- Email: [email protected]
TRPA1 and TRPV1 Antagonists Do Not Inhibit Human Acidosis-Induced Pain
Accepted Manuscript TRPA1 and TRPV1 antagonists do not inhibit human acidosis-induced pain Matthias G. Schwarz, Barbara Namer, Peter W. Reeh, Michael J.M. Fischer PII:
S1526-5900(16)30370-4
DOI:
10.1016/j.jpain.2016.12.011
Reference:
YJPAI 3350
To appear in:
Journal of Pain
Received Date: 15 September 2016 Revised Date:
21 November 2016
Accepted Date: 21 December 2016
Please cite this article as: Schwarz MG, Namer B, Reeh PW, Fischer MJM, TRPA1 and TRPV1 antagonists do not inhibit human acidosis-induced pain, Journal of Pain (2017), doi: 10.1016/ j.jpain.2016.12.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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TRPA1 and TRPV1 antagonists do not inhibit human acidosis-induced pain Matthias G. Schwarza, Barbara Namera, Peter W. Reeha, Michael J.M. Fischera,b a
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Institute of Physiology and Pathophysiology, Friedrich-Alexander University ErlangenNürnberg, Universitätsstrasse 17, 91054 Erlangen, Germany
b
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Center of Physiology and Pharmacology Medical University of Vienna, Schwarzspanierstrasse 17, 1090 Vienna, Austria
Running title: Acidosis-induced pain
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Disclosures: Research funding was provided by the Interdisciplinary Center for Clinical Research
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(IZKF, grant E27) of the University of Erlangen-Nürnberg. There is no conflict of interest.
Corresponding author:
Univ.-Prof. Dr. med. Michael J.M. Fischer Center of Physiology and Pharmacology Medical University of Vienna Schwarzspanierstrasse 17, 1090 Vienna, Austria Phone: +43 1 40160 31410 Fax:
+43 1 40160 31129
Email: [email protected]
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Abstract Acidosis occurs in a variety of pathophysiological and painful conditions where it is thought to excite or contribute to excitation of nociceptive neurons. Despite potential clinical relevance the principal
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receptor for sensing acidosis is unclear, but several receptors have been proposed. We investigated the contribution of the acid sensing ion channels, TRPV1 and TRPA1 to peripheral pain signaling. We first established a human pain model using intra-epidermal injection of TRPA1 agonist carvacrol. This resulted in concentration-dependent pain sensations, which were reduced by experimental TRPA1
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antagonist A-967079. Capsaicin-induced pain was reduced by the TRPV1 inhibitor BCTC. Amiloride was used to block acid sensing ion channels. Testing these antagonists in a double-blind and
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randomized experiment, we probed the contribution of the respective channels to experimental acidosis-induced pain in fifteen healthy human subjects. A continuous intra-epidermal injection of pH 4.3 was employed to counter the buffering capacity of tissue and generate a prolonged painful stimulation. In this model, addition of A-967079, BCTC or amiloride did not reduce the reported pain.
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In conclusion, target-validated antagonists, applied locally in humans, have excluded the main hypothesized targets and the mechanism of the human acidosis-induced pain remains unclear.
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Perspective: An acidic milieu is a trigger of pain in many clinical conditions. The aim of this study was to identify the contribution of the currently hypothesized sensors of acid-induced pain in
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humans, using antagonists with validated actions in human subjects. Surprisingly, inhibition of these receptors did not alter acidosis-induced pain.
Keywords: acidosis; amiloride; A-967079; BCTC; carvacrol; psychophysics
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Introduction The systemic pH level in the human body is maintained within narrow limits. Tissue acidosis can occur in physiological processes like sportive exercise 47. However, it occurs also in conditions which
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are at least potentially harmful, including inflammatory processes where extracellular pH is actively regulated, and muscular ischemia where reduced perfusion or increased demand cause acidosis 24,39. Compared to healthy tissue, tumors are often acidotic with a pH in the range of 5.8–7.4; values
fast-growing tumors
29,35
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below 6.7 occur in about 10% of cases 63. In this context, it is not surprising that pain is a hallmark of . Acidification aggravates pain induced by inflammatory mediators in rats
could be reduced by urine alkalization 58.
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and humans 42,52. Pain in cystitis patients, where the inflamed bladder tissue is exposed to acidic pH,
Several models have been used to study acid-induced pain in humans. A continuous injection of buffered acidic solution into the skin or muscle can generate pain throughout the session
49,50
.
Although tissue acidosis occurs frequently in clinical conditions, the transduction cascade and
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especially the primary receptor(s) activated by protons is/are not yet clear. Several receptors have been considered, including the acid-sensing ion channel (ASIC) family 25, transient receptor potential vanilloid type 1 (TRPV1), transient receptor potential ankyrin type 1 (TRPA1), and proton-sensing G
43
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protein-coupled receptors 56. The discovery of exclusive acid activation of the human TRPA1 receptor poses the translational question of how important this receptor may be in the transduction of
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cutaneous acidosis. The hTRPA1 channel fulfills several criteria to act as an essential detector of acidification. It is expressed in a substantial fraction of peripheral sensory neurons, has a low activation threshold, and it might be co-activated by other irritating substances such as inflammatory mediators and products of oxidative stress
7,33,48,60
. This renders TRPA1 a likely
candidate involved in neurogenic inflammation and sustained pain
62
. In comparison, the pH
threshold of TRPV1 to excite rodent nociceptors in vitro is in the range of pH 6.9–6.1 and controlled by the phosphorylation state 14,18,51Fsuppl.
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In this study, we examined TRPA1, TRPV1 and the ASICs being possible candidates for the detection of acidosis by testing A-967079, BCTC and amiloride as corresponding antagonists for acidosisinduced pain in humans. A requirement to test the relevance of TRPA1 in human pain perception is a
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TRPA1-based model. We established such a model, using carvacrol as a reversible non-covalent agonist and (1E,3E)-1-(4-Fluorophenyl)-2-methyl-1-penten-3-one oxime (A-967079) as a selective antagonist. Validation of the other pertinent channel antagonists allowed addressing the role of
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channels hypothesized to contribute to the acid-induced pain in humans.
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Methods
Human psychophysical experiments. All experiments were performed in accordance with the Declaration of Helsinki. The experimental procedures were approved by the Ethics Committee of the University of Erlangen-Nürnberg (23-16B). Subjects were recruited by announcing the study in an
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electronic student’s lists and board at the University of Erlangen-Nürnberg as well as on a publicly accessible board in the Institute of Physiology at the Erlangen-Nürnberg. Subjects were educated about the purpose of the study and signed the informed consent form at least one day before the experiment. A medical history questionnaire was completed to exclude subjects with chronic disease
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or under medication. Based on a biologically relevant effect size compared to variability in pilot
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experiments, a total of 15 healthy volunteers (eight females and seven males) aged 20–50 years participated in the study from March–June 2016. All subjects completed the first experiment consisting of the TRPA1 and TRPV1 agonist studies, as well as the second experiment using acidosis for stimulation. Five of these subjects were engaged in the Laser-Doppler flowmetry experiment and eight in the experiment to determination concentration-dependent inhibition of A-967079. Pain Rating. Pain intensity was reported verbally by subjects every 5 s by means of a numeric rating scale ranging 0–100, with 0 for ‘no sensation’, the range 0–10 for ‘non-painful sensations’, 10–100 for ‘painful sensations’ and 100 for the ‘maximum imaginable pain’. The rating range 0–10 was
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introduced to allow coding of non-painful sensations, including mechanical distension, itch, warmth and coolness. As an index of the perceived pain, the area under the curve (AUC pain) is the integral above the pain threshold, summing the excess over 10.
