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PubMedDate not supplied

Intracellular pH regulatory mechanism in human atrial myocardium: functional evidence for Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) symporter.

Intracellular pH (pH(i)) exerts considerable influence on cardiac contractility and rhythm. Over the last few years, extensive progress has been made in understanding the system that controls pH(i) in animal cardiomyocytes. In addition to the housekeeping Na(+)-H(+) exchanger (NHE), the Na(+)-HCO(3)(-) symporter (NHS) has been demonstrated in animal cardiomyocytes as another acid extruder. However, whether the NHE and NHS functions exist in human atrial cardiomyocytes remains unclear. We therefore investigated the mechanism of pH(i) recovery from intracellular acidosis (induced by NH(4)Cl prepulse) using intracellular 2',7'-bis(2-carboxethyl)-5(6)-carboxy-fluorescein fluorescence in human atrial myocardium. In HEPES (nominally HCO(3)(-)-free) Tyrode solution, pH(i) recovery from induced intracellular acidosis could be blocked completely by 30 microM 3-methylsulfonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE 694), a specific NHE inhibitor, or by removing extracellular Na(+). In 3% CO(2)-HCO(3)(-) Tyrode solution, HOE 694 only slowed the pH(i) recovery, while addition of HOE 694 together with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (an NHS inhibitor) or removal of extracellular Na(+) inhibited the acid extrusion entirely. Therefore, in the present study, we provided evidence that two acid extruders involved in acid extrusion in human atrial myocytes, one which is HCO(3)(-) independent and one which is HCO(3)(-) dependent, are mostly likely NHE and NHS, respectively. When we checked the percentage of contribution of these two carriers to pH(i) recovery following induced acidosis, we found that the activity of NHE increased steeply in the acid direction, while that of NHS did not change. Our present data indicate for the first time that two acid extruders, NHE and NHS, exist functionally and pH(i) dependently in human atrial cardiomyocytes.

Fluoresceins↗
PubMed1980

Isolation and identification of Clara cells from rabbit lung.

A procedure has been developed for the isolation of nonciliated bronchiolar epithelial cells (Clara cells) from rabbit lung. Following pulmonary lavage to eliminate macrophages, cells (5% Clara cells) were released by digestion with 0.1% Protease I in HEPES-buffered balanced salt solution containing 0.5 mM ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid instilled through the trachea. These cells were then separated on the basis of size using the Beckman JE-6 elutriator rotor. The fourth fraction collected from the elutriator contained about 30% Clara cells. This fraction was then layered on a two-polymer aqueous phase system consisting of 5% dextran T500 (DT) and 3.8% polyethylene glycol 6000 (PEG) in sodium phosphate buffer. A cell fraction was obtained from the PEG phase, which included approximately 70% Clara cells. These cells were found to be greater than 90% viable by trypan blue dye exclusion. Identification of isolated Clara cells was confirmed by light microscopic observation of nitro blue tetrazolium staining and by ultrastructural characteristics as observed by electron microscopy.

Animals↗
PubMed2000

Diffusion of small solutes in the lateral intercellular spaces of MDCK cell epithelium grown on permeable supports.

The diffusion coefficients of four solutes ranging in molecular weight from 238 to 10,000 in the lateral intercellular spaces (LIS) of cultured kidney cells (MDCK) grown on permeable supports were determined from the spread of fluorescence produced after the release of caged compounds by a pulse from a UV laser. Two types of experiments were performed: measurement of the rate of change of fluorescence after releasing a caged fluorophore, and measurement of the change in fluorescence of a relatively static fluorescent dye produced by the diffusion of an uncaged ligand for the dye. Fluorescence intensity was determined by photon-counting the outputs of a multichannel photomultiplier tube. Diffusion coefficients were determined in free solution as well as in the LIS of MDCK cells grown on permeable supports and the hindrance factor, theta, determined from the ratio of the free solution diffusivity to that in the LIS. The hindrance factors for 3000-MW dextran, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, MW 524) and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, MW 238) were not significantly different from 1. The diffusion of 10,000-MW dextran was substantially reduced in the LIS with a theta of 5.6 +/- 0.3. Enzymatic digestion by neuraminidase of the sialic acid residues of the glycosylation groups in the LIS increased the diffusivity of the 10,000-MW dextran 1.8-fold indicating hindrance by the glycocalyx. We conclude that small solutes, such as Na(+) and Cl(-), would not be significantly restricted in their diffusion in the LIS and that solute concentration gradients could not develop along the LIS under physiologic conditions.

