A modified semipermeable membrane technique for carbonic anhydrase histochemistry of the nervous system

We present a modification of Hansson's method for the demonstration of carbonic anhydrase activity. Using a semipermeable membrane together with a fluid incubation medium, frozen sections of aldehyde-fixed tissue were incubated without floating or dipping. Thin sections (thickness, 20-40 microns) were mounted on the outer surface of a tubular-shaped, semipermeable cellophane dialysis membrane containing the incubation fluid. After incubation for 25-30 min at room temperature, the sections were rinsed in buffer and treated with 0.5% (NH4)2S solution. The histochemical reaction was fully inhibited by 10(-4) M acetazolamide.     

Novel method for performing carbonic anhydrase histochemistry and immunocytochemistry on cryosections.

It is difficult to correlate structure with function in the kidney because of the extensive cell heterogeneity. Carbonic anhydrase (CA) is an enzyme that mediates renal acidification and is found predominantly in proximal tubule and collecting duct cells. We modified Hansson's method for histochemically identifying cellular CA activity on PLP-fixed rabbit kidney sections mounted on Millipore filters, and then removed the filters to perform peanut lectin and antibody labeling on the same sections. There was adequate preservation of morphology, and individual cells could be identified with CA activity in the cytosol and specific antibody or lectin labeling on the cell surfaces.     

Light microscopic histochemistry of the postnatal development and localization of carbonic anhydrase activity in glial and neuronal cell types of the rat central nervous system

Summary The postnatal expression of carbonic anhydrase (CA) activity in glia and neurons was investigated by a modified light microscopical method of Hansson. Strong CA activity was observed during the first postnatal week in ameboid microglia, clustering in the cingulum, the periaqueductal region, the roof of the lateral ventricles and the white matter of the cerebellum. The intensity of staining gradually decreased during the second week and finally disappeared. From the 9th postnatal day on, cerebellar Purkinje cells expressed strong CA activity, which completely disappeared by the end of the investigation period. CA staining of the oligodendrocytes and pericytes could be observed from the first postnatal day on. The present results raised the possibility that carbonic anhydrase activity may play a role in the regulation of the development and proliferation of some cell types, perhaps via intracellular pH changes.     

Mitochondrial carbonic anhydrase in the nervous system: expression in neuronal and glial cells

Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C-terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30-kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell-specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion-induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.     

Light microscopic histochemistry of the postnatal development and localization of carbonic anhydrase activity in glial and neuronal cell types of the rat central nervous system

The postnatal expression of carbonic anhydrase (CA) activity in glia and neurons was investigated by a modified light microscopical method of Hansson. Strong CA activity was observed during the first postnatal week in ameboid microglia, clustering in the cingulum, the periaqueductal region, the roof of the lateral ventricles and the white matter of the cerebellum. The intensity of staining gradually decreased during the second week and finally disappeared. From the 9th postnatal day on, cerebellar Purkinje cells expressed strong CA activity, which completely disappeared by the end of the investigation period. CA staining of the oligodendrocytes and pericytes could be observed from the first postnatal day on. The present results raised the possibility that carbonic anhydrase activity may play a role in the regulation of the development and proliferation of some cell types, perhaps via intracellular pH changes.     

A proposal for the mechanism of carbonic anhydrase action

A new catalytic mechanism is proposed for the hydration of CO₂ by the zinc metalloenzyme carbonic anhydrase. This mechanism identifies the group controlling catalytic activity as an active site histidine, in which the protonated imidazole ring coordinates to zinc, losing a proton. Geometric constraints on the histidine unit make the metal-ligand bond a strained and, therefore, labile one. In the hydration reaction, the metal-bound neutral histidine moiety serves as a proton acceptor for the transient ionization of metal-bound water. Zinc-bound hydroxide attacks the carbon of the substrate to generate metal-bound bicarbonate, and the system regenerates itself by losing the elements of carbonic acid.     

