Article Figures & Data
Figures
- Figure 1.
NGF-responsive adult sensory neurons have a large cytoplasmic pool of high-affinity NGF receptors. Six micrometer cryostat sections of L5 DRG processed for high-affinity NGF binding (left) or trkA immunohistochemistry (right) reveal a large cytoplasmic pool of proteins that are able to bind 20 pm radio-iodinated NGF with high affinity or that are immunoreactive to trkA-selective antibodies. Scale bar, 20 μm.
- Figure 2.
Exposure of sensory neurons to an acidic environment results in mobilization of trkA to the membrane. A, B, Fluorescence photomicrographs depicting the relative level of membrane-associated trkA expression in representative adult DRG neurons exposed to control (pH 7.4, A) or acidic (pH 6.5, B) media for 30 min, then processed to detect trkA under nonpermeabilizing conditions. Scale bar, 20 μm. C, Scatter plot depicts the relationship between trkA immunofluorescence signal labeling intensity (y-axis; normalized to the mean signal intensity from the pH 7.4 neurons; open circles, pH 6.5; closed circles, pH 7.4) and perikaryal diameter (x-axis) for all neurons analyzed from three separate experiments. D, Summary bar graph of data (from C) depicts relative mean levels of membrane-associated trkA on adult DRG neurons exposed to acidic (pH 6.5) or control (pH 7.4) media for 30 min. Note the elevated level of trkA detected under acidic conditions, most notable in small to medium size neurons. Asterisks indicate significant differences between experimental groups (Mann–Whitney U test; ***p < 0.001; pH 7.4, n = 213; pH 6.5, n = 239).
- Figure 3.
pH challenge rapidly mobilizes trkA to the membrane. A, Temporal quantitative analysis of relative levels of membrane-associated trkA immunofluorescence signal in adult DRG neurons exposed to control (pH 7.4) or acidic (pH 6.5) media for times as indicated and normalized to the mean signal intensity from the control pH group. B, Quantitative analysis of trkA immunofluorescence signal levels on sensory neurons exposed to acidosis and then reintroduced to control pH as normalized to the mean signal intensity from the control pH group. Summary bar graph reveals that trkA mobilized to the membrane in response to acidosis is not likely internalized when exposed back to pH 7.4 medium for 30 min, a condition required for biotinylation of cell-surface membrane-associated proteins in C. C, Representative Western blot where equivalent amounts of eluted biotinylated surface proteins (Memb trkA) or cytoplasmic proteins (Cyto trkA) from the same sensory neurons exposed to physiological pH 7.4 or acidic pH 6.5 media were loaded and then probed to detect levels of trkA protein. GAPDH served as a loading control for the cytoplasmic proteins (Cyto GAPDH). D, Summary bar graphs of densitometry performed on membrane and cytoplasmic eluents from the biotinylation experiments with conditions as indicated reveal a significant increase in the amount of cell-surface trkA protein detected following exposure to acidic conditions (one-way ANOVA with post hoc Tukey's; **p < 0.01; ***p < 0.001).
- Figure 4.
Acidic pH challenge rapidly mobilizes trkA to the membrane from internal stores. A, Bar graphs summarize relative changes in neuronal cell-surface trkA expression from three separate experiments (detected with immunofluorescence) over neurons exposed to control (pH 7.4) or acidic (pH 6.5) media for 30 min with or without exposure to Golgi collapsing compound BFA and as normalized to the mean signal intensity from the control pH group. Note: A significant increase is observed in the mean levels of trkA mobilized to the neuronal membranes of sensory neurons in response to acidosis when compared with the control pH, a response that is blocked with BFA treatment. B, Immunofluorescence photomicrographs and summary histograms (C) depict degree of FM 1–43FX-stained neuronal membrane internalization in response to conditions as indicated. Note: Significant membrane internalization was only observed in the NGF challenge control group and not in response to acidic pH challenge with or without BFA treatment. (Data normalized to the control pH of each experimental condition and pooled from three separate experiments; A, C, one-way ANOVA with post hoc Tukey's, **p < 0.01; ***p < 0.001). Scale bar, 20 μm.
- Figure 5.
