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.

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.

Carbonic anhydrase II promotes cardiomyocyte hypertrophy

2012 ◽  
Vol 90 (12) ◽  
pp. 1599-1610 ◽  
Author(s):  
Brittany F. Brown ◽  
Anita Quon ◽  
Jason R.B. Dyck ◽  
Joseph R. Casey
Keyword(s):  
Carbonic Anhydrase ◽  
Ventricular Myocytes ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Carbonic Anhydrase Ii ◽  
Cardiomyocyte Hypertrophy ◽  
Wild Type ◽  
Over Expression ◽  
Pathological Cardiac Hypertrophy ◽  
Neonatal Rat Ventricular Myocytes

Pathological cardiac hypertrophy, the maladaptive remodelling of the myocardium, often progresses to heart failure. The sodium–proton exchanger (NHE1) and chloride–bicarbonate exchanger (AE3) have been implicated as important in the hypertrophic cascade. Carbonic anhydrase II (CAII) provides substrates for these transporters (protons and bicarbonate, respectively). CAII physically interacts with NHE1 and AE3, enhancing their respective ion transport activities by increasing the concentration of substrate at their transport sites. Earlier studies found that a broad-spectrum carbonic anhydrase inhibitor prevented cardiomyocyte hypertrophy (CH), suggesting that carbonic anhydrase is important in the development of hypertrophy. Here we investigated whether cytosolic CAII was the CA isoform involved in hypertrophy. Neonatal rat ventricular myocytes (NRVMs) were transduced with recombinant adenoviral constructs to over-express wild-type or catalytically inactive CAII (CAII-V143Y). Over-expression of wild-type CAII in NRVMs did not affect CH development. In contrast, CAII-V143Y over-expression suppressed the response to hypertrophic stimuli, suggesting that CAII-V143Y behaves in a dominant negative fashion over endogenous CAII to suppress hypertrophy. We also examined CAII-deficient (Car2) mice, whose hearts exhibit physiological hypertrophy without any decrease in cardiac function. Moreover, cardiomyocytes from Car2 mice do not respond to prohypertrophic stimulation. Together, these findings support a role of CAII in promoting CH.

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.

Synthesis and Evaluation of 1-hydroxybenzotriazole Derivatives: Dual Inhibitors of Carbonic Anhydrase II and Sodium Hydrogen Exchanger I

2020 ◽  
Vol 17 ◽  
Author(s):  
Dhandeep Singh ◽  
Nirmal Singh
Keyword(s):  
Carbonic Anhydrase ◽  
Reperfusion Injury ◽  
Intracellular Ph ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Carbonic Anhydrase Ii ◽  
Dual Inhibitor ◽  
Sodium Hydrogen ◽  
Sodium Hydrogen Exchanger ◽  
Dual Inhibitors

: Ischemia reperfusion injury is responsible for impaired graft functioning in organ transplants, cerebral dysfunction, ischemic heart diseases, systemic inflammatory response syndrome, gastrointestinal dysfunction, and multiple organ dysfunction syndromes. Intracellular pH is critical for cell survival in ischemia reperfusion injury. Sodium hydrogen exchanger I and carbonic anhydrase II are critical in regulation of intracellular pH. Inhibition of sodium hydrogen exchanger I and carbonic anhydrase II during reprfusion is found to ameliorate ischemia reperfusion injury separately. An attempt is made to synthesize dual inhibitors of sodium hydrogen exchanger and carbonic anhydrase to have better potential drug molecule in ischemia reperfusion injury treatment. The hydroxybenzotriazole is considered as a central pharmacophore for this dual activity and 12 derivatives are synthesized. All derivatives are tested for sodium hydrogen exchanger I and carbonic anhydrase II inhibitory activity. The tosylate derivative (12) is found to be the most potent derivative with IC50 158.7± 8.4 µM for carbonic anhydrase II and 31.07 ± 1.06 µM for sodium hydrogen exchanger I. Although the potency is less than standard drugs but this is the first report of dual inhibitor of carbonic anhydrase II and sodium hydrogen exchanger.

scholarly journals Kidney Proximal Tubule Lipoapoptosis Is Regulated by Fatty Acid Transporter-2 (FATP2)

2017 ◽  
Vol 29 (1) ◽  
pp. 81-91 ◽  
Author(s):  
Shenaz Khan ◽  
Pablo D. Cabral ◽  
William P. Schilling ◽  
Zachary W. Schmidt ◽  
Asif N. Uddin ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Epithelial Cells ◽  
Proximal Tubule ◽  
Proximal Tubules ◽  
Proximal Tubule Cell ◽  
Apical Surface ◽  
Wild Type ◽  
Ckd Progression ◽  
Fatty Acid Transporter ◽  
Acid Transporter

