Combination of a Rapidly Penetrating Agonist and a Slowly Penetrating Antagonist Affords Agonist Action of Limited Duration at the Cellular Level

Abstract

The capsaicin receptor TRPV1 (transient receptor potential vanilloid 1) has been an object of intense interest for pharmacological development on account of its critical role in nociception. In the course of structure activity analysis, it has become apparent that TRPV1 ligands may vary dramatically in the rates at which they interact with TRPV1, presumably reflecting differences in their abilities to penetrate into the cell. Using a fast penetrating agonist together with an excess of a slower penetrating antagonist, we find that we can induce an agonist response of limited duration and, moreover, the duration of the agonist response remains largely independent of the absolute dose of agonist, as long as the ratio of antagonist to agonist is held constant. This general approach for limiting agonist duration under conditions in which absolute agonist dose is variable should have more general applicability.

Keywords: Capsaicin; Pain; Pharmacodynamics; Resiniferatoxin; TRPV1; Vanilloid.

Fig. 1.

Structures of the agonist capsaicin and the antagonists BCTC, I-RTX, JYL-827, AMG9810, and capsazepine.

Fig. 2.

Calcium signal following simultaneous addition of capsaicin together with an antagonist. At 90 s 300 nM capsaicin was added together with the indicated concentrations of the indicated antagonist, and intracellular calcium levels in CHO cells stably expressing rat TRPV1 were monitored as a function of time using Fura2 as a calcium reporter. The antagonists were (A) BCTC, (B) I-RTX, (C) JYL-827, (D) AMG9810, (E) Capsazepine. Results shown represent the averaged signal from 35–50 cells measured simultaneously in a single experiment and are representative of the 3 experiments performed.

Fig. 3.

Comparison of the time course of decrease in calcium signal at different capsaicin concentrations with fixed ratios of antagonist to capsaicin. Capsaicin at 100 nM, 300 nM, and 1,000 nM was added simultaneously with the indicated ratios of antagonist to capsaicin (expressed ratio values rounded to one significant figure). The initial calcium response and that after 100 s and 500 s are plotted. AMG 9810 showed the most rapid rate of apparent penetration, with complete inhibition at T=0 at sufficiently high ratios of antagonist to capsaicin. Values at 100 nM and 300 nM represent the mean ± SE of triplicate experiments. Values at 1,000 nM represent the mean ± SE for the data within the single experiment. Note that the time scale is shifted relative to that in Fig. 2.

Fig. 4.

Comparison of the time course of decrease in calcium signal at different capsaicin concentrations with fixed ratios of antagonist to capsaicin. Capsaicin at 100 nM, 300 nM, and 1,000 nM was added simultaneously with the indicated ratios of antagonist to capsaicin (expressed ratio values rounded to one significant figure). The initial calcium response and that after 100 s and 500 s are plotted. I-RTX showed the apparent slowest rate of penetration, with little decrease at T=0 at any ratio of antagonist to capsaicin. Values at 100 nM and 300 nM represent the mean ± SE of triplicate experiments. Values at 1,000 nM represent the mean ± SE for the data within the single experiment. Note that the time scale is shifted relative to that in Fig. 2.

Fig. 5.

Comparison of the time course of decrease in calcium signal at different capsaicin concentrations with fixed ratios of antagonist to capsaicin. Capsaicin at 100 nM, 300 nM, and 1,000 nM was added simultaneously with the indicated ratios of antagonist to capsaicin (expressed ratio values rounded to one significant figure). The initial calcium response and that after 100 s and 500 s are plotted. BCTC represents an antagonist with an apparent intermediate rate of penetration. Note the partial inhibition at T=0 at the highest ratio of antagonist to capsaicin. Values at 100 nM and 300 nM represent the mean ± SE of triplicate experiments. Values at 1,000 nM represent the mean ± SE for the data within the single experiment. Note that the time scale is shifted relative to that in Fig. 2.