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Purpose: In vivo 31P MRSI enables noninvasive mapping of absolute pH values via the pH‐dependent chemical shifts of inorganic phosphates (Pi). A particular challenge is the quantification of extracellular Pi with low SNR in vivo. The purpose of this study was to demonstrate feasibility of assessing both intra‐ and extracellular pH across the whole human brain via volumetric 31P MRSI at 7T. Methods: 3D 31P MRSI data sets of the brain were acquired from three healthy volunteers and three glioma patients. Low‐rank denoising was applied to enhance the SNR of 31P MRSI data sets that enables detection of extracellular Pi at high spatial resolutions. A robust two‐compartment quantification model for intra‐ and extracellular Pi signals was implemented. Results: In particular low‐rank denoising enabled volumetric mapping of intra‐ and extracellular pH in the human brain with voxel sizes of 5.7 mL. The average intra‐ and extracellular pH measured in white matter of healthy volunteers were 7.00 ± 0.00 and 7.33 ± 0.03, respectively. In tumor tissue of glioma patients, both the average intra‐ and extracellular pH increased to 7.12 ± 0.01 and 7.44 ± 0.01, respectively, compared to normal appearing tissue. Conclusion: Mapping of pH values via 31P MRSI at 7T using the proposed two‐compartment quantification model improves reliability of pH values obtained in vivo, and has the potential to provide novel insights into the pH heterogeneity of various tissues. [ABSTRACT FROM AUTHOR]

The sea urchin embryo develops a calcitic endoskeleton through intracellular formation of amorphous calcium carbonate (ACC). Intracellular precipitation of ACC, requires HCO3−/CO32− concentrating as well as proton export mechanisms to promote calcification. These processes are of fundamental importance in biological mineralization, but remain largely unexplored. Here, we demonstrate that the calcifying primary mesenchyme cells (PMCs) use Na+/H+-exchange (NHE) mechanisms to control cellular pH homeostasis during maintenance of the skeleton. During skeleton re-calcification, pHi of PMCs is increased accompanied by substantial elevation in intracellular [HCO3−] mediated by the Na+/HCO3− cotransporter Sp_Slc4a10. However, PMCs lower their pHi regulatory capacities associated with a reduction in NHE activity. Live-cell imaging using green fluorescent protein reporter constructs in combination with intravesicular pH measurements demonstrated alkaline and acidic populations of vesicles in PMCs and extensive trafficking of large V-type H+-ATPase (VHA)-rich acidic vesicles in blastocoelar filopodial cells. Pharmacological and gene expression analyses underline a central role of the VHA isoforms Sp_ATP6V0a1, Sp_ATP6V01_1 and Sp_ATPa1-4 for the process of skeleton re-calcification. These results highlight novel pH regulatory strategies in calcifying cells of a marine species with important implications for our understanding of the mineralization process in times of rapid changes in oceanic pH. [ABSTRACT FROM AUTHOR]

6‐Phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase isoform 3 (PFKFB3) is a key enzyme of the glycolytic pathway, and it plays an essential role in angiogenesis. 3‐(3‐Pyridinyl)‐1‐(4‐pyridinyl)‐2‐propen‐1‐one (3PO) is frequently used as a glycolysis inhibitor and is thought to inhibit PFKFB3. However, this latter effect of 3PO has never been investigated in detail and was the aim of the present study. To demonstrate binding of 3PO to PFKFB3, we used isothermal titration calorimetry. However, 3PO did not bind to PFKFB3, even up to 750 µm, in contrast to 3 µm of AZ67, which is a potent and specific PFKFB3 inhibitor. Instead, 3PO accumulated lactic acid inside the cells, leading to a decrease in the intracellular pH and an inhibition of enzymatic reactions of the glycolytic pathway. [ABSTRACT FROM AUTHOR]

Background: Changes in transcellular bioelectrical patterns are known to play important roles during developmental and regenerative processes. The Drosophila follicular epithelium has proven to be an appropriate model system for studying the mechanisms by which bioelectrical signals emerge and act. Fluorescent indicator dyes in combination with various inhibitors of ion-transport mechanisms have been used to investigate the generation of membrane potentials (Vmem) and intracellular pH (pHi). Both parameters as well as their anteroposterior and dorsoventral gradients were affected by the inhibitors which, in addition, led to alterations of microfilament and microtubule patterns equivalent to those observed during follicle-cell differentiation. Results: We expressed two genetically-encoded fluorescent sensors for Vmem and pHi, ArcLight and pHluorin-Moesin, in the follicular epithelium of Drosophila. By means of the respective inhibitors, we obtained comparable effects on Vmem and/or pHi as previously described for Vmem- and pHi-sensitive fluorescent dyes. In a RNAi-knockdown screen, five genes of ion-transport mechanisms and gap-junction subunits were identified exerting influence on ovary development and/or oogenesis. Loss of ovaries or small ovaries were the results of soma knockdowns of the innexins inx1 and inx3, and of the DEG/ENaC family member ripped pocket (rpk). Germline knockdown of rpk also resulted in smaller ovaries. Soma knockdown of the V-ATPase-subunit vha55 caused size-reduced ovaries with degenerating follicles from stage 10A onward. In addition, soma knockdown of the open rectifier K+channel 1 (ork1) resulted in a characteristic round-egg phenotype with altered microfilament and microtubule organisation in the follicular epithelium. Conclusions: The genetic tool box of Drosophila provides means for a refined and extended analysis of bioelectrical phenomena. Tissue-specifically expressed Vmem- and pHi-sensors exhibit some practical advantages compared to fluorescent indicator dyes. Their use confirms that the ion-transport mechanisms targeted by inhibitors play important roles in the generation of bioelectrical signals. Moreover, modulation of bioelectrical signals via RNAi-knockdown of genes coding for ion-transport mechanisms and gap-junction subunits exerts influence on crucial processes during ovary development and results in cytoskeletal changes and altered follicle shape. Thus, further evidence amounts for bioelectrical regulation of developmental processes via the control of both signalling pathways and cytoskeletal organisation. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus causes chronic and relapsing infections, which may be difficult to treat. So-called small colony variants (SCVs) have been associated with chronic infections and their occurrence has been shown to increase under antibiotic pressure, low pH and intracellular localization. In clinics, S. aureus isolated from invasive infections often show a dysfunction in the accessory gene regulator (agr), a major virulence regulatory system in S. aureus. To assess whether intracellular environment and agr function influence SCV formation, an infection model was established using lung epithelial cells and skin fibroblasts. This allowed analyzing intracellular survival and localization of a panel of S. aureus wild type strains and their isogenic agr knock out mutants as well as a natural dysfunctional agr strain by confocal laser scanning microscopy (CLSM). Furthermore, bacterial colonies were quantified after 1, 3, and 5 days of intracellular survival by time-lapse analysis to determine kinetics of colony appearance and SCV formation. Here, we show that S. aureus strains with an agr knock out predominantly resided in a neutral environment, whereas wild type strains and an agr complemented strain resided in an acidic environment. S. aureus agr mutants derived from an intracellular environment showed a higher percentage of SCVs as compared to their corresponding wild type strains. Neutralizing acidic phagolysosomes with chloroquine resulted in a significant reduction of SCVs in S. aureus wild type strain 6850, but not in its agr mutant indicating a pH dependent formation of SCVs in the wild type strain. The in-depth understanding of the interplay between intracellular persistence, agr function and pH should help to identify new therapeutic options facilitating the treatment of chronic S. aureus infections in the future. [ABSTRACT FROM AUTHOR]