Increased mobilization of trkA to neuronal membrane enhances response to NGF challenge. A, Fluorescence photomicrographs depicting relative levels of activated/phosphorylated trkA (phospho-trkA) detected under permeabilizing conditions in representative adult DRG neurons exposed to acidic (pH 6.5) or control (pH 7.4) media (in the presence of anti-BDNF) for 30 min, followed by a 15 min challenge with pH-specific medium + anti-BDNF alone or with anti-BDNF + 50 ng/ml NGF. Scale bar, 10 μm. Scatter plots illustrate the relationship between phospho-trkA-like labeling intensity (y-axis; normalized to the mean signal intensity from the control pH group) and perikaryal diameter (x-axis) for all sensory neurons analyzed from three separate experiments exposed to control (pH 7.4, B) or acidic (pH 6.5, C) media as above and then challenged with 50 ng/ml NGF (open circles) or not (filled circles). D, Summary bar graph depicting relative levels of activated trkA (phospho-trkA; normalized to pH 7.4 control) in representative adult DRG neurons exposed to acidic (pH 6.5) or control (pH 7.4) media for 30 min and then challenged with 50 ng/ml NGF and as normalized to the mean signal intensity from the control pH group. E, Fluorescence photomicrographs of sensory neurons exposed to identical conditions as in A and processed to detect activated/phosphorylated p38MAPK Note: A significant increase is observed in the levels of phospho-trkA detected in sensory neurons in response to acidosis with a parallel response observed for phospho-p38MAPK (E) (one-way ANOVA with post hoc Tukey's; ***p < 0.001; n = 131–210 neurons analyzed per experimental condition). Scale bar, 20 μm.
- Figure 6.
Neuronal activity is linked to mobilization of trkA to membrane. A, Acid-induced [Ca2+]i increases in DRG cells. Typical sample trace exhibiting reversible repeatable Ca2+ transients upon exposure of sensory neurons to a lowered pH of 6.5. The six-point asterisk in the trace refers to the pseudocolor image seen in B (pH 7.4) exhibiting baseline fluorescence. The five-point asterisk highlights a typical peak Ca2+ response seen in the corresponding pseudocolor image seen in B (pH 6.5). C, Bar graph representing the mean Ca2+ increase observed upon repeated exposure to a lowered pH of 6.5 (n = 6 neurons; ±SEM). D, E, Summary bar graphs of data (normalized to pH 7.4 controls), depicting relative changes in the level of membrane-associated trkA expression in adult DRG neurons exposed to acidic (pH 6.5) or control (pH 7.4) media for 30 min in the presence or absence of Ca2+ chelator BAPTA-AM (50 μm), DMSO, or the acid-sensing ion channel blocker amiloride (250 μm). Note: Blocking proton-sensitive channels or chelating Ca2+ greatly attenuates the amount of trkA detected on the cell surface in response to acidosis when compared with pH 6.5 alone. F, Summary bar graphs from experiments (normalized to pH 7.4 controls), depicting relative changes in membrane-associated trkA expression in adult DRG neurons exposed for 30 min to acidic (pH 6.5) or control (pH 7.4) media for 30 min with or without KCl depolarization (15 mm or 45 mm) as indicated. Note: KCl-induced depolarization of neurons exposed to pH 7.4 results in mobilization of trkA to the membrane in a manner equivalent to that observed in response to a lowered pH of 6.5; but KCl does not induce further mobilization in neurons already exposed to pH 6.5. (one-way ANOVA with post hoc Tukey's; ***p < 0.001; n = 101–140 neurons analyzed per experimental condition).
Tables
- Table 1.
Effects of acidosis on incidence of sensory neurons expressing cell surface-associated trkA
pH trkA+ve neurons trkA−ve neurons % trkA+ve R1 7.4 531 643 45.2 R2 7.4 875 1121 43.8 R3 7.4 369 483 43.3 R4 7.4 908 1140 44.3 R5 7.4 680 962 41.4 R6 7.4 536 736 42.1 X̄ ± SEM 43.4 ± 0.6 R1 6.5 568 657 46.3 R2 6.5 383 529 41.9 R3 6.5 648 864 42.8 R4 6.5 1042 1390 42.8 R5 6.5 532 676 44.0 R6 6.5 452 556 44.8 X̄ ± SEM 43.8 ± 0.7 Data summarizing incidence of trkA immunopositive and trkA immunonegative sensory neurons in cultures exposed to acidic (pH 6.5) or control (pH 7.4) media for 30 min, followed by processing for trkA immunocytochemistry under nonpermeabilizing conditions. Note: Exposure to acidic conditions does not discernibly alter the incidence of neurons displaying membrane-associated trkA [six replicates (R)].