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TRPA1 and TRPV1 pain model. In nine injection sites on the volar forearm carvacrol and capsaicin were injected intradermally in different concentrations with and without the respective blockers A967079 and BCTC using a 0.3 mm canula (30G, BD Micro-Fine, Le Pont de Claix Cedex, France). Injection solutions had a final volume of 50 µl and included carvacrol 200 µM, carvacrol 500 µM,
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carvacrol 1000 µM, A-967079 10 µM, carvacrol 500 µM + A-967079 10 µM, capsaicin 3.2 µM, BCTC 1 µM and capsaicin 3.2 µM + BCTC 1 µM and a phosphate-buffered synthetic interstitial fluid (PB-SIF,
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see below) which served as reference. The different solutions were injected in random order and in double-blind manner. Injections alternated between the left and right forearm and were at least 3 cm apart. To determine inhibition by A-967079, we used microinjections of 50 µl in eight subjects, who had participated in the experiment described above. Carvacrol 500 µM, carvacrol 500 µM
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combined with A-967079 (0.1, 1 and 10 µM), and A-967079 (10 µM) alone, all dissolved in PB-SIF pH 7.4, were injected double-blind and in random order as described above. Pain intensity was reported by subjects every 5 s for 120 s using the numeric rating scale described above. Acidosis pain model. To investigate acidosis-induced pain, we first compared the published human
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models. Iontophoresis, driving protons into the skin as described, was tested varying the hydrochloric acid concentration, contact diameter, current and duration
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tissue damage in our hands, even at neutral pH
50
25
. However, this caused
. Thus continuous acid buffer injection was
preferred as a model. Based on pilot experiments presented in the supplement, we selected a continuous infusion of PB-SIF pH 4.3 over 3 minutes to elicit acid-induced pain. A butterfly cannula with an outer diameter of 0.4 mm was inserted about 7 mm intradermally along the skin of the volar forearm. The cannula with 30 cm of flexible tubing (0.3 ml volume, B. Braun, Melsungen, Germany) was connected to a syringe mounted on a syringe pump (SP101i, World Precision Instruments, Sarasota, FL). Solutions were injected with a constant rate of 0.66 ml/min for 3 min, resulting in a
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total volume of 2 ml. Modulation of acid induced pain was tested by four injections, PB-SIF pH 4.3 alone or combined with A-967079 10 µM or BCTC 1 µM or amiloride 200 µM. All solutions were applied in random order and in a double-blind fashion at 4 different application sites. Pain intensity
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was reported by subjects every 10 s using the numeric rating scale described above. Skin perfusion. The increase in skin perfusion around the whitish injection bleb was used as index of sensory activation by carvacrol. To this end, skin blood flow was measured by a moorLDI2-VR LaserDoppler scanner, positioned at a distance of 30 cm and scanning line-wise within one minute a 2.4 x
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5.0 cm skin area centered at the injection site. The area of hyperperfusion did not exceed the long axis of this area. Four injections were applied to the volar forearm in random order and double-blind
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manner as describe above. These included PB-SIF pH 7.4 as control, and carvacrol 200, 500 and 1000 µM diluted in PB-SIF pH 7.4. Image scans took one minute and were repeated every minute. After two baseline scans, test substances were injected and followed by 12 further scans. A constant background signal, obtained from measurements on arms with a blood pressure cuff inflated to a
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pressure above the systolic blood pressure (250 mmHg), was subtracted. The area of increased skin blood flow was calculated from the number of pixels with a signal exceeding mean plus two standard deviations of the baseline images.
Chemicals and solutions. The original synthetic interstitial fluid by Bretag was modified to a
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phosphate buffered synthetic interstitial fluid (PB-SIF) version by replacing the sodium bicarbonate by sodium phosphate buffer as described
9,51
. The basic SIF contains (in mM) sodium chloride 108,
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potassium chloride 3.5, magnesium sulfate 0.7 calcium chloride 1.5, sodium gluconate 9.6, glucose 5.5, and sucrose 7.6; for PB-SIF pH 7.4 disodium hydrogen phosphate 23 mM was used, for pH 4.3 sodium dihydrogen phosphate 27.9 mM, and the resulting solution was titrated with sodium hydroxide or hydrogen chloride. Sterile solutions were obtained by using a 0.22 µM syringe filter from Sarstedt (Nürmbrecht, Germany). PB-SIF was used as control solution for all experiments and to dilute sterile stock solutions. Carvacrol, capsaicin, amiloride and lidocaine were purchased from Sigma-Aldrich (Taufkirchen, Germany). Salts and sugars were purchased from Carl Roth (Karlsruhe,
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Germany) except for sodium-gluconate purchased from VWR Chemicals (Leuven, Belgium). A967079 was obtained from Tocris (Wiesbaden-Nordenstadt, Germany) and 4-(3-Chloro-2-pyridinyl)N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide
(BCTC)
from
Sigma
(Taufkirchen,
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Germany). Stock solutions were carvacrol 100 mM and A-967079 10 mM in dimethyl sulfoxide, capsaicin 10 mM and BCTC 10 mM in ethanol. In all experiments, the final concentration of dimethyl sulfoxide or ethanol was 0.1% or less. All substances were dissolved in PB-SIF pH 7.4. Solutions were aliquoted per subject and frozen at -20°C for a maximum of 3 months until experimental use.
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Statistics. Data are presented as mean ± SEM. Two experimental groups were compared by t-tests for dependent or independent samples. Bonferroni correction was used to adjust p-levels in case of
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linked tests. Multiple groups were evaluated by analysis of variance (ANOVA). This was followed by Dunnett’s test for comparison with one group or HSD post-hoc test in case pair-wise comparisons were of interest. Analysis was performed using Statistica 8 (StatSoft, Tulsa, USA). All results were tested for differences by sex or correlation to age. All statistical tests were two-sided and a p-value
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below 0.05 was considered statistically significant.
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Results In initial experiments, the actions of TRPA1 and TRPV1 agonists and antagonists were validated in human skin. Each experiment started with an intradermal control injection of phosphate buffered-
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synthetic interstitial fluid (PB-SIF) which did not induce painful sensations (p = 0.27, single sample ttest vs. zero, no pain in 13/15 and marginal pain in 2/15 subjects), followed by eight further injections in randomized order (Figure 1a). Intradermal injection of carvacrol 200 µM caused pain
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ratings in 80% of the subjects (12/15), and carvacrol 500 µM and 1000 µM in all subjects (individual peak ratings: 47 ± 6 and 48 ± 5). Subjects experienced a fast onset of burning pain which rapidly
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subsided and fell below pain threshold for carvacrol 500 and 1000 µM in a median period of 30 s (Figure 1b, Supplemental Figure 1). All three tested carvacrol concentrations caused pain in contrast to PB-SIF (p = 0.006, p = 0.002 and p < 0.001, t-test dependent samples, Figure 1c). Carvacrol 200 µM caused mild pain, whereas carvacrol 500 µM caused substantially more pain (p = 0.003, t-test dependent samples). Higher concentrations tested in the senior author indicated that pain ratings
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caused by carvacrol 1000 µM were not close to saturation (Supplemental Figure 2). Next, we quantified carvacrol-induced nociceptor activation by measurement of the resulting skin perfusion using the Laser-Doppler scanner. The spatial integral of the blood flow change (AUC)
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within a period of 12 minutes after injection showed that carvacrol increased skin blood flow compared to PB-SIF injection (ANOVA, F(3,12)=11.9, n = 5 subjects, Figure 1d). The peak flow increase
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for carvacrol was +291% for 500 µM and +351% for 1000 µM compared to +69% by PB-SIF (p = 0.021 and 0.001, Dunnett’s post-hoc test, Figure 1e). Injection of Carvacrol 200, 500 and 1000 µM increased the area of skin hyperperfusion to a maximum of 2.9 ± 0.5 cm2, 7.4 ± 1.2 cm2 and 8.7 ± 0.5 cm2 compared to 3.3 ± 1.2 cm2 in the control injection (p = 0.62, 0.008 and 0.004, t-test dependent samples, Supplemental Figure 3a,b). Validating the antagonists, the injection of the TRPA1 antagonist A-967079 10 µM alone compared to PB-SIF did not increase pain ratings (p = 0.19, t-test dependent samples). The co-application of carvacrol 500 µM and A-967079 10 µM induced 31% lower pain ratings compared to carvacrol alone
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(p = 0.028, t-test dependent samples, Figure 1f,g). The co-applications of carvacrol 500 µM and A967079 0.1, 1 and 10 µM were performed to optimize the antagonist concentration. The results are indicative of a concentration-dependent inhibition which did not reach significance in the eight
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tested subjects (r = 0.28, p = 0.12, Spearman correlation). The AUC of the pain ratings was 85%, 73% and 59% of the carvacrol response in the presence of A-967079 0.1 µM, 1 µM, 10 µM, respectively (Supplemental Figure 4).
The TRPV1 antagonist BCTC 1 µM alone did not increase pain ratings compared to PB-SIF (p = 0.32, t-
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test dependent samples). The TRPV1 agonist capsaicin 3.2 µM produced an immediate onset of pain which rapidly fell below the pain threshold in a median period of 35 seconds. The co-application of
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capsaicin 3.2 µM and BCTC 1 µM induced 87% lower pain ratings compared to capsaicin alone (p < 0.001, t-test dependent samples, Figure 1h,i).
To exclude that the acidic environment affects the stability or inhibitory efficiency of the antagonists, we repeated the experiments in an injection solutions of pH 4.3. In 12 subjects an
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injection of PB-SIF pH 7.4 was followed by then seven injections at pH 4.3 in a randomized and double blind manner. These 50 µl injections consisted of PB-SIF, capsaicin 3.2 µM, capsaicin 3.2 µM + BCTC 1 µM, BCTC 1 µM, carvacrol 500 µM, carvacrol 500 µM + A-967079 10 µM, A-967079 10 µM. Compared to solutions of pH 7.4, the pH 4.3 solutions generated an acute and intense pain matching
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the duration of the injection of about 2 seconds. This effect was independently rated by subjects as 47.5 ± 5 for pH 4.3, and similar for the other acidic bolus injections (range 41–50). Compared to PB-
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SIF pH 7.4, application of capsaicin 3.2 µM at pH 4.3 caused pain (p < 0.024, t-test dependent samples). As observed at neutral pH, also at pH 4.3 the co-application of capsaicin 3.2 µM and BCTC 1 µM induced lower pain ratings compared to capsaicin alone (p < 0.040, t-test dependent samples, Supplemental Figure 5a,b). Carvacrol 500 µM at pH 4.3 induced higher pain ratings compared to PBSIF pH 7.4 (p < 0.003, t-test dependent samples). In pH 4.3, results with A-967079 do not allow to asses a potential action against carvacrol due to a much increased interindividual variability (Supplemental Figure 5c,d).
