Angiotensin II type 2 receptor (AT2R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons

Background The angiotensin II (AngII) receptor subtype 2 (AT2R) 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 AT2R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2R 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 AT2R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2R 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 AT1R antagonist losartan had no effect on capsaicin responses. AT2R 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 AT2R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas. Conclusions AT2R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting.


Introduction
The octapeptide angiotensin II (AngII) is known to regulate blood pressure, fluid balance and other functions via two known membrane bound G proteincoupled receptors, angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) (De Gasparo et al., 2000;Paul et al., 2006). There is increasing evidence that AngII may play a significant role in the nervous system, including pain mechanisms. AngII and AT2R protein expression have been detected in rat dorsal root ganglion (rDRG), human dorsal root gan-glion (hDRG) and trigeminal ganglia (Chakrabarty et al., 2008;Imboden et al., 2009;Patil et al., 2010), and AT2R mRNA in hDRG extracts, indicating the existence of an intrinsic angiotensinergic system. Furthermore, co-localization of AngII with substance P and calcitonin gene-related peptide containing DRG neurons (Patil et al., 2010) suggests a role for AngII in nociception. AT2R antagonists have shown efficacy in rodent neuropathic pain models (see Smith, 2011;Smith and Wyse, 2011), and the clinical efficacy and safety of AT2R antagonist EMA401 was reported recently in post-herpetic neuralgia (McCarthy et al., 2012).
While AngII and its metabolite AngIII both act at the AT1R and the AT2R in the brain (Zini et al., 1996;Wright et al., 2003, Pelegrini-Da-Silva et al., 2005, and have important effects in the central nervous system (CNS) on pain mechanisms (see the Discussion section), we have focused on peripheral mechanisms since the AT2R antagonist EMA401 used in our study does not have significant CNS distribution after oral dosing.
We have examined the functional effects of the AT2R antagonist EMA401 in cultured human and rat DRG neurons on the responses to capsaicin. EMA401 is a member of the tetrahydroisoquinoline class of AT2R antagonists (Supporting Information Fig. S1). Capsaicin is the pungent ingredient of chilli peppers, which acts on the TRPV1 receptor in nociceptive neurons (Smith et al., 2002;Facer et al., 2007) to activate calcium influx, leading to the sensation of pain. TRPV1 is activated by a variety of noxious stimuli, including capsaicin, heat, protons and leukotrienes (Tominaga et al., 1998;Hwang et al., 2000). Furthermore, there is evidence that shows that TRPV1 expression is up-regulated in clinical conditions of chronic pain (Mathews et al., 2004;Apostolidis et al., 2005;Yilmaz et al., 2007;Akbar et al., 2008). We have used an in vitro model described previously (Anand et al., 2006(Anand et al., , 2010 for determining the antinociceptive effects of EMA401 on capsaicin responses and its morphological effects on neurite outgrowth. We also studied the expression of AT2R in a range of normal and post-nerve injury human tissues using immunocytochemistry.

In vitro studies
Preparation of cultured neurons was as described previously; briefly, avulsed human cervical DRG (hDRG) were obtained as a necessary part of the surgical nerve repair procedure from three patients, with fully informed consent and approval of the Local Research Ethics Committee, Royal National Orthopaedic Hospital, Stanmore, UK. Tissue was enzyme-digested and mechanically dissociated to yield a neuronal suspension, which was plated on collagen and laminin-coated glass bottomed MatTek dishes (MatTek Corp., Ashland, MA, USA) in Ham's F12 medium containing 10% heat-inactivated fetal calf serum (HIFCS), penicillin and streptomycin (100 mg/mL each), and neurotrophic factors (NTFs) such as nerve growth factor (NGF) (100 ng/mL), GDNF and NT3 (50 ng/mL each). Cultures were incubated at 37°C in a humid environment for 48 h before being treated with AngII, or EMA401 for determining the effects on neurite density or used for Ca 2+ imaging studies. Similarly, bilateral DRG from all levels were harvested from 12 adult female Wistar rats (Charles River UK Ltd, Margate, Kent, UK) and neuronal cultures were prepared as described above, and incubated in BSF2 medium [containing 2% HIFCS, 0.1 mg/mL transferrin, 60 ng/mL pro-

What's already known about this topic?
• The angiotensin II type 2 receptor (AT2R) is expressed in sensory neurons, and in rat DRG AT2R mRNA co-localises with substance P, suggesting an involvement in nociception.

