Extracellular pH Research Articles

Heterogeneous Fenton's-like catalyst potentiation of hydrogen peroxide disinfection: an investigation into mechanisms of action.

This study aimed to establish the mechanisms of action (MOA) of a novel surface functionalised polyacrylonitrile (PAN) catalyst, which was previously shown to have potent antimicrobial activity in conjunction with hydrogen peroxide (H2O2). Bactericidal activity was determined using a disinfectant suspension test. The MOA was investigated by measuring loss of 260nm absorbing material, membrane potential and permeability assays, analysis of intra- and extracellular ATP and pH, and tolerance to sodium chloride and bile salts.The catalyst lowered sub-lethal concentrations of H2O2 from 0.2% to 0.09%. H2O2 ± 3g PAN catalyst significantly (P≤0.05) reduced sodium chloride and bile salt tolerance, suggesting the occurance of sublethal cell membrane damage. The catalyst significantly increased (P≤0.05) N-Phenyl-l-napthylamine uptake (1.51-fold) and leakage of nucleic acids, demonstrating increased membrane permeability. A significant (P≤0.05) loss of membrane potential (0.015 a.u.), coupled with pertubation of intracellular pH homeostasis and depletion of intracellular ATP, suggests potentiation of H2O2-mediated cell membrane damage. This is the first study to investigate the catalyst's antimicrobial mechanism of action, with the cytoplasmic membrane being a target for cellular injury.

Real Time and Spatiotemporal Quantification of pH and H2O2 Imbalances with a Multiplex Surface-Enhanced Raman Spectroscopy Nanosensor

Oxidative stress is involved in many aging-related pathological disorders and is the result of defective cellular management of redox reactions. Particularly, hydrogen peroxide (H2O2), is a major byproduct and a common oxidative stress biomarker. Monitoring its dynamics and a direct correlation to diseases remains a challenge due to the complexity of redox reactions. Sensitivity and specificity are major drawbacks for H2O2 sensors regardless of their readout. Luminiscent boronate-based probes such as 3-mercaptophenylboronic acid (3-MPBA) are emerging as the most effective quantitation tool due to their specificity and sensitivity. Problems associated with these probes are limited intracellular sensing, water solubility, selectivity, and quenching. We have synthesized a boronate-based nanosensor with a surface-enhanced Raman spectroscopy (SERS) readout to solve these challenges. Furthermore, we found out that environmental pH gradients, as found in biological samples, affect the sensitivity of boronate-based sensors. When the sensor is in an alkaline environment, the oxidation of 3-MPBA by H2O2 is more favored than in an acidic environment. This leads to different H2O2 measurements depending on pH. To solve this issue, we synthesized a multiplex nanosensor capable of concomitantly quantifying pH and H2O2. Our nanosensor first measures the local pH and based on this value, provides the amount of H2O2. It seems that this pH-dependent sensitivity effect applies to all boronic acid based probes. We tested the multiplexing ability by quantitatively measuring intra- and extracellular pH and H2O2 dynamics under physiological and pathological conditions on healthy cells and cells in which H+ and/or H2O2 homeostasis has been altered.

Carbogen-Induced Respiratory Acidosis Blocks Experimental Seizures by a Direct and Specific Inhibition of NaV1.2 Channels in the Axon Initial Segment of Pyramidal Neurons.

