TMEM16A in smooth muscle cells acts as a pacemaker channel in the internal anal sphincter.

Maintenance of fecal continence requires a continuous or basal tone of the internal anal sphincter (IAS). Paradoxically, the basal tone results largely from high-frequency rhythmic contractions of the IAS smooth muscle. However, the cellular and molecular mechanisms that initiate these contractions remain elusive. Here we show that the IAS contains multiple pacemakers. These pacemakers spontaneously generate propagating calcium waves that drive rhythmic contractions and establish the basal tone. These waves are myogenic and act independently of nerve, paracrine or autocrine signals. Using cell-specific gene knockout mice, we further found that TMEM16A Cl- channels in smooth muscle cells (but not in the interstitial cells of Cajal) are indispensable for pacemaking, rhythmic contractions, and basal tone. Our results identify TMEM16A in smooth muscle cells as a critical pacemaker channel that enables the IAS to contract rhythmically and continuously. This study provides cellular and molecular insights into fecal continence.

Bitter taste receptors in the reproductive system: Function and therapeutic implications.

Type 2 taste receptors (TAS2Rs), traditionally known for their role in bitter taste perception, are present in diverse reproductive tissues of both sexes. This review explores our current understanding of TAS2R functions with a particular focus on reproductive health. In males, TAS2Rs are believed to play potential roles in processes such as sperm chemotaxis and male fertility. Genetic insights from mouse models and human polymorphism studies provide some evidence for their contribution to male infertility. In female reproduction, it is speculated that TAS2Rs influence the ovarian milieu, shaping the functions of granulosa and cumulus cells and their interactions with oocytes. In the uterus, TAS2Rs contribute to uterine relaxation and hold potential as therapeutic targets for preventing preterm birth. In the placenta, they are proposed to function as vigilant sentinels, responding to infection and potentially modulating mechanisms of fetal protection. In the cervix and vagina, their analogous functions to those in other extraoral tissues suggest a potential role in infection defense. In addition, TAS2Rs exhibit altered expression patterns that profoundly affect cancer cell proliferation and apoptosis in reproductive cancers. Notably, TAS2R agonists show promise in inducing apoptosis and overcoming chemoresistance in these malignancies. Despite these advances, challenges remain, including a lack of genetic and functional studies. The application of techniques such as single-cell RNA sequencing and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated endonuclease 9 gene editing could provide deeper insights into TAS2Rs in reproduction, paving the way for novel therapeutic strategies for reproductive disorders.

Phenanthroline relaxes uterine contractions induced by diverse contractile agents by decreasing cytosolic calcium concentration.

Uterine contractions during labor and preterm labor are influenced by a complex interplay of factors, including hormones and inflammatory mediators. This complexity may contribute to the limited efficacy of current tocolytics for preterm labor, a significant challenge in obstetrics with 15 million cases annually and approximately 1 million resulting deaths worldwide. We have previously shown that the myometrium expresses bitter taste receptors (TAS2Rs) and that their activation leads to uterine relaxation. Here, we investigated whether the selective TAS2R5 agonist phenanthroline can induce relaxation across a spectrum of human uterine contractions and whether the underlying mechanism involves changes in intracellular Ca2+ signaling. We performed experiments using samples from pregnant women undergoing scheduled cesarean delivery, assessing responses to various inflammatory mediators and oxytocin with and without phenanthroline. Our results showed that phenanthroline concentration-dependently inhibited contractions induced by PGF2α, U46619, 5-HT, endothelin-1 and oxytocin. Furthermore, in hTERT-infected human myometrial cells exposed to uterotonics, phenanthroline effectively suppressed the increase in intracellular Ca2+ concentration induced by PGF2α, U46619, oxytocin, and endothelin-1. These results suggest that the selective TAS2R5 agonist may not only significantly reduce uterine contractions but also decrease intracellular Ca2+ levels. This study highlights the potential development of TAS2R5 agonists as a new class of uterine relaxants, providing a novel avenue for improving the management of preterm labor.

