Effect of PCMBS on CO2permeability of Xenopus oocytes expressing aquaporin 1 or its C189S mutant

1998 ◽  
Vol 275 (6) ◽  
pp. C1481-C1486 ◽  
Author(s):  
Gordon J. Cooper ◽  
Walter F. Boron
Keyword(s):  
Carbonic Anhydrase ◽  
Intracellular Ph ◽  
Xenopus Oocytes ◽  
Membrane Lipid ◽  
Wild Type ◽  
Aquaporin 1 ◽  
Gas Channel ◽  
A Cell ◽  
Pass Through ◽  
Rule Out

A recent study on Xenopus oocytes [N. L. Nakhoul, M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 ( Cell Physiol. 43): C543–548, 1998] injected with carbonic anhydrase showed that expressing aquaporin 1 (AQP1) increases by ∼40% the rate at which exposing the cell to CO2 causes intracellular pH to fall. This observation is consistent with several interpretations. Overexpressing AQP1 might increase apparent CO2 permeability by 1) allowing CO2 to pass through AQP1, 2) stimulating injected carbonic anhydrase, 3) enhancing the CO2 solubility of the membrane’s lipid, or 4) increasing the expression of a native “gas channel.” The purpose of the present study was to distinguish among these possibilities. We found that expressing the H2O channel AQP1 in Xenopus oocytes increases the CO2 permeability of oocytes in an expression-dependent fashion, whereas expressing the K+ channel ROMK1 has no effect. The mercury derivative p-chloromercuriphenylsulfonic acid (PCMBS), which inhibits the H2O movement through AQP1, also blocks the AQP1-dependent increase in CO2 permeability. The mercury-insensitive C189S mutant of AQP1 increases the CO2 permeability of the oocyte to the same extent as does the wild-type channel. However, the C189S-dependent increase in CO2permeability is unaffected by treatment with PCMBS. These data rule out options 2–4 listed above. Thus our results suggest that CO2passes through the pore of AQP1 and are the first data to demonstrate that a gas can enter a cell by a means other than diffusing through the membrane lipid.

scholarly journals Effect of expressing the water channel aquaporin-1 on the CO2 permeability ofXenopus oocytes

1998 ◽  
Vol 274 (2) ◽  
pp. C543-C548 ◽  
Author(s):  
Nazih L. Nakhoul ◽  
Bruce A. Davis ◽  
Michael F. Romero ◽  
Walter F. Boron
Keyword(s):  
Xenopus Oocytes ◽  
Water Channel ◽  
Membrane Lipid ◽  
Hydration Reaction ◽  
Extracellular Solution ◽  
Aquaporin 1 ◽  
Gas Channel ◽  
Rate Limiting Step ◽  
Aqp1 Expression ◽  
Rate Limiting

It is generally accepted that gases such as CO2 cross cell membranes by dissolving in the membrane lipid. No role for channels or pores in gas transport has ever been demonstrated. Here we ask whether expression of the water channel aquaporin-1 (AQP1) enhances the CO2 permeability of Xenopus oocytes. We expressed AQP1 in Xenopus oocytes by injecting AQP1 cRNA, and we assessed CO2permeability by using microelectrodes to monitor the changes in intracellular pH (pHi) produced by adding 1.5% CO2/10 mM[Formula: see text] to (or removing it from) the extracellular solution. Oocytes normally have an undetectably low level of carbonic anhydrase (CA), which eliminates the CO2 hydration reaction as a rate-limiting step. We found that expressing AQP1 (vs. injecting water) had no measurable effect on the rate of CO2-induced pHi changes in such low-CA oocytes: adding CO2 caused pHi to fall at a mean initial rate of 11.3 × 10−4 pH units/s in control oocytes and 13.3 × 10−4 pH units/s in oocytes expressing AQP1. When we injected oocytes with water, and a few days later with CA, the CO2-induced pHi changes in these water/CA oocytes were more than fourfold faster than in water-injected oocytes (acidification rate, 53 × 10−4 pH units/s). Ethoxzolamide (ETX; 10 μM), a membrane-permeant CA inhibitor, greatly slowed the pHi changes (16.5 × 10−4 pH units/s). When we injected oocytes with AQP1 cRNA and then CA, the CO2-induced pHi changes in these AQP1/CA oocytes were ∼40% faster than in the water/CA oocytes (75 × 10−4 pH units/s), and ETX reduced the rates substantially (14.7 × 10−4 pH units/s). Thus, in the presence of CA, AQP1 expression significantly increases the CO2 permeability of oocyte membranes. Possible explanations include 1) AQP1 expression alters the lipid composition of the cell membrane, 2) AQP1 expression causes overexpression of a native gas channel, and/or 3) AQP1 acts as a channel through which CO2 can permeate. Even if AQP1 should mediate a CO2 flux, it would remain to be determined whether this CO2movement is quantitatively important.

