Method of moments framework for differential expression analysis of single-cell RNA sequencing data.

Differential expression analysis of single-cell RNA sequencing (scRNA-seq) data is central for characterizing how experimental factors affect the distribution of gene expression. However, distinguishing between biological and technical sources of cell-cell variability and assessing the statistical significance of quantitative comparisons between cell groups remain challenging. We introduce Memento, a tool for robust and efficient differential analysis of mean expression, variability, and gene correlation from scRNA-seq data, scalable to millions of cells and thousands of samples. We applied Memento to 70,000 tracheal epithelial cells to identify interferon-responsive genes, 160,000 CRISPR-Cas9 perturbed T cells to reconstruct gene-regulatory networks, 1.2 million peripheral blood mononuclear cells (PBMCs) to map cell-type-specific quantitative trait loci (QTLs), and the 50-million-cell CELLxGENE Discover corpus to compare arbitrary cell groups. In all cases, Memento identified more significant and reproducible differences in mean expression compared with existing methods. It also identified differences in variability and gene correlation that suggest distinct transcriptional regulation mechanisms imparted by perturbations.

Subcellular trafficking of FGF controls tracheal invasion of Drosophila flight muscle.

To meet the extreme oxygen demand of insect flight muscle, tracheal (respiratory) tubes ramify not only on its surface, as in other tissues, but also within T-tubules and ultimately surrounding every mitochondrion. Although this remarkable physiological specialization has long been recognized, its cellular and molecular basis is unknown. Here, we show that Drosophila tracheoles invade flight muscle T-tubules through transient surface openings. Like other tracheal branching events, invasion requires the Branchless FGF pathway. However, localization of the FGF chemoattractant changes from all muscle membranes to T-tubules as invasion begins. Core regulators of epithelial basolateral membrane identity localize to T-tubules, and knockdown of AP-1γ, required for basolateral trafficking, redirects FGF from T-tubules to surface, increasing tracheal surface ramification and preventing invasion. We propose that tracheal invasion is controlled by an AP-1-dependent switch in FGF trafficking. Thus, subcellular targeting of a chemoattractant can direct outgrowth to specific domains, including inside the cell.

The Drosophila tracheal terminal cell as a model for branching morphogenesis.

The terminal cells of the Drosophila larval tracheal system are perhaps the simplest delivery networks, providing an analogue for mammalian vascular growth and function in a system with many fewer components. These cells are a prime example of single-cell morphogenesis, branching significantly over time to adapt to the needs of the growing tissue they supply. While the genetic mechanisms governing local branching decisions have been studied extensively, an understanding of the emergence of a global network architecture is still lacking. Mapping out the full network architecture of populations of terminal cells at different developmental times of Drosophila larvae, we find that cell growth follows scaling laws relating the total edge length, supply area, and branch density. Using time-lapse imaging of individual terminal cells, we identify that the cells grow in three ways: by extending branches, by the side budding of new branches, and by internally growing existing branches. A generative model based on these modes of growth recapitulates statistical properties of the terminal cell network data. These results suggest that the scaling laws arise from the coupled contributions of branching and internal growth. This study establishes the terminal cell as a uniquely tractable model system for further studies of transportation and distribution networks.

Glaesserella parasuis serotype 4 exploits fibronectin via RlpA for tracheal colonization following porcine circovirus type 2 infection.

Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality, but the role played by PCV2 and bacterial and host factors contributing to this process have not been defined. Bacterial attachment is assumed to occur via specific receptor-ligand interactions between adhesins on the bacterial cell and host proteins adsorbed to the implant surface. Mass spectrometry (MS) analysis of PCV2-infected swine tracheal epithelial cells (STEC) revealed that the expression of Extracellular matrix protein (ECM) Fibronectin (Fn) increased significantly on the infected cells surface. Importantly, efficient G. parasuis serotype 4 (GPS4) adherence to STECs was imparted by interactions with Fn. Furthermore, abrogation of adherence was gained by genetic knockout of Fn, Fn and Integrin ß1 antibody blocking. Fn is frequently exploited as a receptor for bacterial pathogens. To explore the GPS4 adhesin that interacts with Fn, recombinant Fn N-terminal type I and type II domains were incubated with GPS4, and the interacting proteins were pulled down for MS analysis. Here, we show that rare lipoprotein A (RlpA) directly interacts with host Fibronectin mediating GPS4 adhesion. Finally, we found that PCV2-induced Fibronectin expression and adherence of GPS4 were prevented significantly by TGF-ß signaling pathway inhibitor SB431542. Our data suggest the RlpA-Fn interaction to be a potentially promising novel therapeutic target to combat PCV2 and GPS4 coinfection.

