Localization Of Carbonic Anhydrase Research Articles

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.

Role of carbonic anhydrase in calcification in the gorgonian Leptogorgia virgulata

AbstractThe relationship between carbonic anhydrase and calcification in the gorgonian Leptogorgia virgulata was examined by analysis of the enzyme's activity, its cellular locations and its response to inhibition by Diamox. Enzyme activity was 2.7 nmol μg−1 protein s−1 at 4°C. Carbonic anhydrase localization at the light level using immunofluorescent techniques and at the ultrastructural level using Hansson's method showed activity in the scleroblasts and axial epithelium. Specifically, at the electron microscopic level this activity was found to be very high on the membranes of the spicule‐forming vacuole and electron‐dense bodies. Membranes of the microvilli and plasma membranes of the desmocytes showed high activity of this enzyme. The activity on the scleroblast plasma membranes was variable. This enzyme is possibly present in the axis. Regenerated branch tips incubated in seawater containing 10−4 M and 10−6 M Diamox showed an increase in 45Ca uptake into the spicules and axes. The tissue o fbranch tips incubated in 10−6 M Diamox showed a decrease in 45Ca uptake. The observation that carbonic anhydrase is located adjacent to calcifying structures and organelles associated with spicule formation suggests that the enzyme is involved in calcification. The increase in 45Ca uptake from enzyme inhibition may suggest that carbonic anhydrase is involved in the regulation of HCO3.

Carbonic anhydrase inhibitors.

The purpose of the present review is to describe the use of carbonic anhydrase inhibitors in clinical medicine and renal physiology. We first describe the localization of carbonic anhydrase within the kidney and then discuss evidence for its role in renal acidification and NaCl absorption. This is then followed by a description of clinical uses for carbonic anhydrase inhibitors. Many of the uses and effects of carbonic anhydrase inhibitors can be predicted from an understanding of renal physiology and the role of carbonic anhydrase. The limited potency of carbonic anhydrase inhibitors correlates well with the large magnitude of carbonic anhydrase-independent bicarbonate absorption. While theories for carbonic anhydrase-independent bicarbonate absorption are presented, the exact mechanisms remain unresolved.

Chapter 8 Carbonic Anhydrase: The First Marker of Glial Development

Publisher Summary This chapter describes localization of carbonic anhydrase (CA). Localization of CA in cells is fundamental in understanding the function of this enzyme in the nervous tissue. Two qualitative techniques—histochemistry and immunocytochemistry—have independently confirmed the original finding of a selective localization of CA in glial cells, as demonstrated by a quantitative cytochemical technique. All three approaches (histochemistry, immunocytochemistry, and cytochemistry) demonstrate a high concentration of CA activity in oligodendrocytes and its presence in myelin. The glial localization of CA holds true for both the central nervous system (CNS) and the peripheral nervous system (PNS), thereby suggesting a general role of this enzyme in the conversion of carbonic acid formed from metabolic CO2 back to CO2. In an investigation presented in the chapter, a new microdiver technique has been applied to the samples of single nerve cells and surrounding glial cells. Single cell preparations (neurons and oligodendrocytes) have been isolated under the dissection microscope from fresh unstained sections of the CNS of the rat. In the same investigation, the activity of single-cell samples isolated from neural tissue, choroid plexus, and single erythrocytes are compared. This novel approach offered the possibilities of obtaining direct information on the localization of CA in the CNS and of exploring the functional relationship between glia and neurons at a cellular level.

Histochemical localization of carbonic anhydrase in the pig's exocrine pancreas.

Carbonic anhydrase is essential for the pancreatic secretion of NaHCO3. To localize the distribution of carbonic anhydrase in the exocrine gland and, hence, identify the potential cellular source of secreted NaHCO3, histochemical staining of carbonic anhydrase was carried out on pancreatic biopsies obtained from six pigs at both supramaximal intravenous secretin stimulation and at secretory rest. Tissue staining, using Hansson's technique, revealed a strong staining both in membranes and cytoplasm in the duct cells and a weaker cytoplasmic staining in the acinar cells. Staining reaction was abolished by 10(-5) mol l-1 acetazolamide. Duct cells, accordingly, seem to be responsible for NaHCO3 secretion, while acinar cells appear unlikely to contribute substantially to NaHCO3 secretion.