scholarly journals Transgenic expression of carbonic anhydrase III in cardiac muscle demonstrates a mechanism to tolerate acidosis

2019 ◽  
Vol 317 (5) ◽  
pp. C922-C931 ◽  
Author(s):  
Han-Zhong Feng ◽  
J.-P. Jin
Keyword(s):  
Carbonic Anhydrase ◽  
Transgenic Mice ◽  
Intracellular Ph ◽  
Skeletal Muscles ◽  
Ex Vivo ◽  
Physiological Role ◽  
Wild Type Mouse ◽  
Carbonic Anhydrases ◽  
Wild Type ◽  
Carbonic Anhydrase Iii

Carbonic anhydrase III (CAIII) is abundant in liver, adipocytes, and skeletal muscles, but not heart. A cytosolic enzyme that catalyzes conversions between CO2 and [Formula: see text] in the regulation of intracellular pH, its physiological role in myocytes is not fully understood. Mouse skeletal muscles lacking CAIII showed lower intracellular pH during fatigue, suggesting its function in stress tolerance. We created transgenic mice expressing CAIII in cardiomyocytes that lack endogenous CAIII. The transgenic mice showed normal cardiac development and life span under nonstress conditions. Studies of ex vivo working hearts under normal and acidotic conditions demonstrated that the transgenic and wild-type mouse hearts had similar pumping functions under normal pH. At acidotic pH, however, CAIII transgenic mouse hearts showed significantly less decrease in cardiac function than that of wild-type control as shown by higher ventricular pressure development, systolic and diastolic velocities, and stroke volume via elongating the time of diastolic ejection. In addition to the effect of introducing CAIII into cardiomyocytes on maintaining homeostasis to counter acidotic stress, the results demonstrate the role of carbonic anhydrases in maintaining intracellular pH in muscle cells as a potential mechanism to treat heart failure.

scholarly journals Carbonic anhydrase II binds to and increases the activity of the epithelial sodium-proton exchanger, NHE3

2015 ◽  
Vol 309 (4) ◽  
pp. F383-F392 ◽  
Author(s):  
Devishree Krishnan ◽  
Lei Liu ◽  
Shane A. Wiebe ◽  
Joseph R. Casey ◽  
Emmanuelle Cordat ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Proximal Tubule ◽  
Intracellular Ph ◽  
Solid Phase ◽  
Carbonic Anhydrase Ii ◽  
Proximity Ligation Assay ◽  
Physical Interaction ◽  
Proximal Tubule Cell ◽  
Wild Type ◽  
Sodium Proton Exchanger

Two-thirds of sodium filtered by the renal glomerulus is reabsorbed from the proximal tubule via a sodium/proton exchanger isoform 3 (NHE3)-dependent mechanism. Since sodium and bicarbonate reabsorption are coupled, we postulated that the molecules involved in their reabsorption [NHE3 and carbonic anhydrase II (CAII)] might physically and functionally interact. Consistent with this, CAII and NHE3 were closely associated in a renal proximal tubular cell culture model as revealed by a proximity ligation assay. Direct physical interaction was confirmed in solid-phase binding assays with immobilized CAII and C-terminal NHE3 glutathione- S-transferase fusion constructs. To assess the effect of CAII on NHE3 function, we expressed NHE3 in a proximal tubule cell line and measured NHE3 activity as the rate of intracellular pH recovery, following an acid load. NHE3-expressing cells had a significantly greater rate of intracellular pH recovery than controls. Inhibition of endogenous CAII activity with acetazolamide significantly decreased NHE3 activity, indicating that CAII activates NHE3. To ascertain whether CAII binding per se activates NHE3, we expressed NHE3 with wild-type CAII, a catalytically inactive CAII mutant (CAII-V143Y), or a mutant unable to bind other transporters (CAII-HEX). NHE3 activity increased upon wild-type CAII coexpression, but not in the presence of the CAII V143Y or HEX mutant. Together these studies support an association between CAII and NHE3 that alters the transporter's activity.

scholarly journals The Lipid Lysyl-Phosphatidylglycerol Is Present in Membranes of Rhizobium tropici CIAT899 and Confers Increased Resistance to Polymyxin B Under Acidic Growth Conditions

2007 ◽  
Vol 20 (11) ◽  
pp. 1421-1430 ◽  
Author(s):  
Christian Sohlenkamp ◽  
Kanaan A. Galindo-Lagunas ◽  
Ziqiang Guan ◽  
Pablo Vinuesa ◽  
Sally Robinson ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Minimal Medium ◽  
Polymyxin B ◽  
Growth Conditions ◽  
Membrane Lipid ◽  
Low Ph ◽  
Wild Type ◽  
Rhizobium Tropici ◽  
Gram Negative ◽  
Inducible Genes

