2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein and Acidosis

The role of metabolic acidosis in the progression of chronic kidney disease (CKD) remains unclear. The aim of the present study was to investigate the direct effects of acid loading on the proliferation of rat glomerular mesangial cells (GMCs) in vitro and the possible role of sodium-hydrogen ion exchanger isoform 1 (NHE1). Rat GMCs were treated with acidic medium as acid loading. Growth and proliferation of GMCs was studied by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, thymidine ((3)H-TdR) incorporation, and flow cytometry. NHE1 protein expression and activity were quantified by Western blot and dual wavelength epifluorescent illumination with 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. 5-(N,N-dimethyl) amiloride hydrochloride (DMA), a specific inhibitor of NHE1, was used to investigate the possible involvement of NHE1 in the proliferation of GMCs. The MTT assay, (3)H-TdR incorporation, and cell cycle distribution analysis indicated that acid loading stimulated the proliferation of GMCs. Acid loading increased NHE1 activity, but had no effects on NHE1 expression at the protein level. The effects of acid loading on the proliferation of GMCs were inhibited by DMA. Acid loading induced GMC proliferation through NHE1-dependent pathways. Our findings may contribute to the understanding of metabolic acidosis in the progression of CKD.

Topics: Acidosis; Amiloride; Animals; Blotting, Western; Cell Cycle; Cell Proliferation; Disease Progression; Flow Cytometry; Fluoresceins; Hydrogen-Ion Concentration; Mesangial Cells; Rats; Renal Insufficiency, Chronic; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Staining and Labeling; Tetrazolium Salts; Thiazoles

Metabolic acidosis is thought to exacerbate chronic kidney disease in part by stimulating the release of potentially injurious substances. To define the genes whose expression is affected by exposure to an acidic milieus, we examined the effect of exposure of MDCK cells to pH 7.4 and pH 7.0 for 24 h on gene expression using a canine derived microarray. Exposure to this pH stress for 24 h led to increased expression of 278 genes (2.2% of the transcriptome) by at least 2-fold and 60 of these (21%) were upregulated by >3-fold. On the other hand, 186 genes (1.5% of the transcriptome) were downregulated by at least 2-fold and 16 of these (9%) were downregulated by 3-fold or more. Ten percent of the genes upregulated by at least threefold encode proinflammatory cytokine proteins, including colony stimulating factor 2, chemokine ligand 7, chemokine ligand 20, chemokine ligand 8, and interleukin-1α. Two others encode metallopeptidases. The most highly upregulated gene encodes a protein, lubricin, shown to be important in preventing cartilage damage and in tissue injury or repair. Upregulation of four genes was confirmed by quantitative PCR. Housekeeping genes were not increased. To examine the effect of decreasing medium pH, we measured intracellular pH (pH(i)) using 2,7-bis (2-carboxyethyl)5-carboxyfluorescein. With extracellular pH (pH(o)) of 7.0, pH(i) fell and remained depressed. These findings suggest that a pH stress alone can increase renal expression of proinflammatory and other genes that contribute to renal injury.

Topics: Acidosis; Animals; Cytokines; Dogs; Down-Regulation; Fluoresceins; Hydrogen-Ion Concentration; Kidney; Madin Darby Canine Kidney Cells; Microfilament Proteins; Myosin-Light-Chain Kinase; Protein Array Analysis; Transcription Factor CHOP; Transcriptome; Up-Regulation

Changing intracellular pH (pHi) exerts considerable influence on many cellular functions. Different pHi regulators, such as the Na-H exchanger (NHE), Na/(Equation is included in full-text article.)symporter, and Cl/OH exchanger (CHE), have been identified in mature mammalian cells. The aims of the present study were to investigate the physiological mechanisms of pHi recovery and to further explore the effects of alcohol on the pHi in human umbilical cord blood CD34 stem cell-like cells (HUCB-CD34STs). HUCB-CD34STs were loaded with the pH-sensitive dye, 2',7'-bis(2-carboxethyl)-5(6)-carboxyfluorescein, to examine pHi. In isolated HUCB-CD34STs, we found that (1) the resting pHi is 7.03 ± 0.02; (2) 2 Na-dependent acid extruders and a Cl-dependent acid loading carrier exist and are functional; (3) alcohol functions in a concentration-dependent manner to reduce pHi and increase NHE activity, but it does not affect CHE activity; and (4) fomepizole, a specific alcohol dehydrogenase inhibitor, does not change the intracellular acidosis and NHE activity-induced by alcohol, whereas 3-amino-1, 2,4-trizole, a specific catalase inhibitor, entirely abolishes these effects. In conclusion, we demonstrate that 2 acid extruders and 1 acid loader (most likely NHE, NBC, and CHE, respectively) functionally existed in HUCB-CD34STs. Additionally, the intracellular acidosis is mainly caused by catalase-mediated alcohol metabolites, which provoke the activity of NHE.

