Increase In pHi Research Articles

Loss-of-activity-mutation in the cardiac chloride-bicarbonate exchanger AE3 causes short QT syndrome

Patients with short QT syndrome (SQTS) may present with syncope, ventricular fibrillation or sudden cardiac death. Six SQTS susceptibility genes, encoding cation channels, explain <25% of SQTS cases. Here we identify a missense mutation in the anion exchanger (AE3)-encoding SLC4A3 gene in two unrelated families with SQTS. The mutation causes reduced surface expression of AE3 and reduced membrane bicarbonate transport. Slc4a3 knockdown in zebrafish causes increased cardiac pHi, short QTc, and reduced systolic duration, which is rescued by wildtype but not mutated SLC4A3. Mechanistic analyses suggest that an increase in pHi and decrease in [Cl−]i shortened the action potential duration. However, other mechanisms may also play a role. Altered anion transport represents a mechanism for development of arrhythmia and may provide new therapeutic possibilities.

A genetic variant of the sperm-specific SLO3 K+ channel has altered pH and Ca2+ sensitivities

To fertilize an oocyte, sperm must first undergo capacitation in which the sperm plasma membrane becomes hyperpolarized via activation of potassium (K+) channels and resultant K+ efflux. Sperm-specific SLO3 K+ channels are responsible for these membrane potential changes critical for fertilization in mouse sperm, and they are only sensitive to pH i However, in human sperm, the major K+ conductance is both Ca2+- and pH i -sensitive. It has been debated whether Ca2+-sensitive SLO1 channels substitute for human SLO3 (hSLO3) in human sperm or whether human SLO3 channels have acquired Ca2+ sensitivity. Here we show that hSLO3 is rapidly evolving and reveal a natural structural variant with enhanced apparent Ca2+ and pH sensitivities. This variant allele (C382R) alters an amino acid side chain at a principal interface between the intramembrane-gated pore and the cytoplasmic gating ring of the channel. Because the gating ring contains sensors to intracellular factors such as pH and Ca2+, the effectiveness of transduction between the gating ring and the pore domain appears to be enhanced. Our results suggest that sperm-specific genes can evolve rapidly and that natural genetic variation may have led to a SLO3 variant that differs from wild type in both pH and intracellular Ca2+ sensitivities. Whether this physiological variation confers differences in fertility among males remains to be established.

The Effect of SLC4A11 Mutations on Intracellular pH

SLC4A11 is a ubiquitously expressed transporter showing Na+‐OH−, NH3‐H+ cotransport, and water permeability. SLC4A11 mutations have been associated with Fuchs' endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED2) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). To address the role of known human SLC4A11 mutations on cellular physiology we measured intracellular pH regulation in PS120 fibroblasts that have been stably transfected with SLC4A11 mutant variants.Mutations were made in the Wild‐Type (WT) construct phCMV2‐hSLC4A11 by site‐directed mutagenesis. Mutants with a single amino acid change were obtained and referred to as R125H, C386R, V507I and N639A respectively. PS120 cells were stably transfected with those constructs and empty vector (EV) using SuperFect, and selected by Geneticin. For intracellular pH (pHi) measurement, PS120 cells grown on coverslips were loaded with a pH‐sensitive fluorescent dye, 2′7′‐Bis‐(2‐Carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein (BCECF‐AM). The coverslip was mounted in a perfusion chamber (37°C) on the stage of a fluorescence microscope. Perfusion was performed with a Bicarbonate‐free solution (BF) to obtain stable baseline pH, followed by pulsing cells with 10 mM NH4Cl for 5 minutes, and changed back to BF perfusion until pHi was stable. Fluorescence ratio (495/440 nm) was converted to pHi following calibration with high K+/Nigericin. Results are means ± SE from 3 trials.The baseline pHi was 7.211 ± 0.136, 7.877 ± 0.056, 7.391 ± 0.069, 7.226 ± 0.232, 7.504 ± 0.104 and 7.359 ± 0.221 for WT, EV, R125H, C386R, V507I and N639A, respectively. The pHi increase from NH4Cl pulse was 0.486 ± 0.014, 0.161 ± 0.018, 0.298 ± 0.05, 0.307 ± 0.07, 0.304 ± 0.011 and 0.442 ± 0.084 respectively. The rate of pHi increase (ΔpH/min) was 2.184 ± 0.33, 0.469 ± 0.037, 1.004 ± 0.124, 1.024 ± 0.362, 1.17 ± 0.142 and 1.818 ± 0.405, respectively. The subsequent rate of pHi decrease in the presence of NH4Cl was (ΔpH/min) 0.153 ± 0.038, 0.006 ± 0.012, 0.012 ± 0.005, 0.011 ± 0.003, 0.033 ± 0.002 and 0.009 ± 0.007, respectively. Removal of NH4Cl‐BF resulted in maximum acidification of 1.205 ± 0.122, 0.343 ± 0.074, 0.671 ± 0.019, 0.409 ± 0.122, 0.747 ± 0.041 and 0.748 ± 0.048 pH units. NH4Cl induced pHi changes were WT &gt; N639A &gt; V507I &gt; R125H &gt; C386R &gt; EV.Expression of SLC4A11 WT and mutants lower resting pHi relative to EV, consistent with a H+ permeability. NH4Cl induced pHi changes were consistent with reduction of NH3:H+ linked cotransport in SLC4A11 mutants.Support or Funding InformationNIH R01 EY08834‐23 &amp; P30EY019008