Mammalian Na+/H+ exchanger type I isoform (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH (pHi) by removing one intracellular proton in exchange for one extracellular sodium ion. Abnormal activity of the protein occurs in cardiovascular disease and breast cancer. The purpose of this study is to examine the role of negatively charged amino acids of extracellular loop 3 (EL3) in the activity of the NHE protein. We mutated glutamic acid 217 and aspartic acid 226 to alanine, and to glutamine and asparagine, respectively. We examined effects on expression levels, cell surface targeting and activity of NHE1, and also characterized affinity for extracellular sodium and lithium ions. Individual mutation of these amino acids had little effect on protein function. However, mutation of both these amino acids together impaired transport, decreasing the Vmax for both Na+ and Li+ ions. We suggested that amino acids E217 and D226 form part of a negatively charged coordination sphere, which facilitates cation transport in the NHE1 protein. [ABSTRACT FROM AUTHOR]

Swelling of secretory vesicles is critical for the regulated release of intra-vesicular contents from cells during secretion. At the secretory vesicle membrane of the exocrine pancreas and neurons, GTP-binding G proteins, vH+-ATPase, potassium channels and AQP water channels, are among the players implicated in vesicle volume regulation. Here we report in the endocrine insulin-secreting MIN6 cells, the similar requirement of vH+-ATPase-mediated intracellular acidification on glucose-stimulated insulin release. MIN6 cells exposed to the vH+-ATPase inhibitor Bafilomycin A show decreased acidification of the cytosolic compartment that include insulin-carrying granules. Additionally, a loss of insulin granules near the cell plasma membrane following Bafilomycin A treatment, suggests impaired transport of insulin granules and consequent decrease in glucose-stimulated insulin secretion and accumulation of intracellular insulin. These results suggest that vH+-ATPase-mediated intracellular acidification is required for insulin secretion in beta cells. [ABSTRACT FROM AUTHOR]

Temperature-dependency on cell membrane injury and inactivation of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles (MBCO2) was investigated. The number of surviving S. pastorianus cells after MBCO2 treatment detected with yeast and mould agar (YMA, an optimum agar) was higher than that with YMA adding 2.5 g/L sodium chloride and yeast nitrogen base agar (a minimum agar). However, the decrease of the surviving number by thermal treatment was not changed among above agars used. The fluorescence polarization (FP), which indicated the phase transition of the membrane of S. pastorianus cells treated with MBCO2 increased with increasing temperature. The activity of the alkaline phosphatase (AP), a periplasmic enzyme, in S. pastorianus cells after MBCO2 and thermal treatments increased with the FP but was reduced by further increasing temperature. The FP and AP activities after MBCO2 treatment increased at a temperature lower than the temperature of the thermal treatment. In addition, intracellular pH of S. pastorianus decreased by the MBCO2 treatment at lower temperature with increasing pressure. Therefore, it was revealed that phase transition of the cell membrane and inactivation of S. pastorianus was caused by MBCO2 treatment at lower temperature than thermal treatment and that the effect was induced by the dissolved CO2 and increased with increasing pressure. [ABSTRACT FROM AUTHOR]

Soluble adenylyl cyclase (sAC) is the evolutionarily most ancient of a set of 10 adenylyl cyclases (Adcys). While Adcy1 to Adcy9 are cAMP-producing enzymes that are activated by G-protein coupled receptors (GPCRs), Adcy10 (sAC) is an intracellular adenylyl cyclase. sAC plays a pivotal role in numerous cellular processes, ranging from basic physiological functions to complex signaling cascades. As a distinct member of the adenylyl cyclase family, sAC is not activated by GPCRs and stands apart due to its unique characteristics, regulation, and localization within cells. This minireview aims to honour Ulli Brandt, the outgoing Executive Editor of our journal, Biochimica Biophysica Acta (BBA), and longstanding Executive Editor of the BBA section Bioenergetics. We will therefore focus this review on bioenergetic aspects of sAC and, in addition, review some important recent general developments in the field of research on sAC. • This is a mini-review to honour Ulli Brandt, the resigning Editor in Chief • The review emphasizes regulatory bioenergetic roles of sAC and other important novel findings on this enzyme [ABSTRACT FROM AUTHOR]

Bifidobacterium bifidum rarely utilises yeast extract, which causes significant challenges in its industrial production. This study characterised the utilisation of amino acids by B. bifidum to improve the viability of the strain. In this study, the proliferation effect of B. bifidum in different nitrogen sources was determined, and the amino acid contents of different nitrogen sources were analysed, which indicated that hydrophobic amino acids may inhibit B. bifidum growth. Among different hydrophobic amino acids, methionine was identified as the strongest growth inhibitor. Furthermore, the intracellular pH and NH 4 + contents of B. bifidum CCFM16 were higher in methionine-free medium. Among the NH 3 -producing enzymes related to amino acid metabolism, the metC gene was significantly transcribed in methionine-free medium. In addition, tcyP , metB , metC and metE genes encoding cysteine transporter proteins and the conversion pathway of cysteine to methionine were transcribed 2.03-fold, 7.03-fold, 3.05-fold and 2.92-fold, respectively, in methionine-free medium compared to methionine-containing medium. Compared with methionine-containing medium, the activity of tricarboxylic acid cycle rate-limited enzyme isocitrate dehydrogenase in methionine-free medium also increased from 198.89 to 410.56 nmol/min/mg protein, which probably enhanced the carbon flux and promoted growth. Meanwhile, the activity of pyrroline-5-carboxylate reductase, an enzyme that synthesises proline, increased from 14.76 to 35.28 nmol/min/mg protein, which promoted proline synthesis in response to osmotic stress. Cyclopropane fatty acids were also significantly higher in methionine-free medium, which may promote growth by decreasing cell membrane permeability to protons and enhancing cell membrane fluidity. This study provides a theoretical basis for high-density culture of B. bifidum. [Display omitted] • Methionine decreases the intracellular pH of Bifidobacterium bifidum. • Methionine-free medium enhanced carbon metabolism and proline anabolism. • Proline is a main compatible solute synthesized to resist acid stress. • Cyclopropane fatty acids stimulate better adaptation of strains to acid stress. [ABSTRACT FROM AUTHOR]