Topics: Acidosis; Amitrole; Antigens, CD34; Antiporters; Catalase; Cells, Cultured; Dose-Response Relationship, Drug; Ethanol; Fetal Blood; Fluoresceins; Fomepizole; Humans; Hydrogen-Ion Concentration; Intracellular Space; Pyrazoles; Sodium-Bicarbonate Symporters; Sodium-Hydrogen Exchangers; Stem Cells

In experiments reported here, we tested the ability of CGP-48506 to reverse the depressed cardiac contractility associated with hypercapnic acidosis in isolated rat cardiac myocytes. CGP-48506 is a cardiotonic agent that directly and specifically promotes the actin-cross-bridge reaction. Myocytes superfused at pH 6.8 demonstrated a significantly reduced extent of cell shortening, but an increase in the peak amplitude of the Ca2+ transient. Moreover, cells in acidosis showed small, but significant, decreases in time to peak shortening to 50 percent relaxation. Superfusion of the cells with 3, 7, and 10 micro-molar CGP-48506 restored the inhibited contractility as a function of concentration with no significant effects on the Ca2+-transient. Moreover, 10 micro-molar CGP-48506 completely reversed the depressed myocyte contraction associated with an increase in time to peak shortening and time to 50 percent and 75 percent relaxation. Our results indicate that the depression of contractility associated with acidosis is due to a reduced myofilament response to Ca2+, which can be overcome by agents working downstream from troponin C through a direct effect on the actin-myosin interaction.

Topics: Acidosis; Animals; Azocines; Calcium; Cell Separation; Fluoresceins; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Muscle Cells; Myocardial Contraction; Rats

The effect of Tris-Hydroxymethyl Aminomethane (THAM) on intracellular pH (pHi) is unknown. We previously demonstrated that the effect of sodium bicarbonate on pHi depends on the non-bicarbonate buffering system. First, human hepatocytes from hepatocytes cell culture (HepG2) were perfused with an acidotic artificial medium containing 5-mmol/L (H5) or 30-mmol/L (H30) concentrations of 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES), a non-bicarbonate buffer. We studied the effect of THAM on the pHi in both conditions. We repeated the same protocol using an acidotic human blood with a 5% or 40% hematocrit. The pHi was measured with the pH-sensitive fluorescent dye bis-carboxyethyl carboxy-fluorescein (BCECF). Gas analysis was performed before and during the alkaline infusion. The results showed that THAM caused an intracellular alkalization that was higher when the non-bicarbonate buffer concentration was low (0.45 +/- 0.21 and 0.22 +/- 0.14 pH units with H5 and H30, respectively). A significant relationship was found between changes in pHi and changes in PCO(2). Similar results were obtained with the human blood. In conclusion, the intracellular alkalizing effect of THAM is caused by the induced decrease of PCO(2) linked to the extracellular non-bicarbonate buffer capacity: The smaller the concentration of extracellular non-bicarbonate buffer, the higher the PCO(2) decrease caused by THAM.

Topics: Acid-Base Equilibrium; Acidosis; Bicarbonates; Blood; Blood Gas Analysis; Buffers; Cells, Cultured; Cytoplasm; Dose-Response Relationship, Drug; Drug Combinations; Extracellular Space; Fluoresceins; Fluorescent Dyes; Hepatocytes; HEPES; Humans; Intracellular Fluid; Tromethamine

