Bitter tastants relax the mouse gallbladder smooth muscle independent of signaling through tuft cells and bitter taste receptors

Keshavarz, Maryam; Ruppert, Anna-Lena; Meiners, Mirjam; Poharkar, Krupali; Liu, Shuya; Mahmoud, Wafaa; Winterberg, Sarah; Hartmann, Petra; Mermer, Petra; Perniss, Alexander; Offermanns, Stefan; Kummer, Wolfgang; Schütz, Burkhard

doi:10.1038/s41598-024-69287-6

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Published: 08 August 2024

Bitter tastants relax the mouse gallbladder smooth muscle independent of signaling through tuft cells and bitter taste receptors

Maryam Keshavarz^1,2^nAff6,
Anna-Lena Ruppert³,
Mirjam Meiners^1,2,
Krupali Poharkar^1,2,
Shuya Liu⁵^nAff4,
Wafaa Mahmoud^1,2^nAff7,
Sarah Winterberg³,
Petra Hartmann^1,2,
Petra Mermer^1,2,
Alexander Perniss^1,2^nAff8,
Stefan Offermanns^2,5,
Wolfgang Kummer^1,2 &
…
Burkhard Schütz³

Scientific Reports volume 14, Article number: 18447 (2024) Cite this article

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Abstract

Disorders of gallbladder motility can lead to serious pathology. Bitter tastants acting upon bitter taste receptors (TAS2R family) have been proposed as a novel class of smooth muscle relaxants to combat excessive contraction in the airways and other organs. To explore whether this might also emerge as an option for gallbladder diseases, we here tested bitter tastants for relaxant properties and profiled Tas2r expression in the mouse gallbladder. In organ bath experiments, the bitter tastants denatonium, quinine, dextromethorphan, and noscapine, dose-dependently relaxed the pre-contracted gallbladder. Utilizing gene-deficient mouse strains, neither transient receptor potential family member 5 (TRPM5), nor the Tas2r143/Tas2r135/Tas2r126 gene cluster, nor tuft cells proved to be required for this relaxation, indicating direct action upon smooth muscle cells (SMC). Accordingly, denatonium, quinine and dextromethorphan increased intracellular calcium concentration preferentially in isolated gallbladder SMC and, again, this effect was independent of TRPM5. RT-PCR revealed transcripts of Tas2r108, Tas2r126, Tas2r135, Tas2r137, and Tas2r143, and analysis of gallbladders from mice lacking tuft cells revealed preferential expression of Tas2r108 and Tas2r137 in tuft cells. A TAS2R143-mCherry reporter mouse labeled tuft cells in the gallbladder epithelium. An in silico analysis of a scRNA sequencing data set revealed Tas2r expression in only few cells of different identity, and from in situ hybridization histochemistry, which did not label distinct cells. Our findings demonstrate profound tuft cell- and TRPM5-independent relaxing effects of bitter tastants on gallbladder smooth muscle, but do not support the concept that these effects are mediated by bitter receptors.

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Introduction

The gallbladder stores and modifies bile between meals, and releases its content into the duodenum in response to feeding and the subsequent action of hormonal and neural signaling¹. One critical factor in this dynamic process, contraction and relaxation of the gallbladder SMC layer, must be tightly controlled, and disturbances in gallbladder motility may lead to serious pathology, including inflammation and gallstone disease². Endogenous factors known to contract the gallbladder smooth muscle include cholecystokinin (CCK) released from intestinal I-cells and nerve-derived acetylcholine (ACh), while e.g. bile salts and noradrenaline represent relaxing factors². Recently, tuft cells, a minor cell type in the gallbladder epithelium, emerged as a further regulator of gallbladder tone. In the murine gallbladder, they responded to the bacterial metabolite, propionate, with a concomitant release of ACh and cysteinyl leukotrienes, which resulted in enhanced mucus secretion from gallbladder epithelial cells, and in smooth muscle contraction, respectively³.

In addition to such endogenous mediators of known cellular origin, bitter tasting substances have been identified as potent regulators of smooth muscle tone in other organs including airways and intestine⁴. Although some constrictor effects have been reported at select locations like the pulmonary vasculature⁵, relaxing effects prevail and exploiting bitter tastants as a new class of therapeutics to combat bronchoconstriction in asthma has been suggested⁶. Generally, these effects are attributed to activation of classical bitter taste receptors of the taste receptor type 2 (Tas2r) gene family, which, in mice, comprises about 35 functional genes⁷. Against initial expectation, their expression is not restricted to sensory (type II) cells of oropharyngeal taste buds, but they are widely expressed in extraoral tissues including smooth muscle, suggesting additional non-gustatory functions for these receptors^4,8,9,10. Besides smooth muscle^11,12, they have received particular attention in tuft cells of the respiratory tract and urethra as sensors of bacterial products, triggering innate immune effector mechanisms and protective neural reflexes^13,14,15,16.

Downstream signaling has been originally characterized in type II taste cells of oropharyngeal taste buds^17,18. Here, TAS2R are coupled to the α-subunit of gustducin (α-gust; gene name Gnat3). Upon binding of the ligand, the βγ-subunits are released, and a downstream signaling cascade is activated that comprises phospholipase C beta 2 (PLCβ2), inositol trisphosphate receptor 3 (IP₃R3), and TRPM5¹⁹. During this process, the release of calcium ions from intracellular stores leads to an activation of the membrane-bound TRPM5, and this channel then allows an influx of sodium ions, ultimately resulting in membrane depolarization and release of transmitter substances²⁰. Remarkably, this canonical taste transduction cascade is neither unique for taste receptor signaling, nor is it found in all cells expressing TAS2R family members. Tuft cells link this signaling pathway also to the succinate receptor SUCNR1 in the intestine and airways^21,22,23,24 and to the free fatty acid receptor 2 in the gallbladder³, which here serve to detect microbial and danger-associated metabolites. In smooth muscle, on the other hand, expression of Gnat3 and of Trpm5 have been reported for the mouse gastric fundus²⁵, but not for human airways, in which an alternative pathway comprising of the G protein G_αi1,2,3, an unspecified phospholipase C beta, proteinase activated receptor 3, LIM kinase and cofilin shall mediate relaxation by TAS2R14^26,27.

On this background, we hypothesized that prototypic bitter tastants exert relaxing effects, either directly on smooth muscle cells or secondary through action on tuft cells, and that TAS2R family members and components of the canonical downstream signaling cascade are expressed in the gallbladder. Relaxing properties of bitter tastants were tested on explanted gallbladders using appropriate genetic models to dissect the roles of tuft cells and of TRPM5, the central ion channel of the taste transduction cascade, in this process. In addition, we profiled the expression of Tas2r in the mouse gallbladder by RT-PCR, in silico analysis of available scRNA-seq data, in situ hybridization (ISH), and using a Tas2r143 reporter mouse strain. The use of tuft cell-deficient mice allowed to determining the extent of Tas2r expression in non-tuft cells.

Results

Bitter tastants dose-dependently relax the pre-contracted gallbladder independent of the canonical taste transduction cascade

We tested the capacity of typical bitter taste receptor agonists to relax gallbladder smooth muscle in an organ bath setup. Gallbladders were pre-contracted with either muscarine (10 µM) or CCK (100 nM). Both led to a rapid increase (0.30–0.35 g) in the contraction force, which then plateaued for at least 40 min (Sup. Fig. 1A) without being affected by further vehicle (ethanol or methanol) treatment (Sup. Fig. 2). To test for potential effects of bitter tastants, we employed compounds that previously have been described as relaxants in other organs^28,29. Denatonium (1–1000 µM, Fig. 1A), agonist at e.g. TAS2R105, TAS2R108, TAS2R123, TAS2R135, TAS2R140 and TAS2R144^7,30; quinine (1–1000 µM, Fig. 1B), an agonist at e.g. TAS2R108, TAS2R126, and TAS2R137⁷; noscapine (1–1000 µM, Fig. 1C), a broad TAS2R agonist acting upon human TAS2R14³¹ with unknown specificity for mouse TAS2Rs; and dextromethorphan (1–1000 µM, Fig. 1D), also a broadly active agonist acting upon human TAS2R1 and Tas2R10^5,28, all relaxed the pre-contracted gallbladder in a concentration-dependent manner. All these relaxing effects occurred independent of signaling through TRPM5, because they were still present in Trpm5^–/– mice (Fig. 1). Notably, denatonium exhibited an increased relaxing effect at higher concentrations (100 µM and 1 mM) after maximal pre-contraction with muscarine in Trpm5^–/– mice (Fig. 1A). These experiments indicated that the tuft cell canonical taste transduction cascade was not involved in bitter tastant-induced gallbladder smooth muscle relaxation.