Animals↗
PubMed2005

Bicarbonate exporting transporters in the ovine ruminal epithelium.

In order to stabilize the intraruminal pH, bicarbonate secretion by the ruminal epithelium seems to be an important prerequisite. The present study therefore focussed on the characterization of bicarbonate exporting systems in ruminal epithelial cells. Intracellular pH (pH(i)) was measured spectrofluorometrically in primary cultured ruminal epithelial cells loaded with the pH-sensitive fluorescent dye, 2,7-bis(carboxyethyl)-5(6')-carboxyfluorescein acetomethyl ester. Switching from CO2/HCO3- -buffered to HEPES-buffered solution caused a rapid intracellular alkalinization followed by a counter-regulation towards initial pH(i). The recovery of pH(i) was dependent upon extracellular chloride, but independent of extracellular sodium. Adding 500 microM H2DIDS significantly reduced the increase of pH(i). For further characterization of the bicarbonate exporting systems, we tested the ability to reverse the direction from HCO3- export to import in the absence of sodium and chloride. Under sodium and chloride-free conditions, counter-regulation after CO2-induced pH(i) decrease did not differ from pH(i) recovery in the presence of sodium and chloride. Existence of bicarbonate exporting systems in cultured ruminal epithelial cells and intact ruminal epithelium was verified by reverse transcription polymerase chain reaction (RT-PCR). Using RT-PCR and subsequent sequencing, expression of mRNA encoding for AE2, DRA and PAT1 could be found. Bicarbonate exporting systems could therefore be detected both on the functional and structural level.

Animals↗
PubMed2005

Voltammetric studies of Zn and Fe complexes of EDTA: evidence for the push mechanism.

The 'push' hypothesis for the antioxidant action of Zn2+ is based on its displacement of iron from a low molecular weight pro-oxidant complex. In this study, the chemical plausibility of that proposed function is investigated by cyclic voltammetry. As a model for a pro-oxidative low molecular weight iron complex the Fe(II/III)/EDTA couple was examined. This complex was selected for its well-defined electrochemical, iron stability constants, and similarity to other low molecular weight chelates in physiological fluids in terms of logical binding sites, i.e. amino, and carboxylate groups. Also investigated were iron complexes of nitrilotriacetic acid and DL-glutamic acid. Results demonstrate that approximately 90% of the cyclic voltammetric peak current for Fe(III)EDTA reduction and the EC' current for the mediated reduction of H2O2 by Fe(II/III)EDTA (Fenton Reaction) are lost when Zn2+ is introduced to a 1:1 molar ratio relative to iron. All experiments were conducted in HEPES buffered solutions at pH 7.4. Iron (II/III) complexes of nitrilotriacetic acid and DL-glutamic acid followed the same trends. Cyclic voltammetric experiments indicate that Zn2+ displaces Fe(III) from EDTA despite the much larger stability constant for the iron complex (10(25.1)) versus zinc (10(16.50)). The hydrolysis aided displacement of Fe(III) from EDTA by Zn2+ is considered by the equilibria modeling program, HySS. With Fe(III) hydrolysis products included, Zn2+ is able to achieve 90% displacement of iron from EDTA, a result consistent with cyclic voltammetric observations.

Catalysis↗
PubMed1984

A modified direct phosphate assay for studying ATPases.