The alteration of the fine-tuned balance of phospho/dephosphorylation reactions in the cell often results in functional disturbance. In the yeast Saccharomyces cerevisiae, the overexpression of Ser/Thr phosphatase Ppz1 drastically blocks cell proliferation, with a profound change in the transcriptomic and phosphoproteomic profiles. While the deleterious effect on growth likely derives from the alteration of multiple targets, the precise mechanisms are still obscure. Ppz1 is a negative effector of potassium influx. However, we show that the toxic effect of Ppz1 overexpression is unrelated to the Trk1/2 high-affinity potassium importers. Cells overexpressing Ppz1 exhibit decreased K+ content, increased cytosolic acidification, and fail to properly acidify the medium. These effects, as well as the growth defect, are counteracted by the deletion of NHA1 gene, which encodes a plasma membrane Na+, K+/H+ antiporter. The beneficial effect of a lack of Nha1 on the growth vanishes as the pH of the medium approaches neutrality, is not eliminated by the expression of two non-functional Nha1 variants (D145N or D177N), and is exacerbated by a hyperactive Nha1 version (S481A). All our results show that high levels of Ppz1 overactivate Nha1, leading to an excessive entry of H+ and efflux of K+, which is detrimental for growth.

Cancer stem cells (CSCs) are known for their superior tumor-initiating and tumor-repopulating potential, partly reflecting their pronounced ability to evade immune recognition. Liu and colleagues recently identified a new aldehyde dehydrogenase (ALDH)-dependent mechanism whereby triple-negative breast CSCs evade immunosurveillance upon recruitment of myeloid-derived suppressor cells. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) patients and CF mouse models have increased risk for gastrointestinal tumors. CF mice show augmented intestinal proliferation of unknown etiology and an altered intestinal environment. We examined the role of the cystic fibrosis transmembrane conductance regulator (Cftr) in Wnt/β-catenin signaling, stem cell proliferation, and its functional expression in the active intestinal stem cell (ISC) population. Dysregulation of intracellular pH (pHi) in CF ISCs was investigated for facilitation of Wnt/β-catenin signaling. Methods: Crypt epithelia from wild-type (WT) and CF mice were compared ex vivo and in intestinal organoids (enteroids) for proliferation and Wnt/β-catenin signaling by standard assays. Cftr in ISCs was assessed by immunoblot of sorted Sox9enhanced green fluorescent protein(EGFP) intestinal epithelia and pHi regulation by confocal microfluorimetry of leucine-rich G-protein–coupled receptor 5 ISCs. Plasma membrane association of the Wnt transducer Dishevelled 2 (Dvl2) was assessed by fluorescence imaging of live enteroids from WT and CF mice crossed with Dvl2-EGFP/ACTB-tdTomato,-EGFP)Luo/J (RosamT/mG) mice. Results: Relative to WT, CF intestinal crypts showed an ∼30% increase in epithelial and Lgr5+ ISC proliferation and increased Wnt/β-catenin signaling. Cftr was expressed in Sox9EGFPLo ISCs and loss of Cftr induced an alkaline pHi in ISCs. CF crypt-base columnar cells showed a generalized increase in plasma membrane Dvl2-EGFP association as compared with WT. Dvl2-EGFP membrane association was charge- and pH-dependent and increased in WT crypt-base columnar cells by Cftr inhibition. Conclusions: CF intestine shows increased ISC proliferation and Wnt/β-catenin signaling. Loss of Cftr increases pHi in ISCs, which stabilizes the plasma membrane association of the Wnt transducer Dvl, likely facilitating Wnt/β-catenin signaling. Absence of Cftr-dependent suppression of ISC proliferation in the CF intestine may contribute to increased risk for intestinal tumors.

Neutrophils provide rapid and efficient defense mechanisms against invading pathogens. Upon stimulation with proinflammatory mediators, including complement factors and bacterial peptides, neutrophils respond with changes in their membrane potential, intracellular pH, and cellular size. This study provides an approach to quantify these important changes simultaneously using multiparametric flow cytometry, thereby revealing a typical sequence of neutrophil activation consisting of depolarization, alkalization, and increase in cellular size. Additionally, the time resolution of the flow cytometric measurement is improved in order to allow changes that occur within seconds to be monitored, and thus to enhance the kinetic analysis of the neutrophil response. The method is appropriate for the reliable semiquantitative detection of small variations with respect to an increase, no change, and decrease in those parameters as demonstrated by the screening of various proinflammatory mediators. As a translational outlook, the findings are put into context in inflammatory conditions in vitro as well as in a clinically relevant whole blood model of endotoxemia. Taken together, the multiparametric analysis of neutrophil responsiveness regarding depolarization, alkalization, and changes in cellular size may contribute to a better understanding of neutrophils in health and disease, thus potentially yielding innovative mechanistic insights and possible novel diagnostic and/or prognostic approaches.

Intracellular pH (pHi) plays a crucial role in mammalian sperm physiology. However, it is a challenging task to acquire quantitative single sperm pHi images due to their small size and beating flagella. In this study, we established a robust pHi imaging system using the dual-emission ratiometric pH indicator, SNARF-5F. Simultaneous good signal/noise ratio fluorescence signals were obtained exciting with a green high-power LED (532 nm) and acquiring with an EM-CCD camera through an image splitter with two band-pass filters (550–600 nm, channel 1; 630–650 nm, channel 2). After in vivo calibration, we established an imaging system that allows determination of absolute pHi values in spermatozoa, minimizing cell movement artifacts. Using this system, we determined that bicarbonate increases non-capacitated human pHi with slower kinetics than in mouse spermatozoa. This difference suggests that distinct ionic transporters might be involved in the bicarbonate influx into human and mouse spermatozoa. Alternatively, pHi regulation downstream bicarbonate influx into spermatozoa could be different between the two species.

Sulfur dioxide (SO2) is a colorless and irritating gas. Recent studies indicate that SO2 acts as the gas signal molecule and inhibits vascular smooth muscle cell (VSMC) proliferation. Cell proliferation depends on intracellular pH (pHi). Transmembrane cystein mutation of Na+- independent Cl-/HCO3- exchanger (anion exchanger, AE) affects pHi. However, whether SO2 inhibits VSMC proliferation by reducing pHi is still unknown. Here, we investigated whether SO2 reduced pHi to inhibit the proliferation of VSMCs and explore its molecular mechanisms. Within a range of 50–200 μM, SO2 was found to lower the pHi in VSMCs. Concurrently, NH4Cl pre-perfusion showed that SO2 significantly activated AE, whereas the AE inhibitor 4,4′-diisothiocyanatostilbene- 2,20-disulfonic acid (DIDS) significantly attenuated the effect of SO2 on pHi in VSMCs. While 200 μM SO2 sulphenylated AE2, while dithiothreitol (DTT) blocked the sulphenylation of AE2 and subsequent AE activation by SO2, thereby restoring the pHi in VSMCs. Furthermore, DIDS pretreatment eliminated SO2-induced inhibition of PDGF-BB-stimulated VSMC proliferation. We report for the first time that SO2 inhibits VSMC proliferation in part by direct activation of the AE via posttranslational sulphenylation and induction of intracellular acidification.

