Effects of pH on Ca2+i, Na+i, and pHi of MDCK cells: Na(+)-Ca2+ and Na(+)-H+ antiporter interactions

1991 ◽  
Vol 261 (3) ◽  
pp. C482-C489 ◽  
Author(s):  
A. B. Borle ◽  
C. Bender
Keyword(s):  
Mdck Cells ◽  
Extracellular Ph ◽  
Intracellular Acidosis ◽  
Experimental Conditions ◽  
Fluorescent Indicators ◽  
Reverse Mode ◽  
Effects Of Ph ◽  
Ph Stat ◽  
Stat Method ◽  
Free Media

The effects of high and low extracellular pH (8.0 and 6.8) on the intracellular concentration of Ca2+, Na+, and H+ were measured in perfused Madin-Darby canine kidney (MDCK) cells cast in agarose gel threads. Cytosolic free Ca2+ (Ca2+i) was measured with aequorin, while intracellular Na+ (Na+i) and H+ (Hi+ or pHi) were determined with the fluorescent indicators SBFI and BCECF, respectively. In addition, H+ secretion was assayed by the pH-stat method, and Na+ or Ca2+ fluxes were measured with 22Na or 45Ca, respectively. H+ secretion was significantly depressed by several experimental conditions that are known to inhibit the Na(+)-H+ antiporter: H+ secretion decreased 44% in the presence of 10(-5) M ethylisopropylamiloride, 49% in Na+o-free media, 44% in the presence of 10(-4) M ouabain, and 32% in the presence of 10(-4) M 8-bromoadenosine 3',5'-cyclic monophosphate. In addition, pHi decreased by 0.2 pH units in Na+o-free media. Finally, recovery from an intracellular acidosis evoked by 20 mM NH4Cl pulse required the presence of extracellular Na+. When the extracellular pH (pHo) was increased from 7.4 to 8.0, H+ secretion increased 58% from 17.5 to 27.7 nmol.min-1.mg protein-1 and Na+ influx increased 48%. As a result, pHi rose from 7.43 to 7.71 and Na+i increased from 15.6 to 19.7 mM. Finally, Ca2+i rose from 120 to 268 nM. These results suggest that the high pHo stimulated the Na(+)-H+ antiporter, and the subsequent rise in Na+i decreased the Na+ electrochemical potential, thereby activating the reverse mode of Na(+)-Ca2+ exchange (Ca2+ influx vs. Na+ efflux) which led to the rise in Ca2+i.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Ferroxidase activity of ferritin: effects of pH, buffer and Fe(II) and Fe(III) concentrations on Fe(II) autoxidation and ferroxidation

1999 ◽  
Vol 338 (3) ◽  
pp. 615-618 ◽  
Author(s):  
Xiaoke YANG ◽  
N. Dennis CHASTEEN
Keyword(s):  
Ph Control ◽  
Alternative Mechanism ◽  
Experimental Conditions ◽  
Iron Storage ◽  
Ferroxidase Activity ◽  
Effects Of Ph ◽  
Ph Stat ◽  
Ph Buffer ◽  
Horse Spleen Ferritin

It is widely accepted that iron deposition in the iron storage protein ferritin in vitro involves Fe(II) oxidation, and that ferritin facilitates this oxidation at a ferroxidase site on the protein. However, these views have recently been questioned, with the protein ferroxidase activity instead being attributed to autoxidation from the buffer alone. Ligand exchange between another protein with ferroxidase activity and ferritin has been proposed as an alternative mechanism for iron incorporation into ferritin. In the present work, a pH stat apparatus is used to eliminate the influence of buffers on iron(II) oxidation. Here we show that the recent experiments questioning the ferroxidase activity of ferritin were flawed by inadequate pH control, that buffers actually retard rather than facilitate iron(II) oxidation, and that horse spleen ferritin has ferroxidase activity when measured under proper experimental conditions. Furthermore, high pH (7.0), a high Fe(II) concentration and the presence of Fe(III) all favour Fe(II) autoxidation in the presence or absence of ferritin.