Development, validation, and use of a monitoring method for fipronil and its metabolites in chicken eggs by QuEChERS with online-SPE-LC-Q/Orbitrap analysis.

RATIONALE: The residues of fipronil and its metabolites in chicken eggs pose a threat to human health, so regular monitoring is necessary. However, the pretreatments of the existing detection methods are complex and time-consuming. A simple and streamlined pretreatment method is needed to improve the detection efficiency. METHOD: A rapid, efficient, and facile approach employing the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method with online solid-phase extraction liquid chromatography tandem Q Exactive Orbitrap high-resolution mass spectrometry (online-SPE-LC-HRMS) was established and evaluated for the determination of fipronil, fipronil-desulfinyl, fipronil-sulfone, and fipronil-sulfide in chicken eggs. An improved sample preparation technique combining QuEChERS and online-SPE was developed. Negative targeted ion fragmentation scanning and targeted-selected ion monitoring of HRMS were adopted to identify and quantify the target analytes. RESULTS: The proposed pretreatment method took a few steps in <13 min to achieve excellent recoveries and negligible interference. High selectivity was acquired with the adoption of Q/Orbitrap HRMS. The limit of quantification (LOQ) of the analytes was 2.5 µg kg-1 , meeting the detection requirements of the maximum residue level enacted by the Codex Alimentarius Commission, Japan, and the United States for the sum of fipronil and its metabolites. Extraction recoveries at three spiked concentration levels were within 84.56% to 93.84%, with relative standard deviation ≤5.87%. CONCLUSION: The established method is efficient and easy to operate and displays satisfactory LOQs, recoveries, accuracy, and precision. This approach serves as a reference method for monitoring eggs while providing potential solutions for fipronil determination in more complicated matrices.

Mode Switch of Ca2 + Oscillation-Mediated Uterine Peristalsis and Associated Embryo Implantation Impairments in Mouse Adenomyosis.

Adenomyosis is a debilitating gynecological disease of the uterus with no medicinal cure. The tissue injury and repair hypothesis for adenomyosis suggests that uterine hyperperistalsis or dysperistalsis plays a pivotal role in establishing adenomyotic lesions. However, specific impairments in uterine peristalsis and the underlying cellular signals for these changes in adenomyosis remain elusive. Here, we report a precision-cut uterine slice preparation that preserves in vivo uterine architecture and generates peristalsis similar to that seen in the whole uterus. We found that uterine peristalsis in neonatal mice at day 14 and adult mice at day 55 presents as bursts with multiple peaks induced by intracellular Ca2+ oscillations. Using a mouse model of adenomyosis induced by tamoxifen, a selective estrogen receptor modulator, we discovered that uterine peristalsis and Ca2+ oscillations from adenomyotic uteri on days 14 and 55 become spikes (single peaks) with smaller amplitudes. The peak frequency of Ca2+ oscillations or peristalsis does not show a difference between control and adenomyotic mice. However, both the estimated force generated by uterine peristalsis and the total Ca2+ raised by Ca2+ oscillations are smaller in uteri from adenomyotic mice. Uteri from adenomyotic mice on day 14, but not on day 55, exhibit hyperresponsiveness to oxytocin. Embryo implantations are decreased in adenomyotic adult mice. Our results reveal a mode switch from bursts to spikes (rather than an increased peak frequency) of uterine Ca2+ oscillations and peristalsis and concurrent hyperresponsiveness to oxytocin in the neonatal stage are two characteristics of adenomyosis. These characteristics may contribute to embryo implantation impairments and decreased fertility in adenomyosis.

Genetic deletion of the Tas2r143/Tas2r135/Tas2r126 cluster reveals that TAS2Rs may not mediate bitter tastant-induced bronchodilation.