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Next, a possible action against experimental tissue acidosis was tested. The limitations of the chosen model, constant acid buffer infusion, were probed in pilot experiments. Prolonged infusion of the acid for 7 minutes revealed considerable desensitization, which also showed up as tachyphylaxis
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upon repeated 3 minute infusions. Coinjection of lidocaine (2 mM) demonstrated that acid induced pain could be inhibited (Supplemental Figure 6). The injection of PB-SIF pH 4.3 caused robust pain ratings throughout the three minutes of buffer-flow in 13/15 subjects; two subject’s ratings fell below the pain threshold during the injection. The maximum rating of about 30 was observed within
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the first minute. This was followed by a slow rating decrease until the end of the injection. The ratings returned to baseline values within three minutes after the injection in all subjects (Figure 2A).
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The addition of either A-967079, BCTC or amiloride in effective concentrations did not make any difference with respect to magnitude or time course of the pain ratings as compared to the painful control infusion (ANOVA, F(3,42)= 0.77, Figure 2B-D). Intra-individual comparison of the four experimental arms did not indicate cross-over effects (Figure 2E). Individual time-course of all 15
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subjects to the different stimuli are shown in Supplemental Figure 7. For all presented results, no
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differences by sex and no correlation to age was observed.
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Discussion Inhibition of TRPA1, TRPV1 and ASIC channels by respective antagonists did not alter intradermal human acidosis-induced pain. The TRP channel antagonists and their effective concentrations were
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validated by the inhibition of established channel agonist effects in the same human subjects.
Carvacrol pain model. TRPA1 channels are essential detectors of potentially harmful chemicals, especially those of electrophilic nature. The TRPA1 receptor is mainly expressed on C-fibers
including cinnamaldehyde and mustard oil
1,37
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throughout the body 55. Previous human studies examining TRPA1 have only used covalent agonists, . However, these substances show a slow activation 5,26
. This is due to the slow
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kinetic, a substantial desensitization and a partially irreversible action
covalent modification of intracellular N-terminal cysteines 7. These properties led us to favor the use of the non-covalent TRPA1 agonist carvacrol 4. The non-covalent agonism has the advantage of a rapid onset of activation and recovery of inward current responses after carvacrol application with a time constant of 1.5 seconds 34. We established carvacrol in human testing, making use of the low
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toxicity and widespread use of this substance which is the main flavor component of oregano and thyme. In heterologous expression systems, human TRPA1 channels were activated by carvacrol with an EC50 of 7 µM
31
. The unknown carvacrol concentration at the nociceptor membrane upon
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intradermal injection may explain the higher concentration required in psychophysical compared to cellular experiments. Carvacrol 500 µM was considered suitable for pharmacological inhibition as all
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subjects showed a fast pain onset and a substantial pain level, which subsided within two minutes after injection. In pilot experiments, higher carvacrol concentrations caused substantially higher pain ratings; suggesting saturation was not reached at 500 µM. In the rating range of 0–10, no uniform non-painful sensations were reported by subjects. Carvacrol can also activate TRPV3 channels
61
.
Based on several considerations, we conclude that TRPV3 has no relevant contribution to the human carvacrol-induced pain model. First, the concentration of carvacrol required to activate TRPV3 is much higher (mouse TRPV3, EC50 of 2-4 mM) compared to TRPA1 (7 µM). This determination is
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complicated by an initially weak TRPV3 activation, which increases in response to repeated or prolonged exposure 64. Second, cellular responses to carvacrol depend largely on TRPA1
4,16
. Third,
isopentyl pyrophosphate, which occurs in the cholesterol synthesis pathway, can inhibit TRPV3. It
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can also inhibit TRPA1 but with lower potency (IC50 values: 0.24 µM for TRPV3 and 7.5 µM for TRPA1) . In pilot experiments, isopentyl pyrophosphate (0.1–3.3 µM) did not inhibit carvacrol-induced
cutaneous pain (data not shown). Overall, the few existing studies indicate a limited role of TRPV3 in
reported before for mechanoinsensitive C-fibers 46.
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pain 41. For carvacrol 500 and 1000 µM, the area of hyperperfusion was in the range of what was
Inhibitors of TRPA1 and TRPV1 activation. For TRPA1, the first widely available antagonist HC-030031
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had a micromolar IC50 15 and the required concentrations were close to off-target effects and came with solubility issues 11,20. With A-967079 an antagonist with an IC50 of 51 nM for the human TRPA1 channel became available 12. The limited extent of the observed inhibition of carvacrol-induced pain by A-967079 at micromolar concentrations indicates the contribution of other mechanisms. Testing
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A-967079 in acidic milieu evoked high ratings in a few subjects, resulting in an enhanced variability that did not allow to judge the action of A-967079 under this condition. The use of capsaicin in human experimental pain models is established, and the prominent amount of inhibition by BCTC (87%) in the current study is in line with previous observations 59. BCTC has become the standard
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TRPV1 antagonist for laboratory use, since it replaced the partial antagonist capsazepine that recently turned out to be an effective TRPA1 agonist in addition 30. Therefore, we chose to test BCTC
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for TRPV1 inhibition; this abrogated capsaicin-induced pain in 9/15 subjects and largely inhibited this pain on average. Capsaicin is highly specific for TRPV1 at low concentrations, therefore other receptors, especially TRPM8 which is also blocked by BCTC, have at best a marginal contribution to acidosis-induced pain.
Experimental pain by acidosis. A contribution of tissue acidosis to the pain caused by muscular ischemia and inflammation is widely accepted 51,52. Continuous injection of acidic extracellular buffer solution into the skin was repeatedly used as a human experimental pain model 50. The composition
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of the injection solution is critical. The first study reported an isotonic phosphate buffer in distilled water, especially lacking calcium, which required far lower injection rates compared to all subsequent studies where the phosphate-buffered acidic PB-SIF solution was designed to resemble
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physiological extracellular ion compositions 49,50. The latter PB-SIF was also used in the current study. Continuous injection is necessary to generate a steady pH-profile, coping with the continuous replenishment of buffering capacity by the local blood perfusion 49. It should be noted that acidosis also inhibits conductivity of many ion channels 51, which may be the reason why higher pain ratings
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are better achieved by higher infusion rates rather than by lower pH. An alternative approach has been proposed using iontophoresis, driving the hydronium ions of hydrochloric acid solution into the
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skin using a constant current 25. We have tried this model, including systematic variations, but could not get rid of local tissue alteration lasting for a few days. The skin was immediately swollen around pores and subsequent minimal lesions appeared as burns, possibly caused by inhomogeneous and therefore high local current flow. Discussion with the authors indicated that this does not occur with
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saline solutions of hydrochloric acid. However, this generates uncertainty about the relative transport of hydronium versus sodium ions. For the current study, we lowered the pH from 5.2 to 4.3 to increase the pain ratings, producing a small effect because of the limited capacity of the phosphate buffer in the range of pH 4.3–5.2. A further decrease of pH was avoided, as pH 3.4 may
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damage neurons, indicated by experiments showing an irreversible leakage of neuropeptides from
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isolated nerve in vitro 19.
Receptors detecting painful tissue acidosis. We have recently described acid sensitivity of exclusively the human TRPA1 43. TRPA1 responsiveness is species-specific, and low pH activated neither rodent nor rhesus monkey TRPA1. In acidotic conditions, TRPA1 channel activation is reported to contribute to neuropathology by causing myelin damage 21. ASICs are also discussed as candidates for sensors of acidosis. There are six acid sensing ion channel subtypes, occuring as monomers and in various heteromeric assemblies with different pH sensitivity and response kinetics; most of these receptors share extensive desensitization within seconds, only
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few heteromers generate sustained currents 22. A bolus injection of acidic calcium-free phosphatebuffered saline hypodermal into human forearms was reported to induce pain that could be blocked by amiloride 59. In our experiments the pain caused by all bolus-injections at pH 4.3 was immediate
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and subsided at the end of the injection. The fast kinetic and the high intensity seemed to reduce differentiation between the experimental conditions compared to bolus-injections at pH 7.4. The same paper also presented a ‘cross check’ using capsazepine, which only partially blocked the pain, but this is not conclusive, as this partial antagonist is unable to block the proton response of TRPV1 13
. We stimulated for three minutes with a physiological extracellular
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(in corneal nociceptors)
solution (PB-SIF) and observed no inhibition by amiloride (200 µM). In contrast, with proton
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iontophoresis a weak effect of amiloride 1 mM was seen, stimulating for 4 minutes with no effect observed in the first minute 28. However, Diclofenac topically applied was as effective as amiloride and showed an effect in the first minute, as demonstrated previously
53
. Such high amiloride
concentration may exert non-specific anti-nociceptive effects as observed in mice, where amiloride blocked capsaicin and formalin-induced pain behavior
17
. In rat skin in vitro, amiloride 1 mM
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massively increased and prolonged the nociceptor proton responses, while 10 µM was ineffective 54. A well-known side effect of micromolar amiloride is to block the sodium-hydrogen antiporter (HNE) which leads to intracellular acidification and block of K2P channels, resulting in depolarization 40.
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Amiloride was also found to block TRPA1 which we suspected to explain the reduced acidosisinduced pain 25. However, in the present study, the TRPA1 antagonists A-967079 and amiloride were
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both unable to reduce the acidosis-induced pain. Importantly, mild and slowly accumulating tissue acidosis, as it is developing naturally, inactivates ASIC currents, because their threshold for inactivation is at higher pH than for activation. That is why ASICs close their gates when the pH sinks 2
. In addition, at body temperature compared to laboratory temperature, ASIC currents show a
further increased rate of desensitization
8,38
. Hardly any changes in nociceptor responses to
acidification (pH 5) in ASIC1a/2/3 triple-knockout mice also argue against a principal role of ASICs 28.