The antifungal activity of fisetin against Candida albicans is explored, elucidating a mechanism centered on membrane permeabilization and ensuing disruption of pH homeostasis. The Minimum Inhibitory Concentration (MIC) of fisetin, indicative of its interaction with the fungal membrane, increases in the presence of ergosterol. Hoechst 33342 and propidium-iodide staining reveal substantial propidium-iodide accumulation in fisetin-treated C. albicans cells at their MIC, with crystal violet uptake assays confirming fisetin-induced membrane permeabilization. Leakage analysis demonstrates a significant release of DNA and proteins in fisetin-treated cells compared to controls, underscoring the antifungal effect through membrane disruption. Green fluorescence, evident in both the cytoplasm and vacuoles of fisetin-treated cells under BCECF, AM staining, stands in contrast to controls where only acidic vacuoles exhibit staining. Ratiometric pH measurements using BCECF, AM reveal a noteworthy reduction in intracellular pH in fisetin-treated cells, emphasizing its impact on pH homeostasis. DiBAC 4 (3) uptake assays demonstrate membrane hyperpolarization in fisetin-treated cells, suggesting potential disruptions in ion flux and cellular homeostasis. These results provide comprehensive insights into the antifungal mechanisms of fisetin, positioning it as a promising therapeutic agent against Candida infections.

Climate change threatens the survival of symbiotic cnidarians by causing photosymbiosis breakdown in a process known as bleaching. Direct effects of temperature on cnidarian host physiology remain difficult to describe because heatwaves depress symbiont performance, leading to host stress and starvation. The symbiotic sea anemone Exaiptasia diaphana provides an opportune system to disentangle direct versus indirect heat effects on the host, as it can survive indefinitely without symbionts. We tested the hypothesis that heat directly impairs cnidarian physiology by comparing symbiotic and aposymbiotic individuals of two laboratory subpopulations of a commonly used clonal strain of E. diaphana, CC7. We exposed anemones to a range of temperatures (ambient, +2°C, +4°C and +6°C) for 15-18 days, then measured their symbiont population densities, autotrophic carbon assimilation and translocation, photosynthesis, respiration and host intracellular pH (pHi). Symbiotic anemones from the two subpopulations differed in size and symbiont density and exhibited distinct heat stress responses, highlighting the importance of acclimation to different laboratory conditions. Specifically, the cohort with higher initial symbiont densities experienced dose-dependent symbiont loss with increasing temperature and a corresponding decline in host photosynthate accumulation. In contrast, the cohort with lower initial symbiont densities did not lose symbionts or assimilate less photosynthate when heated, similar to the response of aposymbiotic anemones. However, anemone pHi decreased at higher temperatures regardless of cohort, symbiont presence or photosynthate translocation, indicating that heat consistently disrupts cnidarian acid-base homeostasis independent of symbiotic status or mutualism breakdown. Thus, pH regulation may be a critical vulnerability for cnidarians in a changing climate., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Much evidence supports the view that hypoxic/ischemic injury is largely due to increased intracellular Ca concentration ([Ca](i)) resulting from 1) decreased intracellular pH (pH(i)), 2) stimulated Na/H exchange that increases Na uptake and thus intracellular Na (Na(i)), and 3) decreased Na gradient that decreases or reverses net Ca transport via Na/Ca exchange. The Na/H exchanger (NHE) is also stimulated by hypertonic solutions; however, hypertonic media may inhibit NHE's response to changes in pH(i) (Cala PM and Maldonado HM. J Gen Physiol 103: 1035-1054, 1994). Thus we tested the hypothesis that hypertonic perfusion attenuates acid-induced increases in Na(i) in myocardium and, thereby, decreases Ca(i) accumulation during hypoxia. Rabbit hearts were Langendorff perfused with HEPES-buffered Krebs-Henseleit solution equilibrated with 100% O(2) or 100% N(2). Hypertonic perfusion began 5 min before hypoxia or normoxic acidification (NH(4)Cl washout). Na(i), [Ca](i), pH(i), and high-energy phosphates were measured by NMR. Control solutions were 295 mosM, and hypertonic solutions were adjusted to 305, 325, or 345 mosM by addition of NaCl or sucrose. During 60 min of hypoxia (295 mosM), Na(i) rose from 22+/-1 to 100+/-10 meq/kg dry wt while [Ca](i) rose from 347+/-11 to 1,306+/-89 nM. During hypertonic hypoxic perfusion (325 mosM), increases in Na(i) and [Ca](i) were reduced by 65 and 60%, respectively (P

Objective: This study examined the effects of methyl mercaptan, a product of the bacterial putrefaction of protein in periodontal pockets, on the function of cells in culture. Method and materials: Human gingival fibroblasts and periodontal ligament cells were exposed to a constant, continuous flow of methyl mercaptan in vitro. Control and test cultures were then examined for changes in intracellular pH, an event often associated with alterations in cellular function. Intracellular pH was determined by single-cell image analysis of cells loaded with a fluorescent, pH-sensitive dye. Periodontal ligament cells were also tested for changes in synthesis of total protein and fibronectin. Results: Test cells exhibited a consistent decrease in intracellular pH following exposure to methyl mercaptan. Measurements of total protein production showed that test periodontal ligament cell cultures produced approximately 30% less protein than control cultures (P < 0.05). Western-blot analysis of fibronectin in medium demonstrated that abnormal monomeric fibronectins were a major protein in test, but not in control, cell cultures. Conclusion: Exposure to methyl mercaptan induced alterations in intracellular events that paralleled changes in extracellular matrix proteins. The observed changes in extracellular matrix proteins support the hypothesis that methyl mercaptan contributes to the progression of periodontal disease. [ABSTRACT FROM AUTHOR]

