Pharmacological differences between the human and rat vanilloid receptor 1 (VR1)

Abstract

Vanilloid receptors (VR1) were cloned from human and rat dorsal root ganglion libraries and expressed in Xenopus oocytes or Chinese Hamster Ovary (CHO) cells. Both rat and human VR1 formed ligand gated channels that were activated by capsaicin with similar EC(50) values. Capsaicin had a lower potency on both channels, when measured electrophysiologically in oocytes compared to CHO cells (oocytes: rat=1.90+/-0.20 microM; human=1.90+/-0.30 microM: CHO cells: rat=0.20+/-0.06 microM; human=0.19+/-0.08 microM). In CHO cell lines co-expressing either rat or human VR1 and the calcium sensitive, luminescent protein, aequorin, the EC(50) values for capsaicin-induced responses were similar in both cell lines (rat=0.35+/-0.06 microM, human=0.53+/-0.03 microM). The threshold for activation by acidic solutions was lower for human VR1 channels than that for rat VR1 (EC(50) pH 5.49+/-0.04 and pH 5.78+/-0.09, respectively). The threshold for heat activation was identical (42 degrees C) for rat and human VR1. PPAHV was an agonist at rat VR1 (EC(50) between 3 and 10 microM) but was virtually inactive at the human VR1 (EC(50)>10 microM). Capsazepine and ruthenium red were both more potent at blocking the capsaicin response of human VR1 than rat VR1. Capsazepine blocked the human but not the rat VR1 response to low pH. Capsazepine was also more effective at inhibiting the noxious heat response of human than of rat VR1.

Figure 1

Alignment of the putative protein sequences of human and rat VR1. The predicted sequences of the human (above, this paper) and rat (below, Caterina et al., 1997, Accession number AF029310) were aligned using the GCG GAP program (University of Wisconsin, Genetics Computer Group). Solid vertical bars indicate identity, a colon indicates strong similarity, a period indicates weak similarity and a gap indicates no similarity. Putative ankyrin repeats are underlined and predicted transmembrane domains are in bold and underlined. The human VR1 sequence has been deposited in the EMBL database and been assigned accession number AJ272063.

Figure 2

The human VR1 clone codes for a functional capsaicin receptor. Human and rat VR1 were expressed in oocytes. (a) Capsaicin log (concentration)-response curve for the human clone (n=10) compared to that for the rat (n=6). Currents recorded at −60 mV by sequential application of increasing concentrations of capsaicin and normalized to the response elicited by 30 μM capsaicin. (b) Current voltage relationships produced using voltage steps from the holding value of −60 mV in the presence of 1 μM capsaicin, normalized to the current at +80 mV. Leak current in the absence of capsaicin subtracted (n=8, human; n=3, rat). (c) Reversible antagonism of the response to 1 μM capsaicin by 300 nM or 10 μM capsazepine as indicated. Wash period of 5 min indicated by gap in trace. (d) Percentage inhibition of the response to 1 μM capsaicin by increasing concentration of capsazepine, expressed as a percentage of the response in the absence of antagonist (n=12 human; n=9 rat). The inset in (d) shows mean currents with 1 μM capsaicin (values, 136±27 nA (n=12), and 112±23 nA (n=9)). This shows that the differences seen with capsazepine are not due to differences in expression level.

Figure 3

Agonism of capsaicin and PPAHV in rat- and human-VR1 expressing CHO cells measures using aequorin. (a) Capsaicin log(concentration)-response curves for CHO cells stably expressing human and rat VR1 clones. The current elicited by increasing concentrations of capsaicin was measured at +30 mV and normalized to that elicited by 10 μM capsaicin for each cell (n=4 for each). EC₅₀ values, 0.20±0.06 μM rat and 0.19±0.08 μM human. (b) CHO cells expressing aequorin and either human or rat VR1 were challenged with a range of capsaicin concentrations and the calcium signal averaged over the first 10 s for rat VR1 and the first 20 s for human VR1 cells. Graphs represent mean of three separate log (concentration) response experiments. EC₅₀ values were similar using this protocol, 0.53±0.03 μM for human VR1 and 0.35±0.06 μM for rat VR1. Results are expressed as the percentage of maximal response. (c) CHO cells expressing aequorin and rat VR1 were challenged with 1, 3 and 10 μM of PPAHV and the luminescence was averaged over the first 10 s. CHO cells expressing aequorin and human VR1 were challenged with 10 μM PPAHV and the luminescence was averaged over the first 20 s. Results are expressed as the percentage of maximal response to 3 μM capsaicin of cells assayed in parallel.

