American journal of physiology. Cell physiology最新文献

Cancer-associated cachexia decreases the quality of life, reduces therapy response, and diminishes survival prospects. Effective cachexia countermeasures remain a significant unmet need. Research into cancer cachexia has made extensive use of models of colon, lung, and pancreatic cancers. However, although cachexia also affects people with metastatic breast cancer, the mechanisms underlying breast cancer-associated cachexia are relatively understudied. Thus, we sought to investigate orthotopic mouse models of metastatic breast cancer for the progression of cachexia, with a focus on muscle wasting given its role in the frailty that is a hallmark of the condition. Female Balb/c mice received an intramammary fat pad injection of 4T1.2 or EMT6.5 cells, and NOD.SCID.γ (NSG) mice received MDA-MB-231-HM (231-HM) cells, to induce primary breast tumors that were subsequently excised. The resultant metastatic burden after approximately 4 wk led to variable loss of muscle mass (tibialis anterior: EMT6.5: -17.1%, 231-HM: -13.5%, and 4T1.2: -9.5%) and fat mass (gonadal fat: EMT6.5: -75.1%, 231-HM: -62.5%, and 4T1.2: -30.2%). Muscle protein synthesis markers were decreased in EMT6.5 tumor-bearing mice. Distinct increases in the abundance of mRNA for E3-ubiquitin ligase and autophagy-related genes were observed between models. Neuromuscular junction perturbations were observed in EMT6.5 and 4T1.2 tumor-bearing mice. Neutrophilia was noted in the muscles of EMT6.5 tumor-bearing mice. The findings show that muscle mass and function are reduced in mouse models of metastatic breast cancer. Further study of these models could provide useful insights with which to better understand the diversity of cachexia progression across different cancer types.NEW & NOTEWORTHY Cancer-associated cachexia is a significant contributor to poor prognosis for many cancer patients. However, mechanisms underlying cachexia associated with breast cancer are relatively understudied. This project examined mouse models of metastatic triple-negative breast cancer and found that different models exhibited varying degrees of cachexia severity, which were associated with distinct effects upon markers of protein synthesis and breakdown. Further study of these models could help to better understand the diversity of cachexia across cancer types.

Extracellular vesicles (EVs), composed of proteins, lipids, and nucleic acids, are key mediators of intercellular communication across various tissues. However, research on EVs isolated directly from bone tissue remains limited. Here, we identified for the first time and characterized two EV subpopulations extracted from bone tissue and enhanced our understanding of their roles in bone physiology. These two EV subpopulations were reproducibly isolated: a large EV population (lEV; 237.7 ± 8.8 nm) and a small EV population (sEV; 109.2 ± 8.3 nm), both exhibiting the expected shape and presence of EV and bone cell markers. Comparative analysis of their cargos revealed unique or enriched proteins and miRNA profiles for each, suggesting shared functional characteristics with bone cells, including osteocytes, osteoblasts, and osteoclasts. Notably, lEVs contained proteins such as FHL2 and pleiotrophin, along with miRNAs including miR-15b-5p, miR-29a-3p, and miR-128-3p, all of which are involved in early osteogenic signaling pathways such as Hippo, TGF-β, and Wnt. Furthermore, sEVs contained ALP, PAPSS2, and miR-125b, both known regulators of matrix mineralization. Both EV subpopulations were internalized by stromal ST2 and preosteoblastic MC3T3-E1 cells. We present evidence that lEVs significantly enhanced ALP activity in ST2 cells, indicating early osteogenic stimulation, whereas sEVs partially promoted matrix mineralization in primary osteoblasts. Our findings provide novel insights into the role of bone-derived EVs as possible complementary mediators of osteogenesis in vivo and highlight the potential of their cargos in advancing bone regeneration strategies.NEW & NOTEWORTHY This study identifies for the first time two extracellular vesicle (EV) subpopulations isolated from young male mice bone tissue, with distinct mean size and molecular signature. Analysis of the protein and miRNA cargos of these EVs indicates that the large and small EVs are involved in different stages of osteogenesis, from commitment to the formation of mineralized tissue. These findings provide new insights into the role of bone-derived EVs in bone physiology.

