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.     

Distribution of acid and alkaline deoxyribonucleases in white and grey matter regions of growing and aging chick brain

The activities of acid and alkaline deoxyribonucleases in the white and grey matter areas of growing and old chick cerebrum were measured. Two marker enzymes for glial cells, butyrylcholinesterase and carbonic anhydrase were also measured in these regions. Higher specific activities of both butyrylcholinesterase and carbonic anhydrase were found in the white matter region at all the stages studied. Acid and alkaline deoxyribonuclease activities were observed in both white and grey matter. The decrease in the specific activity of acid deoxyribonuclease with advancement of age was more pronounced as compared to the alkaline deoxyribonuclease Marked reduction in total acid deoxyribonuclease activity in white matter, beyond the age of 130 days, was observed. On the other hand, total alkaline deoxyribonuclease activity in both white and grey matter continued to increase with age Further, the activity per mg of DNA also increased in white matter of the old brain. These results indirectly suggest a continued role for alkaline deoxyribonuclease in glial cells formed at a later age.     

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.     

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.     

Subcellular distribution of carbonic anhydrase and Na+,K+-ATPase in the brain of the hyt/hyt hypothyroid mice

Activities of carbonic anhydrase and Na⁺,K⁺-ATPase in tissue homogenates and in subcellular fractions from different brain regions were studied in inherited primary hypothyroid (hyt/hyt) mice. The body weight, the weight of different brain regions, and the plasma thyroxine and triiodothyronine levels of hyt/hyt mice were significantly lower than those of the age-matched hyt/+ controls. In tissue homogenates of cerebral cortex, brain stem and cerebellum of hypothyroid mice, the activity of carbonic anhydrase (units/mg protein) was 59.2, 57.6, and 43.2%, and the activity of Na⁺,K⁺-ATPase (nmol Pi/mg protein/min) was 73.7, 74.4 and 68.7%, respectively, of that in corresponding regions of euthyroid littermates. The decrease in enzyme activity in tissue homogenates was also reflected in different subcellular fractions. In cerebral cortex and brain stem, carbonic anhydrase activity in cytosol, myelin and mitochondrial fractions of hypothyroid mice was about 45–50% of that in euthyroid mice, while in cerebellum the carbonic anhydrase activity in these subcellular fractions of hyt/hyt mice was only 33–38% of that in hyt/+ controls. Na⁺,K⁺-ATPase activity in myelin fraction of different brain regions of hyt/hyt mice was about 34–42% of that in hyt/+ mice, while in mitochondria, synaptosome and microsome fractions were about 44–52, 46–53, and 66–68%, respectively of controls. These data indicate that the activity of both carbonic anhydrase and Na⁺,K⁺-ATPase was affected more in the myelin than other subcellular fractions and more in the cerebellum than cerebral cortex and brain stem by deficiency of thyroid hormones. A reduction in the activity of transport enzymes in brain tissues as a result of thyroid hormone deficiency during the critical period of development may underlie permanent nervous disorders in primary hypothyroidism.     

Carbonic anhydrase C in white-skeletal-muscle tissue.

We investigated the activity of carbonic anhydrase in blood-free perfused white skeletal muscles of the rabbit. Carbonic anhydrase activities were measured in supernatants and in Triton extracts of the particulate fractions of white-skeletal-muscle homogenate by using a rapid-reaction stopped-flow apparatus equipped with a pH electrode. An average carbonic anhydrase concentration of about 0.5 microM was determined for white skeletal muscle. This concentration is about 1% of that inside the erythrocyte. Some 85% of the muscle enzyme was found in the homogenate supernatant, and only 15% appeared to be associated with membranes and organelles. White-skeletal-muscle carbonic anhydrase was characterized in terms of its Michaelis constant and catalytic-centre activity (turnover number) for CO2 and its inhibition constant towards ethoxzolamide. These properties were identical with those of the rabbit erythrocyte carbonic anhydrase C, suggesting that a type-C enzyme is present in white skeletal muscle. Affinity chromatography of muscle supernatant and of lysed erythrocytes showed that, whereas rabbit erythrocytes contain about equal amounts of carbonic anhydrase isoenzymes B and C, the B isoenzyme is practically absent from white skeletal muscle. Similarly, ethoxzolamide-inhibition curves suggested that white skeletal muscle contains no carbonic anhydrase A. It is concluded that white skeletal muscle contains essentially one carbonic anhydrase isoenzyme, the C form, most of which is probably of cytosolic origin.     

