American journal of physiology. Lung cellular and molecular physiology最新文献

Intracellular calcium (Ca²⁺) release via phospholipase C (PLC) following G-protein-coupled receptor (GPCR) activation is typically linked to membrane depolarization and airway smooth muscle (ASM) contraction. However, recent findings show that bitter taste receptor agonists, such as chloroquine (CQ), induce a paradoxical and potent relaxation response despite activating the Ca²⁺ signaling pathway. This relaxation has been hypothesized to be driven by a distinct compartmentalization of calcium ions toward the cellular periphery, subsequently leading to membrane hyperpolarization, in contrast to the contractile effects of histamine. In this study, we further investigate the spatiotemporal dynamics of Ca²⁺ signaling in ASM cells using single-cell microscopy and deep learning-based segmentation, integrating the results into a comprehensive model of ASM ion channel dynamics to compare the effects of histamine, CQ, and flufenamic acid (FFA). Our results show that histamine induces a strong, synchronized calcium release, nearly twice as high as that of CQ, which produces a sustained but lower-magnitude response. Per-cell analysis reveals more variable and asynchronous Ca²⁺ signaling for CQ and FFA, with higher entropy compared with histamine. Integrating these findings into an ASM ion channel model, we observed that histamine-mediated Ca²⁺ release activates voltage-gated Ca²⁺ and Na⁺ channels (leading to depolarization). In contrast, CQ preferentially engages BKCa, SKCa, and chloride channels (promoting hyperpolarization). These findings provide insights into the unique mechanisms by which bitter taste receptor agonists can modulate ASM tone, offering potential therapeutic strategies for relaxing ASM and alleviating airway hyperresponsiveness in conditions such as asthma.NEW & NOTEWORTHY Using machine-learning methods, these studies identify spatiotemporal differences in calcium responses between agonists of Gq-coupled receptors and bitter taste receptors in airway smooth muscle cells. The findings provide deeper insights into the mechanism of action of bitter tastant-induced airway smooth muscle relaxation.

Premature infants are at higher risk for developing chronic lung diseases, especially following supplemental oxygen (hyperoxia) in early life. We previously demonstrated that moderate hyperoxia (<60% O₂) induces cellular senescence in fetal airway smooth muscle cells (fASM) and fibroblasts. However, the mechanisms underlying O₂-induced senescence are still under investigation. In this study we investigated the role of endoplasmic reticulum (ER) stress and mitochondrial dysfunction, using fASM cells exposed to 21% O₂ (normoxia) vs. ∼50% O₂ (hyperoxia). Normoxia or hyperoxia-exposed fASM were treated with the ER stress inhibitor salubrinal [12.5 μM], the antioxidant MitoQ [100 nM], or the mitochondrial fission inhibitor Mdivi-1 [10 μM]. Samples were harvested at day 2, 3, and 7 and analyzed for markers of senescence, oxidative stress, ER stress response, and mitochondrial dynamics using protein analysis and fluorescence microscopy. Hyperoxia enhanced senescence, upregulating multiple markers of DNA damage in particular, cyclin-dependent cell cycle regulator p21, cytosolic and mitochondrial reactive oxygen species (ROS) levels, mitochondria fragmentation, and anti-apoptosis B-cell lymphoma-extra large (Bcl-xL), while downregulating the proliferation marker Ki-67. Hyperoxia also activated all three ER stress pathways. However, the level of p21 and/or Bcl-xL was decreased in hyperoxia-exposed cells treated with the ER stress inhibitor salubrinal or the antioxidant MitoQ, but not the fission inhibitor Mdivi-1. These findings highlight the role of mitochondrial ROS and ER stress in hyperoxia-induced senescence of fASM and suggest that mitochondrial-targeted antioxidants and/or inhibitors of ER stress pathways can blunt the detrimental effects of hyperoxia in developing lungs.NEW & NOTEWORTHY Supplemental O₂ (hyperoxia) in premature infants detrimentally affects bronchial airways leading to increased senescence. Understanding the mechanisms by which hyperoxia initiates senescence in developing airways is critical for future therapeutic strategies. The current study showed that hyperoxia-induced senescence is mediated through increased mitochondrial reactive oxygen species and endoplasmic reticulum (ER) stress. ER stress inhibitors or mitochondria-targeted antioxidants may represent future therapies to blunt detrimental effects of supplemental oxygen in developing lungs.