In vivo Production of Soluble Inorganic Pyrophosphatases in Two Strains of Vibrio cholerae

1970 ◽  
Vol 24 (1) ◽  
pp. 38-41
Author(s):  
Taslima Taher Lina ◽  
Mohammad Ilias
Keyword(s):  
Vibrio Cholerae ◽  
Specific Activity ◽  
Experimental Conditions ◽  
Environmental Strain ◽  
Cell Extracts ◽  
Atcc Strain ◽  
The Family ◽  
Effects Of Ph ◽  
Vibrio Cholerae O1

The in vivo production of soluble inorganic pyrophosphatases (PPases) was investigated in two strains, namely, Vibrio cholerae EM 004 (environmental strain) and Vibrio cholerae O1 757 (ATCC strain). V. cholerae is known to contain both family I and family II PPase coding sequences. The production of family I and family II PPases were determined by measuring the enzyme activity in cell extracts. The effects of pH, temperature, salinity of the growth medium on the production of soluble PPases were studied. In case of family I PPase, V. cholerae EM 004 gave the highest specific activity at pH 9.0, with 2% NaCl + 0.011% NaF and at 37°C. The strain V. cholerae O1 757 gave the highest specific activity at pH 9.0, with media containing 0% NaCl and at 37°C. On the other hand, under all the conditions family II PPase did not give any significant specific activity, suggesting that the family II PPase was not produced in vivo in either strains of V. cholerae under different experimental conditions. Keywords: Vibrio cholerae, Pyrophosphatases (PPases), Specific activityDOI: http://dx.doi.org/10.3329/bjm.v24i1.1235 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 38-41

scholarly journals Kinetics and Mechanism of Hydrolysis of Acetylthiocholine by Butyrylcholine Esterase

2002 ◽  
Vol 57 (11-12) ◽  
pp. 1072-1077 ◽  
Author(s):  
Karel Komers ◽  
Alexandr Čegan ◽  
Marek Link
Keyword(s):  
Acetic Acid ◽  
Kinetics And Mechanism ◽  
Kinetics Parameters ◽  
Mechanism Of Hydrolysis ◽  
Ph Stat ◽  
Stat Method ◽  
Ellman’S Method ◽  
Hydrolysis Of

Kinetics and mechanism of hydrolysis of acetylthiocholine by the enzyme butyrylcholine esterase was studied. The spectrophotometric Ellman’s method and potentiometric pH-stat method were used for continuous determination of the actual concentration of the products thiocholine and acetic acid in the reaction mixture. The validity of the Michaelis-Menten (Briggs-Haldane) equation in the whole course of the reaction under used conditions was proved. The corresponding kinetics parameters (Vm and KM) were calculated from the obtained dependences of concentration of thiocholine or acetic acid vs. time and compared. From this comparison the deciding kinetic role of the step producing thiocholine was derived. The values of initial molar concentration of the enzyme and of the rate constants of the kinetic model were estimated.

scholarly journals Carbonic anhydrase IX is a pH-stat that sets an acidic tumour extracellular pH in vivo

2018 ◽  
Vol 119 (5) ◽  
pp. 622-630 ◽  
Author(s):  
Shen-Han Lee ◽  
Dominick McIntyre ◽  
Davina Honess ◽  
Alzbeta Hulikova ◽  
Jesús Pacheco-Torres ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Extracellular Ph ◽  
Carbonic Anhydrase Ix ◽  
Ph Stat

Bicarbonate Transport Systems in the Intestine of the Seawater EEL

1990 ◽  
Vol 150 (1) ◽  
pp. 381-394 ◽  
Author(s):  
MASAAKI ANDO ◽  
M. V. SUBRAMANYAM
Keyword(s):  
Latent Period ◽  
Water Transport ◽  
Basolateral Membrane ◽  
Transport Systems ◽  
Bicarbonate Transport ◽  
Ph Stat ◽  
Stat Method

Utilizing a pH-stat method, the rates of mucosal and serosal alkalinization were measured separately in the seawater eel intestine. These two rates were dependent on contralateral HCO3− concentration and were inhibited by contralateral application of DIDS, an inhibitor of HCO3− transport, indicating that the mucosal and serosal alkalinization are due to HCO3− secretion and absorption, respectively. The mucosal alkalinization was enhanced after inhibiting Na+/K+/Cl− cotransport by treatment with bumetanide, furosemide or Ba2+, with a latent period of more than lOmin, suggesting that HCO3− absorption from mucosa to serosa depends on Na+/K+/Cl− cotransport. The serosal alkalinization caused by HCO3− absorption was completely abolished after mucosal application of bumetanide. After pretreatment with bumetanide, mucosal omission of Cl− halved the enhanced rate of mucosal alkalinization, and Na+ omission had no effect on it; this indicates that the exit of HCO3− into the lumen depends on luminal Cl−, i.e. on the existence of the usual C1−/HCO3− exchange on the brushborder membrane. When serosal Na+ was removed under the same conditions, mucosal alkalinization was reduced, indicating that HCO3− entry from the serosal fluid depends on Na+. Serosal omission of Cl− did not reduce mucosal alkalinization. In addition, serosal alkalinization was enhanced by serosal removal of Na+ but not of Cl−. These results suggest that there is a Na+/HCO3− cotransport on the basolateral membrane. A possible model for HCO3− transport systems in the seawater eel intestine is proposed, and a possible role for these transport systems is discussed in relation to Na+, Cl− and water transport.

