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. 2011 Jul 20;31(29):10516-28.
doi: 10.1523/JNEUROSCI.2992-10.2011.

TRPV1 and TRPA1 function and modulation are target tissue dependent

Sacha Malin  1 , Derek Molliver, Julie A Christianson, Erica S Schwartz, Pam Cornuet, Kathryn M Albers, Brian M Davis
Affiliations

TRPV1 and TRPA1 function and modulation are target tissue dependent

Sacha Malin et al. J Neurosci. .

Abstract

The nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) families of growth factors regulate the sensitivity of sensory neurons. The ion channels transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential channel, subfamily A, member 1 (TRPA1), are necessary for development of inflammatory hypersensitivity and are functionally potentiated by growth factors. We have shown previously that inflamed skin exhibits rapid increases in artemin mRNA with slower, smaller increases in NGF mRNA. Here, using mice, we show that, in inflamed colon, mRNA for both growth factors increased with a pattern distinct from that seen in skin. Differences were also seen in the pattern of TRPV1 and TRPA1 mRNA expression in DRG innervating inflamed skin and colon. Growth factors potentiated capsaicin (a specific TRPV1 agonist) and mustard oil (a specific TRPA1 agonist) behavioral responses in vivo, raising the question as to how these growth factors affect individual afferents. Because individual tissues are innervated by afferents with unique properties, we investigated modulation of TRPV1 and TRPA1 in identified afferents projecting to muscle, skin, and colon. Muscle and colon afferents are twice as likely as skin afferents to express functional TRPV1 and TRPA1. TRPV1 and TRPA1 responses were potentiated by growth factors in all afferent types, but compared with skin afferents, muscle afferents were twice as likely to exhibit NGF-induced potentiation and one-half as likely to exhibit artemin-induced potentiation of TRPV1. Furthermore, skin afferents showed no GDNF-induced potentiation of TRPA1, but 43% of muscle and 38% of colon afferents exhibited GDNF-induced potentiation. These results show that interpretation of afferent homeostatic mechanisms must incorporate properties that are specific to the target tissue.