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TRPV1 channels reliably respond to acidic pH in cellular models and the pH response can be substantially sensitized
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. TRPV1 activation by agonists can be considered cooperative, such that
acidification also sensitizes against inflammatory mediators and capsaicin in mice and humans 27,36.
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There is little doubt that TRPV1 activation can cause pain, which would position TRPV1 well as an acidosis sensor in sensory neurons. In addition, pH-sensitivity of TRPV1 knockout neurons is largely absent
10,32
. Although the stomach environment is an exception with respect to pH, it should be
noted that here, also, neither ASIC3 nor TRPV1 receptors are responsible for nociceptor activation.
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In fact, using proton-stimulated gastric neuropeptide release as an index, the respective knockouts were not different from wild-type 3. It should be noted that all ischemic forms of acidosis lead to
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accumulation of lactate, which directly inhibits TRPV1 44.
In summary, the currently hypothesized transducers do not seem to explain tissue acidosis-induced pain in humans. Other mechanisms need to be explored. This might include testing experimentally presensitized skin to mimick inflammation
45
and considering the involvement of further channels,
e.g. proton-sensing G-protein coupled receptors GPR132 (G2A), GPR4, GPR68 (OGR1) and GPR65 23
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(TDAG8), which are expressed in nociceptors
. However, only limited pharmacological tools for
these receptors are available. Given the clinical importance of the topic, further effort has the
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potential to alleviate the highly prevalent acidosis-associated pain.
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Acknowledgements: The authors thank Marion Strupf and Birgit Vogler for support. M.G.S. received an IZKF scholarship. M.J.M.F. received financial support by the IZKF grant E27. M.G.S. performed the study, analyzed data, prepared the figures and drafted the manuscript. The present work was
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performed in fulfillment of the requirements for obtaining the degree ‘Dr. med.’ at the FriedrichAlexander-Universität Erlangen-Nürnberg (FAU). B.N. and P.W.R. provided experimental guidance and contributed to the manuscript. M.J.M.F. conceived the study, supervised data analysis and
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wrote the manuscript. The authors declare no conflict of interest.
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Abbreviations
A-967079: (1E,3E)-1-(4-Fluorophenyl)-2-methyl-1-penten-3-one oxime ASIC:
acid sensing ion channel
AUC pain: area under the curve for painful sensation
4-(3-Chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide
hTRPA1:
transient receptor potential ankyrin 1, human isoform
TRPV1:
transient receptor potential vanilloid, type 1
TRPV3:
transient receptor potential vanilloid, type 3
PB-SIF:
phosphate buffered synthetic interstitial fluid
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BCTC:
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Figure legends
Figure 1. Carvacrol induces TRPA1-dependent cutaneous pain and erythema. A) The study design contained one intradermal control injection, followed by eight injections applied in randomized
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order and double-blind. Substances were injected every 5 minutes and pain intensity was rated for 2 minutes (vertical arrows indicate injections; horizontal arrows indicate rating periods). B) Time course of numerical rating by 15 healthy subjects on a numerical rating scale 0–100. Dotted line indicates the pain threshold at 10. In contrast to PB-SIF control injection, carvacrol 200–1000 µM
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caused concentration-dependent burning pain. C) Area under the curve above the pain threshold (AUC pain) shows concentration-dependence. D) Objective measure of the carvacrol-induced flow
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increase in 5 subjects. The skin blood flow was quantified in arbitrary units once per minute by Laser-Doppler scanning. Insets show representative images for carvacrol 500 µM from one subject at 0, 2, 4, 6, 8 and 10 min. E) Area under the curve calculated for a period of 12 min after application. Compared to PB-SIF injection (open circle), carvacrol 500 and 1000 µM increased the skin blood flow. F,G) Co-application of carvacrol and TRPA1 antagonist A-967079 (10 µM) reduced pain by 31%
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compared to carvacrol alone (15 subjects). H,I) Capsaicin (3.2 µM) caused substantial pain in all subjects. Co-application of capsaicin and the TRPV1 antagonist BCTC reduced pain by 87% compared
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to capsaicin alone (15 subjects). *p < 0.05 vs. control, # p < 0.05 vs. agonist.
Figure 2. Acidosis-induced pain is not altered by TRPA1, TRPV1 and ASIC antagonists. A) The study
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design consisting of four continuous intradermal injections (black bars, 3 minutes) in randomized and double blinded order. Pain intensity was rated every 10 seconds from one minute before the injection until 3 minutes after the end (grey arrows). B-E) Time courses of numerical ratings by 15 healthy subjects. The horizontal dotted line indicates the pain threshold at a rating of 10. A continuous intradermal acidic injection at pH 4.3 with a flow rate of 0.66 ml/min applied for 3 minutes (black bar) generated moderate burning pain. Neither A-967079 10 µM nor BCTC 1 µM nor amiloride 200 µM could inhibit the induced pain compared to the control injection of PB-SIF pH 4.3; this control response is shown as dotted line in panels A-C for comparison. F) Comparison of the AUC
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pain for all tested subjects, comparing each of the three test substances to PB-SIF. Each line connects the AUC pain value of one subject, open symbols indicated the mean.
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Supplemental Figure legends
Supplemental Figure 1. Individual ratings in response to carvacrol injections. Individual time courses of the ratings of 15 healthy subjects injected intradermally with 50 µl of the indicated
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solution. For 2 minutes every 5 seconds rating on a numerical rating scale 0–100 was acquired. Open symbols indicate the mean rating of Carvacrol induced concentration-dependent burning pain.
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Supplemental Figure 2. Pilot experiment for the carvacrol concentration-response. Carvacrol 200– 4000 µM was injected in the volar forearm of the senior author. Time course (A) and area under the curve of values above the pain threshold (B) show that the dose-response relationship extends far beyond the 500 µM chosen for testing the antagonist.
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Supplemental Figure 3. Size of the skin erythema. A) Objective measure of the area of the carvacrol-induced axon reflex erythema in 5 subjects. Experiments presented in Figure 1D were analyzed with focus on the area of hyperperfusion, calculated from the number of pixels exceeding mean plus two standard deviations of the baseline. B) Maximal area of the erythema, measured one
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minute after injection of the respective substances.
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Supplemental Figure 4. Concentration-response relationship of the inhibition by A-967079. Time courses of numerical ratings by eight healthy subjects on a numerical rating scale 0–100. Dotted line indicates the pain threshold at rating 10. A) The pain ratings in the presence of A-967079 indicate a concentration-dependent inhibitory effect on the pain elicited by carvacrol 500 µM. B) The areas under the curve of pain ratings were normalized to the carvacrol 500 µM control response. Supplemental Figure 5. TRP agonists and antagonists in acidic injection solutions. A) Time course of numerical ratings by 12 healthy subjects on a numerical rating scale 0–100. Dotted line indicates the
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pain threshold at 10. Capsaicin (3.2 µM) pH 4.3 caused higher pain ratings compared to PB-SIF pH 7.4. Co-application of capsaicin and the TRPV1 antagonist BCTC reduced pain by 66% compared to capsaicin alone. B) Area under the curve above the pain threshold (AUC pain). Note the variability of
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ratings after acidic injections compared to non-acidic injections in Figure 1. C,D) Carvacrol 500 µM pH 4.3 induced higher pain ratings compared to control injections of PB-SIF pH 7.4. Results with A967079 (10 µM) were highly variable. *p < 0.05 vs. pH 7.4, # p < 0.05 vs. agonist alone.
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Supplemental Figure 6. Pilot experiments for prolonged acid injections. Syringe pump-controlled continuous injections of PB-SIF pH 4.3 intradermal into the volar forearm. Time courses of numerical
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ratings of two healthy subjects on a numerical rating scale 0–100. Dotted lines indicate the pain threshold at 10. A) Seven minutes of continuous injection of PB-SIF pH 4.3 at a rate of 0.66 ml/min. The pain ratings decreased despite continuous injection (AUC pain 54.5 within the second minute compared to 8.5 in the last minute). Addition of lidocaine 2 mM almost abolished the pain sensation, which is compared to control in the inset. B) Three consecutive intradermal injections of PB-SIF pH
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4.3. Each intradermal injection lasted three minutes and was applied through the same needle at a flow rate of 0.66 ml/min. The resulting pain is quantified in the inset (AUC pain). Desensitization appears as tachyphylaxis, in particular from the first to the second stimulus. C) Consecutive
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injections with increasing flow rates. PB-SIF pH 4.3 was continuously injected intradermally for seven minutes each through the same needle with increasing flow rates ranging from 0.083–0.66 ml/min.
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Supplemental Figure 7. Individual ratings in response to continuous acid intradermal injection. A) Individual time courses of numerical ratings by 15 healthy subjects. A continuous intradermal acidic injection at pH 4.3 with a flow rate of 0.66 ml/min was applied for 3 minutes (black bar). The dotted line indicates the pain threshold at rating 10. Intradermal injection of PB-SIF, plus A-967079 10 µM, BCTC 1 µM or amiloride 200 µM as indicated. Means are represented by colored lines and are superimposed in panel B.
