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

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

Olfactory receptor Olfr78 (prostate-specific G protein-coupled receptor PSGR) expression in arterioles supplying skeletal and cardiac muscles and in arterioles feeding some murine organs

The olfactory receptor Olfr78 (prostate-specific G protein-coupled receptor PSGR) is a member of the G protein-coupled receptor family mediating olfactory chemosensation, but it is additionally expressed in other tissues. Olfr78 expressed in kidney participates in blood pressure regulation, and in prostate it plays a role in the development of cancer. We here screened many organs/tissues of transgenic mice co-expressing β-galactosidase with Olfr78. X-gal-positive cells were detectable in smooth muscle cells of numerous arterioles of striated muscles (heart ventricles and skeletal muscles of various embryological origin). In addition, in most organs where we found expression of Olfr78 mRNA, X-gal staining was restricted to smooth muscle cells of small blood vessels. The dominant expression of Olfr78 in arteriolar smooth muscle cells supports the concept of an important role in blood pressure regulation and suggests a participation in the fine tuning of blood supply especially of striated muscles. This should be considered when targeting Olfr78 in other contexts such as prostate cancer

Avertin®, but Not Volatile Anesthetics Addressing the Two-Pore Domain K+ Channel, TASK-1, Slows Down Cilia-Driven Particle Transport in the Mouse Trachea.

Volatile anesthetics inhibit mucociliary clearance in the airways. The two-pore domain K+ channel, TASK-1, represents one of their molecular targets in that they increase its open probability. Here, we determine whether particle transport speed (PTS) at the mucosal surface of the mouse trachea, an important factor of the cilia-driven mechanism in mucociliary clearance, is regulated by TASK-1.RT-PCR analysis revealed expression of TASK-1 mRNA in the manually dissected and laser-assisted microdissected tracheal epithelium of the mouse. Effects of anesthetics (isoflurane and Avertin®) and TASK-1 inhibitors (anandamide and A293) on ciliary activity were investigated by assessment of PTS at the mucosal surface of the explanted and opened murine trachea. Neither TASK-1 inhibitors nor isoflurane had any impact on basal and ATP-stimulated PTS. Avertin® reduced basal PTS, and ATP-stimulated PTS decreased in its presence in wild-type (WT) mice. Avertin®-induced decrease in basal PTS persisted in WT mice in the presence of TASK-1 inhibitors, and in two different strains of TASK-1 knockout mice.Our findings indicate that TASK-1 is expressed by the tracheal epithelium but is not critically involved in the regulation of tracheal PTS in mice. Avertin® reduces PTS independent of TASK-1

Inhibitors of TASK-1 channel have no effect on basal and subsequent ATP-stimulated particle transport speed (PTS).

<p><b>(A)</b> Cumulative increase of anandamide concentration did not affect PTS. N = 3 tracheas from 3 animals. <b>(B)</b> Ethanol (solvent of anandamide) alone had no effect on PTS. N = 2 tracheas from 2 animals. <b>(C)</b> Cumulative increase of A293 concentration had no effect on PTS. N = 3 tracheas from 3 animals. <b>(D)</b> DMSO (solvent of A293) alone did not affect PTS. N = 3 tracheas from 3 animals (paired t-test). Effects of drugs were comparable to those of their solvents (ANOVA). n.s: not significant.</p

Anesthetics differentially decrease cilia-driven particle transport speed (PTS).

