Mathematical Modelling of the Role of Intra- and Extracellular Activity of Carbonic Anhydrase and Membrane Permeabilities of HCO3, H2O and CO2in18O Exchange

Wunder, M.; Böllert, P.; Gros, Gerolf

doi:10.1080/10256019808036371

Cited by 14 publications

(27 citation statements)

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“…This property has been well studied in previous reports [26, 27, 30]. In these methods, the membrane inlet is immersed in cell suspension or in contact with cell suspension and measures the 18 O content of extracellular CO 2 .…”

Section: Discussionmentioning

confidence: 98%

“…This has been done for red blood cells assessing intracellular CA activity and flux rates of CO 2 and bicarbonate across the membranes [26, 27, 30]. The advantage of the 18 O depletion method measured by MIMS is that it sensitively measures CA activity in whole cell suspensions, manifests the second phase which is sensitive to extracellular CA activity, and provides a visual demonstration of the inhibition of the extracellular and total CA activity.…”

Section: Discussionmentioning

confidence: 99%

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Detecting extracellular carbonic anhydrase activity using membrane inlet mass spectrometry

Delacruz

Mikulski

et al. 2010

Analytical Biochemistry

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Current research into the function of carbonic anhydrases in cell physiology emphasizes the role of membrane-bound carbonic anhydrases, such as carbonic anhydrase IX that has been identified in malignant tumors and is associated with extracellular acidification as a response to hypoxia. We present here a mass spectrometric method to determine the extent to which total carbonic anhydrase activity is due to extracellular carbonic anhydrase in whole cell preparations. The method is based on the biphasic rate of depletion of 18 O from CO 2 measured by membrane inlet mass spectrometry. The slopes of the biphasic depletion are a sensitive measure of the presence of carbonic anhydrase outside and inside of the cells. This property is demonstrated here using suspensions of human red cells in which external carbonic anhydrase was added to the suspending solution. It is also applied to breast and prostate cancer cells which both express exofacial carbonic anhydrase IX. Inhibition of external carbonic anhydrase is achieved by use of a membrane impermeant inhibitor that was synthesized for this purpose, p-aminomethylbenzenesulfonamide attached to a polyethyleneglycol polymer.Keywords carbonic anhydrase; mass spectrometry; carbon dioxide; stable isotope; enzyme activityThe zinc metalloenzyme carbonic anhydrase (CA) catalyzes the hydration of carbon dioxide to form bicarbonate and a proton [1,2]. There are 16 isozymes of CA in the α-class of mammalian carbonic anhydrases that play a role in respiration, formation of secretory fluids, acid-base balance, and other physiological functions [3]. The isozyme CA IX is of particular interest since it is found in few normal tissues but its expression is induced in certain tumor cells by hypoxia [4,5]. CA IX is a transmembrane protein the catalytic site of which is external to the cell and is shown to play a role in extracellular acidification associated with cell Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. proliferation and metastasis [6][7][8][9]. Its inhibition may be of benefit in anticancer therapies for several types of cancer, including breast and prostate cancer. NIH Public AccessThe inhibition of carbonic anhydrases by sulfonamides is well studied in terms of its biochemical, physiological, and medical aspects [3,10,11]. Oxygen-18 and carbon-13 labeled bicarbonate was prepared by dissolving KH 13 CO 3 (99% 13 C) in enriched water (up to 90% 18 O enrichment). This solution was allowed to come to isotopic equilibrium overnight, after which water was removed by vacuum distillation. The resulting product was stored at room temp...

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Detecting extracellular carbonic anhydrase activity using membrane inlet mass spectrometry

Delacruz

Mikulski

et al. 2010

Analytical Biochemistry

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“…When applying the mathematical model that describes this process in terms not only of CA activity and HCO − 3 permeability, but also of CO 2 permeability, as first introduced by Wunder and Gros (1997, 1998) and Wunder et al (1998), they concluded that DIDS is a powerful inhibitor of red cell membrane CO 2 permeability. Since DIDS is an ε-amino group reagent affecting a variety of proteins, they concluded that CO 2 permeation in the red cell membrane must be a process mediated by a membrane protein.…”

Section: Protein Channels For Molecular Co2 In Membranes Of High Co2 mentioning

confidence: 99%

How does carbon dioxide permeate cell membranes? A discussion of concepts, results and methods

