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An application of CO2/HCO3−-free solution (Zero-CO2) did not increase intracellular pH (pHi) in ciliated human nasal epithelial cells (c-hNECs), leading to no increase in frequency (CBF) or amplitude (CBA) of the ciliary beating. This study demonstrated that the pHi of c-hNECs expressing carbonic anhydrase IV (CAIV) is high (7.64), while the pHi of ciliated human bronchial epithelial cells (c-hBECs) expressing no CAIV is low (7.10). An extremely high pHi of c-hNECs caused pHi, CBF and CBA to decrease upon Zero-CO2 application, while a low pHi of c-hBECs caused them to increase. An extremely high pHi was generated by a high rate of HCO3− influx via interactions between CAIV and Na+/HCO3− cotransport (NBC) in c-hNECs. An NBC inhibitor (S0859) decreased pHi, CBF and CBA and increased CBF and CBA in c-hNECs upon Zero-CO2 application. In conclusion, the interactions of CAIV and NBC maximize HCO3− influx to increase pHi in c-hNECs. This novel mechanism causes pHi to decrease, leading to no increase in CBF and CBA in c-hNECs upon Zero-CO2 application, and appears to play a crucial role in maintaining pHi, CBF and CBA in c-hNECs periodically exposed to air (0.04% CO2) with respiration. [ABSTRACT FROM AUTHOR]

Single cilia, 100 nm in diameter and 10 µm in length, were isolated from mouse tracheae with Triton X-100 (0.02%) treatment, and the effects of pH on ciliary beating were examined by measuring the ciliary beat frequency (CBF) and the ciliary bend distance (CBD—an index of amplitude) using a high-speed video microscope (250 fps). ATP (2.5 mM) plus 8Br-cAMP (10 µM) reactivated the CBF and CBD in the isolated cilia, similar to the cilia of in vivo tracheae. In the reactivated isolated cilia, an elevation in pH from 7.0 to 8.0 increased the CBF from 3 to 15 Hz and the CBD from 0.6 to 1.5 µm. The pH elevation also increased the velocity of the effective stroke; however, it did not increase the recovery stroke, and, moreover, it decreased the intervals between beats. This indicates that H+ (pHi) directly acts on the axonemal machinery to regulate CBF and CBD. In isolated cilia priorly treated with 1 µM PKI-amide (a PKA inhibitor), 8Br-cAMP did not increase the CBF or CBD in the ATP-stimulated isolated cilia. pH modulates the PKA signal, which enhances the axonemal beating generated by the ATP-activated inner and outer dyneins. [ABSTRACT FROM AUTHOR]

Introduction: Bacteria frequently encounter nutrient limitation in nature. The ability of living in this nutrient shortage environment is vital for bacteria to preserve their population and important for some pathogenic bacteria to cause infectious diseases. Usually, we study how bacteria survive after nutrient depletion, a total starvation condition when bacteria almost cease growth and try to survive. However, nutrient limitation may not always lead to total starvation. Methods: Bacterial adaptation to nutrient shortage was studied by determining bacterial growth curves, intracellular pH, intracellular amino acid contents, gene transcription, protein expression, enzyme activity, and translation and replication activities. Results: No exogenous supply of methionine results in growth attenuation of Streptococcus pneumoniae, a human pathogen. In this paper, we refer to this inhibited growth state between ceased growth under total starvation and fullspeed growth with full nutrients as semi-starvation. Similar to total starvation, methionine semi-starvation also leads to intracellular acidification. Surprisingly, it is intracellular acidification but not insufficient methionine synthesis that causes growth attenuation under methionine semi-starvation. With excessive glutamine supply in the medium, intracellular methionine level was not changed, while bacterial intracellular pH was elevated to ~ 7.6 (the optimal intracellular pH for pneumococcal growth) by glutamine deamination, and bacterial growth under semi-starvation was restored fully. Our data suggest that intracellular acidification decreases translation level and glutamine supply increases intracellular pH to restore translation level, thus restoring bacterial growth. Discussion: This growth with intracellular pH adjustment by glutamine is a novel strategy we found for bacterial adaptation to nutrient shortage, which may provide new drug targets to inhibit growth of pathogenic bacteria under semi-starvation. [ABSTRACT FROM AUTHOR]

Cell-to-cell communication is fundamental to the organization and functionality of multicellular organisms. Intercellular signals orchestrate a variety of cellular responses, including gene expression and protein function changes, and contribute to the integrated functions of individual tissues. Dictyostelium discoideum is a model organism for cell-to-cell interactions mediated by chemical signals and multicellular formation mechanisms. Upon starvation, D. discoideum cells exhibit coordinated cell aggregation via cyclic adenosine 3′,5′-monophosphate (cAMP) gradients and chemotaxis, which facilitates the unicellular-to-multicellular transition. During this process, the calcium signaling synchronizes with the cAMP signaling. The resulting multicellular body exhibits organized collective migration and ultimately forms a fruiting body. Various signaling molecules, such as ion signals, regulate the spatiotemporal differentiation patterns within multicellular bodies. Understanding cell-to-cell and ion signaling in Dictyostelium provides insight into general multicellular formation and differentiation processes. Exploring cell-to-cell and ion signaling enhances our understanding of the fundamental biological processes related to cell communication, coordination, and differentiation, with wide-ranging implications for developmental biology, evolutionary biology, biomedical research, and synthetic biology. In this review, I discuss the role of ion signaling in cell motility and development in D. discoideum. [ABSTRACT FROM AUTHOR]

A unique feature of the tumor microenvironment is extracellular acidosis in relation to intracellular milieu. Metabolic reprogramming in tumors results in overproduction of H+ ions (and lactate), which are extruded from the cells to support tumor survival and progression. As a result, the transmembrane pH gradient (ΔpH), representing the difference between intracellular pH (pHi) and extracellular pH (pHe), is posited to be larger in tumors compared with normal tissue. Controlling the transmembrane pH difference has promise as a potential therapeutic target in cancer as it plays an important role in regulating drug delivery into cells. The current study shows successful development of an MRI/MRSI‐based technique that provides ΔpH imaging at submillimeter resolution. We applied this technique to image ΔpH in rat brains with RG2 and U87 gliomas, as well as in mouse brains with GL261 gliomas. pHi was measured with Amine and Amide Concentration‐Independent Detection (AACID), while pHe was measured with Biosensor Imaging of Redundant Deviation in Shifts (BIRDS). The results indicate that pHi was slightly higher in tumors (7.40–7.43 in rats, 7.39–7.47 in mice) compared with normal brain (7.30–7.38 in rats, 7.32–7.36 in mice), while pHe was significantly lower in tumors (6.62–6.76 in rats, 6.74–6.84 in mice) compared with normal tissue (7.17–7.22 in rats, 7.20–7.21 in mice). As a result, ΔpH was higher in tumors (0.64–0.81 in rats, 0.62–0.65 in mice) compared with normal brain (0.13–0.16 in rats, 0.13–0.16 in mice). This work establishes an MRI/MRSI‐based platform for ΔpH imaging at submillimeter resolution in gliomas. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM), in which collagen is the most abundant protein, impacts many aspects of tumor physiology, including cellular metabolism and intracellular pH (pHi), as well as the efficacy of chemotherapy. Meanwhile, the role of collagen in differential cell responses to treatment within heterogeneous tumor environments remains poorly investigated. In the present study, we simultaneously monitored the changes in pHi and metabolism in living colorectal cancer cells in vitro upon treatment with a chemotherapeutic combination, FOLFOX (5-fluorouracil, oxaliplatin and leucovorin). The pHi was followed using the new pH-sensitive probe BC-Ga-Ir, working in the mode of phosphorescence lifetime imaging (PLIM), and metabolism was assessed from the autofluorescence of the metabolic cofactor NAD(P)H using fluorescence lifetime imaging (FLIM) with a two-photon laser scanning microscope. To model the ECM, 3D collagen-based hydrogels were used, and comparisons with conventional monolayer cells were made. It was found that FOLFOX treatment caused an early temporal intracellular acidification (reduction in pHi), followed by a shift to more alkaline values, and changed cellular metabolism to a more oxidative state. The presence of unstructured collagen markedly reduced the cytotoxic effects of FOLFOX, and delayed and diminished the pHi and metabolic responses. These results support the observation that collagen is a factor in the heterogeneous response of cancer cells to chemotherapy and a powerful regulator of their metabolic behavior. [ABSTRACT FROM AUTHOR]