<p><b>Isoflurane has no effect on PTS, whereas Avertin® reduces PTS. (A)</b> Isoflurane did not affect PTS. N = 3 tracheas from 3 animals. <b>(B)</b> HEPES (solvent of isoflurane) alone also had no effect on PTS. N = 2 tracheas from 2 animals. <b>(C)</b> Avertin® (0.4 mM) decreased PTS significantly. N = 3 tracheas from 3 animals. <b>(D)</b> Avertin® (1 mM) decreased PTS. N = 5 tracheas from 5 animals. <b>(E)</b> Avertin® (4 mM) robustly decreased basal PTS and ATP could not induce increase in PTS. N = 3 tracheas from 3 animals. <b>(F)</b> Water (solvent of Avertin®) had no effect on PTS. N = 4 tracheas from 4 animals. <b>(G)</b> Washing away of Avertin® from the chamber resulted in an increase in PTS, thus demonstrating viability of the trachea. N = 5 tracheas from 5 animals. P values ≤ 0.05 (paired t-test) are indicated. <b>(H)</b> Avertin® dose-dependently decreased basal PTS, and influenced ATP-stimulated PTS. The effects of different concentrations of Avertin® on PTS and their influence on ATP-stimulated PTS were compared with that of water after 1 min of their application. The y-axis shows the changes in PTS (ΔPTS) that occurred within 1 min after Avertin® application (left) and 1 min after ATP (100 μM) application in the continuous presence of Avertin® at different concentrations (right). Water was added instead of Avertin® in the solvent control, labelled here as “0 mM Avertin®”. P values ≤ 0.05 (ANOVA) are indicated. n.s: not significant.</p

TASK-1 mRNA, but not TASK-3 mRNA, is expressed in the tracheal epithelium of mice.

<p><b>(A)</b> RT-PCR from manually dissected tracheal epithelium (TE) using laboratory scissors, agarose gel. Irrelevant lanes from the gel were removed. Solid white lines indicate parts of the gel that were cut and pasted but from the same gel. <b>(B)</b> Laser-assisted microdissection of the tracheal epithelium. Area of the epithelium to be picked was first marked (left panel), the green lines show laser tracing. The marked piece of tissue (missing in right panel) was catapulted into the lid of a reaction tube and processed for RT-PCR. <b>(C)</b> RT-PCR from laser-microdissected (LMD) samples, agarose gel. β-Actin served as a housekeeping gene to control for PCR efficacy. Heart was used as a positive control for RT-PCR detection of TASK-1 mRNA. ØRT: control run without reverse transcriptase, H₂O: control run without template, M: 100 base pair size marker.</p

TASK-1 potassium channel is not critically involved in mediating hypoxic pulmonary vasoconstriction of murine intra-pulmonary arteries

<div><p>The two-pore domain potassium channel KCNK3 (TASK-1) is expressed in rat and human pulmonary artery smooth muscle cells. There, it is associated with hypoxia-induced signalling, and its dysfunction is linked to pathogenesis of human pulmonary hypertension. We here aimed to determine its role in hypoxic pulmonary vasoconstriction (HPV) in the mouse, and hence the suitability of this model for further mechanistic investigations, using appropriate inhibitors and TASK-1 knockout (KO) mice. RT-PCR revealed expression of TASK-1 mRNA in murine lungs and pre-acinar pulmonary arteries. Protein localization by immunohistochemistry and western blot was unreliable since all antibodies produced labelling also in TASK-1 KO organs/tissues. HPV was investigated by videomorphometric analysis of intra- (inner diameter: 25–40 μm) and pre-acinar pulmonary arteries (inner diameter: 41–60 μm). HPV persisted in TASK-1 KO intra-acinar arteries. Pre-acinar arteries developed initial HPV, but the response faded earlier (after 30 min) in KO vessels. This HPV pattern was grossly mimicked by the TASK-1 inhibitor anandamide in wild-type vessels. Hypoxia-provoked rise in pulmonary arterial pressure (PAP) in isolated ventilated lungs was affected neither by TASK-1 gene deficiency nor by the TASK-1 inhibitor A293. TASK-1 is dispensable for initiating HPV of murine intra-pulmonary arteries, but participates in sustained HPV specifically in pre-acinar arteries. This does not translate into abnormal rise in PAP. While there is compelling evidence that TASK-1 is involved in the pathogenesis of pulmonary arterial hypertension in humans, the mouse does not appear to serve as a suitable model to study the underlying molecular mechanisms.</p></div