Reduction Of Extracellular pH Research Articles

Volatile organic compounds from Stenotrophomonas geniculata J-0 as potential biofumigants manage bulb rot caused by Fusarium oxysporum in postharvest Lanzhou lily.

The Lanzhou lily bulbs are often vulnerable to postharvest infections by pathogenic fungi, leading to lily bulb rot. This study investigated the ability of volatile organic compounds (VOCs) produced by Stenotrophomonas geniculata J-0 to control the highly pathogenic fungus Fusarium oxysporum BH-7 in postharvest Lanzhou lily bulbs. VOCs of S. geniculata J-0 showed inhibitory effect on the mycelial growth of F. oxysporum BH-7, with a maximum inhibition of 100%. Scanning electron microscope (SEM) observed that VOCs caused a shift in mycelial morphology from elongated and uniform tubular to collapsed and wrinkled. Moreover, VOCs of J-0 significantly reduced pathogenic fungal spore germination and sporulation. Through headspace gas chromatography-ion mobility spectrometry analysis, J-0 emitted 15 volatile compounds. The fumigation test of BH-7 with single pure synthetic compounds showed that 1-penten-3-one had excellent antifungal activity, with an inhibition rate of 100% at 4 μL/L. Additionally, our results revealed 1-penten-3-one destroyed the integrity and increased the permeability of BH-7 mycelial cell membranes, leading to leakage of intracellular electrolytes and substances, a reduction in extracellular pH, a blockage of ergosterol synthesis and an elevation in malondialdehyde content. In vivo experiments, fumigation of 1-penten-3-one at an exceptionally low concentration (4 μL/L) for a very short period of time (0.5h) was effective in delaying the onset and prevalence of postharvest diseases. Hence, this study provides novel antifungal agents to control disease in postharvest Lanzhou lily and enhances our understanding of the biocontrol potential of volatiles from S. geniculata.

Deficiency of G protein-coupled receptor Gpr111/Adgrf2 causes enamel hypomineralization in mice by alteration of the expression of kallikrein-related peptidase 4 (Klk4) during pH cycling process.

Enamel is formed by the repetitive secretion of a tooth-specific extracellular matrix and its decomposition. Calcification of the enamel matrix via hydroxyapatite (HAP) maturation requires pH cycling to be tightly regulated through the neutralization of protons released during HAP synthesis. We found that Gpr115, which responds to changes in extracellular pH, plays an important role in enamel formation. Gpr115-deficient mice show partial enamel hypomineralization, suggesting that other pH-responsive molecules may be involved. In this study, we focused on the role of Gpr111/Adgrf2, a duplicate gene of Gpr115, in tooth development. Gpr111 was highly expressed in mature ameloblasts. Gpr111-KO mice showed enamel hypomineralization. Dysplasia of enamel rods and high carbon content seen in Gpr111-deficient mice suggested the presence of residual enamel matrices in enamel. Depletion of Gpr111 in dental epithelial cells induced the expression of ameloblast-specific protease, kallikrein-related peptidase 4 (Klk4), suggesting that Gpr111 may act as a suppressor of Klk4 expression. Moreover, reduction of extracellular pH to 6.8 suppressed the expression of Gpr111, while the converse increased Klk4 expression. Such induction of Klk4 was synergistically enhanced by Gpr111 knockdown, suggesting that proper enamel mineralization may be linked to the modulation of Klk4 expression by Gpr111. Furthermore, our in vitro suppression of Gpr111 and Gpr115 expression indicated that their suppressive effect on calcification was additive. These results suggest that both Gpr111 and Gpr115 respond to extracellular pH, contribute to the expression of proteolytic enzymes, and regulate the pH cycle, thereby playing important roles in enamel formation.