An application of CO2/HCO3−-free solution (Zero-CO2) did not increase intracellular pH (pHi) in ciliated human nasal epithelial cells (c-hNECs), leading to no increase in frequency (CBF) or amplitude (CBA) of the ciliary beating. This study demonstrated that the pHi of c-hNECs expressing carbonic anhydrase IV (CAIV) is high (7.64), while the pHi of ciliated human bronchial epithelial cells (c-hBECs) expressing no CAIV is low (7.10). An extremely high pHi of c-hNECs caused pHi, CBF and CBA to decrease upon Zero-CO2 application, while a low pHi of c-hBECs caused them to increase. An extremely high pHi was generated by a high rate of HCO3− influx via interactions between CAIV and Na+/HCO3− cotransport (NBC) in c-hNECs. An NBC inhibitor (S0859) decreased pHi, CBF and CBA and increased CBF and CBA in c-hNECs upon Zero-CO2 application. In conclusion, the interactions of CAIV and NBC maximize HCO3− influx to increase pHi in c-hNECs. This novel mechanism causes pHi to decrease, leading to no increase in CBF and CBA in c-hNECs upon Zero-CO2 application, and appears to play a crucial role in maintaining pHi, CBF and CBA in c-hNECs periodically exposed to air (0.04% CO2) with respiration.

Single cilia, 100 nm in diameter and 10 µm in length, were isolated from mouse tracheae with Triton X-100 (0.02%) treatment, and the effects of pH on ciliary beating were examined by measuring the ciliary beat frequency (CBF) and the ciliary bend distance (CBD—an index of amplitude) using a high-speed video microscope (250 fps). ATP (2.5 mM) plus 8Br-cAMP (10 µM) reactivated the CBF and CBD in the isolated cilia, similar to the cilia of in vivo tracheae. In the reactivated isolated cilia, an elevation in pH from 7.0 to 8.0 increased the CBF from 3 to 15 Hz and the CBD from 0.6 to 1.5 µm. The pH elevation also increased the velocity of the effective stroke; however, it did not increase the recovery stroke, and, moreover, it decreased the intervals between beats. This indicates that H+ (pHi) directly acts on the axonemal machinery to regulate CBF and CBD. In isolated cilia priorly treated with 1 µM PKI-amide (a PKA inhibitor), 8Br-cAMP did not increase the CBF or CBD in the ATP-stimulated isolated cilia. pH modulates the PKA signal, which enhances the axonemal beating generated by the ATP-activated inner and outer dyneins.

Cell-to-cell communication is fundamental to the organization and functionality of multicellular organisms. Intercellular signals orchestrate a variety of cellular responses, including gene expression and protein function changes, and contribute to the integrated functions of individual tissues. Dictyostelium discoideum is a model organism for cell-to-cell interactions mediated by chemical signals and multicellular formation mechanisms. Upon starvation, D. discoideum cells exhibit coordinated cell aggregation via cyclic adenosine 3′,5′-monophosphate (cAMP) gradients and chemotaxis, which facilitates the unicellular-to-multicellular transition. During this process, the calcium signaling synchronizes with the cAMP signaling. The resulting multicellular body exhibits organized collective migration and ultimately forms a fruiting body. Various signaling molecules, such as ion signals, regulate the spatiotemporal differentiation patterns within multicellular bodies. Understanding cell-to-cell and ion signaling in Dictyostelium provides insight into general multicellular formation and differentiation processes. Exploring cell-to-cell and ion signaling enhances our understanding of the fundamental biological processes related to cell communication, coordination, and differentiation, with wide-ranging implications for developmental biology, evolutionary biology, biomedical research, and synthetic biology. In this review, I discuss the role of ion signaling in cell motility and development in D. discoideum.

Fluorescent probes, typically based on the intramolecular charge transfer (ICT) mechanism, have received considerable research attention in cell detection due to their non-invasiveness, fast response, easy regulation, high sensitivity, and low damage tolerance for in vivo bio-samples. Generally, intracellular pH and biological species such as various gases, metal ions, and anions constitute the foundation of cells and participate in the basic physiological processes, whose abnormal level can lead to poisoning, cardiovascular disease, and cancer in living organisms. Therefore, monitoring of their quantity plays an essential role in understanding the status of organisms and preventing, diagnosing, and treating diseases. In the last decades, remarkable progress has been made in developing ICT probes forthe detection of biological elements. In this review, we highlight the recent ICT probes focusing primarily on the detection of intracellular pH, various gases (H2S, CO, H2O2, and NO), metal ions (Cu2+, Hg2+, Pb2+, Zn2+, and Al3+), and anions (ClO-, CN-, SO32-, and F-). In addition, we discuss the issues and limitations of ICT-based fluorescent probes for in vivo detection and explore the clinical translational potential and challenges of these materials, providing valuable guidance and insights for the design of fluorescent materials. [ABSTRACT FROM AUTHOR]

Ambroxol (ABX), a frequently prescribed secretolytic agent which enhances the ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of amplitude) by 30%, activates a voltage-dependent Ca2+ channel (CaV1.2) and a small transient Ca2+ release in the ciliated lung airway epithelial cells (c-LAECs) of mice. The activation of CaV1.2 alone enhanced the CBF and CBA by 20%, mediated by a pHi increasei and a [Cl−]i decrease in the c-LAECs. The increase in pHi, which was induced by the activation of the Na+-HCO3− cotransporter (NBC), enhanced the CBF (by 30%) and CBA (by 15–20%), and a decrease in [Cl−]i, which was induced by the Cl− release via anoctamine 1 (ANO1), enhanced the CBA (by 10–15%). While a Ca2+-free solution or nifedipine (an inhibitor of CaV1.2) inhibited 70% of the CBF and CBA enhancement using ABX, CaV1.2 enhanced most of the CBF and CBA increases using ABX. The activation of the CaV1.2 existing in the cilia stimulates the NBC to increase pHi and ANO1 to decrease the [Cl−]i in the c-LAECs. In conclusion, the pHi increase and the [Cl−]i decrease enhanced the CBF and CBA in the ABX-stimulated c-LAECs. [ABSTRACT FROM AUTHOR]

