Membrane-associated Carbonic Anhydrase Research Articles

Differential carbonic anhydrase activities control EBV-induced B-cell transformation and lytic cycle reactivation.

Epstein-Barr virus (EBV) contributes to ~1% of all human cancers including several B-cell neoplasms. A characteristic feature of EBV life cycle is its ability to transform metabolically quiescent B-lymphocytes into hyperproliferating B-cell blasts with the establishment of viral latency, while intermittent lytic cycle induction is necessary for the production of progeny virus. Our RNA-Seq analyses of both latently infected naïve B-lymphocytes and transformed B-lymphocytes upon lytic cycle replication indicate a contrasting expression pattern of a membrane-associated carbonic anhydrase isoform CA9, an essential component for maintaining cell acid-base homeostasis. We show that while CA9 expression is transcriptionally activated during latent infection model, lytic cycle replication restrains its expression. Pharmacological inhibition of CA-activity using specific inhibitors retards EBV induced B-cell transformation, inhibits B-cells outgrowth and colony formation ability of transformed B-lymphocytes through lowering the intracellular pH, induction of cell apoptosis and facilitating degradation of CA9 transcripts. Reanalyses of ChIP-Seq data along with utilization of EBNA2 knockout virus, ectopic expression of EBNA2 and sh-RNA mediated knockdown of CA9 expression we further demonstrate that EBNA2 mediated CA9 transcriptional activation is essential for EBV latently infected B-cell survival. In contrast, during lytic cycle reactivation CA9 expression is transcriptionally suppressed by the key EBV lytic cycle transactivator, BZLF1 through its transactivation domain. Overall, our study highlights the dynamic alterations of CA9 expression and its activity in regulating pH homeostasis act as one of the major drivers for EBV induced B-cell transformation and subsequent B-cell lymphomagenesis.

Potential Novel Role of Membrane-Associated Carbonic Anhydrases in the Kidney.

Carbonic anhydrases (CAs), because they catalyze the interconversion of carbon dioxide (CO2) and water into bicarbonate (HCO3-) and protons (H+), thereby influencing pH, are near the core of virtually all physiological processes in the body. In the kidneys, soluble and membrane-associated CAs and their synergy with acid-base transporters play important roles in urinary acid secretion, the largest component of which is the reabsorption of HCO3- in specific nephron segments. Among these transporters are the Na+-coupled HCO3- transporters (NCBTs) and the Cl--HCO3- exchangers (AEs)-members of the "solute-linked carrier" 4 (SLC4) family. All of these transporters have traditionally been regarded as "HCO3-" transporters. However, recently our group has demonstrated that two of the NCBTs carry CO32- rather than HCO3- and has hypothesized that all NCBTs follow suit. In this review, we examine current knowledge on the role of CAs and "HCO3-" transporters of the SLC4 family in renal acid-base physiology and discuss how our recent findings impact renal acid secretion, including HCO3- reabsorption. Traditionally, investigators have associated CAs with producing or consuming solutes (CO2, HCO3-, and H+) and thus ensuring their efficient transport across cell membranes. In the case of CO32- transport by NCBTs, however, we hypothesize that the role of membrane-associated CAs is not the appreciable production or consumption of substrates but the minimization of pH changes in nanodomains near the membrane.

Blood and Gill Carbonic Anhydrase in the Context of a Chondrichthyan Model of CO2 Excretion.

Pacific spiny dogfish (Squalus suckleyi) have been widely used as a representative species for chondrichthyan CO2 excretion. Pacific spiny dogfish have a slower red blood cell (RBC) carbonic anhydrase (CA) isoform than teleost fishes, extracellular CA activity, no endogenous plasma CA inhibitor, and plasma-accessible CA IV at the gills. Thus, both the RBC and plasma compartments contribute to bicarbonate ion () dehydration at the gills for CO2 excretion in contrast to teleost fishes, in which dehydration is restricted to RBCs. We compared CA activity levels, subcellular localization, and presence of plasma CA inhibitors in the blood and gills of 13 chondrichthyans to examine the hypothesis that the dogfish model of CO2 excretion applies broadly to chondrichthyans. In general, blood samples from the 12 other chondrichthyans examined had lower RBC CA activity than teleosts, some extracellular CA activity, and no endogenous plasma CA inhibitor. While type IV-like membrane-associated CA was found in the gills in all four of the chondrichthyans examined, S. suckleyi had three times more CA activity ( μmol CO2 min-1 mg protein-1) in the microsomal (membrane) fraction of gills than the other three. In addition, unexpected variation in CA characteristics was observed between chondrichthyan species. Thus, in general, it appears that the pattern of CA distribution in fishes can be generally categorized as either chondrichthyan or teleost models. However, further studies should examine the functional significance of the within-chondrichthyan differences we observed and investigate whether CO2 excretion patterns exist along a continuum or in discrete groups.