A simple, rapid assay for purified ATPases is presented, based upon the formation of phosphomolybdate and its extraction into butyl acetate. The inclusion of imidazole makes the assay more sensitive and reproducible apparently because of the formation of an imidazole-phosphomolybdate complex. Protein (100 micrograms), Hepes buffer [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (0.1 M) and nucleotides (1 mM) were all shown to cause interference. The interference by nucleotides could be counteracted by using more molybdate. Butyl acetate was shown to extract virtually all of the phosphomolybdate almost instantaneously upon vortex mixing.

Acetates↗
PubMed1985

Solid-phase purification and analysis of dicarboxylic porphyrins extracted from cultured tumor cells.

We describe here a sensitive method for the purification and analysis of porphyrins present in hematoporphyrin derivative. Hematoporphyrin derivative is a solution containing a complex mixture of dicarboxylic porphyrins such as hematoporphyrin IX, monohydroxyethyl monovinyl deuteroporphyrin isomers, and protoporphyrin IX in addition to porphyrin aggregates of variable molecular sizes. This mixture is known for its ability to be selectively retained by tumor cells and for its cytotoxicity in the presence of light. In order to study the mechanisms of hematoporphyrin derivative uptake and its cellular metabolism, extraction methods are required that combine high recoveries with minimum changes of very labile components. Extraction with perchloric acid: methanol mixtures recovered only some 60% of the porphyrins taken up by tumor cells and artifactual fluorescent spots were seen on thin-layer chromatograms. Improved yields were obtained upon extraction with dimethyl sulfoxide or Triton X-100:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer mixture, but the extracts were not suitable for reverse-phase thin-layer chromatography (RTLC). The procedure described here consists of extracting porphyrins from cultured tumor cells with a buffered detergent followed by sequential chromatography on DEAE-cellulose columns and on reverse-phase octadecylsilyl cartridges. Identification of the isolated free dicarboxylic porphyrins is conveniently done by RTLC.

Animals↗
PubMed1991

Carbonic anhydrase assay: strong inhibition of the leaf enzyme by CO2 in certain buffers.

Apparent carbonic anhydrase activity in leaf extracts, measured as the rate of H+ production associated with the CO2 hydration reaction, varied by as much as 25-fold when the assay buffer was varied. Highest activities were usually recorded in barbitone buffer, with lower activities in imidazole, Tricine, Hepes, Tris, and phosphate buffers. The greatest differences were observed with the enzyme isolated from leaves of the monocotyledonous plants Zea mays (maize) and Triticum aestivum (wheat). Smaller differences were observed with carbonic anhydrase from dicotyledonous species and there was no effect on the erythrocyte enzyme. Leaf carbonic anhydrase activity measured by the mass spectrometric procedure was unaffected by varying the assay buffer. The low activity in certain buffers observed with the former assay system was found to be due to inhibition of the enzyme-catalyzed reaction by higher concentrations of CO2. Carbonic anhydrase from some sources was also strongly inhibited by certain inorganic and organic anions.

Barbital↗
PubMed1992

Rapid analysis of mitotic histone H1 phosphorylation by cationic disc electrophoresis at neutral pH in minigels.

A new method is described for analysis of histone H1 and other basic proteins by cationic disc electrophoresis in polyacrylamide gels at neutral pH. The multiphasic buffer (disc) system uses Na+ as leading ion, L-histidine as trailing ion, and Hepes as buffering counterion. These "Hepes/histidine gels" have three advantages over conventional acid-urea gels for studies of H1 phosphorylation and dephosphorylation: speed, convenience, and the need for only small amounts of cells or chromatin. Core histones and their acetylated forms can also be separated in gels containing 0.4% Triton X-100. The difference in electrophoretic mobility between mitotic (superphosphorylated) and interphase H1 from HeLa cells is approximately twice as great at neutral pH as at pH 4.5, making it possible to separate these two H1 forms rapidly and easily in Hepes/histidine "minigels" only 5-cm long. Total histones can be rapidly prepared by simply neutralizing 0.2 N HCl extracts, and the entire analysis, from harvesting cells to destaining gels, can be carried out in 1 day. The stacking effect of the disc system produces sharp bands and high resolution even with relatively dilute samples.