Brain pH is a critical factor for determining neuronal activity, with alkalosis increasing and acidosis reducing excitability. Acid shifts in brain pH through the breathing of carbogen (5% CO2/95% O2) reduces seizure susceptibility in animal models and patients. The molecular mechanisms underlying this seizure protection remain to be fully elucidated. Here, we demonstrate that male and female mice exposed to carbogen are fully protected from thermogenic-triggered seizures. Whole-cell patch-clamp recordings revealed that acid shifts in extracellular pH (pHo) significantly reduce action potential firing in CA1 pyramidal neurons but did not alter firing in hippocampal inhibitory interneurons. In real-time dynamic clamp experiments, acidification reduced simulated action potential firing generated in hybrid model neurons expressing the excitatory neuron predominant NaV1.2 channel. Conversely, acidification had no effect on action potential firing in hybrid model neurons expressing the interneuron predominant NaV1.1 channel. Furthermore, knockdown of Scn2a mRNA in vivo using antisense oligonucleotides reduced the protective effects of carbogen on seizure susceptibility. Both carbogen-mediated seizure protection and the reduction in CA1 pyramidal neuron action potential firing by low pHo were maintained in an Asic1a knock-out mouse ruling out this acid-sensing channel as the underlying molecular target. These data indicate that the acid-mediated reduction in excitatory neuron firing is mediated, at least in part, through the inhibition of NaV1.2 channels, whereas inhibitory neuron firing is unaffected. This reduction in pyramidal neuron excitability is the likely basis of seizure suppression caused by carbogen-mediated acidification.SIGNIFICANCE STATEMENT Brain pH has long been known to modulate neuronal excitability. Here, we confirm that brain acidification reduces seizure susceptibility in a mouse model of thermogenic seizures. Extracellular acidification reduced excitatory pyramidal neuron firing while having no effect on interneuron firing. Acidification also reduced dynamic clamp firing in cells expressing the NaV1.2 channel but not in cells expressing NaV1.1 channels. In vivo knockdown of Scn2a mRNA reduced seizure protection of acidification. In contrast, acid-mediated seizure protection was maintained in the Asic1a knock-out mouse. These data suggest NaV1.2 channel as an important target for acid-mediated seizure protection. Our results have implications on how natural variations in pH can modulate neuronal excitability and highlight potential antiseizure drug development strategies based on the NaV1.2 channel.

Electron Paramagnetic Resonance Implemented with Multiple Harmonic Detections Successfully Maps Extracellular pH In Vivo.

Extracellular acidification indicates a metabolic shift in cancer cells and is, along with tissue hypoxia, a hallmark of tumor malignancy. Thus, non-invasive mapping of extracellular pH (pHe) is essential for researchers to understand the tumor microenvironment and to monitor tumor response to metabolism-targeting drugs. While electron paramagnetic resonance (EPR) has been successfully used to map pHe in mouse xenograft models, this method is not sensitive enough to map pHe with a moderate amount of exogenous pH-sensitive probes. Here, we show that a modified EPR system achieves twofold higher sensitivity by using the multiple harmonic detection (MHD) method and improves the robustness of pHe mapping in mouse xenograft models. Our results demonstrate that treatment of a mouse xenograft model of human-derived pancreatic ductal adenocarcinoma cells with the carbonic anhydrase IX (CAIX) inhibitor U-104 delays tumor growth with a concurrent tendency toward further extracellular acidification. We anticipate that EPR-based pHe mapping can be expanded to monitor the response of other metabolism-targeting drugs. Furthermore, pHe monitoring can also be used for the development of improved metabolism-targeting cancer treatments.

Calcium-dependent membrane interactions of tumor-targeting peptide pHLIP.

Cancerous tissues undergo extensive changes to their cellular environments that differentiate them from healthy tissues. These changes include changes in extracellular pH and Ca2+ concentrations, and the exposure of phosphatidylserine (PS) to the extracellular environment, which can modulate the interaction of peptides and proteins with the plasma membrane. Deciphering the molecular mechanisms of such interactions is critical for advancing the knowledge-based design of cancer-targeting molecular tools, such as pHLIP (pH-Low Insertion Peptide). Here, we explore the effects of PS, Ca2+, and peptide protonation state on the interactions of pHLIP with lipid membranes. Cellular studies demonstrate that exposed PS on the plasma membrane promotes pHLIP targeting. The magnitude of this effect is dependent on extracellular Ca2+ concentration, indicating that divalent cations play an important role in pHLIP targeting in vivo. The targeting mechanism is further explored with a combination of fluorescence and circular dichroism experiments in model membranes and microsecond-timescale all-atom molecular dynamics simulations. Our results demonstrate that Ca2+ is engaged in coupling peptide-lipid interactions in the unprotonated transmembrane conformation of pHLIP. The simulations reveal that while the pH-induced insertion leads to a strong depletion of PS around pHLIP, the Ca2+-induced insertion has the opposite effect. Thus, extracellular levels of Ca2+ are crucial to link cellular changes in membrane lipid composition with the selective targeting and insertion of pHLIP. The characterized Ca2+-dependent coupling between pHLIP sidechains and PS provides atomistic insights into the general mechanism for lipid-coupled regulation of protein-membrane insertion by divalent cations.