Intrinsic thermodynamics of high affinity inhibitor binding to recombinant human carbonic anhydrase IV.

Membrane-associated carbonic anhydrase (CA) isoform IV participates in carbon metabolism and pH homeostasis and is implicated in the development of eye diseases such as retinitis pigmentosa and glaucoma. A series of substituted benzenesulfonamides were designed and their binding affinity to CA IV was determined by fluorescent thermal shift assay and isothermal titration calorimetry (ITC). Compound [(4-chloro-2-phenylsulfanyl-5-sulfamoyl-benzoyl)amino]propyl acetate (19) bound CA IV with the K d of 1.0nM and exhibited significant selectivity over the remaining 11 human CA isoforms. The compound could be developed as a drug targeting CA IV. Various forms of recombinant CA IV were produced in Escherichia coli and mammalian cell cultures. Comparison of their temperature stability in various buffers and salt solutions demonstrated that CA IV is most stable at slightly alkaline conditions and at elevated sodium sulfate concentrations. High-resolution X-ray crystallographic structures of ortho-Cl and meta-thiazole-substituted benzene sulfonamide in complex with CA IV revealed the position of and interactions between the ligand and the protein. Sulfonamide inhibitor binding to CA IV is linked to several reactions-the deprotonation of the sulfonamide amino group, the protonation of CA-Zn(II)-bound hydroxide at the active site of CA IV, and the compensating reactions of the buffer. The dissection of binding-linked reactions yielded the intrinsic thermodynamic parameters, characterizing the interaction between CA IV and the sulfonamides in the binding-able protonation forms, including Gibbs energy, enthalpy, and entropy, that could be used for the characterization of binding to any CA in the process of drug design.

Bicarbonate Recycling by HIF-1-Dependent Carbonic Anhydrase Isoforms 9 and 12 Is Critical in Maintaining Intracellular pH and Viability of Nucleus Pulposus Cells.

Intervertebral disc degeneration is a ubiquitous condition closely linked to chronic low-back pain. The health of the avascular nucleus pulposus (NP) plays a crucial role in the development of this pathology. We tested the hypothesis that a network comprising HIF-1α, carbonic anhydrase (CA) 9 and 12 isoforms, and sodium-coupled bicarbonate cotransporters (NBCs) buffer intracellular pH through coordinated bicarbonate recycling. Contrary to the current understanding of NP cell metabolism, analysis of metabolic-flux data from Seahorse XF analyzer showed that CO2 hydration contributes a significant source of extracellular proton production in NP cells, with a smaller input from glycolysis. Because enzymatic hydration of CO2 is catalyzed by plasma membrane-associated CAs we measured their expression and function in NP tissue. NP cells robustly expressed isoforms CA9/12, which were hypoxia-inducible. In addition to increased mRNA stability under hypoxia, we observed binding of HIF-1α to select hypoxia-responsive elements on CA9/12 promoters using genomic chromatin immunoprecipitation. Importantly, in vitro loss of function studies and analysis of discs from NP-specific HIF-1α null mice confirmed the dependency of CA9/12 expression on HIF-1α. As expected, inhibition of CA activity decreased extracellular acidification rate independent of changes in HIF activity or lactate/H+ efflux. Surprisingly, CA inhibition resulted in a concomitant decrease in intracellular pH that was mirrored by inhibition of sodium-bicarbonate importers. These results suggested that extracellular bicarbonate generated by CA9/12 is recycled to buffer cytosolic pH fluctuations. Importantly, long-term intracellular acidification from CA inhibition lead to compromised cell viability, suggesting that plasma-membrane proton extrusion pathways alone are not sufficient to maintain homeostatic pH in NP cells. Taken together, our studies show for the first time that bicarbonate buffering through the HIF-1α-CA axis is critical for NP cell survival in the hypoxic niche of the intervertebral disc. © 2017 American Society for Bone and Mineral Research.