Chromatin↗
PubMed1994

Aminosulfonic acid buffer preserves myocardium during prolonged ischemia.

Prevention of myocardial acidosis during global ischemia in operative cardiopreservation was explored in two series of dogs where acid-base control was the only variable. A specifically designed aminosulfonic acid buffer composition, 3:1 molar equivalents NaMOPS to HEPES, 0.2 mol/L, was compared with NaHCO3 (pH 8). Dissolved in standard cardioplegic solution it was given every 30 minutes by coronary infusion at 20 degrees C during 3 hours of global ischemia. Glass electrode intramyocardial pH, adenosine triphosphate (ATP) level, left ventricular contractility (Dp/Dt) and compliance (-Dp/Dt), and other cardiovascular parameters were measured frequently throughout ischemia and for 75 minutes thereafter. In the buffer group (n = 6) myocardial pH remained above entry levels throughout the study period, adenosine triphosphate level remained normal during ischemia, and Dp/Dt and -Dp/Dt at 75 minutes of reperfusion were above entry levels. In the NaHCO3 group (n = 6) pH declined and remained depressed throughout ischemia, adenosine triphosphate level fell steadily and significantly throughout the experiment, and Dp/Dt and -Dp/Dt never regained entry levels. The difference in each parameter between the two groups was statistically significant (p < 0.05). We conclude that control of myocardial acid-base equilibrium alone during global ischemia will preserve myocardial function and minimize reperfusion injury.

Acidosis↗
PubMed1989

Relationship of hemolysis buffer structure, pH and ionic strength to spontaneous contour smoothing of isolated erythrocyte membranes.

Isolated human erythrocyte membranes crenate when suspended in isotonic medium, but can use MgATP to reduce their net positive curvature, yielding smooth discs and cup forms that eventually undergo endocytosis. An earlier report from this laboratory (Patel, V.P. and Fairbanks, G. (1981) J. Cell Biol. 88, 430-440), has described a phenomenon of ATP-independent shape change in which ghosts prepared by hemolysis and washing in synthetic zwitterionic buffers crenated at 0 degree C, but underwent conversion to smooth discs and cups when warmed in the absence of MgATP. We have further explored the effect of the hemolysis condition on the requirement for ATP in ghost shape change. 25 hemolysis buffers were applied at 10 mM (pH 7.4, 0 degree C). Eight anionic buffers with relatively high ionic strength (e.g., phosphate and diethylmalonic acid (DMA] yielded ghosts requiring ATP for shape change, while two cationic buffers (Bistris and imidazole) and ten synthetic zwitterionic buffers (e.g., Tricine and Hepes) with lower ionic strength produced ghosts that smoothed spontaneously at 30 degrees C. Hemolysis at intermediate ionic strength yielded mixed populations in which spontaneous smoothing was expressed in all-or-none fashion. Maximal ATP-independent shape change was induced by hemolysis at pH 7.3-7.7, while ATP was required after hemolysis at pH less than or equal to 7.1 even when the ionic strength at hemolysis was low. Ghosts requiring ATP could be converted to ATP independence by washing at low ionic strength, but ATP independence could not be reversed readily by washing at high ionic strength. Exposure to low ionic strength at pH greater than 7.1 presumably changes membrane organization in a way that alters the temperature dependence of tensions within the bilayer or skeleton of the composite membrane.

Adenosine Triphosphate↗
PubMed1995

Taurine analog modulation of taurine uptake by two different mechanisms in cultured glial cells.