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Background: During Drosophila oogenesis, the follicular epithelium differentiates into several morphologically distinct follicle-cell populations. Characteristic bioelectrical properties make this tissue a suitable model system for studying connections between electrochemical signals and the organisation of the cytoskeleton. Recently, we have described stage-specific transcellular antero-posterior and dorso-ventral gradients of intracellular pH (pHi) and membrane potential (Vmem) depending on the asymmetrical distribution and/or activity of various ion-transport mechanisms. In the present study, we analysed the patterns of basal microfilaments (bMF) and microtubules (MT) in relation to electrochemical signals. Results: The bMF- and MT-patterns in developmental stages 8 to 12 were visualised using labelled phalloidin and an antibody against acetylated α-tubulin as well as follicle-cell specific expression of GFP-actin and GFP-α-tubulin. Obviously, stage-specific changes of the pHi- and Vmem-gradients correlate with modifications of the bMF- and MT-organisation. In order to test whether cytoskeletal modifications depend directly on bioelectrical changes, we used inhibitors of ion-transport mechanisms that have previously been shown to modify pHi and Vmem as well as the respective gradients. We inhibited, in stage 10b, Na+/H+-exchangers and Na+-channels with amiloride, V-ATPases with bafilomycin, ATP-sensitive K+-channels with glibenclamide, voltage-dependent L-type Ca2+-channels with verapamil, Cl−-channels with 9-anthroic acid and Na+/K+/2Cl−-cotransporters with furosemide, respectively. The correlations between pHi, Vmem, bMF and MT observed in different follicle-cell types are in line with the correlations resulting from the inhibition experiments. While relative alkalisation and/or hyperpolarisation stabilised the parallel transversal alignment of bMF, acidification led to increasing disorder and to condensations of bMF. On the other hand, relative acidification as well as hyperpolarisation stabilised the longitudinal orientation of MT, whereas alkalisation led to loss of this arrangement and to partial disintegration of MT. Conclusions: We conclude that the pHi- and Vmem-changes induced by inhibitors of ion-transport mechanisms simulate bioelectrical changes occurring naturally and leading to the cytoskeletal changes observed during differentiation of the follicle-cell epithelium. Therefore, gradual modifications of electrochemical signals can serve as physiological means to regulate cell and tissue architecture by modifying cytoskeletal patterns. [ABSTRACT FROM AUTHOR]

Heart failure is the leading cause of death among diabetic people. Cellular and molecular entities leading to diabetic cardiomyopathy are, however, poorly understood. Coupling of cardiac carbonic anhydrase II (CAII) and Na+/H+ exchanger 1 (NHE1) to form a transport metabolon was analyzed in obese type 2 diabetic mice (ob −/− ) and control heterozygous littermates (ob +/− ). Echocardiography showed elevated systolic interventricular septum thickness and systolic posterior wall thickness in ob −/− mice at 9 and 16 weeks. ob −/− mice showed increased left ventricular (LV) weight/tibia length ratio and increased cardiomyocyte cross sectional area as compared to controls, indicating cardiac hypertrophy. Immunoblot analysis showed increased CAII expression in LV samples of ob −/− vs. ob +/− mice, and augmented Ser703 phosphorylation on NHE1 in ob −/− hearts. Reciprocal co-immunoprecipitation analysis showed strong association of CAII and NHE1 in LV samples of ob −/− mice. NHE1-dependent rate of intracellular pH (pH i) normalization after transient acid loading of isolated cardiomyocytes was higher in ob −/− mice vs. ob +/− . NHE transport activity was also augmented in cultured H9C2 rat cardiomyoblasts treated with high glucose/high palmitate, and it was normalized after CA inhibition. We conclude that the NHE1/CAII metabolon complex is exacerbated in diabetic cardiomyopathy of ob −/− mice, which may lead to perturbation of pH i and [Na+] and [Ca2+] handling in these diseased hearts. • Coupling of cardiac carbonic anhydrase II and Na+/H+ exchanger 1, analyzed in diabetic ob −/− mice. • ob −/− diabetic/obese mice showed altered echocardiographic recordings, cardiac hypertrophy • ob −/− hypertrophic and functionally impaired mice hearts displayed increased CAII expression • NHE1/CAII form a metabolon complex, which was exacerbated in diabetic cardiomyopathy of ob−/− [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of various cellular processes, including cell survival and apoptosis. Here, we report that Xenopus p42 MAPK becomes phosphorylated in apoptotic eggs, however this modification does not activate the enzyme. Using phosphorylation residue-specific antibodies, we demonstrate that this modification occurs on the Tyr residue in the MAPK activation segment, pinpointing the autophosphorylation mechanism. Notably, MAPK phosphorylation in apoptotic Xenopus eggs coincides with prominent intracellular acidification accompanying apoptosis in these cells. Furthermore, autophosphorylation of recombinant Xenopus MAPK is stimulated and phosphorylation of a protein substrate is inhibited under low pH conditions. Thus, acidic intracellular conditions inactivate MAPK and effectively disable the MAPK-mediated survival pathway in the apoptotic eggs. Given that cell acidification is a rather common feature of apoptosis, we hypothesize that stimulation of MAPK autophosphorylation and shutdown of the MAPK pathway may represent universal traits of apoptotic cell death. Image 1 • MAPK autophosphorylation is stimulated in apoptotic Xenopus eggs. • Phosphorylation of a MAPK substrate protein is inhibited in these cells. • Stimulation of MAPK autophosphorylation coincides with egg acidification. • Rearrangement of the MAPK activation segment occurs under acidic conditions. [ABSTRACT FROM AUTHOR]

Fermented cereal doughs constitute a predominant part of West African diets. The environment of fermented doughs can be hostile for microbial survival due to high levels of microbial metabolites such as weak carboxylic organic acids and ethanol. In order to get a better understanding of the intrinsic factors affecting the microbial successions of yeasts during dough fermentation, survival and physiological responses of the yeasts associated with West African fermented cereal doughs were investigated at exposure to relevant concentrations of microbial inhibitory compounds. Three strains each of the predominant species, i.e. Saccharomyces cerevisiae , Kluyveromyces marxianus , Pichia kudriavzevii as well as the opportunistic pathogen Candida glabrata were studied. The strains were exposed to individual stress factors of cereal doughs, i.e. (i) pH 3.4, (ii) 3% (v /v) ethanol (EtOH pH3.4), (iii) 285 mM lactic acid (LA pH3.4) and (iv) 150 mM acetic acid (AA pH3.4) as well as to combinations of these stress factors, i.e. (v) (LA + AA) pH 3.4 and (vi) (LA + AA+EtOH) pH 3.4. Growth and single cell viability were studied by flow cytometry using combined SYTO 13 and propidium iodide (PI) staining. Intracellular pH (pH i), plasma membrane integrity and micro-colony development of stressed cells were studied by fluorescence microscopy using PI and carboxyfluorescein diacetate succinimidyl ester (CFDA-se). Viability of the yeast strains was not affected by pH 3.4 and 3% (v /v) ethanol (EtOH pH3.4). 285 mM lactic acid (LA pH3.4) reduced the specific growth rate (μ max) from 0.27-0.41 h−1 to 0.11–0.26 h−1 and the viability from 100% to 2.6–41.7% at 72 h of exposure in most yeast strains, except for two strains of C. glabrata. 150 mM acetic acid (AA pH3.4) as well as the combinations (LA + AA) pH 3.4 and (LA + AA+EtOH) pH 3.4 reduced μ max to 0.0 h−1 and induced significant cell death for all the yeast strains. Exposed to (LA + AA+EtOH) pH 3.4 , the most resistant yeast strains belonged to S. cerevisiae followed by P. kudriavzevii , whereas C. glabrata and K. marxianus were more sensitive. Strain variations were observed within all four species. When transferred to non-stress conditions, i.e. MYGP, pH 5.6, after exposure to (LA + AA+EtOH) pH 3.4 for 6 h, 45% of the single cells of the most resistant S. cerevisiae strain kept their plasma membrane integrity, recovered their pH i to near physiological range (pH i = 6.1–7.4) and resumed proliferation after 3–24 h of lag phase. The results obtained are valuable in order to change processing conditions of the dough to favor the survival of preferable yeast species, i.e. S. cerevisiae and K. marxianus and inhibit opportunistic pathogen yeast species as C. glabrata. • The intrinsic stress factors of mawè doughs have a synergistic negative impact on yeast survival • Ethanol and pH did not affect yeast survival, while organic acids with ethanol at low pH strongly affected yeast survival • The sensitivity of the examined yeasts was species, strain and cell dependent • Saccharomyces cerevisiae strains were generally the least sensitive • pH i and membrane integrity are key physiological parameters for yeast cell sensitivity [ABSTRACT FROM AUTHOR]