Bitter taste receptors (TAS2Rs) and their signaling elements are detected throughout the body, and bitter tastants induce a wide variety of biological responses in tissues and organs outside the mouth. However, the roles of TAS2Rs in these responses remain to be tested and established genetically. Here, we employed the CRISPR/Cas9 gene-editing technique to delete three bitter taste receptors-Tas2r143/Tas2r135/Tas2r126 (i.e., Tas2r triple knockout [TKO]) in mice. The fidelity and effectiveness of the Tas2r deletions were validated genetically at DNA and messenger RNA levels and functionally based on the tasting of TAS2R135 and TAS2R126 agonists. Bitter tastants are known to relax airways completely. However, TAS2R135 or TAS2R126 agonists either failed to induce relaxation of pre-contracted airways in wild-type mice and Tas2r TKO mice or relaxed them dose-dependently, but to the same extent in both types of mice. These results indicate that TAS2Rs are not required for bitter tastant-induced bronchodilation. The Tas2r TKO mice also provide a valuable model to resolve whether TAS2Rs mediate bitter tastant-induced responses in many other extraoral tissues.

Oscillating calcium signals in smooth muscle cells underlie the persistent basal tone of internal anal sphincter.

A persistent basal tone in the internal anal sphincter (IAS) is essential for keeping the anal canal closed and fecal continence; its inhibition via the rectoanal inhibitory reflex (RAIR) is required for successful defecation. However, cellular signals underlying the IAS basal tone remain enigmatic. Here we report the origin and molecular mechanisms of calcium signals that control the IAS basal tone, using a combination approach including a novel IAS slice preparation that retains cell arrangement and architecture as in vivo, 2-photon imaging, and cell-specific gene-modified mice. We found that IAS smooth muscle cells generate two forms of contractions (i.e., phasic and sustained contraction) and Ca2+ signals (i.e., synchronized Ca2+ oscillations [SCaOs] and asynchronized Ca2+ oscillations [ACaOs]) that last for hours. RyRs, TMEM16A, L-type Ca2+ channels, and gap junctions are required for SCaOs, which account for phasic contraction and 75% of sustained contraction. Nevertheless, only RyRs are required for ACaOs, which contribute 25% of sustained contraction. Nitric oxide, the primary neurotransmitter mediating the RAIR, blocks both types of Ca2+ signals, leading to IAS's full relaxation. Our results show that the oscillating nature of Ca2+ signals generates and maintains the basal tone without causing cytotoxicity to IAS. Our study provides insight into fecal continence and normal defecation.

Smooth muscle cell-specific TMEM16A deletion does not alter Ca2+ signaling, uterine contraction, gestation length, or litter size in mice†.

Ion channels in myometrial cells play critical roles in spontaneous and agonist-induced uterine contraction during the menstrual cycle, pregnancy maintenance, and parturition; thus, identifying the genes of ion channels in these cells and determining their roles are essential to understanding the biology of reproduction. Previous studies with in vitro functional and pharmacological approaches have produced controversial results regarding the presence and role of TMEM16A Ca2+-activated Cl- channels in myometrial cells. To unambiguously determine the function of this channel in these cells, we employed a genetic approach by using smooth muscle cell-specific TMEM16A deletion (i.e. TMEM16ASMKO) mice. We found that myometrial cells from TMEM16ASMKO mice generated the same pattern and magnitude in Ca2+ signals upon stimulation with KCl, oxytocin, and PGF2α compared to the isogenic control myometrial cells. At the uterine tissue level, TMEM16A deletion also did not cause detectable changes in either spontaneous or agonist (i.e. KCl, oxytocin, and PGF2α)-induced contractions. Moreover, in vivo the TMEM16ASMKO mice gave birth at full term with the same litter size as genetically identical control mice. Finally, TMEM16A immunostaining in both control and TMEM16ASMKO mice revealed that this protein was highly expressed in the endometrial stroma, but did not co-localize with a smooth muscle specific marker MYH11. Collectively, these results unequivocally demonstrate that TMEM16A does not serve as a pacemaking channel for spontaneous uterine contraction, neither does it function as a depolarizing channel for agonist-evoked uterine contraction. Yet these two functions could underlie the normal gestation length and litter size in the TMEM16ASMKO mice.