Autonomic imbalance is a key driver of cardiovascular disease progression, arising from disrupted interactions between sympathetic and parasympathetic signaling. This review explores the molecular mechanisms underpinning autonomic dysfunction, emphasizing the roles of β-adrenergic receptor (βAR) signaling, cyclic AMP (cAMP) compartmentation, and cholinergic regulation. Dysregulated cAMP nanodomain signaling, βAR desensitization, impaired vagal tone, and maladaptive autonomic nerve remodeling collectively promote structural, electrophysiological, and functional deterioration. Advances in high-resolution imaging and molecular mapping have revealed previously unrecognized pathways governing second-messenger compartmentation and neuromodulatory feedback loops. These insights are driving the development of next-generation therapeutics designed to selectively restore autonomic balance. Promising strategies include isoform-specific phosphodiesterase inhibitors, vagus nerve stimulation, and axonal modulation therapy, which target norepinephrine and acetylcholine pathways while preserving physiological responsiveness. Integrating pharmacological, neuromodulatory, and molecular approaches represents an evolving frontier for cardiovascular therapeutics. Future strategies will benefit from precision mapping of autonomic circuits, patient-specific profiling, and optimization of therapeutic timing. By linking fundamental molecular signaling with translational advances, this review highlights opportunities to improve treatment precision and efficacy for autonomic dysfunction in cardiovascular disease.

Zinc is a commonly used antidiarrheal supplement; however, its exact mechanism of action is not well understood. Calcium-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for secretory diarrhea. CaSR is activated by various cations and here we investigated the roles of CaSR in the antidiarrheal effects of the divalent metal zinc (Zn²⁺). In human intestinal T84 cells expressing CaSR, zinc (100 µM) inhibited forskolin-induced secretory short-circuit current (I_sc) by 60% and its effect was comparable with CaSR activator cinacalcet. Zinc effect was via inhibition of apical CFTR Cl^- channel and basolateral K⁺ channels. In cell models, zinc was a CaSR agonist and its antisecretory effects were CaSR-dependent. Similarly, 100 µM zinc inhibited forskolin-induced secretory I_sc by 40% in wild-type mouse intestine with no antisecretory effects in intestinal epithelia-specific CaSR knockout mice (Casr^flox/flox;Vil1-cre). Zinc inhibited I_sc induced by clinically-relevant cAMP agonists (cholera toxin and vasoactive intestinal peptide) by 65% in T84 cells. Interestingly, zinc had no effect on cGMP agonists (heat-stable E. coli enterotoxin and linaclotide)-induced secretory I_sc, suggesting that its antisecretory effects are specific to cAMP. The mechanisms of zinc effect in T84 cells involved intracellular Ca²⁺ release via ryanodine receptors and inhibition of cAMP synthesis. Our findings suggest that CaSR activation is a major mechanism for the antidiarrheal effects of zinc, which specifically reduces cAMP levels. In addition to its use in cholera, zinc can be effective in other cAMP-mediated secretory diarrheas.NEW & NOTEWORTHY Zinc is a commonly used antidiarrheal supplement; however, its exact mechanisms of antisecretory effects remain unknown. In this study, we demonstrated that zinc is an agonist of the extracellular calcium-sensing receptor (CaSR) and its antidiarrheal effects are via reduced cAMP synthesis in intestinal epithelial cells. In addition to elucidating the mechanism of action of this antidiarrheal supplement, our data support the use of zinc as a simple and effective treatment option for all cAMP-mediated diarrheas.