Lysyl-phosphatidylglycerol (LPG) is a well-known membrane lipid in several gram-positive bacteria but is almost unheard of in gram-negative bacteria. In Staphylococcus aureus, the gene product of mprF is responsible for LPG formation. Low pH-inducible genes, termed lpiA, have been identified in the gram-negative α-proteobacteria Rhizobium tropici and Sinorhizobium medicae in screens for acid-sensitive mutants and they encode homologs of MprF. An analysis of the sequenced bacterial genomes reveals that genes coding for homologs of MprF from S. aureus are present in several classes of organisms throughout the bacterial kingdom. In this study, we show that the expression of lpiA from R. tropici in the heterologous hosts Escherichia coli and Sinorhizobium meliloti causes formation of LPG. A wild-type strain of R. tropici forms LPG (about 1% of the total lipids) when the cells are grown in minimal medium at pH 4.5 but not when grown in minimal medium at neutral pH or in complex tryptone yeast (TY) medium at either pH. LPG biosynthesis does not occur when lpiA is deleted and is restored upon complementation of lpiA-deficient mutants with a functional copy of the lpiA gene. When grown in the low-pH medium, lpiA-deficient rhizobial mutants are over four times more susceptible to the cationic peptide polymyxin B than the wild type.

CPX, a selective A1-adenosine-receptor antagonist, regulates intracellular pH in cystic fibrosis cells

1995 ◽  
Vol 269 (1) ◽  
pp. C226-C233 ◽  
Author(s):  
V. Casavola ◽  
R. J. Turner ◽  
C. Guay-Broder ◽  
K. A. Jacobson ◽  
O. Eidelman ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Receptor Antagonist ◽  
Intracellular Ph ◽  
Adenosine Receptor ◽  
Ph Regulation ◽  
Cystic Fibrosis Transmembrane Regulator ◽  
Wild Type ◽  
A1 Adenosine Receptor ◽  
Adenosine Receptor Antagonist ◽  
Cystic Fibrosis Transmembrane

The selective A1-adenosine-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX), has been reported to activate Cl- efflux from cystic fibrosis cells, such as pancreatic CFPAC-1 and lung IB3 cells bearing the cystic fibrosis transmembrane regulator(delta F508) mutation, but has little effect on the same process in cells repaired by transfection with wild-type cystic fibrosis transmembrane regulator (O. Eidelman, C. Guay-Broder, P. J. M. van Galen, K. A. Jacobson, C. Fox, R. J. Turner, Z. I. Cabantchik, and H. B. Pollard. Proc. Natl. Acad. Sci. USA 89: 5562-5566, 1992). We report here that CPX downregulates Na+/H+ exchange activity in CFPAC-1 cells but has a much smaller effect on cells repaired with the wild-type gene. CPX also mildly decreases resting intracellular pH. In CFPAC-1 cells, this downregulation is dependent on the presence of adenosine, since pretreatment of the cells with adenosine deaminase blocks the CPX effect. We also show that, by contrast, CPX action on these cells does not lead to alterations in intracellular free Ca2+ concentration. We conclude that CPX affects pH regulation in CFPAC-1 cells, probably by antagonizing the tonic action of endogenous adenosine.

scholarly journals Inhibition of Carbonic Anhydrase IX Promotes Apoptosis through Intracellular pH Level Alterations in Cervical Cancer Cells

2021 ◽  
Vol 22 (11) ◽  
pp. 6098
Author(s):  
Ebru Temiz ◽  
Ismail Koyuncu ◽  
Mustafa Durgun ◽  
Murat Caglayan ◽  
Ataman Gonel ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Intracellular Ph ◽  
Hela Cells ◽  
Protein Level ◽  
Caspase 3 ◽  
Solid Tumours ◽  
Carbonic Anhydrase Ix ◽  
Parp Activation ◽  
Ca Ix ◽  
Cervical Cancer Cells