The quinine-induced gallbladder smooth muscle relaxation occurs independent of TAS2R126, TAS2R135, and TAS2R143

Next, we analyzed if the quinine-induced relaxation was affected in mice that lack expression from the Tas2r143/135/126 gene cluster encoding for one of the known quinine receptors (TAS2R126)⁷. Mice with genetic deletion of other quinine-responsive bitter receptors (TAS2R105, TAS2R108, TAS2R115, TAS2R137, TAS2R140, TAS2R144)⁷ are not available yet. In Tas2r143/135/126 triple knockout mice³² expression of Tas2r126, Tas2r135, and Tas2r143 was abolished, shown here for taste buds in the vallate papilla by ISH (Fig. 2A–D). In organ bath experiments, however, quinine (1–1000 µM) dose-dependently relaxed the pre-contracted gallbladder smooth muscle of triple knockout mice to a similar extent as in their corresponding wildtypes (Fig. 2E), indicating that TAS2R expression from this gene-cluster was not involved in this bitter tastant-induced relaxation. Also, the EC₅₀ values were not significantly different between the genotypes (p = 0.1681, Fig. 2E).

The quinine-induced gallbladder smooth muscle relaxation occurs independent of the presence of tuft cells

We then asked whether signaling through tuft cells was needed in the bitter tastant-induced relaxation, and tested the effect of quinine in Pou2f3 knockout mice that lack gallbladder tuft cells^3,33. Quinine (1–1000 µM) dose-dependently relaxed the pre-contracted gallbladder to a similar extent when compared to wildtype (Fig. 2F), indicating that gallbladder tuft cells were not involved in this bitter tastant-induced relaxation. The EC₅₀ values were not significantly different between the genotypes (p = 0.2068, Fig. 2F).

Denatonium, quinine, and dextromethorphan increase [Ca²⁺]_i in isolated gallbladder cells

In human airways, denatonium and quinine have been proposed to be potent bronchodilators, and this effect was paradoxically associated with increases in [Ca²⁺]_i in airway smooth muscle cells in vitro¹². In mice, on the other hand, both Ca²⁺-rising and Ca²⁺-lowering effects of bitter tastants have been described, with the airway smooth muscle cells being at rest or contracted, respectively³⁴. Along this line we here analyzed the effect of the three bitter tastants, denatonium, quinine, and dextromethorphan on [Ca²⁺]_i in dissociated gallbladder cells loaded with the Ca²⁺-indicator, Fluo-4. Vehicle alone (methanol 0.5 × 10^–3% for denatonium; ethanol 0.5 × 10^–3% M for dextromethorphan and quinine) evoked fluctuations in [Ca²⁺]_i of less than 10% (data not shown). Therefore, we took a response of 10% change at minimum to define cells as responders to a test stimulus. Cells responsive to 1 µM CCK (CCK⁺) were considered as smooth muscle cells (Acta2-expressing), since expression of the CCK receptor A (Cckar) was confined to cluster #5 in an in silico-analysis of a scRNA-seq data set (GSM5888949 https://krupalipoharkar.shinyapps.io/gallbladder_data/³³), while the Cckbr subtype was not expressed. An additional Acta2^high cell population (cluster #3) did not express CCK receptors and probably represented vascular smooth muscle cells (Fig. 3A–C).

Both CCK⁺ and CCK^– cells reacted with a brief increase in [Ca²⁺]_i to denatonium and dextromethorphan over a concentration range from 10^–6 to 10^–3 M, and to quinine over a concentration range from 10^–6 to 10^–4 M (Fig. 3D–F). Reactivity declined at higher concentrations when agonists were given successively, indicating desensitization. This was further supported for denatonium and quinine by experiments in which agonists were given at highest dose directly after the initial CCK stimulus, showing stronger responses than in the cumulative administration series (Sup. Fig. 3A, B). In case of dextromethorphan at 10^–3 M, however, such a desensitizing effect was not observed (Sup. Fig. 3C). The proportion of responders (≥ 10% increase) to denatonium varied from 5 to 25% over the tested concentration range without significant differences between CCK⁺ and CCK^– cells (Fig. 3G), neither were there significant differences in the magnitude of the increase in [Ca²⁺]_i between various denatonium concentration, nor between responding CCK⁺ and CCK^– cells (Fig. 3H). These data indicated non-specific effects of denatonium and thus this bitter taste stimulus was not further applied. In contrast, responders to the lowest concentration (10^–6 M) of quinine and DXM were much more frequent (about 50%) than those to denatonium in the CCK⁺ population, and significantly less responders were found in the CCK^– group (Fig. 3I, K). Also, the magnitude of [Ca²⁺]_i increase was significantly higher in CCK⁺ than in CCK^– cells at this bitter tastant concentration (Fig. 3J, L). In the cumulative administration experiments, responsiveness of CCK⁺ and CCK^– cells swapped with increasing concentration of quinine, with significantly more responders and higher reactivity of CCK^– cells at 10^–5 and 10^–4 M (Fig. 3J).

To analyze whether quinine and dextromethorphan exerted their effects through the canonical taste transduction signaling cascade, experiments were conducted with the Trpm5 gene-deficient mouse line. Notably, WT mice from this line (Trpm5^+/+) were overall less reactive in calcium imaging experiments than C57BL/6J mice, including their response to CCK at 10^–6 M (Sup. Fig. 4). Genetic deletion of Trpm5 did not further affect the CCK response (Sup. Fig. 4), and neither the rate of responders nor the magnitude of the increase in [Ca²⁺]_i evoked by dextromethorphan and quinine was reduced in Trpm5^–/– compared to Trpm5^+/+ mice both in CCK⁺ (Fig. 4) and CCK^– (Sup. Fig. 5) cells. Collectively, these data show that quinine and dextromethorphan increase [Ca²⁺]_i preferentially, but not exclusively, in gallbladder smooth muscle cells (CCK⁺), but this effect was independent of the canonical taste transduction signaling cascade involving TRPM5.

Bitter taste receptors are differentially expressed in gallbladder and vallate papilla

Our findings of bitter tastant-induced smooth muscle relaxation prompted us to identify their molecular targets and the corresponding cell type identity. The mouse gallbladder mucosal epithelium harbors chemosensory tuft cells^3,35,36,37. Since both, tuft cell-dependent paracrine and cell-autonomous autocrine actions of TAS2R agonists have been described, e.g. in lung and gut⁹, we here assessed the presence and cellular localization of Tas2r gene expression in the mouse gallbladder. First, the expression pattern of all 35 mouse Tas2r genes was studied in whole gallbladder preparations taken from C57BL/6mice by RT-PCR, and compared with samples from vallate papilla. While all Tas2r family members were found to be expressed in the vallate papilla, a limited expression profile was noted in the gallbladder. Here, especially Tas2r108, Tas2r126, Tas2r135, Tas2r137 and Tas2r143 were consistently detected, whereas transcripts of Tas2r138 were found only in half of the gallbladder samples (3/6) and all others even less frequent or not at all (Sup. Table 2 and Fig. 5A). To estimate the contribution of tuft and type II taste cells to Tas2r expression in the gallbladder and tongue, respectively, we compared samples from Pou2f3^–/– mice, which lack tuft and type II taste cells³⁸, with those from their wildtype littermates (Pou2f3^+/+) by real-time RT-PCR. In both the vallate papilla (Fig. 5B) and in the gallbladder (Fig. 5C), absence of tuft/taste cells resulted in complete loss of detectable expression of Trpm5 and Gnat3, coding for two components of the canonical taste transduction signal cascade. The expression of Plcb2, on the other hand, was lost in the vallate papilla, but not in the gallbladder, indicating expression in additional cell types beyond the tuft cell lineage in this organ. Similarly, expression levels of Tas2r126, Tas2r135, and Tas2r143 were found unchanged between the two genotypes, both in tongue and in gallbladder, while Tas2r108 and Tas2r137 expression levels were largely reduced in gallbladder samples from Pou2f3^–/– mice (Fig. 5B, C).