What this study adds?
• The AT2R is expressed in human peripheral somatic and visceral nerves, and is co-localised with TRPV1 in human DRG neurons. The AT2R antagonist EMA401 inhibits capsaicin responses and angiotensin II (AngII)-induced cyclic adenosine monophosphate (cAMP) increases in human and rat cultured DRG neurons. AngII causes calcium influx in DRG neurons and sensitizes capsaicin-mediated calcium influx.
[Correction added on 9th January 2013, after first online publication: the statements in two text boxes above have been previously misprinted and are now revised to the original accepted version.] gesterone, 0.16 mg/mL sodium selenite, 3 mg/mL bovine serum albumen (BSA), penicillin/streptomycin 100 mg/mL each, 16 mg/mL putrescine, 10 mg/mL insulin], and NTFs for 48 h before being studied.

Calcium imaging
Functional effects of acute EMA401 treatment on capsaicin responses were determined as previously described in Fura2 AM (Molecular Probes Life Technologies, Paisley, UK) loaded neurons (Anand et al., 2006. Responses to paired capsaicin stimuli, with and without EMA401, AngII or 8-bromo-cAMP (Calbiochem, Merck Chemicals Ltd, Beeston, Nottingham, UK), were measured in Fura2 AM loaded neurons as a change in the baseline 340/380lex nm ratio before, during and after addition. Experiments were conducted at 37°C in a humidified environment on an inverted Nikon microscope (Diaphot 300; Nikon, UK Ltd, Kingston Upon Thames, Surrey, UK) and alternately excited at 340 and 380 nm wavelengths. Forskolin was used as a surrogate source of cyclic adenosine monophosphate (cAMP) to test for adenylyl cyclase involvement, and confirmed with 8-bromo cAMP (details in Supporting Information Method S1).

Neurite outgrowth assay
Neuronal cultures were treated with AngII, EMA401 or both, and compared with NTF-treated controls in duplicate for 48 h, followed by 4% PFA fixation and Gap43 immunostaining. Lyophylized AngII (Sigma, Poole, UK) was reconstituted in sterile distilled water, aliquoted and stored at -20°C. EMA401 and Losartan (Sigma) were dissolved in HBSS containing 0.1% BSA (pH 7.4) and stored at 4°C.

Immunostaining and morphological assessment
Neurons were permeabilized with methanol (-20°C, Diameters of AT2R positive and negative neurons were measured as the average of the widest and longest axes perpendicular to each other and plotted as a frequency distribution, and the average diameters for AT2R positive and negative neurons were calculated. Gap43 immunostained images were analysed for mean neurite density (mean fluorescence intensity) from individual neurons after background subtraction; averages of five measurements (arbitrary units) within a fixed area of 100 mm 2 were obtained from the neuritecontaining area surrounding each neuron, from 50 neurons in each preparation using IMAGEJ software (NIH, National Institutes of Health, Bethesda, MD, USA). Maximum neurite lengths were measured from Tiff images of individual neurons using IMAGEJ software. Similarly, Tiff images at a fixed exposure were acquired from neuronal cultures immunostained for cAMP, after confirming the absence of immunostaining in negative controls, in which the primary antibody had been omitted. After background subtraction, the mean fluorescence intensity in each neuron was measured from a demarcated circular region of interest from the brightest region in the cell body, and the values were averaged for each group, using IMAGEJ software. Neurite density, length and cAMP fluorescence intensity are expressed as percent of control Ϯ standard error of the mean (SEM). Student's unpaired t-test was used to compare between groups; p < 0.05 was considered to be statistically significant.