Influence of tetrapeptide tuftsin on intracellular pH of mouse peritoneal macrophages

It has been shown by the method of probe microfluorimetry of single cells that tuftsin at concentrations of 0.1 and 1.0 μg/mL, increasing the phagocytic activity of murine peritoneal macrophages, causes biphasic changes in the intracellular pH (pHi) over time. First there was a decrease in pHi, reaching the limit in 5 min of incubation. Then, the pHi value increased until reaching the maximum 30 min after the interaction between the cells and the agent. Afterwards, the observed parameter (pHi) did not vary up to minute 55. It was shown that there was no increase in intracellular pH on the addition of the Na+/H+-exchange blocker ethylisopropylamiloride to the incubation medium in the presence of tuftsin. This fact suggests that the observed increase in pHi during tuftsin treatment in the second phase of cell response is associated with the Na+/H+ exchange system.

Block of CDK1-dependent polyadenosine elongation of Cyclin B mRNA in metaphase-i-arrested starfish oocytes is released by intracellular pH elevation upon spawning.

Meiotic progression requires the translation of maternal mRNAs in a strict temporal order. In isolated animal oocytes, translation of maternal mRNAs containing a cytoplasmic polyadenylation element (CPE), such as cyclin B, is activated by in vitro stimulation of meiotic resumption which induces phosphorylation of CPEB (CPE-binding protein) and elongation of their polyadenosine (poly(A)) tails; whether or not this model can be applied in vivo to oocytes arrested at metaphase of meiosis I in ovaries is unknown. In this study, we found that active CDK1 (cyclin-dependent kinase 1) phosphorylated CPEB in ovarian oocytes arrested at metphase I in the starfish body cavity, but phosphorylation of CPEB was not sufficient for elongation of cyclin B poly(A) tails. Immediately after spawning, however, mRNA was polyadenylated, suggesting that an increase in intracellular pH (pHi ) upon spawning triggers the elongation of poly(A) tails. Using a cell-free system made from maturing oocytes at metaphase I, we demonstrated that polyadenylation was indeed suppressed at pH below 7.0. These results suggest that a pH-sensitive process, functioning after CPEB phosphorylation, is blocked under physiologically low pHi (<7.0) in metaphase-I-arrested oocytes. The increase in pHi (>7.0) that occurs after spawning triggers polyadenylation of cyclin B mRNA and progression into meiosis II.

Single cell imaging reveals that the motility regulator speract induces a flagellar alkalinization that precedes and is independent of Ca2+ influx in sea urchin spermatozoa

Speract, a peptide from the egg jelly coat of certain sea urchin species, modulates sperm motility through a signaling pathway involving several ionic fluxes leading to pHi and [Ca2+]i increases. [Ca2+]i oscillations in the flagellum regulate its beating pattern modulating sperm swimming. Recent evidence showed the importance of pHi in controlling Ca2+ influx and chemotaxis. However, spatio-temporal characterization of the flagellar pHi increase triggered by speract, and its correlation to that of [Ca2+]i is lacking. Here, we show for the first time in single sea urchin spermatozoa that the speract-induced flagellar pHi increase precedes and is independent of [Ca2+]i increase. Our results support a leading role of pHi in modulating the Ca2+ signals that govern sperm swimming.