OR14I1 is a receptor for the human cytomegalovirus pentameric complex and defines viral epithelial cell tropism.

A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH-gL-UL128-UL130-UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT-mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies.

Azithromycin inhibits muscarinic 2 receptor-activated and voltage-activated Ca2+ permeant ion channels and Ca2+ sensitization, relaxing airway smooth muscle contraction.

Azithromycin (AZM) has been used for the treatment of asthma and chronic obstructive pulmonary disease (COPD); however, the effects and underlying mechanisms of AZM remain largely unknown. The effects of AZM on airway smooth muscles (ASMs) and the underlying mechanisms were studied using isometric muscle force measurements, the examination of lung slices, imaging, and patch-clamp techniques. AZM completely inhibited acetylcholine (ACH)-induced precontraction of ASMs in animals (mice, guinea pigs, and rabbits) and humans. Two other macrolide antibiotics, roxithromycin and Klaricid, displayed a decreased inhibitory activity, and the aminoglycoside antibiotics penicillin and streptomycin did not have an inhibitory effect. Precontractions were partially inhibited by nifedipine (selective inhibitor of L-type voltage-dependent Ca2+ channels (LVDCCs)), Pyr3 (selective inhibitor of TRPC3 and/or STIM/Orai channels, which are nonselective cation channels (NSCCs)), and Y-27632 (selective inhibitor of Rho-associated kinase (ROCK)). Moreover, LVDCC- and NSCC-mediated currents were inhibited by AZM, and the latter were suppressed by the muscarinic (M) 2 receptor inhibitor methoctramine. AZM inhibited LVDCC Ca2+ permeant ion channels, M2 receptors, and TRPC3 and/or STIM/Orai, which decreased cytosolic Ca2+ concentrations and led to muscle relaxation. This relaxation was also enhanced by the inhibition of Ca2+ sensitization. Therefore, AZM has potential as a novel and potent bronchodilator. The findings of this study improve the understanding of the effects of AZM on asthma and COPD.

Inflammatory mediators mediate airway smooth muscle contraction through a G protein-coupled receptor-transmembrane protein 16A-voltage-dependent Ca2+ channel axis and contribute to bronchial hyperresponsiveness in asthma.

BACKGROUND: Allergic inflammation has long been implicated in asthmatic hyperresponsiveness of airway smooth muscle (ASM), but its underlying mechanism remains incompletely understood. Serving as G protein-coupled receptor agonists, several inflammatory mediators can induce membrane depolarization, contract ASM, and augment cholinergic contractile response. We hypothesized that the signal cascade integrating on membrane depolarization by the mediators might involve asthmatic hyperresponsiveness. OBJECTIVE: We sought to investigate the signaling transduction of inflammatory mediators in ASM contraction and assess its contribution in the genesis of hyperresponsiveness. METHODS: We assessed the capacity of inflammatory mediators to induce depolarization currents by electrophysiological analysis. We analyzed the phenotypes of transmembrane protein 16A (TMEM16A) knockout mice, applied pharmacological reagents, and measured the Ca2+ signal during ASM contraction. To study the role of the depolarization signaling in asthmatic hyperresponsiveness, we measured the synergistic contraction by methacholine and inflammatory mediators both ex vivo and in an ovalbumin-induced mouse model. RESULTS: Inflammatory mediators, such as 5-hydroxytryptamin, histamine, U46619, and leukotriene D4, are capable of inducing Ca2+-activated Cl- currents in ASM cells, and these currents are mediated by TMEM16A. A combination of multiple analysis revealed that a G protein-coupled receptor-TMEM16A-voltage-dependent Ca2+ channel signaling axis was required for ASM contraction induced by inflammatory mediators. Block of TMEM16A activity may significantly inhibit the synergistic contraction of acetylcholine and the mediators and hence reduces hypersensitivity. CONCLUSIONS: A G protein-coupled receptor-TMEM16A-voltage-dependent Ca2+ channel axis contributes to inflammatory mediator-induced ASM contraction and synergistically activated TMEM16A by allergic inflammatory mediators with cholinergic stimuli.