Regional differences in the histochemical localization of carbonic anhydrase in the midgut of tobacco hornworm (Manduca sexta)

AbstractCarbonic anhydrase (CA) activity was assayed by a histochemical method in mounted cryostat sections of larval midgut of Manduca sexta (Insecta: Sphingidae). In anterior and middle midgut most CA activity was associated with apical microvilli of goblet cells; with occasional patchy staining of the columnar cell brush border in anterior midgut. In contrast, in the posterior midgut, the apical microvilli of goblet cells showed no CA activity, but intense staining was associated with the brush border of columnar cells. Staining of mitochondria and nuclei was seen in all midgut regions. In addition, midguts from late fifth instar larvae showed intense staining of spherites (mineral concretions). Acetazolamide (10−5M) completely inhibited staining at all sites except nuclei and spherites. Thiocyanate (100 mM) completely inhibited staining at all sites except anterior and middle goblet cell microvilli, which remained partially reactive. These results suggest that the anteriormiddle and posterior regions play different roles in generating and managing the alkalinization of midgut contents characteristic of lepidopteran midgut. The different sensitivities of CA activity in anterior‐middle and posterior midgut to thiocyanate raises the possibility that more than one form of the enzyme may be involved.

Immunohistochemical localization of carbonic anhydrase I and II in submandibular salivary glands of the mouse, rat, hamster and guinea pig

Tissues were fixed in Bouin and Carnoy solutions, and the peroxidase, anti-peroxidase (PAP) method was used. Serous acinar cells of the guinea pig were positive for carbonic anhydrase (CA), but acinar cells of rodents were negative. Granular convoluted tubule (GCT) cells of the mouse, rat and hamster had varying degrees of staining for CA isoenzyme I and II. Striated and excretory ducts from all four species were usually positive for CA. Scattered cells containing a high concentration of CA I and CA II were present in striated ducts and GCT of the hamster, GCT of the rat and striated and excretory ducts of the guinea pig.

Immunoelectron microscopic localization of carbonic anhydrase III in rat skeletal muscle

The subcellular distribution of carbonic anhydrase III in rat soleus and vastus lateralis muscles was studied using an immunogold technique. The enzyme protein was found to be distributed diffusely in the cytoplasm of skeletal muscle cells. Red skeletal muscle (mainly type I fibers) revealed very strong immunogold staining whereas in white muscle (mainly type II fibers) gold particles were almost completely absent. No immunoreaction was observed in mitochondria or in other intracellular organelles.

Immunohistochemical localization of carbonic anhydrase in submandibular salivary glands of mice and hamsters treated with phenylephrine, testosterone or duct-ligation

Using the peroxidase antiperoxidase (PAP) technique, the submandibular glands (SMG) from normal mice showed positive carbonic anhydrase (CA) staining in both striated duct (SD) and granular convoluted tubule (GCT) cells, which varied from moderate to strong. In normal hamsters, the GCT showed strong focal staining. Phenylephrine in mice and hamsters resulted in a decrease in CA in GCT cells; staining was much reduced in the GCT segments of the mouse. One hour after a phenylephrine injection into testosterone-treated mice, there was no change in the staining intensity of GCT cells but an increase in cell size. Only a slight decrease in immunostaining occurred in mouse and hamster SD cells. CA staining in duct-ligated glands of mice decreased within 3 days but regenerated duct-like structures at later stages showed moderate staining.

Distribution of carbonic anhydrase in cells and membranes isolated from pig gastric mucosa.

Mucosal cells were isolated from pig stomachs and then fractionated on linear density gradients of Percoll. Different types of cells were identified by their typical staining and morphology. In disrupted cell fractions, hydration of CO2 by carbonic anhydrase was measured by means of pH-stat technique. Localization of carbonic anhydrase to certain cell fractions was also studied by histochemical staining. Both parietal cells and carbonic anhydrase were confined to the low and intermediate density fractions of the gradients. Purified membranes from pig gastric mucosa, which contained the acid pump of the stomach, the H,K-ATPase, also contained a firmly bound carbonic anhydrase of high activity. The enzyme activity in the membranes was inhibited by acetazolamide, furosemide and KSCN. The molecular mass of the carbonic anhydrase was 33 kDA as estimated by its binding of [14C]furosemide followed by polyacrylamide gel electrophoresis. Previous suggestions of a role of carbonic anhydrase as a supplier of H+ in the secretion of acid are supported by its high activity and its localization to the same membrane as the acid pump of the stomach.

Localization of Carbonic Anhydrase in Mesophyll Cells of Terrestrial C<sub>3</sub> Plants in Relation to CO<sub>2</sub> Assimilation

Localization of Carbonic Anhydrase in Mesophyll Cells of Terrestrial C<sub>3</sub> Plants in Relation to CO<sub>2</sub> Assimilation