Cell-Type-Specific Effects of the Ovarian Cancer G-Protein Coupled Receptor (OGR1) on Inflammation and Fibrosis; Potential Implications for Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by irreversible lung scarring. The pathophysiology is not fully understood, but the working hypothesis postulates that a combination of epithelial injury and myofibroblast differentiation drives progressive pulmonary fibrosis. We previously demonstrated that a reduction in extracellular pH activates latent TGF-β1, and that TGF-β1 then drives its own activation, creating a feed-forward mechanism that propagates myofibroblast differentiation. Given the important roles of extracellular pH in the progression of pulmonary fibrosis, we sought to identify whether pH mediates other cellular phenotypes independent of TGF-β1. Proton-sensing G-protein coupled receptors are activated by acidic environments, but their role in fibrosis has not been studied. Here, we report that the Ovarian Cancer G-Protein Coupled Receptor1 (OGR1 or GPR68) has dual roles in both promoting and mitigating pulmonary fibrosis. We demonstrate that OGR1 protein expression is significantly reduced in lung tissue from patients with IPF and that TGF-β1 decreases OGR1 expression. In fibroblasts, OGR1 inhibits myofibroblast differentiation and does not contribute to inflammation. However, in epithelial cells, OGR1 promotes epithelial to mesenchymal transition (EMT) and inflammation. We then demonstrate that sub-cellular localization and alternative signaling pathways may be responsible for the differential effect of OGR1 in each cell type. Our results suggest that strategies to selectively target OGR1 expression may represent a novel therapeutic strategy for pulmonary fibrosis.

Extracellular acidosis stimulates breast cancer cell motility through aryl hydrocarbon receptor and c-src kinase activation.

A reduction in extracellular pH (pHe) is a characteristic of most malignant tumors. The aryl hydrocarbon receptor (AhR) is a transcription factor localized in a cytosolic complex with c-Src, which allows it to trigger nongenomic effects through c-Src. Considering that the slightly acidic tumor microenvironment promotes breast cancer progression in a similar way to the AhR/c-Src axis, our aim was to evaluate whether this pathway could be activated by low pHe. We examined the effect of pHe 6.5 on AhR/c-Src axis using two breast cancer cell lines (MDA-MB-231 and LM3) and mammary epithelial cells (NMuMG) and found that acidosis increased c-Src phosphorylation only in tumor cells. Moreover, the presence of AhR inhibitors prevented c-Src activation. Low pHe reduced intracellular pH (pHi), while amiloride treatment, which is known to reduce pHi, induced c-Src phosphorylation through AhR. Analyses were conducted on cell migration and metalloproteases (MMP)-2 and -9 activities, with results showing an acidosis-induced increase in MDA-MB-231 and LM3 cell migration and MMP-9 activity, but no changes in NMuMG cells. Moreover, all these effects were blocked by AhR and c-Src inhibitors. In conclusion, acidosis stimulates the AhR/c-Src axis only in breast cancer cells, increasing cell migration and MMP-9 activity. Although the AhR activation mechanism still remains elusive, a reduction in pHi may be thought to be involved. These findings suggest a critical role for the AhR/c-Src axis in breast tumor progression stimulated by an acidic microenvironment.

Determining Arachidonic Acid Interaction Sites for Acid-Sensing Ion Channels using MD Simulations

Acid-sensing ion channels (ASICs) are trimeric, proton-gated cation channels found in both the central and peripheral nervous system. ASICs are important to pain sensation in relation to tissue acidification (Wemmie et al. Nat Rev Neurosci 2013). Although known to be activated by a reduction in extracellular pH through binding of protons (Waldmann et al. Nature 1997), there is evidence that ASICs are also modulated by membrane lipids. Arachidonic acid (AA) (Smith et al. J Neurosci 2007) and lysophosphatidylcholine (LPC) (Marra et al. EMBO J 2016) have been shown to activate the ASIC3 subtype without requiring a change in pH. Evidence suggest that lipid activation is likely a direct effect of lipid binding, rather than an effect of membrane distortion (Marra et al. EMBO J 2016). However, this mechanism of activation and possible binding sites are unknown. Structures of chicken ASIC1 have been determined in the resting (Yoder et al. Nature 2018), open (Baconguis et al. Cell 2014), and desensitized (Gonzales et al. Nature 2009) states. Using computational homology modelling and molecular dynamics (MD) simulations, we have simulated models of human ASIC1 and ASIC3 channels in a membrane environment containing AA, LPC and POPC. Exploiting the MARTINI coarse grained model and force field (Marrink et al., J. Phys. Chem. B 2007), we have compared lipid interactions with the two subtypes of ASICs for different functional states. We have identified putative binding sites for AA through analysing lipid contacts and residence times. Our data propose that specific residues, which differ between hASIC1 and hASIC3, are crucial for the AA interaction patterns observed in hASIC3. The identified binding sites are further explored through free energy calculations and atomistic resolution MD simulations.