Tissues
A range of tissues were used in this study for which fully informed consent was obtained with approval of the Local Ethics Committee. Specimens were snap-frozen in liquid nitrogen and stored at -70°C until use or immersed in Zamboni's fixative (2% w/v formalin, 0.1 mol/L phosphate and 15% v/v saturated picric acid) for 2 h and stored in phosphate buffered saline (PBS) containing 15% sucrose and 0.01% azide.

Skin
Skin samples (4-mm punch biopsies) were obtained under local anaesthesia from the leg (calf) of normal control subjects (n = 6; mean age 41 years, range 25-54 years; 4 women).

Intestine
Samples of full thickness, surgically resected large bowel were obtained from patients undergoing surgery for carcinoma or disease unrelated to inflammation, where uninvolved normal regions were used as controls (n = 8; age mean age = 63 years; range = 17-77 years; 3 women).
Sites of primary antibody attachment were revealed using nickel-enhanced, avidin-biotin peroxidase (ABC; Vector Laboratories, Peterborough, UK) as described (Shu et al., 1988). Sections were counter-stained for nuclei in 0.1% w/v aqueous neutral red, dehydrated and mounted in xylene-based mount (DPX; BDH/ Merck, Poole, UK) prior to photomicrography.
AT2R-immunoreactive, nucleated neurons in sensory ganglia (DRG) were counted throughout the sections and their diameter was assessed using a calibrated microscope eyepiece graticule. Nerve samples were assessed quantitatively using computerized image analysis from images captured using an Olympus DP70 camera mounted to an Olympus BX50 microscope, and analysed using analySIS (version 5.0 Soft Imaging System GmbH, Munster, Germany) software. Positive immunostaining was highlighted by setting the greylevel detection limits to threshold and the area of highlighted immunoreactivity obtained as % area of the field scanned and five random fields per tissue section were scanned at the same magnification (¥40). Results were expressed as % area, and to correct for variation in overall nerve density, they were also expressed as the ratio AT2R : neurofilaments. The Mann-Whitney test was used for statistical analysis (p-values <0.05 were considered statistically significant).

Western blotting
Protein extracts were prepared by homogenizing tissue in standard RIPA buffer at 4°C. Homogenates from human bladder and peripheral nerve were available for use with the AT2R antibody (details in Supporting Information Method S2).

In vitro
rDRG neurons were observed to be uniformly labelled with Gap43 (Fig. 1A), smaller-sized neurons were double-labelled for AT2R and Gap43 ( Fig. 1B and C). Cultured hDRG neurons were also double-labelled for Gap43 and AT2R (Fig. 1E-G); analysis of the cell body diameters showed that the AT2R positive neurons in hDRG were also of small diameter (average 36 Ϯ 2.6 mm), while AT2R negative neurons had a larger average diameter of 55.9 Ϯ 4 mm (measurements taken from 42 neurons).

Effect of AngII and EMA401 (AT2R antagonist) on capsaicin responses
Representative traces are shown in Fig.. 2A-H. Recordings of the 340/380 ratio showed a stable baseline, with a rapid increase in capsaicin stimulation in hDRG neuron ( Fig. 2A). AngII application (100 nmol/L and 10 mmol/L) stimulated calcium influx in capsaicin responsive neurons of hDRG [response to 10 mmol/L AngII after first capsaicin application (200 nmol/mL), as shown in Fig. 2B] and significant enhancement of subsequent capsaicin response (Fig. 2C). Preincubation with EMA401 reduced the subsequent capsaicin response shown in a different hDRG neuron (response to capsaicin first stimulus, Fig. 2D; inhibitory effect of EMA401, Fig. 2E). In a rDRG neuron, sample traces demonstrate the response to the first capsaicin stimulus (Fig. 2F), lack of response on addition of 100 nmol/L AngII (Fig. 2G) and enhanced response to second capsaicin stimulus (Fig. 2H). , followed by washout and response to 10 mmol/L AngII (B), followed by washout and increased response to second capsaicin stimulus (1 mmol/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.