An Investigation of the Impact of NHE1 Phosphorylation on Steady State Intracellular pH

The Sodium‐Hydrogen Exchanger 1 (NHE1) is involved in cell scaffolding, mobility and intracellular pH (pHi) homeostasis in most cells. The C‐terminus of NHE1 is phosphorylated by several protein kinases, each contributing to NHE1 function. While investigation of one or two sites have been conducted with various agonists, we do not have an understanding of how these sites coordinate or if there is a hierarchical phosphorylation regulation of NHE1. To determine the potential phosphorylation‐dependent regulation of NHE1 on pHi, we focus on four protein kinases with known sites on NHE1; ERK 1⁄2, protein kinase B (AKT), RhoA kinase (Rock) and ribosomal s6 kinase (RSK). To measure the impact of these four kinases we will treat cells with three different agonist; lysophophatidic acid (LPA), platelet‐derived growth factor (PDGF), and phenylephrine (PE). We first determined the dose response for each agonist in fibroblasts deficient in NHE1 expression (PS120 cells) and in stable expressing human NHE1 in PS120 cells (PSN cells). To determine the role of each kinase on NHE1 basal activity, cells were treated with each agonist and teady state pHi was determined 15‐20 min after addition of agonist. Increases in pHi of 0.18 to 0.2 were observed for agonists. The role of each kinase was determined by treating cells prior to agonist with a specific cell permeable kinase inhibitor (1uM MK‐2206, 10uM BI‐D1870, 10uM Y27632 and 0.5uM SCH772984). The impact of direct phosphorylation was determined for each agonist in cells expressing human NHE1 with Ser/Thr to Ala mutations for each kinase site. This work begins to identify the coordinated and role of multiple phosphorylation events on NHE1.

P2X receptors trigger intracellular alkalization in isolated perfused mouse medullary thick ascending limb.

AimsExtracellular ATP is an important regulator of renal tubular transport. Recently, we found that basolateral ATP markedly inhibits Na+ and Cl− absorption in mouse medullary thick ascending limb (mTAL) via a P2X receptor. The underlying mechanism that mediates this ATP-dependent transport inhibition in mTAL is, however, unclear. The renal outer medullary K+ channel (ROMK) is sensitive to intracellular pH where a reduction leads to closing of ROMK. We speculated that P2X receptor stimulation in the TAL could lead to changes in pHi, leading to a reduction in NaCl transport.MethodsTo test this hypothesis, we measured pHi in single perfused mouse mTALs using the fluorescent ratiometric dye 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethylester.ResultsInterestingly, basolateral ATP (100 μm) caused a prominent, reversible intracellular alkalization of mTAL, with an average pHi increase of 0.14 ± 0.02 (n = 14). This was completely abolished by the P2X receptor antagonist periodate-oxidized ATP (50 μm). The P2X receptor-mediated intracellular alkalization required the activity of the apical Na+/H+ exchanger (NHE3). Typically, Gq-coupled receptors cause a significant acidification of tubular epithelial cells, which was confirmed in this study, by P2Y2 and Ca2+ sensing receptor stimulation.ConclusionThis study reports that stimulation of basolateral P2X receptors causes a substantial intracellular alkalization in the isolated perfused mouse mTAL. This intracellular alkalization is mediated through an increased apical NHE3 activity, similar to what we previously observed when tubular transport is inhibited with furosemide. This increased NHE3 activity causes H+ secretion in the mTAL and provides further support that the TAL is a site of urinary acidification.

Effects of Adipokines and Insulin on Intracellular pH, Calcium Concentration, and Responses to Hypo-Osmolarity in Human Articular Chondrocytes from Healthy and Osteoarthritic Cartilage.