We further analyzed a publicly available single cells RNA-sequencing data set (GSM5888949 https://krupalipoharkar.shinyapps.io/gallbladder_data/³³) of mouse gallbladder for the expression of the canonical taste transduction pathway and of Tas2r family members. Unbiased clustering revealed 14 distinct cell clusters, among which cluster #9 represented tuft cells, identified by Pou2f3 expression (Fig. 6), and clusters #3 and #5 smooth muscle cells, identified by high Acta2 expression (Fig. 3A–C). In this data set, Tas2r expression was restricted to only those 6 family members, which also had been found consistently in the previous RT-PCR screen (Tas2r108, Tas2r126, Tas2r135, Tas2r137, Tas2r143), or in 50% of samples (Tas2r138). In all these cases only very few cells or even only single hits within a cluster were noted. Specifically, in the smooth muscle cell populations, only one Tas2r143 expressing cell was found in the Cckar⁺ (CCK-responsive) population (cluster #5) and only one Tas2r135 expressing cell in the Cckar^- population (cluster #3) (Fig. 6).

Among the crucial elements of the canonical taste transduction pathway, expression of the taste-specific G protein Gnat3 and of the cation channel Trpm5 was enriched in tuft cells, although not being fully exclusive for this cluster (Fig. 6H, I), although real-time RT-PCR had not revealed detectable levels in Pou2f3^–/– mice (Fig. 5C). Specifically, some Trpm5 hits were found in both smooth muscle cell populations (Fig. 6I). To determine, whether this low expression might result in detectable TRPM5 protein, gallbladders from a Chat^BAC-GFP tuft cell-reporter mouse strain were subjected to TRPM5-immunolabeling. In support of the RT-PCR data, TRPM5-immunoreactivity was restricted to the cholinergic, i.e. Chat^BAC-eGFP-immunoreactive tuft cells, but not seen in α-smooth-muscle-actin-immunoreactive smooth muscle cells (Sup. Fig. 6). In accordance with the real-time RT-PCR data on Pou2f3^–/– mice, expression of Plcb2 showed no preference for tuft cells in the sequencing data set but was scattered across gallbladder cell populations with particularly few positive smooth muscle cells (Fig. 6H).

Next, we studied cellular localization of Tas2r mRNA expression by ISH. Guided by the results from the RT-PCR and the in-silico analysis of scRNA sequencing data, we generated gene-specific riboprobes for those Tas2r family members which had been consistently found by these techniques (Tas2r108, Tas2r126, Tas2r135, Tas2r137, Tas2r143). Initially, the sensitivities and specificities of all riboprobes were assessed in sections containing vallate papilla taste buds taken from Pou2f3^+/+ and from Pou2f3^–/– mice that lack type II taste cells³⁸. As expected, Pou2f3 mRNA was detected by ISH in taste buds from Pou2f3^+/+, but not from Pou2f3^–/– mice (Fig. 7). Likewise, transcripts for components of the canonical bitter taste transduction cascade³⁹, namely Gnat3, Plcb2, and Trpm5 were present in vallate papilla from Pou2f3^+/+, but not from Pou2f3^–/– mice (Sup. Fig. 7). To prove that taste buds were still present in Pou2f3^–/– mice, expression of the type II + III taste cell structural marker, Vil1, was examined. Vil1 gene expression was found both in Pou2f3^+/+³⁷, and in Pou2f3^–/– taste buds (Sup. Fig. 7). Sense probes for all transcripts never resulted in specific staining in ISH experiments (Sup. Figs. 7 + 8). When testing riboprobes for the above mentioned five Tas2r genes in ISH experiments, we observed signals for all genes in taste buds from Pou2f3^+/+ mice, but not in taste buds from Pou2f3^–/– mice (Fig. 7), indicating expression restricted to type II taste cells in the vallate papilla. Again, use of sense riboprobes did not result in specific staining (Sup. Fig. 8). Next, we assessed the expression patterns of Pou2f3, Tas2r108, Tas2r126, Tas2r135, Tas2r137, and Tas2r143 in the mouse gallbladder by ISH. The gallbladder wall consists of a mucosa, a muscle layer, and a serosa¹. Besides columnar gallbladder epithelial cells, the mucosal epithelial layer harbors tuft cells, which express and depend on Pou2f3 (Fig. 7M, S). Probing for Tas2r108 (Fig. 7N, T), Tas2r126 (Fig. 7O, U), Tas2r135 (Fig. 7P, V), Tas2r137 (Fig. 7Q, W), and Tas2r143 (Fig. 7R, X) did not result in detectable signals in any of the three layers, neither in Pou2f3^+/+nor in Pou2f3^–/– mice.

Finally, we took advantage of a newly generated reporter mouse line that expressed the fluorophore mCherry under the control of the Tas2r143 promoter (TAS2R143-mCherry) (Fig. 8A). When analyzing tissue sections from this mouse line for mCherry fluorescence, we observed intense fluorescent labeling of vallate papilla taste buds (Fig. 8B), columnar cells at the squamo-columnar junction in the stomach (Fig. 8C), and single cells in the gallbladder mucosal epithelium (Fig. 8E), but not in the small intestine, i.e. duodenum (Fig. 8D). Double-fluorescence analysis for mCherry and 5-lipoxygenase-activating protein (FLAP), a marker for tuft cells, showed that expression from the TAS2R143-mCherry transgene was confined to tuft cells in gallbladder sections (Fig. 2F–H).

Discussion

This study demonstrates profound relaxing effects of some prototypic bitter tastants on the mouse gallbladder, together with the expression of a select panel of Tas2r and of members of the canonical taste transduction signaling cascade in this organ, but does not support the concept of a causal link between them, i.e. bitter receptor-mediated relaxation.

In general, bitter receptor expression appeared to be widespread, although not ubiquitous among cell types in the murine gallbladder. A limited cell type specificity was noted for Tas2r108 and Tas2r137, whose expression was largely reduced, albeit not fully abrogated, in Pou2f3^–/– mice lacking tuft cells. At the single cell level, neither in gallbladder tuft cells, nor in other cell types their expression was high enough to be detected by ISH, despite strong signals were obtained in taste buds, and only one positive cell showed up in the scRNA-seq data set. TAS2R108 and TAS2R137 can be activated by quinine and denatonium^7,9, two of the bitter tastants used in this study, and we recently identified tuft cells as potential regulators of gallbladder tone³. Hence, activation of TAS2R108 and TAS2R137 on tuft cells appeared as one possibility how bitter tastants might exert effects on muscle tone. However, the relaxant response to quinine remained fully intact in Pou2f3^–/– mice, excluding a role of tuft cells and making an involvement of TAS2R108 and TAS2R137, in view of their minimal residual expression in these mice, unlikely.