Loss of vision caused by an alkaline shift in the pH-dependence of the corneal H+/OH--conducting membrane protein Slc4a11.

SLC4A11 is the only member of the solute carrier 4 family of membrane transport proteins that transports H+ (or its thermodynamic equivalent OH−) rather than bicarbonate or carbonate. Mutations in SLC4A11 result in the loss of the fluid pumping function of cells that line the posterior of the cornea (corneal endothelial cells), resulting in corneal swelling and vision loss (corneal dystrophy). SLC4A11-mediated H+/OH− conductance is exquisitely sensitive to both intracellular and extracellular pH (pHi and pHe), being activated by a rise in either, and could help to regulate the pH of both compartments to maintain transport processes. One mutation in SLC4A11 ‘Arg125His’ that causes corneal dystrophy appears to render SLC4A11 inactive under physiological conditions. We expressed wild-type and R125H mutant SLC4A11 in Xenopus oocytes. We measured the membrane conductance of those cells under voltage clamp and monitored pHi with a H+-selective microelectrode as we caused pHi and/or pHe to rise. We found that, at fixed pHe = 8.50, the pKi that describes the pHi-dependence of mutant SLC4A11 activity is alkaline shifted (greater than 7.60, n = 8) compared to wild-type (7.04 +/− 0.01, n = 7), explaining the loss of function. However, as we increased pHe to 10.00, mutant pKi rose to a value (7.06 +/− 0.08, n = 6) close to that of wild-type SLC4A11. These observations indicate that the molecular defect in SLC4A11/R125H lies with the pHe-dependence of pKi (presumably the ability of pHe to cause the appropriate long-range, transmembrane conformational-changes in SLC4A11) and suggest that the defect in the SLC4A11/R125H mutant may be amenable to pharmacological correction.

Inhibition of Cav1.4 channels by protons depends on the identity of the α2δ subunit.

In retinal photoreceptors, CaV1.4 channels coassemble with the β2 and α2δ-4 subunits and regulate the release of neurotransmitter. Like other CaV channels, CaV1.4 undergoes a prominent inhibition by extracellular protons with low pH decreasing the maximal current amplitude (Imax) and depolarizing the half maximal activation voltage (Vh). However, the mechanisms underlying this form of regulation remain controversial. Here, we analyzed the role of subunit identity on proton-mediated inhibition of CaV1.4 (coexpressed with β2x13) in transfected HEK 293T cells. In cells cotransfected with α2δ-4, changing the extracellular pH from 8.0 to 6.25 caused a significantly greater inhibition of Imax (ΔIMax) and shift in Vh (ΔVh) (ΔIMax = 89.8 ± 1.1 %, ΔVh = 22.7 ± 1.7 mV) than in cells cotransfected with α2δ-1 (ΔIMax = 64.6 ± 4.9 %, ΔVh = 9.0 ± 1.4 mV) or expressing CaV1.4 alone (ΔIMax = 48.3 ± 3.0 %, ΔVh = 9.4 ± 2.1 mV). Notably, this effect of α2δ-4 was selectively diminished for a C-terminal variant of CaV1.4 channels with hyperpolarized voltage-dependence of activation due to a disease-causing mutation (K1591X) but not alternative splicing (CaV1.4 Δ47). The increase in ΔIMax due to α2δ-4 was significantly lower in cells expressing K1591X (11.0 ± 9.9 %) as compared to CaV1.4 (46.2 ± 0.7 %) and CaV1.4 Δ47 (28.7 ± 2.4 %). We conclude that α2δ-4 potentiates proton-mediated inhibition of CaV1.4 in a manner that is modified by pathological variations in the C-terminal domain of CaV1.4.