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Highlights Carvacrol-induced pain is inhibited by TRPA1 antagonist A-967079 in human subjects
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Capsaicin-induced pain is inhibited by TRPV1 antagonist BCTC in human subjects
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TRPA1, TRPV1 and ASIC channels do not explain tissue acidosis-induced pain
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S1526-5900(16)30370-4
DOI:
10.1016/j.jpain.2016.12.011
Reference:
YJPAI 3350
To appear in:
Journal of Pain
Received Date: 15 September 2016 Revised Date:
21 November 2016
Accepted Date: 21 December 2016
Please cite this article as: Schwarz MG, Namer B, Reeh PW, Fischer MJM, TRPA1 and TRPV1 antagonists do not inhibit human acidosis-induced pain, Journal of Pain (2017), doi: 10.1016/ j.jpain.2016.12.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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TRPA1 and TRPV1 antagonists do not inhibit human acidosis-induced pain Matthias G. Schwarza, Barbara Namera, Peter W. Reeha, Michael J.M. Fischera,b a
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Institute of Physiology and Pathophysiology, Friedrich-Alexander University ErlangenNürnberg, Universitätsstrasse 17, 91054 Erlangen, Germany
b
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Center of Physiology and Pharmacology Medical University of Vienna, Schwarzspanierstrasse 17, 1090 Vienna, Austria
Running title: Acidosis-induced pain
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Disclosures: Research funding was provided by the Interdisciplinary Center for Clinical Research
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(IZKF, grant E27) of the University of Erlangen-Nürnberg. There is no conflict of interest.
Corresponding author:
Univ.-Prof. Dr. med. Michael J.M. Fischer Center of Physiology and Pharmacology Medical University of Vienna Schwarzspanierstrasse 17, 1090 Vienna, Austria Phone: +43 1 40160 31410 Fax:
+43 1 40160 31129
Email: [email protected]
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Abstract Acidosis occurs in a variety of pathophysiological and painful conditions where it is thought to excite or contribute to excitation of nociceptive neurons. Despite potential clinical relevance the principal
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receptor for sensing acidosis is unclear, but several receptors have been proposed. We investigated the contribution of the acid sensing ion channels, TRPV1 and TRPA1 to peripheral pain signaling. We first established a human pain model using intra-epidermal injection of TRPA1 agonist carvacrol. This resulted in concentration-dependent pain sensations, which were reduced by experimental TRPA1
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antagonist A-967079. Capsaicin-induced pain was reduced by the TRPV1 inhibitor BCTC. Amiloride was used to block acid sensing ion channels. Testing these antagonists in a double-blind and
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randomized experiment, we probed the contribution of the respective channels to experimental acidosis-induced pain in fifteen healthy human subjects. A continuous intra-epidermal injection of pH 4.3 was employed to counter the buffering capacity of tissue and generate a prolonged painful stimulation. In this model, addition of A-967079, BCTC or amiloride did not reduce the reported pain.
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In conclusion, target-validated antagonists, applied locally in humans, have excluded the main hypothesized targets and the mechanism of the human acidosis-induced pain remains unclear.
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Perspective: An acidic milieu is a trigger of pain in many clinical conditions. The aim of this study was to identify the contribution of the currently hypothesized sensors of acid-induced pain in
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humans, using antagonists with validated actions in human subjects. Surprisingly, inhibition of these receptors did not alter acidosis-induced pain.
Keywords: acidosis; amiloride; A-967079; BCTC; carvacrol; psychophysics
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Introduction The systemic pH level in the human body is maintained within narrow limits. Tissue acidosis can occur in physiological processes like sportive exercise 47. However, it occurs also in conditions which
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are at least potentially harmful, including inflammatory processes where extracellular pH is actively regulated, and muscular ischemia where reduced perfusion or increased demand cause acidosis 24,39. Compared to healthy tissue, tumors are often acidotic with a pH in the range of 5.8–7.4; values
fast-growing tumors
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below 6.7 occur in about 10% of cases 63. In this context, it is not surprising that pain is a hallmark of . Acidification aggravates pain induced by inflammatory mediators in rats
could be reduced by urine alkalization 58.
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and humans 42,52. Pain in cystitis patients, where the inflamed bladder tissue is exposed to acidic pH,
Several models have been used to study acid-induced pain in humans. A continuous injection of buffered acidic solution into the skin or muscle can generate pain throughout the session
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.
Although tissue acidosis occurs frequently in clinical conditions, the transduction cascade and
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especially the primary receptor(s) activated by protons is/are not yet clear. Several receptors have been considered, including the acid-sensing ion channel (ASIC) family 25, transient receptor potential vanilloid type 1 (TRPV1), transient receptor potential ankyrin type 1 (TRPA1), and proton-sensing G
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protein-coupled receptors 56. The discovery of exclusive acid activation of the human TRPA1 receptor poses the translational question of how important this receptor may be in the transduction of
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cutaneous acidosis. The hTRPA1 channel fulfills several criteria to act as an essential detector of acidification. It is expressed in a substantial fraction of peripheral sensory neurons, has a low activation threshold, and it might be co-activated by other irritating substances such as inflammatory mediators and products of oxidative stress
7,33,48,60
. This renders TRPA1 a likely
candidate involved in neurogenic inflammation and sustained pain
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. In comparison, the pH
threshold of TRPV1 to excite rodent nociceptors in vitro is in the range of pH 6.9–6.1 and controlled by the phosphorylation state 14,18,51Fsuppl.
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In this study, we examined TRPA1, TRPV1 and the ASICs being possible candidates for the detection of acidosis by testing A-967079, BCTC and amiloride as corresponding antagonists for acidosisinduced pain in humans. A requirement to test the relevance of TRPA1 in human pain perception is a
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TRPA1-based model. We established such a model, using carvacrol as a reversible non-covalent agonist and (1E,3E)-1-(4-Fluorophenyl)-2-methyl-1-penten-3-one oxime (A-967079) as a selective antagonist. Validation of the other pertinent channel antagonists allowed addressing the role of
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channels hypothesized to contribute to the acid-induced pain in humans.
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Methods
Human psychophysical experiments. All experiments were performed in accordance with the Declaration of Helsinki. The experimental procedures were approved by the Ethics Committee of the University of Erlangen-Nürnberg (23-16B). Subjects were recruited by announcing the study in an
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electronic student’s lists and board at the University of Erlangen-Nürnberg as well as on a publicly accessible board in the Institute of Physiology at the Erlangen-Nürnberg. Subjects were educated about the purpose of the study and signed the informed consent form at least one day before the experiment. A medical history questionnaire was completed to exclude subjects with chronic disease
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or under medication. Based on a biologically relevant effect size compared to variability in pilot
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experiments, a total of 15 healthy volunteers (eight females and seven males) aged 20–50 years participated in the study from March–June 2016. All subjects completed the first experiment consisting of the TRPA1 and TRPV1 agonist studies, as well as the second experiment using acidosis for stimulation. Five of these subjects were engaged in the Laser-Doppler flowmetry experiment and eight in the experiment to determination concentration-dependent inhibition of A-967079. Pain Rating. Pain intensity was reported verbally by subjects every 5 s by means of a numeric rating scale ranging 0–100, with 0 for ‘no sensation’, the range 0–10 for ‘non-painful sensations’, 10–100 for ‘painful sensations’ and 100 for the ‘maximum imaginable pain’. The rating range 0–10 was
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introduced to allow coding of non-painful sensations, including mechanical distension, itch, warmth and coolness. As an index of the perceived pain, the area under the curve (AUC pain) is the integral above the pain threshold, summing the excess over 10.
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TRPA1 and TRPV1 pain model. In nine injection sites on the volar forearm carvacrol and capsaicin were injected intradermally in different concentrations with and without the respective blockers A967079 and BCTC using a 0.3 mm canula (30G, BD Micro-Fine, Le Pont de Claix Cedex, France). Injection solutions had a final volume of 50 µl and included carvacrol 200 µM, carvacrol 500 µM,
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carvacrol 1000 µM, A-967079 10 µM, carvacrol 500 µM + A-967079 10 µM, capsaicin 3.2 µM, BCTC 1 µM and capsaicin 3.2 µM + BCTC 1 µM and a phosphate-buffered synthetic interstitial fluid (PB-SIF,
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see below) which served as reference. The different solutions were injected in random order and in double-blind manner. Injections alternated between the left and right forearm and were at least 3 cm apart. To determine inhibition by A-967079, we used microinjections of 50 µl in eight subjects, who had participated in the experiment described above. Carvacrol 500 µM, carvacrol 500 µM
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combined with A-967079 (0.1, 1 and 10 µM), and A-967079 (10 µM) alone, all dissolved in PB-SIF pH 7.4, were injected double-blind and in random order as described above. Pain intensity was reported by subjects every 5 s for 120 s using the numeric rating scale described above. Acidosis pain model. To investigate acidosis-induced pain, we first compared the published human
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models. Iontophoresis, driving protons into the skin as described, was tested varying the hydrochloric acid concentration, contact diameter, current and duration
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tissue damage in our hands, even at neutral pH
50
25
. However, this caused
. Thus continuous acid buffer injection was
preferred as a model. Based on pilot experiments presented in the supplement, we selected a continuous infusion of PB-SIF pH 4.3 over 3 minutes to elicit acid-induced pain. A butterfly cannula with an outer diameter of 0.4 mm was inserted about 7 mm intradermally along the skin of the volar forearm. The cannula with 30 cm of flexible tubing (0.3 ml volume, B. Braun, Melsungen, Germany) was connected to a syringe mounted on a syringe pump (SP101i, World Precision Instruments, Sarasota, FL). Solutions were injected with a constant rate of 0.66 ml/min for 3 min, resulting in a
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total volume of 2 ml. Modulation of acid induced pain was tested by four injections, PB-SIF pH 4.3 alone or combined with A-967079 10 µM or BCTC 1 µM or amiloride 200 µM. All solutions were applied in random order and in a double-blind fashion at 4 different application sites. Pain intensity
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was reported by subjects every 10 s using the numeric rating scale described above. Skin perfusion. The increase in skin perfusion around the whitish injection bleb was used as index of sensory activation by carvacrol. To this end, skin blood flow was measured by a moorLDI2-VR LaserDoppler scanner, positioned at a distance of 30 cm and scanning line-wise within one minute a 2.4 x
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5.0 cm skin area centered at the injection site. The area of hyperperfusion did not exceed the long axis of this area. Four injections were applied to the volar forearm in random order and double-blind
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manner as describe above. These included PB-SIF pH 7.4 as control, and carvacrol 200, 500 and 1000 µM diluted in PB-SIF pH 7.4. Image scans took one minute and were repeated every minute. After two baseline scans, test substances were injected and followed by 12 further scans. A constant background signal, obtained from measurements on arms with a blood pressure cuff inflated to a
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pressure above the systolic blood pressure (250 mmHg), was subtracted. The area of increased skin blood flow was calculated from the number of pixels with a signal exceeding mean plus two standard deviations of the baseline images.