Immunohistochemical localization of carbonic anhydrase isoenzymes II and III in quail kidney

The immunohistochemical localization of carbonic anhydrase isoenzymes has never been investigated in avian renal tissue previously. Enzyme activity has largely been documented by histochemical and physiological reports. In this investigation, specific antisera were used to study the distribution of the cytosolic carbonic anhydrase II and III isoenzymes in the quail kidney. Comparison between the present findings and the corresponding histochemical patterns, previously obtained in the same species by a cobalt phosphate precipitation method, resulted in the bulk of renal carbonic anhydrase activity being attributed to the carbonic anhydrase II isoenzyme. Conversely, moderate carbonic anhydrase III immunostaining appeared to be confined to the smooth muscle cells of ureteral and arteriolar walls. Indirect evidence of the occurrence, in the quail kidney, of a membrane-associated carbonic anhydrase form, antigenically distinct from the II and III isoforms, was inferred.     

Use of a rat Y chromosome probe to determine the long-term survival of glial cells transplanted into areas of CNS demyelination

A lack of suitable markers for cells which undergo division following transplantation has hindered studies assessing the long-term survival of glial cell grafts in the CNS. A probe specific to the rat Y chromosome was used to identify male glial cells grafted into an area of ethidium bromide-induced demyelination in syngeneic adult female rat spinal cord 4 weeks, 6 months and 12 months post-transplantation. At all time points there was extensive oligodendrocyte remyelination of transplanted lesions, and graft-derived cells were present within the lesion up to 12 months post-transplantation. In order to demonstrate graft-derived oligodendrocytes in the remyelinated region at 6 and 12 months, double-labelling studies were performed using the oligodendrocyte-specific antibodies carbonic anhydrase II or phosphatidyl ethanolamine-binding protein in combination with the Y chromosome probe. It was found that the majority of oligodendrocytes in the transplanted region were graft-derived. Graft-mediated remyelination was associated with a reduction in myelin sheath thickness and increase in nodal frequency similar to that observed in spontaneous remyelination, suggesting that, like axons remyelinated spontaneously, axons remyelinated by grafted cells will be capable of secure conduction. An alteration in the immunoreactivity of oligodendrocytes from carbonic anhydrase II-negative in the unlesioned dorsal funiculus to carbonic anhydrase II-positive in the remyelinated dorsal funiculus was considered to reflect a reduction in the amount of myelin supported by each oligodendrocyte, leading to the proposal that carbonic anhydrase II immunoreactivity may provide a means of identifying areas of remyelination in normally carbonic anhydrase II-negative white matter tracts.     

Modeling of microstructural kinematics during simple elongation of central nervous system tissue

Damage to axons and glial cells in the central nervous system (CNS) white matter is a nearly universal feature of traumatic brain injury, yet it is not clear how the tissue mechanical deformations are transferred to the cellular components of the CNS. Defining how cellular deformations relate to the applied tissue deformation field can both highlight cellular populations at risk for mechanical injury, and define the fraction of cells in a specific population that will exhibit damage. In this investigation, microstructurally based models of CNS white matter were developed and tested against measured transformations of the CNS tissue microstructure under simple elongation. Results show that axons in the unstretched optic nerves were significantly wavy or undulated, where the measured axonal path length was greater than the end-to-end distance of the axon. The average undulation parameter--defined as the true axonal length divided by the end-to-end length--was 1.13. In stretched nerves, mean axonal undulations decreased with increasing applied stretch ratio (lambda)--the mean undulation values decreased to 1.06 at lambda = 1.06, 1.04 at lambda = 1.12, and 1.02 at lambda = 1.25. A model describing the gradual coupling, or tethering, of the axons to the surrounding glial cells best fit the experimental data. These modeling efforts indicate the fraction of the axonal and glial populations experiencing deformation increases with applied elongation, consistent with the observation that both axonal and glial cell injury increases at higher levels of white matter injury. Ultimately, these results can be used in conjunction with computational simulations of traumatic brain injury to aid in establishing the relative risk of cellular structures in the CNS white matter to mechanical injury.     