The genes of the other three bitter receptors consistently detected by RT-PCR, Tas2r126, Tas2r135 and Tas2r143, are grouped in a cluster on mouse chromosome 6, which may facilitate co-expression. Still, expression of only Tas2r126 and not of the others was noted in the mouse stomach in a previous study utilizing another Tas2r143-reporter strain⁴⁰. Expression of these three Tas2r was not reduced in gallbladders of Pou2f3^–/– mice, indicating that non-tuft cells contribute most to their transcript levels in the gallbladder. Accordingly, scRNA-seq data analysis revealed hits scattered across two (Tas2r126), six (Tas2r135) and seven (Tas2r143) different cell clusters. Again, only few cells per cluster were positive for all these receptors, and transcripts of these genes were undetectable at single cell level by ISH. Consistent with some Tas2r143 hits in the main epithelial cell cluster, single epithelial cells were noted in the TAS2R143-mCherry reporter mouse line. These cells, however, were found FLAP-immunoreactive and thus tuft cells. Transcriptional activation of this gene cluster thus may be more complex or selective than previously suggested, mRNA stabilities may vary, or their expression level was underestimated in the scRNA data set due to inherent limitations. Nevertheless, the TAS2R143-mCherry reporter mouse line introduced in this study represents a new tool to study bitter taste-related tuft cell biology in mice.

Among this group of bitter receptors, the ligand profile of TAS2R143 is largely unknown, but TAS2R126 can be activated by quinine and TAS2R135 by denatonium⁷, making this cluster a candidate for causing the relaxant response observed in this study. Indeed, quinine relaxed the pre-contracted gallbladder, but genetic deletion of the entire gene cluster had no impact on this quinine-induced relaxation, similar to what has been reported for mouse bronchi⁴¹.

The possibility that low-level expression of other TAS2R, including those that were not analyzed by ISH, also linked to the canonical signaling pathway, may account for the relaxant responses led us to interrupt the canonical taste transduction cascade at the level of the critical ion channel TRPM5 (Trpm5^–/–). Again, this left the responses unaffected, still leaving the possibility that TAS2R with low-level expression utilize intra-cellular signaling pathways that are different from the classical cascade. Along this line, Woo et al. recently demonstrated Tas2R-mediated signaling inducing relaxation of human airway smooth muscle through destabilizing filamentous actin and dephosphorylating cofilin²⁷.

Tuft cells were not required, because the relaxing effect of quinine was found unchanged in gallbladders lacking tuft cells (Pou2f3^–/–). This finding argued for direct actions of bitter tastants on gallbladder SMC, which was confirmed by recordings of [Ca²⁺]_i in isolated cells. Quinine and DXM preferentially, but not exclusively increased [Ca²⁺]_i in the gallbladder SMC population. Intriguingly, the maximum calcium response in presumed SMC was significantly higher in C57BL/6 mice, when compared to wildtype and knockout littermates of our TRPM5-deficient mouse line. Responses to CCK stimulus in SMC taken from Trpm5^–/– mice, on the other hand, were not different from Trpm5^+/+ littermates, highlighting that the selection of appropriate controls is central to these experiments. Since TRPM5 is not expressed by gallbladder SMC, a decreased calcium response in Trpm5^–/– mice may be attributable to effects by the genetic background of this mouse line, but not by the knockout itself.

Similar paradoxical [Ca²⁺]_i-increasing effects have been observed for airway smooth muscle cells in culture and localized compartmentalization was considered as the reason why this did not translate into contraction¹². In a subsequent study, Zhang and colleagues proposed a model where global [Ca²⁺]_i increases occur in resting cells to an extent that is not sufficient to cause contraction, but may reverse the increase in [Ca²⁺]_i evoked by bronchoconstrictors, ultimately leading to bronchodilation³⁴.

Since we did not observe Tas2r expression in SMC in ISH experiments, low-level expression of Tas2r may be sufficient, or, more likely, bitter tastants elicit their activity on SMC in a TAS2R-independent way. Collectively, the genetic models used in this study strongly argue against a role of those five TAS2R family members, for which we regularly found mRNA expression, in the relaxant responses to the bitter tasting compounds tested, pointing towards effects being mediated through other pathways than bitter receptors^{3,21,22,32,42}. Also, alternative modes of action are known for all bitter compounds used in this study. For example, dextromethorphan is an agonist at sigma-1 receptors and an antagonist at NMDA receptors⁴³. Quinine can cause activation of RalA, a RAS p21-related small G protein⁴⁴ and denatonium has toxic effects on mitochondria⁴⁵. Of course, formally it cannot be excluded that other TAS2R family members with low mRNA abundancy and long half-life at protein level may have escaped detection and drive these responses.

Materials and methods

Mouse strains and handling

C57BL/6JRj mice were purchased from Janvier Labs (stock number #5751862). Chat^BAC-eGFP (#007902), and Trpm5^–/– mice (#005848) were obtained from The Jackson Laboratory (Bar Habor, ME, USA). Pou2f3^–/– mice³⁸ were obtained from the Monell Chemical Senses Center (Philadelphia, PA, USA). Tas2r143/135/126^–/– mice³² were obtained from the Max Planck Institute for Heart and Lung Research (Department of Pharmacology, Bad Nauheim, Germany). Transgenic mice expressing the fluorophore mCherry under the control of the Tas2r143/135/126 promoter (TAS2R143-mCherry) were generated using the BAC clone RP23-316O11 (CHORI, CA, USA) from mouse chromosome 6 containing the Tas2r143, Tas2r135, and Tas2r126 genes. The genomic sequence between the start codon of Tas2r143 and the stop codon of Tas2r126 (35.2 kb) on the BAC was replaced by a cassette carrying the mCherry cDNA followed by a polyadenylation (pA) signal and an FRT-flanked ampicillin resistance cassette using Red/ET recombination kit (Gene Bridges, Heidelberg, Germany) (Fig. 2). Correct targeting was verified by restriction digests and DNA sequencing. After Flp-mediated excision of the ampicillin resistance cassette and linearization, the recombined BAC was injected into C57BL/6 oocytes. Transgenic offspring was genotyped for BAC insertion by genomic PCRs. One founder was used to generate the Tas2r143 reporter line. Animals were kept on a C57BL/6 background. For genotyping by PCR, the following primers were used: forward: 5′-caggagtcattgaactgggag-3′; reverse: 5′-aggatgtcccaggcgaagg-3′; PCR products of 423 bp indicated the transgenic allele.

All transgenic and corresponding wildtype or heterozygous littermates were housed in groups of 2–6 individuals under specified pathogen-free conditions. They were kept on a 12 h light/12 h dark cycle and had access to food and water ad libitum. The genotypes of all mice were verified by PCR using genomic DNA obtained from ear biopsies, according to published protocols supplied by the vendor (The Jackson Laboratory) or donators (I. Matsumoto, Tokyo, Japan, for Pou2f3^–/– mice; S. Offermanns, Bad Nauheim, Germany, for Tas2r143/135/126^–/– and Tas2r143-mCherry mice). Animals were anesthetized by isoflurane (5%) (Abbott) or carbon dioxide and euthanized by transecting the inferior vena cava.

All animals and all experimental protocols were handled in accordance with the guidelines established by the European Community for the care and use of animals and were approved by the local committee at the Regierungspräsidium Giessen, Hesse, Germany. Breeding and use of samples of euthanized mice for further experiments were registered by local animal welfare officers, and the responsible authorities at the Regierungspräsidium Giessen and Darmstadt (Hesse), Germany (registration numbers Ex-15-2016, 571_M, 573_M, 624_M, 641_M, 692_M, 693_M, 793_M). All experiments and methods were in compliance with ARRIVE guidelines.

