Carbonic Anhydrase Immunostaining in Astrocytes in the Rat Cerebral Cortex

Carbonic anhydrase is known to occur in the choroid plexus, oligodendrocytes, and myelin, and to be virtually absent from neurons, in the mammalian CNS; however, there is significant controversy whether it is also present in astrocytes. When brain sections from adult rats were stained for simultaneous immunofluorescence of carbonic anhydrase and the astrocyte marker glutamine synthetase, both antigens were detected in the same glial cells in the cortical gray matter, whereas the oligodendrocytes and myelinated fibers in and adjacent to the white matter showed immunofluorescence only for carbonic anhydrase. Some glial cells in the gray matter also showed double immunofluorescence for carbonic anhydrase and glial fibrillary acidic protein. These results indicate that there is carbonic anhydrase in some astrocytes in the mammalian CNS.     

Carbonic Anhydrase, 5''-Nucleotidase, and 2'',3''-Cyclic Nucleotide-3''-Phosphodiesterase Activities in Oligodendrocytes, Astrocytes, and Neurons Isolated from the Brains of Developing Rats

The activities of three myelin-associated enzymes, carbonic anhydrase, 5'-nucleotidase, and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNP), were measured in oligodendrocytes, neurons, and astrocytes isolated from the brain of rats 10, 20, 60, and 120 days old. The carbonic anhydrase specific activity in oligodendrocytes was three- to fivefold higher than that in brain homogenates at each age, and, at all the ages, low activities of this enzyme were measured in neurons and astrocytes. The oligodendrocytes and astrocytes from the brains of rats at all ages had higher activities of the membrane-bound enzyme 5'-nucleotidase than was observed in neurons. In oligodendrocytes from 10- and 20-day-old rats, the 5'-nucleotidase activity was two-to threefold the activity in the homogenates (i.e., relative specific activity = 2.0-3.0), and the relative specific activity of this enzyme in the oligodendrocytes declined to less than 1.0 at the later ages, concomitant with the accumulation of 5'-nucleotidase in myelin. The CNP activity was always higher in oligodendrocytes than in neurons, but not appreciably different from that in astrocytes from 20 days of age onward. The relative specific activity of CNP was highest in the oligodendrocytes from 10-day-old rats but was lower, at all ages, than we had observed in bovine oligodendrocytes. These enzyme activities in oligodendroglia are quite different in amount and developmental pattern from those reported previously for myelin.     

Development of the various glial cell types in the cerebral cortex of postnatal rats

The present quantitative study in the postnatal rats showed the rapid growth of the various glial cell types in the cerebral cortex. Among them, the increase of microglia was most dramatic. The increase was about 15 times, covering a period of 15 days extending from 5 days of age to 20 days. The majority of the microglia observed were in the outer third of the cortex. During the same period, the number of oligodendrocytes and astrocytes also showed a steady but moderate increase. The increase of oligodendrocytes was most significant between 5 and 10 days. Their density was greater in the inner third of the cortex. Astrocytes were distributed uniformly throughout. Examination of the cerebral cortex in 1- to 3-day-old rats by electron microscopy showed sporadic ameboid microglia cells and glioblasts. The possibility that they served as the precursor cells of microglia and macroglia (astrocytes and oligodendrocytes), respectively, was considered.     

Carbonic anhydrase and oligodendroglia in developing rat cerebellum: A biochemical and immunohistological study

In the cerebellum, the isoenzyme II of carbonic anhydrase (CAII) appears to be a specific oligodendrocyte marker. Formation and maturation of oligodendrocytes during the postnatal development of rat cerebellum were followed by using specific immune serum directed against CAII for immunohistofluorescence and radioimmunoassay. Few oligodendrocytes are present in the cerebellum of the newborn rat. Their number increases rapidly between the fourth and the tenth days after birth and then more slowly until the end of the third week. Sequential changes in oligodendrocyte morphology have been observed. Determinations of CAII in cerebellar homogenate by radioimmunoassay show that the CAII level is low from birth to the end of the second week, the increase in the number of oligodendrocytes being accompanied by a small increase in the CAII level. Subsequent cell maturation is accompanied by significant accumulation of CAII.     

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.     

Localization of the multifunctional protein CAD in astrocytes of rodent brain.

