American journal of physiology. Cell physiology最新文献

The bilateral vagus nerves play a critical role in autonomic control and feeding behavior. The left and right vagi innervate different portions of the gastrointestinal tract with recent reports suggesting functional differences between left and right vagal afferents. Vagal afferents originating in the nodose ganglia (NG) detect mechanical and chemical cues, including gut peptides such as cholecystokinin (CCK), which promotes satiation via the CCK1 receptor (CCK1R). Recent work demonstrates that CCK1R-expressing afferents are more abundant and responsive in right NG, but the cellular mechanisms underlying this asymmetry remain unclear. Most CCK1R-containing vagal afferent neurons co-express the cation channel transient receptor potential vanilloid subtype 1 (TRPV1), which contributes to CCK-induced activation of vagal afferent neurons and may explain differences in signaling between the left and right vagal populations. Moreover, the lateralized expression and function of other receptors important to feeding remains to be investigated. Here, we assessed receptor and ion channel expression in left and right NG using NanoString mRNA profiling and examined functional responses to CCK and TRPV1 agonist capsaicin with fluorescent calcium imaging. We found that the left NG showed greater gene expression for TRP channels, including TRPV1, and contained a greater number of TRPV1+ afferents. Meanwhile, right NG had greater expression of the hormone receptor CCK1R and exhibited enhanced calcium responses to CCK. Together, these results identify key differences in receptor/ion channel expression and function between left and right vagal afferents, advancing our understanding of the cellular mechanisms behind lateralized vagal signaling.NEW & NOTEWORTHY The left and right vagus nerves control feeding but innervate different parts of the gastrointestinal tract, suggesting differences between left and right vagal afferents and their signaling. We assessed gene expression and functional responses from left and right vagus nerves. We found the left vagus had greater expression of TRP channels, whereas the right vagus had more hormone receptors and exhibited enhanced responses to CCK. These differences advance our understanding of lateralized vagal signaling.

Children with cerebral palsy (CP) have muscle growth impairments (muscle contractures), altered walking patterns and show markers of inflammation. During muscle repair, macrophages coordinate with muscle stem cells-satellite cells (MuSCs), which have previously been shown to be altered in abundance and function in children with CP. We investigated whether 1) macrophage populations in contractured muscles of children with CP are similar to typically developing (TD) children with a chronic anterior cruciate ligament (ACL)-tear and 2) macrophages, capillaries, MuSC, myonuclei, centrally nucleated fibers were associated with each other, indicative of repair. Thirty-six subjects participated in this study (CP: 11.2 ± 0.7 yr, 18 M/12F, TD: 13.5 ± 0.8 yr, 3 M/3 F). Muscle biopsies were obtained during surgical correction for muscle contractures-adductors/gastrocnemius (CPCon), or vastus lateralis (TD-ACL and CP NonCon). Muscle cross sections were immunohistochemically labeled for total, anti-inflammatory (M2) macrophages, capillaries, myofiber boundaries, whereas MuSC abundance, activation, and proliferation information were used from a prior study. Macrophage subpopulations in CPCon were similar to TD-ACL muscles. Within CPCon, there were positive associations between total, M1 macrophages, and MuSC content (r = 0.54, r = 0.70, P < 0.05, respectively), but not in the CP NonCon muscles. Centrally nucleated fibers, myonuclear abundance, and MuSC content were also positively associated with each other only in the CPCon muscles (r = 0.65, r = 0.46, r = 0.66, P < 0.05, respectively). In TD-ACL injured muscles, similar associations were seen between macrophages and MuSC, central nucleation and myonuclear abundance. Collectively, our data suggest that contractured muscles in children with CP may be in a state of repair, similar to ACL-injured TD children.NEW & NOTEWORTHY Muscle growth impairments seen as muscle contractures are common in children with cerebral palsy. Here we show these muscles appear to have altered macrophage populations, which are similar to joint injury-induced muscle inflammation in typically developing children. Only within contractured muscles, macrophage content, muscle stem cell abundance, capillary density and fiber characteristics are positively associated with each other, indicative of repair, similar to that seen in joint injury-induced muscle inflammation.