RT-PCR

Whole gallbladder and a piece of tongue containing the vallate papilla were shock-frozen in RLT buffer plus (51304, QIAamp DNA Mini Kit, Qiagen, Hilden, Germany) and stored at -80^◦C until use. Total RNA was isolated by using the RNeasy kit according to the manufacturer’s protocol (Qiagen). Genomic DNA contamination was eliminated using DNase-I (79254, Qiagen) and a reverse transcription was done for 50 min at 42^◦C using quantiscript reverse transcriptase (205313, Qiagen), with random hexamer primer. The subsequent PCR was performed by adding 1 μl cDNA, 0.5 μl of each primer (20 pM; MWG Biotech, Ebersberg, Germany) (Supplemental Table 1), 2.5 μl 10× PCR buffer II (100 mM Tris–HCl, 500 mM KCl, pH 8.3), 2 μl MgCl₂ (15 mM), 0.5 μl dNTP (10 mM each), 0.1 μl AmpliTaq Gold polymerase (5 U/μl; all reagents from Applied Biosystems, Darmstadt, Germany) and 17.9 μl H₂O. The cycling conditions were 12 min at 95 °C, 40 cycles with 30 s at 95 °C, 30 s at 59 °C, 30 s at 72 °C, and a final extension at 72 °C for 7 min. Control reactions included the absence of DNA template and the absence of reverse transcriptase. The PCR products were separated by gel electrophoresis on a 2% TRIS–acetate-EDTA agarose gel with a 100 bp DNA ladder (15628050, Invitrogen, Karlsruhe, Germany) as length marker, and finally detected by UV illumination using a spectrophotometer (Ultrospec 2100 Pro, Biochrom, Cambridge, UK).

Real-time PCR (iCycler, Bio-Rad, Germany; iTaq Universal SYBR Green Supermix, Bio-Rad Laboratories Inc., CA, USA) was conducted on gallbladders and tongues from Pou2f3^+/+ and Pou2f3^−/− mice to quantify Trpm5, PLCβ2, Gnat3, Tas2r108, Tas2r126, Tas2r135 and Tas2r143 expression, taking β2-microglobulin (B2m) as housekeeping gene. PCR primers are specified in Supplemental Table 1. The PCR conditions included initial denaturation for 10 min at 95 °C followed by 45 cycles of 30 s at 95 °C, 30 s at 61 °C, and 30 s at 72 °C for Trpm5, and initial denaturation for 3 min at 95 °C followed by 45 cycles of 15 s at 95 °C, 15 s at 61 °C, and 15 s at 72 °C for the other targets. Relative expression was calculated as 2^−ΔCT, with CT = cycle threshold and ΔCT = CT (target) − CT (β2m).

ScRNA data set analysis

A mouse gallbladder gene expression dataset was downloaded from GEO datasets at NCBI (GSM5888949) and re-analyzed. The Seurat R package (version 2.3.4) was employed for analysis and re-clustering, utilizing principal component analysis for initial assessment. Subsequently, Uniform Manifold Approximation and Projection (UMAP) was applied for non-linear dimensional reduction, and cells were visualized in a two-dimensional UMAP plot. Cluster identification and annotation were based on typical marker genes. After the initial clustering, to explore the gene expression patterns within each identified cluster, violin plots were created by using the Seurat R package (version 2.3.4) and its built-in functions for plotting. The data, however, comes with inherent limitations. The dataset was generated using short reads and exhibited a relatively low sequencing depth. This aspect of the experimental design has the potential to lead to an underrepresentation of low-expressed genes, including those encoding G protein-coupled receptors (GPCRs). GPCRs, known for their involvement in various physiological processes, often demonstrate lower expression levels compared to other genes. To complement the experimental findings, an in-silico approach was employed. The in-silico method involved computational simulations, leveraging predictive algorithms to analyze the transcriptome dynamics of gallbladder cells. This computational perspective enhanced the comprehensive examination of the dataset. Despite the robustness of the in-silico method, it is essential to acknowledge that computational analyses also have inherent limitations, and results should be interpreted in the context of these considerations.

In situ hybridization

For in situ hybridization (ISH), the vallate papilla was identified at the back of the tongue and removed with surrounding muscle tissue. The gallbladder was left attached to a piece of liver tissue during dissection to minimize the risk of damage. The tissue used for ISH was submerged in TissueTek OCT compound (Sakura Finetek Europe B.V, Alphen aan den Rijn, The Netherlands) and quickly frozen in isopentane (2-methylbutane, 78-78-4, Sigma-Aldrich, Steinheim, Germany) at −40 °C. After an initial storage at −70 °C, 16 µm thick tissue sections were cut at −15 °C using a cryostat (CM3050S, Leica Microsystems, Nussloch, Germany) and mounted on silanized (3-(Triethoxysilyl)-propylamine, 919-30-2, Merck Millipore, Darmstadt, Germany) glass slides. Tissue sections on glass slides were air-dried at room temperature for 30 min and subjected to the following procedure (all procedures at room temperature unless otherwise stated): 1 h fixation in freshly prepared 4% (w/v) paraformaldehyde (PFA) in phosphate-buffered saline (10 mM PBS, pH 7.5), three times 10 min each washing in PBS, permeabilization for 10 min in 0.4% (v/v) Triton X-100 (9002-93-1, Carl Roth) in PBS. After additional washes in PBS, the sections were acetylated for 10 min with triethanolamine/acetic anhydride (Sigma-Aldrich), washed again, dehydrated in 50% and 70% isopropanol, and finally air-dried. Complementary anti-sense (as) RNA probes for the detection of mouse transcripts in tissue sections were generated in the following way: first, gene-specific DNA fragments for Gnat3 (GeneBank acc. no. NM_001081143.1, nucleotides (nt) 86–840), Plcβ2 (NM_177568.2, nt 309–1192), Pou2f3 (NM_011139.2, nt 110–921), Trpm5 (NM_020277.2, nt 461–1292), Villin (Vil) (NM_009509.2, nt 1531–2318), Tas2r108 (NM_020502.1, nt 44–848), Tas2r126 (NM_207028, nt 68–879), Tas2r135 (NM_199159.1, nt 147–946), Tas2r137 (NM_001025149.1, nt 193–1000), and Tas2r143 (NM_001025061.1, nt 80–795) were amplified by PCR (for primer and amplicon length details see Supplemental Table 1) using mouse C57BL/6J ileum cDNA as template, and subcloned into pGEM-T (pGEM-T Vector System, Promega, Mannheim, Germany). The identity of the cloned gene fragments was confirmed by double-stranded sequencing (Microsynth Seqlab GmbH, Göttingen, Germany). Antisense and sense riboprobes were generated by in vitro transcription using T7 RNA Polymerase (Sigma-Aldrich) and SP6 RNA polymerase (Roche Diagnostics, Mannheim, Germany), respectively, and radioactive (UTPαS [35S], NEG039C001MC, PerkinElmer, Waltham, USA)-labeled nucleotides. The ISH procedure was essentially performed as described previously³⁶, with a few modifications. The tissue sections on microscopic slides were covered with 30–40 μl of hybridization solution, containing 50% formamide (75-12-7, VWR International S.A.S, Briare, France), 0.6 M NaCl (AppliChem, Darmstadt, Germany), 10 mM Tris/HCl (pH 7.4) (Carl Roth), 1 mM Na₂EDTA (Carl Roth), 1× Denhardt’s solution (Sigma-Aldrich), 10% dextran sulfate (dextran sulfate sodium salt from Leuconostoc spp., Sigma-Aldrich), 100 mg/ml sonicated salmon sperm DNA (Agilent, Santa Clara, USA), 0.05% (w/v) E. coli MRE600 tRNA (Sigma-Aldrich), 20 mM dithiothreitol (3483-12-3, Roche), 50,000 d.p.m./µl S35-labeled riboprobe, and cover-slipped. Hybridization was carried out overnight at 60 °C in a humid chamber (Nunc Square BioAssay Dishes, Thermo Scientific). After hybridization, coverslips were removed in 2 × standard saline/sodium citrate (SSC) (20×: 3 M NaCl, AppliChem, 0.3 M Na₃Citrate × 2 H₂O, Carl Roth, 5 M HCl, Carl Roth) and the sections washed in the following order: 15 min in 2 × SSC, 15 min in 1 × SSC, 30 min at 37 °C in RNase solution [20 mg/ml RNase A (AppliChem) and 1 U/ml RNase T1 (Roche Diagnostics)], 30 min at RT in RNase-solution, 15 min in 1 × SSC, 15 min in 0.5 × SSC, 15 min in 0.2 × SSC, 60 min at 60 °C in 0.2 × SSC, 15 min in 0.2 × SSC, and finally 15 min in distilled water. For the visualization of radioactive hybridization signals, sections were exposed to Carestream BIOMAX MR autoradiography film (Sigma-Aldrich) for 2–3 days to estimate further exposure times, then coated under absence of light with nuclear emulsion (Ilford K5, Harman Tech., Mobberley, UK), exposed for 2 weeks at 4 °C in the dark and finally developed using Ilford Phenisol developer (Harman) and Ilford Hypam fixer (Harman). Sections were counterstained with methyl green (Sigma-Aldrich), and cover-slipped. Antisense and sense RNA probes were run in parallel in the same experiment to ensure equivalent conditions. In each experiment, vallate papillae containing taste buds were used as positive control.