Effects of nitrite at different temperatures on physiological parameters and growth in clown knifefish (Chitala ornata, Gray 1831)

The clown knifefish (Chitala ornata) is a facultative air breather that is cultured in freshwater farms in Mekong Delta, Vietnam. We report the effects of different temperatures (27 °C, 30 °C, and 33 °C) and nitrite exposure (1 mM) at these temperatures on haemoglobin derivatives, plasma ions, and acid-base status over 14 days. We also tested the effects of nitrite exposure at different temperatures on growth over 90 days of culture. An increase in temperature alone did not affect Na+, Cl−, HCO3−, and osmolality concentrations, whereas K+ was temporarily increased. Acid-base status was significantly affected by an increased partial pressure of carbon dioxide in the blood (PCO2) and reduction of extracellular pH (pHe) during elevated temperatures. Plasma nitrite levels and methaemoglobin (metHb) percentage reached the highest values at 33 °C compared to those at 27 °C and 30 °C with nitrite exposure after 3 days, but they decreased significantly at experimental termination by the detoxification process converting nitrite to nitrate. Plasma NO3− and the sum of [NO2−] and [NO3−] continuously increased during nitrite exposure at all three temperatures, reaching the highest values at 33 °C. Weight gain and survival rate increased at elevated temperatures with the highest value at 33 °C (29 g and 85%, respectively), while feed conversion ratio (FCR) values were unchanged at all temperatures. In contrast, elevated temperature caused a significant lowering of survival rate in nitrite exposure, but weight gain was highest with nitrite exposure at 30 °C and 33 °C.

Subunit composition critically determines pH sensitivity of epithelial sodium channels in Xenopus laevis

The Epithelial Sodium Channel (ENaC) mediates the selective flux of sodium ions across the apical membrane of epithelial cells. ENaC consists of three subunits (α,β,γ) and its activity at the cell surface is subject to a multitude of regulatory mechanisms, including proteolytic cleavage, extracellular pH and Na+ ions. Channel regulation may also depend on ENaC subunit composition, as an additional δ‐subunit can replace α‐ENaC in the heterotrimeric configuration. As the physiological implications of ENaC containing the δ‐subunit are poorly understood, we characterized the regulation of Xenopus δβγ‐ENaC by extracellular pH and Na+.Regulation of Xenopus δβγ‐ENaC by extracellular Na+ and pH was investigated by two‐electrode voltage‐clamp and cell‐attached Patch‐Clamp electrophysiology using the Xenopus oocyte expression system. Successive reduction of extracellular pH from pH 8 – 6 increased amiloride‐sensitive transmembrane currents of oocytes expressing δβγ‐ENaC by > 7 fold. In cell‐attached Patch‐Clamp recordings the pH of the pipette solution (pHpip = extracellular) affected δβγ‐ENaC open probability and kinetics. Compared to pHpip at 7.4, alkaline conditions (pHpip 8) decreased open probability as well as the mean channel open time, whereas under acidic conditions (pHpip 6) an increased open probability was accompanied by a reduction of average channel closed times. Transmembrane currents mediated by Xenopus δβγ‐ENaC are sensitive to sodium self‐inhibition, a phenomenon describing the fast inhibition of ENaC in the presence of high extracellular Na+ concentrations. Channel activation under acidic conditions (pH 7 & 6) was accompanied by a decrease in magnitude and apparent Na+‐affinity of self‐inhibition, whereas these effects were reversed under alkaline conditions (pH 8). Hypothesizing mutual regulation of δβγ‐ENaC activity by extracellular Na+ and pH, we examined whether neutralization of pH‐sensitive aspartates in the δ‐subunit affected channel self‐inhibition and regulation by pH. Specific aspartates in a subunit domain corresponding to a putative Na+‐sensing site of mouse α‐ENaC (Kashlan et al., 2015, J Biol Chem 290:568–76) were replaced by asparagines via site‐directed mutagenesis (δD293N, δD296N, δD293,6N). All mutations decreased pH‐mediated channel activation. Notably, ENaC containing the δD296N mutation also displayed a decreased sodium self‐inhibition, which was not observed in channels bearing a single δD293N mutation. Furthermore, human δβγ‐ENaCs which have a reduced sodium self‐inhibition were not activated by acidic pH as observed with the Xenopus orthologue displaying a strong sodium self‐inhibition.In conclusion, these results demonstrate that activity of ENaC containing the δ‐subunit in Xenopus laevis is sensitive to changes in the extracellular pH, whereas ENaC containing the α‐subunit is not. Proton‐mediated activation of ENaC involves changes in single channel kinetics and a reduction of sodium self‐inhibition. Thus, sensitivity of this amphibian ENaC to extracellular pH is critically determined by its subunit composition.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Decreased extracellular pH inhibits osteogenesis through proton-sensing GPR4-mediated suppression of yes-associated protein