We investigated acid-base permeability properties of electrically resistive monolayers of alveolar epithelial cells (AEC) grown in primary culture. AEC monolayers were grown on tissue culture-treated polycarbonate filters. Filters were mounted in a partitioned cuvette containing two fluid compartments (apical and basolateral) separated by the adherent monolayer, cells were loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was determined. Monolayers in HCO-free Na(+) buffer (140 mM Na(+), 6 mM HEPES, pH 7.4) maintained a transepithelial pH gradient between the two fluid compartments over 30 min. Replacement of apical fluid by acidic (6.4) or basic (8.0) buffer resulted in minimal changes in intracellular pH. Replacement of basolateral fluid by acidic or basic buffer resulted in transmembrane proton fluxes and intracellular acidification or alkalinization. Intracellular alkalinization was blocked > or =80% by 100 microM dimethylamiloride, an inhibitor of Na(+)/H(+) exchange, whereas acidification was not affected by a series of acid/base transport inhibitors. Additional experiments in which AEC monolayers were grown in the presence of acidic (6.4) or basic (8.0) medium revealed differential effects on bioelectric properties depending on whether extracellular pH was altered in apical or basolateral fluid compartments bathing the cells. Acid exposure reduced (and base exposure increased) short-circuit current from the basolateral side; apical exposure did not affect short-circuit current in either case. We conclude that AEC monolayers are relatively impermeable to transepithelial acid/base fluxes, primarily because of impermeability of intercellular junctions and of the apical, rather than basolateral, cell membrane. The principal basolateral acid exit pathway observed under these experimental conditions is Na(+)/H(+) exchange, whereas proton uptake into cells occurs across the basolateral cell membrane by a different, undetermined mechanism. These results are consistent with the ability of the alveolar epithelium to maintain an apical-to-basolateral (air space-to-blood) pH gradient in situ.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acid-Base Equilibrium; Acidosis; Alkalosis; Animals; Cell Membrane Permeability; Cell Polarity; Epithelial Cells; Extravascular Lung Water; Fluoresceins; Fluorescent Dyes; Male; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Respiratory Mucosa; Sodium; Sodium-Hydrogen Exchangers

The chemosensitive response of locus coeruleus (LC) neurones to changes in intracellular pH (pH(i)), extracellular pH (pH(o)) and molecular CO(2) were investigated using neonatal rat brainstem slices. A new technique was developed that involves the use of perforated patch recordings in combination with fluorescence imaging microscopy to simultaneously measure pH(i) and membrane potential (V(m)). Hypercapnic acidosis (15 % CO(2), pH(o) 6.8) resulted in a maintained fall in pH(i) of 0.31 pH units and a 93 % increase in the firing rate of LC neurones. On the other hand, isohydric hypercapnia (15 % CO(2), 77 mM HCO(3)(-), pH(o) 7.45) resulted in a smaller and transient fall in pH(i) of about 0.17 pH units and an increase in firing rate of 76 %. Acidified Hepes (N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid)-buffered medium (pH(o) 6.8) resulted in a progressive fall in pH(i) of over 0.43 pH units and an increase in firing rate of 126 %. Isosmotic addition of 50 mM propionate to the standard HCO(3)(-)-buffered medium (5 % CO(2), 26 mM HCO(3)(-), pH(o) 7.45) resulted in a transient fall in pH(i) of 0.18 pH units but little increase in firing rate. Isocapnic acidosis (5 % CO(2), 7 mM HCO(3)(-), pH(o) 6.8) resulted in a slow intracellular acidification to a maximum fall of about 0.26 pH units and a 72 % increase in firing rate. For all treatments, the changes in pH(i) preceded or occurred simultaneously with the changes in firing rate and were considerably slower than the changes in pH(o). In conclusion, an increased firing rate of LC neurones in response to acid challenges was best correlated with the magnitude and the rate of fall in pH(i), indicating that a decrease in pH(i) is a major part of the intracellular signalling pathway that transduces an acid challenge into an increased firing rate in LC neurones.

Topics: Acidosis; Animals; Animals, Newborn; Carbon Dioxide; Electrophysiology; Extracellular Space; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Hypercapnia; Kinetics; Locus Coeruleus; Membrane Potentials; Microscopy, Fluorescence; Neurons; Patch-Clamp Techniques; Pons; Propionates; Rats; Rats, Sprague-Dawley; Tetrodotoxin

