Mechanisms of ATP release in airway epithelial cells

The mucociliary clearance (MCC) process that removes foreign particles and pathogens is the primary innate defense mechanism in the airways. Major components of MCC, i.e., ion transport, mucin secretion, and ciliary beat frequency, are regulated by extracellular ATP and adenosine, acting on cell surface purinergic receptors. Given the physiological importance of purinergic regulation of MCC activities, the objective of this dissertation was to elucidate signaling elements and pathways relevant for ATP release from airway epithelial cells. The protease activated receptor (PAR) agonist thrombin elicited a rapid Ca2+-dependent release of ATP. In contrast, the P2Y2 receptor agonist UTP caused negligible ATP release, despite promoting a robust Ca2+ response. Thrombin-elicited ATP release was associated with Rho activation, was accompanied by enhanced cellular uptake of the hemichannel fluorescence probe propidium iodide in a Ca2+ - and Rho kinase-dependent manner, and was inhibited by connexin/pannexin hemichannel blockers. These studies suggested that thrombin promotes ATP release from airway epithelial cells via Rho- and Ca2+-dependent activation of connexin/pannexin hemichannels. Similarly to thrombin, hypotonic challenge triggered ATP release, which was accompanied by RhoA activation, MLC phosphorylation, and dye uptake. ATP release and dye uptake in hypotonic challenge-stimulated cells were inhibited by transfecting cells with a dominant negative mutant of RhoA, and by inhibiting or knocking-down pannexin 1. Transient receptor potential 4 (TRPV4) inhibitors reduced RhoA activation, dye uptake, and ATP release. Thus, hypotonic stress-induced ATP release occurs via Rho-dependent pannexin 1 hemichannel opening, and TRPV4 likely transduces osmotic stress into Rho-mediated ATP release. In goblet cells, PAR agonists stimulated the concomitant release of mucins and ATP, which was dependent on intracellular Ca2+-mobilization and cytoskeletal reorganization. Mucin granules contained ATP, but levels of ADP and AMP within granules exceeded those of ATP. Direct release of ADP/AMP from mucin granules likely represents an important source of ASL adenosine, promoting A2b receptor-dependent ion/water secretion necessary for mucin hydration. In sum, this dissertation suggests a major mechanism for ATP release from non-mucous cells, i.e., Rho-dependent pannexin 1 opening. These studies also reveal that PARs promote Ca2+-regulated secretion of ATP/ADP/AMP-rich mucin granules from goblet cells

Thrombin Promotes Release of ATP from Lung Epithelial Cells through Coordinated Activation of Rho- and Ca 2+ -dependent Signaling Pathways

Extracellular ATP controls key aspects of lung function via activation of epithelial cell purinergic receptors, but how ATP is released from cells remains poorly understood. To identify mechanistic components upstream of ATP release, we examined the effect of selected G protein coupled-receptor activation on ATP release from lung epithelial cells. The protease-activated receptor (PAR) agonist thrombin elicited a rapid Ca2+-dependent release of ATP from A549 cells. In contrast, the P2Y2 receptor agonist UTP caused negligible ATP release, despite promoting a robust Ca2+ response. Agonist-elicited ATP release was associated with Rho activation and was reduced in cells transfected with dominant negative mutants of p115-Rho GEF or RhoA, and by inhibitors of Rho kinase (ROCK). However, RhoA activation alone did not promote ATP release if temporally separated from Ca2+ mobilization. PAR3 was the only PAR subtype detected in A549 cells by reverse transcription-PCR. Transfection of cells with human PAR3 cDNA increased thrombin-promoted ATP release, inositol phosphate formation, and RhoA activation. Conversely, small interference RNA against PAR3 diminished thrombin-evoked responses. Thrombin-elicited ATP release was accompanied by an enhanced cellular uptake of propidium iodide in a Ca2+- and ROCK-dependent manner and was inhibited by connexin/pannexin hemichannel blockers. Our data suggest that thrombin promotes ATP release from A549 cells via Rho- and Ca2+-dependent activation of connexin/pannexin hemichannels. The relevance of these findings is highlighted by the observation that exposure of primary cultures of well differentiated human bronchial epithelial cells to thrombin resulted in robust ATP release, which was inhibited by ROCK inhibitors and by connexin/pannexin hemichannel blockers