Type 2 diabetes is caused by dysfunction of pancreatic β cells. Sympathetic neurons innervate pancreatic β cells and noradrenaline inhibits insulin secretion and proliferation of pancreatic β cells in vitro. Previously, we have generated a genetically engineered mice exhibiting inducible and sympathetic neuron-selective loss of the tyrosine hydroxylase gene (Th-cKO mice), resulting in approximately 70% decline of pancreatic noradrenaline levels. In this study, we investigated the effects of sustained and downregulated sympathetic noradrenergic signaling on the pancreatic β cells using Th-cKO mice. Intraperitoneal glucose tolerance test revealed higher glucose tolerance in Th-cKO mice than the control mice without any difference in insulin tolerance test. Th-cKO mice also exhibited higher circulating insulin levels under glucose challenge. We also found that proliferative activity of pancreatic β cells increased in Th-cKO mice. These results indicate that partial and sustained suppression of sympathetic noradrenaline signaling could enhance insulin secretion and proliferative activity of pancreatic β cells. In addition, we found that Th-cKO mice showed partial alleviation of streptozotocin-induced hyperglycemia accompanied by increased β-cell proliferative response. Our data suggest that sympathetic noradrenaline synthesis can be a potential therapeutic target for diabetes.NEW & NOTEWORTHY In this paper, we found that inducible and partial reduction of noradrenaline synthesis in the sympathetic neurons increased circulating insulin levels under glucose challenges and upregulated proliferative activity of pancreatic β cells in mice. Moreover, inducible downregulation of sympathetic noradrenaline signaling also partially attenuated streptozotocin-induced hyperglycemia. These results suggest that sympathetic noradrenaline signaling should control insulin secretion and proliferative activity of β cells and that sympathetic noradrenaline synthesis can be a therapeutic target for diabetes.

Astrocytes play a crucial role in maintaining central nervous system homeostasis, supporting neuronal function and regulating oxidative stress. The placenta, through the secretion of small extracellular vesicles (sEVs), facilitates communication between the maternal and fetal environments, potentially mitigating external stressors. Bisphenol A (BPA), an endocrine disruptor, has been implicated in oxidative stress and mitochondrial dysfunction, particularly in the developing brain. However, the mechanisms by which placental sEVs influence astrocyte responses to BPA remain unclear. This study investigates the effects of BPA on astrocyte oxidative stress and mitochondrial activity and explores how placental sEVs modulate these responses. Human glioblastoma astrocytoma (U-373 MG) cells were exposed to environmentally relevant concentrations of BPA (10 nM), with or without placental sEVs isolated from human term placental explants. Reactive oxygen species (ROS) levels, mitochondrial activation, and antioxidant enzyme expression (SOD1, GCLC, and GSTA) were assessed. Direct BPA exposure increased astrocyte ROS levels and mitochondrial activation, indicative of oxidative stress. Placental sEVs were rapidly internalized by astrocytes and counteracted BPA-induced ROS accumulation, restoring mitochondrial homeostasis. Notably, sEVs from BPA-exposed placental explants were more efficiently incorporated into astrocytes, suggesting an adaptive response. sEVs treatment also upregulated antioxidant enzyme expression and reduced inflammatory cytokine markers (CCL2 and IL-1β), indicating a potential protective mechanism. These findings suggest that placental sEVs play a critical role in modulating astrocyte responses to oxidative stress and mitochondrial dysfunction. The ability of sEVs to restore redox homeostasis highlights their potential physiological function in fetal neuroprotection against environmental stressors.NEW & NOTEWORTHY The study demonstrates that BPA induces oxidative stress and mitochondrial dysfunction in human astrocytes. It introduces a novel role of sEVs in counteracting these effects by reducing ROS, restoring mitochondrial activity, and upregulating antioxidant enzymes. Notably, sEVs from BPA-exposed placental explants were more efficiently incorporated into astrocytes, suggesting an adaptive protective mechanism. These findings highlight a potential fetal neuroprotective role of placental sEVs against environmental stressors.

Cardiovascular homeostasis is an adaptive process shaped by the interplay between the nervous system and the heart. Emerging evidence demonstrates that immune cells integrate within and across these signaling pathways to modulate inflammatory responses to injury. This neuroimmune interface has a fundamental role in cardiovascular homeostasis and, when dysregulated, may contribute to the pathogenesis of several cardiovascular diseases. Despite its importance, research on the neuroimmune mechanisms in cardiovascular disease is scarce, possibly because it stands upon an intersection of two traditionally separate fields: immunology and neuroscience. This review provides a comprehensive overview of the role of autonomic nervous system within the brain-heart axis, with a focus on signaling pathways that regulate immune function. We detail the bidirectional afferent and efferent connections among central autonomic centers, cardiovascular, and immune tissues in maintaining homeostasis. Finally, we discuss how these neuroimmune circuits are altered in three major cardiovascular diseases, representative of both low-degree chronic inflammation and autonomic dysfunction: heart failure with preserved ejection fraction, hypertension, and atrial fibrillation. Our synthesis of current literature highlights the necessity for a deeper understanding of neuroimmune interactions. Advancing this knowledge may be crucial for developing targeted therapies that improve patient outcomes.