N-Sulfamoylphenyl- and N-sulfamoylphenyl-N-thiazolyl-β-alanines and their derivatives as inhibitors of human carbonic anhydrases

A series of N-substituted and N,N-disubstituted β-amino acids and their derivatives bearing benzenesulfonamide moiety were designed and synthesized in search of compounds that would be high-affinity and selective inhibitors of human carbonic anhydrases (CA). There are 12 catalytically active human CA isoforms, the cytosolic CA I, CA II, CA III, CA VII, and CA XIII, secreted CA VI, the mitochondrial CA VA and CA VB, membrane-associated CA IV, and transmembrane CA IX, CA XII, and CA XIV. The di-bromo meta-substituted compounds exhibited low nanomolar dissociation constants and over 10-fold selectivity for mitochondrial isozyme CA VB, implicated in diseases of the central nervous system and obesity. These compounds can be used for further development as inhibitors of significant binding affinity and selectivity towards CA VB isozyme.

Design, Synthesis, Molecular Docking Analysis, and Carbonic Anhydrase IX Inhibitory Evaluations of Novel N-Substituted-β-d-Glucosamine Derivatives that Incorporate Benzenesulfonamides.

A series of novel N-substituted-β-d-glucosamine derivatives that incorporate benzenesulfonamides were designed using a fragment-based drug design strategy. Each derivative was synthesized and evaluated in vitro for its inhibitory activity against human carbonic anhydrase (hCA) IX; several derivatives displayed desirable potency profiles against this enzyme. The molecular docking studies provided the design rationale and predicted potential binding modes for carbonic anhydrase (CA) IX and three target compounds, including the most potent inhibitor, compound 7f (IC50 = 10.01 nM). Moreover, the calculated Log P (cLog P) values showed that all the compounds tended to be hydrophilic. In addition, topological polar surface area (TPSA) value-based predictions highlighted the selectivity of these carbohydrate-based inhibitors for membrane-associated CA IX.

Amorphous polyphosphate/amorphous calcium carbonate implant material with enhanced bone healing efficacy in a critical-size defect in rats

In this study the effect of amorphous calcium carbonate (ACC) microparticles and amorphous calcium polyphosphate (polyP) microparticles (termed aCa-polyP-MP) on bone mineral forming cells/tissue was investigated in vitro and in vivo. The ACC particles (termed ACC-P10-MP) were prepared in the presence of Na-polyP. Only the combinations of polyP and ACC microparticles enhanced the proliferation rate of human mesenchymal stem cells (MSCs). Gene expression studies revealed that ACC causes an upregulation of the expression of the cell membrane-associated carbonic anhydrase IX (CA IX; formation of ACC), while the transcript level of the alkaline phosphatase (ALP; liberation of orthophosphate from polyP) changes only relatively little. In contrast, aCa-polyP-MP primarily induces ALP expression. If both components are applied together a strong stimulation of expression of both marker genes is observed. In order to investigate whether ACC also enhances bone regeneration induced by polyP in vivo, the particles were encapsulated into PLGA (poly(d,l-lactide-co-glycolide)) microspheres (diameter ~800 μm) and implanted into rat critical-size calvarial defects. The studies revealed that animals that received aCa-polyP-MP microspheres showed an increased rate of regeneration compared to β-tri-calcium phosphate (β-TCP) controls. This effect is even accelerated if microspheres with both aCa-polyP-MP and ACC-P10-MP (1 : 1 weight ratio) are applied, resulting in an almost complete restoration of the defect area after 12 weeks. qRT-PCR analyses of tissue sections through the regeneration zone with microspheres containing both aCa-polyP-MP and ACC-P10-MP revealed a significantly higher upregulation of expression of the marker genes compared to each of the components alone. The Young’s moduli for microspheres containing aCa-polyP-MP (1.74 MPa) and aCa-polyP-MP/ACC-P10-MP (2.38 MPa) were markedly higher compared to β-TCP-controls (0.63 mPa). Our results show that the combined application of ACC and Ca-polyP (both in the amorphous state) opens new strategies for the development of regenerative implants for the reconstruction of bone defects.