The CAD multidomain protein, which includes active sites of carbamyl phosphate synthetase II (CPS II, glutamine-dependent), aspartate transcarbamylase, and dihydroorotase, was immunostained in normal rat brains, the gliotic brains of myelin-deficient mutant rats, and brains from normal weanling hamsters. In each of these tissues CAD was observed in cells resembling astrocytes. In hamster brain, CAD immunofluorescence was also found in cells closely related to astrocytes, i.e., the Bergmann glia in cerebellum and the tanycytes surrounding the third ventricle. The astrocytic identity of the CAD-positive cells in rat brain was confirmed by double immunofluorescence staining with antibodies against glial fibrillary acidic protein (GFAP). The two enzymes carbonic anhydrase and glutamine synthetase occur in the cytoplasm of normal astrocytes in gray matter and of reactive astrocytes during gliosis. Products of each enzyme, i.e., bicarbonate and glutamine, are required for the CPS II reaction, which is the first step in the biosynthesis of pyrimidines. Therefore, the present results suggest roles for carbonic anhydrase and glutamine synthetase, as well as CAD, in pyrimidine biosynthesis in brain and a role for the astrocytes in the de novo synthesis of pyrimidines.     

A transport metabolon. Functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers

The cytoplasmic carboxyl-terminal domain of AE1, the plasma membrane chloride/bicarbonate exchanger of erythrocytes, contains a binding site for carbonic anhydrase II (CAII). To examine the physiological role of the AE1/CAII interaction, anion exchange activity of transfected HEK293 cells was monitored by following the changes in intracellular pH associated with AE1-mediated bicarbonate transport. AE1-mediated chloride/bicarbonate exchange was reduced 50-60% by inhibition of endogenous carbonic anhydrase with acetazolamide, which indicates that CAII activity is required for full anion transport activity. AE1 mutants, unable to bind CAII, had significantly lower transport activity than wild-type AE1 (10% of wild-type activity), suggesting that a direct interaction was required. To determine the effect of displacement of endogenous wild-type CAII from its binding site on AE1, AE1-transfected HEK293 cells were co-transfected with cDNA for a functionally inactive CAII mutant, V143Y. AE1 activity was maximally inhibited 61 +/- 4% in the presence of V143Y CAII. A similar effect of V143Y CAII was found for AE2 and AE3cardiac anion exchanger isoforms. We conclude that the binding of CAII to the AE1 carboxyl-terminus potentiates anion transport activity and allows for maximal transport. The interaction of CAII with AE1 forms a transport metabolon, a membrane protein complex involved in regulation of bicarbonate metabolism and transport.     

Expression of carbonic anhydrase II mRNA and protein in oligodendrocytes during toxic demyelination in the young adult mouse

The aim of this study was to identify events that might take place in oligodendrocytes early in the process of demyelination, i.e., before the occurrence of massive loss of myelin. It was considered important to focus on demyelination and remyelination in young adults, in whose brains there would be relatively few juvenile glial precursor cells. CAII mRNA and protein were used to monitor changes in oligodendrocytes during cuprizone intoxication in the mice. After four or eight weeks of cuprizone feeding CAII message became less plentiful in oligodendrocyte processes. Two days after removal of cuprizone CAII message had appeared in those cell processes. Four or eight weeks after beginning cuprizone feeding CAII protein had decreased∼25% in forebrain homogenates. The loss of CAII protein was reversible after four weeks on cuprizone, but not after eight weeks. After four weeks of cuprizone feeding the numbers of CAII mRNA-prositive oligodendrocytes had decreased by ∼50%m and after eight weeks, by ∼80%. By 12 weeks, however, the number of oligodendrocytes expressing CAII mRNA had spontaneously returned to normal levels. Before eight weeks of cuprizone feeding, loss of myelinated tracts in the corpus striatum was reversible. Demyelination appreared to become irreversible after nine weeks of intoxication, although expression of CAII mRNA remained reversible. The results suggest that in the brain of the young adult, oligodendrocytes expressing message for CAII can be generated spontaneously shortly before demyelination becomes irreversible, and can survive and continue to express CAII mRNA but not CAII protein. Special issue dedicated to Dr. Marion E. Smith.     