Previous data have shown that HEPES, a taurine structural analog, inhibits the uptake of taurine by cultured cells differently, depending on its addition either to the culture medium or to the Krebs-Ringer buffer used for cell incubation during taurine uptake measurements (Lleu and Rebel, J Neurosci Res 23: 78-86, 1989). An extensive study of the effect of numerous other taurine structural analogs on taurine uptake by cultured glial cells was carried out. Our results show that taurine uptake modulation by structural analogs follows two different mechanisms. For the first mechanism, observable after the simultaneous presence of taurine and of its analog during the incubation time of the uptake experiment (10 min), the amine function on the molecule is essential. The sulfonate group could be replaced either by a sulfinic group or by a carboxylic group. beta-Alanine, hypotaurine, acetyltaurine, guanidinoethanesulfonate and guanidinopropionate are the most potent inhibitors in this first mechanism. For the second mechanism, which requires the presence of the analog in the culture medium during the 48 hr preceding the taurine uptake measurement, the simultaneous presence of an amine and of a sulfonate group or of an amine and a sulfinate group is required. Carboxylates are ineffective in modulating taurine uptake in this mechanism. The sulfonate buffers synthesized by Good et al. (Biochemistry 5: 467-477, 1966) also affect taurine uptake in both mechanisms.

Animals↗
PubMed1991

Chloride efflux from isolated choroid plexus.

Chloride efflux was analyzed in adult rat lateral ventricle choroid plexus (LVCP) incubated in artificial CSF (aCSF) at 37 degrees C. Following steady-state loading of 36Cl in LVCP, the tracer release from plexus to aCSF was quantified by the efflux coefficient (k, s-1), equal to ln 2/t/2. Cl efflux could be described by a 2-component model, with a t/2 for the 'fast' component matching well that for [3H]sucrose (extracellular marker) and a slower, drug-inhibitable component of 36Cl release thought to reflect cellular washout. The cellular Cl efflux was more than twice as fast as 37 degrees C than at 15 degrees C. There was progressively more rapid efflux (k) of 36Cl from cells as the aCSF was altered over a range of several pH values from 6.7 (k = 0.026 s-1) to 8.2 (0.070 s-1). CSF medium anion replacement (isethionate and HEPES for Cl and HCO3, respectively) reduced the k for 36Cl by 57%. Acetazolamide (0.1 mM) and other Cl transport inhibitors (disulfonic stilbenes and loop diuretic) reduced Cl efflux by 35-55%. Acetazolamide inhibited Cl release from LVCP into aCSF whether the latter contained Cl and HCO3, or not. Overall, the findings suggest that Cl extrusion from choroid plexus is by way of an anion exchanger and via channels.

Acetazolamide↗
PubMed1992

Specific interactions of mercury chloride with membranes and other ligands as revealed by mercury-NMR.

High resolution mercury nuclear magnetic resonance (199Hg-NMR) experiments have been performed in order to monitor mercury chemical speciation when HgCl2 is added to water solutions and follow mercury binding properties towards biomembranes or other ligands. Variations of 199Hg chemical shifts by several hundred ppm depending upon pH and/or pCl changes or upon ligand or membrane addition afforded to determine the thermodynamic parameters which describe the equilibria between the various species in solution. By comparison to an external reference, the decrease in concentration of mercury species in solution allowed to estimate the amount as well as the thermodynamic parameters of unlabile mercury-ligand or mercury-membrane complexes. Hence, some buffer molecules can be classified in a scale of increasing complexing power towards Hg(II): EGTA greater than Tris greater than HEPES. In contrast, MOPS, Borax, phosphates and acetates show little complexation properties for mercury, in our experimental conditions. Evidence for complexation with phosphatidylethanolamine (PE), phosphatidylserine (PS) and human erythrocyte membranes has been found. Hg(II) does not form complexes with egg phosphatidylcholine membranes. Interaction with PE and PS model membranes can be described by the presence of two mercury sites, one labile, the other unlabile, in the NMR time scale. In the labile site Hg(PE) and Hg(PS)2 would be formed whereas in the unlabile site Hg(II) would establish bridges between three PE or PS molecules. Calculated thermodynamic data clearly indicate that PE is a better complexing agent than PS. Evidence is also found that complexation with lipids uses at first the HgCl2 species. Interestingly, mercury complexation with ligands or membranes can be completely reversed by addition of decimolar NaCl solutions. Minute mechanisms for mercury complexation with the primary amine of PE or PS membrane head groups are discussed.