Demonstration of acetylcholinesterase by semipermeable membrane technique: Estimation of soluble and fixation-labile portions in different regions of the central nervous system

Summary A histochemical method for the demonstration of acetylcholinesterase using semipermeable membranes is described. This technique prevents any loss of enzyme activity caused by dissolution and/or fixation. The soluble and fixation-labile portions of acetylcholinesterase were estimated in several regions of the central nervous system of the rat and differences were found. The method improves the accuracy of the histochemical demonstration of the acetylcholinesterase on the light microscopical level.The research reported in this paper was supported by the Ministerium für Wissenschaft und Technik der DDR     

A novel carbonic anhydrase from the giant clam Tridacna gigas contains two carbonic anhydrase domains

This report describes the presence of a unique dual domain carbonic anhydrase (CA) in the giant clam, Tridacna gigas. CA plays an important role in the movement of inorganic carbon (Ci) from the surrounding seawater to the symbiotic algae that are found within the clam's tissue. One of these isoforms is a glycoprotein which is significantly larger (70 kDa) than any previously reported from animals (generally between 28 and 52 kDa). This alpha-family CA contains two complete carbonic anhydrase domains within the one protein, accounting for its large size; dual domain CAs have previously only been reported from two algal species. The protein contains a leader sequence, an N-terminal CA domain and a C-terminal CA domain. The two CA domains have relatively little identity at the amino acid level (29%). The genomic sequence spans in excess of 17 kb and contains at least 12 introns and 13 exons. A number of these introns are in positions that are only found in the membrane attached/secreted CAs. This fact, along with phylogenetic analysis, suggests that this protein represents the second example of a membrane attached invertebrate CA and it contains a dual domain structure unique amongst all animal CAs characterized to date.     

A systematic quantification of carbonic anhydrase transcripts in the mouse digestive system

Background  

Carbonic anhydrases (CAs) are physiologically important enzymes which participate in many gastrointestinal processes such as acid and bicarbonate secretion and metabolic pathways including gluconeogenesis and ureagenesis. The genomic data suggests that there are thirteen enzymatically active members of the mammalian CA isozyme family. In the present study, we systematically examined the mRNA expression levels of all known CA isozymes by quantitative real-time PCR in eight tissues of the digestive system of male and female mice.     

Evidence for the presence of carbonic anhydrase in the plasma membrane of rat hepatocytes

The cellular distribution of carbonic anhydrase is a key characteristic for the role of the enzyme in cell function. In several epithelia involved in bicarbonate transport this enzyme is located in the plasma membrane. Because bicarbonate secretion is an important mechanism in bile formation by the liver, we investigated the presence of carbonic anhydrase activity in isolated plasma membranes from rat hepatocytes. Carbonic anhydrase activity was enriched 1.79-fold in plasma membrane preparations. This activity was inhibited by acetazolamide and activated by Triton X-100, but was insensitive to Cl- or CNO-. It is highly unlikely that the low contamination of cytoplasm and intracellular membranes could account for the presence of carbonic anhydrase activity in plasma membrane preparations. Moreover, the results from resuspension/washing of plasma membrane fractions in ionic media suggest an absence of soluble carbonic anhydrase adsorption upon plasma membrane. Accordingly, the present findings provide strong evidence for the presence of carbonic anhydrase in the plasma membrane of rat hepatocytes.     

Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CA VI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension [Fernley, R. T., Wright, R. D., & Coghlan, J. P. (1988b) Biochemistry 27, 2815]. Overall the human CA VI protein has a sequence identity of 35% with human CA II, while residues involved in the active site of the enzymes have been conserved. The human sheep secreted carbonic anhydrases have a sequence identity of 72%. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.     

A role for iron in an ancient carbonic anhydrase

Since 1933, carbonic anhydrase research has focused on enzymes from mammals (alpha class) and plants (beta class); however, two additional classes (gamma and delta) were discovered recently. Cam, from the procaryote Methanosarcina thermophila, is the prototype of the gamma class and the first carbonic anhydrase to be characterized from either an anaerobic organism or the Archaea domain. All of the enzymes characterized from the four classes have been purified aerobically and are reported to contain a catalytic zinc. Herein, we report the anaerobic reconstitution of apo-Cam with Fe2+, which yielded Cam with an effective kcat that exceeded that for the Zn2+-reconstituted enzyme. M?ssbauer spectroscopy showed that the Fe2+-reconstituted enzyme contained high spin Fe2+ that, when oxidized to Fe3+, inactivated the enzyme. Reconstitution with Fe3+ was unsuccessful. Reconstitution with Cu2+, Mn2+, Ni2+, or Cd2+ yielded enzymes with effective kcat values that were 10% or less than the value for the Zn2+-reconstituted Cam. Cam produced in Escherichia coli and purified anaerobically contained iron with effective kcat and kcat/Km values exceeding the values for Zn2+-reconstituted Cam. The results identify a previously unrecognized biological function for iron.     