The administration of opioid receptor antagonists is believed to overcome ventilatory depressant effects of opioids. Here we show that many ventilatory depressant effects of morphine are converted to excitatory responses after µ₁-opioid receptor blockade, and that these responses are accompanied by ventilatory instability. In this study, we report 1) ventilatory responses elicited by morphine (10 mg/kg, iv) and 2) ventilatory responses elicited by a subsequent hypoxic-hypercapnic (HH) gas challenge and return to room air in male Sprague Dawley rats pretreated with 1) vehicle, 2) the centrally acting selective µ₁-opioid receptor antagonist, naloxonazine (1.5 mg/kg, iv), or 3) the centrally acting (delta 1,2) δ_1,2-opioid receptor antagonist, naltrindole (1.5 mg/kg, iv). The morphine-induced decreases in frequency of breathing, peak inspiratory flow, peak expiratory flow, expiratory flow at 50% expired TV, inspiratory drive, and expiratory drive in vehicle-treated rats were converted to profound increases in naloxonazine-treated rats. Additionally, the adverse effects of morphine on expiratory delay and apneic pause were augmented in naloxonazine-treated rats, and administration of morphine increased ventilatory instability (i.e., noneupneic breathing index) in naloxonazine-treated rats, which was not due to increases in ventilatory drive. Subsequent exposure to a HH gas challenge elicited qualitatively similar responses in both groups, whereas the responses upon return to room air (e.g., frequency of breathing, inspiratory time, expiratory time, end expiratory pause, relaxation time, expiratory delay, and noneupneic breathing index) were substantially different in naloxonazine-treated versus vehicle-treated rats. The above mentioned effects of morphine were only marginally affected in naltrindole-treated rats. These novel data highlight the complicated effects that µ₁-opioid receptor antagonism exerts on the ventilatory effects of morphine.NEW & NOTEWORTHY This study shows that the systemic injection of morphine elicits a pronounced overshoot in ventilation in freely-moving Sprague Dawley rats pretreated with the centrally-acting selective µ₁-opioid receptor antagonist, naloxonazine, but not with the centrally-acting δ_1,2-opioid receptor antagonist, naltrindole. This suggests that morphine can recruit a non-µ₁-opioid receptor system that promotes breathing.

Bronchopulmonary dysplasia (BPD) is a common chronic lung disease in pediatrics. Neonatal mice placed in hyperoxia (85% oxygen, HYP) develop lung injury reminiscent of BPD. We tested the hypothesis that mice deficient in arginase-2 (Arg2KO) exposed to HYP would have attenuated lung inflammation and injury compared with similarly exposed wild-type mice. Arg2KO and C57BL/6 (WT) mice were placed in either room air (NORM) or HYP on postnatal day 0 (P0) and exposed for up to 14 days. RNAseq data on P1 and P14 showed that HYP differentially upregulated genes, particularly those related to development and inflammation, between the two genotypes. Neonatal mice exposed to HYP had evidence of alveolar simplification at P7 and P14, which was slightly attenuated in Arg2KO mice. After 14 days in HYP, mice were moved to NORM, and methacholine challenge testing was performed at 6, 8, or 12 wk of age. WT mice exposed to neonatal hyperoxia showed greater methacholine-induced respiratory system resistance (R_RS) at 6 and 8 wk of age compared with WT mice exposed to NORM. The methacholine-induced increase in R_RS in Arg2KO mice exposed to neonatal hyperoxia was not different from normoxia-exposed mice of either genotype. At 6, 8, and 12 wk, alveolar simplification was evident in both WT and Arg2KO mice exposed to neonatal hyperoxia with no differences between genotypes. These data demonstrate that Arg2KO attenuated both the hyperoxia-induced lung inflammation at P1 and P14 and the airway hyperreactivity at 6 and 8 wk of age.NEW & NOTEWORTHY Our findings suggest that inhibiting arginase 2 may be a potential therapeutic target for mitigating short-term and long-term adverse outcomes related to airway reactivity in bronchopulmonary dysplasia (BPD) that deserves further study. Furthermore, our results suggest that airway reactivity and lung architecture may be differentially regulated in neonates and may require specific and different targeting to prevent the specific outcome in neonates at risk for developing BPD.