The pH of extracellular fluids is a basic property of the tissue microenvironment and is normally maintained at 7.40 ± 0.05 in humans. Many pathological circumstances, such as ischemia, inflammation, and tumorigenesis, result in the reduction of extracellular pH in the affected tissues. In this study, we reported that the osteogenic differentiation of BMSCs was significantly inhibited by decreases in the extracellular pH. Moreover, we demonstrated that proton-sensing GPR4 signaling mediated the proton-induced inhibitory effects on the osteogenesis of BMSCs. Additionally, we found that YAP was the downstream effector of GPR4 signaling. Our findings revealed that the extracellular pH modulates the osteogenic responses of BMSCs by regulating the proton-sensing GPR4-YAP pathway.

Acidosis differently modulates the inflammatory program in monocytes and macrophages

Inflammation, ischemia or the microenvironment of solid tumors is often accompanied by a reduction of extracellular pH (acidosis) that stresses the cells and acts on cellular signaling and transcription. The effect of acidosis on the expression of various inflammatory markers, on functional parameters (migration, phagocytic activity) and on signaling pathways involved was studied in monocytic cells and macrophages. In monocytic cell lines acidosis led to a reduction in expression of most of the inflammatory mediators, namely IL-1ß, IL-6, TNF-α, MCP-1, COX-2 and osteopontin. In primary human monocytes MCP-1 and TNF-α were reduced but COX-2 and IL-6 were increased. In RAW264.7 macrophage cell line IL-1ß, COX-2 and iNOS expression was increased, whereas MCP-1 was reduced similar to the effect in monocytic cells. For primary human monocyte-derived macrophages the regulation of inflammatory markers by acidosis depended on activation state, except for the acidosis-induced downregulation of MCP-1 and TNF-α. Acidosis affected functional immune cell behavior when looking at phagocytic activity which was increased in a time-dependent manner, but cellular motility was not changed. Neither ERK1/2 nor CREB signaling was stimulated by the reduction of extracellular pH. However, p38 was activated by acidosis in RAW264.7 cells and this activation was critical for the induction of IL-1ß, COX-2 and iNOS expression. In conclusion, acidosis may impede the recruitment of immune cells, but fosters inflammation when macrophages are present by increasing the level of COX-2 and iNOS and by functionally forcing up the phagocytic activity.