Small molecule discovery for modulation of Otopetrin proton channels.

Proton conductance across the plasma membrane is involved in many biological roles, ranging from sour taste reception to regulation of intracellular and extracellular pH. Recently, a eukaryotic family of membrane-bound proteins, named Otopetrins, were characterized as proton channels. Otopetrins elicit inward conducting proton currents in response to low extracellular pH levels. Cryo-electron microscopy structures of Otopetrins revealed that they bear no structural similarity to other proton channels, like the voltage-gated proton channel Hv1 or the influenza proton channel M2, marking them a new architecture for proton channels. Since their discovery, OTOP1 was shown to be the sour-taste receptor for vertebrates; however, the biological roles of other members, OTOP2 and OTOP3, remain uninvestigated. Moreover, current research on Otopetrins falls short of elucidating their mechanisms of proton conductance and gating and there are no known molecular modulators of these channels - agonistic or antagonistic. Here, using molecular docking with AutoDock Vina, we virtually screened a diversity set of the Chembridge small molecule library against the available cryo-EM structure of Danio rerio OTOP1. Functional testing of the resulting hits with electrophysiology identified six small molecules that block OTOP1 currents to varying degrees. Based on the chemical scaffold of the best identified blocker, we then screened a filtered subset of the Chembridge library and successfully identified several more blockers, improving upon the results of the initial screening. These small molecules, which are predicted to bind in a potential proton conductance pathway, can be used as chemical probes in structural and functional characterization of OTOP1 to provide insight on the mechanisms of proton conductance and gating. Furthermore, the small molecules we identified provide us with a chemical scaffold for developing a pharmacophore to find better ligands for use in future in vitro and in vivo experiments.

Revealing the allosteric mechanism of pH-dependence in the proton-activated chloride channel.

The ability of the Proton-activated chloride (PAC) channel to perform its chloride transfer function across the membrane was shown to be dependent on the pH in the extracellular environment. Thus, an increase of extracellular pH from pH 4.5 to pH 8 results in a conformational change in transmembrane domain (TMD) of PAC and abolishes chloride conductivity. Mutagenesis of various extracellular domain (ECD) regions of PAC, including the Acidic Pocket, the beta 3 sheet of the “finger region” and the distal end of the ECD, altered the pH-sensitivity of PAC without revealing how such changes affect the pH sensitivity and its effect on the structure and dynamics of the distal end of the TMD. To understand this mechanism we used molecular pH-specific molecular dynamics (MD) simulations of PAC protein with the ECpH-MD method, capturing the pH-dependent conformational transitions between the active and inactive forms of PAC protein. The effect of changes in protonation states of a number of experimentally determined pH-sensor residues revealed their specific structural roles in the mechanism of conformational change underlying the changes in PAC activity. The allosteric pathways connecting these pH-sensors in the ECD to the TMD at different pH values revealed and quantified from analyses of the various MD trajectories with the rigorous N-body information theory (NbIT) method. Together with the structural data determined for various states of PAC (inactive, active, desensitized), the atomistic level of the dynamics driving the allosteric communication offers a comprehensive mechanistic understanding of the pH-dependence in PAC function.