Exposure to hypoxic, acidic, ion-shifted Ringer (HAIR) for 15-40 min has been shown to cause rapid astrocyte death upon reperfusion with normal media. The ion shifts of the HAIR solution included a rise in extracellular K(+) (e.g., [K(+)](o)) and a fall in [Na(+)](o), [Cl(-)](o), and [Ca(2+)](o), characteristic of ischemic-traumatic brain insults. We investigated the ionic basis of the HAIR-induced injury. After HAIR exposure, reperfusion in 0 Ca(2+)/EGTA media completely protected astrocytes. Preincubation of cells in BAPTA-AM ester was also protective, indicating that the injury was triggered by Ca(2+) influx during reperfusion. Neither nimodipine, CNQX, APV, nor TTX reduced injury. Astrocyte death could be blocked by 100 microM Ni(2+) or 100 microM benzamil, suggesting involvement of Na(+)-Ca(2+) exchange. KB-R7943, which preferentially inhibits reverse Na(+)-Ca(2+) exchange, also protected astrocytes. Elevation of [K(+)](o) was not necessary for astrocyte death. However, when [Na(+)](o) was maintained at 151 mM throughout the HAIR protocol, cell death was markedly reduced. We postulate that [Na(+)](o) shifts aid reversal of Na(+)-Ca(2+) exchange by favoring cytosolic Na(+) loading. Possible means of astrocytic Na(+) accumulation are discussed.

Topics: Acidosis; Animals; Astrocytes; Biological Transport; Calcium; Cell Death; Cell Hypoxia; Cells, Cultured; Chelating Agents; Chlorides; Egtazic Acid; Extracellular Space; Fluoresceins; Fluorescent Dyes; Ischemic Attack, Transient; Isotonic Solutions; Potassium; Rats; Ringer's Solution; Sodium

We examined intracellular pH (pH(i)) regulation in the retrotrapezoid nucleus (RTN), a CO(2)-sensitive site, and the hypoglossal nucleus, a nonchemosensitive site, during development (postnatal days 2-18) in rats. Respiratory acidosis [10% CO(2), extracellular pH (pH(o)) 7.18] caused acidification without pH(i) recovery in the RTN at all ages. In the hypoglossal nucleus, pH(i) recovered in young animals, but as animal age increased, the slope of pH(i) recovery diminished. In animals older than postnatal day 11, the pH(i) responses to hypercapnia were identical in the hypoglossal nucleus and the RTN, but hypoglossal nucleus and RTN neurons could regulate pH(i) during intracellular acidification at constant pH(o) at all ages. Recovery of pH(i) from acidification in the RTN depended on extracellular Na+ and was inhibited by amiloride but was unaffected by DIDS, suggesting a role for Na+/H+ exchange. Hence, pH(i) regulation during acidosis is more effective in the hypoglossal nucleus in younger animals, possibly as a requirement of development, but in older juvenile animals (older than postnatal day 11), pH(i) regulation is relatively poor in chemosensitive (RTN) and nonchemosensitive nuclei (hypoglossal nucleus).

Topics: Acidosis; Aging; Amiloride; Ammonia; Animals; Animals, Newborn; Carbon Dioxide; Female; Fluoresceins; Fluorescent Dyes; Homeostasis; Hydrogen-Ion Concentration; Hypoglossal Nerve; In Vitro Techniques; Intracellular Fluid; Male; Medulla Oblongata; Neurons; Nigericin; Rats; Rats, Sprague-Dawley

Activity-related shifts in intracellular pH (pHi) can exert potent neuromodulatory actions. Different states of neuronal activity of thalamocortical neurons were found to differentially modulate pHi. Tonic activity evoked by injection of depolarizing current led to a reversible rise in [H+]i which was nearly abolished in the presence of TTX. Block of voltage-gated calcium channels with I mM Ni2+ reduced the [H+]i transients related to tonic activity. Rhythmic activation of burst discharges caused changes of [H+]i which were decreased by TTX, whereas I mM Ni2+ almost abolished the [H+]i transients. The present results show that different forms of neuronal activity can lead to intracellular acidification caused by different mechanisms, i.e. Na+ and Ca2+ influx through sodium and Ca2+ channels, respectively, and the subsequent activation of a Ca2+/H+ pump. The resulting acidosis is suggested to reduce further Ca2+ influx and prevent excessive neuronal excitation.