Cadherins in the Developing Central Nervous System: An Adhesive Code for Segmental and Functional Subdivisions

In this review, we describe general features of the expression of cadherins in the developing central nervous system (CNS) of vertebrates. In the early neuroepithelium, the expression of several cadherins is restricted to specific regions corresponding to segmental domains. Segmental boundaries often coincide with changes in cadherin expression, subdividing the primordial CNS into different adhesive domains. In the different neuromeric domains, early neurons are generated which differentially express cadherins. In the mantle layer, these early neurons seem to sort out according to which cadherin they express, and they aggregate into various gray matter regions (brain nuclei and cortical lamina and regions). The gray matter structures expressing a given cadherin become connected to one another to form parts of particular functional systems or neuronal circuits. Together, these findings show that cadherins provide a molecular system reflecting both early embryonic and mature nervous system architecture. The possible roles of cadherins in the formation and maintenance of segmental and functional nervous system structures are discussed.     

Immunocytochemical localization of carbonic anhydrase in myelinated fibers in peripheral nerves of rat and mouse

Rat sciatic nerve, spinal root, and cranial nerve were immunostained with an antibody against rat brain carbonic anhydrase II (ca), to determine the localization of ca in the rat peripheral nervous system (PNS). Similar methods were applied to mouse nerves to see if that antigen could be detected in the PNS of this species. In rat nerves, intense immunostaining was observed in the axoplasm of many of the myelinated fibers, whereas others were stained less intensely or were negative. A heterogeneous pattern of immunostaining was also found in neuronal perikarya within the ganglia, and in some regions of the ganglia ca immunostaining was found in putative satellite cells and their processes. Ca in rat PNS therefore appears to occur at both neuronal and glial sites, whereas it is exclusively glial in the CNS. In longitudinal sections of some fibers within rat nerves, ca immunostaining could be detected at the inner boundaries of the myelin sheaths. In mouse nerves, axoplasmic staining was observed but it was fainter than in rat nerves. Interspecies differences were most obvious in the dorsal columns of the spinal cord. In rat, intensely stained axons proceeded through the roots into the gracilis or cuneate and often into the gray matter. In mouse, there was much less immunostaining of axons but more intense ca immunostaining in CNS myelin than in the CNS myelin in the rat cord. The implications concerning putative functions of ca in the rodent nervous system are discussed.     

Axial mesendoderm refines rostrocaudal pattern in the chick nervous system.

There has long been controversy concerning the role of the axial mesoderm in the induction and rostrocaudal patterning of the vertebrate nervous system. Here we investigate the neural inducing and regionalising properties of defined rostrocaudal regions of head process/prospective notochord in the chick embryo by juxtaposing these tissues with extraembryonic epiblast or neural plate explants. We localise neural inducing signals to the emerging head process and using a large panel of region-specific neural markers, show that different rostrocaudal levels of the head process derived from headfold stage embryos can induce discrete regions of the central nervous system. However, we also find that rostral and caudal head process do not induce expression of any of these molecular markers in explants of the neural plate. During normal development the head process emerges beneath previously induced neural plate, which we show has already acquired some rostrocaudal character. Our findings therefore indicate that discrete regions of axial mesendoderm at headfold stages are not normally responsible for the establishment of rostrocaudal pattern in the neural plate. Strikingly however, we do find that caudal head process inhibits expression of rostral genes in neural plate explants. These findings indicate that despite the ability to induce specific rostrocaudal regions of the CNS de novo, signals provided by the discrete regions of axial mesendoderm do not appear to establish regional differences, but rather refine the rostrocaudal character of overlying neuroepithelium.     

Carbonic anhydrase activity in Madin Darby canine kidney cells. Evidence for intercalated cell properties

Madin Darby canine kidney (MDCK) renal epithelial cell cultures have been investigated with respect to their potency to express carbonic anhydrase activity using histochemical methods. Acetazolamide inhibitable carbonic anhydrase activity could be detected in the cytoplasmic compartment as well as in the apical membrane of cells when grown on solid culture supports. Cells forming domes in MDCK monolayers exhibit the highest histochemically detectable enzyme activity. The attempt to subculture clonal cell lines from MDCK monolayer cultures resulted in the establishment of 5 clones, slightly different with respect to size and shape of cells and their potency to form domes. Scanning electron microscopy ensured the identification of one clone (1A4), which distinctly differed from the others with respect to the apical membrane architecture. Co-localization of peanut agglutinin and carbonic anhydrase activity at the plasma membrane always revealed a combined occurrence of enzyme reactivity and lectin binding in the apical membrane domain. Both, lectin binding and carbonic anhydrase activity were distinctly more intense in plasma membrane regions equipped with microvilli. From the results it is concluded that MDCK cells in tissue culture retained properties of intercalated cells of the nephron collecting duct segment.     