Objective:To evaluate the effects of adipokines and insulin on intracellular calcium concentration ([Ca2+]i) and pH (pHi) in human articular chondrocytes from healthy (CHC) and osteoarthritic cartilage (COC).Design:pHi and [Ca2+]i were measured using BCECF and Fura-2 fluorometric probes in CHC and COC under control conditions and following a hypotonic shock. The effects of interleukin-1β (IL1β), tumor necrosis factor-α (TNFα), insulin, leptin, resistin, and adiponectin were assessed.Results:pHi was lower in COC than in CHC. Only IL1β β decreased pHi in both cell types; all the agents enhanced pHi recovery following an ammonium prepulse in CHC, effect that was attenuated by Na+–H+ exchanger inhibitors, but they had no effect in COC. Hypotonic shock (HTS) caused a pHi increase, which was significantly smaller in COC. All the hormones attenuated this response and the effect of IL1β was greater. The basal [Ca2+]i was similar in COC and CHC; IL1β, TNFα, and insulin increased the [Ca2+]i, but leptin, resistin, and adiponectin did not. These effects were greater in COC. This [Ca2+]i increase was dependent on extracellular Ca2+ and attenuated by Na+–Ca2+ exchanger inhibitors. HTS caused a [Ca2+]i increase, which was inhibited by transient receptor potential vanilloid blockers and attenuated by all the hormones tested with the exception of adiponectin.Conclusions:These findings may help explain the association between obesity and osteoarthritis, in which these hormones are altered. The responses of CHC and COC are different, which suggests that a modification of pH and Ca2+ homeostasis is part of the osteoarthritis pathophysiology.

Preferential intracellular pH regulation represents a general pattern of pH homeostasis during acid-base disturbances in the armoured catfish, Pterygoplichthys pardalis.

Preferential intracellular pH (pHi) regulation, where pHi is tightly regulated in the face of a blood acidosis, has been observed in a few species of fish, but only during elevated blood PCO2. To determine whether preferential pHi regulation may represent a general pattern for acid-base regulation during other pH disturbances we challenged the armoured catfish, Pterygoplichthys pardalis, with anoxia and exhaustive exercise, to induce a metabolic acidosis, and bicarbonate injections to induce a metabolic alkalosis. Fish were terminally sampled 2-3 h following the respective treatments and extracellular blood pH, pHi of red blood cells (RBC), brain, heart, liver and white muscle, and plasma lactate and total CO2 were measured. All treatments resulted in significant changes in extracellular pH and RBC pHi that likely cover a large portion of the pH tolerance limits of this species (pH 7.15-7.86). In all tissues other than RBC, pHi remained tightly regulated and did not differ significantly from control values, with the exception of a decrease in white muscle pHi after anoxia and an increase in liver pHi following a metabolic alkalosis. Thus preferential pHi regulation appears to be a general pattern for acid-base homeostasis in the armoured catfish and may be a common response in Amazonian fishes.

Oviduct binding and elevated environmental ph induce protein tyrosine phosphorylation in stallion spermatozoa.

Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca(2+), and albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 × 10(6) spermatozoa/ml), during which it transpired that the highest density (per mm(2)) of oviduct-bound spermatozoa was achieved under noncapacitating conditions. In subsequent experiments, sperm-oviduct incubations were performed for 6 h under noncapacitating versus capacitating conditions. The oviduct-bound spermatozoa showed a time-dependent protein tyrosine phosphorylation response, which was not observed in unbound spermatozoa or spermatozoa incubated in oviduct explant conditioned medium. Both oviduct-bound and unbound sperm remained motile with intact plasma membrane and acrosome. Since protein tyrosine phosphorylation can be induced in equine spermatozoa by media with high pH, the intracellular pH (pHi) of oviduct explant cells and bound spermatozoa was monitored fluorometrically after staining with BCECF-AM dye. The epithelial secretory cells contained large, alkaline vesicles. Moreover, oviduct-bound spermatozoa showed a gradual increase in pHi, presumably due to an alkaline local microenvironment created by the secretory epithelial cells, given that unbound spermatozoa did not show pHi changes. Thus, sperm-oviduct interaction appears to facilitate equine sperm capacitation by creating an alkaline local environment that triggers intracellular protein tyrosine phosphorylation in bound sperm.

2,4-Dichlorophenoxyacetic acid alters intracellular pH and ion transport in the outer mantle epithelium of the bivalve Anodonta cygnea

