The arginine-rich hexapeptide R4W2 is a stereoselective antagonist at the vanilloid receptor 1: a Ca2+ imaging study in adult rat dorsal root ganglion neurons.

Vanilloid receptors (VR) integrate various painful stimuli, e.g., noxious heat, acidic pH, capsaicin, and resiniferatoxin (RTX). Although VR antagonists may be useful analgesics, the available agents capsazepine and ruthenium red lack the necessary potency and selectivity. Recently, submicromolar concentrations of the arginine-rich hexapeptide RRRRWW-NH2(R4W2) blocked VR-mediated ionic currents in a Xenopus expression system in a noncompetitive and nonstereoselective manner. Here, VR-antagonistic effects ofl-R4W2 andd-R4W2, hexapeptides consisting entirely of l- and d-amino acids, were characterized in native adult rat dorsal root ganglion neurons using [Ca2+]i imaging (Fura-2/acetoxymethyl ester). Fura-2 fluorescence ratio (R) was increased by RTX and capsaicin by 0.473 ± 0.098 unit above basal levels of 0.903 ± 0.011 (Rmax, 2.289 ± 0.031; Rmin, 0.657 ± 0.007) in a concentration-dependent manner (log EC50: RTX, −10.04 ± 0.05, n = 10; capsaicin, −6.60 ± 0.10, n = 11). Agonist concentration-response curves were shifted to the right byl- and d-R4W2 (0.1, 1, and 10 μM each) and by capsazepine (3, 10, 30, and 100 μM), whereas their maximal effects and slopes remained unaffected, indicating competitive antagonism. Schild analysis forl-R4W2 yielded apparent dissociation constants of 4.0 nM (RTX) and 3.7 nM (capsaicin), and slopes smaller than unity (RTX, 0.38; capsaicin, 0.42). Apparent dissociation constants and slopes ford-R4W2 and capsaicin were 153 nM and 0.67 versus 4.1 μM and 1.19 for capsazepine and capsaicin. Thus, VR-mediated effects in native dorsal root ganglion neurons were antagonized by l-R4W2 >d-R4W2 > capsazepine (order of potency). In conclusion, the R4W2hexapeptide is a potent, stereospecific, and (probably) competitive VR antagonist, although an allosteric interaction cannot be completely ruled out.