SOLUBLE AND MEMBRANE BOUND CARBONIC ANHYDRASES FROM RAT CNS: REGIONAL DEVELOPMENT

Abstract— The distribution of the soluble, membrane bound and myelin carbonic anhydrase in different regions of the rat CNS was examined as a function of age. A neuraxial progression from spinal cord to upper brain stem was observed for all three enzyme fractions in the 90 day old rat: upper brain stem > lower brain stem and cerebellum > spinal cord. The membrane bound fraction accounted for close to 60% of the total carbonic anhydrase in all regions except the cerebellum where it accounted for only 40%. The developmental pattern of the total membrane bound and soluble fractions were virtually parallel in all regions studied suggesting that they are derived from a common enzyme pool. The myelin enzyme accounts for a small but significant part of the membrane bound fraction and is present at adult levels by 16 days of age indicating it is an early and specific myelin component.     

A comparative electrophoretic analysis of mammalian carbonic anhydrase isozymes: evidence for a third isozyme in red skeletal muscles.

1. Starch gel electrophoretic patterns of carbonic anhydrase (CA) isozymes were examined from tissue extracts of cats, sheep, rabbits and mice. 2. In addition to the widely distributed and extensively studied B and C isozymes, an additional isozyme (called CA-A) was observed. 3. Tissue distribution studies showed the A isozyme to be predominantly localized in red skeletal muscles, although this activity was also observed in white and "mixed" skeletal muscles of the cat, sheep and rabbit, as well as sheep lung and rabbit liver. 4. A, B and C isozymes of carbonic anhydrase from cat, sheep and mice exhibited independent variations in nett surface charge. In terms of decreasing anodal migration, the following results are reported: cat A greater than C greater than B; sheep C greater than B greater than A; and mouse B greater than C greater than A. 5. These results are consistent with the existence of 3 genetic loci encoding carbonic anhydrase in mammalian tissues.     

High-affinity GABA uptake and GABA-metabolizing enzymes in pig forebrain white matter: a quantitative study

GABA receptor activation in central nervous white matter may be protective during white matter hypoxia in the adult, and it may modify axonal conduction, especially in the developing brain. GABA uptake is important for the shaping of the GABA signal, but quantitative data are lacking for GABA uptake and GABA-metabolizing enzymes in central nervous white matter. We report that high-affinity uptake of GABA in adult pig corpus callosum, fimbria, subcortical pyramidal tracts, and occipital white matter is approximately 20% of that in temporal cortex gray matter. Tiagabine (0.1 microM), an antiepileptic drug that specifically inhibits the GAT-1 GABA transporter inhibited GABA uptake 50% in temporal cortex and 60-68% in white structures. This finding indicates that GAT-1 is an important GABA transporter in white matter and suggests that white matter GABA uptake is inhibited during tiagabine therapy. GABA transaminase activity in white structures was approximately 20% of neocortical values. Glutamate decarboxylase (GAD) activity in white structures was only 4% of that in neocortex (7-12 pmol/mg tissue x min(-1) versus approximately 200 pmol/mg tissue x min(-1)). Since white matter activity of citrate synthase of the tricarboxylic acid cycle was approximately 25% of neocortical values ( approximately 0.4 nmol/mg tissue x min(-1) versus approximately 1.5 nmol/mg tissue x min(-1)), the low GAD activity suggests a slower metabolic turnover of GABA in white than in gray matter.     