The Osiris family genes function as novel regulators of the tube maturation process in the Drosophila trachea.

Drosophila trachea is a premier model to study tube morphogenesis. After the formation of continuous tubes, tube maturation follows. Tracheal tube maturation starts with an apical secretion pulse that deposits extracellular matrix components to form a chitin-based apical luminal matrix (aECM). This aECM is then cleared and followed by the maturation of taenidial folds. Finally, air fills the tubes. Meanwhile, the cellular junctions are maintained to ensure tube integrity. Previous research has identified several key components (ER, Golgi, several endosomes) of protein trafficking pathways that regulate the secretion and clearance of aECM, and the maintenance of cellular junctions. The Osiris (Osi) gene family is located at the Triplo-lethal (Tpl) locus on chromosome 3R 83D4-E3 and exhibits dosage sensitivity. Here, we show that three Osi genes (Osi9, Osi15, Osi19), function redundantly to regulate adherens junction (AJ) maintenance, luminal clearance, taenidial fold formation, tube morphology, and air filling during tube maturation. The localization of Osi proteins in endosomes (Rab7-containing late endosomes, Rab11-containing recycling endosomes, Lamp-containing lysosomes) and the reduction of these endosomes in Osi mutants suggest the possible role of Osi genes in tube maturation through endosome-mediated trafficking. We analyzed tube maturation in zygotic rab11 and rab7 mutants, respectively, to determine whether endosome-mediated trafficking is required. Interestingly, similar tube maturation defects were observed in rab11 but not in rab7 mutants, suggesting the involvement of Rab11-mediated trafficking, but not Rab7-mediated trafficking, in this process. To investigate whether Osi genes regulate tube maturation primarily through the maintenance of Rab11-containing endosomes, we overexpressed rab11 in Osi mutant trachea. Surprisingly, no obvious rescue was observed. Thus, increasing endosome numbers is not sufficient to rescue tube maturation defects in Osi mutants. These results suggest that Osi genes regulate other aspects of endosome-mediated trafficking, or regulate an unknown mechanism that converges or acts in parallel with Rab11-mediated trafficking during tube maturation.

Regulation of Archease by the mTOR-vATPase axis.

Larval terminal cells of the Drosophila tracheal system generate extensive branched tubes, requiring a huge increase in apical membrane. We discovered that terminal cells compromised for apical membrane expansion - mTOR-vATPase axis and apical polarity mutants - were invaded by the neighboring stalk cell. The invading cell grows and branches, replacing the original single intercellular junction between stalk and terminal cell with multiple intercellular junctions. Here, we characterize disjointed, a mutation in the same phenotypic class. We find that disjointed encodes Drosophila Archease, which is required for the RNA ligase (RtcB) function that is essential for tRNA maturation and for endoplasmic reticulum stress-regulated nonconventional splicing of Xbp1 mRNA. We show that the steady-state subcellular localization of Archease is principally nuclear and dependent upon TOR-vATPase activity. In tracheal cells mutant for Rheb or vATPase loci, Archease localization shifted dramatically from nucleus to cytoplasm. Further, we found that blocking tRNA maturation by knockdown of tRNAseZ also induced compensatory branching. Taken together, these data suggest that the TOR-vATPase axis promotes apical membrane growth in part through nuclear localization of Archease, where Archease is required for tRNA maturation.

An essential function for autocrine hedgehog signaling in epithelial proliferation and differentiation in the trachea.

The tracheal epithelium is a primary target for pulmonary diseases as it provides a conduit for air flow between the environment and the lung lobes. The cellular and molecular mechanisms underlying airway epithelial cell proliferation and differentiation remain poorly understood. Hedgehog (HH) signaling orchestrates communication between epithelial and mesenchymal cells in the lung, where it modulates stromal cell proliferation, differentiation and signaling back to the epithelium. Here, we reveal a previously unreported autocrine function of HH signaling in airway epithelial cells. Epithelial cell depletion of the ligand sonic hedgehog (SHH) or its effector smoothened (SMO) causes defects in both epithelial cell proliferation and differentiation. In cultured primary human airway epithelial cells, HH signaling inhibition also hampers cell proliferation and differentiation. Epithelial HH function is mediated, at least in part, through transcriptional activation, as HH signaling inhibition leads to downregulation of cell type-specific transcription factor genes in both the mouse trachea and human airway epithelial cells. These results provide new insights into the role of HH signaling in epithelial cell proliferation and differentiation during airway development.