β-Alanine Metabolism Leads to Increased Extracellular pH during the Heterotrophic Ammonia Oxidation of Pseudomonas putida Y-9

The mechanisms underlying the increase in external pH caused by heterotrophic nitrification and aerobic denitrification microorganisms during ammonia oxidation were unclear. This work demonstrated that after culturing Pseudomonas putida Y-9 for 60 h in a medium with ammonium nitrogen as the sole nitrogen source at an initial pH of 7.20, the pH value increased to 9.21. GC-TOF-MS analysis was used to compare the significantly regulated metabolites and related metabolic pathways between different time points. The results showed that the consumption of H+ in the conversion of malonic acid to 3-hydroxypropionic acid in the β-alanine metabolic pathway was the main reason for the increase in pH. RT-qPCR confirmed that the functional gene ydfG dominated the consumption of H+. This study provides new research ideas for the change of external pH caused by bacterial metabolism and further expands the understanding of the interaction between bacteria and the environment.

TASK inhibition by mild acidosis increases Ca2+ oscillations to mediate pH sensing in rat carotid body chemoreceptor cells.

Severe levels of acidosis (pH < 6.8) have been shown to cause a sustained rise in cytosolic Ca2+ concentration in carotid body Type 1 (glomus) cells. To understand how physiologically relevant levels of acidosis regulate Ca2+ signaling in glomus cells, we studied the effects of small changes in extracellular pH (pHo) on the kinetics of Ca2+ oscillations. A decrease in pHo from 7.4 to 7.3 (designated mild) and 7.2 (designated moderate) acidosis produced significant increases in the frequency and amplitude of Ca2+ oscillations. These effects of acidosis on Ca2+ oscillations were not blocked by NS383 and amiloride [acid-sensing ion channel (ASIC) inhibitors]. Mild and moderate levels of acidosis, however, caused a small but significant inhibition of two-pore domain acid-sensing K+ channels (TASK) (TASK-1- and TASK-3-like channels) and depolarized the cell by 6-13 mV. Acidosis-induced increase in Ca2+ oscillations was inhibited by nifedipine (1 µM; L-type Cav inhibitor) and by TTA-P2 (20 µM; T-type Cav inhibitor). Mild inhibition of TASK activity by N-[(2,4-difluorophenyl)methyl]-2'-[[[2-(4methoxyphenyl)acetyl]amino]methyl][1,1'-biphenyl]-2-carboxamide (A1899) (0.3 µM) and 1-[1-[6-[[1,1'-biphenyl]-4-ylcarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine-4-yl]-4-piperidinyl]-1-butanon (PK-THPP) (0.1 µM) increased Ca2+ oscillation frequency to levels similar to those observed with mild-moderate acidosis. Mild acidosis (pHo 7.3) and mild hypoxia (∼5%O2) produced similar levels of changes in the kinetics of Ca2+ oscillations. Block of tetraethylammonium (TEA)-sensitive Kv channels did not affect acid-induced increase in Ca2+ oscillations. Our study shows that mild and moderate levels of acidosis increase the frequency and amplitude of Ca2+ oscillations primarily by inhibition of TASK without involving ASICs, and suggests a major role of TASK for signal transduction in response to a physiological change in pHo.

Multiple Effects of Echinochrome A on Selected Ion Channels Implicated in Skin Physiology.

Echinochrome A (Ech A), a naphthoquinoid pigment from sea urchins, is known to have anti-inflammatory and analgesic effects that have been suggested to be mediated by antioxidant activity and intracellular signaling modulation. In addition to these mechanisms, the ion channels in keratinocytes, immune cells, and nociceptive neurons may be the target for the pharmacological effects. Here, using the patch clamp technique, we investigated the effects of Ech A on the Ca2+-permeable TRPV3, TRPV1 and Orai1 channels and the two-pore domain K+ (K2P) channels (TREK/TRAAK, TASK-1, and TRESK) overexpressed in HEK 293 cells. Ech A inhibited both the TRPV3 and Orai1 currents, with IC50 levels of 2.1 and 2.4 μM, respectively. The capsaicin-activated TRPV1 current was slightly augmented by Ech A. Ech A alone did not change the amplitude of the TREK-2 current (ITREK2), but pretreatments with Ech A markedly facilitated ITREK2 activation by 2-APB, arachidonic acid (AA), and acidic extracellular pH (pHe). Similar facilitation effects of Ech A on TREK-1 and TRAAK were observed when they were stimulated with 2-APB and AA, respectively. On the contrary, Ech A did not affect the TRESK and TASK-1 currents. Interestingly, the ITREK2 maximally activated by the combined application of 2-APB and Ech A was not inhibited by norfluoxetine but was still completely inhibited by ruthenium red. The selective loss of sensitivity to norfluoxetine suggested an altered molecular conformation of TREK-2 by Ech A. We conclude that the Ech A-induced inhibition of the Ca2+-permeable cation channels and the facilitation of the TREK/TRAAK K2P channels may underlie the analgesic and anti-inflammatory effects of Ech A.