Action of numerous adverse environmental factors on higher plants is spatially-heterogenous; it means that induction of a systemic adaptive response requires generation and transmission of the stress signals. Electrical signals (ESs) induced by local action of stressors include action potential, variation potential, and system potential and they participate in formation of fast physiological changes at the level of a whole plant, including photosynthetic responses. Generation of these ESs is accompanied by the changes in activity of H+-ATPase, which is the main system of electrogenic proton transport across the plasma membrane. Literature data show that the changes in H+-ATPase activity and related changes in intra- and extracellular pH play a key role in the ES-induced inactivation of photosynthesis in non-irritated parts of plants. This inactivation is caused by both suppression of CO2 influx into mesophyll cells in leaves, which can be induced by the apoplast alkalization and, probably, cytoplasm acidification, and direct influence of acidification of stroma and lumen of chloroplasts on light and, probably, dark photosynthetic reactions. The ES-induced inactivation of photosynthesis results in the increasing tolerance of photosynthetic machinery to the action of adverse factors and probability of the plant survival. [ABSTRACT FROM AUTHOR]

Disruption of acid–base balance is linked to various diseases and conditions. In the heart, intracellular acidification is associated with heart failure, maladaptive cardiac hypertrophy, and myocardial ischemia. Previously, we have reported that the ratio of the in‐cell lactate dehydrogenase (LDH) to pyruvate dehydrogenase (PDH) activities is correlated with cardiac pH. To further characterize the basis for this correlation, these in‐cell activities were investigated under induced intracellular acidification without and with Na+/H+ exchanger (NHE1) inhibition by zoniporide. Male mouse hearts (n = 30) were isolated and perfused retrogradely. Intracellular acidification was performed in two ways: (1) with the NH4Cl prepulse methodology; and (2) by combining the NH4Cl prepulse with zoniporide. 31P NMR spectroscopy was used to determine the intracellular cardiac pH and to quantify the adenosine triphosphate and phosphocreatine content. Hyperpolarized [1‐13C]pyruvate was obtained using dissolution dynamic nuclear polarization. 13C NMR spectroscopy was used to monitor hyperpolarized [1‐13C]pyruvate metabolism and determine enzyme activities in real time at a temporal resolution of a few seconds using the product‐selective saturating excitation approach. The intracellular acidification induced by the NH4Cl prepulse led to reduced LDH and PDH activities (−16% and −39%, respectively). This finding is in line with previous evidence of reduced myocardial contraction and therefore reduced metabolic activity upon intracellular acidification. Concomitantly, the LDH/PDH activity ratio increased with the reduction in pH, as previously reported. Combining the NH4Cl prepulse with zoniporide led to a greater reduction in LDH activity (−29%) and to increased PDH activity (+40%). These changes resulted in a surprising decrease in the LDH/PDH ratio, as opposed to previous predictions. Zoniporide alone (without intracellular acidification) did not change these enzyme activities. A possible explanation for the enzymatic changes observed during the combination of the NH4Cl prepulse and NHE1 inhibition may be related to mitochondrial NHE1 inhibition, which likely negates the mitochondrial matrix acidification. This effect, combined with the increased acidity in the cytosol, would result in an enhanced H+ gradient across the mitochondrial membrane and a temporarily higher pyruvate transport into the mitochondria, thereby increasing the PDH activity at the expense of the cytosolic LDH activity. These findings demonstrate the complexity of in‐cell cardiac metabolism and its dependence on intracellular acidification. This study demonstrates the capabilities and limitations of hyperpolarized [1‐13C]pyruvate in the characterization of intracellular acidification as regards cardiac pathologies. [ABSTRACT FROM AUTHOR]

Many cancer cells, regardless of their tissue origin or genetic landscape, have increased expression or activity of the plasma membrane Na-H exchanger NHE1 and a higher intracellular pH (pHi) compared with untransformed cells. A current perspective that remains to be validated is that increased NHE1 activity and pHi enable a Warburg-like metabolic reprogramming of increased glycolysis and decreased mitochondrial oxidative phosphorylation. We tested this perspective and find it is not accurate for clonal pancreatic and breast cancer cells. Using the pharmacological reagent ethyl isopropyl amiloride (EIPA) to inhibit NHE1 activity and decrease pHi, we observe no change in glycolysis, as indicated by secreted lactate and intracellular pyruvate, despite confirming increased activity of the glycolytic enzyme phosphofructokinase-1 at higher pH. Also, in contrast to predictions, we find a significant decrease in oxidative phosphorylation with EIPA, as indicated by oxygen consumption rate (OCR). Decreased OCR with EIPA is not associated with changes in pathways that fuel oxidative phosphorylation or with mitochondrial membrane potential but occurs with a change in mitochondrial dynamics that includes a significant increase in elongated mitochondrial networks, suggesting increased fusion. These findings conflict with current paradigms on increased pHi inhibiting oxidative phosphorylation and increased oxidative phosphorylation being associated with mitochondrial fusion. Moreover, these findings raise questions on the suggested use of EIPA-like compounds to limit metabolic reprogramming in cancer cells.

Fluorescent probes, typically based on the intramolecular charge transfer (ICT) mechanism, have received considerable research attention in cell detection due to their non-invasiveness, fast response, easy regulation, high sensitivity, and low damage tolerance for in vivo bio-samples. Generally, intracellular pH and biological species such as various gases, metal ions, and anions constitute the foundation of cells and participate in the basic physiological processes, whose abnormal level can lead to poisoning, cardiovascular disease, and cancer in living organisms. Therefore, monitoring of their quantity plays an essential role in understanding the status of organisms and preventing, diagnosing, and treating diseases. In the last decades, remarkable progress has been made in developing ICT probes for the detection of biological elements. In this review, we highlight the recent ICT probes focusing primarily on the detection of intracellular pH, various gases (H2S, CO, H2O2, and NO), metal ions (Cu2+, Hg2+, Pb2+, Zn2+, and Al3+), and anions (ClO−, CN−, SO32−, and F−). In addition, we discuss the issues and limitations of ICT-based fluorescent probes for in vivo detection and explore the clinical translational potential and challenges of these materials, providing valuable guidance and insights for the design of fluorescent materials.