Bitter taste receptors as targets for tocolytics in preterm labor therapy.

Preterm birth (PTB) is the leading cause of neonatal mortality and morbidity, with few prevention and treatment options. Uterine contraction is a central feature of PTB, so gaining new insights into the mechanisms of this contraction and consequently identifying novel targets for tocolytics are essential for more successful management of PTB. Here we report that myometrial cells from human and mouse express bitter taste receptors (TAS2Rs) and their canonical signaling components (i.e., G-protein gustducin and phospholipase C ß2). Bitter tastants can completely relax myometrium precontracted by different uterotonics. In isolated single mouse myometrial cells, a phenotypical bitter tastant (chloroquine, ChQ) reverses the rise in intracellular Ca2+ concentration ([Ca2+]i) and cell shortening induced by uterotonics, and this reversal effect is inhibited by pertussis toxin and by genetic deletion of α-gustducin. In human myometrial cells, knockdown of TAS2R14 but not TAS2R10 inhibits ChQ's reversal effect on an oxytocin-induced rise in [Ca2+]i Finally, ChQ prevents mouse PTBs induced by bacterial endotoxin LPS or progesterone receptor antagonist mifepristone more often than current commonly used tocolytics, and this prevention is largely lost in α-gustducin-knockout mice. Collectively, our results reveal that activation of the canonical TAS2R signaling system in myometrial cells produces profound relaxation of myometrium precontracted by a broad spectrum of contractile agonists, and that targeting TAS2Rs is an attractive approach to developing effective tocolytics for PTB management.-Zheng, K., Lu, P., Delpapa, E., Bellve, K., Deng, R., Condon, J. C., Fogarty, K., Lifshitz, L. M., Simas, T. A. M., Shi, F., ZhuGe, R. Bitter taste receptors as targets for tocolytics in preterm labor therapy.

Airway smooth muscle dysfunction in Pompe (Gaa-/- ) mice.

Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA), an enzyme responsible for hydrolyzing lysosomal glycogen. Deficiency of GAA leads to systemic glycogen accumulation in the lysosomes of skeletal muscle, motor neurons, and smooth muscle. Skeletal muscle and motor neuron pathology are known to contribute to respiratory insufficiency in Pompe disease, but the role of airway pathology has not been evaluated. Here we propose that GAA enzyme deficiency disrupts the function of the trachea and bronchi and this lower airway pathology contributes to respiratory insufficiency in Pompe disease. Using an established mouse model of Pompe disease, the Gaa-/- mouse, we compared histology, pulmonary mechanics, airway smooth muscle (ASM) function, and calcium signaling between Gaa-/- and age-matched wild-type (WT) mice. Lysosomal glycogen accumulation was observed in the smooth muscle of both the bronchi and the trachea in Gaa-/- but not WT mice. Furthermore, Gaa-/- mice had hyporesponsive airway resistance and bronchial ring contraction to the bronchoconstrictive agents methacholine (MCh) and potassium chloride (KCl) and to a bronchodilator (albuterol). Finally, calcium signaling during bronchiolar smooth muscle contraction was impaired in Gaa-/- mice indicating impaired extracellular calcium influx. We conclude that GAA enzyme deficiency leads to glycogen accumulation in the trachea and bronchi and impairs the ability of lower ASM to regulate calcium and respond appropriately to bronchodilator or constrictors. Accordingly, ASM dysfunction may contribute to respiratory impairments in Pompe disease.

Extraoral bitter taste receptors in health and disease.