Serine metabolism contributes to cell survival by regulating extracellular pH and providing an energy source in Saccharomyces cerevisiae.

Changes in extracellular pH affect the homeostasis and survival of unicellular organisms. Supplementation of culture media with amino acids can extend the lifespan of budding yeast, Saccharomyces cerevisiae, by alleviating the decrease in pH. However, the optimal amino acids to use to achieve this end, and the underlying mechanisms involved, remain unclear. Here, we describe the specific role of serine metabolism in the regulation of pH in a medium. The addition of serine to synthetic minimal medium suppressed acidification, and at higher doses increased the pH. CHA1, which encodes a catabolic serine hydratase that degrades serine into ammonium and pyruvate, is essential for serine-mediated alleviation of acidification. Moreover, serine metabolism supports extra growth after glucose depletion. Therefore, medium supplementation with serine can play a prominent role in the batch culture of budding yeast, controlling extracellular pH through catabolism into ammonium and acting as an energy source after glucose exhaustion.

Isolation and Identification of Limosilactobacillus reuteri PSC102 and Evaluation of Its Potential Probiotic, Antioxidant, and Antibacterial Properties.

We isolated and characterized Limosilactobacillus reuteri PSC102 and evaluated its probiotic, antioxidant, and antibacterial properties. We preliminarily isolated 154 candidates from pig feces and analyzed their Gram nature, morphology, and lactic acid production ability. Based on the results, we selected eight isolates and tested their ability to produce digestive enzymes. Finally, we identified one isolate using 16S rRNA gene sequencing, namely, L. reuteri PSC102. We tested its probiotic properties in vitro, including extracellular enzyme activities, low pH and bile salt tolerance, autoaggregation and coaggregation abilities, adhesion to Caco-2 cells, antibiotic susceptibility, and hemolytic and gelatinase activities. Antioxidant activity was determined using 1-diphenyl-2-picrylhydrazyl and 2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical scavenging and reducing power assays. The antibacterial activity of this strain and its culture supernatant against enterotoxigenic Escherichia coli were evaluated using a time-kill assay and disk diffusion method, respectively. L. reuteri PSC102 exhibited tolerance toward low pH and bile salt and did not produce harmful enzymes or possess hemolytic and gelatinase activities. Its intact cells and cell-free extract exhibited potential antioxidant activities, and significantly inhibited the growth of enterotoxigenic E. coli. Our results demonstrate that L. reuteri PSC102 is a potential probiotic candidate for developing functional feed.

Half-Sandwich Rhodium Complexes with Releasable N-Donor Monodentate Ligands: Solution Chemical Properties and the Possibility for Acidosis Activation.