Immunocytochemical localization of carbonic anhydrase in the spinal cords of normal and mutant (shiverer) adult mice with comparisons among fixation methods

The peroxidase-antiperoxidase technique was used for immunocytochemical localization of carbonic anhydrase in the mouse spinal cord to detect whether this antigen was normally present in myelinated fibers, in oligodendrocytes in both white and gray matter, and in astrocytes, and to determine where the carbonic anhydrase might be localized in the spinal cords of dysmyelinating mutant (shiverer) mice. The most favorable methods for treating tissue were: 1) immersion in formalin-ethanol-acetic acid followed by paraffin embedding, or 2) light fixation with paraformaldehyde and preparation of vibratome sections. Carnoy's solution, followed by paraffin embedding, extracted myelin from the tissue, while aqueous aldehydes, when used before paraffin embedding, reduced staining everywhere except at sites of compact myelin. The latter conclusion was based, in part, on the almost complete loss of this antigen from the shiverer cord, where compact myelin is known to be virtually absent but where membrane-bound carbonic anhydrase was demonstrated enzymatically. When the optimal methods were used with normal mouse cords, carbonic anhydrase was found throughout the white matter columns and in the oligodendrocytes in gray and white matter. The staining of the white matter was attributed to myelinated fibers because of the similarity in distribution to both a histological myelin stain and the immunocytochemical staining for myelin basic protein. In the mutant mice the oligodendrocyte cell bodies and processes, which were stained in all areas of the spinal cord, were particularly numerous at the periphery of the sections. In contrast to the oligodendrocytes, the fibrous astrocytes appeared to lack carbonic anhydrase, or to have lower than detectable levels, since the astrocyte marker, glial fibrillary acidic protein, had a very different distribution from that of carbonic anhydrase. Even finer localization was obtained in vibratome sections, where the antibody against carbonic anhydrase permitted visualization of the processes connecting oligodendrocytes to myelinated fibers in the normal adult spinal cord.     

The extracellular component of a transport metabolon. Extracellular loop 4 of the human AE1 Cl-/HCO3- exchanger binds carbonic anhydrase IV

Cytosolic carbonic anhydrase II (CAII) and the cytoplasmic C-terminal tails of chloride/bicarbonate anion exchange (AE) proteins associate to form a bicarbonate transport metabolon, which maximizes the bicarbonate transport rate. To determine whether cell surface-anchored carbonic anhydrase IV (CAIV) interacts with AE proteins to accelerate the bicarbonate transport rate, AE1-mediated bicarbonate transport was monitored in transfected HEK293 cells. Expression of the inactive CAII V143Y mutant blocked the interaction between endogenous cytosolic CAII and AE1, AE2, and AE3 and inhibited their transport activity (53 +/- 3, 49 +/- 10, and 35 +/- 1% inhibition, respectively). However, in the presence of V143Y CAII, expression of CAIV restored full functional activity to AE1, AE2, and AE3 (AE1, 101 +/- 3; AE2, 85 +/- 5; AE3, 108 +/- 1%). In Triton X-100 extracts of transfected HEK293 cells, resolved by sucrose gradient ultracentrifugation, CAIV recruitment to the position of AE1 suggested a physical interaction between CAIV and AE1. Gel overlay assays showed a specific interaction between CAIV and AE1, AE2, and AE3. Glutathione S-transferase pull-down assays revealed that the interaction between CAIV and AE1 occurs on the large fourth extracellular loop of AE1. We conclude that AE1 and CAIV interact on extracellular loop 4 of AE1, forming the extracellular component of a bicarbonate transport metabolon, which accelerates the rate of AE-mediated bicarbonate transport.     

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

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 K_d = 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 (pH_i) recovery rate by 31 ± 3, or 38 ± 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pH_i 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 [-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. pH regulation; bicarbonate transport; metabolon     