Topics: Acidosis; Animals; Calcium Channels; Electric Stimulation; Electrophysiology; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; In Vitro Techniques; Membrane Potentials; Neurons; Patch-Clamp Techniques; Rats; Rats, Long-Evans; Sodium Channels; Tetrodotoxin; Thalamus

The cellular mechanism responsible for the reduction of tension in cerebral small arteries to acidosis is not known. In this study the role of smooth muscle intracellular Ca2+ concentration ([Ca2+]i) and membrane potential for the relaxation to acidosis was investigated in isolated rat cerebral small arteries. Isometric force was measured simultaneously with [Ca2+]i (fura 2) or with membrane potential (intracellular microelectrodes), and acidosis was induced by increasing PCO2 or reducing HCO3- of the bathing solution. Both hypercapnic and normocapnic acidosis were associated with a reduction of intracellular pH [measured with 2',7'-bis-(carboxyethyl)-5 (and -6)-carboxyfluorescein], caused relaxation, and reduced [Ca2+]i. However, whereas hypercapnic acidosis caused hyperpolarization, normocapnic acidosis was associated with depolarization. It is concluded that a reduction of [Ca2+]i is in part responsible for the direct effect of the acidosis on the vascular smooth muscle both during normo- and hypercapnia. The mechanism responsible for the reduction of [Ca2+]i differs between the hypercapnic and normocapnic acidosis, being partly explained by hyperpolarization during hypercapnic acidosis, whereas it is seen despite depolarization during normocapnic acidosis.

Topics: Acidosis; Animals; Bicarbonates; Calcium; Carbon Dioxide; Cerebral Arteries; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Hypercapnia; In Vitro Techniques; Male; Membrane Potentials; Muscle Contraction; Muscle, Smooth, Vascular; Rats; Rats, Wistar

The fluorescent dye BCECF was used to simultaneously determine the intracellular pH (ratio 495 : 450 nm) and changes in relative tissue volume (fluorescence at the 450 nm isosbestic wavelength) in rat hippocampal slices. Anoxia in the presence of glucose caused tissue swelling and subsequent intracellular acidosis after a short and small transient alkaline peak. Reoxygenation reversed tissue swelling only partly and ended in persistent tissue swelling. The intracellular pH was initially further acidified before restoration to the normoxic intracellular pH occurred. Omitting glucose during anoxia caused similar but more marked changes of relative tissue volume. However, acidosis during anoxia was less marked and subsequently converted to alkalosis. Reoxygenation also caused initial acidification but the intracellular pH was not completely restored afterwards.

Topics: Acidosis; Animals; Calibration; Fluoresceins; Fluorescent Dyes; Glucose; Hippocampus; Hydrogen-Ion Concentration; Hypoxia, Brain; In Vitro Techniques; Rats; Rats, Wistar

Cytosolic pH (pHi) and Na+/H+ exchange activity were measured in lymphocytes from 22 patients with mild chronic renal failure, and 21 age- and sex-matched normotensive healthy control subjects using the fluorescent dye technique. The basal pHi in resting lymphocytes was not significantly different in both groups tested (control, pHi 7.18 +/- 0.04; patients with mild chronic renal failure, 7.17 +/- 0.05). The initial rate of pHi recovery immediately after intracellular acidification with 100 mmol/l propionic acid, representing the maximum Na+/H+ exchange activity, was significantly higher in lymphocytes from patients with mild chronic renal failure (7.10 +/- 0.52 dpHi/s, mean +/- SEM) when compared with control subjects (5.42 +/- 0.47 dpHi/s; p < 0.05). No significant correlation between Na+/H+ exchange activity and blood pressure could be obtained in patients with mild chronic renal failure. Furthermore, there was no relationship of Na+/H+ exchange activity to cytosolic pH or extracellular pH. It is concluded that an enhanced Na+/H+ exchange activity can be detected in patients with mild chronic renal failure and may not be related to the significant abnormalities of electrolyte and acid-base metabolism commonly observed in patients with end-stage renal failure or on hemodialysis.

Topics: Acid-Base Equilibrium; Acidosis; Adult; Bicarbonates; Blood Pressure; Female; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Kidney Failure, Chronic; Kidney Function Tests; Lymphocytes; Male; Middle Aged; Propionates; Sodium-Hydrogen Exchangers