Cancer chemotherapeutics usually have serious side effects. Targeting the special properties of cancer and activation of the anticancer drug in the tumor microenvironment in situ may decrease the intensity of the side effects and improve the efficacy of therapy. In this study, half-sandwich Rh complexes are introduced, which may be activated at the acidic, extracellular pH of the tumor tissue. The synthesis and aqueous stability of mixed-ligand complexes with a general formula of [Rh(η5-Cp*)(N,N/O)(N)]2+/+ are reported, where (N,N/O) indicates bidentate 8-quinolate, ethylenediamine and 1,10-phenanthroline and (N) represents the releasable monodentate ligand with a nitrogen donor atom. UV-visible spectrophotometry, 1H NMR, and pH-potentiometry were used to determine the protonation constants of the monodentate ligands, the proton dissociation constants of the coordinated water molecules in the aqua complexes, and the formation constants of the mixed-ligand complexes. The obtained data were compared to those of the analogous Ru(η6-p-cymene) complexes. The developed mixed-ligand complexes were tested in drug-sensitive and resistant colon cancer cell lines (Colo205 and Colo320, respectively) and in four bacterial strains (Gram-positive and Gram-negative, drug-sensitive, and resistant) at different pH values (5-8). The mixed-ligand complexes with 1-methylimidazole displayed sufficient stability at pH 7.4, and their activation was found in cancer cells with decreasing pH; moreover, the mixed-ligand complexes demonstrated antimicrobial activity in Gram-positive and Gram-negative bacteria, including the resistant MRSA strain. This study proved the viability of incorporating releasable monodentate ligands into mixed-ligand half-sandwich complexes, which is supported by the biological assays.

PH-Selective Reactions to Selectively Reduce Cancer Cell Proliferation: Effect of CaS Nanostructures in Human Skin Melanoma and Benign Fibroblasts.

An acidic extracellular pH value (pHe) is characteristic of many cancers, in contrast to the physiologic pHe found in most benign cells. This difference in pH offers a unique opportunity to design and engineer chemicals that can be employed for pH-selective reactions in the extracellular fluid of cancer cells. The viability of human skin melanoma and corresponding fibroblasts exposed to CaS dispersions is reported. The viability of melanoma cells decreases with CaS dispersion concentration and reaches 57% at 3%, a value easily distinguishable from melanoma control experiments. In contrast, the viability of benign fibroblasts remains nearly constant within experimental error over the range of dispersion concentrations studied. The CaS dispersions facilitate vinculin delocalization in the cytoplasmic fluid, a result consistent with improved focal adhesion kinase (FAK) regulation in melanoma cells. Thermodynamic considerations are consistent with the formation of from CaS in the presence of protons. The thermodynamic prediction is verified in independent experiments with solid CaS and acidic aqueous solutions. The amount of formed decreases with pH. An activation energy for the process of (30 ± 10) kJ/mol in the temperature range of 280 to 330 K is estimated from initial rate measurements as a function of temperature. The total Gibbs energy minimization approach was employed to establish the distribution of sulfides-including in the gas and aqueous phases-from the dissociation of CaS as a function of pH to mimic physiologically relevant pH values. Theoretical calculations suggest that partially protonated CaS in solution can be stable until the sulfur atom bonds to two hydrogen atoms, resulting in the formation of Ca2+ and , which can be solvated and/or released to the gas phase. Our results are consistent with a model in which CaS is dissociated in the extracellular fluid of melanoma cells selectively. The results are discussed in the context of the potential biomedical applications of CaS dispersions in cancer therapies.

Vasopressin V1a receptor and oxytocin receptor regulate murine sperm motility differently.

Specific receptors for the neurohypophyseal hormones, arginine vasopressin (AVP) and oxytocin, are present in the male reproductive organs. However, their exact roles remain unknown. To elucidate the physiological functions of pituitary hormones in male reproduction, this study first focused on the distribution and function of one of the AVP receptors, V1a. In situ hybridization analysis revealed high expression of the Avpr1a in Leydig cells of the testes and narrow/clear cells in the epididymis, with the expression pattern differing from that of the oxytocin receptor (OTR). Notably, persistent motility and highly proportional hyperactivation were observed in spermatozoa from V1a receptor-deficient mice. In contrast, OTR blocking by antagonist atosiban decreased hyperactivation rate. Furthermore, AVP stimulation could alter the extracellular pH mediated by the V1a receptor. The results highlight the crucial role of neurohypophyseal hormones in male reproductive physiology, with potential contradicting roles of V1a and OTR in sperm maturation. Our findings suggest that V1a receptor antagonists are potential therapeutic drugs for male infertility.