Carbonic Anhydrase IX Promotes the Tumorigenicity of Adult T-Cell Leukemia/Lymphoma-Derived Cells in NOG Mice

Carbonic anhydrase IX (CA9) is a membrane-associated carbonic anhydrase that regulates pH through its extra-cellular catalytic domain and thereby fosters tumor cell survival, growth, and invasion. The expression of CA9 is induced by hypoxia via HIF-1α. CA9 overexpression is known to be closely associated with aggressive behaviors and poor prognoses in various types of human solid tumors. In particular, CA9 is a critical mediator of the expansion of breast cancer stem cells in hypoxic niches by sustaining the mesenchymal and 'stemness' phenotypes of these cells. However, as for hematological malignancies, there are few reports suggesting a relation between its tumorigenecity and CA9. Here we demonstrated that CA9 plays a crucial role in promoting the tumorigenicity of the ATL-derived cells.Recently we established a highly tumorigenic subline, ST1-N6, from an ATL-derived cell line, ST1, by serial transplantation in immunodeficient NOG mice. ST1-N6 cells developed subcutaneous tumors under a shorter duration and smaller cell number than parental ST1 cells. DNA microarray gene expression analysis revealed an upregulated expression of CA9 in ST1-N6 cells as compared with ST1 cells. After serial sorting of ST1 cells by CA9 expression, we obtained ST1-CA9high and ST1-CA9low sublines. Like ST1-N6 cells, ST1-CA9high cells showed stronger tumorigenicity than ST1-CA9low or parental ST1 cells. Knockdown of CA9 expression with shRNAs suppressed the tumorigenicity of ST1-CA9high cells, whereas ST1 cells introduced with CA9 significantly showed enhanced tumorigenicity. Another ATL-derived cell line, TL-Om1 also showed increased tumorigenicity in NOG mice when introduced with CA9. These observations indicate that CA9 contributes to promoting the tumorigenic potential of ATL-derived cells in xenotransplantation.To investigate CA9 expression on fresh ATL cells, we prepared peripheral blood from six acute ATL patients, and tested for quantitative expression of CA9 in sera of six ATL patients and three healthy donors by ELISA. The amounts of serum CA9 were 31.1~95.4 pg/ml in the healthy donors, while they were 175.6~1, 971.6 pg/ml in the ATL patients. We next prepared ATL tissues from nine ATL patients, and two of them contained a few CA9high cells, which were cells expressing CD25, suggesting that a part of ATL lymphoma cells expressed CA9. These results suggested that the amount of serum CA9 tends to be higher in ATL patients than healthy donors.Based on these findings, we suppose that CA9 plays an important role in the malignant development of ATL. DisclosuresNo relevant conflicts of interest to declare.

Mineralization of bone-related SaOS-2 cells under physiological hypoxic conditions.

Inorganic polyphosphate (polyP) is a physiological energy-rich polymer with multiple phosphoric anhydride bonds. In cells such as bone-forming osteoblasts, glycolysis is the main pathway generating metabolic energy in the form of ATP. In the present study, we show that, under hypoxic culture conditions, the growth/viability of osteoblast-like SaOS-2 cells is not impaired. The addition of polyP to those cells, administered as amorphous calcium polyP nanoparticles (aCa-polyP-NP; approximate size 100 nm), significantly increased the proliferation of the cells. In the presence of polyP, the cells produce significant levels of lactate, the end product of anaerobic glycolysis. Under those conditions, an eight-fold increase in the steady-state level of the membrane-associated carbonic anhydrase IX is found, as well as a six-fold induction of the hypoxia-inducible factor 1. Consequently, biomineral formation onto the SaOS-2 cells decreases under low oxygen tension. If the polyP nanoparticles are added to the cells, the degree of mineralization is enhanced. These changes had been measured also in human mesenchymal stem cells. The assumption that the bicarbonate, generated by the carbonic anhydrase in the presence of polyP under low oxygen, is deposited as a constituent of the bioseeds formed during initial hydroxyapatite formation is corroborated by the identification of carbon besides of calcium, oxygen and phosphorus in the initial biomineral deposit onto the cells using the sensitive technology of high-resolution energy dispersive spectrometry mapping. Based on these data, we conclude that polyP is required for the supply of metabolic energy during bone mineral formation under physiological, hypoxic conditions, acting as a 'metabolic fuel' for the cells to grow.