In this study, we investigated the role of Na+/H+ antiport in regulating cytosolic (intracellular) pH (pHi) in isolated and cultured ferret pulmonary arterial smooth muscle cells (PSMC). We also studied the effects of modulating pHi on the cytosolic (intracellular) calcium concentration ([Ca2+]i) in the PSMC and on the pulmonary arterial pressure (Ppa) of isolated ferret lungs. pHi was modulated by the NH4Cl washout method. To eliminate the contribution of Cl-/HCO3- exchangers, the PSMC and isolated lungs were perfused in HCO3- free buffer. Blocking the Na+/H+ antiporter decreased baseline pHi and prevented the recovery from NH4Cl washout-induced intracellular acidosis. Intracellular alkalinization caused an initial transient increase in both [Ca2+]i and Ppa that were dependent on extracellular Ca2+ entry. Maintaining cytosolic alkalinization caused another increase in Ppa that was not associated with an increase in [Ca2+]i. Intracellular acidosis also caused an increase in [Ca2+]i and Ppa. The cytosolic acidosis-induced increase in [Ca2+]i and Ppa were mediated by both extracellular Ca2+ influx and release of stored intracellular Ca2+. Cytosolic acidosis also appears to have a direct effect on the smooth muscle contractile elements. Both cytosolic alkalosis and acidosis increased vascular reactivity.

Topics: Acidosis; Alkalosis; Animals; Calcium; Cytosol; Ferrets; Fluoresceins; Fluorescence; Fluorescent Dyes; Homeostasis; Hydrogen-Ion Concentration; In Vitro Techniques; Indoles; Male; Muscle Contraction; Muscle Tonus; Muscle, Smooth; Potassium Chloride; Pulmonary Artery; Sodium; Sodium-Hydrogen Exchangers; Vasoconstriction

1. Rat brainstem slices were taken for simultaneous measurements of intracellular pH (pHi) and membrane currents or potentials in dorsal vagal neurons, dialysed with the pH-sensitive dye BCECF. 2. Intrinsic intracellular buffering power was 18 mM per pH unit, as determined by exposure to trimethylamine in CO2/HCO3(-)-free, Hepes-buffered saline. 3. Tonic spike activity led to a stable fall in pHi of 0.05-0.2 pH units from a baseline of 7.19 in current-clamp mode, whereas depolarization from -60 to 0 mV for 1 min in voltage-clamp mode produced an intracellular acidification of 0.3 pH units. The depolarization-evoked fall in pHi was suppressed by 1 mM Ni2+ or 0.2 mM Cd2+, but not by 0.5 microM TTX or CO2/HCO3(-)-free saline. 4. Kainate (100 microM) led to an an inward current of -620 pA and a threefold increase in membrane conductance, accompanied by a fall in pHi of 0.33 pH units. 5. GABA (1 mM) evoked a bicuculline-blockable conductance increase and fall in pHi of up to 0.5 pH units. The GABA-induced pHi decrease, but not the conductance increase, was suppressed in Hepes solution. 6. Neither tonic spike activity, nor resting current or conductance were markedly changed upon Hepes-induced intracellular alkalinizations of up to 0.35 pH units, or by an anoxia-induced fall in pHi of a maximum of 0.36 pH units. 7. The data show that neuronal activity produces profound changes in pHi. It appears that spontaneous spike discharge of dorsal vagal neurons is rather tolerant of major perturbations in pHi.

Topics: Acidosis; Animals; Brain Stem; Dialysis; Electric Conductivity; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; In Vitro Techniques; Kainic Acid; Membrane Potentials; Neurons; Nigericin; Patch-Clamp Techniques; Rats; Rats, Wistar; Time Factors; Vagus Nerve

Contact lens wear causes significant epithelial and stromal acidosis. In this study, we tested whether lens wear can cause endothelial acidosis as well. Rabbit corneas were isolated and perfused in vitro. The endothelial intracellular pH (pHi) was measured with a pH sensitive fluorescent probe (BCECF). Three conditions were examined: 1) Polymethylmethacrylate (PMMA) and rigid gas-permeable (RGP) contact lens wear using a range of oxygen transmissibility (Dk/L) from 0 to 121, 2) epithelial hypoxia produced by exposure to oligomycin/sodium azide solution or epithelial perfusion with 100% N2 equilibrated Ringer's solution, and 3) epithelial exposure to Ringer's equilibrated with 5% CO2, balance air. PMMA and RGP contact lens wear acidified endothelial cells by 0.23 +/- 0.01 (n = 23) and 0.11 +/- 0.01 pH units (n = 23), respectively, within twenty min of lens insertion. Epithelial hypoxia, induced by sodium azide and oligomycin, reversibly acidified the endothelium by 0.04 +/- 0.01 pH units (n = 4). However, epithelial hypoxia induced by perfusion with 100% N2 equilibrated Ringer's did not have a significant effect on endothelial pHi. Introduction of 5% CO2 to the epithelium, acidified the endothelium by 0.15 +/- 0.02 pH units (n = 7) within 10 min. We conclude that contact lens wear can significantly acidify corneal endothelial cells. The endothelial pHi change is caused almost exclusively by a build up of CO2 behind the lens; hypoxia having very little contribution. As expected, RGP contact lenses induced less endothelial acidosis than PMMA controls.