Acid–base and ion balance in fishes with bimodal respiration

The evolution of air breathing during the Devonian provided early fishes with bimodal respiration with a stable O2 supply from air. This was, however, probably associated with challenges and trade-offs in terms of acid-base balance and ionoregulation due to reduced gill:water interaction and changes in gill morphology associated with air breathing. While many aspects of acid-base and ionoregulation in air-breathing fishes are similar to water breathers, the specific cellular and molecular mechanisms involved remain largely unstudied. In general, reduced ionic permeability appears to be an important adaptation in the few bimodal fishes investigated but it is not known if this is a general characteristic. The kidney appears to play an important role in minimizing ion loss to the freshwater environment in the few species investigated, and while ion uptake across the gut is probably important, it has been largely unexplored. In general, air breathing in facultative air-breathing fishes is associated with an acid-base disturbance, resulting in an increased partial pressure of arterial CO2 and a reduction in extracellular pH (pHE ); however, several fishes appear to be capable of tightly regulating tissue intracellular pH (pHI ), despite a large sustained reduction in pHE , a trait termed preferential pHI regulation. Further studies are needed to determine whether preferential pHI regulation is a general trait among bimodal fishes and if this confers reduced sensitivity to acid-base disturbances, including those induced by hypercarbia, exhaustive exercise and hypoxia or anoxia. Additionally, elucidating the cellular and molecular mechanisms may yield insight into whether preferential pHI regulation is a trait ultimately associated with the early evolution of air breathing in vertebrates.

Temperature sensitivity of acid-sensitive outwardly rectifying (ASOR) anion channels in cortical neurons is involved in hypothermic neuroprotection against acidotoxic necrosis

The acid-sensitive outwardly rectifying (ASOR) anion channel has been found in non-neuronal cell types and was shown to be involved in acidotoxic death of epithelial cells. We have recently shown that the ASOR channel is sensitive to temperature. Here, we extend those results to show that temperature-sensitive ASOR anion channels are expressed in cortical neurons and involved in acidotoxic neuronal cell death. In cultured mouse cortical neurons, reduction of extracellular pH activated anionic currents exhibiting phenotypic properties of the ASOR anion channel. The neuronal ASOR currents recorded at pH 5.25 were augmented by warm temperature, with a threshold temperature of 26 °C and the Q10 value of 5.6. After 1 h exposure to acidic solution at 37 °C, a large population of neurons suffered from necrotic cell death which was largely protected not only by ASOR channel blockers but also by reduction of temperature to 25 °C. Thus, it is suggested that high temperature sensitivity of the neuronal ASOR anion channel provides, at least in part, a basis for hypothermic neuroprotection under acidotoxic situations associated with a number of pathological brain states.

A Link between Glial Ca2+ Signaling and Hypoxia in Aging?

Most neuroscientists now acknowledge that there is more to the brain than neurons. A flurry of studies in the past decade have documented the importance of glia in complex processes that were previously believed to be strictly neuronal.1 The majority of these studies have pointed to a key role of astrocytic Ca2+ signaling in the modulation of synaptic transmission. In fact, Ca2+ signaling in electrically silent astrocytes can in many ways be compared with electrical excitability in neurons: Astrocytes integrate multiple inputs into dynamic Ca2+ signals and in turn modulate the activity of surrounding neurons. Perhaps, the most interesting discovery is that astrocytic Ca2+ signaling appears to be intimately involved in neural homeostatic regulation. For example, Ca2+ signaling in brain stem astrocytes may help to control breathing: A reduction in extracellular pH leads to astrocytic Ca2+-dependent ATP release. ATP binds to purinergic (ATP) receptors on neighboring respiratory neurons, depolarizing them and resulting in an increase in respiration.2

Metabolic changes associated with acid–base regulation during hypercarbia in the CO 2-tolerant chondrostean, white sturgeon ( Acipenser transmontanus)