Effect of AngII and EMA401 on neurite density
Gap43 immunostaining showed cell bodies and neurites (Fig. 3A); AngII treatment resulted in dense neurite outgrowth (Fig. 3B), while EMA401 treatment had an opposite effect (Fig. 3C). Neurites appeared to be healthy with no indication of vesicle formation or degeneration and were positively and uniformly immunostained in all preparations ( Fig. 3A-C). In rDRG neurons treated with EMA401 (Fig. 3H), there was a tendency for mean neurite density to be reduced compared with untreated controls, but this was not statistically significant.

Effect of AngII and EMA401 on neurite length
Control rDRG neurons had an average neurite length of 717 Ϯ 25 mm. Neurons treated with 10 nmol/L AngII had exuberant longer, denser neurites with a significantly increased average neurite length (bar 2, Fig. 3J: n = 3 experiments, ***p < 0.001), compared with controls; 100 nmol/L EMA401 reduced this AngII-mediated increase, which was no longer significant (bar 3, in comparison with bar 1). Furthermore, 10 and 100 nmol/L EMA401 by itself reduced the average neurite length compared with neurite length of control neurons by a small but significant extent (bars 4 and 5, Fig. 3J, *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).
did not have significantly increased cAMP levels (i.e., EMA401 prevented the increase; bar 4, Fig. 3K), and treatment with 100 nmol/L EMA401 alone showed a slight reduction, but this was not significant (bar 5, Fig. 3K). Results are from n = 4 experiments, with measurements taken from at least 175 neurons for each treatment group.

Antibody characterization
In the first instance, antibodies to AT2R were evaluated by titration on tissue sections of DRG. Antibody sc-7420 showed immunoreaction with sensory neurons and other structures in which the immunoreactivity diminished with increasing antibody dilution (Supporting Information Fig. S2a-d), and no immunostaining at dilutions greater than 1:500. Abcam antibodies Ab 19134 showed only weak nerve fibres in both pre-and post-fixed tissues, and of the other AT2R antibodies (sc-48452, sc-48451 and sc-9040), sc-48452 gave the best immunostaining of DRG neurons and nerve fibres. Pre-absorption of AT2R antibody sc-48452, with homologous peptide antigen, abolished all neuronal immunostaining in DRG at 5 ¥ 10 -2 mg antigen. A gradual return of immunostaining and intensity was achieved with decreasing concentrations of peptide antigen (Supporting Information Fig. S2e-j). Western blotting with extracts of human urinary bladder or normal human nerve using AT2R antibody sc-48452 at a dilution of 1:500 revealed a single molecular band of approximately 66 kDa (Supporting Information Fig. S2k).

DRG
The AT2R antibody sc-7420 labelled a sub-population of small-/medium-diameter (Յ50 mm) neuronal cells, some of which appeared to be very small and intensely immunoreactive ( Fig. 4A and B) in avulsion-injured DRG (n = 4, see below). For comparison, a serial section showed a range of sizes of sensory neurons immunostained with nerve marker neurofilaments (Fig. 4C). In pre-fixed DRG, background staining was low and only a few intense positive neurons were present, some with axonal processes, although no AT2R-immunoreactive fibres were detected in dorsal nerve roots (Fig. 4D). In post-fixed DRG using antibodies sc-48452 (n = 4; age range 21-23 years; trauma-to-surgery delay = 21 days-5 months; 2 women), strong small-/medium-diameter (Յ50 mm) neurons were detected along with prominent fine and large calibre nerve fibres within the DRG and dorsal roots (Fig. 4E). Since the AT2R antibody sc-48452 showed better immunohistology results for sensory neurons and fibres and appeared specific by preabsorption and Western blot, it was used for most subsequent studies. In avulsed DRG, the AT1 (sc-117) antibody did not label neuronal structures at any of the dilutions tested, but appeared to label vascular structures around the neuronal cells (Fig. 4F). AT2R-immunoreactive, nucleated sensory cells were counted and measured in post-fixed DRG using sc-48452. A total of 519 neurons (n = 4, avulsed DRG) were evaluated, and of these, the mean value (mean % total Ϯ SEM) for immunostained small-/mediumdiameter (Յ50 mm) neurons was 60.4 Ϯ 5.6 and 4.0 Ϯ 1.4 for large (>50 mm) diameter neurons (Fig. 4G). Some very small neurons appeared to be intensely immunoreactive. AT2R immunostaining using sc-48452 in post-fixed, control post-mortem DRG (n = 4) showed that counts of AT2Rimmunoreactive, small/medium diameter (Յ50 mm) nucleated sensory cells were similar to avulsion injured DRG (post-mortem controls 57.3 Ϯ 5.8). In control hDRG frozen sections, fluorescent immunostaining with the AT2R antibody sc48452 showed that AT2R immunostaining neuron counts were similar to the ABC method above; intensely positive AT2R immunoreactivity was co-localized in 41.2 Ϯ 3.7% of TRPV1 positive neurons (Fig. 4G-I: n = 4, 172 neurons).