Figure 4

Capsazepine and ruthenium red inhibit capsaicin responses in rat- and human-VR1 expressing CHO cells. (a) Capsazepine inhibited the capsaicin response in CHO cells expressing aequorin and either rat or human VR1. Various concentrations of capsazepine were added to the cells for 10 min prior to the assay, then the response was measured over 10 s (rat VR1) or 20 s (human VR1) after the addition of an EC₅₀ concentration of capsaicin. Capsazepine effectively inhibited both rat VR1 and human VR1 responses to capsazepine (IC₅₀ values of 0.22±0.02 and 0.04± 0.00 μM respectively). Signal was normalized to that produced by the EC₅₀ concentration of capsaicin with no antagonist as 100%, to allow comparison between rat and human VR1 expressing cell lines. (b) Ruthenium red inhibition of capsaicin responses was measured as in (a). Ruthenium red was an effective blocker of capsaicin responses in rat VR1 and human VR1 (IC₅₀ values of 0.22±0.03 and 0.09±0.02 μM respectively).

Figure 5

Ability of Ruthenium Red and Capsazepine to block low pH-induced responses in rat- and human-VR1 expressing CHO cells. (a) Low pH solutions stimulate uptake of Ca²⁺ in CHO cells that express aequorin and either rat or human VR1. Rat VR1 is more sensitive to low pH with a significant stimulation seen at pH 6.0 whereas human VR1 is not activated until pH is dropped to 5.5. CHO cells that express aequorin but not receptor show no calcium signal at low pH. (b) CHO cells that express aequorin and either rat or human VR1were preincubated with a range of capsazepine concentrations and challenged with pH 5 buffer as in Figure 4a. Capsazepine completely inhibited the human VR1 proton induced calcium signal (IC₅₀=0.31±0.02 μM), but was ineffective against the rat VR1. Signal was normalized to pH5 signal with no antagonist as 100% to allow comparison between rat and human VR1 expressing cell lines. (c) CHO cells that express aequorin and human VR1were preincubated with a range of capsazepine concentrations and challenged with pH 4.0, pH 4.5 or pH 5 buffer. Capsazepine was equally effective at blocking the proton-induced response at each pH used. (d) Ruthenium red was effective at blocking pH 5-induced calcium signal in both rat and human VR1 expressing cells.

Figure 6

Effect of heat on intracellular calcium concentration ([Ca²⁺]_i- expressed as ratio values) in VR1-expressing CHO cells. (a) human and (b) rat VR1 CHO cells show a rapid increase in [Ca²⁺]_i when the temperature rises above around 40°C. Each trace on graphs (a) and (b) represent a single cell in the same representative experiment. The estimated average threshold temperature at which [Ca²⁺]_i began to increase rapidly showed no difference between rat- and human- VR1. (c) Average [Ca²⁺]_i increases in a group of around 30 human VR1 transfected- CHO cells from a single representative experiment in response to heating to 50°C (as in a)) in the absence and presence of capsazepine (1 μM). Responses to heat in human VR1-CHO showed virtually complete and reversible inhibition by capsazepine (1 μM). (d) Comparison of the effect of 1 μM capsazepine (CPZ) on the heat response in human (filled bars) and rat (open bars)- VR1 CHO cells. Heat responses in the presence of capsazepine, and those following capsazepine washoff for approximately 5 min (‘Recovery') were normalized to an initial response to heat in the same cells (‘Control'). Inhibition of the heat response by capsazepine in rat VR1-CHO is markedly weaker than in human VR1-CHO.