The ciliary transport is controlled by two parameters of the ciliary beating, frequency (CBF) and amplitude. In this study, we developed a novel method to measure both CBF and ciliary bend distance (CBD, an index of ciliary beating amplitude) in ciliated human nasal epithelial cells (cHNECs) in primary culture, which are prepared from patients contracting allergic rhinitis and chronic sinusitis. An application of Cl−-free NO3− solution or bumetanide (an inhibitor of Na+/K+/2Cl− cotransport), which decreases intracellular Cl− concentration ([Cl−]i), increased CBD, not CBF, at 37 °C; however, it increased both CBD and CBF at 25 °C. Conversely, addition of Cl− channel blockers (5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and 4-[[4-Oxo-2-thioxo-3-[3-trifluoromethyl]phenyl]-5-thiazolidinylidene]methyl] benzoic acid (CFTR(inh)-172)), which increase [Cl−]i, decreased both CBD and CBF, suggesting that CFTR plays a crucial role for maintaining [Cl−]i in these cells. We speculate that Cl− modulates activities of the molecular motors regulating both CBD and CBF in cHNECs. Moreover, application of the CO2/HCO3−-free solution did not change intracellular pH (pHi), and addition of an inhibitor of carbonic anhydrase (acetazolamide) sustained pHi increase induced by the NH4+ pulse, which transiently increased pHi in the absence of acetazolamide. These results indicate that the cHNEC produces a large amount of CO2, which maintains a constant pHi even under the CO2/HCO3−-free condition. [ABSTRACT FROM AUTHOR]

Despite the improvement of diagnostic methods and anticancer therapeutics, the human population is still facing an increasing incidence of several types of cancers. According to the World Health Organization, this growing trend would be partly linked to our environment, with around 20% of cancers stemming from exposure to environmental contaminants, notably chemicals like polycyclic aromatic hydrocarbons (PAHs). PAHs are widespread pollutants in our environment resulting from incomplete combustion or pyrolysis of organic material, and thus produced by both natural and anthropic sources; notably benzo[a]pyrene (B[a]P), i.e. the prototypical molecule of this family, that can be detected in cigarette smoke, diesel exhaust particles, occupational-related fumes, and grilled food. This molecule is a well-recognized carcinogen belonging to group 1 carcinogens. Indeed, it can target the different steps of the carcinogenic process and all cancer hallmarks. Interestingly, H+ dynamics have been described as key parameters for the occurrence of several, if not all, of these hallmarks. However, information regarding the role of such parameters during environmental carcinogenesis is still very scarce. The present review will thus mainly give an overview of the impact of B[a]P on H+ dynamics in liver cells, and will show how such alterations might impact different aspects related to the finely-tuned balance between cell death and survival processes, thereby likely favoring environmental carcinogenesis. In total, the main objective of this review is to encourage further research in this poorly explored field of environmental molecular toxicology. • Benzo[a]pyrene, a well-known environmental carcinogen, alters H+ dynamics. • NHE1 activation by B[a]P plays a key role in H+ dynamics alterations. • B[a]P-altered pH controls cell death/survival balance via mitochondria and lysosome. • B[a]P impact on H+ dynamics fits with the malignant tumor-related H+ gradient reversal. • Altered H+ dynamics might be universal mechanisms in environmental carcinogenesis. [ABSTRACT FROM AUTHOR]

• A multifunctional single cell analysis platform for microoperation, microspectroscopy and microimaging was built. • We use the characteristic and controllable modification method of the laser induced deposition on the surface of fiber tip. • Cancer cell has a higher intracellular pH than the extracellular pH while the normal cell has a contrary trend. The accurate detection of intracellular and extracellular microenvironment at single cell is important for deep understanding of complicated cellular physiological activities, such as exploring organelle structures, and revealing the spatial heterogeneity of intracellular chemical components. Here, we built a single cell analysis platform by using a silver nanoparticles decorated optical fiber tip (diameter less than 500 nm) integrated with microoperation, microspectroscopy and microimaging, working for light propagation, sensing, cell manipulation, and in-situ spectroscopy and imaging, especially for exploring the subcellular microenvironment of a single cell. In view of the flexibility of fiber tip, the silver nanoparticles and the pH sensitive Raman reporters were both modified on the fiber tip surface by the one-step laser-induced Ag deposition method, and this optical fiber sensor was used for the accurate intracellular and extracellular pH detections by the surface-enhanced Raman spectroscopy (SERS) method. Cell heterogeneity according to their different pH microenvironments was disclosed by this optical fiber-based single cell analysis platform and it is also promising in learning biological processes within single cell, as well as monitoring intracellular responses during various external stimuli and therapies. [ABSTRACT FROM AUTHOR]

Measurements made in the blood, part of the extracellular compartment, are used in the clinical assessment of acid-base disorders; however, intracellular events determine much of the metabolic importance of these disorders. Intracellular and interstitial compartment acid-base balance is complex and varies in different tissues. This review considers the determination of extracellular pH in the context of ion transport processes at the interface of cells and the interstitial fluid, and between epithelial cells lining the transcellular contents of the gastrointestinal and urinary tracts that open to the external environment. A further consideration is the role of these membrane transporters in the generation of acid-base disorders and the associated disruption of electrolyte balance. This review suggests a process of internal and external balance for pH regulation similar to that of potassium, and considers the role of secretory gastrointestinal epithelia and renal epithelia with respect to normal pH homeostasis and clinical disorders. Electroneutrality of electrolytes in the extracellular fluid is a fundamental feature of reciprocal changes in Cl- or non-Cl- anions and HCO3-. Normal mechanisms for protecting cell pH and producing normal gastrointestinal and renal secretions in healthy states also may result in disease when abnormal. In a similar manner, organic anions such as ketoacid anions and lactate, normally transported as fuels between organs, result in acid-base disturbances in disease. Understanding the genomic basis of these transporters may contribute to specific treatments. [ABSTRACT FROM AUTHOR]