Ectopic expression in commonly used transgenic Drosophila GAL4 driver lines.

Transgenic tools such as the GAL4/UAS system in Drosophila have been used extensively to induce spatiotemporally controlled changes in gene expression and tissue-specific expression of a range of transgenes. We previously discovered unexpected expression of the commonly used dilp2-GAL4 line in tracheal tissue which significantly impacted growth phenotypes. We realized that few GAL4 lines have been thoroughly characterized, particularly when considering transient activity that may have significant impact on phenotypic readouts. Here, we characterized a further subset of 12 reportedly tissue-specific GAL4 lines commonly used in genetic studies of development, growth, endocrine regulation, and metabolism. Ten out of 12 GAL4 lines exhibited ectopic activity in other larval tissues, with seven being active in the larval trachea. Since this ectopic activity may result in phenotypes that do not depend on the manipulation in the intended target tissue, it is recommended to carefully analyze the outcome while taking this aspect into consideration.

Obesity impairs ciliary function and mucociliary clearance in the murine airway epithelium.

Obesity is a risk factor for increased morbidity and mortality in viral respiratory infection. Mucociliary clearance (MCC) in the airway is the primary host defense against viral infections. However, the impact of obesity on MCC is unclear, prompting this study. Using murine tracheal tissue culture and in vitro influenza A virus (IAV) infection models, we analyzed cilia-driven flow and ciliary beat frequency (CBF) in the airway epithelium to evaluate MCC. Short-term IAV infection increased cilia-driven flow and CBF in control mice, but not in high-fat diet-induced obese mice. Basal cilia-driven flow and CBF were also lower in obese mice than in control mice. Mechanistically, the increase of extracellular adenosine triphosphate (ATP) release during IAV infection, which was observed in the control mice, was abolished in the obese mice; however, the addition of ATP increased cilia-driven flow and CBF both in control and obese mice to a similar extent. In addition, RNA sequencing and reverse transcription-polymerase chain reaction revealed the downregulation of several cilia-related genes, including Dnah1, Dnal1, Armc4, and Ttc12 (the dynein-related genes); Ulk4 (the polychaete differentiation gene); Cep164 (the ciliogenesis and intraflagellar transport gene); Rsph4a, Cfap206, and Ppil6 (the radial spoke structure and assembly gene); and Drc3(the nexin-dynein regulatory complex genes) in obese murine tracheal tissues compared with their control levels. In conclusion, our studies demonstrate that obesity attenuates MCC under basal conditions and during IAV infection by downregulating the expression of cilia-related genes and suppressing the release of extracellular ATP, thereby increasing the susceptibility and severity of IAV infection.NEW & NOTEWORTHY Our study shows that obesity impairs airway mucociliary clearance (MCC), an essential physical innate defense mechanism for viral infection. Mechanically, this is likely due to the obesity-induced downregulation of cilia-related genes and attenuation of extracellular ATP release. This study provides novel insights into the mechanisms driving the higher susceptibility and severity of viral respiratory infections in individuals with obesity.

Metachrony drives effective mucociliary transport via a calcium-dependent mechanism.

The mucociliary transport apparatus is critical for maintaining lung health via the coordinated movement of cilia to clear mucus and particulates. A metachronal wave propagates across the epithelium when cilia on adjacent multiciliated cells beat slightly out of phase along the proximal-distal axis of the airways in alignment with anatomically directed mucociliary clearance. We hypothesized that metachrony optimizes mucociliary transport (MCT) and that disruptions of calcium signaling would abolish metachrony and decrease MCT. We imaged bronchi from human explants and ferret tracheae using micro-optical coherence tomography (µOCT) to evaluate airway surface liquid depth (ASL), periciliary liquid depth (PCL), cilia beat frequency (CBF), MCT, and metachrony in situ. We developed statistical models that included covariates of MCT. Ferret tracheae were treated with BAPTA-AM (chelator of intracellular Ca2+), lanthanum chloride (nonpermeable Ca2+ channel competitive antagonist), and repaglinide (inhibitor of calaxin) to test calcium dependence of metachrony. We demonstrated that metachrony contributes to mucociliary transport of human and ferret airways. MCT was augmented in regions of metachrony compared with nonmetachronous regions by 48.1%, P = 0.0009 or 47.5%, P < 0.0020 in humans and ferrets, respectively. PCL and metachrony were independent contributors to MCT rate in humans; ASL, CBF, and metachrony contribute to ferret MCT rates. Metachrony can be disrupted by interference with calcium signaling including intracellular, mechanosensitive channels, and calaxin. Our results support that the presence of metachrony augments MCT in a calcium-dependent mechanism.NEW & NOTEWORTHY We developed a novel imaging-based analysis to detect coordination of ciliary motion and optimal coordination, a process called metachrony. We found that metachrony is key to the optimization of ciliary-mediated mucus transport in both ferret and human tracheal tissue. This process appears to be regulated through calcium-dependent mechanisms. This study demonstrates the capacity to measure a key feature of ciliary coordination that may be important in genetic and acquired disorders of ciliary function.