Inflammation Promotes Airway Epithelial ATP Release via Calcium-Dependent Vesicular Pathways

ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)–associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling–promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca2+ chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca2+-dependent vesicular mechanisms not associated with mucin granule secretion

Rho Signaling Regulates Pannexin 1-mediated ATP Release from Airway Epithelia

ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia

Society of Dermatology Hospitalists supportive care guidelines for the management of Stevens-Johnson syndrome/toxic epidermal necrolysis in adults

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening conditions with high morbidity and mortality. Supportive care management of SJS/TEN is highly variable. A systematic review of the literature was performed by dermatologists, ophthalmologists, intensivists, and gynecologists with expertise in SJS/TEN to generate statements for supportive care guideline development. Members of the Society of Dermatology Hospitalists with expertise in SJS/TEN were invited to participate in a modified, online Delphi-consensus. Participants were administered 9-point Likert scale questionnaires regarding 135 statements. The RAND/UCLA Appropriateness Method was used to evaluate and select proposed statements for guideline inclusion; statements with median ratings of 6.5 to 9 and a disagreement index of ≤1 were included in the guideline. For the final round, the guidelines were appraised by all of the participants. Included are an evidence-based discussion and recommendations for hospital setting and care team, wound care, ocular care, oral care, urogenital care, pain management, infection surveillance, fluid and electrolyte management, nutrition and stress ulcer prophylaxis, airway management, and anticoagulation in adult patients with SJS/TEN

Eccrine squamous syringometaplasia in an allogenic stem cell transplant patient undergoing chemotherapy

Eccrine squamous syringometaplasia (ESS) is a rare finding defined as metaplastic change of the cuboidal epithelial cells of eccrine glands into two or more layers of squamous epithelial cells. We present a patient who developed ESS after induction of CLAG chemotherapy [2-Chlorodeoxyadenosine (2-CdA) with cytarabine (Ara-C) and (granulocyte-colony stimulating factor) G-CSF] for management of the blast crisis of his chronic myelogenous leukemia (CML). Our patient’s ESS eruption presented with a variety of morphologies, thus multiple skin biopsies were taken to determine the possible diagnosis(es). All skin biopsies showed ESS and the eruption resolved with topical corticosteroids after CLAG therapy was finished

Eccrine squamous syringometaplasia in an allogenic stem cell transplant patient undergoing chemotherapy

Eccrine squamous syringometaplasia (ESS) is a rare finding defined as metaplastic change of the cuboidal epithelial cells of eccrine glands into two or more layers of squamous epithelial cells. We present a patient who developed ESS after induction of CLAG chemotherapy [2-Chlorodeoxyadenosine (2-CdA) with cytarabine (Ara-C) and (granulocyte-colony stimulating factor) G-CSF] for management of the blast crisis of his chronic myelogenous leukemia (CML). Our patient’s ESS eruption presented with a variety of morphologies, thus multiple skin biopsies were taken to determine the possible diagnosis(es). All skin biopsies showed ESS and the eruption resolved with topical corticosteroids after CLAG therapy was finished

Preferences Towards Electronically Exchanging Digital Images With Healthcare Providers Among US Adults.

BACKGROUND: The rapid expansion of telemedicine, including teledermatology, during the COVID-19 pandemic has required both providers and patients alike to adapt to this digital transition. However, patient attitudes towards electronically shared images with their providers are poorly understood. To address this gap, we assessed digital image sharing preferences and associated determinants in a nationally representative sample. METHODS: We analyzed pooled data from the Health Information National Trends Survey 4, Cycle 3 and 4. Digital image sharing preferences were compared by patient characteristics and beliefs via chi-square at a significance level of p RESULTS: Among 6437 adults, 53.5% reported reluctance in electronically shared images and videos with providers. Greater aversion was observed among adults aged 75 or above (70.9%), retired (67.3%), and those with lower education (65.1%), lower annual income (60.9%), limited English proficiency (63.3%), distrust in health information from doctors (75.4%), and fair or poor health (60.4%). CONCLUSION: Patient hesitancy towards digital image sharing may present challenges for teledermatology adoption. Greater efforts may be needed to address the age and socioeconomic digital divide, multilingual telemedicine tools, and patient-physician dynamics to ensure vulnerable groups receive needed teledermatologic care