Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology

Abstract

The vanilloid receptor (VR1) is a ligand-gated ion channel, which plays an important role in nociceptive processing. Therefore, a pharmacological characterization of the recently cloned rat VR1 (rVR1) was undertaken. HEK293 cells stable expressing rVR1 (rVR1-HEK293) were loaded with Fluo-3AM and then incubated at 25 degrees C for 30 min with or without various antagonists or signal transduction modifying agents. Then intracellular calcium concentrations ([Ca(2+)](i)) were monitored using FLIPR, before and after the addition of various agonists. The rank order of potency of agonists (resiniferatoxin (RTX)>capsaicin>olvanil>PPAHV) was as expected, and all were full agonists. The potencies of capsaicin and olvanil, but not RTX or PPAHV, were enhanced at pH 6.4 (pEC(50) values of 7.47+/-0.06, 7.16+/-0.06, 8.19+/-0.06 and 6.02+/-0.03 respectively at pH 7.4 vs 7.71+/-0.05, 7.58+/-0.14, 8.10+/-0.05 and 6.04+/-0.08 at pH 6.4). Capsazepine, isovelleral and ruthenium red all inhibited the capsaicin (100 nM)-induced Ca(2+) response in rVR1-HEK293 cells, with pK(B) values of 7.52+/-0.08, 6.92+/-0.11 and 8.09+/-0.12 respectively (n=6 each). The response to RTX and olvanil were also inhibited by these compounds. None displayed any agonist-like activity. The removal of extracellular Ca(2+) abolished, whilst inhibition of protein kinase C with chelerythrine chloride (10 microM) partially (approximately 20%) inhibited, the capsaicin (10 microM)-induced Ca(2+) response. However, tetrodotoxin (3 microM), nimodipine (10 microM), omega-GVIA conotoxin (1 microM), thapsigargin (1 microM), U73122 (3 microM) or H-89 (3 microM) had no effect on the capsaicin (100 nM)-induced response. In conclusion, the recombinant rVR1 stably expressed in HEK293 cells acts as a ligand-gated Ca(2+) channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor.

Figure 1

RTX- and capsaicin-induced Ca²⁺ responses have different kinetics in rVR1-HEK293 cells. [Ca²⁺]_i (as fluorescent intensity units) was monitored using Fluo-3AM in HEK293 cells stably expressing rVR1 before and after the addition of RTX (100 nM) or capsaicin (100 nM). Data shown are representative traces, typical of at least n=100.

Figure 2

The agonist-induced Ca²⁺ responses are concentration-dependent. [Ca²⁺] was monitored using Fluo-3AM in rVR1-HEK293 cells before and after the addition of RTX (10 pM–1 μM), capsaicin (100 pM–10 μM), olvanil (300 pM–30 μM), or PPAHV (100 pM–10 μM). Responses were measured as peak increase in fluorescence minus basal, expressed relative to the maximum capsaicin response and are given as mean±s.e.mean, where n=6–8.

Figure 3

Acidic conditions cause a rVR1-mediated Ca²⁺ response. [Ca²⁺]_i was measured (as fluorescent intensity units) using Fluo-3AM in wild type or rVR1-transfected HEK293 cells before and after the addition at 20 s of HCl (4.2 mM, A or 2.1 mM, B), and the associated change in pH from 7.4 to 5.5 (A) and 6.7 to 5.5 (B). Traces shown are typical of at least n=20. (C) Depicts the pH-dependency of the response in rVR1-expressing cells. Data are mean±s.e.mean, where n=5.

Figure 4

rVR1 agonists do not display cooperativity. [Ca²⁺]_i was monitored using Fluo-3AM in rVR1-HEK293 cells before and after the addition of RTX (30 nM or 1 μM), capsaicin (100 nM or 10 μM), olvanil (300 nM), or PPAHV (1 μM), alone or in combination. Responses were measured as peak increase in fluorescence minus basal, expressed relative to the maximum capsaicin response and are given as mean±s.e.mean, where n=5.

Figure 5

VR1 antagonists inhibit the capsaicin-induced Ca²⁺ response. In (A) Fluo-3AM-loaded rVR1-HEK293 cells were preincubated with buffer (control), capsazepine (0.1 nM–10 μM), isovelleral (1 nM–10 μM) or ruthenium red (0.1 nM–10 μM) for 30 min at 37°C, and then [Ca²⁺]_i was monitored before and after the addition of capsaicin (100 nM). In (B), the concentration-response relationship for capsaicin (0.1 nM–30 μM) was assessed in the presence or absence of capsazepine (CZP, 0.1 μM) or isovelleral (1–10 μM). Responses were measured as peak increase in fluorescence minus basal, expressed relative to the control capsaicin response and are given as mean±s.e.mean, where n=6.

Figure 6

The role of extracellular Ca²⁺ and PKC in the rVR1-mediated Ca²⁺ response. [Ca²⁺]_i was monitored (as fluorescent intensity units) using Fluo-3AM in rVR1-HEK293 cells, incubated in calcium-containing or calcium-free buffer, in the presence or absence of chelerythrine chloride (Ch. Cl, 10 μM), before and after a capsaicin (1 μM) challenge at 20 s. Data are representative traces, typical of at least n=5.