et al. 2014

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We review briefly how the thinking about the permeation of gases, especially CO2, across cell and artificial lipid membranes has evolved during the last 100 years. We then describe how the recent finding of a drastic effect of cholesterol on CO2 permeability of both biological and artificial membranes fundamentally alters the long-standing idea that CO2—as well as other gases—permeates all membranes with great ease. This requires revision of the widely accepted paradigm that membranes never offer a serious diffusion resistance to CO2 or other gases. Earlier observations of “CO2-impermeable membranes” can now be explained by the high cholesterol content of some membranes. Thus, cholesterol is a membrane component that nature can use to adapt membrane CO2 permeability to the functional needs of the cell. Since cholesterol serves many other cellular functions, it cannot be reduced indefinitely. We show, however, that cells that possess a high metabolic rate and/or a high rate of O2 and CO2 exchange, do require very high CO2 permeabilities that may not be achievable merely by reduction of membrane cholesterol. The article then discusses the alternative possibility of raising the CO2 permeability of a membrane by incorporating protein CO2 channels. The highly controversial issue of gas and CO2 channels is systematically and critically reviewed. It is concluded that a majority of the results considered to be reliable, is in favor of the concept of existence and functional relevance of protein gas channels. The effect of intracellular carbonic anhydrase, which has recently been proposed as an alternative mechanism to a membrane CO2 channel, is analysed quantitatively and the idea considered untenable. After a brief review of the knowledge on permeation of O2 and NO through membranes, we present a summary of the 18O method used to measure the CO2 permeability of membranes and discuss quantitatively critical questions that may be addressed to this method.

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“…Therefore, we solved the set of six linear differential equations describing the change with time of the intra-and extracellular concentrations of the labeled species CO 2 , HCO 3 Ϫ and HOH (8). Each of these equations contains a term for the chemical reactions and the transmembrane flux affecting the species concentration.…”

Section: Methodsmentioning

confidence: 99%

The effect of 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate on CO ₂ permeability of the red blood cell membrane

Forster

Gros

Lin

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in P m,CO 2 would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO 2 and HCO 3 ؊ , we found that DIDS inhibited both P m,HCO ؊ 3 and P m,CO 2 , whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO 2 permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO 2 exchange. Ϫ and H 2 16 O in a red cell suspension at chemical equilibrium can be used in principle to measure the proportional intracellular acceleration (A) of CO 2 hydration produced inside intact red blood cells by carbonic anhydrase (CA) compared with the uncatalyzed rate (1) and the self-exchange permeability of the membrane to HCO 3 (P m,HCO Ϫ 3 ). As predicted, A was found to be the same in intact erythrocytes as in hemolysate, both under normal conditions and when exposed to varying concentrations of a membrane-permeable CA sulfonamide inhibitor (ethoxzolamide), whereas P m,HCO Ϫ 3 was not affected (2). Validation of the ability of the technique to differentiate between CA activity and HCO 3 Ϫ permeability in intact red cells was extended by exposing them to phlorizin (3), an established Band III inhibitor (1); this drug decreased P m,HCORecently we exposed red cells to a more specific inhibitor of Band III protein, 4,4Ј-diisothiocyanato-stilbene-2,2Ј-disulfonate (DIDS), to decrease P m,HCO Ϫ 3 without inhibiting CA. However, we found that whereas DIDS lowered HCO 3 Ϫ transport, it also lowered the membrane permeability to CO 2 . METHODSTwo groups of experiments were carried out, one in Philadelphia and one in Hannover, Germany. Blood from healthy adults, freshly drawn into heparin, was washed three times with 145 mM NaCl at 4°C; the red cells were diluted to approximately 40% hematocrit and used the same day. Hemolysates were prepared by freezing and thawing the cell suspension.The fractional water volume, v, of the red cells in the reaction suspension was calculated as the hematocrit of the stock cell suspension ϫ water content of red cells [0.61 (1) for the Philadelphia experiments and 0.65 (4) for the Hannover experiments] ϫ its dilution in the reaction mixture. In later experiments, v was calculated as 55.5 ϫ [cyanmethemoglobin] in mM in the reactant mixture. NaHCO 3 was prepared by incubating 2% and 5% 18O labeled HOH with unlabeled NaHCO 3 at 150°C in a pressure bomb for several days, after which the water was removed by lyophylization. DIDS was obtained from Research Organics and phlorizin (phloridzin) was obtained from Sigma.The technique of the ...

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Mathematical Modelling of the Role of Intra- and Extracellular Activity of Carbonic Anhydrase and Membrane Permeabilities of HCO₃, H₂O and CO₂in¹⁸O Exchange

Cited by 14 publications

References 6 publications

Detecting extracellular carbonic anhydrase activity using membrane inlet mass spectrometry

Detecting extracellular carbonic anhydrase activity using membrane inlet mass spectrometry

How does carbon dioxide permeate cell membranes? A discussion of concepts, results and methods

The effect of 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate on CO ₂ permeability of the red blood cell membrane

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Mathematical Modelling of the Role of Intra- and Extracellular Activity of Carbonic Anhydrase and Membrane Permeabilities of HCO3, H2O and CO2in18O Exchange

Cited by 14 publications

References 6 publications

Detecting extracellular carbonic anhydrase activity using membrane inlet mass spectrometry

Detecting extracellular carbonic anhydrase activity using membrane inlet mass spectrometry

How does carbon dioxide permeate cell membranes? A discussion of concepts, results and methods

The effect of 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate on CO 2 permeability of the red blood cell membrane

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Mathematical Modelling of the Role of Intra- and Extracellular Activity of Carbonic Anhydrase and Membrane Permeabilities of HCO₃, H₂O and CO₂in¹⁸O Exchange

The effect of 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate on CO ₂ permeability of the red blood cell membrane