Chemicals and solutions. The original synthetic interstitial fluid by Bretag was modified to a
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phosphate buffered synthetic interstitial fluid (PB-SIF) version by replacing the sodium bicarbonate by sodium phosphate buffer as described
9,51
. The basic SIF contains (in mM) sodium chloride 108,
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potassium chloride 3.5, magnesium sulfate 0.7 calcium chloride 1.5, sodium gluconate 9.6, glucose 5.5, and sucrose 7.6; for PB-SIF pH 7.4 disodium hydrogen phosphate 23 mM was used, for pH 4.3 sodium dihydrogen phosphate 27.9 mM, and the resulting solution was titrated with sodium hydroxide or hydrogen chloride. Sterile solutions were obtained by using a 0.22 µM syringe filter from Sarstedt (Nürmbrecht, Germany). PB-SIF was used as control solution for all experiments and to dilute sterile stock solutions. Carvacrol, capsaicin, amiloride and lidocaine were purchased from Sigma-Aldrich (Taufkirchen, Germany). Salts and sugars were purchased from Carl Roth (Karlsruhe,
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Germany) except for sodium-gluconate purchased from VWR Chemicals (Leuven, Belgium). A967079 was obtained from Tocris (Wiesbaden-Nordenstadt, Germany) and 4-(3-Chloro-2-pyridinyl)N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide
(BCTC)
from
Sigma
(Taufkirchen,
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Germany). Stock solutions were carvacrol 100 mM and A-967079 10 mM in dimethyl sulfoxide, capsaicin 10 mM and BCTC 10 mM in ethanol. In all experiments, the final concentration of dimethyl sulfoxide or ethanol was 0.1% or less. All substances were dissolved in PB-SIF pH 7.4. Solutions were aliquoted per subject and frozen at -20°C for a maximum of 3 months until experimental use.
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Statistics. Data are presented as mean ± SEM. Two experimental groups were compared by t-tests for dependent or independent samples. Bonferroni correction was used to adjust p-levels in case of
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linked tests. Multiple groups were evaluated by analysis of variance (ANOVA). This was followed by Dunnett’s test for comparison with one group or HSD post-hoc test in case pair-wise comparisons were of interest. Analysis was performed using Statistica 8 (StatSoft, Tulsa, USA). All results were tested for differences by sex or correlation to age. All statistical tests were two-sided and a p-value
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below 0.05 was considered statistically significant.
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Results In initial experiments, the actions of TRPA1 and TRPV1 agonists and antagonists were validated in human skin. Each experiment started with an intradermal control injection of phosphate buffered-
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synthetic interstitial fluid (PB-SIF) which did not induce painful sensations (p = 0.27, single sample ttest vs. zero, no pain in 13/15 and marginal pain in 2/15 subjects), followed by eight further injections in randomized order (Figure 1a). Intradermal injection of carvacrol 200 µM caused pain
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ratings in 80% of the subjects (12/15), and carvacrol 500 µM and 1000 µM in all subjects (individual peak ratings: 47 ± 6 and 48 ± 5). Subjects experienced a fast onset of burning pain which rapidly
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subsided and fell below pain threshold for carvacrol 500 and 1000 µM in a median period of 30 s (Figure 1b, Supplemental Figure 1). All three tested carvacrol concentrations caused pain in contrast to PB-SIF (p = 0.006, p = 0.002 and p < 0.001, t-test dependent samples, Figure 1c). Carvacrol 200 µM caused mild pain, whereas carvacrol 500 µM caused substantially more pain (p = 0.003, t-test dependent samples). Higher concentrations tested in the senior author indicated that pain ratings
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caused by carvacrol 1000 µM were not close to saturation (Supplemental Figure 2). Next, we quantified carvacrol-induced nociceptor activation by measurement of the resulting skin perfusion using the Laser-Doppler scanner. The spatial integral of the blood flow change (AUC)
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within a period of 12 minutes after injection showed that carvacrol increased skin blood flow compared to PB-SIF injection (ANOVA, F(3,12)=11.9, n = 5 subjects, Figure 1d). The peak flow increase
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for carvacrol was +291% for 500 µM and +351% for 1000 µM compared to +69% by PB-SIF (p = 0.021 and 0.001, Dunnett’s post-hoc test, Figure 1e). Injection of Carvacrol 200, 500 and 1000 µM increased the area of skin hyperperfusion to a maximum of 2.9 ± 0.5 cm2, 7.4 ± 1.2 cm2 and 8.7 ± 0.5 cm2 compared to 3.3 ± 1.2 cm2 in the control injection (p = 0.62, 0.008 and 0.004, t-test dependent samples, Supplemental Figure 3a,b). Validating the antagonists, the injection of the TRPA1 antagonist A-967079 10 µM alone compared to PB-SIF did not increase pain ratings (p = 0.19, t-test dependent samples). The co-application of carvacrol 500 µM and A-967079 10 µM induced 31% lower pain ratings compared to carvacrol alone
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(p = 0.028, t-test dependent samples, Figure 1f,g). The co-applications of carvacrol 500 µM and A967079 0.1, 1 and 10 µM were performed to optimize the antagonist concentration. The results are indicative of a concentration-dependent inhibition which did not reach significance in the eight
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tested subjects (r = 0.28, p = 0.12, Spearman correlation). The AUC of the pain ratings was 85%, 73% and 59% of the carvacrol response in the presence of A-967079 0.1 µM, 1 µM, 10 µM, respectively (Supplemental Figure 4).
The TRPV1 antagonist BCTC 1 µM alone did not increase pain ratings compared to PB-SIF (p = 0.32, t-
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test dependent samples). The TRPV1 agonist capsaicin 3.2 µM produced an immediate onset of pain which rapidly fell below the pain threshold in a median period of 35 seconds. The co-application of
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capsaicin 3.2 µM and BCTC 1 µM induced 87% lower pain ratings compared to capsaicin alone (p < 0.001, t-test dependent samples, Figure 1h,i).
To exclude that the acidic environment affects the stability or inhibitory efficiency of the antagonists, we repeated the experiments in an injection solutions of pH 4.3. In 12 subjects an
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injection of PB-SIF pH 7.4 was followed by then seven injections at pH 4.3 in a randomized and double blind manner. These 50 µl injections consisted of PB-SIF, capsaicin 3.2 µM, capsaicin 3.2 µM + BCTC 1 µM, BCTC 1 µM, carvacrol 500 µM, carvacrol 500 µM + A-967079 10 µM, A-967079 10 µM. Compared to solutions of pH 7.4, the pH 4.3 solutions generated an acute and intense pain matching
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the duration of the injection of about 2 seconds. This effect was independently rated by subjects as 47.5 ± 5 for pH 4.3, and similar for the other acidic bolus injections (range 41–50). Compared to PB-
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SIF pH 7.4, application of capsaicin 3.2 µM at pH 4.3 caused pain (p < 0.024, t-test dependent samples). As observed at neutral pH, also at pH 4.3 the co-application of capsaicin 3.2 µM and BCTC 1 µM induced lower pain ratings compared to capsaicin alone (p < 0.040, t-test dependent samples, Supplemental Figure 5a,b). Carvacrol 500 µM at pH 4.3 induced higher pain ratings compared to PBSIF pH 7.4 (p < 0.003, t-test dependent samples). In pH 4.3, results with A-967079 do not allow to asses a potential action against carvacrol due to a much increased interindividual variability (Supplemental Figure 5c,d).