Cystic fibrosis (CF) is a chronic disease caused by dysfunctional or absent cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is expressed in immune cells and regulates innate immunity, both directly and indirectly. The epithelial sodium channel (ENaC) contributes to dysfunction in CF airway epithelial cells. However, the impact of non-CFTR ion channel dysfunction on CF immune responses is not understood. Improved understanding of how immune function is regulated by ion channels may allow antibiotic- and mutation-agnostic treatment approaches to chronic infection and inflammation. Therefore, we hypothesized that ENaC is aberrantly expressed in CF macrophages and directly contributes to impaired phagocytic and inflammatory functions. ENaC expression was characterized in immune cells isolated from CF and non-CF blood donors. Monocyte-derived macrophage (MDM) function and bacterial killing were tested with ENaC modulation. Baseline ENaC expression in human CF MDMs, lymphocytes, and granulocytes was increased at both the transcript and protein level relative to non-CF and persisted after infection. CFTR inhibition in non-CF MDMs resulted in ENaC overexpression. CFTR modulator treatment reduced but did not eliminate ENaC overexpression in CF MDMs. Interestingly, ENaC inhibition increased CFTR expression. Amiloride-treated CF MDMs also showed normalized reactive oxygen species (ROS) production, improved autophagy, and decreased proinflammatory cytokine production. Sodium channel expression in CF MDMs normalized after amiloride treatment with minimal effect on other ion channels. In summary, ENaC modulation in immune cells is a novel potential therapeutic target for CF infection control, either in combination with CFTR modulators, or as a sole agent for people not eligible for CFTR modulators.NEW & NOTEWORTHY New research reveals that epithelial sodium channel (ENaC) overexpression in cystic fibrosis (CF) immune cells impairs macrophage function. Inhibiting ENaC increases cystic fibrosis transmembrane conductance regulator (CFTR) expression, normalizes reactive oxygen species production, improves autophagy, and reduces proinflammatory cytokine production. This suggests that ENaC modulation could be a novel therapeutic target for CF infection control, either alone or with CFTR modulators, offering new hope for patients not eligible for current treatments.

In patients with chronic obstructive pulmonary disease (COPD), lung-tissue regenerative mechanisms are thought to be exhausted, to which cellular senescence may contribute. Lung-derived mesenchymal stem/stromal cells (LMSCs) constitute a potent supportive cell type able to self-renew and promote alveolar regeneration. We hypothesized that LMSCs are less sensitive to senescence induction in COPD than other supportive cells, for example, lung fibroblasts (LFs), and therefore more promising in regenerative strategies. We compared senescence markers in LMSCs and LFs from the same subjects with/without replicative- and stress-induced senescence. LMSCs and LFs were isolated from COPD and non-COPD lung tissue using cell-specific protocols and expanded for multiple passages under the same culture conditions. Proliferation, senescence-associated β-galactosidase (SA-β-gal) activity, expression of senescence markers (CDKN2A/P16, CDKN1A/P21, and LMNB1), P21 protein levels, secretion of senescence markers (IL-6 and IL-8), and alveolar growth factors [hepatocyte growth factor (HGF) and fibroblast growth factor 10 (FGF10)] were assessed in the absence/presence of paraquat (PQ). We observed higher population doublings, and lower SA-β-gal positive cells and P21 protein levels in LMSCs compared with LFs at baseline. COPD-derived LFs had lower population doublings and higher cellular size than controls, which was not observed for COPD-derived LMSCs. LMSCs displayed lower sensitivity to PQ-induced senescence compared with LFs (COPD and control combined). Senescence induction was accompanied by increased IL-6 and IL-8 secretion, to which fibroblasts were more sensitive, and by reduced FGF10 but not HGF expression in both cell types. This study demonstrates that LMSCs have lower levels of senescence and lower sensitivity toward senescence induction compared with LFs, affecting cell expansion and FGF10 expression. This suggests that LMSCs are better suited for cell-based therapies.NEW & NOTEWORTHY We demonstrate that LMSCs are less sensitive to senescence induction by oxidative stress and replication than LFs, which was accompanied by an increased ability to expand. This makes LMSCs more suitable for cell-based therapies in COPD. As senescence affected growth factors involved in alveolar repair, specifically FGF10 expression in both LMSCs and LFs, we additionally suggest that the development of anti-senescence strategies may promote endogenous tissue repair in COPD.