The fermentation process of milk to yoghurt using Lactobacillus delbrueckii subsp. bulgaricus in co-culture with Streptococcus thermophilus is hallmarked by the breakdown of lactose to organic acids such as lactate. This leads to a substantial decrease in pH - both in the medium, as well as cytosolic. The latter impairs metabolic activities due to the pH-dependence of enzymes, which compromises microbial growth. To quantitatively elucidate the impact of the acidification on metabolism of L. bulgaricus in an integrated way, we have developed a proton-dependent computational model of lactose metabolism and casein degradation based on experimental data. The model accounts for the influence of pH on enzyme activities as well as cellular growth and proliferation of the bacterial population. We used a machine learning approach to quantify the cell volume throughout fermentation. Simulation results show a decrease in metabolic flux with acidification of the cytosol. Additionally, the validated model predicts a similar metabolic behaviour within a wide range of non-limiting substrate concentrations. This computational model provides a deeper understanding of the intricate relationships between metabolic activity and acidification and paves the way for further optimization of yoghurt production under industrial settings. • Kinetic model that takes cell culture growth into account. • Integration of new experimental data sets. • Potential to predict acidification during batch culture growth. [ABSTRACT FROM AUTHOR]

Purpose: The accumulation of carbon dioxide during large-scale culture of animal cells brings adverse effects, appropriate aeration strategies alleviate CO2 accumulation while improper reactor operation may lead to the presence of low CO2 partial pressure (pCO2) condition as occurs in many industrial cases. Thus, this study aims to reveal the in-depth influence of low pCO2 on Chinese Hamster Ovary (CHO) cells for providing a reference for design space determination of CO2 control with regard to the Quality by Design (QbD) guidelines. Methods and results: The headspace air over purging caused the ultra-low pCO2 (ULC) where the monoclonal antibody production as well as the aerobic metabolic activity were reduced. Intracellular metabolomics analysis indicated a less efficient aerobic glucose metabolic state under ULC conditions. Based on the increase of intracellular pH and lactate dehydrogenase activity, the shortage of intracellular pyruvate could be the cause of the deficient aerobic metabolism, which could be partially mitigated by pyruvate addition under ULC conditions. Finally, a semi-empirical mathematical model was used to better understand, predict and control the occurrence of extreme pCO2 conditions during the cultures of CHO cells. Conclusion: Low pCO2 steers CHO cells into a defective metabolic state. A predictive relation among pCO2, lactate, and pH control was applied to get new insights into CHO cell culture for better and more robust metabolic behavior and process performance and the determination of QbD design space for CO2 control. [ABSTRACT FROM AUTHOR]

The functioning of the immune and reproductive systems is crucial for the fitness and survival of species and is strongly influenced by the environment. To evaluate the effects of short-term heat stress (HS) on these systems, confirming and deepening previous studies, female sea urchin Paracentrotus lividus were exposed for 7 days to 17 °C, 23 and 28 °C. Several biomarkers were detected such as the ferric reducing power (FRAP), ABTS-based total antioxidant capacity (TAC-ABTS), nitric oxide metabolites (NOx), total thiol levels (TTL), myeloperoxidase (MPO) and protease (PA) activities in the coelomic fluid (CF) and mitochondrial membrane potential (MMP), H2O2 content and intracellular pH (pHi) in eggs and coelomocytes, in which TAC-ABTS and reactive nitrogen species (RNS) were also analyzed. In the sea urchins exposed to HS, CF analysis showed a decrease in FRAP levels and an increase in TAC-ABTS, TTL, MPO and PA levels; in coelomocytes, RNS, MMP and H2O2 content increased, whereas pHi decreased; in eggs, increases in MMP, H2O2 content and pHi were found. In conclusion, short-term HS leads to changes in five out of the six CF biomarkers analyzed and functional alterations in the cells involved in either reproductive or immune activities. [ABSTRACT FROM AUTHOR]

Cancer cells exhibit increased glycolytic flux and adenosine triphosphate (ATP) hydrolysis. These processes increase the acidic burden on the cells through the production of lactate and protons. Nonetheless, cancer cells can maintain an alkaline intracellular pH (pHi) relative to untransformed cells, which sets the stage for optimal functioning of glycolytic enzymes, evasion of cell death, and increased proliferation and motility. Upregulation of plasma membrane transporters allows for H+ and lactate efflux; however, recent evidence suggests that the acidification of organelles can contribute to maintenance of an alkaline cytosol in cancer cells by siphoning off protons, thereby supporting tumor growth. The Golgi is such an acidic organelle, with resting pH ranging from 6.0 to 6.7. Here, we posit that the Golgi represents a "proton sink" in cancer and delineate the proton channels involved in Golgi acidification and the ion channels that influence this process. Furthermore, we discuss ion channel regulators that can affect Golgi pH and Golgi-dependent processes that may contribute to pHi homeostasis in cancer.

Abstract Intracellular pH critically affects various biological processes, and an appropriate cytoplasmic pH is essential for ensuring bacterial growth. Glucose is the preferred carbon source for most heterotrophs; however, excess glucose often causes the accumulation of acidic metabolites, lowering the intracellular pH and inhibiting bacterial growth. Bacillus thuringiensis can effectively cope with glucose‐induced stress; unfortunately, little is known about the regulators involved in this process. Here, we document that the target of the dual‐function sRNA YhfH, the lipR gene, encodes a LacI‐family transcription factor LipR as an intracellular pH regulator when B. thuringiensis BMB171 is suddenly exposed to glucose. Under glucose conditions, lipR deletion leads to early growth arrest by causing a rapid decrease in intracellular pH (~5.4). Then, the direct targets and a binding motif (GAWAWCRWTWTCAT) of LipR were identified based on the electrophoretic mobility shift assay, the DNase‐I footprinting assay, and RNA sequencing, and the gapN gene encoding a key enzyme in glycolysis was directly inhibited by LipR. Furthermore, Ni2+ is considered a possible effector for LipR. In addition to YhfH, the lipR expression was coregulated by itself, CcpA, and AbrB. Our study reveals that LipR plays a balancing role between glucose metabolism and intracellular pH in B. thuringiensis subjected to glucose stress.

Gloeobacter violaceus ligand‐gated ion channel (GLIC) is a prokaryotic orthologue of brain pentameric neurotransmitter receptors. Using whole‐cell patch‐clamp electrophysiology in a host cell line, we show that short‐chain dicarboxylate compounds are positive modulators of pHo 5‐evoked GLIC activity, with a rank order of action fumarate > succinate > malonate > glutarate. Potentiation by fumarate depends on intracellular pH, mainly as a result of a strong decrease of the pHo 5‐evoked current when intracellular pH decreases. The modulating effect of fumarate also depends on extracellular pH, as fumarate is a weak inhibitor at pHo 6 and shows no agonist action at neutral pHo. A mutational analysis of residue dependency for succinate and fumarate effects, based on two carboxylate‐binding pockets previously identified by crystallography (Fourati et al., 2020), shows that positive modulation involves both the inter‐subunit pocket, homologous to the neurotransmitter‐binding orthotopic site, and the intra‐subunit (also called vestibular) pocket. An almost similar pattern of mutational impact is observed for the effect of caffeate, a known negative modulator. We propose, for both dicarboxylate compounds and caffeate, a model where the inter‐subunit pocket is the actual binding site, and the region corresponding to the vestibular pocket is required either for inter‐subunit binding itself, or for binding‐to‐gating coupling during the allosteric transitions involved in pore‐gating modulation. Key points: Using a bacterial orthologue of brain pentameric neurotransmitter receptors, we show that the orthotopic/orthosteric agonist site and the adjacent vestibular region are functionally interdependent in mediating compound‐elicited modulation. We propose that the two sites in the extracellular domain are involved 'in series', a mechanism which may have relevance for eukaryote receptors.We show that short‐chain dicarboxylate compounds are positive modulators of the Gloeobacter violaceus ligand‐gated ion channel (GLIC). The most potent compound identified is fumarate, known to occupy the orthotopic/orthosteric site in previously published crystal structures.We show that intracellular pH modulates GLIC allosteric transitions, as previously known for extracellular pH.We report a caesium to sodium permeability ratio (PCs/PNa) of 0.54 for GLIC ion pore. [ABSTRACT FROM AUTHOR]