CO 2 tolerance in white sturgeon is associated with the ability to tightly regulate intracellular pH (pHi) despite a large reduction in extracellular pH (pHe) termed preferential pHi regulation. How this regulatory response affects whole animal metabolic rate is unknown. Accordingly, we characterized oxygen consumption rate ( M ˙ O 2 ) and metabolically-relevant organismal and cellular responses in white sturgeon during exposure to hypercarbia. White sturgeon were able to protect intracellular pH (pHi) in liver and white muscle as early as 6 h (the earliest time period investigated) following exposure to severe (sub-lethal) hypercarbia (45 and 90 mmHg PCO 2). Sturgeon exposed to 15 and 30 mmHg PCO 2 exhibited pHe compensation and significant increases in M ˙ O 2 (up to 80% greater than control values). In contrast, severe hypercarbia (≥ 45 mmHg PCO 2) elicited an uncompensated reduction in pHe (up to ~ 1.0 pH units) and red blood cells (as great as ~ 0.5 pH units), and was accompanied by 30 and 60% reductions in M ˙ O 2 , respectively. While behavioral, respiratory and cellular responses to hypercarbia were observed, none corresponded well with the pattern or magnitude of changes in M ˙ O 2 . The findings of this research provide empirical support for the hypothesis that preferential pHi regulation is not metabolically costly, and thus may have been a strategy strongly selected for in fishes encountering short-term hypercarbia.

Activation of P-glycoprotein (Pgp)-mediated drug efflux by extracellular acidosis: in vivo imaging with 68Ga-labelled PET tracer

In vitro it has been shown that the functional activity of P-glycoprotein (Pgp), an important drug transporter responsible for multidrug resistance, can be strongly increased by extracellular acidosis. Here mitogen-activated protein kinases (MAPK) (p38, ERK1/2) seem to play an important role for signal transduction. However, it is unclear whether these effects are also relevant in vivo. With the newly developed PET tracer Schiff base-based (68)Ga-MFL6.MZ the functional Pgp activity was visualized under acidic conditions and during inhibition of MAPKs non-invasively by means of microPET in rat tumours. Tumours were acidified either by inspiratory hypoxia (8% O(2)) or by injection of lactic acid. Inhibitors of the MAPK were injected intratumourally. With increasing tumour volume the tumour pH changed from 7.0 to 6.7 and simultaneously the Pgp activity increased almost linearly. When the tumour was acidified by direct lactic acid injection the PET tracer uptake was reduced by 20% indicating a higher transport rate out of the cells. Changing the inspiratory O(2) fraction to 8% dynamically led to a reduction of extracellular pH and in parallel to a decrease of tracer concentration. While inhibition of the p38 pathway reduced the Pgp transport rate, inhibition of ERK1/2 had practically no impact. An acidic extracellular environment significantly stimulates the Pgp activity. The p38 MAPK pathway plays an important role for Pgp regulation in vivo, whereas ERK1/2 is of minor importance. From these results new strategies for overcoming multidrug resistance (e.g. reducing tumour acidosis, inhibition of p38) may be developed.

Evaluation of endogenous acidic metabolic products associated with carbohydrate metabolism in tumor cells

Tumor cells have a high tolerance for acidic and hypoxic microenvironments, also producing abundant lactic acid through accelerated glycolysis in the presence or absence of O(2). While the accumulation of lactate is thought to be a major contributor to the reduction of pH-circumscribing aggressive tumors, it is not known if other endogenous metabolic products contribute this acidity. Furthermore, anaerobic metabolism in cancer cells bears similarity to homo-fermentative lactic acid bacteria, however very little is known about an alternative pathway that may drive adenosine triphosphate (ATP) production independent of glycolysis. In this study, we quantify over 40 end-products (amines, acids, alcohols, aldehydes, or ketones) produced by malignant neuroblastoma under accelerated glycolysis (+glucose (GLU) supply 1-10 mM) +/- mitochondrial toxin; 1-methyl-4-phenylpyridinium (MPP(+)) to abate aerobic respiration to delineate differences between anaerobic vs. aerobic cell required metabolic pathways. The data show that an acceleration of anaerobic glycolysis prompts an expected reduction in extracellular pH (pH(ex)) from neutral to 6.7 +/- 0.006. Diverse metabolic acids associated with this drop in acidity were quantified by ionic exchange liquid chromatography (LC), showing concomitant rise in lactate (Ctrls 7.5 +/- 0.5 mM; +GLU 12.35 +/- 1.3 mM; +GLU + MPP 18.1 +/- 1.8 mM), acetate (Ctrl 0.84 +/- 0.13 mM: +GLU 1.3 +/- 0.15 mM; +GLU + MPP 2.7 +/- 0.4 mM), fumarate, and a-ketoglutarate (<10 microM) while a range of other metabolic organic acids remained undetected. Amino acids quantified by o-phthalaldehyde precolumn derivatization/electrochemical detection-LC show accumulation of L: -alanine (1.6 +/- .052 mM), L: -glutamate (285 +/- 9.7 microM), L: -asparagine (202 +/- 2.1 microM), and L: -aspartate (84.2 +/- 4.9 microM) produced during routine metabolism, while other amino acids remain undetected. In contrast, the data show no evidence for accumulation of acetaldehyde, aldehydes, or ketones (Purpald/2,4-dinitrophenylhydrazine-Brady's reagent), acetoin (Voges-Proskauer test), or alcohols (NAD(+)-linked alcohol dehydrogenase). In conclusion, these results provide preliminary evidence to suggest the existence of an active pyruvate-alanine transaminase or phosphotransacetylase/acetyl-CoA synthetase pathway to be involved with anaerobic energy metabolism of cancer cells.

