The putative role of vanilloid receptor-like protein-1 in mediating high threshold noxious heat-sensitivity in rat cultured primary sensory neurons.

High threshold noxious heat-activated currents and vanilloid receptor-like protein-1 expression were studied in rat cultured primary sensory neurons to find out the molecule(s) responsible for high threshold noxious heat-sensitivity. The average temperature threshold and amplitude of high threshold noxious heat-activated currents were 51.6 +/- 0.13 degrees C and -2.0 +/- 0.1nA (at a holding potential of -60 mV), respectively. The current-voltage relationship of high threshold noxious heat-activated currents was linear at positive membrane potentials, while it showed a weak inward rectification at negative membrane potentials. The average reversal potential measured in control intracellular and extracellular solutions was 4.5 +/- 0.9 mV (n = 6). Ionic substitutions revealed that the high threshold noxious heat-activated current is a nonselective cationic current with calculated ionic permeabilities of Cs+ : Na+ : Ca2+ (1 : 1.3 : 4.5). Consecutive stimuli reduced the heat threshold from 52.2 +/- 1 to 48.4 +/- 1.4 degrees C and then to 44 +/- 0.7 degrees C (n = 3). High threshold noxious heat-activated currents could dose-dependently and reversibly be reduced by ruthenium red (100 nm-10 micro m) but not by capsazepine (10 micro m). The average longest diameter of high threshold noxious heat-sensitive neurons was 31.48 +/- 0.5 micro m (A = approximately 778 micro m2; n = 77). Twenty-three percent of the total neuronal population expressed vanilloid receptor-like protein-1. The average area of the vanilloid receptor-like protein-1-immunopositive cells was 1,696 +/- 65.3 micro m2 (d = approximately 46 micro m). Vanilloid receptor-like protein-1-expressing neurons did not express the vanilloid receptor 1. Comparison of our data with results obtained in vanilloid receptor-like protein-1-expressing non-neuronal cells and previous immunohistochemical findings suggests that high threshold noxious heat-activated currents are produced by vanilloid receptor-like protein-1 and that high threshold heat-sensitive dorsal root ganglion neurons are the perikarya of type I noxious heat-sensitive fibers.

Cloning and functional characterization of the guinea pig vanilloid receptor 1.

We have cloned a guinea pig Vanilloid receptor 1 (VR1) from a dorsal root ganglion cDNA library and expressed it in CHO cells. The receptor has been functionally characterized by measuring changes in intracellular calcium produced by capsaicin, low pH and noxious heat. Capsaicin produced a concentration-dependent increase in intracellular calcium in guinea pig VR1-CHO cells with an estimated EC(50) of 0.17 +/- 0.0065 micro M, similar to that previously reported for rat and human VR1. Olvanil and resiniferatoxin were also effective agonists (EC(50) values of 0.0087 +/- 0.0035 micro M and 0.067 +/- 0.014 micro M, respectively), but 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) and anandamide showed little agonist activity up to 10 micro M. As with human and rat VR1, guinea pig VR1 was also activated by pH below 6.0 and by noxious heat (>42 degrees C). Capsazepine acted as an antagonist of capsaicin responses in guinea pig VR1-CHO cells (IC(50) of 0.324 +/- 0.041 micro M ), as seen at rat VR1. However, in contrast to its lack of activity against pH and heat responses at rat VR1, capsazepine was an effective antagonist of these responses at guinea pig VR1. Capsazepine displayed an IC(50) of 0.355 +/- 25 micro M against pH 5.5, and provided complete blockade of heat responses at 1 micro M. Thus, capsazepine can significantly inhibit calcium influx due to heat and pH 5.5 at guinea pig VR1 and human VR1 but is inactive against these activators at rat VR1.

Nonpeptide bradykinin B2 receptor antagonists: conversion of rodent-selective bradyzide analogues into potent, orally-active human bradykinin B2 receptor antagonists.

The 1-(2-nitrophenyl)thiosemicarbazide (TSC) derivative, (S)-1-[4-(4-benzhydrylthiosemicarbazido)-3-nitrobenzenesulfonyl]pyrrolidine-2-carboxylic acid [2-[(2-dimethylaminoethyl)methylamino]ethyl]amide (bradyzide; (S)-4), was recently disclosed as a novel, potent, orally active nonpeptide bradykinin (BK) B2 receptor antagonist. The compound inhibited the specific binding of [3H]BK to NG108-15 cell membrane preparations (rodent neuroblastoma-glioma) expressing B2 receptors with a K(i) of 0.5 +/- 0.2 nM. Compound (S)-4 also demonstrated oral efficacy against Freund's complete adjuvant (FCA)-induced mechanical hyperalgesia in rats with an ED50 value of 0.84 micromol/kg. After we optimized the terminal binding determinants projecting from the TSC framework, we found that it was possible to replace the potentially toxicophoric nitro and divalent sulfur moieties with only a 15-fold loss in binding affinity ((S)-14a). However, bradyzide and its congeners were found to have much lower affinities for cloned human B2 receptors, expressed in Cos-7 cells. The hitherto synthesized TSC series was screened against the human B2 receptor, and the dibenzosuberane (DBS) pharmacophore emerged as the key structural requirement for potency. Incorporation of this group resulted in a series of derivatives ((S)-14d,e and 19b-d) with K(i) ranges of 10.7-176 nM in NG108-15 cells (expressing the rodent B2 receptor) and 0.79-253 nM in Cos-7 cells (expressing the human B2 receptor). There was no evidence of agonist activity with any of the nonpeptides in any of the cell lines tested. In vivo, oral administration of compound 19c reversed FCA-induced and turpentine-induced mechanical hyperalgesia in rodents with ED50 values of 0.027 and 0.32 micromol/kg, respectively. The selectivity profiles of compounds (S)-14f and (S)-14g were also assessed to determine the conformational and/or steric preferences of the double-ring arrangement. The affinity of (S)-14 g for the human B2 receptor suggested that it may be a hydrophobic interaction with the ethane bridge of the DBS moiety that accounts for the increased potency of compounds (S)-14d,e and 19b,c at this receptor, by favoring a binding mode inaccessible to the unsubstituted diphenylmethyl derivative, (S)-4.