Albuminuria and tubular atrophy are among the highest risks for CKD progression to ESRD. A parsimonious mechanism involves leakage of albumin-bound nonesterified fatty acids (NEFAs) across the damaged glomerular filtration barrier and subsequent reabsorption by the downstream proximal tubule, causing lipoapoptosis. We sought to identify the apical proximal tubule transporter that mediates NEFA uptake and cytotoxicity. We observed transporter-mediated uptake of fluorescently labeled NEFA in cultured proximal tubule cells and microperfused rat proximal tubules, with greater uptake from the apical surface than from the basolateral surface. Protein and mRNA expression analyses revealed that kidney proximal tubules express transmembrane fatty acid transporter-2 (FATP2), encoded by Slc27a2, but not the other candidate transporters CD36 and free fatty acid receptor 1. Kidney FATP2 localized exclusively to proximal tubule epithelial cells along the apical but not the basolateral membrane. Treatment of mice with lipidated albumin to induce proteinuria caused a decrease in the proportion of tubular epithelial cells and an increase in the proportion of interstitial space in kidneys from wild-type but not Slc27a2−/−mice. Ex vivo microperfusion and in vitro experiments with NEFA-bound albumin at concentrations that mimic apical proximal tubule exposure during glomerular injury revealed significantly reduced NEFA uptake and palmitate-induced apoptosis in microperfused Slc27a2−/− proximal tubules and Slc27a2−/− or FATP2 shRNA-treated proximal tubule cell lines compared with wild-type or scrambled oligonucleotide–treated cells, respectively. We conclude that FATP2 is a major apical proximal tubule NEFA transporter that regulates lipoapoptosis and may be an amenable target for the prevention of CKD progression.

Regulation of the human NBC3 Na+/HCO3− cotransporter by carbonic anhydrase II and PKA

2004 ◽  
Vol 286 (6) ◽  
pp. C1423-C1433 ◽  
Author(s):  
Frederick B. Loiselle ◽  
Patricio E. Morgan ◽  
Bernardo V. Alvarez ◽  
Joseph R. Casey
Keyword(s):  
Carbonic Anhydrase ◽  
Recovery Rate ◽  
Dominant Negative ◽  
Carbonic Anhydrase Ii ◽  
Transport Activity ◽  
Wild Type ◽  
Hek 293 ◽  
Transfected Cells ◽  
293 Cells ◽  
Optimal Function

Human NBC3 is an electroneutral Na+/HCO3− cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO3− metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO2 + H2O ⇆ HCO3− + H+ in many tissues. We investigated whether NBC3, like some Cl−/HCO3− exchange proteins, could bind CAII and whether PKA could regulate NBC3 activity through modulation of CAII binding. CAII bound the COOH-terminal domain of NBC3 (NBC3Ct) with Kd = 101 nM; the interaction was stronger at acid pH. Cotransfection of HEK-293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAII binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for optimal function, because the NBC3 D1135N/D1136N retained only 29 ± 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 μM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pHi) recovery rate by 31 ± 3, or 38 ± 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pHi recovery rate by 69 ± 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [γ-32P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO3− transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct.

Carbonic anhydrase II/sodium-proton exchanger 1 metabolon complex in cardiomyopathy of ob type 2 diabetic mice

2019 ◽  
Vol 136 ◽  
pp. 53-63 ◽  
Author(s):  
Carolina Jaquenod De Giusti ◽  
Paula G. Blanco ◽  
Paula A. Lamas ◽  
Fernanda Carrizo Velasquez ◽  
Juan M. Lofeudo ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Carbonic Anhydrase Ii ◽  
Diabetic Mice ◽  
Sodium Proton Exchanger ◽  
Type 2 Diabetic

scholarly journals Increased water flux induced by an aquaporin-1/carbonic anhydrase II interaction

2015 ◽  
Vol 26 (6) ◽  
pp. 1106-1118 ◽  
Author(s):  
Gonzalo Vilas ◽  
Devishree Krishnan ◽  
Sampath Kumar Loganathan ◽  
Darpan Malhotra ◽  
Lei Liu ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Close Association ◽  
Water Flux ◽  
Membrane Vesicles ◽  
Carbonic Anhydrase Ii ◽  
Physical Interaction ◽  
Aquaporin 1 ◽  
Amino Terminal

Aquaporin-1 (AQP1) enables greatly enhanced water flux across plasma membranes. The cytosolic carboxy terminus of AQP1 has two acidic motifs homologous to known carbonic anhydrase II (CAII) binding sequences. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII in Xenopus oocytes or mammalian cells increased water flux relative to AQP1 expression alone. This required the amino-terminal sequence of CAII, a region that binds other transport proteins. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring the second acidic cluster of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins.