Ezrin knockdown reduces procaterol-stimulated ciliary beating without morphological changes in mouse airway cilia.

Mucociliary clearance, which is conducted by beating cilia cooperating with the surface mucous layer, is a major host defense mechanism of the airway epithelium. Ezrin, a crosslinker between membrane proteins and the actin cytoskeleton, is located in microvilli and around the basal bodies in airway ciliary cells. It is also likely that ezrin plays an important role in apical localization of ß2 adrenergic receptor (ß2AR) in airway ciliary cells. Here, we studied the physiological roles of ezrin by using trachea and airway epithelial cells prepared from ezrin-knockdown (Vil2kd/kd) mice. The trachea and airway ciliary cells of Vil2kd/kd mice presented a normal morphology and basal body orientation, suggesting that ezrin is not directly involved in development and planar cell polarity of cilia. Procaterol stimulates ciliary beating (frequency and amplitude) via ß2AR in the airway ciliary cells. In the Vil2kd/kd mice, airway ciliary beating stimulated with procaterol was partly inhibited due to the impairment of cell surface expression of ß2AR. These results suggest that ezrin regulates the beating of airway ciliary cells by promoting the apical surface localization of ß2AR. This article has an associated First Person interview with the first author of the paper.

Element accumulation in the tracheal and bronchial cartilages of monkeys.

Compositional changes in the tracheal and bronchial cartilages can affect respiratory ventilation and lung function. We aimed to elucidate element accumulation in the tracheal and bronchial cartilages of monkeys and divided it into four sites: the tracheal, tracheal bifurcation, left bronchial, and right bronchial cartilages. The elemental content was analyzed using inductively coupled plasma atomic emission spectrometry. The average calcium content was two to three times higher in the tracheal cartilage than in the other three cartilages. The trends of phosphorus and zinc were similar to those of calcium. The average calcium, phosphorus, and zinc cartilage contents were the highest in the tracheal cartilage and decreased in the following order: the left bronchial, right bronchial, and tracheal bifurcation cartilages. These findings revealed that differences existed in element accumulation between different sites within the same airway cartilage and that calcium, phosphorus, and zinc accumulation mainly occurred in the tracheal cartilage. A substantial direct correlation was observed between age and calcium content in the tracheal and bronchial cartilages and all such monkeys with high calcium content were > four years of age. These results suggest that calcium accumulation occurs in the tracheal and bronchial cartilages after reaching a certain age. An extremely substantial direct correlation was observed between calcium and phosphorus contents in the tracheal and bronchial cartilages. This finding is similar to the previously published calcium and phosphorus correlations in several other cartilages, suggesting that the calcium and phosphorus contents of cartilage exist in a certain ratio.

New mouse models for high resolution and live imaging of planar cell polarity proteins in vivo.

The collective polarization of cellular structures and behaviors across a tissue plane is a near universal feature of epithelia known as planar cell polarity (PCP). This property is controlled by the core PCP pathway, which consists of highly conserved membrane-associated protein complexes that localize asymmetrically at cell junctions. Here, we introduce three new mouse models for investigating the localization and dynamics of transmembrane PCP proteins: Celsr1, Fz6 and Vangl2. Using the skin epidermis as a model, we characterize and verify the expression, localization and function of endogenously tagged Celsr1-3xGFP, Fz6-3xGFP and tdTomato-Vangl2 fusion proteins. Live imaging of Fz6-3xGFP in basal epidermal progenitors reveals that the polarity of the tissue is not fixed through time. Rather, asymmetry dynamically shifts during cell rearrangements and divisions, while global, average polarity of the tissue is preserved. We show using super-resolution STED imaging that Fz6-3xGFP and tdTomato-Vangl2 can be resolved, enabling us to observe their complex localization along junctions. We further explore PCP fusion protein localization in the trachea and neural tube, and discover new patterns of PCP expression and localization throughout the mouse embryo.