Catalysis and pH Control by Membrane-associated Carbonic Anhydrase IX in MDA-MB-231 Breast Cancer Cells

Carbonic anhydrase IX (CAIX) is a membrane-bound, tumor-related enzyme whose expression is often considered a marker for hypoxia, an indicator of poor prognosis in the majority of cancer patients, and is associated with acidification of the tumor microenvironment. Here, we describe for the first time the catalytic properties of native CAIX in MDA-MB-231 breast cancer cells that exhibit hypoxia-inducible CAIX expression. Using (18)O exchange measured by membrane inlet mass spectrometry, we determined catalytic activity in membrane ghosts and intact cells. Exofacial carbonic anhydrase activity increases with exposure to hypoxia, an activity which is suppressed by impermeant sulfonamide CA inhibitors. Inhibition by sulfonamide inhibitors is not sensitive to reoxygenation. CAIX activity in intact cells increases in response to reduced pH. Data from membrane ghosts show that the increase in activity at reduced pH is largely due to an increase in the dehydration reaction. In addition, the kinetic constants of CAIX in membrane ghosts are very similar to our previous measurements for purified, recombinant, truncated forms. Hence, the activity of CAIX is not affected by the proteoglycan extension or membrane environment. These activities were measured at a total concentration for all CO(2) species at 25 mm and close to chemical equilibrium, conditions which approximate the physiological extracellular environment. Our data suggest that CAIX is particularly well suited to maintain the extracellular pH at a value that favors the survival fitness of tumor cells.

TGF‐β upregulates tumor‐associated carbonic anhydrase IX gene expression in Hep3B cells

Carbonic anhydrase IX (CAIX) is a membrane-associated carbonic anhydrase (CA) that is overexpressed in a variety of tumor types and associated with increased metastasis, giving a poor prognosis. Transcriptional regulation of transmembrane protein CAIX is complex. We describe further characterization of the 1.2kb hCA9 promoter, and the effect of TGF-beta on the transcriptional activity and expression of hCAIX in Hep3B cells. Transcriptional activity of different promoter regions of hCA9 promoter showed the presence of negative regulatory region between -300bp and -500bp of hCAIX promoter. The -116/+38 region was enough for basal transcriptional activity in Hep3B cells. TGF-beta upregulates all promoter regions of hCA9 with the highest beig for -466/+38 that has a negatively regulated region. The transcriptional activation of hCA9 promoter by TGF-beta is consistent with hCAIX mRNA levels revealed by RT-PCR and hCAIX protein expression levels by flow cytometry in Hep3B cells.

Carbonic anhydrase activators. Activation of the membrane-associated isoform XV with amino acids and amines

An activation study of the membrane-associated carbonic anhydrase (CA, EC 4.2.1.1) isoform XV with a series of natural and non-natural amino acids and aromatic/heterocyclic amines is reported. Murine CA XV was strongly activated by some amino acids ( d-Phe, l-/ d-DOPA, d-Trp, l-Tyr) and amines (dopamine, serotonin, l-adrenaline and 4-(2-aminoethyl)-morpholine) with activation constants in the range of 4.0–9.5 μM. l-/ d-His, l-Phe, histamine and several other heterocyclic amines showed less efficient activation ( K As in the range of 11.6–33.4 μM). The activation profile of CA XV is quite different from that of the cytosolic isoforms CA I and II or the membrane-associated CA IV. All mammalian isoforms CA I–XV are thus characterized for their interaction with this set of amino acid and amine activators, some of which are biogenic amines or neurotransmitters present in sufficiently high amounts in various tissues for exerting significant biologic responses.