Topics: Acidosis; Animals; Carbon Dioxide; Cell Hypoxia; Contact Lenses; Cornea; Endothelium, Corneal; Epithelium; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Methylmethacrylate; Methylmethacrylates; Oxygen; Rabbits

Based on contact lens-induced stromal acidification of the cornea, it has been suggested that the corneal epithelial and endothelial cells also become acidotic during contact lens wear. This alleged acidification may have a role in altered cell appearance and metabolism during contact lens wear. This study investigated the effects of anoxia, carbon dioxide retention, and contact lens gas transmissibility on the epithelial and aqueous humor pH in living rabbits.. Epithelial intracellular pH (pHi) and aqueous humor pH were fluorophotometrically measured with a pH sensitive-dye (BCECF) during contact lens wear or exposure to various gas mixtures.. Polymethylmethacrylate (PMMA) lens wear acidified epithelial cells by preventing CO2 efflux and by inducing hypoxia. Increasing lens oxygen transmissibility decreased epithelial acidification. After initiation of rigid, gas-permeable (RGP) lens wear or CO2-air exposure, pHi dropped transiently and then recovered partially. This recovery of pHi was not observed during anoxia, whether induced by PMMA lens wear or exposure to 100% N2. The aqueous humor also acidified during PMMA lens wear, a phenomenon not observed during RGP lens wear. Changes in aqueous pH were smaller, slower, and delayed when compared to their epithelial counterparts.. Hypoxic contact lens wear acidifies the corneal epithelium and aqueous humor. The aqueous humor pH change indicates a probable endothelial acidification during hypoxic contact lens wear; the pH changes are caused by two separate and additive effects, CO2 retention and hypoxic acidosis. Increases in the oxygen transmissibility of the lens decrease the cellular acidosis, which might minimize cellular complications arising from contact lens wear. We estimate that a lens with an oxygen transmissibility (Dk/L) of 300 x 10(-11) (cm/sec)(ml O2/ml x mm Hg) is needed to prevent epithelial pHi changes in the open eye. In contrast, lenses with Dk/L as low as 18 x 10(-9) (cm/sec)(ml O2/ml x mm Hg) can prevent aqueous humor pH changes.

Topics: Acidosis; Animals; Aqueous Humor; Cell Hypoxia; Contact Lenses; Cornea; Epithelium; Fluoresceins; Fluorescent Dyes; Fluorophotometry; Hydrogen-Ion Concentration; Methylmethacrylate; Methylmethacrylates; Oxygen Consumption; Rabbits

The effect of acid-base disturbances on sodium/proton (Na+/H+) exchange has been examined in animal models; however, few data are available from human studies. To test the effect of metabolic acidosis on Na+/H+ exchange in man, as well as to examine the relationship between Na+/H+ exchange and cytosolic calcium ([Ca2+]i), we measured both variables in patients with decreased renal function with mild metabolic acidosis (pH 7.34 +/- 0.06), in normal control subjects (pH 7.41 +/- 0.02), and in subjects before (pH 7.40 +/- 0.01), and after (pH 7.26 +/- 0.04) ammonium chloride (NH4Cl) 15 g for 5 d. Lymphocytes and platelets were loaded with the cytosolic pH (pHi) indicator 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein and acidified to pH approximately 6.6 with propionic acid. To quantitate Na+/H+ exchange, dpHi/dt was determined at 1 min. [Ca2+]i was measured with fura-2. Na+/H+ exchange was significantly increased only in lymphocytes of patients with renal insufficiency. Neither intracellular pH (pHi) nor [Ca2+]i was different from controls. NH4Cl resulted in a significant increase in Na+/H+ exchange in lymphocytes, but not in platelets of normal subjects. Values of pHi and [Ca2+]i in either cell type remained unaffected. Since metabolic acidosis influenced Na+/H+ only in lymphocytes, but not in platelets, it is possible that protein synthesis may be involved in increasing Na+/H+ exchange.