Immunohistochemical analysis and distribution of epithelial mast cells in the rat larynx and trachea.

Mast cells (MCs) in rat airways have been classified into two subtypes: epithelial MCs and connective tissue MCs (CTMCs). However, the immunohistochemical characteristics, cellular morphology, and distribution of epithelial MCs in the upper airways remain unclear. The present study investigated the morphological characteristics and distribution of epithelial MCs using 5-hydroxytryptamine (5-HT) and other immunohistochemical markers in sectioned or whole-mount preparations of the rat larynx and trachea. A double immunofluorescence analysis revealed the colocalization of 5-HT immunoreactivity with c-kit, a stem cell factor receptor commonly used as a MC marker, in both epithelial MCs and CTMCs. Dopa decarboxylase, an enzyme involved in 5-HT synthesis, was detected in both subtypes, suggesting their ability to synthesize and release 5-HT. Tryptase and histidine decarboxylase (a biosynthetic enzyme of histamine), which are well-known mediators of MCs, were exclusive to CTMCs. Epithelial MCs were pleomorphic with long cytoplasmic processes, whereas CTMCs were round and lacked cytoplasmic processes. The density of epithelial MCs was significantly higher in the glottis and cranial part of the trachea than in the epiglottis and other parts of the trachea. The present results showed that the morphology and immunohistochemical characteristics of epithelial MCs were different from those of CTMCs in the rat larynx and trachea, and variform epithelial MCs were predominantly located at the entrance of the upper airways. Epithelial MCs may release 5-HT to regulate innate immune responses by modulating epithelial cell functions at the entrance gate of the upper airways.

Chronic changes in developmental oxygen have little effect on mitochondria and tracheal density in the endothermic moth Manduca sexta.

Oxygen availability during development is known to impact the development of insect respiratory and metabolic systems. Drosophila adult tracheal density exhibits developmental plasticity in response to hypoxic or hyperoxic oxygen levels during larval development. Respiratory systems of insects with higher aerobic demands, such as those that are facultative endotherms, may be even more responsive to oxygen levels above or below normoxia during development. The moth Manduca sexta is a large endothermic flying insect that serves as a good study system to start answering questions about developmental plasticity. In this study, we examined the effect of developmental oxygen levels (hypoxia: 10% oxygen, and hyperoxia: 30% oxygen) on the respiratory and metabolic phenotype of adult moths, focusing on morphological and physiological cellular and intercellular changes in phenotype. Mitochondrial respiration rate in permeabilized and isolated flight muscle was measured in adults. We found that permeabilized flight muscle fibers from the hypoxic group had increased mitochondrial oxygen consumption, but this was not replicated in isolated flight muscle mitochondria. Morphological changes in the trachea were examined using confocal imaging. We used transmission electron microscopy to quantify muscle and mitochondrial density in the flight muscle. The respiratory morphology was not significantly different between developmental oxygen groups. These results suggest that the developing M. sexta trachea and mitochondrial respiration have limited developmental plasticity when faced with rearing at 10% or 30% oxygen.

Seihaito, a Kampo medicine, attenuates IL-13-induced mucus production and goblet cell metaplasia.

Goblet cell hyperplasia and increased mucus production are features of airway diseases, including asthma, and excess airway mucus often worsens these conditions. Even steroids are not uniformly effective in mucus production in severe asthma, and new therapeutic options are needed. Seihaito is a Japanese traditional medicine that is used clinically as an antitussive and expectorant. In the present study, we examined the effect of Seihaito on goblet cell differentiation and mucus production. In in vitro studies, using air-liquid interface culture of guinea-pig tracheal epithelial cells, Seihaito inhibited IL-13-induced proliferation of goblet cells and MUC5AC, a major component of mucus production. Seihaito suppressed goblet cell-specific gene expression, without changing ciliary cell-specific genes, suggesting that it inhibits goblet cell differentiation. In addition, Seihaito suppressed MUC5AC expression in cells transfected with SPDEF, a transcription factor activated by IL-13. Furthermore, Seihaito attenuated in vivo goblet cell proliferation and MUC5AC mRNA expression in IL-13-treated mouse lungs. Collectively, these findings demonstrated that Seihaito has an inhibitory effect on goblet cell differentiation and mucus production, which is at least partly due to the inhibition of SPDEF.

Intratracheally administered LNA gapmer antisense oligonucleotides induce robust gene silencing in mouse lung fibroblasts.