Although students initially learn of ionic buffering in basic chemistry, buffering and acid-base transport in biology often is relegated to specialized classes, discussions, or situations. That said, for physiology, nephrology, pulmonology, and anesthesiology, these basic principles often are critically important for mechanistic understanding, medical treatments, and assessing therapy effectiveness. This short introductory perspective focuses on basic chemistry and transport of buffers and acid-base equivalents, provides an outline of basic science acid-base concepts, tools used to monitor intracellular pH, model cellular responses to pH buffer changes, and the more recent development and use of genetically encoded pH-indicators. Examples of newer genetically encoded pH-indicators (pHerry and pHire) are provided, and their use for in vitro, ex vivo, and in vivo experiments are described. The continued use and development of these basic tools provide increasing opportunities for both basic and potentially clinical investigations. [ABSTRACT FROM AUTHOR]

• Photodynamic therapy combined with temozolomide can inhibit the expression of NHE1 protein. • The expression of NHE1 was dose-dependent on temozolomide. • Photodynamic therapy combined with temozolomide can inhibit the migration and invasion of C6 glioma cells. • Photodynamic therapy combined with temozolomide can influence mitochondrial associated apoptosis of C6 glioma cells. As a targeted therapeutic technique for glioma inhibition, photodynamic therapy (PDT) has gradually become a focus of basic research related to glioma treatment. The capacity of PDT to kill glioma cells involves varieties of pathways. In glioma cells, activated sodium-hydrogen exchanger isoform 1 (NHE1) can inhibit the cytotoxic effect of temozolomide (TMZ), promote cell migration and invasion, and inhibit cell apoptosis by changing the acid-base equilibrium. The purpose of our study was to explore if PDT combined with TMZ can effectively inhibit glioma cells by influencing NHE1 in vitro. We analyzed the expression levels of proteins such as NHE1, ezrin, vimentin, Bcl-2, and Bax by Western blot analysis, we assessed the migration and invasion of rat C6 glioma cells by Transwell assay, and we evaluated C6 cell apoptosis in vitro by flow cytometry. Western blot results indicated that NHE1, ezrin and vimentin were downregulated after cotreatment of C6 cells, and intracellular acidification was detected by a fluorometric intracellular pH assay. The migration and invasion capacities of C6 cells were significantly hindered after cotreatment, as shown by the Transwell assay. Experimental data also revealed a significant increase in cell apoptosis after cotreatment, as detected by flow cytometry; corresponding proapoptotic changes in Bcl-2, Bax and caspase-3 were also observed in vitro. These results demonstrate that PDT combined with TMZ can inhibit C6 cell migration and invasion and promote mitochondrial-associated apoptosis by inhibiting NHE1. Therefore, this study provides supporting evidence for a potential method for the treatment of glioma. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A lysosomal targeting naphthalimide-benzimidazole based probe has been designed and synthesized for detecting intracellular pH. • The portable kits are also designed to demonstrate the real time application of probe. • A cell based model study has been performed using lysomotropic regent chloroquine for determining lysosomal alkalinisation. • Probe is successfully applied to cells and nematodes results of which demonstrated the potential of probe for monitoring dynamic pH changes in living systems. Abstract A lysosomal targeting naphthalimide-benzimidazole based probe has been designed, synthesized and evaluated for detecting lysosomal pH changes inside living cells. The probe displays highly selective and sensitive response towards pH changes in aqueous medium. The sensing mechanism involves intramolecular charge transfer from benzimidazole moiety to naphthalimide core. The portable kits were also designed to demonstrate real time application of the probe. Probe 2 was successfully applied to cells and nematodes and all the findings emphasized that it can easily detect the lysosomal pH changes in living cells. To emphasize the ability of probe 2 for determining the lysosomal alkalinisation, a cell based model study was performed using lysomotropic regent chloroquine, results of which demonstrated that probe 2 has the potential for monitoring dynamic pH changes in living systems. [ABSTRACT FROM AUTHOR]

Acidic Golgi pH plays an important role in protein glycosylation, one of the critical quality attributes of therapeutic proteins. To determine the intracellular Golgi pH during culture, stable Chinese hamster ovary (CHO) cell clones expressing pHluorin2, a ratiometric pH‐sensitive fluorescent protein (FP), in the cis‐ and trans‐Golgi, were constructed by fusing pHluorin2 with specific targeting proteins, acetylglucosaminyltransferase, and a galactosyltransferase, respectively. Stable CHO cell clones expressing pHluorin2 in the cytoplasm were also constructed. The subcellular localization of FPs was confirmed by immunofluorescence analysis. Live‐cell imaging revealed that the intracellular pH (pHi) of clones expressing the ratiometric pH‐sensitive FPs converged to a specific pH range (cis‐Golgi: 6.4–6.5; trans‐Golgi: 5.9–6.0; and cytoplasm: 7.1–7.2). The pHi was successfully evaluated in various culture conditions. Although culture pH was maintained at 7.2 in a bioreactor, the Golgi pH increased with culture time. Elevated ammonia concentration and osmolality were partially responsible for the increased Golgi pH during bioreactor cultures. Taken together, the application of ratiometric pH‐sensitive FPs in monitoring the Golgi pH of CHO cells during culture provides a new perspective to improve protein glycosylation through pHi control. Acidic Golgi pH plays an important role in protein glycosylation, one of the critical quality attributes of therapeutic proteins. To determine the intracellular Golgi pH during culture, stable Chinese hamster ovary (CHO) cell clones expressing pHluorin2, a ratiometric pH‐sensitive fluorescent protein (FP), in the cis‐ and trans‐Golgi, were constructed by fusing pHluorin2 with specific targeting proteins, acetylglucosaminyltransferase, and a galactosyltransferase, respectively. Stable CHO cell clones expressing pHluorin2 in the cytoplasm were also constructed. [ABSTRACT FROM AUTHOR]

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca2+ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline‐rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2‐dependent pTyr cascade serves as a sensor for Ca 2+ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N‐[2‐[[[2‐[(2,3‐dihydro‐2‐oxo‐1 H‐indol‐5‐yl)amino]‐5‐(trifluoromethyl)‐4‐pyrimidinyl]amino]methyl]phenyl]‐ N‐methyl‐methanesulfonamide hydrate (PF431396) produced a significant and concentration‐dependent reduction in intracellular Ca 2+ levels during capacitation. Further studies revealed that PF431396‐treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca 2+ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca 2+ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca 2+ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH 4Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca 2+ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation. The present work reveals a novel mechanism of regulation of CatSper, a key polymodal sperm‐specific Ca2+ channel proved to be essential for mammalian sperm function and male fertility. Our results show that capacitation‐associated protein tyrosine phosphorylation (pTyr) acts as an intracellular sensor for Ca 2+ entry in human sperm through regulation of pH‐induced activation of CatSper. [ABSTRACT FROM AUTHOR]