Topics: Acidosis; Acidosis, Renal Tubular; Acute Disease; Blood Platelets; Carbon Dioxide; Carrier Proteins; Chronic Disease; Electrolytes; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Lymphocytes; Male; Sodium; Sodium-Hydrogen Exchangers

Intracellular pH was estimated from the fluorescence of 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) in isolated renal cortical tubules from control, potassium-depleted (KD) and NH4Cl-induced metabolic acidosis (MA) rats. While pHi was not different among control, short- and long-term KD, and NH4Cl MA rats, in vitro rates of ammonium production were increased in rats with metabolic acidosis and both short- and long-term potassium depletion. Mitochondrial matrix pH, and the pH gradient across the mitochondrial membrane were not different in tubules from KD rats compared to those from controls. These results are interpreted to indicate that a signal other than intracellular acidosis maintains the high rate of renal ammoniagenesis seen in KD.

Topics: Acidosis; Ammonia; Animals; Cytosol; Fluoresceins; Fluorescent Dyes; Glutamine; Hydrogen-Ion Concentration; Kidney Tubules; Male; Mitochondria; Nutritional Requirements; Potassium; Potassium Deficiency; Rats; Rats, Inbred Strains

The relationships between extracellular pH (pHo), intracellular pH (pHi), and loss of cell viability were evaluated in cultured rat hepatocytes after ATP depletion by metabolic inhibition with KCN and iodoacetate (chemical hypoxia). pHi was measured in single cells by ratio imaging of 2',7'-biscarboxy-ethyl-5,6-carboxyfluorescein (BCECF) fluorescence using multiparameter digitized video microscopy. During chemical hypoxia at pHo of 7.4, pHi decreased from 7.36 to 6.33 within 10 min. pHi remained at 6.1-6.5 for 30-40 min (plateau phase). Thereafter, pHi began to rise and cell death ensued within minutes, as evidenced by nuclear staining with propidium iodide and coincident leakage of BCECF from the cytoplasm. An acidic pHo produced a slightly greater drop in pHi, prolonged the plateau phase of intracellular acidosis, and delayed the onset of cell death. Inhibition of Na+/H+ exchange also prolonged the plateau phase and delayed cell death. In contrast, monensin or substitution of gluconate for Cl- in buffer containing HCO3- abolished the pH gradient across the plasma membrane and shortened cell survival. The results indicate that intracellular acidosis after ATP depletion delays the onset of cell death, whereas reduction of the degree of acidosis accelerates cell killing. We conclude that intracellular acidosis protects against hepatocellular death from ATP depletion, a phenomenon that may represent a protective adaptation against hypoxic and ischemic stress.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acidosis; Amiloride; Animals; Bicarbonates; Carrier Proteins; Cell Survival; Cells, Cultured; Chlorides; Fluoresceins; Gluconates; Hydrogen-Ion Concentration; Liver; Male; Monensin; Oxygen; Rats; Rats, Inbred Strains; Sodium-Hydrogen Exchangers

We have investigated the role of the Na-H antiport in the regulation of intracellular pH (pHi) in vascular smooth muscle. Experiments were conducted on contractile-state rat aortic smooth muscle cells grown in primary culture and loaded with the pH-sensitive, fluorescent indicator 2',7',-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Cells equilibrated in a normal physiological salt solution (PSS) containing 135 mM Na, pH 7.4 at 37 degrees C, had a pHi of 7.16 +/- 0.04 (means +/- SE; n = 8). 5-(N-ethyl-N-isopropyl)amiloride (EIPA) caused a concentration-dependent fall in pHi. Removal of extracellular Na caused an intracellular acidification that was rapidly reversed on replacement of Na. The rate of recovery from NH4Cl-induced intracellular acidosis was dependent on extracellular Na concentration (Km 14.6 +/- 2.8 mM) and was accelerated by increasing the transmembrane Na gradient and slowed by decreasing it. Recovery from acidosis was completely abolished by either EIPA or the absence of extracellular Na. These results demonstrate that the Na-H antiport is an important mechanism for the maintenance and regulation of pHi in vascular smooth muscle cells. The BCECF fluorescence technique provides an ideal method for further studies on the mechanisms for pHi regulation in these cells.

Topics: Acidosis; Animals; Aorta; Carrier Proteins; Cells, Cultured; Cytoplasm; Fluoresceins; Hydrogen; Hydrogen-Ion Concentration; Intracellular Membranes; Muscle, Smooth; Rats; Rats, Inbred Strains; Sodium-Hydrogen Exchangers