Purinergic activation of a leak potassium current in freshly dissociated myocytes from mouse thoracic aorta

Exogenous ATP elicits a delayed calcium-independent K(+) current on freshly isolated mouse thoracic aorta myocytes. We investigated the receptor, the intracellular pathway and the nature of this current. The patch-clamp technique was used to record ATP-elicited delayed K(+) current in freshly dissociated myocytes. ATP-elicited delayed K(+) current was not inhibited by a 'cocktail' of K(+) channel blockers (4-AP, TEA, apamin, charybdotoxin, glibenclamide). The amplitude of the delayed K(+) current decreased after the reduction of extracellular pH from 7.4 to 6.5. These two characteristics suggest that this current could be carried by the TASK subfamily of 'twin-pore potassium channels' (K2P). Purinergic agonists including dATP, but not ADP, activated the delayed K(+) current, indicating that P2Y(11) is the likely receptor involved in its activation. The PKC activator phorbol ester 12,13-didecanoate stimulated this current. In addition, the PKC inhibitor Gö 6850 partially inhibited it. Real-time quantitative PCR showed that the genes encoding TASK-1 and TASK-2 are expressed. Our results indicate that blocker cocktail-insensitive delayed K(+) current in freshly dissociated aortic myocytes is probably carried by the TASK subfamily of twin-pore channels.

Acidosis inhibits spontaneous activity and membrane currents in myocytes isolated from the rabbit atrioventricular node

Recent evidence from intact hearts suggests that the function of cardiac nodal tissue may be particularly susceptible to acidosis. Little is currently known, however, about the effects of acidosis on the cellular electrophysiology of the atrioventricular node (AVN). This study was conducted, therefore, to determine the effect of acidosis on the spontaneous activity and membrane currents of myocytes isolated from the rabbit AVN, recorded at 35–37 °C using whole-cell patch-clamp. Reduction of extracellular pH (pH e; from 7.4 to 6.8 or 6.3) produced pH-dependent slowing of spontaneous action potential rate and upstroke velocity, and reductions in maximum diastolic potential and action potential amplitude. Ionic current recordings under voltage-clamp indicated that acidosis (pH e 6.3) decreased L-type Ca current ( I Ca,L), without significant changes in voltage-dependent activation or inactivation. Acidosis reduced the E-4031-sensitive, rapid delayed rectifier current ( I Kr) tail amplitude at − 40 mV following command pulses to between − 30 and + 50 mV, and accelerated tail-current deactivation. In contrast, the time-dependent hyperpolarisation-activated current, I f, was unaffected by acidosis. Background current insensitive to E-4031 and nifedipine was reduced by acidosis. Measurement of intracellular pH (pH i) from undialysed cells using BCECF showed a reduction in mean pH i from 7.24 to 6.45 ( n = 17) when pH e was lowered from 7.4 to 6.3. We conclude that I f is unlikely to be involved in the response of the AVN to acidosis, whilst inhibition of I Ca,L and I Kr by acidosis are likely to play a significant role in effects on AVN cellular electrophysiology.