Carbonic anhydrase II binds to and enhances activity of the Na+/H+ exchanger

We examined the ability of carbonic anhydrase II to bind to and affect the transport efficiency of the NHE1 isoform of the mammalian Na(+)/H(+) exchanger. The C-terminal region of NHE1 was expressed in Escherichia coli fused with an N-terminal glutathionine S-transferase or with a C-terminal polyhistidine tag. Using a microtiter plate binding assay we showed that the C-terminal region of NHE1 binds carbonic anhydrase II (CAII) and binding was stimulated by low pH and blocked by antibodies against the C-terminal of NHE1. The binding to NHE1 was confirmed by demonstrating protein-protein interaction using affinity blotting with CAII and immobilized NHE1 fusion proteins. CAII co-immunoprecipitated with NHE1 from CHO cells suggesting the proteins form a complex in vivo. In cells expressing CAII and NHE1, the H(+) transport rate was almost 2-fold greater than in cells expressing NHE1 alone. The CAII inhibitor acetazolamide significantly decreased the H(+) transport rate of NHE1 and transfection with a dominant negative CAII inhibited NHE1 activity. Phosphorylation of the C-terminal of NHE1 greatly increased the binding of CAII. Our study suggests that NHE1 transport efficiency is influenced by CAII, likely through a direct interaction at the C-terminal region. Regulation of NHE1 activity by phosphorylation could involve modulation of CAII binding.     

The bicarbonate transport metabolon

To allow cells to control their pH and bicarbonate levels, cells express bicarbonate transport proteins that rapidly and selectively move bicarbonate across the plasma membrane. Physical interactions have been identified between the carbonic anhydrase isoform, CAII, and the erythrocyte membrane Cl- /HCO3(-) anion exchanger, AE1, mediated by an acidic motif in the AE1 C-terminus. We have found that the presence of CAII attached to AE1 accelerates AE1 HCO3(-) transport activity, as AE1 moves bicarbonate either into or out of the cell. In efflux mode the presence of CAII attached to AE1 will increase the local concentration of bicarbonate at the AE1 transport site. As bicarbonate is transported into the cell by AE1, the presence of CAII on the cytosolic surface accelerates transport by consumption of bicarbonate, thereby maximizing the transmembrane bicarbonate concentration gradient experienced by the AE1 molecule. Functional and physical interactions also occur between CAII and Na+/HCO3(-) co-transporter isoforms NBC1 and NBC3. All examined bicarbonate transport proteins, except the DRA (SLC26A3) Cl-/HCO3(-) exchange protein, have a consensus CAII binding site in their cytoplasmic C-terminus. Interestingly, CAII does not bind DRA. CAIV is anchored to the extracellular surface of cells via a glycosylphosphatidyl inositol linkage. We have identified extracellular regions of AE1 and NBC1 that directly interact with CAIV, to form a physical complex between the proteins. In summary, bicarbonate transporters directly interact with the CAII and CAIV carbonic anhydrases to increase the transmembrane bicarbonate flux. The complex of a bicarbonate transporter with carbonic anhydrase forms a "Bicarbonate Transport Metabolon."     

Radioimmunoassay of carbonic anhydrase I and II in rats. Application to the central nervous system during ontogeny]

A specific radioimmunoassay method for rat erythrocyte carbonic anhydrases I and II was developed using a double antibody system. Its sensitivity was in the nanogram range for each of the two isozymes. The method has been applied to the assay of cerebral carbonic anhydrase. Only CAII has been found in brain extracts of perfused rats. Accordingly, the assay of CAI in cerebral tissue can be used to quantify erythrocyte contamination on condition that the ratio CAII/CAI in blood had been worked out. The developmental change in the soluble and the Triton X-100 solubilized brain CAII from birth to adult is reported.     

Biochemical and Immunocytochemical Evidence for a Deficiency of Normal Interfascicular Oligodendroglia in the CNS of the Dysmyelinating Mutant (md) Rat

Carbonic anhydrase was assayed and carbonic anhydrase and 5'-nucleotidase were localized in the CNS of myelin-deficient mutant rats and normal littermates. The carbonic anhydrase specific activities were reduced by 61% and 29% in the mutants' forebrains and cerebella, respectively, and the total carbonic anhydrase activity in the spinal cords was reduced by 35%. Immunostained cells were found in gray matter from both normal and mutant rats, but, in the mutants, there was a marked deficiency of interfascicular oligodendrocytes in the regions that are normally occupied by white matter. It is suggested that a developmental study could indicate the step(s) at which normal differentiation of interfascicular oligodendroglia is blocked in this mutant.     

