from the third donor 24 hours after incubation with 10 µM scrambled peptide or PY(A)-H peptide. There was a reduction of Na V 1.8 immunofluorescence in PY(A)-H peptide–treated human DRG neurons compared to scrambled peptide. A marked reduction in immunofluorescence was noted particularly in the processes (indicated by white arrows). (G) Quantification of Na V 1.8 staining fluorescence in DRGs. Significance determined using unpaired Student t test * P < 0.05. (H) Representative I Na at +20 mV of human DRG neurons after 24-hour incubation with 10 µM scrambled (top) or PY(A) peptide (bottom). (I) Quantification of I Na current density at +20 mV pooled from donors 1, 2, and 3. Significance determined using unpaired Student t test *** P < 0.005 (n = 12). ANOVA, analysis of variance; DRG, dorsal root ganglion.

Article Snippet: Cultured human DRG neuronal suspensions were purchased from Anabios (San Diego, CA).

Techniques: Expressing, Sequencing, Incubation, Immunofluorescence, Fluorescence, Staining