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Next, a possible action against experimental tissue acidosis was tested. The limitations of the chosen model, constant acid buffer infusion, were probed in pilot experiments. Prolonged infusion of the acid for 7 minutes revealed considerable desensitization, which also showed up as tachyphylaxis
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upon repeated 3 minute infusions. Coinjection of lidocaine (2 mM) demonstrated that acid induced pain could be inhibited (Supplemental Figure 6). The injection of PB-SIF pH 4.3 caused robust pain ratings throughout the three minutes of buffer-flow in 13/15 subjects; two subject’s ratings fell below the pain threshold during the injection. The maximum rating of about 30 was observed within
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the first minute. This was followed by a slow rating decrease until the end of the injection. The ratings returned to baseline values within three minutes after the injection in all subjects (Figure 2A).
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The addition of either A-967079, BCTC or amiloride in effective concentrations did not make any difference with respect to magnitude or time course of the pain ratings as compared to the painful control infusion (ANOVA, F(3,42)= 0.77, Figure 2B-D). Intra-individual comparison of the four experimental arms did not indicate cross-over effects (Figure 2E). Individual time-course of all 15
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subjects to the different stimuli are shown in Supplemental Figure 7. For all presented results, no
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differences by sex and no correlation to age was observed.
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Discussion Inhibition of TRPA1, TRPV1 and ASIC channels by respective antagonists did not alter intradermal human acidosis-induced pain. The TRP channel antagonists and their effective concentrations were
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validated by the inhibition of established channel agonist effects in the same human subjects.
Carvacrol pain model. TRPA1 channels are essential detectors of potentially harmful chemicals, especially those of electrophilic nature. The TRPA1 receptor is mainly expressed on C-fibers
including cinnamaldehyde and mustard oil
1,37
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throughout the body 55. Previous human studies examining TRPA1 have only used covalent agonists, . However, these substances show a slow activation 5,26
. This is due to the slow
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kinetic, a substantial desensitization and a partially irreversible action
covalent modification of intracellular N-terminal cysteines 7. These properties led us to favor the use of the non-covalent TRPA1 agonist carvacrol 4. The non-covalent agonism has the advantage of a rapid onset of activation and recovery of inward current responses after carvacrol application with a time constant of 1.5 seconds 34. We established carvacrol in human testing, making use of the low
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toxicity and widespread use of this substance which is the main flavor component of oregano and thyme. In heterologous expression systems, human TRPA1 channels were activated by carvacrol with an EC50 of 7 µM
31
. The unknown carvacrol concentration at the nociceptor membrane upon
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intradermal injection may explain the higher concentration required in psychophysical compared to cellular experiments. Carvacrol 500 µM was considered suitable for pharmacological inhibition as all
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subjects showed a fast pain onset and a substantial pain level, which subsided within two minutes after injection. In pilot experiments, higher carvacrol concentrations caused substantially higher pain ratings; suggesting saturation was not reached at 500 µM. In the rating range of 0–10, no uniform non-painful sensations were reported by subjects. Carvacrol can also activate TRPV3 channels
61
.
Based on several considerations, we conclude that TRPV3 has no relevant contribution to the human carvacrol-induced pain model. First, the concentration of carvacrol required to activate TRPV3 is much higher (mouse TRPV3, EC50 of 2-4 mM) compared to TRPA1 (7 µM). This determination is
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complicated by an initially weak TRPV3 activation, which increases in response to repeated or prolonged exposure 64. Second, cellular responses to carvacrol depend largely on TRPA1
4,16
. Third,
isopentyl pyrophosphate, which occurs in the cholesterol synthesis pathway, can inhibit TRPV3. It
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can also inhibit TRPA1 but with lower potency (IC50 values: 0.24 µM for TRPV3 and 7.5 µM for TRPA1) . In pilot experiments, isopentyl pyrophosphate (0.1–3.3 µM) did not inhibit carvacrol-induced
cutaneous pain (data not shown). Overall, the few existing studies indicate a limited role of TRPV3 in
reported before for mechanoinsensitive C-fibers 46.
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pain 41. For carvacrol 500 and 1000 µM, the area of hyperperfusion was in the range of what was
Inhibitors of TRPA1 and TRPV1 activation. For TRPA1, the first widely available antagonist HC-030031
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had a micromolar IC50 15 and the required concentrations were close to off-target effects and came with solubility issues 11,20. With A-967079 an antagonist with an IC50 of 51 nM for the human TRPA1 channel became available 12. The limited extent of the observed inhibition of carvacrol-induced pain by A-967079 at micromolar concentrations indicates the contribution of other mechanisms. Testing
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A-967079 in acidic milieu evoked high ratings in a few subjects, resulting in an enhanced variability that did not allow to judge the action of A-967079 under this condition. The use of capsaicin in human experimental pain models is established, and the prominent amount of inhibition by BCTC (87%) in the current study is in line with previous observations 59. BCTC has become the standard
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TRPV1 antagonist for laboratory use, since it replaced the partial antagonist capsazepine that recently turned out to be an effective TRPA1 agonist in addition 30. Therefore, we chose to test BCTC
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for TRPV1 inhibition; this abrogated capsaicin-induced pain in 9/15 subjects and largely inhibited this pain on average. Capsaicin is highly specific for TRPV1 at low concentrations, therefore other receptors, especially TRPM8 which is also blocked by BCTC, have at best a marginal contribution to acidosis-induced pain.
Experimental pain by acidosis. A contribution of tissue acidosis to the pain caused by muscular ischemia and inflammation is widely accepted 51,52. Continuous injection of acidic extracellular buffer solution into the skin was repeatedly used as a human experimental pain model 50. The composition
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of the injection solution is critical. The first study reported an isotonic phosphate buffer in distilled water, especially lacking calcium, which required far lower injection rates compared to all subsequent studies where the phosphate-buffered acidic PB-SIF solution was designed to resemble
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physiological extracellular ion compositions 49,50. The latter PB-SIF was also used in the current study. Continuous injection is necessary to generate a steady pH-profile, coping with the continuous replenishment of buffering capacity by the local blood perfusion 49. It should be noted that acidosis also inhibits conductivity of many ion channels 51, which may be the reason why higher pain ratings
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are better achieved by higher infusion rates rather than by lower pH. An alternative approach has been proposed using iontophoresis, driving the hydronium ions of hydrochloric acid solution into the
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skin using a constant current 25. We have tried this model, including systematic variations, but could not get rid of local tissue alteration lasting for a few days. The skin was immediately swollen around pores and subsequent minimal lesions appeared as burns, possibly caused by inhomogeneous and therefore high local current flow. Discussion with the authors indicated that this does not occur with
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saline solutions of hydrochloric acid. However, this generates uncertainty about the relative transport of hydronium versus sodium ions. For the current study, we lowered the pH from 5.2 to 4.3 to increase the pain ratings, producing a small effect because of the limited capacity of the phosphate buffer in the range of pH 4.3–5.2. A further decrease of pH was avoided, as pH 3.4 may
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damage neurons, indicated by experiments showing an irreversible leakage of neuropeptides from
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isolated nerve in vitro 19.
Receptors detecting painful tissue acidosis. We have recently described acid sensitivity of exclusively the human TRPA1 43. TRPA1 responsiveness is species-specific, and low pH activated neither rodent nor rhesus monkey TRPA1. In acidotic conditions, TRPA1 channel activation is reported to contribute to neuropathology by causing myelin damage 21. ASICs are also discussed as candidates for sensors of acidosis. There are six acid sensing ion channel subtypes, occuring as monomers and in various heteromeric assemblies with different pH sensitivity and response kinetics; most of these receptors share extensive desensitization within seconds, only
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few heteromers generate sustained currents 22. A bolus injection of acidic calcium-free phosphatebuffered saline hypodermal into human forearms was reported to induce pain that could be blocked by amiloride 59. In our experiments the pain caused by all bolus-injections at pH 4.3 was immediate
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and subsided at the end of the injection. The fast kinetic and the high intensity seemed to reduce differentiation between the experimental conditions compared to bolus-injections at pH 7.4. The same paper also presented a ‘cross check’ using capsazepine, which only partially blocked the pain, but this is not conclusive, as this partial antagonist is unable to block the proton response of TRPV1 13
. We stimulated for three minutes with a physiological extracellular
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(in corneal nociceptors)
solution (PB-SIF) and observed no inhibition by amiloride (200 µM). In contrast, with proton
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iontophoresis a weak effect of amiloride 1 mM was seen, stimulating for 4 minutes with no effect observed in the first minute 28. However, Diclofenac topically applied was as effective as amiloride and showed an effect in the first minute, as demonstrated previously
53
. Such high amiloride
concentration may exert non-specific anti-nociceptive effects as observed in mice, where amiloride blocked capsaicin and formalin-induced pain behavior
17
. In rat skin in vitro, amiloride 1 mM
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massively increased and prolonged the nociceptor proton responses, while 10 µM was ineffective 54. A well-known side effect of micromolar amiloride is to block the sodium-hydrogen antiporter (HNE) which leads to intracellular acidification and block of K2P channels, resulting in depolarization 40.
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Amiloride was also found to block TRPA1 which we suspected to explain the reduced acidosisinduced pain 25. However, in the present study, the TRPA1 antagonists A-967079 and amiloride were
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both unable to reduce the acidosis-induced pain. Importantly, mild and slowly accumulating tissue acidosis, as it is developing naturally, inactivates ASIC currents, because their threshold for inactivation is at higher pH than for activation. That is why ASICs close their gates when the pH sinks 2
. In addition, at body temperature compared to laboratory temperature, ASIC currents show a
further increased rate of desensitization
8,38
. Hardly any changes in nociceptor responses to
acidification (pH 5) in ASIC1a/2/3 triple-knockout mice also argue against a principal role of ASICs 28.