Glial-Cell Cultures from Brains of Carbonic Anhydrase II-Deficient Mutant Mice: Delay in Oligodendrocyte Maturation

Carbonic anhydrase II (CAII) is a multifunctional enzyme found in oligodendrocytes and astrocytes in normal mouse brains. We have begun to compare the glial cells in primary cultures from neonatal genetically CAII-deficient (Car) mice to those from normal (con) mice in order to detect developmental defects, if any, in Car glial cells. In con cultures intensely CAII-positive cells costained with antibodies against the oligodendrocytic markers, O4 and myelin basic protein (MBP), respectively. Most (82%) of the CAII-positive cells were O4-positive, but only 60% were MBP-positive. Some clumps of GFAP-positive cells were CAII-positive. At each respective number of days in vitro (DIV) total numbers of O4-positive cells were similar in Car and con cultures, and total numbers of galactocerebroside-positive cells also were similar in Car and con cultures. However, compared to cells in con cultures at 7 DIV, a lower percent of Car cells in the oligodendrocyte lineage expressed MBP, and morphological differentiation also was subnormal in that the Car cells showed fewer processes and membrane sheets. Car and con cultures expressed similar numbers of MBP-positive cells by 10 DIV. The results suggest a temporary delay in the maturation of Car oligodendrocytes.     

Polymorphism of human erythrocyte carbonic anhydrase in the population of Moscow and in the native populations of Siberia detected by polyacrylamide gel electrophoresis

Carbonic anhydrase of human erythrocytes was separated by polyacrylamide electrophoresis into four fractions determined, obviously, by two loci, CAI and CAII. Investigation of Moscow population sample of 458 men (516 healthy and 42 with schizophrenia) showed monomorphism of carbonic anhydrase for these two loci. Carbonic anhydrase I and carbonic anhydrase II were differentiated with fluorogenic substrates. The polymorphic variant of CAII was discovered while studying the sample of Siberian mongoloids (Evenks and Jakuts) with frequency 0,047 and 0,045, respectively.     

Microglia - insights into immune system structure, function, and reactivity in the central nervous system

Microglia are essential cellular components of a well-functioning central nervous system (CNS). The development and establishment of the microglial population differs from the other major cell populations in the CNS i.e. neurons and macroglia (astrocytes and oligodendrocytes). This different ontogeny gives microglia unique properties. In recent years detailed studies of the microglial population have been greatly facilitated by the use of bone marrow (BM) chimeric animals. Experimental BM transplants have provided the opportunity to trace and investigate how BM cells migrate into the CNS and settle to become microglia. Furthermore various functional properties of microglia in the normal and pathological CNS are now being revealed because of combinations of BM transplantations and experimental disease models. Here, we describe some of the latest findings in microglial biology and discuss the potential for using microglia in therapeutic interventions.     

Glial Cell Markers in the Reeler Mutant Mouse: A Biochemical and Immunohistological Study

The glial cell contents of S100 protein, 2',3'-cyclic AMP, 3'-phosphohydrolase (CNP), isoenzyme II of carbonic anhydrase (CAII) and butyrylcholinesterase (BuChE) were biochemically determined in the cerebellum and cerebrum of the reeler mutant mouse. Astrocytes and oligodendrocytes, shown by this study, contain abnormal amounts of these components. The CAII concentration was significantly increased in the particulate fraction of the reeler cerebellum and cerebrum (by 50% and 89%, respectively). The BuChE specific activity was greatly increased in the reeler, by 120% for cerebellum and by 40% in cerebrum. In contrast, the S100 protein concentration was reduced in the reeler cerebellum by 40% and by 25% in cerebrum, while the CNP specific activity increased by 30% in the reeler cerebellum. In addition, the glial cell distribution was studied by immunohistological techniques with antibodies directed against S100 protein, glial fibrillary acidic protein (GFA) and CAII. Apparently the density of glial cells is not significantly affected. However, the Golgi epithelial cells were usually abnormally placed and their Bergmann fibres were less well developed.     

Carbonic anhydrase II-based metal ion sensing: Advances and new perspectives

Carbonic anhydrases are archetypical zinc metalloenzymes and as such, they have been developed as the recognition element of a family of fluorescent indicators (sensors) to detect metal ions, particularly Zn²⁺ and Cu²⁺. Subtle modification of the structure of human carbonic anhydrase II isozyme (CAII) alters the selectivity, sensitivity, and response time for these sensors. Sensors using CAII variants coupled with zinc-dependent fluorescent ligands demonstrate picomolar sensitivity, unmatched selectivity, ratiometric fluorescence signal, and near diffusion-controlled response times. Recently, these sensors have been applied to measuring the readily exchangeable concentrations of zinc in the cytosol and nucleus of mammalian tissue culture cells and concentrations of free Cu²⁺ in seawater.