Immunohistochemistry

Immunohistochemistry (IHC) was performed according to two different protocols. On the one hand, freshly dissected gallbladders from a Chat^BAC-GFP tuft cell-reporter mouse strain (see Sup. Fig. 6 upper row) were carefully opened with a surgical scissor, pinned on wax plates, and immersion fixed overnight in 4% PFA in 0.1 M phosphate buffer, washed, dehydrated and embedded in paraffin (Tissue-Tek Paraffin Wax, Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands). The specimens were cut at a thickness of 8 μm, mounted on silanized glass slides, and dried overnight at 37 °C. Sections were stored at room temperature until use. Before incubation with the primary antibodies, all sections were deparaffinized (xylene, 100%, 96%, 80%, 70% and 50% ethanol, respectively, each step for 5 min). Next, microwave oven heating for antigen retrieval was done for 10 min in PBS containing 10% citric acid, pH 6 (Merck, Darmstadt, Germany). The slides were incubated for 1 h in 10% normal horse serum, 0.5% Tween-20, 0.1% bovine serum albumin in PBS, pH 7.4, before applying the appropriate primary antibody combination overnight at room temperature. Primary antibodies included combination of rabbit anti-TPRM5 (1:200)^46,47, and chicken anti-GFP (NB100-1614, 1:4000, Novus). After a washing step, the sections were incubated with secondary antibodies, Cy3-conjugated anti-rabbit IgG from donkey (1:2000, Merck Millipore) with FITC-conjugated anti-chicken IgY from Donkey (703-095-155, 1:2000, Dianova) for 1 h at room temperature. The slides were rinsed, postfixed for 10 min in buffered 4% PFA, rinsed again, and coverslipped with carbonate-buffered glycerol (pH 8.6). The sections were evaluated with an epifluorescence microscope (Axioplan Zeiss, Jena, Germany, Jena, Germany) using appropriate filter sets. The specificity of secondary reagents was validated by omission of primary antibodies.

Alternatively, whole gallbladder with a piece of surrounding liver tissue from C57BL/6J mouse strain was immersion-fixed overnight in Bouin Hollande fixative (see Sup. Fig. 6 lower row), then extensively washed in 70% isopropanol, dehydrated, and embedded in paraffin⁴⁸. Paraffin blocks were cut with a microtome at 7 µm thickness, and mounted on silanized microscope glass slides. After deparaffinization and blocking procedures (see above), two primary antibodies from different donor species (rabbit anti-TRPM5, see above; and FITC-conjugated monoclonal mouse anti-α-smooth muscle actin (αSMA, 1:500, clone 1A4; Sigma-Aldrich, Taufkirchen, Germany, RRID: AB_476977) were co-applied in PBS/1% BSA and incubated over night at 16 °C, followed by 2 h at 37 °C. After extensive washing in distilled water followed by PBS, immunoreactions for TRPM5 were visualized by incubation for 2 h at room temperature with a secondary antibody labeled with Alexa Fluor 647 (chicken anti-rabbit-IgG, 1:200, Art.-No. A21443, Life Technologies). Immunofluorescence signals were documented in a surface scan using a BX50WI confocal laser scanning microscope (Olympus Optical, Hamburg, Germany) and Olympus Fluoview 2.1 software, and stored as false color images (8-bit tiff format) without modifications in brightness and contrast.

Visualization of mCherry fluorescence

For the visualization of native mCherry fluorescence, selected tissues, i.e. gallbladder, tongue, stomach and small intestine were immersion fixed in 4% PFA for 4 h, washed several times in 0.01 M phosphate buffer, then cryo-protected by incubation overnight in the same buffer with 20% sucrose (Carl Roth), and frozen in TissueTek OCT compound by placing in isopentane cooled with liquid nitrogen. Sections (16 µm thick) were cut with a cryostat, mounted on glass slides, and cover-slipped using Fluoromount-G (Art.no. 15586276, Thermo Fisher Sci.). Native fluorescence was documented with a Zeiss Imager M2 microscope (Zeiss, Oberkochem, Germany), equipped with a Zeiss AxioCam HRc camera, and Zeiss ZEN 2011 software. To combine the visualization of native mCherry fluorescence with single immunofluorescence, a goat anti-5 lipoxygenase-activating protein (FLAP, NB300-891, 1:50; Novus Biol., Centennial, CO, USA) antiserum was applied in PBS/1% BSA and incubated overnight at 16 °C, followed by 2 h at 37 °C. After extensive washing in distilled water followed by PBS, immunoreactions were visualized by a two-step procedure using a species-specific biotinylated secondary antibody (703-065-155, 1:200, Jackson ImmunoResearch), applied for 45 min, followed by streptavidin conjugated with Alexa Fluor 488 (016-160-084, 1:200, Jackson ImmunoResearch) for 2 h.

Organ bath force recording

Whole gallbladders were harvested and placed in minimum essential medium (MEM; Invitrogen Gibco, Oslo, Norway). They were mounted between two stainless steel clips in a computerized organ-bath chamber system (AD Instruments GmbH, Heidelberg, Germany). The chamber was filled with 37 °C warm MEM, which was supplemented with 1% penicillin/streptomycin (Sigma-Aldrich; P4333) and continuously aerated with a 95% O₂/5% CO₂ gas mixture. The temperature was held at 37 °C using a bath circulator (Thermo Fisher Scientific, Waltham, USA). The upper stainless clip was connected to an isometric force transducer (Power Lab 8.30; AD Instruments GmbH, Heidelberg, Germany) and converted signals were analyzed with LabChart 6 software (AD Instruments GmbH, Heidelberg, Germany). Changes in tension were recorded as force in gram (g). All gallbladders were first equilibrated against a passive load of 1 g and then adjusted at 0.5 g tension. All samples were equilibrated for 30 min until they reached a stable baseline tension. Pre-contraction was achieved by applying either 10 µM muscarine (2303-35-7, Sigma-Aldrich), or 0.1 µM cholecystokinin (CCK; fragment 26–33 amide, 25679-24-7, Sigma-Aldrich). For relaxation studies, dextromethorphan hydrobromide monohydrate (DXM) (6700-34-1), denatonium benzoate (3734-33-6), quinine hydrochloride, and noscapine hydrochloride (912-60-7) were obtained from Sigma-Aldrich. Stock solutions of DXM and quinine were dissolved in 100% ethanol, whereas all other drugs were dissolved in deionized H₂O, and all drugs were further diluted in water or MEM to the desired experimental concentration immediately before use. Each type of experiment was performed on samples from at least three animals. The exact number of animals is indicated in the graphs. The delta force in percent with respect to the maximum force in response to either muscarine or CCK was calculated for each experiment.