Chlorine (Cl₂) is a highly reactive halogen gas that undergoes rapid hydrolysis in lung epithelial lining fluid (ELF) upon inhalation, forming hypochlorous acid (HOCl) and hydrochloric acid (HCl). These products subsequently, through chemical reactions, modify the structure and the function of membrane proteins. Herein, we investigated the effects of Cl₂ on connexin-hemichannels and the release of ATP in the ELF. Adult C57BL/6 mice were subjected to 400 ppm Cl₂ for 30 min. Subsequent analysis revealed a marked increase in ATP levels within the BAL, with concentrations reaching 43.952 ± 9.553 nM at 2 h and 30.554 ± 7.383 nM at 24 h post exposure, relative to control. In addition, at 24 h post exposure, the lung wet/dry (W/D) ratio significantly increased from 4.48 ± 0.142 to 5.067 ± 0.359, whereas alveolar fluid clearance (AFC) decreased from 0.249 ± 0.019 to 0.145 ± 0.018. Electrophysiological recordings in alveolar type 2 (AT2) cells revealed reduced open probabilities (P_o) of both ENaC (4 pS) and a cation channel (18 pS), declining from 0.323 ± 0.021 and 0.202 ± 0.022 to 0.151 ± 0.042 and 0.091 ± 0.019, respectively. Instillation of 50 µL of 100 µg/mL Gap27-a connexin mimetic peptide selectively inhibiting connexin-hemichannels-administered 30 min post exposure, restored ATP to control, normalized the W/D ratio, improved AFC, and reestablished ENaC function. Moreover, Gap27 normalized airway resistance following methacholine challenge. In human airway smooth muscle cells (hASMCs), 100 μM ATP induced [Formula: see text] elevation and depolarized V_m to -40 mV, with both effects partially reversed by P2X₇R inhibitor, A804598.NEW & NOTEWORTHY Inhaled chlorine gas reacts with lung epithelial lining fluid to form hypochlorous and hydrochloric acids that alter membrane protein structure and function. Under oxidative stress, connexin hemichannels open, releasing ions and metabolites, such as ATP. The released ATP signals danger, cell death, and tissue injury. Early administration of Gap27, a connexin-hemichannel inhibitor, at 30 min post exposure preserves ENaC function and prevents the subsequent development of pulmonary edema. These compelling findings underscore a promising therapeutic strategy.

Proliferative retinopathies are associated with abnormal angiogenesis that can result in visual impairment or vision loss. The tight junction complex regulates blood-retinal barrier integrity; however, its role in proliferative retinopathies is still at an early stage. Here, we used human retinal microvascular endothelial cells (HRMVECs) and a mouse model of oxygen-induced retinopathy (OIR) to investigate the impact of interleukin-33 (IL-33) signaling on tight junction disintegration and pathological angiogenesis. Our experimental findings demonstrate that IL-33 induces zonula occludens-1 (ZO-1) serine/threonine phosphorylation and tight junction disruption in HRMVECs. In addition, mass spectroscopy analysis revealed that treating HRMVECs with IL-33 induces ZO-1 phosphorylation at the Thr861 residue. Furthermore, we observed that NOX1-PKCδ (protein kinase C-δ) signaling modulates IL-33-induced ZO-1 phosphorylation and tight junction integrity in HRMVECs. We also observed that IL-33 depletion significantly reduces OIR-induced NOX1-PKCδ-ZO-1 signaling and vascular leakage in the ischemic retina. We also observed that the NOX1-specific inhibitor, fluoflavine (ML-090), attenuated OIR-induced NADPH oxidase activity and pathological retinal neovascularization in the ischemic retina. Thus, we infer that IL-33-mediated NOX1-PKCδ-ZO-1 signaling regulates ischemia-induced retinal endothelial cell tight junction disruption and retinal neovascularization.NEW & NOTEWORTHY The significance of blood-retinal barrier integrity in proliferative retinopathies, including retinopathy of prematurity and diabetic retinopathy, is still in its nascent phase. Using human retinal microvascular endothelial cells and an oxygen-induced retinopathy animal model, we demonstrate that IL-33-mediated NOX1-PKCδ-ZO-1 signaling regulates tight junction disruption and blood-retinal barrier integrity in ischemic retinal diseases.