N-Nitrosulfonamides: A new chemotype for carbonic anhydrase inhibition

A series of N¹-substituted aromatic sulfonamides was obtained by applying a selective sulfonamide nitration synthetic strategy leading to Ar-SO₂NHNO₂ derivatives which were investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Two human (h) hCA isoforms, the cytosolic hCA II and the transmembrane hCA IX, in addition to the fungal enzyme from Malassezia globosa, MgCA, were included in the study. Most of the new compounds reported selectively inhibited hCA IX over hCA II and at the same time showed effective MgCA inhibitory properties, with K_Is ranging between 0.22 and 8.09 μM. The N-nitro sulfonamides are a new chemotype with CA inhibitory effects. As hCA IX was recently validated as antitumor/antimetastatic drug target, its selective inhibition could be exploited for interesting biomedical applications. Moreover, due to the effective MgCAs inhibitory properties of the N-nitro sulfonamides, of considerable interest in the cosmetics field as potential anti-dandruff agents, the N-nitro sulfonamides may be considered as interesting leads for the design of more efficient compounds targeting fungal enzymes.     

The histochemical demonstration of choline acetyltransferase by semipermeable membrane technique

Summary The application of the semipermeable membrane technique in light microscopical demonstration of choline acetyltransferase is described. The method founds upon earlier developed lead salt techniques. Use of semipermeable membranes fully prevents any loss of enzyme by dissolvement or inactivation during fixation. Addition of NaCl to the incubation medium markedly increases the activity of choline acetyltransferase.The research reported in this paper was supported by the Ministerium für Wissenschaft und Technik der DDR     

A quantitative cytochemical method for measuring carbonic anhydrase activity

Synopsis Components of a histochemical method for demonstrating carbonic anhydrase activity have been investigated quantitatively. It was found that it is not necessary to use free-floating sections provided the reaction is done in a reaction medium of controlled depth. This permits the use of normal cryostat sections on glass slides, so making this technique applicable to the cytochemical bioassay of gastrin. The better control of the pH of the reaction, and changes in the concentration of phosphate and of cobalt, have resulted in a quantitatively reproducible reaction in the parietal cells of guinea-pig fundus. The reaction product is measured by microdensitometry. The specificity of the carbonic anhydrase reaction has been tested by the response elicited by gastrin acting on the parietal cellsin vitro and by the use of acetazolamide.     

Rapid separation of carbonic anhydrase isozymes using cellulose acetate membrane electrophoresis

A method is described for the rapid separation of carbonic anhydrase(CA) isozymes by cellulose acetate membrane electrophoresisin which CA activity is detected using the pH-indicating dye,bromcresol purple. This method can detect bovine erythrocyteCA in a 0.3 mm³ sample applied at a concentration of 100 ngcm^–3 (total of 30 pg applied) while at higher concentrationsthree isozymes were observed. It was found, using a potentiometrictechnique, that intact cells of Anabaena flos-aquae (Cyanophyceae)and Chlorella ellipsoidea had no detectable activity while C.saccharophila and Chlamydomonas reinhardtii (Chlorophyceae)had external CA activity. CA activity of the extracts suggestedthe presence of internal CA in all species. After electrophoresisit was found that C. saccharophila and C. reinhardtii had twoisozymes while A. flos-aquae and C. ellipsoidea had only a singledetectable band. Spinach had up to five detectable isozymesthat were difficult to resolve. Incubation of spinach extractwith the CA inhibitor ClO₄^– (500 mol m^–3) inhibitedCA activity by 90% using the potentiometric technique, but afterelectrophoresis had no detectable effect. This technique isuseful in identifying isozymes that are substantially differentin electrical charge and in monitoring CA isozyme activity duringenzyme purification. Key words: Carbonic anhydrase, isozymes, cyanobacteria, microalgae, spinach