Cell separation and intracellular calcium recording

Gallbladder was dissected, cut into small pieces, and enzymatically digested at 37 °C for 30 min with dispase (2 mg/mL; Sigma-Aldrich) in HBSS (Invitrogen), and 5 min in trypsin/PBS (1:1, Invitrogen). After centrifugation (60×g, 5 min) and mechanical dissociation by pipetting, cells were resuspended in PBS. Isolated cells were plated on coverslips in MEM supplemented with 1% penicillin/streptomycin and 5% fetal bovine serum (26140079, Thermo Fisher) for 30–60 min at 37 °C. Fluo-4-AM (F14201, Thermo Fisher) was mixed with pluronic F-127 (P3000MP, Invitrogen, 0.005%) by sonication and diluted to a final concentration of 5 μmol/l in tyrode III solution (8 mM CaCl₂, 130 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES), 10 mM glucose, 10 mM pyruvic acid, and 5 mM NaHCO₃). Plated cells were loaded with the fluorescent calcium indicator in tyrode III solution for 10–30 min at 37 °C. Intracellular calcium concentration ([Ca²⁺]_i) was analyzed with a confocal laser-scanning microscope (Zeiss LSM 710) equipped with an argon laser providing excitation at 488 nm and recording emission at > 514 nm during continuous superfusion (3 ml/min) with tyrode solution. SMC were identified by their response to 1 µM CCK, and cells with at least 10% increase in [Ca²⁺]_i evoked responses were considered as responder SMC. Fluorescence intensities at the start of the recording period were set arbitrarily at 100%.

Statistical analysis

Data in the graphs depicting time courses or dose responses are presented as mean ± standard error of the mean (SEM) or medians ± interquartile ranges (IQR). Statistical analyses were performed using GraphPad Prism 7 software (La Jolla, CA, USA). An assessment of the normality of the data was done using Kolmogorov–Smirnov normality test and revealed significant deviation from a normal distribution. Therefore, nonparametric tests, i.e. Mann–Whitney U-test or Kruskall-Wallis test followed by Dunn’s multiple comparisons test, were applied. The difference in relative frequencies of [Ca²⁺]_i responses was evaluated using the Chi-square test. Values for EC₅₀of agonist responses were determined using non-linear regression sigmoidal curve analysis according to the Hill equation. Differences were considered as statistically significant when p < 0.05.

Data availability

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials. A mouse gallbladder gene expression dataset was downloaded from GEO datasets at NCBI (GSM5888949) and re-analyzed.

References

Housset, C. et al. Functions of the gallbladder. Compr. Physiol. 6(3), 1549–1577 (2016).
Article PubMed Google Scholar
Portincasa, P., Di Ciaula, A. & van Berge-Henegouwen, G. P. Smooth muscle function and dysfunction in gallbladder disease. Curr. Gastroenterol. Rep. 6(2), 151–162 (2004).
Article PubMed Google Scholar
Keshavarz, M. et al. Cysteinyl leukotrienes and acetylcholine are biliary tuft cell cotransmitters. Sci. Immunol. 7(69), eabf6734 (2022).
Article CAS PubMed Google Scholar
Avau, B. & Depoortere, I. The bitter truth about bitter taste receptors: Beyond sensing bitter in the oral cavity. Acta Physiol. (Oxf) 216(4), 407–420 (2016).
Article CAS PubMed Google Scholar
Upadhyaya, J. D. et al. Dextromethorphan mediated bitter taste receptor activation in the pulmonary circuit causes vasoconstriction. PLoS One 9(10), e110373 (2014).
Article ADS PubMed PubMed Central Google Scholar
Conaway Jr, S., Nayak, A. P. & Deshpande, D. A. Therapeutic potential and challenges of bitter taste receptors on lung cells. Curr. Opin. Pharmacol. 51, 43–49 (2020).
Article PubMed Google Scholar
Lossow, K. et al. Comprehensive analysis of mouse bitter taste receptors reveals different molecular receptive ranges for orthologous receptors in mice and humans. J. Biol. Chem. 291(29), 15358–15377 (2016).
Article CAS PubMed PubMed Central Google Scholar
Behrens, M. & Meyerhof, W. Gustatory and extragustatory functions of mammalian taste receptors. Physiol. Behav. 105(1), 4–13 (2011).
Article CAS PubMed Google Scholar
Lu, P. et al. Extraoral bitter taste receptors in health and disease. J. Gen. Physiol. 149(2), 181–197 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Ki, S. Y. & Jeong, Y. T. Taste receptors beyond taste buds. Int. J. Mol. Sci. 23(17), 33 (2022).
Article Google Scholar
Kim, D. et al. Coupling of airway smooth muscle bitter taste receptors to intracellular signaling and relaxation is via G(alphai1,2,3). Am. J. Respir. Cell Mol. Biol. 56(6), 762–771 (2017).
Article CAS PubMed PubMed Central Google Scholar
Deshpande, D. A. et al. Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction. Nat. Med. 16(11), 1299–1304 (2010).
Article CAS PubMed PubMed Central Google Scholar
Tizzano, M. et al. Expression of taste receptors in solitary chemosensory cells of rodent airways. BMC Pulm. Med. 11, 3 (2011).
Article CAS PubMed PubMed Central Google Scholar
Lee, R. J. & Cohen, N. A. Taste receptors in innate immunity. Cell Mol. Life Sci. 72(2), 217–236 (2015).
Article CAS PubMed Google Scholar
Krasteva, G. et al. Cholinergic chemosensory cells in the trachea regulate breathing. Proc. Natl. Acad. Sci. USA 108(23), 9478–9483 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Deckmann, K. et al. Bitter triggers acetylcholine release from polymodal urethral chemosensory cells and bladder reflexes. Proc. Natl. Acad. Sci. USA 111(22), 8287–8292 (2014).
Article ADS CAS PubMed PubMed Central Google Scholar
Chaudhari, N. & Roper, S. D. The cell biology of taste. J. Cell Biol. 190(3), 285–296 (2010).
Article CAS PubMed PubMed Central Google Scholar
Adler, E. et al. A novel family of mammalian taste receptors. Cell 100(6), 693–702 (2000).
Article CAS PubMed Google Scholar
Roper, S. D. & Chaudhari, N. Taste buds: Cells, signals and synapses. Nat. Rev. Neurosci. 18(8), 485–497 (2017).
Article CAS PubMed PubMed Central Google Scholar
Huang, Y. A. & Roper, S. D. Intracellular Ca(2+) and TRPM5-mediated membrane depolarization produce ATP secretion from taste receptor cells. J. Physiol. 588(Pt 13), 2343–2350 (2010).
Article CAS PubMed PubMed Central Google Scholar
Nadjsombati, M. S. et al. Detection of Succinate by Intestinal Tuft Cells Triggers a Type 2 Innate Immune Circuit. Immunity 49(1), 33-41e7 (2018).
Article CAS PubMed PubMed Central Google Scholar
Perniss, A. et al. A succinate/SUCNR1-brush cell defense program in the tracheal epithelium. Sci. Adv. 9(31), eadg8842 (2023).
Article CAS PubMed PubMed Central Google Scholar
Lei, W. et al. Activation of intestinal tuft cell-expressed Sucnr1 triggers type 2 immunity in the mouse small intestine. Proc. Natl. Acad. Sci. USA 115(21), 5552–5557 (2018).
Article ADS CAS PubMed PubMed Central Google Scholar
Schneider, C. et al. A metabolite-triggered tuft cell-ILC2 circuit drives small intestinal remodeling. Cell 174(2), 271-284e14 (2018).
Article CAS PubMed PubMed Central Google Scholar
Avau, B. et al. The gustatory signaling pathway and bitter taste receptors affect the development of obesity and adipocyte metabolism in mice. PLoS One 10(12), e0145538 (2015).
Article PubMed PubMed Central Google Scholar
Kim, D. et al. Biased TAS2R bronchodilators inhibit airway smooth muscle growth by downregulating phosphorylated extracellular signal-regulated kinase 1/2. Am. J. Respir. Cell Mol. Biol. 60(5), 532–540 (2019).
Article CAS PubMed PubMed Central Google Scholar
Woo, J. A. et al. A Par3/LIM kinase/cofilin pathway mediates human airway smooth muscle relaxation by TAS2R14. Am. J. Respir. Cell Mol. Biol. 68(4), 417–429 (2023).
Article CAS PubMed PubMed Central Google Scholar
Manson, M. L. et al. Bitter taste receptor agonists mediate relaxation of human and rodent vascular smooth muscle. Eur. J. Pharmacol. 740, 302–311 (2014).
Article CAS PubMed Google Scholar
Pulkkinen, V. et al. The bitter taste receptor (TAS2R) agonists denatonium and chloroquine display distinct patterns of relaxation of the guinea pig trachea. Am. J. Physiol. Lung Cell Mol. Physiol. 303(11), L956–L966 (2012).
Article CAS PubMed Google Scholar
Chandrashekar, J. et al. T2Rs function as bitter taste receptors. Cell 100(6), 703–711 (2000).
Article CAS PubMed Google Scholar
Kertesz, Z. et al. Agonists for bitter taste receptors T2R10 and T2R38 attenuate LPS-induced permeability of the pulmonary endothelium in vitro. Front. Physiol. 13, 794370 (2022).
Article PubMed PubMed Central Google Scholar
Perniss, A. et al. Chemosensory cell-derived acetylcholine drives tracheal mucociliary clearance in response to virulence-associated formyl peptides. Immunity 52(4), 683-699.e11 (2020).
Article CAS PubMed Google Scholar
O’Leary, C. E. et al. Bile acid-sensitive tuft cells regulate biliary neutrophil influx. Sci. Immunol. 7(69), eabj1080 (2022).
Article CAS PubMed PubMed Central Google Scholar
Zhang, C. H. et al. The cellular and molecular basis of bitter tastant-induced bronchodilation. PLoS Biol. 11(3), e1001501 (2013).
Article CAS PubMed PubMed Central Google Scholar
Luciano, L. & Reale, E. A new cell type (‘brush cell’) in the gall bladder epithelium of the mouse. J. Submicrosc. Cytol. 1, 43–52 (1969).
Google Scholar
Schutz, B. et al. Chemical coding and chemosensory properties of cholinergic brush cells in the mouse gastrointestinal and biliary tract. Front. Physiol. 6, 87 (2015).
PubMed PubMed Central Google Scholar
Ruppert, A. L. et al. Advillin is a tuft cell marker in the mouse alimentary tract. J. Mol. Histol. 51(4), 421–435 (2020).
Article CAS PubMed PubMed Central Google Scholar
Matsumoto, I. et al. Skn-1a (Pou2f3) specifies taste receptor cell lineage. Nat. Neurosci. 14(6), 685–687 (2011).
Article CAS PubMed PubMed Central Google Scholar
Finger, T. E. & Kinnamon, S. C. Taste isn’t just for taste buds anymore. F1000 Biol. Rep. 3, 20 (2011).
Article PubMed PubMed Central Google Scholar
Liu, S. et al. Members of bitter taste receptor cluster. Front. Physiol. 8, 849 (2017).
Article PubMed PubMed Central Google Scholar
Lu, P. et al. Genetic deletion of the Tas2r143/Tas2r135/Tas2r126 cluster reveals that TAS2Rs may not mediate bitter tastant-induced bronchodilation. J. Cell Physiol. 236(9), 6407–6423 (2021).
Article CAS PubMed PubMed Central Google Scholar
Howitt, M. R. et al. The taste receptor TAS1R3 regulates small intestinal tuft cell homeostasis. Immunohorizons 4(1), 23–32 (2020).
Article CAS PubMed Google Scholar
Keam, S. J. Dextromethorphan/bupropion: First approval. CNS Drugs 36(11), 1229–1238 (2022).
Article CAS PubMed Google Scholar
Bhatia, V., Esmati, L. & Bhullar, R.P. Regulation of Ras p21 and RalA GTPases activity by quinine in mammary epithelial cells. Mol. Cell Biochem. (2023).
Wen, X. et al. Denatonium inhibits growth and induces apoptosis of airway epithelial cells through mitochondrial signaling pathways. Respir. Res. 16(1), 13 (2015).
Article CAS PubMed PubMed Central Google Scholar
Kaske, S. et al. TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells. BMC Neurosci. 8, 49 (2007).
Article PubMed PubMed Central Google Scholar
Hofmann, T. et al. TRPM5 is a voltage-modulated and Ca(2+)-activated monovalent selective cation channel. Curr. Biol. 13(13), 1153–1158 (2003).
Article CAS PubMed Google Scholar
Schutz, B. et al. Sweat gland innervation is pioneered by sympathetic neurons expressing a cholinergic/noradrenergic co-phenotype in the mouse. Neuroscience 156(2), 310–318 (2008).
Article CAS PubMed Google Scholar