Cancer-induced skeletal muscle wasting (cachexia) is responsible for over 20% of cancer-related deaths, yet much about the pathophysiology of the condition remains unknown. Importantly, cancer cachexia does not seem wholly responsive to traditional anabolic stimuli such as nutritional interventions. It is possible that tumors directly or indirectly target skeletal muscle for their dynamic and abundant pool of amino acids that can be reliably used by tumors to supplement energy production and biomass synthesis. Therefore, understanding how the presence of a tumor alters circulating and tissue-level amino acid pools could provide valuable insight into tumor-induced muscle wasting. The purpose of this review was to examine the current body of research that has studied amino acids in the context of cancer cachexia to better understand how amino acids behave during the development of cancer, cachexia, and various cancer chemotherapies. Distinct heterogeneity was observed in the behavior of amino acids when comparing weight-stable versus patients with cachectic cancer, and there may be important temporal considerations based on rhythmic changes in amino acid turnover and mealtimes. Overall, there was very little consistency in the reported fluctuations of amino acids from study to study, suggesting that there may be heterogeneous pathophysiology based on tumor type, stage, patient age, and comorbidities. Further work is required to characterize longitudinal changes in amino acid metabolism with consideration for these factors. Enhancing our understanding of amino acid metabolism during cancer cachexia could provide opportunities for advancement in practical methodologies in cachexia research and treatment strategies.

Aging causes changes to arterial contractility and tissue microstructure, resulting in arterial stiffening, a strong risk factor for cardiovascular diseases. Because the interaction between these effects is largely unexplored, this study aims to investigate how aging-induced changes in contractility and wall constituent microstructure impact arterial biomechanics in murine aortas. Vasoreactive responses of thoracic descending aortas of adult (5-mo-old, n = 5) and old (24-mo-old, n = 5) C57Bl/6J mice to phenylephrine, N^ω-nitro-l-arginine methyl ester (l-NAME), and sodium nitroprusside were measured under dynamic pressurization conditions. Whole vessel and individual-constituent biaxial viscoelastic properties were characterized during contraction and relaxation while mimicking physiological dynamic loading conditions. In addition, elastin fibers, collagen fibers, and smooth muscle nuclei microstructural organization and morphological properties were quantified in pressurized aortas using two-photon laser scanning microscopy. Compared with adult mice, aortas of old mice displayed thicker walls but similar pressure-diameter behaviors in the absence of contraction. Vasoconstriction in aortas of adult mice 1) significantly increased wall thickness, 2) reduced pulse wave velocity at physiologically high pressure ranges, 3) reduced circumferential and axial stresses and stiffnesses, and 4) altered constituent load bearing. Conversely, aortas of old mice exhibited reduced contractility, altered vasoreactive responses, and reduced cell density. As a result, they were uncapable to alter any of their biomechanical properties through vasoconstriction. In conclusion, vasoconstriction enables modulation of axial and circumferential stresses and stiffnesses in the adult mouse aorta. With aging, this modulatory capacity was impaired.NEW & NOTEWORTHY Using a biaxial pressure myograph that mimics physiological loading conditions, this study demonstrates the capacity of vascular smooth muscle cells in male C57BL/6J mice at 5 mo of age to modulate aortic stiffness through vasoconstriction and dilation. In addition, this capacity is shown to be lost in male C57BL/6J mice at 24 mo of age.