The lung is a complex organ with various cell types having distinct roles. Antisense oligonucleotides (ASOs) have been studied in the lung, but it has been challenging to determine their effectiveness in each cell type due to the lack of appropriate analytical methods. We employed three distinct approaches to study silencing efficacy within different cell types. First, we used lineage markers to identify cell types in flow cytometry, and simultaneously measured ASO-induced silencing of cell-surface proteins CD47 or CD98. Second, we applied single-cell RNA sequencing (scRNA-seq) to measure silencing efficacy in distinct cell types; to the best of our knowledge, this is the first time scRNA-seq has been applied to measure the efficacy of oligonucleotide therapeutics. In both approaches, fibroblasts were the most susceptible to locally delivered ASOs, with significant silencing also in endothelial cells. Third, we confirmed that the robust silencing in fibroblasts is broadly applicable by silencing two targets expressed mainly in fibroblasts, Mfap4 and Adam33. Across independent approaches, we demonstrate that intratracheally administered LNA gapmer ASOs robustly induce gene silencing in lung fibroblasts. ASO-induced gene silencing in fibroblasts was durable, lasting 4-8 weeks after a single dose. Thus, lung fibroblasts are well aligned with ASOs as therapeutics.

Pluronic F127 hydrogel-loaded extracellular vesicles from adipose-derived mesenchymal stem cells promote tracheal cartilage regeneration via SCNN1B delivery.

Extracellular vesicles (EVs) derived from adipose-derived mesenchymal stem cells (AMSC-EVs) have been highlighted as a cell-free therapy due to their regenerative capability to enhance tissue and organ regeneration. Herein, we aimed to examine the mechanism of PF127-hydrogel@AMSC-EVs in promoting tracheal cartilage defect repair. Based on bioinformatics methods, SCNN1B was identified as a key gene for the osteogenic differentiation of AMSCs induced by AMSC-EVs. EVs were isolated from rat AMSCs and then loaded onto thermo-sensitive PF-127 hydrogel to develop PF127-hydrogel@AMSC-EVs. It was established that PF127-hydrogel@AMSC-EVs could effectively deliver SCNN1B into AMSCs, where SCNN1B promoted AMSC osteogenic differentiation. The promotive effect was evidenced by enhanced ALP activity, extracellular matrix mineralization, and expression of s-glycosaminoglycan, RUNX2, OCN, collagen II, PERK, and ATF4. Furthermore, the in vivo experiments revealed that PF127-hydrogel@AMSC-SCNN1B-EVs stimulated tracheal cartilage regeneration in rats through PERK/ATF4 signaling axis activation. Therefore, PF127-hydrogel@AMSC-SCNN1B-EVs may be a novel cell-free biomaterial to facilitate tracheal cartilage regeneration and cartilage injury repair.

The Increase in the Frequency and Amplitude of the Beating of Isolated Mouse Tracheal Cilia Reactivated by ATP and cAMP with Elevation in pH.

Single cilia, 100 nm in diameter and 10 µm in length, were isolated from mouse tracheae with Triton X-100 (0.02%) treatment, and the effects of pH on ciliary beating were examined by measuring the ciliary beat frequency (CBF) and the ciliary bend distance (CBD-an index of amplitude) using a high-speed video microscope (250 fps). ATP (2.5 mM) plus 8Br-cAMP (10 µM) reactivated the CBF and CBD in the isolated cilia, similar to the cilia of in vivo tracheae. In the reactivated isolated cilia, an elevation in pH from 7.0 to 8.0 increased the CBF from 3 to 15 Hz and the CBD from 0.6 to 1.5 µm. The pH elevation also increased the velocity of the effective stroke; however, it did not increase the recovery stroke, and, moreover, it decreased the intervals between beats. This indicates that H+ (pHi) directly acts on the axonemal machinery to regulate CBF and CBD. In isolated cilia priorly treated with 1 µM PKI-amide (a PKA inhibitor), 8Br-cAMP did not increase the CBF or CBD in the ATP-stimulated isolated cilia. pH modulates the PKA signal, which enhances the axonemal beating generated by the ATP-activated inner and outer dyneins.

Testosterone Enhances KV Currents and Airway Smooth Muscle Relaxation Induced by ATP and UTP through P2Y4 Receptors and Adenylyl Cyclase Pathway.

Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5'-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y2 and P2Y4 receptors and K+ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K+ (KV) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K+ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K+ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). KV channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y2), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K+ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y4 receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of KV1.2 and KV1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y4 signaling and KV channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males.