Transient changes in intracellular pH (pHi) regulate normal cell behaviors, but roles for spatiotemporal pHi dynamics in single-cell behaviors remain unclear. Here, we mapped single-cell spatiotemporal pHi dynamics during mammalian cell cycle progression both with and without cell cycle synchronization. We found that single-cell pHi is dynamic throughout the cell cycle: pHi decreases at G1/S, increases in mid-S, decreases at late S, increases at G2/M and rapidly decreases during mitosis. Importantly, although pHi is highly dynamic in dividing cells, non-dividing cells have attenuated pHi dynamics. Using two independent pHi manipulation methods, we found that low pHi inhibits completion of S phase whereas high pHi promotes both S/G2 and G2/M transitions. Our data also suggest that low pHi cues G1 exit, with decreased pHi shortening G1 and increased pHi elongating G1. Furthermore, dynamic pHi is required for S phase timing, as high pHi elongates S phase and low pHi inhibits S/G2 transition. This work reveals that spatiotemporal pHi dynamics are necessary for cell cycle progression at multiple phase transitions in single human cells. [ABSTRACT FROM AUTHOR]

The International Society of Cancer Metabolism (ISCaM) meeting on Cancer Metabolic Rewiring, held in Braga Portugal in October 2019, provided an outstanding forum for investigators to present current findings and views, and discuss ideas and future directions on fundamental biology as well as clinical translations. The first session on Cancer pH Dynamics was preceded by the opening keynote presentation from our group entitled Intracellular pH Regulation of Protein Dynamics: From Cancer to Stem Cell Behaviors. In this review we introduce a brief background on intracellular pH (pHi) dynamics, including how it is regulated as well as functional consequences, summarize key findings included in our presentation, and conclude with perspectives on how understanding the role of pHi dynamics in stem cells can be relevant for understanding how pHi dynamics enables cancer progression.

Abstract Background The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is predominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. Results Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. Conclusions Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

Abstract Deviation of the H+ concentration from optimum values within the organelles is closely associated with irregular cellular functions that cause the onset of various diseases. Therefore, determining subcellular pH values in live cells and tissues is valuable for diagnostic purposes. In this study, we report a novel ratiometric fluorescence probe 1H-pyrazole-3-carboxylic acid, 4-(benzo[d]thiazol-2-yl)-3-(2,4-dihydroxy-3-methylphenyl)-1H-pyrazole-5-carboxylicacid4-(2-benz othiazolyl)-5-(2,4-dihydroxy-3-methylphenyl), to which we will refer as ThiAKS Green (Thiazole AKyol shifting green), that is pH sensitive. The results presented here show that the probe can penetrate the cell membrane in less than 30 minutes and does not show any detectable toxicity. The measured color shifts up on pH change are linear and most significant around physiological pH (pKa=7.45), thus making this probe suitable for live-cell imaging and intracellular pH measurements. During the long-incubation periods following the application of the probe and the fluorescent microscopy measurements, it shows stable properties and is easy to detect in live cells. In conclusion, the results suggest that ThiAKS Green can be used to obtain precise information on the H+ distribution at various compartments of the live cells.

Acidic lysosomes are indispensable for cancer development and linked to chemotherapy resistance. Chloroquine (CQ) and functional analogues have been considered as a potential solution to overcome the cancer progression and chemoresistance by inhibiting the lysosome-mediated autophagy and multidrug exocytosis. However, their anti-cancer efficacy in most clinical trials demonstrated modest improvement. In this study, we investigated the detailed mechanisms underlying the acquired resistance of K562 leukemic cells to CQ treatment. In response to 5–80 μM CQ, the lumen pH of endosomal-lysosomal system immediately increased and gradually reached dynamic equilibrium within 24 h. Leukemic cells produced more acidic organelles to tolerate 5–10 μM CQ. CQ (20–80 μM) concentration-dependently triggered cytosolic pH (pHi) rise, G0/G1 arrest, mitochondrial depolarization/fragmentation, and necrotic/apoptotic cell death. Oxidant induction by CQ was responsible for the mitochondria-dependent cytotoxicity and partial pHi elevation. Cells that survived the CQ cytotoxicity were accompanied with increased mitochondria. Under the 80 μM CQ challenge, co-treatment with the inhibitor of F 0 part of mitochondrial H+-ATP synthase, oligomycin (40 nM), prevented the elevation of oxidants as well as pHi, and attenuated stresses on mitochondria, cell survival, and cell proliferation. Besides, oligomycin-treated cells obviously displayed the lysosomal peripheralization and plasma membrane blebbing, suggesting that these cells were in process of lysosomal exocytosis and microvesicle release. Enhanced motion of these secretory processes allowed the cells to exclude CQ and repair necrotic injury. Together, the oxidant production and the proton dynamic interconnection among lysosomes, mitochondria, and cytosol are crucial for leukemic susceptibility to lysosomotropic chemotherapeutics. [Display omitted] • CQ causes the elevation of cytosolic pH (pHi) in parallel with increases of G0/G1 arrest, mitochondrial depolarization, and cell death. • RONS induction by CQ is responsible for the mitochondria-dependent cytotoxicity and partial pHi elevation, but not the proliferation arrest. • Blockade of proton flux through the mitochondrial ATP synthase by oligomycin completely prevents the CQ-induced rise in RONS and pHi. • Reduction of RONS and pHi potentiates the secretory expulsion of lysosomal and cytosolic CQ, and attenuates the CQ-induced leukemic cell loss. [ABSTRACT FROM AUTHOR]

Herein, a novel pH‐responsive phosphorescent probe based on cyclometalated iridium(III) complex is reported. To prevent oxygen quenching of phosphorescence and to improve the probe biocompatibility, the complex is covalently conjugated with a water‐soluble block‐copolymer that also increases its pH sensitivity. The resulting polymeric nanoprobe demonstrates a strong response of the phosphorescence lifetime onto pH variations in physiological range. Cellular experiments with Chinese hamster ovary (CHO‐K1) cells show the predominant internalization of the probe in acidified cell compartments, endosomes and lysosomes. The analysis of phosphorescence lifetime imaging microscopy data confirms applicability of the sensor for monitoring of intra‐ and extracellular pH in cell cultures. [ABSTRACT FROM AUTHOR]

Deviation of the H+ concentration from optimum values within the organelles is closely associated with irregular cellular functions that cause the onset of various diseases. Therefore, determining subcellular pH values in live cells and tissues is valuable for diagnostic purposes. In this study, we report a novel ratiometric fluorescence probe 1H-pyrazole-3-carboxylic acid, 4-(benzo[d]thiazol-2-yl)-3-(2,4-dihydroxy-3-methylphenyl)-1H-pyrazole-5-carboxylicacid4-(2-benz othiazolyl)-5-(2,4-dihydroxy-3-methylphenyl), to which we will refer as ThiAKS Green (Thiazole AKyol shifting green), that is pH sensitive. The results presented here show that the probe can penetrate the cell membrane in less than 30 minutes and does not show any detectable toxicity. The measured color shifts up on pH change are linear and most significant around physiological pH (pKa=7.45), thus making this probe suitable for live-cell imaging and intracellular pH measurements. During the long-incubation periods following the application of the probe and the fluorescent microscopy measurements, it shows stable properties and is easy to detect in live cells. In conclusion, the results suggest that ThiAKS Green can be used to obtain precise information on the H+ distribution at various compartments of the live cells. [ABSTRACT FROM AUTHOR]

