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Differential Sensitivity of Cinnamaldehyde-Evoked Calcium

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Differential Sensitivity of Cinnamaldehyde-Evoked Calcium Bahia and Taylor‑Clark BMC Res Notes (2021) 14:127 https://doi.org/10.1186/s13104‑021‑05539‑2 BMC Research Notes RESEARCH NOTE Open Access Diferential sensitivity of cinnamaldehyde‑evoked calcium fuxes to ruthenium red in guinea pig and mouse trigeminal sensory neurons Parmvir K. Bahia and Thomas E. Taylor‑Clark* Abstract Objective: Transient receptor potential ankyrin 1 (TRPA1) is an excitatory ion channel expressed on a subset of sen‑ sory neurons. TRPA1 is activated by a host of noxious stimuli including pollutants, irritants, oxidative stress and infam‑ mation, and is thought to play an important role in nociception and pain perception. TRPA1 is therefore a therapeutic target for diseases with nociceptive sensory signaling components. TRPA1 orthologs have been shown to have dif‑ ferential sensitivity to certain ligands. Cinnamaldehyde has previously been shown to activate sensory neurons via the selective gating of TRPA1. Here, we tested the sensitivity of cinnamaldehyde‑evoked responses in mouse and guinea pig sensory neurons to the pore blocker ruthenium red (RuR). Results: Cinnamaldehyde, the canonical TRPA1‑selective agonist, caused robust calcium fuxes in trigeminal neurons dissociated from both mice and guinea pigs. RuR efectively inhibited cinnamaldehyde‑evoked responses in mouse neurons at 30 nM, with complete block seen with 3 μM. In contrast, responses in guinea pig neurons were only par‑ tially inhibited by 3 μM RuR. We conclude that RuR has a decreased afnity for guinea pig TRPA1 compared to mouse TRPA1. This study provides further evidence of diferences in ligand afnity for TRPA1 in animal models relevant for drug development. Keywords: TRPA1, Trigeminal, Ruthenium red, Guinea pig, Mouse Introduction important role in initiating or sensitizing noxious sen- Transient Receptor Potential Ankyrin 1 (TRPA1) is a sations and nocifensive refexes in the skin and visceral homo-tetrameric, non-selective cation channel belonging organs such as the airways [3–7]. As such, TRPA1 has to the superfamily of TRP channels. Mammalian TRPA1 been proposed as a target for therapeutic intervention in is expressed primarily in a subset (~ 20–50%) of nocicep- the treatment of pain and respiratory disorders. tive sensory nerves, with cell bodies in dorsal root gan- TRPA1 have been studied using recombinant channels glia (DRG), trigeminal ganglia, and vagal ganglia [1, 2]. and various animal models. With respect to respiratory TRPA1 is activated by a range of electrophilic chemical physiology, activation of TRPA1 by irritants, oxidative irritants and products of oxidative stress, and plays an stress and pollutants causes cough, apnea, refex bron- chospasm and refex modulation of heart rate [6, 8–14]. Furthermore, knockout of TRPA1 has been shown to be *Correspondence: [email protected] Molecular Pharmacology & Physiology, Morsani College of Medicine, protective in allergen-associated airway hyperreactiv- University of South Florida, 12901 Bruce B Downs Blvd, Tampa, FL 33612, ity [15]. Te guinea pig (Cavia porcellus) is of particular USA interest for developing therapeutic targets for respiratory © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Bahia and Taylor‑Clark BMC Res Notes (2021) 14:127 Page 2 of 5 diseases as airway smooth muscle physiology, acute were immediately isolated and enzymatically dissociated responses to allergen exposure, and ability to cough in using previously described methods [5]. Isolated neurons response to inhaled irritant aerosols are more representa- were plated onto poly-D-lysine and laminin-coated cov- tive of human airways than those of rats or mice [16, 17]. erslips, incubated at 37 °C in L-15 (supplemented with However, despite the many studies examining TRPA1 10% fetal bovine serum) and used within 24 h. Neurons 2+ function in guinea pig tissues, the characterization of the were studied for changes in [Ca ]i with Fura-2AM, as channel itself remains incomplete. Tis is an important before [36]. Neuron-covered coverslips were incubated defcit to address given the marked diferences in amino (37 °C) with Fura-2AM (4 μM, for 30 min) in L-15 media acid sequence and functional disparities between rodent containing 10% fetal bovine serum. For imaging, the cov- and human TRPA1 [18, 19]. erslip was