Proton-Activated Chloride Channel: Physiology and Disease.

The maintenance of intracellular and extracellular pH relies on multiple ion transporters/channels. Proton-activated chloride channel (PAC) precisely regulates extracellular and early/late endosomal pH by transporting chloride ion (Cl-) across membranes and has been shown to be implicated in pH imbalance under hypoxic conditions, such as the acidic microenvironments of cancer and ischemia. In this article, the phenotypic characteristics, molecular mechanisms, physiology of PAC and its role in cancer, ischemic stroke and hypoxia will be discussed in order to provide some clues for developing potential therapeutic strategies.

Alkaline pH, Low Iron Availability, Poor Nitrogen Sources and CWI MAPK Signaling Are Associated with Increased Fusaric Acid Production in Fusarium oxysporum

Fusaric acid (FA) is one of the first secondary metabolites isolated from phytopathogenic fungi belonging to the genus Fusarium. This molecule exerts a toxic effect on plants, rhizobacteria, fungi and animals, and it plays a crucial role in both plant and animal pathogenesis. In plants, metal chelation by FA is considered one of the possible mechanisms of action. Here, we evaluated the effect of different nitrogen sources, iron content, extracellular pH and cellular signalling pathways on the production of FA siderophores by the pathogen Fusarium oxysporum (Fol). Our results show that the nitrogen source affects iron chelating activity and FA production. Moreover, alkaline pH and iron limitation boost FA production, while acidic pH and iron sufficiency repress it independent of the nitrogen source. FA production is also positively regulated by the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway and inhibited by the iron homeostasis transcriptional regulator HapX. Collectively, this study demonstrates that factors promoting virulence (i.e., alkaline pH, low iron availability, poor nitrogen sources and CWI MAPK signalling) are also associated with increased FA production in Fol. The obtained new insights on FA biosynthesis regulation can be used to prevent both Fol infection potential and toxin contamination.

Intraperitoneal Delivery of Iopamidol to Assess Extracellular pH of Orthotopic Pancreatic Tumor Model by CEST-MRI.

The extracellular pH (pHe) of solid tumors is often acidic, as a consequence of the Warburg effect, and an altered metabolic state is often associated with malignancy. It has been shown that acidosis can promote tumor progression; thus, many therapeutic strategies have been adopted against tumor metabolism; one of these involves alkalinization therapies to raise tumor pH to inhibit tumor progression, improve immune surveillance, and overcome resistance to chemotherapies. Chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) is a noninvasive technique that can measure pH in vivo using pH-sensitive contrast agents. Iopamidol, an iodinated contrast agent, clinically used for computed tomography (CT), contains amide group protons with pH-dependent exchange rates that can reveal the pHe of the tumor microenvironment. In this study, we optimized intraperitoneal (IP) delivery of iopamidol to facilitate longitudinal assessments of orthotopic pancreatic tumor pHe by CEST-MRI. Following IV-infusion and IP-bolus injections, we compared the two protocols for assessing tumor pH. Time-resolved CT imaging was used to evaluate the uptake of iopamidol in the tumor, revealing that IP-bolus delivered a high amount of contrast agent 40 min postinjection, which was similar to the amounts reached with the IV-infusion protocol. As expected, both IP and IV injection protocols produced comparable measurements of tumor pHe, showing no statistically significant difference between groups (p=0.16). In addition, we showed the ability to conduct longitudinal monitoring of tumor pHe using CEST-MRI with the IP injection protocol, revealing a statistically significant increase in tumor pHe following bicarbonate administration (p < 0.001). In conclusion, this study shows the capability to measure pHe using an IP delivery of iopamidol into orthotopic pancreatic tumors, which is important to conduct longitudinal studies.