Membrane-associated carbonic anhydrase in the respiratory system of the Pacific hagfish (Eptatretus stouti)

Like other agnathans, the Pacific hagfish (Eptatretus stouti) lacks red blood cell (RBC) Cl(-)/HCO(3)(-) exchange. Despite this absence of anion exchange, the majority (86.7+/-1.4%) of the total CO(2) carried in the blood is found within the plasma as HCO(3)(-), and thus presumably is inaccessible to RBC carbonic anhydrase (CA). As such, a branchial plasma-accessible CA isozyme in hagfish would be beneficial for mobilizing the considerable plasma HCO(3)(-) stores for CO(2) excretion and blood acid-base balance. The current study used a combination of molecular and biochemical methods to identify two membrane-associated CA isozymes in the respiratory system of E. stouti. Using homology cloning methods, CA IV and XV-like isozymes were identified in the gill and RBC, respectively. Real-time PCR analysis of relative mRNA expression revealed that CA IV was specific to the gill, while CA XV was found in several tissues including the RBC, gill, liver, heart and muscle. Isolation of subcellular fractions of gill and RBC verified the presence of membrane-associated CA activity in each tissue that persisted after standard washing protocols. Unlike CA activity associated with the cytoplasmic fractions, the activity in gill membranes was not inhibited by sodium dodecyl sulphate, while RBC membrane activity was inhibited to a lesser degree than the cytoplasmic fraction. Additionally, incubation of gill membrane fractions with phosphatidylinositol-specific phospholipase C released significant CA activity into the supernatant indicating the presence of a glycophosphatidyl inositol-linkage to the membrane, as found with other CA IV and XV isozymes. These results demonstrate that Pacific hagfish possess gill and RBC plasma-accessible membrane-associated CA that may play important roles in respiratory gas exchange and acid-base balance.

Carbonic anhydrase inhibitors: 2-Substituted-1,3,4-thiadiazole-5-sulfamides act as powerful and selective inhibitors of the mitochondrial isozymes VA and VB over the cytosolic and membrane-associated carbonic anhydrases I, II and IV

A series of 2-substituted-1,3,4-thiadiazole-5-sulfamides was prepared and assayed as inhibitors of several carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, the membrane-associated CA IV and the mitochondrial CA VA and VB. The new compounds showed weak inhibitory activity against hCA I ( K Is of 102 nM–7.42 μM), hCA II ( K Is of 0.54–7.42 μM) and hCA IV ( K Is of 4.32–10.05 μM) but were low nanomolar inhibitors of hCA VA and hCA VB, with inhibition constants in the range of 4.2–32 nM and 1.3–74 nM, respectively. Furthermore, the selectivity ratios for inhibiting the mitochondrial enzymes over CA II were in the range of 67.5–415, making these sulfamides the first selective CA VA/VB inhibitors.

Carbonic anhydrases IV and IX: subcellular localization and functional role in mouse skeletal muscle

The subcellular localization of carbonic anhydrase (CA) IV and CA IX in mouse skeletal muscle fibers has been studied immunohistochemically by confocal laser scanning microscopy. CA IV has been found to be located on the plasma membrane as well as on the sarcoplasmic reticulum (SR) membrane. CA IX is not localized in the plasma membrane but in the region of the t-tubular (TT)/terminal SR membrane. CA IV contributes 20% and CA IX 60% to the total CA activity of SR membrane vesicles isolated from mouse skeletal muscles. Our aim was to examine whether SR CA IV and TT/SR CA IX affect muscle contraction. Isolated fiber bundles of fast-twitch extensor digitorum longus and slow-twitch soleus muscle from mouse were investigated for isometric twitch and tetanic contractions and by a fatigue test. The muscle functions of CA IV knockout (KO) fibers and of CA IX KO fibers do not differ from the function of wild-type (WT) fibers. Muscle function of CA IV/XIV double KO mice unexpectedly shows a decrease in rise and relaxation time and in force of single twitches. In contrast, the CA inhibitor dorzolamide, whether applied to WT or to double KO muscle fibers, leads to a significant increase in rise time and force of twitches. It is concluded that the function of mouse skeletal muscle fibers expressing three membrane-associated CAs, IV, IX, and XIV, is not affected by the lack of one isoform but is possibly affected by the lack of all three CAs, as indicated by the inhibition studies.