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Figures

Figure 1.
Figure 1.
Expression of growth factors is altered in inflamed tissue. A, Myeloperoxidase assay reveals that TNBS induces rapid inflammation within 24 h of TNBS instillation in the colon. Inflammation begins to resolve by day 4 but remains elevated for at least 2 weeks. B, The time course and magnitude of changes in growth factor mRNA levels in the colon during TNBS-induced colonic inflammation was measured by real-time PCR. NGF and ARTN exhibit parallel increases in expression that peak at day 4. GDNF increases more slowly and remains elevated at 2 weeks, whereas NRTN is immediately decreased and remains so for at least 2 weeks. *p < 0.05, **p < 0.01, ***p < 0.001 versus naive; two-way ANOVA with Bonferroni's posttest. Error bars indicate SEM.
Figure 2.
Figure 2.
TRP channel expression is modulated during inflammation. The time course and magnitude of changes in TRP channel mRNA expression in L2–L5 DRG during CFA-induced inflammation (A) and in L5–S1 DRG following TNBS-induced colon inflammation (B) was measured by real-time PCR. Following skin inflammation, TRPV1 mRNA increases faster than TRPA1 mRNA expression; however, the pattern is reversed for colon inflammation. *p < 0.05 versus naive; two-way ANOVA with Bonferroni's posttest.
Figure 3.
Figure 3.
Growth factors potentiate TRP function in vivo. Mice were injected with growth factor (0.2 mg/20 μl) or saline (20 μl) in one hindpaw and 30 min later, 1 μg of capsaicin (A) or 10% mustard oil (B) was applied to the growth factor/saline injected hindpaw. Nocifensive behaviors were monitored for 5 min. NGF, ARTN, or NRTN preinjection increased the number of responses elicited by either capsaicin or mustard oil, compared with saline. GDNF was only effective in increasing the capsaicin response and had no effect on mustard oil responses. *p < 0.05, **p < 0.01 versus saline; one-way ANOVA with Dunnett's multiple-comparison test.
Figure 4.
Figure 4.
TRPV1 and TRPA1 expression is target tissue dependent. Afferents identified by injection of retrograde tracers into the saphenous nerve (WGA-488; skin), femoral nerve (WGA-488; muscle), or the colon wall (CTB-488) were characterized by Ca2+ imaging in vitro. Neurons innervating different target tissues showed substantial differences in the response kinetics to capsaicin (A–C; TRPV1) and mustard oil (D–F; TRPA1). Percentage responders for each agonist within the identified populations reveal fewer capsaicin and mustard oil responses in skin, compared with muscle and colon afferents. Representative traces of TRPV1 (A) and TRPA1 (D) responses from skin (black), muscle (red), and colon (blue) afferents are overlaid to illustrate differences in response size. Mean peak and area of capsaicin (B, C) and mustard oil (E, F) responses are significantly smaller in skin afferents compared with muscle and colon afferents. *p < 0.01, ***p < 0.001 versus skin; Mann–Whitney test.
Figure 5.
Figure 5.
Coexpression of TRPV1 and TRPA1 is target tissue dependent. Ca2+ imaging was performed to determine the percentage of retrogradely labeled skin, muscle, and colon neurons that respond to capsaicin and/or mustard oil. The percentage of neurons that express only TRPV1 (capsaicin responsive; black bar) or TRPA1 (mustard oil responsive; white bar) is not significantly different between the three afferent populations. In contrast, the percentage of neurons that express both TRPV1 and TRPA1 (hashed bar) is significantly larger in muscle and colon afferents than in skin afferents (p < 0.01). Significantly more nonresponsive (gray bar) skin neurons are observed than muscle or colon neurons (p < 0.01; one-way ANOVA with Dunnett's multiple-comparison test).
Figure 6.
Figure 6.
Repeated application of TRPV1 or TRPA1 agonist produces tachyphylaxis. Ca2+ imaging of responses to repeated 1 μm capsaicin (A) or 100 μm mustard oil (B) application in isolated mouse DRG neurons reveals profound desensitization of TRPV1 (A) and TRPA1 (B) channels. Agonists were applied at 10 min intervals.
Figure 7.
Figure 7.
Growth factors differentially potentiate TRPV1 in target-defined sensory neurons. Ca2+ imaging was used to determine the efficacy of NGF, ARTN, NRTN, or GDNF to potentiate capsaicin-evoked Ca2+ signals in retrogradely labeled skin, muscle, and colon DRG neurons. Examples of Ca2+ traces in response to capsaicin presentation before and after growth factor exposure are shown in A. Capsaicin (1 μm) was applied at 10 min intervals with a 7 min application of growth factor (100 ng/ml) between the first and second capsaicin presentations. The percentage of capsaicin-responsive neurons potentiated by each growth factor is given above each representative trace. Fold changes in response area (ΔFarea) following growth factor application are shown for skin (B), muscle (C), and colon (D) neurons. All four growth factors tested potentiate capsaicin responses in skin, muscle, and colon afferents; however, the prevalence and magnitude of the effects vary with target tissue. The greatest ARTN- and GDNF-induced potentiation is observed among skin afferents. *p < 0.05 versus untreated; Mann–Whitney test.
Figure 8.
Figure 8.
Growth factors differentially potentiate TRPA1 in target-defined sensory neurons. Ca2+ imaging was used to examine the efficacy of NGF, ARTN, NRTN, or GDNF to potentiate mustard oil-evoked Ca2+ signal in retrogradely labeled skin, muscle, and colon DRG neurons. Examples of Ca2+ traces in response to mustard oil presentation before and after growth factor exposure are shown in A. Mustard oil (100 μm) was applied at 10 min intervals with a 7 min application of growth factor (100 ng/ml) between the first and second mustard oil presentations. The percentage of mustard oil-responsive neurons potentiated by each growth factor is given above each representative trace. Fold changes in response area (ΔFarea) following growth factor application are shown for skin (B), muscle (C), and colon (D) neurons. Potentiation of TRPA1 by NGF, ARTN, or NRTN is comparable between all populations, whereas GDNF is ineffective at potentiating TRPA1 responses in skin neurons. *p < 0.05 versus untreated; Mann–Whitney test.
Figure 9.
Figure 9.
Growth factor potentiation profile of TRPV1 responses is target tissue dependent. Ca2+ imaging was used to determine the overlap of NGF, ARTN, NRTN, and GDNF potentiating effects on TRPV1 responses in retrogradely labeled skin, muscle, and colon DRG neurons. Capsaicin (1 μm) was applied to obtain a baseline response followed by growth factor (100 ng/ml) treatment for 7 min and a second application of capsaicin (1 μm). Following a 30 min rest period to allow for recovery from the first growth factor treatment, the same protocol was repeated using a second growth factor. Fold changes in response area (ΔFarea) were calculated and the percentage of potentiated cells (>1-fold change in ΔFarea) was determined. Pie charts show the percentage of TRPV1-expressing afferents potentiated by each pair of growth factors for each target-defined population (n = 811 capsaicin-responsive neurons). The percentage of cells that responded to both NGF and ARTN (purple wedge) is similar (∼40%) in all target tissues (A). Interestingly, TRPV1 responses in skin neurons were more likely to be potentiated by both growth factors in all pairs than in either muscle or colon afferents (B–F). The majority of TRPV1 responses in skin and colon neurons can be potentiated by ARTN (E, pink wedge) or GDNF (green wedge), and the overlap between the two growth factors (yellow wedge) is robust in skin afferents, constituting nearly every GDNF-responsive neuron, yet is completely absent in colon afferents.
Figure 10.
Figure 10.
Growth factor potentiation profile of TRPA1 is target tissue dependent. Ca2+ imaging was used to determine the overlap of NGF, ARTN, NTRN, and GDNF potentiating effects on TRPA1 responses in isolated skin, muscle, and colon DRG neurons. Mustard oil (100 μm) was applied to obtain a baseline response followed by growth factor (100 ng/ml) treatment for 7 min and a second mustard oil (100 μm) application. Following a 30 min rest period to allow for recovery from the first growth factor treatment, the same protocol was repeated using a second growth factor. Fold changes in response area (ΔFarea) were calculated and the percentage of potentiated cells (>1-fold change in ΔFarea) was determined. Pie charts show the percentage of TRPA1-expressing afferents potentiated by each pair of growth factors for each target-defined population (n = 770 mustard oil-responsive neurons). The percentage of TRPA1 responses potentiated by both NGF and ARTN (A, purple wedge), as well as by both NRTN and GDNF (F, lime wedge), is significantly higher in muscle neurons than either skin or colon (p < 0.01; ANOVA with Dunnett's multiple-comparison test). TRPA1 responses potentiated by both NRTN and NGF (B, brown wedge) are most prevalent in colon neurons and completely absent in skin neurons. In fact, among skin neurons, only those TRPA1 responses potentiated by ARTN (A, D, E, pink wedge) are also potentiated by another growth factor, with the exception of GDNF, which had no effect on TRPA1 responses in this population. Overlap between NGF and GDNF potentiation of TRPA1 is also greatest in colon afferents (C, turquoise wedge). Interestingly, even though nearly all TRPA1 responses in colon neurons are potentiated by either ARTN (E, pink wedge) or GDNF (green wedge) alone, no TRPA1 responses in this population are potentiated by both growth factors in the same neuron.
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