Sulfur dioxide (SO2) is a colorless and irritating gas. Recent studies indicate that SO2 acts as the gas signal molecule and inhibits vascular smooth muscle cell (VSMC) proliferation. Cell proliferation depends on intracellular pH (pHi). Transmembrane cystein mutation of Na+- independent Cl-/HCO3- exchanger (anion exchanger, AE) affects pHi. However, whether SO2 inhibits VSMC proliferation by reducing pHi is still unknown. Here, we investigated whether SO2 reduced pHi to inhibit the proliferation of VSMCs and explore its molecular mechanisms. Within a range of 50–200 μM, SO2 was found to lower the pHi in VSMCs. Concurrently, NH4Cl pre-perfusion showed that SO2 significantly activated AE, whereas the AE inhibitor 4,4′-diisothiocyanatostilbene- 2,20-disulfonic acid (DIDS) significantly attenuated the effect of SO2 on pHi in VSMCs. While 200 μM SO2 sulphenylated AE2, while dithiothreitol (DTT) blocked the sulphenylation of AE2 and subsequent AE activation by SO2, thereby restoring the pHi in VSMCs. Furthermore, DIDS pretreatment eliminated SO2-induced inhibition of PDGF-BB-stimulated VSMC proliferation. We report for the first time that SO2 inhibits VSMC proliferation in part by direct activation of the AE via posttranslational sulphenylation and induction of intracellular acidification. [ABSTRACT FROM AUTHOR]

Abstract SLC4A11 mutations are associated with Fuchs' endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). Mice with genetically ablated Slc4a11 recapitulate CHED, exhibiting significant corneal edema and altered endothelial morphology. We recently demonstrated that SLC4A11 functions as an NH 3 sensitive, electrogenic H+ transporter. Here, we investigated the properties of five clinically relevant SLC4A11 mutants: R125H, W240S, C386R, V507I and N693A, relative to wild type, expressed in a PS120 fibroblast cell line. The effect of these mutations on the NH 4 Cl-dependent transporter activity was investigated by intracellular pH and electrophysiology measurements. Relative to plasma membrane expression of Na K ATPase, there were no significant differences in plasma membrane SLC4A11 expression among each mutant and wild type. All mutants revealed a marked decrease in acidification in response to NH 4 Cl when compared to wild type, indicating a decreased H+ permeability in mutants. All mutants exhibited significantly reduced H+ currents at negative holding potentials as compared to wild type. Uniquely, the C386R and W240S mutants exhibited a different inward current profile upon NH 4 Cl challenges, suggesting an altered transport mode. Thus, our data suggest that these SLC4A11 mutants, rather than having impaired protein trafficking, show altered H+ flux properties. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract The activity of many cells is closely related to the pH of their internal environment, and the increase of intracellular pH value is a common feature of many drug-resistant tumors. The intracellular pH needs to be determined as the research basis when studying the intracellular transport protein and ion concentration changes. Nanomaterials are now widely used as the effective imaging and drug delivery vehicles, but it remains to be studied as a sensor for intracellular environment. Based on this, we have constructed a nanoprobe with fluorescence and surface-enhanced Raman scattering (SERS) dual-signal for sensing intracellular pH. The principle of this strategy is to construct fluorescence-SERS dual-signal nanoprobes by modifying pH-responsive fluorescent probes and SERS reporter molecules on the surface of gold nanorods (AuNRs) with the core-shell structure. The fluorescence-SERS dual response of the nanoprobe to pH can be achieved by investigating the fluorescence and SERS spectra of nanoprobes at different pH. Moreover, by incubating the nanoprobes into different cells, different double-signal response results can be obtained, thereby achieving intracellular pH sensing. The nanoprobe has dual responsiveness to fluorescence and SERS, which makes up for many of the deficiencies of single-signal probes, and realizes high sensitivity, accuracy and stability of intracellular pH detection. It is expected to be widely applied in the fields of medicine, chemistry and biology. [ABSTRACT FROM AUTHOR]

β-catenin has roles in cell-cell adhesion and Wnt signaling. We recently showed that β-catenin protein abundance is decreased at higher intracellular pH (pHi), mediated by pH-sensitive interaction with the beta-transducin repeat containing E3 ubiquitin protein ligase (β-TrCP). Increased pHi facilitates β-TrCP binding and degradation of β-catenin. β-catenin mutations that abrogate the pH-sensitive interaction induce significant tumors not seen with other β-catenin stabilizing mutants.

Oocyte maturation is a process that occurs in the ovaries, where an immature oocyte resumes meiosis to attain competence for normal fertilization after ovulation/spawning. In starfish, the hormone 1-methyladenine binds to an unidentified receptor on the plasma membrane of oocytes, inducing a conformational change in the heterotrimeric GTP-binding protein α-subunit (Gα), so that the α-subunit binds GTP in exchange of GDP on the plasma membrane. The GTP-binding protein βγ-subunit (Gβγ) is released from Gα, and the released Gβγ activates phosphatidylinositol-3 kinase (PI3K), followed by the target of rapamycin kinase complex2 (TORC2) and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-dependent phosphorylation of serum- and glucocorticoid-regulated kinase (SGK) of ovarian oocytes. Thereafter, SGK activates Na+/H+ exchanger (NHE) to increase the intracellular pH (pHi) from ~6.7 to ~6.9. Moreover, SGK phosphorylates Cdc25 and Myt1, thereby inducing the de-phosphorylation and activation of cyclin B−Cdk1, causing germinal vesicle breakdown (GVBD). Both pHi increase and GVBD are required for spindle assembly at metaphase I, followed by MI arrest at pHi 6.9 until spawning. Due to MI arrest or SGK-dependent pHi control, spawned oocytes can be fertilized normally

Prostate cancer and osteosarcoma are the second most common type of cancer affecting men and the fifth most common malignancy among adolescents, respectively. The use of non-toxic natural or natural-derived products has been one of the current strategies for cancer therapy, owing to the reduced risks of induced-chemoresistance development and the absence of secondary effects. In this perspective, lactoferrin (Lf), a natural protein derived from milk, emerges as a promising anticancer agent due to its well-recognized cytotoxicity and anti-metastatic activity. Here, we aimed to ascertain the potential activity of bovine Lf (bLf) against highly metastatic cancer cells. The bLf effect on prostate PC-3 and osteosarcoma MG-63 cell lines, both displaying plasmalemmal V-ATPase, was studied and compared with the breast cancer MDA-MB-231 and the non-tumorigenic BJ-5ta cell lines. Cell proliferation, cell death, intracellular pH, lysosomal acidification, and extracellular acidification rate were evaluated. Results show that bLf inhibits proliferation, induces apoptosis, intracellular acidification, and perturbs lysosomal acidification only in highly metastatic cancer cell lines. By contrast, BJ-5ta cells are insensitive to bLf. Overall, our results establish a common mechanism of action of bLf against highly metastatic cancer cells exhibiting plasmalemmal V-ATPase. This study opens promising perspectives for further research on the anticancer role of Lf, which ultimately will contribute to its safer and more rational application in the human therapy of these life-threatening cancers.