As a consequence of altered glucose metabolism, cancer cell intake is increased, producing large amounts of lactate which is pumped out the cytosol by monocarboxylate transporters (MCTs). MCT 1 and MCT4 are frequently overexpressed in tumors, and recently, MCT inhibition has been reported to exert antineoplastic effects. In the present study, MCT1 and MCT4 levels were assessed in esophageal adenocarcinoma (EAC) cells and the effects of the MCT-1 selective inhibitor AZD3965, hypoxia, and a glucose overload were evaluated in vitro. Two EAC cell lines (OE33 and OACM5.1C) were treated with AZD3965 (10–100 nM) under different conditions (normoxia/hypoxia) and also different glucose concentrations, and parameters of cytotoxicity, oxidative stress, intracellular pH (pHi), and lactate levels were evaluated. MCT1 was present in both cell lines whereas MCT4 was expressed in OE33 cells and only in a small proportion of OACM5.1C cells. Glucose addition did not have any effect on apoptosis nor cell proliferation. AZD3965 increased apoptosis and reduced proliferation of OACM5.1C cells, effects which were abrogated when cells were growing in hypoxia. MCT1 inhibition increased intracellular lactate levels in all the cells evaluated, but this increase was higher in cells expressing only MCT1 and did not affect oxidative stress. AZD3965 induced a decrease in pHi of cells displaying low levels of MCT4 and also increased the sodium/hydrogen exchanger 1 (NHE-1) expression on these cells. These data provide in vitro evidence supporting the potential of MCT inhibitors as novel antineoplastic drugs for EAC and highlight the importance of achieving a complete MCT inhibition. [ABSTRACT FROM AUTHOR]

The current viral pandemic, coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), creates health, mental, economic, and other serious challenges that are better to say global crisis. Despite the existence of successful vaccines, the possible mutations which can lead to the born of novel and possibly more dangerous variants of the virus as well as the absence of definitive treatment for this potentially fatal multiple-organ infection in critically ill patients make us keep searching. Theoretically targeting human and viral receptors and enzymes via molecular docking and dynamics simulations can be considered a wise, rational, and efficient way to develop therapeutic agents against COVID-19. In this way, The RNA-dependent RNA polymerase (RdRP), main protease, and spike glycoprotein of SARS-CoV-2 as well as the human angiotensin-converting enzyme 2 receptor and transmembrane serine protease 2 are the most discussed and studied targets that play essential roles in the viral life and infection cycle. In the current in silico investigation, the guanidine functionality containing drugs and medicinal substances such as metformin, famotidine, neuraminidase inhibitors, antimalarial medications, anticancer drug imatinib, CGP compounds, and human serine protease inhibitor camostat were studied against the above-mentioned therapeutic targets and most of them (especially imatinib) have revealed an incredible spectrum of free docking scores and MD results. The current in silico investigation that its novel perspective of view is corroborated by the different experimental and clinical evaluations, confirms that the guanidine moiety can be considered as a missing promising pharmacophore in drug design and development approaches against SARS-CoV-2. Considering the chemical potency of this polyamine group in chemical interaction creation, the observed outcomes in this virtual screening were not surprising. On the other hand, the guanidine functional group has unique physico-chemical properties such as basicity that can make the target cells intracellular pH undesirable for the virus entry, uncoating, and cytosolic lifecycle. According to the obtained results in the current study that are interestingly confirmed by the previously reported efficacy of some the guanidine carrying drugs in COVID-19, guanidine as a potential multi-target anti-SARS-CoV-2 functional scaffold deserves further comprehensive investigations. [ABSTRACT FROM AUTHOR]

ABSTRACT Bacteria are known to cope with amino acid starvation by the stringent response signaling system, which is mediated by the accumulation of the (p)ppGpp alarmones when uncharged tRNAs stall at the ribosomal A site. While a number of metabolic processes have been shown to be regulatory targets of the stringent response in many bacteria, the global impact of amino acid starvation on bacterial metabolism remains obscure. This work reports the metabolomic profiling of the human pathogen Streptococcus pneumoniae under methionine starvation. Methionine limitation led to the massive overhaul of the pneumococcal metabolome. In particular, methionine-starved pneumococci showed a massive accumulation of many metabolites such as glutamine, glutamic acid, lactate, and cyclic AMP (cAMP). In the meantime, methionine-starved pneumococci showed a lower intracellular pH and prolonged survival. Isotope tracing revealed that pneumococci depend predominantly on amino acid uptake to replenish intracellular glutamine but cannot convert glutamine to methionine. Further genetic and biochemical analyses strongly suggested that glutamine is involved in the formation of a “prosurvival” metabolic state by maintaining an appropriate intracellular pH, which is accomplished by the enzymatic release of ammonia from glutamine. Methionine starvation-induced intracellular pH reduction and glutamine accumulation also occurred to various extents under the limitation of other amino acids. These findings have uncovered a new metabolic mechanism of bacterial adaptation to amino acid limitation and perhaps other stresses, which may be used as a potential therapeutic target for infection control. IMPORTANCE Bacteria are known to cope with amino acid starvation by halting growth and prolonging survival via the stringent response signaling system. Previous investigations have allowed us to understand how the stringent response regulates many aspects of macromolecule synthesis and catabolism, but how amino acid starvation promotes bacterial survival at the metabolic level remains largely unclear. This paper reports our systematic profiling of the methionine starvation-induced metabolome in S. pneumoniae. To the best of our knowledge, this represents the first reported bacterial metabolome under amino acid starvation. These data have revealed that the significant accumulation of glutamine and lactate enables S. pneumoniae to form a “prosurvival” metabolic state with a lower intracellular pH, which inhibits bacterial growth for prolonged survival. Our findings have provided insightful information on the metabolic mechanisms of pneumococcal adaptation to nutrient limitation during the colonization of the human upper airway.

The extracellular matrix (ECM), in which collagen is the most abundant protein, impacts many aspects of tumor physiology, including cellular metabolism and intracellular pH (pHi), as well as the efficacy of chemotherapy. Meanwhile, the role of collagen in differential cell responses to treatment within heterogeneous tumor environments remains poorly investigated. In the present study, we simultaneously monitored the changes in pHi and metabolism in living colorectal cancer cells in vitro upon treatment with a chemotherapeutic combination, FOLFOX (5-fluorouracil, oxaliplatin and leucovorin). The pHi was followed using the new pH-sensitive probe BC-Ga-Ir, working in the mode of phosphorescence lifetime imaging (PLIM), and metabolism was assessed from the autofluorescence of the metabolic cofactor NAD(P)H using fluorescence lifetime imaging (FLIM) with a two-photon laser scanning microscope. To model the ECM, 3D collagen-based hydrogels were used, and comparisons with conventional monolayer cells were made. It was found that FOLFOX treatment caused an early temporal intracellular acidification (reduction in pHi), followed by a shift to more alkaline values, and changed cellular metabolism to a more oxidative state. The presence of unstructured collagen markedly reduced the cytotoxic effects of FOLFOX, and delayed and diminished the pHi and metabolic responses. These results support the observation that collagen is a factor in the heterogeneous response of cancer cells to chemotherapy and a powerful regulator of their metabolic behavior.