Bitter taste receptors (TAS2Rs or T2Rs) belong to the superfamily of seven-transmembrane G protein-coupled receptors, which are the targets of >50% of drugs currently on the market. Canonically, T2Rs are located in taste buds of the tongue, where they initiate bitter taste perception. However, accumulating evidence indicates that T2Rs are widely expressed throughout the body and mediate diverse nontasting roles through various specialized mechanisms. It has also become apparent that T2Rs and their polymorphisms are associated with human disorders. In this review, we summarize the physiological and pathophysiological roles that extraoral T2Rs play in processes as diverse as innate immunity and reproduction, and the major challenges in this emerging field.

The molecular basis of the genesis of basal tone in internal anal sphincter.

Smooth muscle sphincters exhibit basal tone and control passage of contents through organs such as the gastrointestinal tract; loss of this tone leads to disorders such as faecal incontinence. However, the molecular mechanisms underlying this tone remain unknown. Here, we show that deletion of myosin light-chain kinases (MLCK) in the smooth muscle cells from internal anal sphincter (IAS-SMCs) abolishes basal tone, impairing defecation. Pharmacological regulation of ryanodine receptors (RyRs), L-type voltage-dependent Ca(2+) channels (VDCCs) or TMEM16A Ca(2+)-activated Cl(-) channels significantly changes global cytosolic Ca(2+) concentration ([Ca(2+)]i) and the tone. TMEM16A deletion in IAS-SMCs abolishes the effects of modulators for TMEM16A or VDCCs on a RyR-mediated rise in global [Ca(2+)]i and impairs the tone and defecation. Hence, MLCK activation in IAS-SMCs caused by a global rise in [Ca(2+)]i via a RyR-TMEM16A-VDCC signalling module sets the basal tone. Targeting this module may lead to new treatments for diseases like faecal incontinence.

Catecholamine exocytosis during low frequency stimulation in mouse adrenal chromaffin cells is primarily asynchronous and controlled by the novel mechanism of Ca2+ syntilla suppression.

Adrenal chromaffin cells (ACCs), stimulated by the splanchnic nerve, generate action potentials (APs) at a frequency near 0.5 Hz in the resting physiological state, at times described as 'rest and digest'. How such low frequency stimulation in turn elicits sufficient catecholamine exocytosis to set basal sympathetic tone is not readily explained by the classical mechanism of stimulus-secretion coupling, where exocytosis is synchronized to AP-induced Ca(2+) influx. By using simulated action potentials (sAPs) at 0.5 Hz in isolated patch-clamped mouse ACCs, we show here that less than 10% of all catecholaminergic exocytosis, measured by carbon fibre amperometry, is synchronized to an AP. The asynchronous phase, the dominant phase, of exocytosis does not require Ca(2+) influx. Furthermore, increased asynchronous exocytosis is accompanied by an AP-dependent decrease in frequency of Ca(2+) syntillas (i.e. transient, focal Ca(2+) release from internal stores) and is ryanodine sensitive. We propose a mechanism of disinhibition, wherein APs suppress Ca(2+) syntillas, which themselves inhibit exocytosis as they do in the case of spontaneous catecholaminergic exocytosis.

The cellular and molecular basis of bitter tastant-induced bronchodilation.

Bronchodilators are a standard medicine for treating airway obstructive diseases, and ß2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than ß2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca(2+) signals, as revealed in cultured ASM cells, to activate large-conductance Ca(2+)-activated K(+) channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca(2+) events nor alter spontaneous local Ca(2+) transients. Interestingly, they increase global intracellular [Ca(2+)]i, although to a much lower level than bronchoconstrictors. We show that these Ca(2+) changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase Cß [PLCß]- inositol 1,4,5-triphosphate receptor [IP3R]), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca(2+)]i induced by bronchoconstrictors, and this lowering of the [Ca(2+)]i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca(2+) channels (VDCCs), resulting in reversal in [Ca(2+)]i, and this inhibition can be prevented by pertussis toxin and G-protein ßγ subunit inhibitors, but not by the blockers of PLCß and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca(2+) signaling pathways via Gßγ to increase [Ca(2+)]i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca(2+)]i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants.