TMEM175 is an AKT-activated lysosomal potassium- and proton-permeable channel that functions to dissipate voltage and pH gradients generated by the V-type H⁺-ATPase. Loss-of-function variants in TMEM175 have been identified as genetic risk factors for Parkinson's disease, highlighting the potential of small-molecule activators as a novel therapeutic strategy for this disease. We developed a high-throughput screening (HTS) assay using HEK-293 cells stably overexpressing TMEM175 at the cell surface and screened 960 Food and Drug Administration (FDA)-approved drugs for TMEM175 potentiators. The screen identified 71 activators, including the cysteinyl leukotriene 1 receptor (CysLT1R) antagonists, pranlukast and montelukast. Because HEK-293 cells lack CysLT1R expression, we suspected these drugs may be direct channel activators. Fluorescence and automated patch-clamp assays were used to evaluate the dose dependency of pranlukast, montelukast, zafirlukast, and the known TMEM175 activator, (2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB). These experiments revealed rank-order potencies and efficacies of DCPIB ∼ zafirlukast > montelukast ≫ pranlukast. DCPIB, zafirlukast, and pranlukast activated TMEM175 independently of AKT activation, whereas the AKT inhibitor MK2206 partially inhibited montelukast-dependent TMEM175 activation. Computer modeling revealed a conformation-dependent solvent-accessible cavity near T119 and H449 that could participate in drug-induced activation, prompting us to examine these sites with mutagenesis. Not only did T119A and H449A mutations decrease apparent potencies of DCPIB, zafirlukast, and montelukast, but the T119A mutation produced a constitutively open channel phenotype. This study adds zafirlukast to the short list of moderately potent TMEM175 activators and identifies a region of the channel that contributes to activation gating.NEW & NOTEWORTHY TMEM175 regulates lysosomal voltage and pH and is genetically linked to Parkinson's disease. Our study identifies zafirlukast and related drugs as activators, defines their AKT dependency, and maps structural determinants of gating. These results open new avenues for targeting TMEM175 in neurodegenerative diseases.

Macrophages are critical cellular mediators within the innate immune system and are the central effectors of chronic inflammation at the cellular level. Here, macrophages regulate the ongoing, simultaneous processes of tissue inflammation, destruction, and repair. They also play an integral role in recruiting key cell types within the inflammatory and wound healing response. Cancer is a chronic inflammatory state and is largely considered a wound that does not heal. As in wound healing, where macrophages engulf and/or destroy foreign insults, macrophages have the potential to also eliminate tumor cells. However, it is now well known that these early proinflammatory, antitumor responses by macrophages are nullified as macrophages repolarize into protumor, anti-inflammatory tumor-associated macrophages (TAMs) in response to tumor cell and microenvironmental-derived factors. After this point, TAMs drive neoplastic progression in multiple distinct ways. This indirect control of tumor progression, where TAMs share great functional overlap with the direct control elicited by neoplastic cells, supports TAMs being central orchestrators and later conductors of the tumor microenvironment (TME)-the focus of our review.

Cardiac Ca²⁺ release channels, type-2 ryanodine receptors (RyR2s), play a pivotal role in cardiac muscle contraction by releasing Ca²⁺ from the sarcoplasmic reticulum. Over 200 missense mutations in humans have been reported to be associated with cardiac diseases. Here, we characterize three RyR2 variants, Q3925E, W4646R, and Q4937K. Q3925E and W4646R mutations are in the Ca²⁺- and caffeine-binding sites, respectively. Our molecular dynamics simulations predicted that the Q4937 residue in the carboxyl terminal domain forms a hydrogen bond with the central domain where the Ca²⁺-binding site is located. Three mutant RyR2s were expressed in heterologous cells, and activities of the recombinant mutant RyR2 channels were determined by [³H]ryanodine binding methods. As expected, Q3925E greatly reduced Ca²⁺-dependent activation and W4646R abolished caffeine activation. Our novel finding is that Q3925E increased inhibitory effects by divalent cations, Ca²⁺ and Mg²⁺, resulting in a strong loss-of-function phenotype. Both W4646R and Q4937K increased affinities for Ca²⁺ activation, and reduced or unchanged Ca²⁺ inhibitions, exhibiting typical gain-of-function phenotypes. Caffeine failed to activate the Q3925E mutant at resting Ca²⁺ but restored its activation at ∼20 µM Ca²⁺, where the Q3925E mutant is in the subactivated state. Computational analysis of the mutated structures suggested that the Q3925E mutation does not reduce Ca²⁺ binding to its site but rearranges domain interface between the central domain involving Ca²⁺-binding site and carboxyl terminal domain, which directly interacts with the channel pore. Thus, it is possible that the Q3925E-RyR2 mutation alters signal transmission between activating Ca²⁺ binding and pore opening.NEW & NOTEWORTHY Q3925E in RyR2 is a part of Ca²⁺-binding site and is known to associate with cardiac sudden death. Our functional and structural modeling data suggested that the Q3925E mutation does not reduce Ca²⁺ binding but alter a domain interaction, causing an impaired Ca²⁺ activation of RyR2. We also found that the Q3925E mutation increases channel inhibition by Mg²⁺ and Ca²⁺, resulting in a strong loss-of-function phenotype.