Factors affecting constancy of acetate concentration and correct determination of methanogenic activity in pH-stat experiments

2003 ◽  
Vol 48 (6) ◽  
pp. 111-118
Author(s):  
M. Mösche ◽  
U. Meyer
Keyword(s):  
Accurate Determination ◽  
Original Method ◽  
Experimental Conditions ◽  
Methanogenic Activity ◽  
Factors Affecting ◽  
Acetate Concentration ◽  
Correct Determination ◽  
Ph Stat ◽  
The Stability

The determination of methanogenic activity with a pH-stat titration bioassay is evaluated utilising a mathematical model of this system. For given kinetic parameters and experimental conditions the model calculates the development of titrant flow and acetate concentration during experiments. Simulations of experiments under various conditions are compared. They show that the original method inherently causes a strong drift of acetate concentration during the experiments and a misestimation of methanogenic activity. As a solution to these disadvantages the addition of sodium hydroxide to the titrant and a careful control of pH during flushing the reactor with gas prior to the experiment are recommended. In this way a better constancy of acetate concentration and a more accurate determination of methanogenic activity should be achievable. The accuracy of this method is limited by the stability of pH-electrode calibration parameters.

pH of isolated resting skeletal muscle and its relation to potassium content

1963 ◽  
Vol 204 (6) ◽  
pp. 1048-1054 ◽  
Author(s):  
Ronald B. Miller ◽  
Ian Tyson ◽  
Arnold S. Relman
Keyword(s):  
Skeletal Muscle ◽  
Intracellular Ph ◽  
Potassium Content ◽  
Ion Concentration ◽  
Detectable Effect ◽  
Rat Diaphragm ◽  
Intracellular Acidosis ◽  
Experimental Conditions ◽  
Hydrogen Ion Concentration

Intracellular pH of isolated rat diaphragm was measured with both a C14-DMO method and a tissue CO2 technique. The values for intracellular pH by each method, although slightly different, changed in parallel under most experimental conditions. Acute, severe potassium depletion in vitro had no detectable effect on intracellular pH, nor did prior depletion in vivo followed by incubation in a potassium-free bath. This was true whether or not the potassium-depleted muscle was exposed to normal or elevated extracellular levels of bicarbonate, and was unaffected by the presence of cationic amino acids in the bath. Acute repletion of previously potassium-depleted muscle resulted in a small rise in cell pH, but this was no greater than that produced by loading normal tissues with potassium. It is concluded that under the conditions of these experiments there is no evidence of intracellular acidosis in potassium-depleted skeletal muscle. Rat diaphragm can lose up to half its potassium content in vitro without detectable increase in hydrogen ion concentration.

scholarly journals How and Why Are Cancers Acidic? Carbonic Anhydrase IX and the Homeostatic Control of Tumour Extracellular pH

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1616 ◽  
Author(s):  
Shen-Han Lee ◽  
John R. Griffiths
Keyword(s):  
Carbonic Anhydrase ◽  
Imaging Techniques ◽  
Tumour Microenvironment ◽  
Extracellular Ph ◽  
Solid Tumours ◽  
Carbonic Anhydrase Ix ◽  
Carbonic Anhydrases ◽  
Somatic Evolution ◽  
Ph Imaging ◽  
Ph Stat

The acidic tumour microenvironment is now recognized as a tumour phenotype that drives cancer somatic evolution and disease progression, causing cancer cells to become more invasive and to metastasise. This property of solid tumours reflects a complex interplay between cellular carbon metabolism and acid removal that is mediated by cell membrane carbonic anhydrases and various transport proteins, interstitial fluid buffering, and abnormal tumour-associated vessels. In the past two decades, a convergence of advances in the experimental and mathematical modelling of human cancers, as well as non-invasive pH-imaging techniques, has yielded new insights into the physiological mechanisms that govern tumour extracellular pH (pHe). In this review, we examine the mechanisms by which solid tumours maintain a low pHe, with a focus on carbonic anhydrase IX (CAIX), a cancer-associated cell surface enzyme. We also review the accumulating evidence that suggest a role for CAIX as a biological pH-stat by which solid tumours stabilize their pHe. Finally, we highlight the prospects for the clinical translation of CAIX-targeted therapies in oncology.