Single-cell RNA sequencing (scRNA-Seq) studies identified a novel subpopulation of epithelial cells along the rostrocaudal axis of human intestine, specifically marked by bestrophin 4 (BEST4), that are enriched for genes regulating pH, GPCR acid-sensing receptors, satiety, cGMP signaling, [Formula: see text] secretion, ion transport, neuropeptides, and paracrine hormones. Interestingly, BEST4⁺ cells in the proximal small intestine express CFTR but have not been widely linked to the previously described CFTR high-expresser cell (CHE) subpopulation in rat and human intestine. ScRNA-Seq studies in rat jejunum identified CHEs and a gene expression profile consistent with human small intestinal BEST4+ and neuropod cells. Protein immunolocalization confirmed that CHEs express CFTR, BEST4, neuropod proteins, high levels of intracellular uroguanylin (UGN), guanylyl cyclase-C (GC-C), and the proton channel otopetrin 2 (OTOP2), and display long basal processes connecting to neurons, confirming that Best4⁺ cells in the proximal small intestine are CHEs. OTOP2, GC-C, and CFTR traffic robustly into the apical domain of CHEs in response to acidic luminal conditions, indicating their roles in luminal pH regulation. In the ΔF508 cystic fibrosis (CF) rat jejunum, the loss of apical CFTR did not affect BEST4 protein expression in CHEs. However, there was an increased abundance of CHE cells in the ΔF508 rat jejunum compared with wild-type animals. Furthermore, ΔF508 rat CHEs expressed higher levels of GC-C at the apical domain compared with wild-type. These data implicate CHEs in intestinal CF disease pathogenesis.NEW & NOTEWORTHY This is the first study to identify CFTR high-expresser cells in the rat small intestine as neuropod cells capable of sensing and responding to luminal pH, and confirms that Best4⁺ cells are CHEs in the proximal small intestine. This study also provides the first characterization of CFTR and relevant mRNA and proteins in CHEs in CF rat models that provide insights into the significance of CHEs to CF intestinal disease.

Piezo1 is an evolutionally conserved mechanosensitive ion channel implicated in the regulation of development, differentiation, and growth of multiple tissues. Despite its importance, the pathways induced downstream of Piezo1 activation remain incompletely defined. Here, we report that the selective Piezo1 agonists, Yoda1, Yaddle1, and Yoda2, stimulate Protein kinase D (PKD) family activation in a concentration-dependent manner, in human aortic endothelial cells (ECs), as shown by an increase in PKD phosphorylation at Ser⁹¹⁰, an autophosphorylation site in the C-terminus, and Ser^738/742 located in the activation loop. Depletion of extracellular Ca²⁺ by EGTA abolished PKD phosphorylation stimulated by Piezo1 agonists, and exposure to the Ca²⁺ ionophore ionomycin potently stimulated PKD. The PKD activation induced by Yoda1, Yaddle1, or Yoda2 was prevented by inhibitors of PKDs (CRT0066101) and protein kinase C (Go6983) and abrogated by siRNA-mediated knockdown of Piezo1. Treatment of ECs with H-067047 or apyrase did not alter the increase in the phosphorylation of PKD at either Ser⁹¹⁰ or Ser^738/742 induced by Yoda1, implying that stimulation of Piezo1 induces PKD activation independently from TRPV4 and autocrine ATP signaling. Exposure of ECs to Yoda1 increased the histone deacetylase (HDAC)7 phosphorylation and migration of ECs into the denuded area of the monolayer, as shown using a scratch wound assay. Treatment of ECs with CRT0066101 prevented HDAC7 phosphorylation and migration of these cells induced by Yoda1, suggesting that Yoda1-stimulated Piezo1 promotes EC migration through a PKD. Our results identify PKD as a novel downstream mediator of effects produced by agonist-induced activation of the Piezo1 mechanosensitive channel in ECs.NEW & NOTEWORTHY Protein kinase D (PKD) emerged as a key node in cellular signaling, but its role in mechanobiology remains unknown. Here, we show that stimulation of the mechanosensitive Piezo1 ion channel markedly increases PKD multisite phosphorylation, indicative of activation. Opening of Piezo1 also promotes histone deacetylase (HDAC)7 phosphorylation and endothelial cell migration in a PKD-dependent manner. The results implicate PKD signaling in the function of Piezo 1 and identify a Piezo1/Ca²⁺/PKC/PKD/HDA7 signaling pathway in endothelial cells.