Ambroxol (ABX), a frequently prescribed secretolytic agent which enhances the ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of amplitude) by 30%, activates a voltage-dependent Ca2+ channel (CaV1.2) and a small transient Ca2+ release in the ciliated lung airway epithelial cells (c-LAECs) of mice. The activation of CaV1.2 alone enhanced the CBF and CBA by 20%, mediated by a pHi increasei and a [Cl−]i decrease in the c-LAECs. The increase in pHi, which was induced by the activation of the Na+-HCO3− cotransporter (NBC), enhanced the CBF (by 30%) and CBA (by 15–20%), and a decrease in [Cl−]i, which was induced by the Cl− release via anoctamine 1 (ANO1), enhanced the CBA (by 10–15%). While a Ca2+-free solution or nifedipine (an inhibitor of CaV1.2) inhibited 70% of the CBF and CBA enhancement using ABX, CaV1.2 enhanced most of the CBF and CBA increases using ABX. The activation of the CaV1.2 existing in the cilia stimulates the NBC to increase pHi and ANO1 to decrease the [Cl−]i in the c-LAECs. In conclusion, the pHi increase and the [Cl−]i decrease enhanced the CBF and CBA in the ABX-stimulated c-LAECs.

Tau, a member of the MAP2/tau family of microtubule-associated proteins, stabilizes and organizes axonal microtubules in healthy neurons. In neurodegenerative tauopathies, tau dissociates from microtubules and forms neurotoxic extracellular aggregates. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. Here, we used molecular dynamics, microtubule-binding experiments, and live-cell microscopy, revealing that highly-conserved histidine residues near the C terminus of each microtubule-binding repeat are pH sensors that can modulate tau-microtubule interaction strength within the physiological intracellular pH range. We observed that at low pH (7.5), tau deprotonation decreased binding to microtubules both in vitro and in cells. Electrostatic and hydrophobic characteristics of histidine were both required for tau-microtubule binding, as substitutions with constitutively and positively charged nonaromatic lysine or uncharged alanine greatly reduced or abolished tau-microtubule binding. Consistent with these findings, tau-microtubule binding was reduced in a cancer cell model with increased intracellular pH but was rapidly restored by decreasing the pH to normal levels. These results add detailed insights into the intracellular regulation of tau activity that may be relevant in both normal and pathological conditions.

HCO3− secretion in distal airways is critical for airway mucosal defense. HCO3−/H+ transport across the apical membrane of airway surface epithelial cells was studied by measuring intracellular pH in luminally microperfused freshly dissected mice bronchioles. Functional studies demonstrated that CFTR, ENaC, Cl−–HCO3− exchange, Na+-H+ exchange, and Na+–HCO3− cotransport are involved in apical HCO3−/H+ transport. RT-PCR of isolated bronchioles detected fragments from Cftr, α, β, γ subunits of ENaC, Ae2, Ae3, NBCe1, NBCe2, NBCn1, NDCBE, NBCn2, Nhe1, Nhe2, Nhe4, Nhe5, Slc26a4, Slc26a6, and Slc26a9. We assume that continuous decline of intracellular pH following alkaline load demonstrates time course of HCO3− secretion into the lumen which is perfused with a HCO3−-free solution. Forskolin-stimulated HCO3− secretion was substantially inhibited by luminal application of CFTRinh-172 (5 μM), H2DIDS (200 μM), and amiloride (1 μM). In bronchioles from a cystic fibrosis mouse model, basal and acetylcholine-stimulated HCO3− secretion was substantially impaired, but forskolin transiently accelerated HCO3− secretion of which the magnitude was comparable to wild-type bronchioles. In conclusion, we have characterized apical HCO3−/H+ transport in native bronchioles. We have demonstrated that cAMP-mediated and Ca2+-mediated pathways are involved in HCO3− secretion and that apical HCO3− secretion is largely mediated by CFTR and H2DIDS-sensitive Cl−–HCO3− exchanger, most likely Slc26a9. The impairment of HCO3− secretion in bronchioles from a cystic fibrosis mouse model may be related to the pathogenesis of early lung disease in cystic fibrosis. [ABSTRACT FROM AUTHOR]

Background: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is predominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. Results: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. Conclusions: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment. [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate the optimal pH for acyl-glucuronidation formation with carboxylic acid-containing compounds in human and rat liver microsomes to improve the predictability of their hepatic clearance. The optimal pH for acyl-glucuronidation of all 17 compounds was around pH 6.0 in human and rat liver microsomes. Correlation analysis was done with the predicted in vitro intrinsic clearance (CL int, in vitro) and in vivo intrinsic clearance (CL int, in vivo) calculated from available reported data of total clearance (CL tot) of 11 compounds in humans. For 8 of the 11 compounds, under the pH 6.0 condition, the CL int, in vitro were within 1/3 to 3-fold error of the observed CL int, in vivo whereas, the error was within 1/3 to 3-fold of the observed CL int, in vivo for only 3 of the 11 under the pH 7.4 condition. The intracellular pH in human and rat hepatocytes decreased in the presence of a carboxylic acid-containing compound. These findings suggest that acyl-glucuronidation in liver microsomes at pH 6.0 is closer to physiological conditions in the presence of carboxylic acid compounds, and thus, use of this pH condition is important for physiological interpretation and predictability of intrinsic clearance. [ABSTRACT FROM AUTHOR]

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen that employs several secreted and surface-bound virulence factors to manipulate its environment, allowing it to cause a variety of disease outcomes. One such virulence factor is Streptolysin S (SLS), a ribosomally- produced peptide toxin that undergoes extensive post-translational modifications. The activity of SLS has been studied for over 100 years owing to its rapid and potent ability to lyse red blood cells, and the toxin has been shown to play a major role in GAS virulence in vivo. We have previously demonstrated that SLS induces hemolysis by targeting the chloride-bicarbonate exchanger Band 3 in erythrocytes, indicating that SLS is capable of targeting host proteins to promote cell lysis. However, the possibility that SLS has additional protein targets in other cell types, such as keratinocytes, has not been explored. Here, we use bioinformatics analysis and chemical inhibition studies to demonstrate that SLS targets the electroneutral sodium-bicarbonate cotransporter NBCn1 in keratinocytes during GAS infection. SLS induces NF-kB activation and host cytotoxicity in human keratinocytes, and these processes can be mitigated by treating keratinocytes with the sodium-bicarbonate cotransport inhibitor S0859. Furthermore, treating keratinocytes with SLS disrupts the ability of host cells to regulate their intracellular pH, and this can be monitored in real time using the pH-sensitive dye pHrodo Red AM in live imaging studies. These results demonstrate that SLS is a multifunctional bacterial toxin that GAS uses in numerous context-dependent ways to promote host cell cytotoxicity and increase disease severity. Studies to elucidate additional host targets of SLS have the potential to impact the development of therapeutics for severe GAS infections. [ABSTRACT FROM AUTHOR]