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Acknowledgements

We thank Vladimir Chubanov and Thomas Gudermann for providing a TRPM5 antiserum. We also thank Ichiro Matsumoto (Monell Chemical Senses Center, Philadelphia, PA, USA) for providing the Pou2f3tm1Abek mice. Marion Zibuschka, Barbara Reuter and Martin Bodenbenner are acknowledged for superb technical assistance.

Funding

Open Access funding enabled and organized by Projekt DEAL. This research was funded by the German Research Foundation (grant KU688/8-1 to W.K., grant SCHU1259/10-1 to B.S.).

Author information

Shuya Liu
Present address: III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Maryam Keshavarz
Present address: Anatomy and Cell Biology, Institute of Theoretical Medicine, Faculty of Medicine, University of Augsburg, Augsburg, Germany
Wafaa Mahmoud
Present address: Department of Anatomy, Faculty of Medicine, Jordan University of Science and Technology, Irbid, Jordan
Alexander Perniss
Present address: Division of Allergy and Clinical Immunology, Department of Medicine, Jeff and Penny Vinik Center for Allergic Disease Research, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

Authors and Affiliations

Institute for Anatomy and Cell Biology, German Center for Lung Research, Justus Liebig University, Giessen, Germany
Maryam Keshavarz, Mirjam Meiners, Krupali Poharkar, Wafaa Mahmoud, Petra Hartmann, Petra Mermer, Alexander Perniss & Wolfgang Kummer
Excellence Cluster Cardio-Pulmonary Institute, Justus Liebig University, Giessen, Germany
Maryam Keshavarz, Mirjam Meiners, Krupali Poharkar, Wafaa Mahmoud, Petra Hartmann, Petra Mermer, Alexander Perniss, Stefan Offermanns & Wolfgang Kummer
Institute for Anatomy and Cell Biology, Philipps-University, Marburg, Germany
Anna-Lena Ruppert, Sarah Winterberg & Burkhard Schütz
Department of Pharmacology, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
Shuya Liu & Stefan Offermanns

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Maryam Keshavarz
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Wolfgang Kummer
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Burkhard Schütz
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M.K., A.-L.R., M.M., W.M., S.W., P.H., P.M. and A.P. generated data. M.K., A.-L.R., M.M., K.P., P.H., B.S., and W.K. analyzed data. S.L., S.O., and B.S. designed, generated, and characterized mouse models. M.K., B.S., and W.K. designed the study and wrote the initial draft of the manuscript.

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Correspondence to Maryam Keshavarz, Wolfgang Kummer or Burkhard Schütz.

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Keshavarz, M., Ruppert, AL., Meiners, M. et al. Bitter tastants relax the mouse gallbladder smooth muscle independent of signaling through tuft cells and bitter taste receptors. Sci Rep 14, 18447 (2024). https://doi.org/10.1038/s41598-024-69287-6

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Received: 29 February 2024
Accepted: 02 August 2024
Published: 08 August 2024
DOI: https://doi.org/10.1038/s41598-024-69287-6