Buffers↗
PubMed1983

Intracellular microinjection of alkaline buffers reversibly inhibits the initial phase of hormone action in meiosis reinitiation of starfish oocytes.

Intracellular microinjection of alkaline Hepes-KOH buffers, which increases intracellular pH (pHI) from 6.92 to 7.70 in fully grown prophase-blocked oocytes of the starfish Marthasterias glacialis, like external application of ammonia and other weak bases (M. Doree, K. Sano, and H. Kanatani, 1982, Dev. Biol. 90, 13-17), inhibited meiosis reinitiation induced by 1-methyladenine (1-MeAde) or dithiothreitol (DTT), a mimetic of the hormone. Oocytes could be released from inhibition by raising the concentration of hormone or of its mimetic. Increasing pHI to 7.70 neither inhibited nor delayed meiosis reinitiation when pH was clamped after the end of the hormone-dependent period, the period during which 1-MeAde is required in the external medium for meiosis to occur, whereas it blocked the action of the hormone at low concentration when performed before the end of the hormone-dependent period. When hormone concentration was higher, germinal vesicle breakdown (GVBD) occurred, but duration of the hormone-dependent period was increased. Delay introduced by alkalinization for oocytes to reach GVBD after 1-MeAde addition was smaller at high than at medium concentrations of the hormone. Increasing pHI did not inhibit action of MPF, the cytoplasmic maturation factor which induces GVBD and the subsequent process of meiotic maturation following hormonal treatment of prophase-blocked oocytes.

Adenine↗
PubMed1986

Permeability of rod outer segment disk membranes as probed by Ficoll density gradient centrifugation and by turbidimetry.

Osmotic volume changes of isolated disks prepared from bovine rod outer segments were investigated utilizing the capacity of osmotically intact disks to float on 2.5% (w/v) Ficoll under hypotonic conditions. The amount of floating disks decreases steadily and approximately linearly when the osmolarity is increased stepwise with an osmotically active solute. The slope of the decrease becomes smaller for partially permeable substances and no decrease in observed for fully permeable solutes. All ions assayed by this method as well as sucrose were osmotically fully active to disk membranes. The buffer molecules Tris and imidazole partially penetrated the disk membranes at pH 7, whereas the zwitterionic buffer Hepes was impermeable. Ethanol showed absolutely no osmotic activity to disk membranes and, therefore, can easily penetrate these membranes. concurrent turbidimetric measurements and, therefore, can easily penetrate these membranes. Concurrent turbidimetric measurements confirmed these results. Upon prolonged exposure of disk membranes to 5% (w/v) Ficoll, a considerable modification of the osmotic properties of disk membranes is observed, indicating a significant interaction of 5% (w/v) Ficoll with disk membranes. No influence of 2.5% (w/v) Ficoll on the osmotic properties of disk membranes was detected. The restriction of the use of Ficoll for disk membrane preparative purposes will be discussed.

Animals↗
PubMed1981

Changes in pH with age in Drosophila and the influence of buffers on longevity.

The pH of whole homogenates of Drosophila melanogaster declines from 7.44 during the first instar larval stage to 6.77 during the late pupal stage. In adult flies the pH declines from 6.92 to 6.70 with increasing age. Total carbonic anhydrase activity declines during the first 10 days of the adult stage. The carbonic anhydrase inhibitor acetazolamide failed to shorten life span. Low molecular weight buffers prolong the median life span when fed to flies in the pH range 7.0-8.3. The greatest prolongation occurs when the flies are given the buffers beginning at 2 weeks of age. 0.1 M HEPES (pH 7.5) increased life span by 22.9% and 0.01 M KHCO3 (pH 7.0) by 17.8%.

Aging↗