Carbonic anhydrase XIV in skeletal muscle: subcellular localization and function from wild-type and knockout mice

The expression of carbonic anhydrase (CA) XIV was investigated in mouse skeletal muscles. Sarcoplasmic reticulum (SR) and sarcolemmal (SL) membrane fractions were isolated from wild-type (WT) and CA XIV knockout (KO) mice. The CA XIV protein of 54 kDa was present in SR and SL membrane fractions as shown by Western blot analysis. CA activity measurements of WT and KO membrane fractions showed that CA XIV accounts for approximately 50% and 66% of the total CA activities determined in the SR and SL fractions, respectively. This indicates the presence of at least one other membrane-associated CA isoform in these membranes, e.g., CA IV, CA IX, or CA XII. Muscle fibers of the extensor digitorum longus (EDL) muscle were immunostained with anti-CA XIV/FITC and anti-sarco(endo)plasmic reticulum Ca(2+)-ATPase 1/TRITC, with anti-CA XIV/FITC and anti-ryanodine receptor/TRITC, or with anti-CA XIV/FITC and anti-monocarboxylate transporter-4/TRITC. CA XIV was expressed in the plasma membrane and in the longitudinal SR but not in the terminal SR. Isometric contraction measurements of single twitches and tetani and a fatigue protocol applied to fiber bundles of the fast-twitch EDL and of the slow-twitch soleus muscle from WT and KO mice showed that the lack of SR membrane-associated CA XIV did not affect maximum force, rise and relaxation times, and fatigue behavior. Thus, it is concluded that a reduction of the total SR CA activity by approximately 50% in CA XIV KO mice does not lead to an impairment of SR function.

Inhibition of membrane-associated carbonic anhydrase isozymes IX, XII and XIV with a library of glycoconjugate benzenesulfonamides

A library of glycoconjugate benzenesulfonamides that contain diverse carbohydrate-triazole tails were investigated for their ability to inhibit the enzymatic activity of the three human transmembrane carbonic anhydrase (CA) isozymes hCA IX, hCA XII and hCA XIV. These isozymes have their CA domains located extracellularly, unlike the physiologically dominant hCA II, and are of immense current interest as druggable targets. Elevated expression of isozymes IX and XII is a marker for a broad spectrum of hypoxic tumors—this physiology may facilitate a novel approach to discriminate between healthy cells and cancerous cells. Many of these glycoconjugates were potent inhibitors (low nM), but importantly exhibited different isozyme selectivity profiles. The most potent hCA IX inhibitor was the glucuronic acid derivative 20 ( K i = 23 nM). This compound was uniquely hCA IX selective cf. all other isozymes (16.4-, 16.8- and 4.6-fold selective against hCA II, XII, and XIV, respectively). At hCA XII there were many inhibitors with K is < 10 nM that also demonstrated excellent selectivity (up to 344-fold) against other isozymes. Potent hCA XIV inhibitors were also identified, several with K is ∼ 10 nM, however no hCA XIV-selective derivatives were evidenced from this library. The sugar tails of this study have shown promise as a valuable approach to both solubilize the aromatic sulfonamide CA recognition pharmacophore and to deliver potent inhibition and isozyme differentiation of the transmembrane CAs.

Biochemical and Stone-Risk Profiles With Topiramate Treatment

Topiramate is a novel neuromodulatory agent commonly prescribed for the treatment of seizure disorders and for migraine headache prophylaxis. Calcium phosphate kidney stones have been observed with topiramate treatment, but a comprehensive elucidation of stone-risk profile was not reported previously. This study explores the relationship between topiramate treatment and propensity for kidney stone formation. Thirty-two topiramate-treated subjects and 50 healthy volunteers participated in a cross-sectional study in which serum chemistry test and 24-hour urine collection results were evaluated for stone risk. Furthermore, a short-term longitudinal study was conducted in 7 patients to assess stone risk before and 3 months after topiramate treatment. Serum bicarbonate levels were lower with topiramate treatment. Urinary pH, urinary bicarbonate excretion, and fractional excretion of bicarbonate increased, whereas urinary citrate excretion was significantly lower (737 +/- 329 versus 278 +/- 226 mg/d; P < 0.001). Net acid excretion did not change. The relative saturation ratio for brushite increased with topiramate treatment (3.14 +/- 1.69 versus 1.27 +/- 1.26; P < 0.001) because of urinary alkalinization and decreased urinary citrate levels. Urinary saturation of undissociated uric acid decreased (41 +/- 52 versus 76 +/- 60 mg/d; P < 0.001). Treatment with topiramate causes systemic metabolic acidosis, markedly lower urinary citrate excretion, and increased urinary pH. These changes increase the propensity to form calcium phosphate stones.

Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias)

Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.