Bivalve molluscs, due to their sedentary mode of life and filter-feeding behavior, are very susceptible to pollutant bioaccumulation and used as sentinel organisms in the assessment of environment pollution. Herein we aimed to determine the in vivo, ex vivo and in vitro effects of 2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, in Anodonta cygnea shell growth mechanisms. For that, we evaluated the effect of 2,4-D (100μM) exposure on the transepithelial short-circuit current (Isc), potential (Vt) and conductance (Gt), as well as on OME ion transport systems and intracellular pH (pHi). In vivo exposure to 2,4-D caused an increase of 50% on the Isc generated by OME and ex vivo addition of that compound to the apical side of OME also induced an Isc increase. Furthermore, 2,4-D was able to cause a pHi increase in isolated cells of OME. Noteworthy, when 2,4-D was added following the exposure to specific inhibitors of several membrane transporters identified as responsible for pHi maintenance in these cells, no significant effect was observed on pHi except when the V-type ATPase inhibitor was used, indicating an overlap with the effect of 2,4-D. Thus, we concluded that 2,4-D is able of enhancing the activity of the V-ATPases present on the OME of A. cygnea and that this effect seems to be due to a direct stimulation of those H+ transporters present on the apical portion of the membrane of OME cells, which are vital for shell maintenance and growth. This study allows us to better understand the molecular mechanisms behind 2,4-D toxicity and its deleterious effect in aquatic ecosystems, with particular emphasis on those involved in shell formation of bivalves.

Modulation of the cardiac sodium/bicarbonate cotransporter by the renin angiotensin aldosterone system: pathophysiological consequences

The sodium/bicarbonate cotransporter (NBC) is one of the major alkalinizing mechanisms in the cardiomyocytes. It has been demonstrated the existence of at least two functional isoforms, one that promotes the co-influx of 1 molecule of Na+ per 1 molecule of HCO−3 (electroneutral isoform; NBCn1) and the other one that generates the co-influx of 1 molecule of Na+ per 2 molecules of HCO−3 (electrogenic isoform; NBCe1). Both isoforms are important to maintain intracellular pH (pHi) and sodium concentration ([Na+]i). In addition, NBCe1 generates an anionic repolarizing current that modulates the action potential duration (APD). The renin-angiotensin-aldosterone system (RAAS) is implicated in the modulation of almost all physiological cardiac functions and is also involved in the development and progression of cardiac diseases. It was reported that angiotensin II (Ang II) exhibits an opposite effect on NBC isoforms: it activates NBCn1 and inhibits NBCe1. The activation of NBCn1 leads to an increase in pHi and [Na+]i, which indirectly, due to the stimulation of reverse mode of the Na+/Ca2+ exchanger (NCX), conduces to an increase in the intracellular Ca2+ concentration. On the other hand, the inhibition of NBCe1 generates an APD prolongation, potentially representing a risk of arrhythmias. In the last years, the potentially altered NBC function in pathological scenarios, as cardiac hypertrophy and ischemia-reperfusion, has raised increasing interest among investigators. This review attempts to draw the attention on the relevant regulation of NBC activity by RAAS, since it modulates pHi and [Na+]i, which are involved in the development of cardiac hypertrophy, the damage produced by ischemia-reperfusion and the generation of arrhythmic events, suggesting a potential role of NBC in cardiac diseases.

PHocal adhesion kinase regulation is on a FERM foundation

Increases in intracellular pH (pHi) occur upon integrin receptor binding to matrix proteins and in tumor cells. In this issue, Choi et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201308034) show that pHi increase activates FAK by causing deprotonation of histidine 58 in its FERM (band 4.1, ezrin, radixin, moesin) homology domain, which exposes a region important for FAK autophosphorylation. This model of FAK activation could contribute to motility of tumor cells by promoting focal adhesion turnover.

Comparison between drug-induced and K+-induced changes in molar acid extrusion fluxes (JH +) and in energy consumption rates in astrocytes

Neuronal excitation leads to an increase of the extracellular K(+) concentration ([K(+)]o) in brain. This increase has at least two energy-consuming consequences: (1) a depolarization-mediated change in intracellular pH (pHi) in astrocytes due to depolarization-mediated increased activity of the acid-extruding Na(+)/bicarbonate transporter NBCe1 (driven by secondary active transport, supported by ion gradients established by the Na(+), K(+)-ATPase); and (2) activation of cellular reuptake of K(+) mediated by the Na(+), K(+)-ATPase in both neurons and astrocytes. Astrocytic, but not neuronal increase in NBCe1 activity and pHi is also seen after chronic treatment with either of the two anti-bipolar drugs carbamazepine or valproic acid. The third 'classical' anti-bipolar drug, 'lithium' increases astrocytic pHi by a different mechanism (stimulation of the acid extruding Na(+)/H(+) exchanger NHE1). The acid extruder fluxes, which depend upon the change in pHi per time unit (ΔpHi/Δt) and intracellular buffering power, have not been established in most of these situations. Therefore their stimulatory effects on energy metabolism has not been quantitated. This has been done in the present study in cultured mouse astrocytes. pHi was determined using the fluorescent pH-sensitive indicator BCECF-AM and an Olympus IX71 live cell imaging fluorescence microscope. Molar acid extrusion fluxes (indicating transporter activity) were determined as pHi changes/min during recovery after acid-loading with NH3/NH4 (+), NBCe1 mRNA and protein expression in the cultured cells by, respectively RT-PCR and Western blotting. Drug-induced up-regulation of acid extrusion flux was slow and less than physiologically seen after increase in K(+) concentration. Energetically, K(+) uptake is much costlier than NBCe1 activity.

