Biology subjects

Flores, C. A.

Publications and source records attributed to Flores, C. A..

7 recordsLinked to original sources

bioRxiv2026

Functional differences in electrolyte transport between the mouse proximal and distal trachea

AimThe mammalian tracheal epithelium is composed by different cell types unevenly distributed along the proximal-distal axis. Nevertheless, variations in expression and function of ion channels and transporters participating in fluid absorption and secretion had never been studied separately in proximal and distal sections of the mouse trachea. In this work, we aim to characterize basal and stimulated absorption and secretion of fluid obtained from proximal and distal trachea from the same animal. MethodsUssing chamber experiments were performed using a custom-made tissue slider that allowed the mounting small tracheal sections, where response to agonists and blockers was recorded. The role of the NKCC1 co-transporter was studied using the Slc12a2-/- mouse. A genetically tomato-induced mouse model was used to assess co-expression of NKCC1 and ASCL3 by immunofluorescence. Animals were instilled with different interleukins (ILs) to determine changes in absorption, secretion and mucus properties. ResultsProximal trachea didnt participate in sodium absorption but exhibited higher cAMP- and succinate-induced anion secretion than the distal section. NBCe1-dependent bicarbonate and TMEM16A-driven chloride secretion was significantly higher in the distal section. NKCC1+ cells were found in the submucosal glands (SMGs) and abundant patches of NKCC1+ cells in the distal region. Isolated NKCC1+ cells co-expressing ASCL3 were also detected. ILs treatment changed the electrophysiological properties of the distal but not the proximal trachea. ConclusionsOur experiments determined that the mouse trachea organizes its functions differentially in the proximal and distal sections, based in the functional distribution of channels, transporters and receptors. While the distal trachea drastically changed its responses to agonists inducing anion secretion the proximal trachea was unperturbed by the action of ILs.

physiology↗

bioRxiv2024

Airway succinate chemosensing induces CFTR-dependent anion secretion and mucus clearance which is impaired in cystic fibrosis.

The respiratory tract possesses a highly regulated innate defense system which includes efficient cilia-mediated mucus transport or mucociliary clearance (MCC). This essential process relies on appropriate hydration of airway surfaces which is controlled by a blend of transepithelial sodium and liquid absorption via the epithelial sodium channel (ENaC), and anion and liquid secretion, primarily regulated by the cystic fibrosis transmembrane conductance regulator (CFTR) channel. MCC is tightly regulated by second messenger signalling pathways. Succinate is derived from parasites, microorganisms and inflammatory cells, and its concentration increases in the airway surface liquid (ASL) during infections. Increases in ASL succinate activates the G-protein coupled succinate receptor (SUCNR1), which acts as a succinate sensor. Here, we tested the hypothesis that succinate signalling was linked to CFTR activity, ASL hydration and increased MCC. We observed that SUCNR1 activation stimulated anion secretion, increased mucus transport and induced bronchoconstriction in mouse airways. In parallel, stimulation of human bronchial epithelial cells (HBEC) with succinate activated anion secretion and increased ASL height. All functions activated by succinate/SUCNR1 were impeded when working with tissues and cells isolated from animal models or individuals affected cystic fibrosis (CF) or when CFTR was inhibited. Moreover, when HBECs derived from {Delta}F508 individuals were incubated with the triple drug combination of elexacaftor/tezacaftor/ivacaftor (ETI), succinate-induced anion secretion was restored, confirming the tight relationship between SUCNR1 signalling and CFTR function. Our results identify a novel activation pathway for CFTR that participates in the defence response of the airways, which is defective in CF. We propose that succinate acts as a danger molecule that alerts the airways to the presence of pathogens leading to a flushing out of the airways.

physiology↗

bioRxiv2021

Inhibition of the sodium-dependent HCO3 -transporter SLC4A4, produces a cystic fibrosis-like airway disease phenotype.

Bicarbonate secretion is a fundamental process involved in maintaining acid-base homeostasis. Disruption of bicarbonate entry into airway lumen, as has been observed in cystic fibrosis, produces several defects in lung function due to thick mucus accumulation. Bicarbonate is critical for correct mucin deployment and there is increasing interest in understanding its role in airway physiology, particularly in the initiation of lung disease in children affected by cystic fibrosis, in the absence of detectable bacterial infection. The current model of anion secretion in mammalian airways consists of CFTR and TMEM16A as apical anion exit channels, with limited capacity for bicarbonate transport compared to chloride. However, both channels can couple to SLC26A4 anion exchanger to maximise bicarbonate secretion. Nevertheless, current models lack any details about the identity of the basolateral protein(s) responsible for bicarbonate uptake into airway epithelial cells. We report herein that the electrogenic, sodium-dependent, bicarbonate cotransporter, SLC4A4, is expressed in the basolateral membrane of human and mouse airways, and that its pharmacological inhibition or genetic silencing reduces bicarbonate secretion. In fully differentiated primary human airway cells, SLC4A4 inhibition induced an acidification of the airways surface liquid and markedly reduced the capacity of cells to recover from an acid load. Studies in the Slc4a4-null mice revealed a previously unreported lung phenotype, characterized by mucus accumulation and reduced mucociliary clearance. Collectively, our results demonstrate that the reduction of SLC4A4 function induced a CF-like phenotype, even when chloride secretion remained intact, highlighting the important role SLC4A4 plays in bicarbonate secretion and mammalian airway function.

physiology↗

bioRxiv2020

Tamoxifen reduces inflammatory infiltration of neutrophils in the airways.

ABSTRACTTamoxifen is a drug of choice for treatment of breast cancer but it has also been reported to bear anti-inflammatory activity. Previously, we have observed that tamoxifen treatment successfully reduced inflammation in horses affected by equine asthma syndrome. More notorious, tamoxifen was effective at reducing the infiltration of neutrophils in the inflamed airways by a mechanism that increases efferocytosis, allowing the resolution of inflammation. Due to the emerging increase in patients with chronic inflammatory lung diseases, there is an urgent need for therapies that help reduce airway inflammation. Thus we tested the effect of tamoxifen on airway neutrophil infiltration by using three different mouse models for acute and chronic inflammation of the lung. We found that the drug was able to produce a significant reduction in neutrophils in all scenarios. Ussing chamber experiments demonstrated that tamoxifen has no effect on fluid secretion and absorption, discarding a possible reduction in mucociliary clearance due to the known ion-channel blocking effects of tamoxifen. Since this drug has been largely used in human medicine, tamoxifen might be a “low hanging fruit” for a novel anti-inflammatory therapy for airway diseases characterized by neutrophilic inflammation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2020

Lack of Kcnn4 improves mucociliary clearance in muco-obstructive lung disease.

Airway mucociliary clearance (MCC) is the main mechanism of lung defense keeping the airways free of infection and mucus obstruction. Airways surface liquid (ASL) volume, ciliary beating and mucus are central for proper MCC, and are critically regulated by sodium (Na+) absorption and anion secretion. Impaired MCC is a key feature of muco-obstructive disease. The calcium-activated potassium (K+)channel KCa.3.1, encoded by the Kcnn4 gene, participates in intestinal ion secretion and previous studies showed that its activation increase Na+ absorption in airway epithelia, suggesting that hyperpolarization induced by KCa3.1 was sufficient to drive Na+ absorption. However, its role in airway epithelial function is not fully understood. We therefore aimed to elucidate the role of KCa3.1 in MCC in a genetically engineered mouse model. We show that KCa3.1 inhibition reduced Na+ absorption in mouse and human airway epithelium. Furthermore, the genetic deletion of Kcnn4 enhanced cilia beating frequency (CBF) and MCC ex vivo and in vivo. Kcnn4 was silenced in the Scnn1b-transgenic mouse (Scnn1btg/+), a model of muco-obstructive lung disease triggered by increased epithelial Na+-absorption, leading to improvements in MCC and reduction of Na+-absorption. KCa3.1 deletion did not change the amount of mucus but did reduce mucus adhesion, neutrophil infiltration and emphysema. Our data support that KCa3.1 inhibition attenuated muco-obstructive disease in the Scnn1btg/+ mice. K+-channel modulation may be a novel therapeutic strategy to treat muco-obstuctive lung diseases.

physiology↗

bioRxiv2019

Mental Health, Schooling Attainment and Polygenic Scores: Are There Significant Gene-Environment Associations?

It is well-established that (1) there is a large genetic component to mental health, and (2) higher schooling attainment is associated with better mental health. Given these two observations, we test the hypothesis that schooling may attenuate the genetic predisposition to poor mental health. Specifically, we estimate associations between a polygenic score (PGS) for depressive symptoms, schooling attainment and gene-environment (GxE) interactions with mental health (depressive symptoms and depression), in two distinct United States datasets at different adult ages-29 years old in the National Longitudinal Study of Adolescent Health (Add Health) and 54 years old in the Wisconsin Longitudinal Study (WLS). OLS results indicate that the association of the PGS with mental health is similar in Add Health and the WLS, but the association of schooling attainment is much larger in Add Health than in the WLS. There is some suggestive evidence that the association of the PGS with mental health is lower for more-schooled older individuals in the WLS, but there is no evidence of any significant GxE associations in Add Health. Quantile regression estimates also show that in the WLS the GxE associations are statistically significant only in the upper parts of the conditional depressive symptoms score distribution. We assess the robustness of the OLS results to omitted variable bias by using the siblings samples in both datasets to estimate sibling fixed-effect regressions. The sibling fixed-effect results must be qualified, in part due to low statistical power. However, the sibling fixed-effect estimates show that college education is associated with fewer depressive symptoms in both datasets.

genomics↗

bioRxiv2019

MCT4 is a high affinity transporter capable of exporting lactate in high-lactate environments

MCT4 is an H+-coupled transporter expressed in metastatic cancer cells, macrophages, and other highly glycolytic cells, where it extrudes excess lactate generated by the Warburg phenomenon or by hypoxia. Intriguingly, its reported Km for lactate, obtained with pH-sensitive probes, is more than an order of magnitude higher than physiological lactate. Here we examined MCT4-rich MDA-MB-231 cells using the FRET sensor Laconic and found a median Km for lactate uptake of only 1.7 mM, while parallel estimation in the same cells with a pH probe gave a Km of 27 mM. The median Km of MCT4 for lactate was 0.7 mM in MCT4-expressing HEK293 cells and 1.2 mM in human macrophages, suggesting that high substrate affinity is a robust property of the transporter. Probed with the FRET sensor Pyronic, MCT4 showed a Km for pyruvate of only 4.2 mM in MDA-MB-231 cells, as opposed to > 150 mM reported previously. We conclude that prior estimates of MCT4 affinity based on pH probes were severely biased by the confounding action of pH regulatory mechanisms. Numerical simulation showed that MCT4, but not MCT1 or MCT2, endows cells with the capability of lactate extrusion in high lactate environments. The revised kinetic properties and novel transport assays may help in developing small-molecule MCT4 blockers for research and therapy.

physiology↗