Angiotensin II type 2 receptor (AT2 R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons

Abstract

Background: The angiotensin II (AngII) receptor subtype 2 (AT2 R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration.

Methods: We used immunostaining with characterized antibodies to study the localization of AT2 R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2 R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence.

Results: AT2 R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2 R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50 = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1 R antagonist losartan had no effect on capsaicin responses. AT2 R was localized in sensory neurons of human DRG, and nerve fibres in peripheral nerves, skin, urinary bladder and bowel. A majority sub-population (60%) of small-/medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2 R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas.

Conclusions: AT2 R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting.

Figure 1

Low power image of cultured adult rat dorsal root ganglion (DRG) neurons showing Gap43 neurons and neurites (A), angiotensin II type 2 receptor (AT₂R) positive neuronal cell bodies (B), and merged image with intense co-localization in some neurons (C, arrows indicate AT₂R negative neurons), and nuclei (D, Hoechst dye positive); bar = 100 μm. High power image of cultured adult hDRG neurons positive for Gap43 (E), smaller neuron positive for AT₂R (F), and merged image (G); bar in (F) = 50 μm.

Figure 2

Sample traces showing calcium influx in human dorsal root ganglion (hDRG) neurons in response to 200 nmol/L capsaicin (first stimulus, A), followed by washout and response to 10 μmol/L AngII (B), followed by washout and increased response to second capsaicin stimulus (1 μmol/L) (C). Different hDRG neuron showing calcium influx in response to the first capsaicin stimulus (D), followed by washout, and reduced second response to capsaicin after 10 nmol/L EMA401 application (E). Rat dorsal root ganglion (rDRG) neuron showing response to first capsaicin stimulus (F), followed by washout and no response to 100 nmol/L AngII application (G), followed by enhanced second response to capsaicin (H). Graph (I) showing amplitude of capsaicin responses in the absence of drugs (bars 1 and 2), significant enhancement with AngII treatment (bar 3, ***p < 0.001, in comparison with bar 2), and abolition of this AngII-mediated enhancement with EMA401 application (bar 4). Losartan (AT1 antagonist) had no effect on capsaicin responses (bar 5). EMA401-mediated capsaicin response inhibition (bar 6, **p < 0.005, in comparison with bar 2) was reversed in the presence of forskolin (bar 7, in comparison with bar 6) and 8-bromo-cAMP (bar 8, in comparison with bar 6, ***p < 0.001). (J) shows dose-related inhibition of capsaicin responses in hDRG neurons by EMA401, and reversal with 8-bromo-cAMP (J). (K) shows dose-related capsaicin inhibition by EMA401 in rDRG neurons.

Figure 3

Morphology of Gap43-immunostained cultured rat dorsal root ganglion (rDRG) neurons without AngII treatment (A), with AngII treatment (B), and after treatment with 100 nmol/L EMA401 (C). Examples of cAMP positive, immunofluorescent neurons without AngII (D), with AngII treatment (E), with 8-bromo-cAMP (F), and with 100 nmol/L EMA401 (G). Scale bar in A–C = 50 μm; D–G = 30 μm. Graph showing neurite density in EMA401-treated rDRG neurons (H), significantly reduced neurite density in hDRG neurons treated with EMA401 (I; **p = 0.005. Graph (J) showing neurite length is significantly increased in AngII-treated rDRG neurons (bar 2, ***p < 0.001), but this not significant after EMA401 treatment (bar 3, J). 10 and 100 nmol/L EMA401 by itself reduced average neurite length compared with control neurons (bars 4 and 5, J; *p < 0.05). Graph (K) of cAMP immunofluorescence signal intensity showing significant increase after 8-br-cAMP treatment (bar 2, **p < 0.01), and after 10 nmol/L AngII treatment in rDRG neurons (bar 3, *p < 0.05). Neurons treated with a combination of 10 nmol/L AngII and 100 nmol/L EMA401 did not have significantly increased cAMP levels (bar 4, K). Treatment with 100 nmol/L EMA401 alone showed a slight reduction, but this was not significant (bar 5, K).

Figure 4

Angiotensin II type 2 receptor (AT₂R) (A, B) and the neuronal cell marker, neurofilament (C) immunostaining in an avulsed dorsal root ganglion (DRG). AT₂R-immunoreactive neurons in pre- (D) and post-fixed DRG (E) using antibody sc-48452. AT₁R immunoreactivity is absent from DRG neuron cell bodies but appears to label vascular structures around the neuronal cells (arrows) (F). Scale bars: A, B = 100 μm; C = 200 μm; D, E = 50 μm, F = 100 μm. Immunofluorescent images of TRPV1 positive neurons in hDRG tissue section (G, green), AT₂R positive (H, red) and merged image, with arrows indicating co-localization of AT₂R and TRPV1 (I), bar = 100 μm.

Figure 5

Angiotensin II type 2 receptor (AT₂R) (A, C, E) and neurofilament (B, D, F) immunostaining of a control peripheral nerve (A, B), proximal injured peripheral nerve (C, D) and neuroma (E, F), magnification ×40. AT₂R (G, I) and neurofilament (H, J) immunostaining in a neuroma (G, H) and trunk of proximal injured peripheral nerve (I, J), magnification ×40. Image analysis (% area) of AT₂R (K, **p = 0.003) and neurofilament immunoreactive fibres in control and proximal injured nerve, non-painful and painful neuromas (L). Results (% area) expressed as the ratio AT₂R : neurofilament (NF), values are mean ± standard error of the mean (M, **p = 0.004).