Vol. 301, Issue 3, 981-986, June 2002

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

Herbert M. Himmel¹ , Thomas Kiss, Sebestyén J. Borvendeg, Clemens Gillen and Peter Illes

Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Medizinische Fakultät, Universität Leipzig, Leipzig (H.M.H., T.K., S.J.B., P.I.); and Abteilung Molekulare Pharmakologie, Grünenthal GmbH, Aachen (C.G.), Germany

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 of L-R₄W₂ and D-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 (R_max, 2.289 ± 0.031; R_min, 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 by L- 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 for L-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 for D-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 R₄W₂ hexapeptide is a potent, stereospecific, and (probably) competitive VR antagonist, although an allosteric interaction cannot be completely ruled out.