placed in a custom-built heated chamber and One of the most commonly used pharmacological superfused HEPES-bufered bath solution (composi- tools in the study of cation channels is the non-specifc tion (mM)): 154 NaCl, 4.7 KCl, 1.2 MgCl 2, 2.5 CaCl2, 10 blocker Ruthenium Red (RuR). Tis water soluble, inor- HEPES, 5.6 dextrose adjusted to pH 7.4 with NaOH) for ganic cationic dye was shown by Moore in 1971 [20] to 10 min before and throughout each experiment. Changes 2+ inhibit transmembrane calcium fuxes, and has since in [Ca ]i were monitored by sequential dual excitation, been demonstrated as an efective blocker of calcium 340 and 380 nm (emission 510 nm)(CoolSnap HQ2; Pho- channels and, in particular, TRP channels [1, 21–23]. RuR tometrics, Surrey, BC, Canada) and analyzed by Nikon has been shown to block the outer pore of diferent TRP Elements (Nikon, Melville, NY, USA). Neurons were channels and is thought to bind to specifc aspartic acid exposed to cinnamaldehyde (50 μM) at t = 4 to 6 min, residues [24, 25]. In the case of heterologously-expressed then, following a washout of 7 min, to a second cinnamal- TRPA1, RuR has been shown to block both mamma- dehyde treatment (100 μM) at t = 13–15 min. Neurons lian and non-mammalian orthologs of the channel [3–5, were treated with various concentrations of ruthenium 26–28] and, in accordance with other TRP channels, this red (0, 30, 300, 3000 nM) preceding, during and after block is greatly diminished in human TRPA1 if the D915 the frst cinnamaldehyde treatment at t = 1–8 min. At residue is mutated [29]. Tere has been a report that the end of the studies, all neurons were exposed to KCl heterologously-expressed guinea pig TRPA1 is less sensi- (75 mM, 60 s) to confrm voltage sensitivity and ionomy- tive to RuR block compared to heterologously-expressed cin (5 μM, 60 s) to obtain a maximal response. All agents mouse TRPA1 [30]. Here we have investigated the efect were purchased from Sigma-Aldrich. 2+ of RuR on the activation of trigeminal neurons from mice As before [37], we determined [Ca ]i by measuring and guinea pigs by the electrophilic TRPA1 agonist cin- the 340/380 ratio and relating all measurements to the namaldehyde. Previous studies have shown that cinna- peak positive response in each cell. Tus we have cho- maldehyde-evoked sensory nerve responses in rodents sen to normalize ratiometric responses at each timepoint 2+ 2+ are largely eliminated by TRPA1 knockout or selective in each cell to its maximum [Ca ]i (evoked by the Ca TRPA1 inhibitors [9, 31–34]. We found marked difer- ionophore ionomycin) – data was presented as the per- ences in RuR’s inhibition of cinnamaldehyde-evoked centage change in 340/380 ratio (R): response at time responses, with efective block of mouse responses at (x) = 100 X (Rx-Rbl)/(Rmax-Rbl), where Rx was the 340/380 30 nM but only partial block of guinea pig responses at ratio of the cell at a given time point, R bl was the cell’s 3 μM. We conclude that guinea pig TRPA1 is less sensi- mean baseline 340/380 ratio measured over 60 s, and tive to RuR block compared to mouse TRPA1. Rmax was the cell’s peak 340/380 ratio. Only cells that 2+ had low [Ca ]i at baseline (R < 1.0) and yielded a robust Main text response to the positive control were included in analy- Methods and results ses. Neurons were defned as TRPA1-expressing if a To study the diferences between the sensitivity of guinea positive response was noted for either cinnamaldehyde pig TRPA1 and mouse TRPA1 to RuR we compared its treatments. Only cinnamaldehyde-sensitive neurons efect on the Ca2+ responses of sensory neurons dissoci- were included in the analyses. Ratiometric responses ated from mouse and guinea pig trigeminal ganglia to the were found to pass the D’Agostino & Pearson test for TRPA1 selective agonist cinnamaldehyde [3, 34]. We have normality (p > 0.05). Te concentration response graph 2+ previously shown that TRPA1 agonists increase [Ca ]i represents unpaired observations, which were compared by inducing Ca2+ infux [35, 36]. Male C57BL/6 mice using a 2-way ANOVA with Dunnett’s multiple compari- (6 weeks old, 6 animals, purchased from Envigo) and sons using GraphPad Prism version 8. A p value less than male Hartley guinea pigs (6 weeks old, 3 animals, pur- 0.05 was taken as statistically signifcant. chased from Charles River) were killed by CO 2 asphyxi- Cinnamaldehyde (50 μM and 100 μM) caused an 2+ ation followed by exsanguination.
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