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TRPV1 channels reliably respond to acidic pH in cellular models and the pH response can be substantially sensitized
57
. TRPV1 activation by agonists can be considered cooperative, such that
acidification also sensitizes against inflammatory mediators and capsaicin in mice and humans 27,36.
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There is little doubt that TRPV1 activation can cause pain, which would position TRPV1 well as an acidosis sensor in sensory neurons. In addition, pH-sensitivity of TRPV1 knockout neurons is largely absent
10,32
. Although the stomach environment is an exception with respect to pH, it should be
noted that here, also, neither ASIC3 nor TRPV1 receptors are responsible for nociceptor activation.
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In fact, using proton-stimulated gastric neuropeptide release as an index, the respective knockouts were not different from wild-type 3. It should be noted that all ischemic forms of acidosis lead to
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accumulation of lactate, which directly inhibits TRPV1 44.
In summary, the currently hypothesized transducers do not seem to explain tissue acidosis-induced pain in humans. Other mechanisms need to be explored. This might include testing experimentally presensitized skin to mimick inflammation
45
and considering the involvement of further channels,
e.g. proton-sensing G-protein coupled receptors GPR132 (G2A), GPR4, GPR68 (OGR1) and GPR65 23
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(TDAG8), which are expressed in nociceptors
. However, only limited pharmacological tools for
these receptors are available. Given the clinical importance of the topic, further effort has the
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potential to alleviate the highly prevalent acidosis-associated pain.
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Acknowledgements: The authors thank Marion Strupf and Birgit Vogler for support. M.G.S. received an IZKF scholarship. M.J.M.F. received financial support by the IZKF grant E27. M.G.S. performed the study, analyzed data, prepared the figures and drafted the manuscript. The present work was
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performed in fulfillment of the requirements for obtaining the degree ‘Dr. med.’ at the FriedrichAlexander-Universität Erlangen-Nürnberg (FAU). B.N. and P.W.R. provided experimental guidance and contributed to the manuscript. M.J.M.F. conceived the study, supervised data analysis and
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wrote the manuscript. The authors declare no conflict of interest.
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Abbreviations
A-967079: (1E,3E)-1-(4-Fluorophenyl)-2-methyl-1-penten-3-one oxime ASIC:
acid sensing ion channel
AUC pain: area under the curve for painful sensation
4-(3-Chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide
hTRPA1:
transient receptor potential ankyrin 1, human isoform
TRPV1:
transient receptor potential vanilloid, type 1
TRPV3:
transient receptor potential vanilloid, type 3
PB-SIF:
phosphate buffered synthetic interstitial fluid
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BCTC:
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Figure legends
Figure 1. Carvacrol induces TRPA1-dependent cutaneous pain and erythema. A) The study design contained one intradermal control injection, followed by eight injections applied in randomized
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order and double-blind. Substances were injected every 5 minutes and pain intensity was rated for 2 minutes (vertical arrows indicate injections; horizontal arrows indicate rating periods). B) Time course of numerical rating by 15 healthy subjects on a numerical rating scale 0–100. Dotted line indicates the pain threshold at 10. In contrast to PB-SIF control injection, carvacrol 200–1000 µM
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caused concentration-dependent burning pain. C) Area under the curve above the pain threshold (AUC pain) shows concentration-dependence. D) Objective measure of the carvacrol-induced flow
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increase in 5 subjects. The skin blood flow was quantified in arbitrary units once per minute by Laser-Doppler scanning. Insets show representative images for carvacrol 500 µM from one subject at 0, 2, 4, 6, 8 and 10 min. E) Area under the curve calculated for a period of 12 min after application. Compared to PB-SIF injection (open circle), carvacrol 500 and 1000 µM increased the skin blood flow. F,G) Co-application of carvacrol and TRPA1 antagonist A-967079 (10 µM) reduced pain by 31%
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compared to carvacrol alone (15 subjects). H,I) Capsaicin (3.2 µM) caused substantial pain in all subjects. Co-application of capsaicin and the TRPV1 antagonist BCTC reduced pain by 87% compared
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to capsaicin alone (15 subjects). *p < 0.05 vs. control, # p < 0.05 vs. agonist.
Figure 2. Acidosis-induced pain is not altered by TRPA1, TRPV1 and ASIC antagonists. A) The study
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design consisting of four continuous intradermal injections (black bars, 3 minutes) in randomized and double blinded order. Pain intensity was rated every 10 seconds from one minute before the injection until 3 minutes after the end (grey arrows). B-E) Time courses of numerical ratings by 15 healthy subjects. The horizontal dotted line indicates the pain threshold at a rating of 10. A continuous intradermal acidic injection at pH 4.3 with a flow rate of 0.66 ml/min applied for 3 minutes (black bar) generated moderate burning pain. Neither A-967079 10 µM nor BCTC 1 µM nor amiloride 200 µM could inhibit the induced pain compared to the control injection of PB-SIF pH 4.3; this control response is shown as dotted line in panels A-C for comparison. F) Comparison of the AUC
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pain for all tested subjects, comparing each of the three test substances to PB-SIF. Each line connects the AUC pain value of one subject, open symbols indicated the mean.
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Supplemental Figure legends
Supplemental Figure 1. Individual ratings in response to carvacrol injections. Individual time courses of the ratings of 15 healthy subjects injected intradermally with 50 µl of the indicated
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solution. For 2 minutes every 5 seconds rating on a numerical rating scale 0–100 was acquired. Open symbols indicate the mean rating of Carvacrol induced concentration-dependent burning pain.
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Supplemental Figure 2. Pilot experiment for the carvacrol concentration-response. Carvacrol 200– 4000 µM was injected in the volar forearm of the senior author. Time course (A) and area under the curve of values above the pain threshold (B) show that the dose-response relationship extends far beyond the 500 µM chosen for testing the antagonist.
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Supplemental Figure 3. Size of the skin erythema. A) Objective measure of the area of the carvacrol-induced axon reflex erythema in 5 subjects. Experiments presented in Figure 1D were analyzed with focus on the area of hyperperfusion, calculated from the number of pixels exceeding mean plus two standard deviations of the baseline. B) Maximal area of the erythema, measured one
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Supplemental Figure 4. Concentration-response relationship of the inhibition by A-967079. Time courses of numerical ratings by eight healthy subjects on a numerical rating scale 0–100. Dotted line indicates the pain threshold at rating 10. A) The pain ratings in the presence of A-967079 indicate a concentration-dependent inhibitory effect on the pain elicited by carvacrol 500 µM. B) The areas under the curve of pain ratings were normalized to the carvacrol 500 µM control response. Supplemental Figure 5. TRP agonists and antagonists in acidic injection solutions. A) Time course of numerical ratings by 12 healthy subjects on a numerical rating scale 0–100. Dotted line indicates the
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pain threshold at 10. Capsaicin (3.2 µM) pH 4.3 caused higher pain ratings compared to PB-SIF pH 7.4. Co-application of capsaicin and the TRPV1 antagonist BCTC reduced pain by 66% compared to capsaicin alone. B) Area under the curve above the pain threshold (AUC pain). Note the variability of
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ratings after acidic injections compared to non-acidic injections in Figure 1. C,D) Carvacrol 500 µM pH 4.3 induced higher pain ratings compared to control injections of PB-SIF pH 7.4. Results with A967079 (10 µM) were highly variable. *p < 0.05 vs. pH 7.4, # p < 0.05 vs. agonist alone.
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Supplemental Figure 6. Pilot experiments for prolonged acid injections. Syringe pump-controlled continuous injections of PB-SIF pH 4.3 intradermal into the volar forearm. Time courses of numerical
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ratings of two healthy subjects on a numerical rating scale 0–100. Dotted lines indicate the pain threshold at 10. A) Seven minutes of continuous injection of PB-SIF pH 4.3 at a rate of 0.66 ml/min. The pain ratings decreased despite continuous injection (AUC pain 54.5 within the second minute compared to 8.5 in the last minute). Addition of lidocaine 2 mM almost abolished the pain sensation, which is compared to control in the inset. B) Three consecutive intradermal injections of PB-SIF pH
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4.3. Each intradermal injection lasted three minutes and was applied through the same needle at a flow rate of 0.66 ml/min. The resulting pain is quantified in the inset (AUC pain). Desensitization appears as tachyphylaxis, in particular from the first to the second stimulus. C) Consecutive
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injections with increasing flow rates. PB-SIF pH 4.3 was continuously injected intradermally for seven minutes each through the same needle with increasing flow rates ranging from 0.083–0.66 ml/min.
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Supplemental Figure 7. Individual ratings in response to continuous acid intradermal injection. A) Individual time courses of numerical ratings by 15 healthy subjects. A continuous intradermal acidic injection at pH 4.3 with a flow rate of 0.66 ml/min was applied for 3 minutes (black bar). The dotted line indicates the pain threshold at rating 10. Intradermal injection of PB-SIF, plus A-967079 10 µM, BCTC 1 µM or amiloride 200 µM as indicated. Means are represented by colored lines and are superimposed in panel B.
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Highlights Carvacrol-induced pain is inhibited by TRPA1 antagonist A-967079 in human subjects
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Capsaicin-induced pain is inhibited by TRPV1 antagonist BCTC in human subjects
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TRPA1, TRPV1 and ASIC channels do not explain tissue acidosis-induced pain
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