scholarly journals Association of CD47 with Integrin Mac-1 (αMβ2, CD11b/CD18) Regulates Macrophage Responses

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1109-1109
Author(s):  
Nataly Podolnikova ◽  
Arnat Balabiyev ◽  
Tatiana P. Ugarova
Keyword(s):  
Conflicts Of Interest ◽  
Integrin Αvβ3 ◽  
The Body ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Hek293 Cells ◽  
Proximity Ligation Assay ◽  
Physical Interaction ◽  
Wild Type ◽  
Surface Receptor

Abstract CD47 is a cell surface receptor, which is expressed by virtually all cells in the body, including immune cells. CD47 has originally been identified as an integrin-associated protein (IAP) and shown to associate with several integrins that belong to the β1 and β3 subfamilies. In addition, association of CD47 with a member of the β2 subfamily, integrin αLβ2, has also been reported. In neutrophils, CD47 mediates a number of integrin αvβ3-dependent functions, including adhesion, migration and phagocytosis. Surprisingly, the association of CD47 with integrin αMβ2 (Mac-1, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, has not been documented. Furthermore, while the major focus of recent studies was the mechanism by which CD47 on various host cells prevents phagocytosis by macrophages, the question as to how CD47 expressed on the surface of macrophages influences the responses of these cells has not been addressed. In the present study, we demonstrated that an association of CD47 with Mac-1 regulates Mac-1-dependent macrophage functions. In particular, adhesion of macrophages isolated from CD47-/- mice to fibrinogen and ICAM-1, the established physiological ligands of Mac-1 was significantly decreased compared to wild-type counterparts. In addition, spreading of CD47-deficient macrophages was decreased by four- and two-fold on fibrinogen and ICAM-1, respectively. Compared to wild-type macrophages, migration of CD47-deficient macrophages to the Mac-1 ligand, cathelicidin peptide LL-37 was significantly reduced. The lack of CD47 on the surface of macrophages impaired their ability to fuse in the presence of IL-4. Finally, the deficiency of CD47 also reduced phagocytosis of opsonized latex beads, a process fully dependent on Mac-1. The functional association of CD47 with Mac-1 implied that similar to other integrins, Mac-1 might form a complex with CD47. Indeed, co-immunoprecipitation experiments using peritoneal mouse macrophages, the IC21 murine macrophage cell line and Mac-1-expressing HEK293 cells revealed that Mac-1 forms a complex with CD47. The cis interaction between Mac-1 and CD47 was also detected using the proximity ligation assay. Together, these results indicate that Mac-1 forms a lateral complex with CD47, which regulates important macrophage functions. Studies to determine the structural requirements for the physical interaction between Mac-1 and CD47 are in progress. Disclosures No relevant conflicts of interest to declare.

H(+)-K(+)-ATPase and carbonic anhydrase II gene expression in the developing rat fundus

1990 ◽  
Vol 259 (1) ◽  
pp. G108-G115 ◽  
Author(s):  
L. R. Marino ◽  
B. H. Muglia ◽  
T. Yamada
Keyword(s):  
Carbonic Anhydrase ◽  
Intracellular Ph ◽  
Cellular Proliferation ◽  
Regional Distribution ◽  
Carbonic Anhydrase Ii ◽  
Mrna Levels ◽  
Gastric Parietal Cell ◽  
Mrna Content ◽  
Developing Rat ◽  
Secretory Capacity

H(+)-K(+)-ATPase and carbonic anhydrase II (CA II) are two enzymes that are involved in the production and secretion of the hydrogen ion by the gastric parietal cell and maintenance of intracellular pH therein. The present studies were undertaken to examine whether H(+)-K(+)-ATPase and CA II expression change in the rat fundus in association with the development of acid secretory capacity. Changes in enzyme mRNA content in the gastric fundus of developing rat pups 1-6 wk of age were evaluated using dot blots and ribonuclease protection assays. In additional studies the localization of H(+)-K(+)-ATPase and carbonic anhydrase II mRNA was examined by in situ hybridization in Formalin-fixed gastric tissues from rats 1, 3, 6, and 8 wk of age. We observed that H(+)-K(+)-ATPase mRNA content increased with age in the developing rat fundus while CA II mRNA exhibited a reciprocal decrease. These changes in enzyme mRNA were accompanied by concomitant changes in the regional distribution of the cells expressing the genes for the two enzymes. Although the changes in H(+)-K(+)-ATPase mRNA paralleled the development of acid secretory capacity, CA II mRNA levels might be regulated by the requirement for maintenance of intracellular pH during periods of cellular proliferation and by exposure of the gastric surface epithelium to the highly acidic luminal environment of the stomach.

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