Many calcifying organisms utilize metabolic CO2 to generate CaCO3 minerals to harden their shells and skeletons. Carbonic anhydrases are evolutionary ancient enzymes that have been proposed to play a key role in the calcification process, with the underlying mechanisms being little understood. Here, we used the calcifying primary mesenchyme cells (PMCs) of sea urchin larva to study the role of cytosolic (iCAs) and extracellular carbonic anhydrases (eCAs) in the cellular carbon concentration mechanism (CCM). Molecular analyses identified iCAs and eCAs in PMCs and highlight the prominent expression of a glycosylphosphatidylinositol-anchored membrane-bound CA (Cara7). Intracellular pH recordings in combination with CO2 pulse experiments demonstrated iCA activity in PMCs. iCA activity measurements, together with pharmacological approaches, revealed an opposing contribution of iCAs and eCAs on the CCM. H+-selective electrodes were used to demonstrate eCA-catalyzed CO2 hydration rates at the cell surface. Knockdown of Cara7 reduced extracellular CO2 hydration rates accompanied by impaired formation of specific skeletal segments. Finally, reduced pHi regulatory capacities during inhibition and knockdown of Cara7 underscore a role of this eCA in cellular HCO3- uptake. This work reveals the function of CAs in the cellular CCM of a marine calcifying animal. Extracellular hydration of metabolic CO2 by Cara7 coupled to HCO3- uptake mechanisms mitigates the loss of carbon and reduces the cellular proton load during the mineralization process. The findings of this work provide insights into the cellular mechanisms of an ancient biological process that is capable of utilizing CO2 to generate a versatile construction material. [ABSTRACT FROM AUTHOR]

Clouding of the eye lens or cataract is an age-related anomaly that affects middle-aged humans. Exploration of the etiology points to a great extent to oxidative stress due to different forms of reactive oxygen species/metabolites such as Hydrogen peroxide (H 2 O 2) that are generated due to intracellular metabolism and environmental factors like radiation. If accumulated and left unchecked, the imbalance between the production and degradation of H 2 O 2 in the lens could lead to cataracts. Our objective was to explore ex vivo the effects of H 2 O 2 on lens physiology. We investigated transparency, intracellular pH (pH i), intercellular gap junction coupling (GJC), hydrostatic pressure (HP) and membrane water permeability after subjecting two-month-old C57 wild-type (WT) mouse lenses for 3 h or 8 h in lens saline containing 50 μM H 2 O 2 ; the results were compared with control lenses incubated in the saline without H 2 O 2. There was a significant decrease in lens transparency in H 2 O 2 -treated lenses. In control lenses, pH i decreases from ∼7.34 in the surface fiber cells to 6.64 in the center. Experimental lenses exposed to H 2 O 2 for 8 h showed a significant decrease in surface pH (from 7.34 to 6.86) and central pH (from 6.64 to 6.56), compared to the controls. There was a significant increase in GJC resistance in the differentiating (12-fold) and mature (1.4-fold) fiber cells compared to the control. Experimental lenses also showed a significant increase in HP which was ∼2-fold higher at the junction between the differentiating and mature fiber cells and ∼1.5-fold higher at the center compared to these locations in control lenses; HP at the surface was 0 mm Hg in either type lens. Fiber cell membrane water permeability significantly increased in H 2 O 2 -exposed lenses compared to controls. Our data demonstrate that elevated levels of lens intracellular H 2 O 2 caused a decrease in intracellular pH and led to acidosis which most likely uncoupled GJs, and increased AQP0-dependent membrane water permeability causing a consequent rise in HP. We infer that an abnormal increase in intracellular H 2 O 2 could induce acidosis, cause oxidative stress, alter lens microcirculation, and lead to the development of accelerated lens opacity and age-related cataracts. • H 2 O 2 -exposed lenses showed accelerated opacity. • H 2 O 2 significantly decreased lens intracellular pH and caused acidosis. • Gap Junction Coupling decreased in lenses exposed to H 2 O 2. • H 2 O 2 caused increases in lens hydrostatic pressure and membrane water permeability. [ABSTRACT FROM AUTHOR]

Chronic Mountain Sickness (CMS) occurs in high-altitude residents with major neurological symptoms such as migraine headaches, dizziness and cognitive deficits. Recent work demonstrated that highlanders have increased intracellular pH (pHi) in their brain cells, perhaps for the sake of adaptation to hypoxemia and help to facilitate glycolysis, DNA synthesis, and cell cycle progression. Since there are well adapted (non-CMS) and maladapted (CMS) high-altitude dwellers, it is not clear whether pHi is differently regulated in these two high-altitude populations. In this work, we obtained induced pluripotent stem cell (iPSC)-derived astrocytes from both CMS and non-CMS highlanders who live in the Peruvian Andes (>14,000 ft) and studied pHi regulation in these astrocytes using pH-sensitive dye BCECF. Our results show that the steady-state pHi (ss pHi) is lower in CMS astrocytes compared with non-CMS astrocytes. In addition, the acid extrusion following an acid loading is faster and the pHi dependence of H+ flux rate becomes steeper in CMS astrocytes. Furthermore, the Na+ dependency of ss pHi is stronger in CMS astrocytes and the Na+/H+ exchanger (NHE) inhibitors blunted the acid extrusion in both CMS and non-CMS astrocytes. We conclude that (a) NHE contributes to the ss pHi stabilization and mediates active acid extrusion during the cytosolic acidosis in highlanders; (b) acid extrusion becomes less pHi sensitive in non-CMS (versus CMS) astrocytes which may prevent NHE from over-activated in the hypoxia-induced intracellular acidosis and render the non-CMS astrocytes more resistant to hypoxemia challenges.

The high metabolic activity and insufficient perfusion of tumors leads to the acidification of the tumor microenvironment (TME) that may inhibit the antitumor T cell activity. We found that pharmacological inhibition of the acid loader chloride/bicarbonate anion exchanger 2 (Ae2), with 4,4’-diisothiocyanatostilbene-2,2’-disulfonicacid (DIDS) enhancedCD4+ andCD8+ T cell function upon TCR activation in vitro, especially under low pH conditions. In vivo, DIDS administration delayed B16OVA tumor growth in immunocompetent mice as monotherapy or when combined with adoptive T cell transfer of OVA-specificT cells. Notably, genetic Ae2 silencing in OVA-specificT cells improvedCD4+/CD8+ T cell function in vitro as well as their antitumor activity in vivo. Similarly, genetic modification of OVA-specificT cells to overexpress Hvcn1, a selectiveH+ outward current mediator that prevents cell acidification, significantly improved T cell function in vitro, even at low pH conditions. The adoptive transfer of OVA-specificT cells overexpressing Hvcn1 exerted a better antitumor activity in B16OVA tumor-bearingmice. Hvcn1 overexpression also improved the antitumor activity of CAR T cells specific for Glypican 3 (GPC3) in mice bearing PM299L-GPC3tumors. Our results suggest that preventing intracellular acidification by regulating the expression of acidifier ion channels such as Ae2 or alkalinizer channels like Hvcn1 in tumor-specificlymphocytes enhances their antitumor response by making them more resistant to the acidic TME.