Skin
In pre-fixed skin, intense AT1 immunoreactivity was detected in vascular endothelial/smooth muscle structures, whereas anti-AT2R antibodies showed nerve fibres and fascicles in the sub-epidermis and deeper dermis (sc-7420, Supporting Information Fig. S3a; sc-48452, Supporting Information Fig. S3c). These correlated with nerve marker (neurofilaments) in serial sections (Supporting Information Fig. S3b and d). Intra-epidermal nerve fibres were not seen.

Urinary bladder
AT2R immunoreactivity was detected in nerve fascicles and scattered single fibres (Supporting Information Fig. S3e and g) correlating with nerve marker (neurofilaments) in the adjacent sections of urinary bladder (Supporting Information Fig. S3f and h).

Intestine
Strongly positive AT2R-immunoreactive neurons, some with axonal processes, were detected in the submucous and myenteric plexuses, and a few nerve fibres in the muscularis mucosae of human intestine (Supporting Information Fig. S3i and k). The AT2R immunoreactivity appeared to co-localize with nerve marker (neurofilament cocktail) in adjacent sections (Supporting Information Fig. S3j and l). There was no evidence of AT2R immunoreactivity in nerve fibres in the mucosa.

Discussion
This study shows the expression of the AT2R in small/ medium diameter hDRG and rDRG neurons in vitro, and in hDRG tissues, partly co-localized with TRPV1 expression. Application of AngII enhanced capsaicin responses in rDRG neurons, indicating sensitization of the TRPV1 ion channel, and initial studies showed that TRPV1-sensitive cultured hDRG neurons respond to AngII by transient calcium influx. Functional effects of EMA401 pretreatment were observed as dosedependent inhibition of capsaicin responses in cultured human and rat DRG neurons (IC50 = 10 nmol/ L), which was reversed in the presence of membrane permeant 8-bromo-cAMP and AngII. Specificity at the AT2R was indicated by the reversal of EMA401mediated inhibition in the presence of AngII and the lack of effect of the AT1R antagonist Losartan. Our studies examined the effect of AngII and EMA401 on capsaicin responses in an established in vitro hDRG neuron model, requiring the identification of capsaicin sensitive neurons. The dose-related effects of exogenous AngII alone in human and rat DRG neurons, and levels of endogenous AngII in vitro and in vivo are of importance including any tonic effects, and are part of our ongoing studies. A major factor determining capsaicin sensitivity is phosphorylation of the capsaicin receptor TRPV1, which is sensitized when phosphorylated and desensitized when dephosphorylated (Bhave et al., 2002). Increased cAMP levels produced by inflammatory mediators such as prostaglandins activate protein kinase A (PKA) in nociceptive afferents, resulting in hyperalgesia, and the direct activation of PKA with cAMP analogues is known to cause behavioural hypersensitivity (Taiwo et al., 1989;Taiwo and Levine, 1991). Accordingly, we observed enhanced capsaicin responses in AngII-treated neurons, which were inhibited in the presence of EMA401. This is likely to involve a GPCR mechanism as AngII application resulted in increased intracellular cAMP, and the inhibitory effect of EMA401 on capsaicin responses was reversed in the presence of forskolin (a source of cAMP) and 8-bromo-cAMP. Together, these findings indicate that EMA401 has an inhibitory effect on capsaicin responses by reducing cAMP. It is thus likely that in situations where AngII is increased, in the periphery/DRG, it may lead to sensitization of TRPV1 and pain. While inhibition of capsaicin responses by 100 nmol/L EMA401 was comparable to maximal inhibitory effects of 1 mmol/L gabapentin and 10 mmol/L morphine, in vitro data comparisons should be regarded with caution as they may not reflect in vivo mechanisms of action or potency for these compounds. In vivo rodent studies utilizing this class of AT2R antagonists have shown efficacy in pain models, including neuropathic pain (see Smith, 2011;Smith and Wyse, 2011).