Volume-regulated anion channels (VRACs) are ubiquitous chloride channels that play important roles in cell volume regulation and numerous other physiological processes. VRACs are heteromeric complexes composed of leucine-rich repeat-containing proteins LRRC8A-E. LRRC8 subunit composition determines biophysical properties of VRACs, including permeability to small signaling molecules. Here, we used primary astrocyte cultures from wild-type and genetically modified C57BL/6 mice to investigate 1) subunit composition of native VRACs in the brain and 2) subunit determinants of VRAC permeability to the excitatory neurotransmitter glutamate. Quantitative real-time PCR (qRT-PCR) and RNA-seq analyses revealed high expression of Lrrc8a-d in mouse forebrain and astrocytes. Genetic deletion of the essential LRRC8A protein abolished swelling-activated glutamate release, measured as efflux of the nonmetabolizable d-[³H]aspartate, confirming the crucial role of VRACs in this process. RNAi-mediated knockdown of individual subunits identified LRRC8A and LRRC8C as key components of glutamate-permeable astrocytic VRACs. qRT-PCR and Western blot analyses further showed that knockdown of LRRC8A or LRRC8C reciprocally altered the protein stability of the partner subunit without affecting their mRNA levels. A similar pattern of mutual regulation was observed between LRRC8A and LRRC8D. In contrast to LRRC8C, downregulation of LRRC8D had a more limited impact on glutamate release. Additional double-knockdown experiments demonstrated that LRRC8C- and LRRC8D-containing channels form distinct VRAC populations. This model was further supported by Western blot results showing no reciprocal regulation of LRRC8C and LRRC8D stability. Together, these findings refine our understanding of how the subunit organization of native brain VRACs governs gliotransmitter release, with implications for normal brain function and neurological disease.NEW & NOTEWORTHY Volume-regulated anion channels (VRACs) are ubiquitously expressed chloride channels composed of LRRC8A-E proteins. In human disorders, gain or loss of VRAC function leads to severe neurological phenotypes, potentially due to altered release of amino acid neurotransmitters. Here, we show that glutamate-permeable VRACs in brain astrocytes are primarily composed of LRRC8A and LRRC8C proteins. These findings provide insight into subunit organization of native VRACs in the CNS, with implications for normal brain function and neurological disease.

Vasopressin regulates transcription of the aquaporin-2 gene (Aqp2) in collecting duct principal cells. To investigate regulatory mechanisms in Aqp2 gene transcription, we engineered an Aqp2 reporter cell line using CRISPR/Cas9 to insert a green fluorescent protein (GFP) cassette at the endogenous Aqp2 gene locus in mpkCCD cells. In the absence of dDAVP (1-desamino-8-D-arginine-vasopressin), a vasopressin analog, these cells exhibited low or undetectable GFP and Aqp2 expression in all cells. dDAVP stimulation (1 nM dDAVP for 48 h) markedly increased both GFP and Aqp2 expression together with reversal upon dDAVP removal. These observations demonstrate that GFP faithfully tracks Aqp2 expression. Interestingly, fewer than 50% of cells express GFP and Aqp2 after dDAVP or forskolin, indicating significant variability even though they were clonally derived. We flow-sorted the GFP^- cells (Aqp2^-) and GFP⁺ cells (Aqp2⁺), regrew them, and restimulated them separately with dDAVP. Cells originating from GFP^- cells gave rise to both GFP^- cells and GFP⁺ cells, and GFP⁺ cells similarly regenerated both GFP^- and GFP⁺ populations in the same proportion. Flow cytometry analysis of the DNA content showed variability in cell cycle phases, with most GFP⁺ cells in G0/G1, and most GFP^- cells in G2/S. RNA-seq analysis of the GFP^- and GFP⁺ cells revealed increased abundance of cell cycle-related transcripts in the GFP^- cells. We conclude that: 1) heterogeneity in Aqp2 expression is related to cell cycle state and 2) the newly generated reporter cell line will likely serve as a useful tool to study Aqp2 transcriptional regulation.NEW & NOTEWORTHY To investigate regulatory mechanisms in Aqp2 gene transcription, we engineered an Aqp2 reporter cell line using CRISPR/Cas9 to insert a green fluorescent protein (GFP) cassette at the endogenous Aqp2 gene locus in mpkCCD cells. We demonstrate that the GFP reporter accurately and dynamically tracks the expression and regulation of endogenous Aqp2. We reveal that Aqp2 heterogeneity in mpkCCD cells is at least partly driven by differences in cell cycle phase.