Inhibition of carbonic anhydrase prevents the Na+/H+ exchanger 1-dependent slow force response to rat myocardial stretch

Myocardial stretch is an established signal that leads to hypertrophy. Myocardial stretch induces a first immediate force increase followed by a slow force response (SFR), which is a consequence of an increased Ca(2+) transient that follows the NHE1 Na(+)/H(+) exchanger activation. Carbonic anhydrase II (CAII) binds to the extreme COOH terminus of NHE1 and regulates its transport activity. We aimed to test the role of CAII bound to NHE1 in the SFR. The SFR and changes in intracellular pH (pHi) were evaluated in rat papillary muscle bathed with CO2/HCO3(-) buffer and stretched from 92% to 98% of the muscle maximal force development length for 10 min in the presence of the CA inhibitor 6-ethoxzolamide (ETZ, 100 μM). SFR control was 120 ± 3% (n = 8) of the rapid initial phase and was fully blocked by ETZ (99 ± 4%, n = 6). The SFR corresponded to a maximal increase in pHi of 0.18 ± 0.02 pH units (n = 4), and pHi changes were blocked by ETZ (0.04 ± 0.04, n = 6), as monitored by epifluorescence. NHE1/CAII physical association was examined in the SFR by coimmunoprecipitation, using muscle lysates. CAII immunoprecipitated with an anti-NHE1 antibody and the CAII immunoprecipitated protein levels increased 58 ± 9% (n = 6) upon stretch of muscles, assessed by immunoblots. The p90(RSK) kinase inhibitor SL0101-1 (10 μM) blocked the SFR of heart muscles after stretch 102 ± 2% (n = 4) and reduced the binding of CAII to NHE1, suggesting that the stretch-induced phosphorylation of NHE1 increases its binding to CAII. CAII/NHE1 interaction constitutes a component of the SFR to heart muscle stretch, which potentiates NHE1-mediated H(+) transport in the myocardium.

Receptor-independent effects of 2′(3′)-O-(4-benzoylbenzoyl)ATP triethylammonium salt on cytosolic pH

The effect of the relatively potent P2X7 receptor agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (BzATP-TEA) on cytosolic pH (pHi) was studied using MC3T3-E1 osteoblast-like cells, which endogenously express P2X7 receptors. pHi was measured fluorimetrically using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. BzATP-TEA (0.3-1.5mM) elicited fast-onset alkalinization responses. In contrast, adenosine 5'-triphosphate disodium salt (5mM) failed to reproduce the BzATP-TEA-induced responses, indicating a P2 receptor-independent mechanism. We speculated that triethylamine, which is present in solutions of BzATP-TEA, permeates the plasma membrane, and is protonated intracellularly, leading to an increase in pHi. Consistent with this hypothesis, triethylammonium (TEA) chloride mimicked the effects of BzATP-TEA on pHi. Moreover, measurements using a Cytosensor microphysiometer revealed that TEA chloride transiently suppressed proton efflux from cells, whereas washout of TEA transiently enhanced proton efflux. BzATP-TEA also elicited a sustained increase in proton efflux that was blocked specifically by the P2X7 antagonist A-438079. Taken together, we conclude that BzATP-TEA-induced alkalinization is unrelated to P2X7 activation, but is due to the presence of TEA. This effect may confound assessment of the outcomes of P2X7 activation by BzATP-TEA in other systems. Thus, control experiments using TEA chloride are recommended to distinguish between receptor-mediated and nonspecific effects of this widely used agonist. We performed such a control and confirmed that BzATP-TEA, but not TEA chloride, caused the elevation of cytosolic free Ca(2+) in MC3T3-E1 cells, ruling out the possibility that receptor-independent effects on pHi underlie BzATP-TEA-induced Ca(2+) signaling.