The morphological findings of this study showed that cultured hDRG neurons were positive for Gap43, and double-labelled for the AT2R in small diameter neurons, which are known to comprise nociceptors. Following treatment with EMA401, the average neurite length and density were reduced, indicating that neurite branching and length may be affected by the AT2R antagonist. AngII treatment significantly enhanced neurite outgrowth, which was completely reversed by EMA401 treatment. This is in agreement with previous studies, which also showed neurite promoting effects of AngII that were blocked with the AT2R antagonist PD123319 (Reinecke et al., 2003;Chakrabarty et al., 2008;Alterman, 2010). Neurite vesiculation/disintegration was not observed after EMA401 treatment, indicating the absence of a neurotoxic effect. Treatment with EMA401 appears to have slowed the rate of neurite extension.
Increased cAMP levels observed in AngII-treated neurons were diminished after EMA401 treatment, demonstrating an association with its effect on neurite outgrowth and capsaicin sensitivity. Neurite outgrowth is dependent on cAMP signalling (Kao et al., 2002;Neumann et al., 2002;Murray et al., 2009) and endogenous cAMP levels in neurons decline during development with a concomitant decline in regenerative capacity (Cai et al., 2001). Following nerve injury, NTF expression is up-regulated, resulting in increased cAMP levels, and up-regulation of growth-associated genes actin, tubulin and Gap43 for promotion of axon outgrowth (Gordon, 2009). In our study, cAMP expression was observed to be significantly enhanced in AngII-treated cultures and reduced in the presence of EMA401, indicating a proinflammatory role for AngII in sensory neurons.
The histological results of this study show the presence of AT2R protein in neurons of hDRG and nerve fibres of peripheral nerves, skin, urinary bladder and intestine, using immunohistochemistry. The expression of AT2R was confirmed with different antibodies and is in accord with previous reports of the presence of AT2R mRNA in rDRG and hDRG (Patil et al., 2010), and localization of AT2R in small-and medium-sized rDRG neurons (Chakrabarty et al., 2008). Additionally, a significant finding of our study is the co-localization of AT2R with a proportion of TRPV1 positive neurons, which corresponds to the AngIImediated calcium influx in capsaicin sensitive hDRG neurons and indicates a potential role for AT2R in nociception. In contrast, AT1R immunoreactivity was absent from neurons, but strongly positive in vascular structures, in hDRG and in other tissues. This observation, coupled with the lack of effect of the AT1R antagonist Losartan on capsaicin responses in DRG neurons, suggests low level of expression or absence of AT1R in hDRG neurons. Western blotting with extracts of human urinary bladder or nerve using AT 2R antibody sc-48452 revealed a single molecular band of 66 kDa similar in magnitude to that previously reported for extracts of human myometrium, where the molecular mass of AT2R was 68 Ϯ 4.6 kDa (Servant et al., 1994). Antibody sc-48452 showed best immunohistology results and appeared specific by preabsorption and Western blot.