Carbonic anhydrase IX (CAIX) is a hypoxia-related protein that plays a role in proliferation in solid tumours. However, how CAIX increases proliferation and metastasis in solid tumours is unclear. The objective of this study was to investigate how a synthetic CAIX inhibitor triggers apoptosis in the HeLa cell line. The intracellular effects of CAIX inhibition were determined with AO/EB, AnnexinV-PI, and γ-H2AX staining; measurements of intracellular pH (pHi), reactive oxygen species (ROS), and mitochondrial membrane potential (MMP); and analyses of cell cycle, apoptotic, and autophagic modulator gene expression (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin, and LC3), caspase protein level (pro-caspase 3 and cleaved caspase-3, -8, -9), cleaved PARP activation, and CAIX protein level. Sulphonamide CAIX inhibitor E showed the lowest IC50 and the highest selectivity index in CAIX-positive HeLa cells. CAIX inhibition changed the morphology of HeLa cells and increased the ratio of apoptotic cells, dramatically disturbing the homeostasis of intracellular pHi, MMP and ROS levels. All these phenomena consequent to CA IX inhibition triggered apoptosis and autophagy in HeLa cells. Taken together, these results further endorse the previous findings that CAIX inhibitors represent an important therapeutic strategy, which is worth pursuing in different cancer types, considering that presently only one sulphonamide inhibitor, SLC-0111, has arrived in Phase Ib/II clinical trials as an antitumour/antimetastatic drug.

scholarly journals The Panzar–Rosse H Statistic and Monopoly. Issues on its Use as a Market Power Measure

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Juan Manuel Sanchez-Cartas
Keyword(s):  
Market Power ◽  
Power Measure ◽  
Pass Through ◽  
Rule Out

AbstractThis note emphasizes some flaws of the Panzar–Rosse H statistic to address market power. First, I show that it is more related to the pass-through rate than to a market power measure, which implies reconsidering its interpretation and use. Second, I show that the inclusion of other strategic variables rather than prices or quantities may lead to contradictory predictions about market power. Therefore, competition authorities should refrain from using the H statistic to rule out significant market power because it is not a well-behaved measure of the degree of competition.

scholarly journals Quorum Sensing: a Transcriptional Regulatory System Involved in the Pathogenicity of Burkholderia mallei

2004 ◽  
Vol 72 (11) ◽  
pp. 6589-6596 ◽  
Author(s):  
Ricky L. Ulrich ◽  
David DeShazer ◽  
Harry B. Hines ◽  
Jeffrey A. Jeddeloh
Keyword(s):  
Quorum Sensing ◽  
Virulence Factor ◽  
Regulatory System ◽  
In Silico Analysis ◽  
Homoserine Lactone ◽  
Burkholderia Mallei ◽  
Wild Type ◽  
Transcriptional Regulatory ◽  
A Cell

ABSTRACT Numerous gram-negative bacterial pathogens regulate virulence factor expression by using a cell density mechanism termed quorum sensing (QS). An in silico analysis of the Burkholderia mallei ATCC 23344 genome revealed that it encodes at least two luxI and four luxR homologues. Using mass spectrometry, we showed that wild-type B. mallei produces the signaling molecules N-octanoyl-homoserine lactone and N-decanoyl-homoserine lactone. To determine if QS is involved in the virulence of B. mallei, we generated mutations in each putative luxIR homologue and tested the pathogenicities of the derivative strains in aerosol BALB/c mouse and intraperitoneal hamster models. Disruption of the B. mallei QS alleles, especially in RJ16 (bmaII) and RJ17 (bmaI3), which are luxI mutants, significantly reduced virulence, as indicated by the survival of mice who were aerosolized with 104 CFU (10 50% lethal doses [LD50s]). For the B. mallei transcriptional regulator mutants (luxR homologues), mutation of the bmaR5 allele resulted in the most pronounced decrease in virulence, with 100% of the challenged animals surviving a dose of 10 LD50s. Using a Syrian hamster intraperitoneal model of infection, we determined the LD50s for wild-type B. mallei and each QS mutant. An increase in the relative LD50 was found for RJ16 (bmaI1) (>967 CFU), RJ17 (bmaI3) (115 CFU), and RJ20 (bmaR5) (151 CFU) compared to wild-type B. mallei (<13 CFU). These findings demonstrate that B. mallei carries multiple luxIR homologues that either directly or indirectly regulate the biosynthesis of an essential virulence factor(s) that contributes to the pathogenicity of B. mallei in vivo.

Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway

2004 ◽  
Vol 287 (4) ◽  
pp. H1730-H1739 ◽  
Author(s):  
Ron Zohar ◽  
Baoqian Zhu ◽  
Peter Liu ◽  
Jaro Sodek ◽  
C. A. McCulloch
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Cardiac Fibroblasts ◽  
Chromatin Condensation ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tunel Staining ◽  
Wild Type ◽  
Nuclear Fragmentation ◽  
A Cell

Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN−/−) mice were treated in vitro with H2O2to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN−/−cells but was increased to only 20% in WT cells. In contrast, after 1–8 h of treatment with H2O2, the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN−/−cells. Electron microscopy of WT cells treated with H2O2showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H2O2-treated OPN−/−cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN−/−and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN−/−cells by ∼30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN−/−cells was not altered. Restoration of OPN expression in OPN−/−fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H2O2treatment. Thus H2O2-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.

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