It is well known that exogenous opioids such as morphine and fentanyl can depress breathing by inhibiting brainstem breathing control circuit activity. However, the role of endogenous opioids in breathing control is less clear. Endogenous opioid peptides and opioid receptors are expressed within the embryonic brainstem at the same time as when respiratory rhythm-generating neurons begin to mature. However, the extent to which endogenous opioids participate in respiratory control maturation is not known. Therefore, our goal is to review the current state of knowledge for the role of endogenous opioids in breathing control development. We set the stage by reviewing how endogenous opioid peptides regulate breathing in young and adult mammals. We describe the prenatal and postnatal development of endogenous opioid peptides and receptors in relation to breathing development. In addition, we review the effects of exogenous opioids on breathing during early life and identify areas in need of further study. We also broadly describe pain circuitry development to compare the opioid influence on nociception with how opioids impact breathing. We map the locations of endogenous opioid peptide production in the adult and developing brainstem respiratory network. Last, we propose clinical breathing conditions that may involve the endogenous opioid system. Given advances in tools for detecting endogenous opioid peptide release and the evidence reviewed herein, future research will yield new discoveries in the role of endogenous opioids in breathing across the lifespan.

Endothelial-to-mesenchymal transition (EndMT) is a biological process that converts endothelial cells to mesenchymal cells with increased proliferative and migrative abilities. EndMT has been implicated in the development of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH), a fatal and progressive lung vascular disease. Transforming growth factor β₁ (TGF-β₁), an inflammatory cytokine, is known to induce EndMT in many types of endothelial cells including lung vascular endothelial cells (LVECs). An increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) is a major stimulus for cellular proliferation and phenotypic transition, but it is unknown whether Ca²⁺ signaling is involved in EndMT. In this study, we tested the hypothesis that TGF-β₁-induced EndMT in human LVEC is Ca²⁺-dependent. Treatment of LVEC with TGF-β₁ for 5-7 days resulted in increase in SNAI1/2 expression, induction of EndMT, upregulation of STIM/Orai1, and enhancement of store-operated Ca²⁺ entry (SOCE). Removal (or chelation) of extracellular or intracellular Ca²⁺ with EGTA or BAPTA-AM, respectively, abolished EndMT in response to TGF-β₁. Moreover, EGTA diminished TGF-β₁-induced increase in SNAI in a dose-dependent manner. Knockdown of either STIM1 or Orai1 was sufficient to prevent TGF-β-mediated increase in SNAI1/2 and EndMT but did not rescue the continuous adherent junctions. Blockade of Orai1 channels by AnCoA4 inhibited TGF-β-mediated EndMT and restored PECAM1-positive continuous adherent junctions. In conclusion, intracellular Ca²⁺ signaling plays a critical role in TGF-β-associated EndMT through enhanced SOCE and STIM1-Orai1 interaction. Thus, targeting Ca²⁺ signaling pathways regulating EndMT may be a novel therapeutic approach to treat PAH and other forms of precapillary pulmonary hypertension.NEW & NOTEWORTHY EndMT has been reported to contribute to the pathogenesis of PAH. In this study, we aimed to determine the role of Ca²⁺ signaling in the development of EndMT in human lung vascular endothelial cells. Our data suggest that TGF-β₁ requires store-operated Ca²⁺ entry through STIM1/Orai channels to induce SNAI-mediated EndMT. For the first time, we demonstrated that TGF-β₁-induced EndMT is a Ca²⁺-dependent event, whereas inhibition of STIM1/Orai interaction attenuated EndMT in response to TGF-β₁.