A moderate reduction in extracellular pH protects macrophages against apoptosis induced by oxidized low density lipoprotein

We investigated the effect of pH on macrophage apoptosis induced by oxidized low density lipoprotein (OxLDL), as human atherosclerotic lesions have regions of low pH. Hydroperoxide-rich and oxysterol-rich LDL caused 38% and 74% apoptosis of J774 macrophages, respectively, at 24 h, as measured by the externalization of phosphatidylserine. Native LDL, however, did not cause apoptosis. Reducing the pH of the culture medium from 7.4 to 7.0 inhibited apoptosis induced by hydroperoxide-rich or oxysterol-rich OxLDL by 61% and 46%, respectively (P < 0.001). These data were confirmed by semiquantitative analysis of cytochrome c release from mitochondria. Decreasing the extracellular pH to 7.0 reduced the uptake of hydroperoxide-rich and oxysterol-rich (125)I-labeled LDL by 82% and 42%, respectively, and reduced cell surface binding of oxysterol-rich LDL by 31%. This may explain the reduced apoptosis. Additionally, low pH did not affect OxLDL-induced apoptosis of human monocytes, which do not possess scavenger receptors for OxLDL, but reduced apoptosis of human monocyte-derived macrophages, which do possess them. Our investigations suggest that the presence of areas of low pH within atherosclerotic lesions may reduce the uptake of OxLDL and reduce macrophage apoptosis, thus affecting lesion progression.

In vivored blood cell sickling and mechanism of recovery in whiting,Merlangius merlangus

Haemoglobin concentrations in vertebrate red blood cells are so high that in human sickle cell disease a single surface amino acid mutation can result in formation of large insoluble haemoglobin aggregates at low oxygen levels, causing peculiar cell deformations or 'sickling'. This may cause vascular occlusion and thereby severe pain, organ failure and death. Here, using light and transmission electron microscopy, we demonstrate extensive in vivo sickling of whiting red blood cells after capture stress without any apparent haemolysis and show its subsequent recovery. We show exceptionally high cooperative proton binding during the sickling process in vitro and identify the reduction of extracellular pH below resting values as the primary cause for in vivo sickling, although the response is modulated to a lesser extent also by oxygen tension. Using isotope tracer fluxes, we further show that beta-adrenergic hormones, which are released under capture stress, activate a powerful endogenous Na/H exchanger in these fish red blood cells, which is known to elevate intracellular pH. beta-adrenergic treatment further leads to a marked reduction of acid-induced in vitro sickling, which is impaired when Na/H exchange is inhibited by amiloride. We propose that this mechanism protects red blood cells of some fishes against the problem of haemoglobin aggregation and red blood cell sickling, except under most severe acidosis. This system offers a unique example of how, over evolutionary time, nature may have overcome what is still a deadly disease in humans.

Role of acid-sensitive outwardly rectifying anion channels in acidosis-induced cell death in human epithelial cells

Recently, a novel type of anion channel activated by extracellular acidification has been found in a variety of mammalian cell types. However, the role of this acid-sensitive outwardly rectifying (ASOR) anion channel is not known. In human epithelial HeLa cells, reduction in extracellular pH below 5 rapidly activated anion-selective whole-cell currents. The currents exhibited strong outward rectification, activation kinetics at positive potentials, low-field anion selectivity, and sensitivity to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and phloretin. When outside-out patches were exposed to acidic bathing solution, single-channel events of the anion channel could be observed. The unitary conductance was 4.8 pS in the voltage range between -80 and +80 mV. The single-channel activity prominently increased with depolarization and was suppressed by DIDS or phloretin. After 1-h incubation in acidic solution (pH 4.5), a significant population of HeLa cells suffered from necrotic cell injury characterized by stainability with propidium iodide and lack of caspase-3 activation. Upon exposure to acidic solution, HeLa cells exhibited immediate, persistent swelling. Both the necrotic volume increase and cell injury induced by extracellular acidification were inhibited by DIDS or phloretin. Therefore, it is concluded that the ASOR anion channel is involved in the genesis of necrotic cell injury induced by acidosis in human epithelial cells.