In post-mortem control human DRG, AT2R expression was intense in a large proportion of small/ medium diameter neurons and was similar to avulsion-injured DRG neurons (the latter is the equivalent of 'central axotomy' or spinal nerve root lesion proximal to the DRG). In contrast, AT2R levels were reduced in human nerve segments proximal to injury (lesion distal to the DRG or 'peripheral axotomy'), but they were preserved or high in painful neuromas, suggesting maintained expression in regenerating nerve fibres. The regulation of AT2R expression in injured human sensory neurons, and the role of target-organ derived factors after nerve injury, deserves further study. Normal skin showed AT2R positive nerve fascicles in deep dermis, sub-epidermal fibres and nerve fibres associated with sweat glands and neurovascular structures. In the urinary bladder, AT2R positive nerve fibres were observed in nerve fascicles and as scattered sub-urothelial fibres. In bowel, AT2R immunostaining was present in nerve fibres of the mucosa and circular and longitudinal muscle layers, and also in a subset of myenteric plexus neurons. The localization of AT2R in human tissues suggests a potential role in diverse clinical pain states, including somatic and visceral disorders.
The AT2R was shown to be up-regulated in the skin of neonatal rats following injury (Viswanathan and Saavedra, 1992) and in PC12 cells by the AT2R agonist CGP42112 (Abadir et al., 2011). Oestrogen was shown to affect axonal sprouting in cultured DRG neurons by locally produced AngII via up-regulation of AT2R, and proposed to underlie the increased prevalence of pain conditions common in pre-menopausal women, such as migraine, painful bladder syndrome/interstitial cystitis, and irritable bowel syndrome (Chakrabarty et al., 2008). As NGF-induced neurite outgrowth in rDRG cultures was not affected by AT2R blockade, EMA401, while sharing features in common with anti-NGF antibodies, may provide improved safety compared with anti-NGF therapy. AT2R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting.
AngII may act as a sensory neurotransmitter and on up-regulated AT2R in sensory neurons by autocrine, paracrine and systemic mechanisms, particularly in relation to vascular innervation, via peripheral and central nerve terminals (including pre-synaptic nerve terminals), and DRG neuronal cell bodies. In the CNS, AngII and its metabolite AngIII both act at the AT1R and the AT2R (Martens et al., 1996;Zini et al., 1996;Wright et al., 2003;Pelegrini-da-Silva et al., 2005), playing a role in pain mechanisms (see Georgieva and Georgiev, 1999, Pelegrini-Da-Silva et al., 2005, Sakagawa et al., 2000Marques-Lopes et al., 2009, 2010, by the distinct expression of AT1R in neurons involved in descending pain modulation (Marques-Lopes et al., 2009), and the conversion of AngII to AngIII (Pelegrini-da-Silva et al., 2009). However, our study relates to the effects of EMA401 on peripheral mechanisms, since it does not show significant CNS distribution. Oral gavage dosing of 14 C-EMA401 at 1 mg/kg in rats did not show detectable radioactivity levels in the brain at the lower limit of quantitation (3.66 ng EMA401/g tissue), and there were no significant CNSrelated side effects in either rats dosed at 1000 mg/kg oral gavage, or in humans during the phase 1 programme (Spinifex, unpublished data).
In conclusion, AT2R antagonist EMA401 treatment resulted in a dose-related functional inhibition of capsaicin responses, and reduced neurite length, in cultured human and rat DRG neurons. The distribution of AT2R in human DRG nociceptors, co-localized with TRPV1, along with the functional in vitro studies, indicates that novel drugs such as EMA401 may inhibit pain perception in clinical disorders. cin receptor TRPV1 in nerve fibres correlates with pain score. J Clin Neurosci 14(9), 864-871. Zini, S., Fournie-Zaluski, M.C., Chauvel, E., Roques, B.P., Corvol, P., Llorens-Cortes, C. (1996). Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: Predominant role of angiotensin III in the control of vasopressin release. Proc Natl Acad Sci U S A 93 (21), 11968-11973.

Supporting Information
Additional Supporting Information may be found in the online version of this article: