Search Results (12)

Background/Objectives: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by excessive extracellular matrix (ECM) production and tissue stiffening, resulting in impaired lung function. Sodium hydrogen exchanger isoform 1 (NHE1) is a key mediator of intracellular and extracellular pH regulation, influencing fibroblast activation, motility, and proliferative pathways. This study investigates the role of NHE1 in actin stress fiber formation, fibroblast-to-myofibroblast differentiation, and cytokine secretion in IPF progression. Methods: Fibroblasts were treated with profibrotic agonists, including transforming growth factor-beta (TGFβ), lysophosphatidic acid (LPA), and serotonin (THT), in the presence or absence of the NHE1-specific inhibitor, EIPA. Actin stress fibers were visualized using phalloidin staining, while α-smooth muscle actin (α-SMA) expression and cytokine secretion (TGFβ, IL-6, and IL-8) were quantified using immunostaining and ELISA. Intracellular pH changes were measured using BCECF-AM fluorescence. Results: Profibrotic agonists induced significant actin stress fiber formation and α-SMA expression in fibroblasts, both of which were abolished by EIPA. NHE1 activity was shown to mediate intracellular alkalization, a critical factor for fibroblast activation. Cytokine secretion, including TGFβ, IL-6, and IL-8, was enhanced by agonist treatments but reduced with NHE1 inhibition. Chronic TGFβ exposure increased intracellular pH and sustained myofibroblast differentiation, which was partially reversed by EIPA. Conclusions: NHE1 is indicated to play a novel and potential role in processes supporting profibrotic agonists driving fibroblast activation and IPF progression. Targeting NHE1 could present a potential therapeutic approach to disrupt profibrotic pathways and mitigate IPF severity. Full article

A well-known feature of tumor cells is high glycolytic activity, leading to acidification of the tumor microenvironment through extensive lactate production. This acidosis promotes processes such as metastasis, aggressiveness, and invasiveness, which have been associated with a worse clinical prognosis. Moreover, the function and expression of transporters involved in regulation of intracellular pH might be altered. In this study, the capacity of tumor cells to regulate their intracellular pH when exposed to a range of pH from very acidic to basic was characterized in two glioma cell lines (F98 and U87) using a new recently published method of fluorescence imaging. Our results show that the regulation of acidity in tumors is not the same for the two investigated cell lines; U87 cells are able to reduce their intracellular acidity, whereas F98 cells do not exhibit this property. On the other hand, F98 cells show a higher level of resistance to acidity than U87 cells. Intracellular regulation of acidity appears to be highly cell-dependent, with different mechanisms activated to preserve cell integrity and function. This characterization was performed on 2D monolayer cultures and 3D spheroids. Spatial heterogeneities were exhibited in 3D, suggesting a spatially modulated regulation in this context. Based on the corpus of knowledge available in the literature, we propose plausible mechanisms to interpret our results, together with some new lines of investigation to validate our hypotheses. Our results might have implications on therapy, since the activity of temozolomide is highly pH-dependent. We show that the drug efficiency can be enhanced, depending on the cell type, by manipulating the extracellular pH. Therefore, personalized treatment involving a combination of temozolomide and pH-regulating agents can be considered. Full article

There is a variety of fluorescent probes for pH measurements and which are mainly used for biological systems. In general, they can be classified into two groups. The first group includes fluorescent pH probes which exhibit a single fluorescence emission peak. For these probes, the fluorescence excitation profile is pH-dependent and the shape of the emission spectra remains almost constant. Hence, the ratiometric pH measurement–which makes pH determination independent of the probe concentration-is implemented when the excitation is performed at two excitation wavelengths and the fluorescence emission is measured at one wavelength. The second group exhibits a dual fluorescence emission peak. Here, each protonated or deprotonated form exhibits characteristics emission and/or absorption spectra. Shifts between spectra obtained for protonated and deprotonated species can be exploited in order to perform a ratiometric measurement. In this study we present a methodology that evaluates the precision of the ratiometric measurements based on multiple wavelengths excitation to determine the optimum wavelengths combination for pH determination in biological samples. This methodology using the BCECF probe is applied to measure the pH drift in cell culture medium. It exhibits a high precision and significantly extends the range of validity for pH measurements spanning from very acidic to basic. Full article

iPSCs and their derivatives are the most promising cell sources for creating skin equivalents. However, their properties are not fully understood. In addition, new approaches and parameters are needed for studying cells in 3D models without destroying their organization. Thus, the aim of our work was to study and compare the metabolic status and pH of dermal spheroids created from dermal papilla cells differentiated from pluripotent stem cells (iDP) and native dermal papilla cells (hDP) using fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM). For this purpose, fluorescence intensities of NAD(P)H and FAD, fluorescence lifetimes, and the contributions of NAD(P)H, as well as the fluorescence intensities of SypHer-2 and BCECF were measured. iDP in spheroids were characterized by a more glycolytic phenotype and alkaline intra-cellular pH in comparison with hDP cells. Moreover, the metabolic activity of iDP in spheroids depends on the source of stem cells from which they were obtained. So, less differentiated and condensed spheroids from iDP-iPSDP and iDP-iPSKYOU are characterized by a more glycolytic phenotype compared to dense spheroids from iDP-DYP0730 and iDP-hES. FLIM and fluorescent microscopy in combination with the metabolism and pH are promising tools for minimally invasive and long-term analyses of 3D models based on stem cells. Full article

Brain diseases including Down syndrome (DS/TS21) are known to be characterized by changes in cellular metabolism. To adequately assess such metabolic changes during pathological processes and to test drugs, methods are needed that allow monitoring of these changes in real time with minimally invasive effects. Thus, the aim of our work was to study the metabolic status and intracellular pH of spheroids carrying DS using fluorescence microscopy and FLIM. For metabolic analysis we measured the fluorescence intensities, fluorescence lifetimes and the contributions of the free and bound forms of NAD(P)H. For intracellular pH assay we measured the fluorescence intensities of SypHer-2 and BCECF. Data were processed with SPCImage and Fiji-ImageJ. We demonstrated the predominance of glycolysis in TS21 spheroids compared with normal karyotype (NK) spheroids. Assessment of the intracellular pH indicated a more alkaline intracellular pH in the TS21 spheroids compared to NK spheroids. Using fluorescence imaging, we performed a comprehensive comparative analysis of the metabolism and intracellular pH of TS21 spheroids and showed that fluorescence microscopy and FLIM make it possible to study living cells in 3D models in real time with minimally invasive effects. Full article

Mineralocorticoids (e.g., aldosterone) support chronic inflammatory tissue damage, including glomerular mesangial injury leading to glomerulosclerosis. Furthermore, aldosterone leads to activation of the extracellular signal-regulated kinases (ERK1/2) in rat glomerular mesangial cells (GMC). Because ERK1/2 can affect cellular pH homeostasis via activation of Na⁺/H⁺-exchange (NHE) and the resulting cellular alkalinization may support proliferation, we tested the hypothesis that aldosterone affects pH homeostasis and thereby cell proliferation as well as collagen secretion also in primary rat GMC. Cytoplasmic pH and calcium were assessed by single-cell fluorescence ratio imaging, using the dyes BCECF or FURA2, respectively. Proliferation was determined by cell counting, thymidine incorporation and collagen secretion by collagenase-sensitive proline incorporation and ERK1/2-phosphorylation by Western blot. Nanomolar aldosterone induces a rapid cytosolic alkalinization which is prevented by NHE inhibition (10 µmol/L EIPA) and by blockade of the mineralocorticoid receptor (100 nmol/L spironolactone). pH changes were not affected by inhibition of HCO₃⁻ transporters and were not dependent on HCO₃⁻. Aldosterone enhanced ERK1/2 phosphorylation and inhibition of ERK1/2-phosphorylation (10 µmol/L U0126) prevented aldosterone-induced alkalinization. Furthermore, aldosterone induced proliferation of GMC and collagen secretion, both of which were prevented by U0126 and EIPA. Cytosolic calcium was not involved in this aldosterone action. In conclusion, our data show that aldosterone can induce GMC proliferation via a MR and ERK1/2-mediated activation of NHE with subsequent cytosolic alkalinization. GMC proliferation leads to glomerular hypercellularity and dysfunction. This effect presents a possible mechanism contributing to mineralocorticoid receptor-induced pathogenesis of glomerular mesangial injury during chronic kidney disease. Full article

Both ion fluxes and changes of cytosolic pH take an active part in the signal transduction of different environmental stimuli. Here we studied the anoxia-induced alteration of cytosolic K⁺ concentration, [K⁺]_cyt, and cytosolic pH, pH_cyt, in rice and wheat, plants with different tolerances to hypoxia. The [K⁺]_cyt and pH_cyt were measured by fluorescence microscopy in single leaf mesophyll protoplasts loaded with the fluorescent potassium-binding dye PBFI-AM and the pH-sensitive probe BCECF-AM, respectively. Anoxic treatment caused an efflux of K⁺ from protoplasts of both plants after a lag-period of 300–450 s. The [K⁺]_cyt decrease was blocked by tetraethylammonium (1 mM, 30 min pre-treatment) suggesting the involvement of plasma membrane voltage-gated K⁺ channels. The protoplasts of rice (a hypoxia-tolerant plant) reacted upon anoxia with a higher amplitude of the [K⁺]_cyt drop. There was a simultaneous anoxia-dependent cytosolic acidification of protoplasts of both plants. The decrease of pH_cyt was slower in wheat (a hypoxia-sensitive plant) while in rice protoplasts it was rapid and partially reversible. Ion fluxes between the roots of intact seedlings and nutrient solutions were monitored by ion-selective electrodes and revealed significant anoxia-induced acidification and potassium leakage that were inhibited by tetraethylammonium. The K⁺ efflux from rice was more distinct and reversible upon reoxygenation when compared with wheat seedlings. Full article

Platelets play a crucial role in the immunological response and are involved in the pathological settings of vascular diseases, and their adhesion to the extracellular matrix is important to bring leukocytes close to the endothelial cells and to form and stabilize the thrombus. Currently there are several methods to study platelet adhesion; however, the optimal parameters to perform the assay vary among studies, which hinders their comparison and reproducibility. Here, a standardization and validation of a fluorescence-based quantitative adhesion assay to study platelet-ECM interaction in a high-throughput screening format is proposed. Our study confirms that fluorescence-based quantitative assays can be effectively used to detect platelet adhesion, in which BCECF-AM presents the highest sensitivity in comparison to other dyes. Full article

Many cell types express an acid-sensitive outwardly rectifying (ASOR) anion current of an unknown function. We characterized such a current in BV-2 microglial cells and then studied its interrelation with the volume-sensitive outwardly rectifying (VSOR) Cl⁻ current and the effect of acidosis on cell volume regulation. We used patch clamp, the Coulter method, and the pH-sensitive dye BCECF to measure Cl⁻ currents and cell membrane potentials, mean cell volume, and intracellular pH, respectively. The ASOR current activated at pH ≤ 5.0 and displayed an I⁻ > Cl⁻ > gluconate⁻ permeability sequence. When compared to the VSOR current, it was similarly sensitive to DIDS, but less sensitive to DCPIB, and insensitive to tamoxifen. Under acidic conditions, the ASOR current was the dominating Cl⁻ conductance, while the VSOR current was apparently inactivated. Acidification caused cell swelling under isotonic conditions and prevented the regulatory volume decrease under hypotonicity. We conclude that acidification, associated with activation of the ASOR- and inactivation of the VSOR current, massively impairs cell volume homeostasis. ASOR current activation could affect microglial function under acidotoxic conditions, since acidosis is a hallmark of pathophysiological events like inflammation, stroke or ischemia and migration and phagocytosis in microglial cells are closely related to cell volume regulation. Full article

Biosensors on the membrane of the vascular endothelium are responsible for sensing mechanical and chemical signals in the blood. Transduction of these stimuli into intracellular signaling cascades regulate cellular processes including ion transport, gene expression, cell proliferation, and/or cell death. The primary cilium is a well-known biosensor of shear stress but its role in sensing extracellular pH change has never been examined. As a cellular extension into the immediate microenvironment, the cilium could be a prospective sensor for changes in pH and regulator of acid response in cells. We aim to test our hypothesis that the primary cilium plays the role of an acid sensor in cells using vascular endothelial and embryonic fibroblast cells as in vitro models. We measure changes in cellular pH using pH-sensitive 2′,7′-biscarboxyethy1-5,6-carboxyfluorescein acetoxy-methylester (BCECF) fluorescence and mitogen-activated protein kinase (MAPK) activity to quantify responses to both extracellular pH (pH_o) and intracellular pH (pH_i) changes. Our studies show that changes in pH_o affect pH_i in both wild-type and cilia-less Tg737 cells and that the kinetics of the pH_i response are similar in both cells. Acidic pH_o or pH_i was observed to change the length of primary cilia in wild-type cells while the cilia in Tg737 remained absent. Vascular endothelial cells respond to acidic pH through activation of ERK1/2 and p38-mediated signaling pathways. The cilia-less Tg737 cells exhibit delayed responsiveness to pH_o dependent and independent pH_i acidification as depicted in the phosphorylation profile of ERK1/2 and p38. Otherwise, intracellular pH homeostatic response to acidic pH_o is similar between wild-type and Tg737 cells, indicating that the primary cilia may not be the sole sensor for physiological pH changes. These endothelial cells respond to pH changes with a predominantly K⁺-dependent pH_i recovery mechanism, regardless of ciliary presence or absence. Full article

Malaria is an infectious disease caused by Plasmodium group. The mechanisms of antimalarial drugs DHA/CQ are still unclear today. The inhibitory effects (IC₅₀) of single treatments with DHA/CQ or V-ATPase inhibitor Baf-A1 or combination treatments by DHA/CQ combined with Baf-A1 on the growth of Plasmodium falciparum strain 3D7 was investigated. Intracellular cytoplasmic pH and labile iron pool (LIP) were labeled by pH probe BCECF, AM and iron probe calcein, AM, the fluorescence of the probes was measured by FCM. The effects of low doses of DHA (0.2 nM, 0.4 nM, 0.8 nM) on gene expression of V-ATPases (vapE, vapA, vapG) located in the membrane of DV were tested by RT-qPCR. DHA combined with Baf-A1 showed a synergism effect (CI = 0.524) on the parasite growth in the concentration of IC₅₀. Intracellular pH and irons were effected significantly by different doses of DHA/Baf-A1. Intracellular pH was decreased by CQ combined with Baf-A1 in the concentration of IC₅₀. Intracellular LIP was increased by DHA combined with Baf-A1 in the concentration of 20 IC₅₀. The expression of gene vapA was down-regulated by all low doses of DHA (0.2/0.4/0.8 nM) significantly (p < 0.001) and the expression of vapG/vapE were up-regulated by 0.8 nM DHA significantly (p < 0.001). Interacting with ferrous irons, affecting the DV membrane proton pumping and acidic pH or cytoplasmic irons homeostasis may be the antimalarial mechanism of DHA while CQ showed an effect on cytoplasmic pH of parasite in vitro. Lastly, this article provides us preliminary results and a new idea for antimalarial drugs combination and new potential antimalarial combination therapies. Full article

In this study, a benzimidazole derivative named BMT-1 is revealed as a potential immunomodulatory agent. BMT-1 inhibits the activity of H+/K+-ATPases from anti-CD3/CD28 activated T cells. Furthermore, inhibition the H+/K+-ATPases by use of BMT-1 should lead to intracellular acidification, inhibiting T cell proliferation. To explore this possibility, the effect of BMT-1 on intracellular pH changes was examined by using BCECF as a pH-dependent fluorescent dye. Interestingly, increases in the pHi were observed in activated T cells, and T cells treated with BMT-1 showed a more acidic intracellular pH. Finally, BMT-1 targeted the H+/K+-ATPases and inhibited the proliferative response of anti-CD3/CD28-stimulated T cells. A cell cycle analysis indicated that BMT-1 arrested the cell cycle progression of activated T cells from the G1 to the S phase without affecting CD25 expression or interleukin-2 (IL-2) production; treating IL-2-dependent PBMCs with BMT-1 also led to the inhibition of cell proliferation. Taken together, these findings demonstrate that BMT-1 inhibits the proliferation of T cells by interfering with H+/K+-ATPases and down-regulating intracellular pHi. This molecule may be an interesting lead compound for the development of new immunomodulatory agents. Full article