A segment of 97 amino acids within the translocation domain of Clostridium difficile toxin B is essential for toxicity.

Clostridium difficile toxin B (TcdB) intoxicates target cells by glucosylating Rho GTPases. TcdB (269 kDa) consists of at least 4 functional domains including a glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a translocation domain (TD), and a receptor binding domain (RBD). The function and molecular mode of action of the TD, which is the largest segment of TcdB and comprises nearly 50% of the protein, remain largely unknown. Here we show that a 97-amino-acid segment (AA1756 - 1852, designated as ?97 or D97), located in the C-terminus of the TD and adjacent to the RBD, is essential for the cellular activity of TcdB. Deletion of this segment in TcdB (designated as TxB-D97), did not adversely alter toxin enzymatic activities or its cellular binding and uptake capacity. TxB-D97 bound to and entered cells in a manner similar to TcdB holotoxin. Both wild type and mutant toxins released their GTDs similarly in the presence of inositol hexakisphosphate (InsP6), and showed a similar glucosyltransferase activity in a cell-free glucosylating assay. Despite these similarities, the cytotoxic activity of TxB-D97 was reduced by more than 5 logs compared to wild type toxin, supported by the inability of TxB-D97 to glucosylate Rac1 of target cells. Moreover, the mutant toxin failed to elicit tumor necrosis factor alpha (TNF-α) in macrophages, a process dependent on the glucosyltransferase activity of the toxin. Cellular fractionation of toxin-exposed cells revealed that TxB-D97 was unable to efficiently release the GTD into cytosol. Thereby, we conclude the 97-amino-acid region of the TD C-terminus of TcdB adjacent to the RBD, is essential for the toxicity of TcdB.

The transmembrane protein 16A Ca(2+)-activated Cl- channel in airway smooth muscle contributes to airway hyperresponsiveness.

RATIONALE: Asthma is a chronic inflammatory disorder with a characteristic of airway hyperresponsiveness (AHR). Ca(2+)-activated Cl(-) [Cl((Ca))] channels are inferred to be involved in AHR, yet their molecular nature and the cell type they act within to mediate this response remain unknown. OBJECTIVES: Transmembrane protein 16A (TMEM16A) and TMEM16B are Cl((Ca)) channels, and activation of Cl((Ca)) channels in airway smooth muscle (ASM) contributes to agonist-induced airway contraction. We hypothesized that Tmem16a and/or Tmem16b encode Cl((Ca)) channels in ASM and mediate AHR. METHODS: We assessed the expression of the TMEM16 family, and the effects of niflumic acid and benzbromarone on AHR and airway contraction, in an ovalbumin-sensitized mouse model of chronic asthma. We also cloned TMEM16A from ASM and examined the Cl(-) currents it produced in HEK293 cells. We further studied the impacts of TMEM16A deletion on Ca(2+) agonist-induced cell shortening, and on Cl((Ca)) currents activated by Ca(2+) sparks (localized, short-lived Ca(2+) transients due to the opening of ryanodine receptors) in mouse ASM cells. MEASUREMENTS AND MAIN RESULTS: TMEM16A, but not TMEM16B, is expressed in ASM cells and its expression in these cells is up-regulated in ovalbumin-sensitized mice. Niflumic acid and benzbromarone prevent AHR and contraction evoked by methacholine in ovalbumin-sensitized mice. TMEM16A produces Cl((Ca)) currents with kinetics similar to native Cl((Ca)) currents. TMEM16A deletion renders Ca(2+) sparks unable to activate Cl((Ca)) currents, and weakens caffeine- and methacholine-induced cell shortening. CONCLUSIONS: Tmem16a encodes Cl((Ca)) channels in ASM and contributes to Ca(2+) agonist-induced contraction. In addition, up-regulation of TMEM16A and its augmented activation contribute to AHR in an ovalbumin-sensitized mouse model of chronic asthma. TMEM16A may represent a potential therapeutic target for asthma.