Localization of carbonic anhydrase activity in the developing rat colon

Carbonic anhydrase activity was localized histochemically by light and electron microscopy in the proximal and distal colon of developing rats. Fixed tissue was taken for normal morphology and carbonic anhydrase localization from fetal (20–22 days gestation), suckling (1–19 days postnatal), weanling (20–25 days postnatal), and adult rats. The proximal colon had distinct villi at birth which were diminished between days 5 and 11 postnatally. The distal colon lacked villi at birth but had rudimentary crypts (ridges and furrows) which were replaced during the suckling period by a flat mucosa interspersed with true crypts. Carbonic anhydrase first appeared in both proximal and distal colonic epithelial cells on the day of birth (22 days gestation). Goblet cells were nonreactive at each developmental period. In neonatal rats, epithelial cells in the upper half of the villi of the proximal colon and on the surface and upper crypts of the distal colon were positive for carbonic anhydrase throughout the cytoplasm. Cells at the villar base (proximal colon) or in the deep crypt (distal colon) had reaction product in the intercellular spaces but not the cytoplasm. By 11 days postnatal, cytoplasmic reaction product was present in proximal colonic cells in the upper three-fourths of the crypt and was concentrated in a heavy band in the apical cytoplasm. In the distal colon, cytoplasmic positive cells did not extend as deeply into the crypts and the apical banding pattern was weak. Intercellular spaces in the deeper crypt epithelium were positive in both proximal colon and distal colon, suggesting a membrane-bound carbonic anhydrase. It was concluded that carbonic anhydrase appeared suddenly at birth and was continuously present in mid- to upper-crypt (or upper villus in early neonatal proximal colon) nongoblet cells into adulthood. This suggests a functional role for carbonic anhydrase in chloride-bicarbonate exchange across the neonatal and adult colonic mucosa.

Carbonic Anhydrase Histochemistry: A Potential Diagnostic Method for Peripheral Nerve Repair

Many large-diameter myelinated axons in spinal dorsal roots contain carbonic anhydrase activity, whereas few small-diameter ventral root axons stain for this enzyme. This differential localization of carbonic anhydrase in sensory and motor nerve fibers is indicative of the potential of carbonic anhydrase histochemistry to provide a convenient method for identifying predominantly motor or sensory fascicles in cut ends of peripheral nerves, thereby facilitating coaptation of fascicles in peripheral nerve repair.

A fast screening method for histochemical localization of carbonic anhydrase. Application to kidney, skeletal muscle, and thrombocytes.

A simple method for histochemical localization of carbonic anhydrase using 5-dimethyl-amino-naphthalene-1-sulfonamide (DNSA) is described. Cryosections of tissues, or cell smears, are incubated in 3 to 10 X 10(-5) M DNSA and viewed in a fluorescence microscope. Upon excitation with ultraviolet light, sites of carbonic anhydrase localization can be identified by an intense blue fluorescence, which is due to the emission of blue light (lambda max = 470 nm) by carbonic anhydrase-DNSA complexes. This fluorescence can be largely suppressed by simultaneous incubation with 1 X 10(-4) to 2 X 10(-3) M concentrations of nonfluorescent carbonic anhydrase inhibitors, displacing DNSA from its binding site on the enzyme. Application of the method to kidney, skeletal muscle, and thrombocytes yields patterns of carbonic anhydrase localization that are in good agreement with results that have been obtained with a variety of other techniques.

Carbonic anhydrase in the rat salivary glands: distribution and ultrastructural localization.

Rat salivary glands were studied by Hanson's method to specify the ultrastructural localization of carbonic anhydrase (CA). Two different procedures were used: 1) The embedding of the tissues in water-soluble resins, followed by the incubation of the resin sections on the medium. 2) The embedding in epon-araldite of previously incubated frozen sections. Light and electron microscopy were used to observe the distribution and the ultrastructural localization of the cobalt precipitate. In parotid and mandibular glands, CA was localized in the secretion granules and the hyaloplasma of the secretory endpieces. The enzyme was also detected on the basal and lateral membranes of the striated duct cells in the three glands. In the convoluted granular duct cells of the mandibular gland CA was found in the hyaloplasma only. In the sublingual gland, CA was localized in the hyaloplasma of the serous crescents and no activity was detected in the mucous tubules. As regards the localization of the enzyme in the granules of the secretory endpieces of parotid and mandibular glands, it appears that CA has to be considered as a secretory product of these cells; this localization is consistent with the presence of the enzyme in rat saliva.

Ultrastructural localization of carbonic anhydrase in lysosomes.

Ultrastructural localization of carbonic anhydrase was determined by applying Hansson's histochemical method to glutaraldehyde-fixed frozen sections of guinea pig peritoneal polymorphonuclear leukocytes (PMNs) and lysosomes isolated from rat liver tissue after the animal had been injected with Triton WR-1339. A positive histochemical reaction for carbonic anhydrase in PMNs was found in the matrix of lysosomes. After PMNs phagocytized polystyrene latex particles or emulsified paraffin oil droplets, a positive reactivity for carbonic anhydrase was found in the space between the lysosomal membrane and the particle. Liver lysosomes also revealed positive carbonic anhydrase histochemical reactivity. To confirm the histochemical reaction, indirect immunoferritin labeling was conducted with rabbit antibody to human red blood cell carbonic anhydrase C on glutaraldehyde-fixed, freeze-thawed human PMNs. Immunolabeling was observed in lysosomes. These results suggest that carbonic anhydrase is a constituent of lysosomes of PMNs and liver cells.

