Background: The clinical efficacy of the Angiotensin II (AngII) receptor AT2R antagonist EMA401, a novel peripherallyrestricted
analgesic, was reported recently in post-herpetic neuralgia. While previous studies have shown that AT2R
is expressed by nociceptors in human DRG (hDRG), and that EMA401 inhibits capsaicin responses in cultured hDRG
neurons, the expression and levels of its endogenous ligands AngII and AngIII in clinical neuropathic pain tissues, and
their signalling pathways, require investigation. We have immunostained AngII, AT2R and the capsaicin receptor TRPV1
in control post-mortem and avulsion injured hDRG, control and injured human nerves, and in cultured hDRG neurons.
AngII, AngIII, and Ang-(1-7) levels were quantified by ELISA. The in vitro effects of AngII, AT2R agonist C21, and Nerve
growth factor (NGF) were measured on neurite lengths; AngII, NGF and EMA401 effects on expression of p38 and
p42/44 MAPK were measured using quantitative immunofluorescence, and on capsaicin responses using calcium
imaging.
Results: AngII immunostaining was observed in approximately 75% of small/medium diameter neurons in control
(n = 5) and avulsion injured (n = 8) hDRG, but not large neurons i.e. similar to TRPV1. AngII was co-localised with
AT2R and TRPV1 in hDRG and in vitro. AngII staining by image analysis showed no significant difference between
control (n = 12) and injured (n = 13) human nerves. AngII levels by ELISA were also similar in control human nerves
(4.09 ± 0.36 pmol/g, n = 31), injured nerves (3.99 ± 0.79 pmol/g, n = 7), and painful neuromas (3.43 ± 0.73 pmol/g,
n = 12); AngIII and Ang-(1-7) levels were undetectable (<0.03 and 0.05 pmol/g respectively). Neurite lengths were
significantly increased in the presence of NGF, AngII and C21 in cultured DRG neurons. AngII and, as expected, NGF
significantly increased signal intensity of p38 and p42/44 MAPK, which was reversed by EMA401. AngII mediated sensitization
of capsaicin responses was not observed in the presence of MAP kinase inhibitor PD98059, and the kinase
inhibitor staurosporine.
Conclusion: The major AT2R ligand in human peripheral nerves is AngII, and its levels are maintained in injured
nerves. EMA401 may act on paracrine/autocrine mechanisms at peripheral nerve terminals, or intracrine mechanisms,
to reduce neuropathic pain signalling in AngII/NGF/TRPV1-convergent pathways.