SIZE DISTRIBUTION AND SORTING DEGREE OF THE RECENT SEDIMENTS OF AL-KHAZER RIVER/ NORTHERN IRAQ

Mechanical and statistical analysis were conducted to determine the grain size distribution and sorting degree of the recent sediments of Al-Khazer river at northern of Iraq in order to clarify the sedimentation processes along this river. The study was carried out after the annual flood time of the river. Sediment size distribution was determined mechanically for major grain size fractions [gravel (-2 phi to 0 phi), sand (0 phi to +4 phi), silt (+5 phi to +7 phi)] and clay (+8 phi)] using sieve and pipette technique. Numerical treatment of grain size curves has been done on the basis of some quintiles read from the empirical cumulative distribution function (CDF) curve: phi median size, sorting, skewness and kurtosis. The results showed that the sediments of this river were relatively coarse grained textured (Ø50=0.35 to 1.2 phi), very poorly sorted, showing strongly coarse skewed towards fine particles and are platy to leptokurtic. The increase in phi toward outlet plain is equivalent to decrease in grain diameter and is indicative of a lower energy and higher depositional environment. For this reason, there are some relatively lower phi values in the inlet plain in comparison with the phi values of outlet plain. On the basis of this assumption, the change in sediment-size distribution along the river is a non-linear fashion and can be inferred from that to which the present deposition takes place by bed or graded suspended load.

Effects of acute hypoxia/acidosis on intracellular pH in differentiating neural progenitor cells

The response of differentiating mouse neural progenitor cells, migrating out from neurospheres, to conditions simulating ischemia (hypoxia and extracellular or intracellular acidosis) was studied. We show here, by using BCECF and single cell imaging to monitor intracellular pH (pHi), that two main populations can be distinguished by exposing migrating neural progenitor cells to low extracellular pH or by performing an acidifying ammonium prepulse. The cells dominating at the periphery of the neurosphere culture, which were positive for neuron specific markers MAP-2, calbindin and NeuN had lower initial resting pHi and could also easily be further acidified by lowering the extracellular pH. Moreover, in this population, a more profound acidification was seen when the cells were acidified using the ammonium prepulse technique. However, when the cell population was exposed to depolarizing potassium concentrations no alterations in pHi took place in this population. In contrast, depolarization caused an increase in pHi (by 0.5 pH units) in the cell population closer to the neurosphere body, which region was positive for the radial cell marker (GLAST). This cell population, having higher resting pHi (pH 6.9–7.1) also responded to acute hypoxia. During hypoxic treatment the resting pHi decreased by 0.1 pH units and recovered rapidly after reoxygenation. Our results show that migrating neural progenitor cells are highly sensitive to extracellular acidosis and that irreversible damage becomes evident at pH 6.2. Moreover, our results show that a response to acidosis clearly distinguishes two individual cell populations probably representing neuronal and radial cells.

Activation of ciliary beating by a mucolytic agent, ambroxol, in bronchioles of mice

Ambroxol has been used as a mucolytic agent in pulmonary diseases. In this study, we studied the effects of ambroxol on ciliary beat frequency (CBF) and ciliary bend amplitude (CBA) of the bronchioles. Mice were anestetized by pentobarbital sodium (70mg/kg, ip) and bronchiolar ciliary cells were isolated from lungs by an elastase treatment. In a HCO3−‐containing solution, ambroxol increased CBA and CBF in a concentration dependent manner: ambroxol (10μM) increased CBA and CBF by 30 % within 15 min. However, in a HCO3−‐free solution, it did not induce any increase in CBA and CBF, although the switch from the HCO3−‐containing solution to the HCO3−‐free solution increased them. Measurement of intracellular pH (pHi) demonstrated that ambroxol (10 μM) increased pHi gradually in bronchiolar ciliary cells. These suggest that an increase in pHi induced by ambroxol increases CBA and CBF. To confirm the effects of pHi on CBA and CBF, an NH4+ pulse was applied. An increase in pHi increased CBA and CBF, whereas a decrease in pHi decreased them. Diisothiocyanostilbene‐disulfonic acid (DIDS) abolished the increases in CBA and CBF induced by ambroxol. In bronchiolar ciliary cells, ambroxol increases pHi via activation of HCO3−/Cl− exchanges, which increases CBA and CBF.