Immunocytochemical localization of carbonic anhydrase on ultrathin frozen sections with protein A-gold.

The protein A-gold technique was used to localize carbonic anhydrase isozymes on ultrathin frozen sections of kidney collecting duct epithelial cells and erythrocytes. The particulate nature of the gold marker gives a more precise appreciation of the intracellular distribution of this enzyme than has been previously possible, and allows the intensity of the labeling to be quantified. Intercalated cells showed four times more labeling over the cytosol than adjacent principal cells in collecting ducts from the inner stripe of the outer medulla: by double-labeling using protein A-gold particles of different sizes, carbonic anhydrase isozymes B and C were simultaneously localized in erythrocytes.

Oligodendroglial structures and distribution shown by carbonic anhydrase immunostaining in the spinal cords of developing normal and shiverer mice.

The spinal cords of young and adult normal and dysmyelinating mutant (shiverer) mice were immunostained with anticarbonic anhydrase to investigate the distribution of oligodendroglial populations into the gray- and white-matter regions in the developing normal and mutant animals; the morphology of oligodendrocytes and their processes at the light microscopic level in gray matter and white matter; and the apparent gliosis in the gray matter, as well as the white matter, of the mutants. Immunocytochemistry and enzyme assays revealed consistent increases in carbonic anhydrase antigenicity and specific activity in controls and mutants between the ages of approximately 15 days and approximately 60 days. As shown previously in adult animals, oligodendroglia in larger than normal proportions were situated at the periphery of the "white-matter" columns, as compared to gray matter, in the shiverers, with, however, significant numbers of oligodendroglia were heterogeneous with respect to shapes, configuration of processes, and intensity of carbonic anhydrase immunostaining. In the shiverer "white matter" the oligodendrocytes were smaller than normal, and their shapes and arrangement were relatively irregular. In the normal gray matter short oligodendroglial processes appeared to be associated with neuronal perikarya, and those processes were more pronounced at approximately 90 days than at approximately 20 days of age. Background staining in normal gray matter suggested that oligodendroglial processes were, in addition, tightly wound around many axons. In shiverer gray matter the oligodendrocytes were smaller, and their processes appeared to be wrapped more loosely around smaller numbers of conspicuous axons and to be associated less frequently with neuronal perikarya. This finding suggests that the deficiency in the myelin basic protein in the mutant may affect interactions between oligodendrocytes and neurons in the gray matter as well as in the white matter. The astrocytic "marker," glial fibrillary acidic protein, was detected in gray and white matter of shiverers as young as 16 days, and the differences from carbonic anhydrase localization supported the conclusion that the processes enwrapping axons in the shiverer mouse CNS are derived from oligodendrocytes, not astrocytes.

‘Chloride-cell’ — like mitochondria-rich cells of salamander larva gill epithelium

Two types of mitochondria-rich cells (MRC) are described ultrastructurally in the gill epithelium of salamander larva. They resemble MRC found in larval ventral epidermis. Histochemical localization of carbonic anhydrase indicated numerous positive reacting cells, most of them flask-shaped. Morphological and functional similarities to fish ‘chloride cells’ are discussed.

Carbonic anhydrase in mouse salivary glands and saliva: a histochemical, immunohistochemical, and enzyme activity study.

Mouse parotid gland and saliva were studied by histochemical, immunohistochemical, and activity measurements for carbonic anhydrase. Hansson 's histochemical reaction for carbonic anhydrase revealed positive enzyme activity in the parotid acinar cell cytoplasm and little or no reaction in the secretory granules. The luminal contents in all of the glandular duct systems also reacted positively, but the duct cells themselves were only weakly positive. Ultrastructural observations confirmed the light microscope histochemical localization and, in addition, revealed luminal content activity in intercellular ducts. Purified carbonic anhydrase isolated from mouse salivary glands was used to raise antibodies in rabbits. Localization of carbonic anhydrase by direct immunolabeling with fluorescein-coupled antibody and indirect immunoperoxidase labeling revealed enzyme localization on or in the acinar cell secretory granule membrane and not in the surrounding cytoplasm. The luminal contents of the intercalated and striated ducts were also strongly positive. Stimulation of salivary secretion with phenylephrine or pilocarpine increased the amount of carbonic anhydrase in saliva. Acetatazolamide and potassium cyanate inhibited carbonic anhydrase activity. Reasons underlying the discrepancy between the histochemical and immunolabeling localization of carbonic anhydrase are discussed. It is concluded that the parotid acinar cells of mice appear to be a significant source of carbonic anhydrase in saliva but its role is enigmatic.