Lipase activity measured in serum by a continuous-monitoring pH-Stat technique--an update.

1989 ◽  
Vol 35 (8) ◽  
pp. 1688-1693 ◽  
Author(s):  
N W Tietz ◽  
J R Astles ◽  
D F Shuey
Keyword(s):  
Olive Oil ◽  
Continuous Monitoring ◽  
Hydroxypropyl Methylcellulose ◽  
Reference Interval ◽  
Sample Volume ◽  
Standard Curve ◽  
Activity Measurements ◽  
Ph Stat ◽  
Stat Method ◽  
Assay Conditions

Abstract Using recent knowledge regarding the roles of colipase, bile acids, Ca2+, and emulsifiers, we optimized a previously published pH-Stat method for lipase (EC 3.1.1.3) activity measurements. The recommended assay conditions are: olive oil/triolein, 100 mL/L; sodium glycocholate, 35 mmol/L; Ca2+, 8.5 mmol/L; and colipase, 6.0 mg/L. The sample volume is 0.10 mL, the reaction pH 9.0, the temperature 30 degrees C, and the concentration of titrant 15 mmol/L. Hydroxypropyl methylcellulose, 20 g/L, replaces acacia as emulsifier to avoid inhibition by excess Ca2+. The standard curve is linear to greater than 4566 U/L. The reference interval with olive oil as substrate is 30-235 U/L. Lipase activities with triolein substrate are 9.9% greater than with olive oil. Interference by pancreatic carboxylesterase (EC 3.1.1.1) activity is inhibited by incubating the sample with diisopropylfluorophosphate. Results correlate well with those by the optimized SingleVial method of Boehringer Mannheim Diagnostics (r = 0.997) and the immunochemical assay of Beckman Instruments, Inc. (r = 0.995). Correlation with the aca method (E.I. DuPont de Nemours & Company) is less satisfactory (r = 0.892), probably owing to lack of colipase in the latter method.

scholarly journals Use of Silica Based Materials as Modulators of the Lipase Catalyzed Hydrolysis of Fats under Simulated Duodenal Conditions

Nanomaterials ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1927
Author(s):  
Sara Muñoz-Pina ◽  
Pedro Amorós ◽  
Jamal El Haskouri ◽  
Ana Andrés ◽  
José V. Ros-Lis
Keyword(s):  
Partial Least Square ◽  
Least Square ◽  
Partial Least Square Regression ◽  
Fat Globules ◽  
Silica Materials ◽  
Functionalized Materials ◽  
Ph Stat ◽  
Surface Functionalizations ◽  
Fat Hydrolysis ◽  
Stat Method

The effect of silica materials and their functionalization in the lipase catalyzed fat hydrolysis has been scarcely studied. Fifteen silica materials were prepared and their effect on the fat hydrolysis was measured, under simulated duodenal conditions, using the pH-stat method. The materials are composed of the combination of three supports (Stöber massive silica nanoparticles, Stöber mesoporous nanoparticles and UVM-7) and four surface functionalizations (methyl, trimethyl, propyl and octyl). In addition, the non-functionalized materials were tested. The functional groups were selected to offer a hydrophobic character to the material improving the interaction with the fat globules and the lipase. The materials are able to modulate the lipase activity and their effect depending on the support topology and the organic covering, being able to increase or reduce the fat hydrolysis. Depending of the material, relative fat hydrolysis rates of 75 to 140% in comparison with absence of the material were obtained. The results were analyzed by Partial Least Square Regression and suggest that the alkyl modified mesopores are able to improve the fat hydrolysis, by contrast the non-porous nanoparticles and the textural pores tend to induce inhibition. The effects are more pronounced for materials containing long alkyl chains and/or in absence of taurodeoxycholate.

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