Dysregulated redox signaling contributes to pulmonary hypertension (PH) and vascular depletion of the redox enzyme extracellular superoxide dismutase (EC-SOD) from smooth muscle cells [EC-SOD SMC knockout (KO)] worsens chronic hypoxic PH. Given the important role of macrophages in PH, this study aimed to determine if interstitial macrophages (IMs) and their interactions with hyaluronan (HA), a component of extracellular matrix (ECM), are modulated by vascular EC-SOD. Floxed wild-type, EC-SOD SMC KO, and SOD mimetic- or vehicle-treated mice were exposed to hypobaric hypoxia [∼10% fraction of inspired oxygen ([Formula: see text])], for 4, 14, or 21 days. Using flow cytometry, we demonstrated that the transient increase in IMs at day 4 was exacerbated in EC-SOD SMC KO mice and prevented with SOD mimetic pretreatment. Highlighting the importance of targeting vascular oxidative stress in the early response to hypoxia, pretreatment with a single dose of EC-SOD mimetic decreased right ventricular systolic pressure, right ventricular hypertrophy, and small vessel muscularization at day 21. To assess IM phenotypic reprogramming in hypoxia, RNA-seq was performed on flow-sorted IMs revealing baseline proinflammatory activation and enhanced activation of vascular and ECM remodeling pathways in response to hypoxia in EC-SOD SMC KO IMs compared with controls. To further investigate the ECM remodeling response, we quantified IMs expressing the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), and IM-hyaluronan binding. Lyve1⁺ IMs and Lyve1⁺ HA⁺ IMs were increased in response to hypoxia in EC-SOD SMC KO mice and accumulated in the perivascular space of the lung. In conclusion, vascular EC-SOD limits IM accumulation and proinflammatory profibrotic IM signaling, including perivascular accumulation of Lyve1⁺ IMs and their binding to hyaluronan.NEW & NOTEWORTHY Expression of the redox enzyme EC-SOD limits PH severity. Using vascular-selective EC-SOD depletion and SOD mimetic treatment in chronic hypoxic PH, we demonstrated that EC-SOD limits the hypoxia-induced accumulation of IMs. IMs from mice with low vascular EC-SOD were proinflammatory at baseline and enhanced ECM remodeling pathway activation in response to hypoxia. We identified Lyve1⁺ IMs as a perivascular, ECM-interacting subset that accumulate in hypoxia and could contribute to vascular remodeling in PH.

Type 2 inflammation and epithelial-to-mesenchymal transitions (EMTs) play critical roles in airway repair after damage from allergens or parasites. The matricellular protein periostin (POSTN) has increased expression in inflammatory conditions and has been implicated in fibrosis and EMT, suggesting a role in airway repair. This study investigates the role of periostin in airway epithelial and lung fibroblast wound repair using an in vitro wound model. Our results demonstrate that the type 2 cytokine IL-13 induces periostin secretion from primary human airway epithelial basal cells. Periostin knockdown in human airway epithelial cells (HAEs) and human lung fibroblasts (HLFs) impairs wound closure, indicating that periostin is required for airway repair. In a coculture model of HAE and HLFs, fibroblast-secreted POSTN is required for airway epithelial wound repair, suggesting that periostin is involved in paracrine signaling between the two cell types. These findings highlight periostin's critical function in epithelial and fibroblast-mediated wound repair, suggesting its potential as a therapeutic target for diseases characterized by aberrant wound healing and fibrosis, such as asthma and idiopathic pulmonary fibrosis.NEW & NOTEWORTHY This article highlights the critical role of periostin (POSTN) in airway epithelial and fibroblast-mediated wound repair. Moreover, the study reveals a paracrine signaling loop between airway epithelial basal cells and lung fibroblasts, emphasizing periostin's therapeutic potential for diseases like asthma and idiopathic pulmonary fibrosis.