Sarcolemmal carbonic anhydrase in red and white rabbit skeletal muscle

Sarcolemmal vesicles of white and red skeletal muscles of the rabbit were prepared by consecutive density gradient centrifugations in sucrose and dextran according to Seiler and Fleischer (1982, J. Biol. Chem. 257, 13,862-13,871). White and red muscle membrane fractions enriched in sarcolemma were characterized by high ouabain-sensitive Na+, K(+)-ATPase, by high Mg2(+)-ATPase activity, and by a high cholesterol content. Ca2(+)-ATPase activity, a marker enzyme for sarcoplasmic reticulum, was not detectable in the highly purified white and red muscle sarcolemmal fractions. White and red muscle sarcolemmal fractions exhibited no significant differences with regard to Na+, K(+)-ATPase, Mg2(+)-ATPase, and cholesterol. Specific activity of carbonic anhydrase in white muscle sarcolemmal fractions was 38 U.ml/mg and was 17.6 U.ml/mg in red muscle sarcolemma. Inhibition properties of sarcolemmal carbonic anhydrase were analyzed for acetazolamide, chlorzolamide, and cyanate. White muscle sarcolemmal carbonic anhydrase is characterized by inhibition constants, KI, toward acetazolamide of 4.6 X 10(-8) M, toward chlorzolamide of 0.75 X 10(-8) M, and toward cyanate of 1.3 X 10(-4) M. Red muscle sarcolemmal carbonic anhydrase is characterized by KI values toward acetazolamide of 8.1 X 10(-8) M, toward chlorzolamide of 6.3 X 10(-8) M, and toward cyanate of 0.81 X 10(-4) M. In contrast to the high specific carbonic anhydrase activities in sarcolemma, carbonic anhydrase activity in sarcoplasmic reticulum from white muscle varied between values of only 0.7 and 3.3 U.ml/mg. Carbonic anhydrase of red muscle sarcoplasmic reticulum ranged from 2.4 to 3.7 U.ml/mg.     

THE SUBCELLULAR DISTRIBUTION OF CARBONIC ANHYDRASE IN HOMOGENATES OF PERFUSED RAT BRAIN

Abstract— The distribution of carbonic anhydrase was examined in subcellular fractions of perfused rat brain and compared with those of markers for cytosol (lactic dehydrogenase), mitochondrial matrix (glutamic dehydrogenase), and mitochondrial membranes (succinic dehydrogenase). About half of the total carbonic anhydrase was found in particulate fractions, with the greatest part of this in the crude mitochondrial fraction. This fraction was separated into its components on a discontinuous sucrose gradient either as such or after isotonic mechanical disruption with a French pressure cell, and the resultant fractions were characterized by electron microscopy and by assay of marker enzymes.
Carbonic anhydrase was solubilized by mechanical disruption, but not to the same extent as lactic dehydrogenase. The highest specific activity for carbonic anhydrase was found in the myelin fraction of the gradient. A mitochondrial locus for carbonic anhydrase is unlikely, but the presence of the enzyme in synaptosomes remains in question.
Addition of soluble carbonic anhydrase did not significantly increase the activity of particulate fractions. Treatment of particulate fractions with detergent was necessary to reveal latent activity; this procedure resulted in a more than ten-fold increase in the measurable carbonic anhydrase activity of myelin fragments.     

Esterase activity of carbonic anhydrases serves as surrogate for selecting antibodies blocking hydratase activity

Abstract

Carbonic anhydrase 9 (CA9) and carbonic anhydrase 12 (CA12) were proposed as potential targets for cancer therapy more than 20 years ago. However, to date, there are only very few antibodies that have been described to specifically target CA9 and CA12 and also block the enzymatic activity of their targets. One of the early stage bottlenecks in identifying CA9- and CA12-inhibiting antibodies has been the lack of a high-throughput screening system that would allow for rapid assessment of inhibition of the targeted carbon dioxide hydratase activity of carbonic anhydrases. In this study, we show that measuring the esterase activity of carbonic anhydrase offers a robust and inexpensive screening method for identifying antibody candidates that block both hydratase and esterase activities of carbonic anhydrase’s. To our knowledge, this is the first implementation of a facile surrogate-screening assay to identify potential therapeutic antibodies that block the clinically relevant hydratase activity of carbonic anhydrases.     

Mitochondrial carbonic anhydrase in osteoclasts and two different epithelial acid-secreting cells

Summary Acid secreting cells are rich in mitochondria and contain high levels of cytoplasmic carbonic anhydrase II. We have studied the ultrastructural distribution of a mitochondrial isoenzyme, carbonic anhydrase V, in two different acid-secreting epithelial cells, gastric parietal cells and kidney intercalated cells as well as in osteoclasts, which are the main bone resorbing cells. The mitochondria differ in carbonic anhydrase V content in these three acid-producing cells: gastric parietal cell mitochondria show strong immunolabelling for this isoenzyme, osteoclast mitochondria faint labelling and kidney intercalated cell mitochondria no labelling. The immunolabelling was located in the mitochondrial matrix, often in close contact with the inner mitochondrial membrane. These results show that mitochondrial carbonic anhydrase levels are not related to acid-transporting activity.