Differential expression of cytoplasmic carbonic anhydrases, CA I and II, and membrane-associated isozymes, CA IX and XII, in normal mucosa of large intestine and in colorectal tumors.

This study compares the localization of carbonic anhydrase isozymes (CA) I and II and that of IX and XII in normal large intestine and in colorectal tumors. Immunohistochemical studies were performed on 69 colorectal lesions. While the normal mucosa of the large intestine showed high expression for CA I and II, the intensity of the immunostaining for both isozymes decreased in benign lesions and was very weak in malignant tumors. The reciprocal pattern of expression observed for these cytoplasmic isozymes and transmembrane CA IX and XII in intestinal tissue specimens supports the suggestion that CA IX and XII may be functionally involved in tumor progression to malignancy and/or in invasion. By contrast, while CA I and II are prominent in normal colorectal mucosa, where they play a role in regulation of pH homeostasis and water and ion transport, loss of expression of these cytoplasmic isozymes consistently accompanies progression to malignant transformation.

The identification of secreted carbonic anhydrase VI as a constitutive glycoprotein of human and rat milk.

In addition to essential nutrients, human milk contains several classes of bioactive factors such as enzymes, hormones, and growth factors, many of which are implicated in infantile growth and development. Secretory carbonic anhydrase isoenzyme VI (CA VI) has been identified earlier as an essential component of mammalian saliva, and we demonstrate here by using biochemical and immunohistochemical techniques that it is also an elementary component of milk. The 42-kDa glycopolypeptide purified from human milk in CA inhibitor affinity chromatography shared 100% homology with salivary CA VI in the protein sequence analysis (40% coverage), and its digestion with PNGase F resulted in a polypeptide backbone similar in size to salivary CA VI. Quantification of CA VI in milk by using a time-resolved immunofluorometric assay revealed an approximately eight-times-higher concentration in human colostrum than in mature milk, the latter corresponding to the levels previously detected in human saliva. The high concentration in the colostrum, in particular its functional and structural stability in an acidic milieu, and its growth-supporting role in the taste buds suggest that milk CA VI is an essential factor in normal growth and development of the infant alimentary tract.

An apical membrane Na+/H+ exchanger isoform, NHE-3, is present in the rat epididymal epithelium.

An acidic milieu is required for sperm maturation and for keeping sperm quiescent during storage in the cauda epididymidis. Previous studies have implicated a Na+/H+ exchanger (NHE) in epididymal acidification together with carbonic anhydrase (CA) and vacuolar proton adenosine triphosphatase (H(+)-ATPase). The present studies were undertaken to discover whether the NHE isoform involved is NHE-3, which is known to mediate Na+ and HCO3- absorption in renal tubules. Using the reverse transcription polymerase chain reaction technique (RT-PCR), Northern blot analysis and in situ hybridization, NHE-3 mRNA was detected mainly in the cauda epididymis and to a lesser extent in other regions of the epididymis. Immunohistochemical studies showed that NHE-3 was present in the apical membranes of the epithelial principal cells and confirmed that its expression is strongest in the cauda region, decreasing towards the more proximal regions. Immunoblotting showed a similar expression pattern. These results demonstrate that NHE-3 is expressed in the rat epididymal duct with strongest expression in its cauda region. These findings are thus consistent with the possibility that NHE-3 in the epididymal duct is involved in luminal Na+ and/or HCO3- absorption, as in the renal proximal tubule, and thereby in the regulation of sperm motility and maturation.

Expression of the transmembrane carbonic anhydrases, CA IX and CA XII, in the human male excurrent ducts.

Testicular fluid is concentrated and acidified during its passage through the excurrent ducts. These processes involve bicarbonate absorption, in which carbonic anhydrases are implicated. In this study, the distribution of two transmembrane carbonic anhydrase isozymes (CA IX and CA XII) in the human excurrent ducts was investigated using isozyme-specific antibodies in conjunction with immunohistochemical and immunoblotting techniques. Specific staining for CA XII was present in the basolateral plasma membrane of the epithelial cells in the efferent ducts, predominantly in the non-ciliated cells. In the epididymal duct, CA XII was detected only in sporadic cells, which also contained CA II, thus suggesting that they are apical mitochondria-rich cells. CA IX was also localized to the basolateral plasma membrane of the epithelium in the efferent ducts, but its staining was weaker and less uniform compared to CA XII. No signal for CA IX was detected in the epididymal duct. Western blot analysis from efferent duct samples revealed specific bands for CA IX and CA XII, confirming that the immunohistochemical stainings represent these isozymes. The expression of CA XII and CA IX in the excurrent duct system and co-expression of CA XII with Aquaporin-1 in the same efferent duct epithelial cells suggest their functional involvement in ion transport and concentration processes of testicular fluid.

Secretion of carbonic anhydrase isoenzyme VI (CA VI) from human and rat lingual serous von Ebner's glands.

Salivary carbonic anhydrase VI (CA VI) appears to contribute to taste function by protecting taste receptor cells (TRCs) from apoptosis. The serous von Ebner's glands locating in the posterior tongue deliver their saliva into the bottom of the trenches surrounding the TRC-rich circumvallate and foliate papillae. Because these glands deliver their saliva directly into the immediate vicinity of TRCs, we investigated whether CA VI is secreted by the von Ebner's glands, using immunochemical techniques. The immunohistochemical results showed that CA VI is present in the serous acinar cells, ductal cells, and ductal content of von Ebner's glands and in the demilune and ductal cells plus ductal content of rat lingual mucous glands. More importantly, CA VI was also detected in taste buds and in the taste pores. Western blotting of saliva collected from the orifices of human von Ebner's glands and CAs purified from rat von Ebner's glands confirmed that CA VI is expressed in these glands and secreted to the bottom of the trenches surrounding the circumvallate and foliate papillae. These findings are consistent with the hypothesis that locally secreted CA VI is implicated in the paracrine modulation of taste function and TRC apoptosis. (J Histochem Cytochem 49:657-662, 2001)

Expression of membrane-associated carbonic anhydrase XIV on neurons and axons in mouse and human brain.

Although long suspected from histochemical evidence for carbonic anhydrase (CA) activity on neurons and observations that CA inhibitors enhance the extracellular alkaline shifts associated with synaptic transmission, an extracellular CA in brain had not been identified. A candidate for this CA was suggested by the recent discovery of membrane CA (CA XIV) whose mRNA is expressed in mouse and human brain and in several other tissues. For immunolocalization of CA XIV in mouse and human brain, we developed two antibodies, one against a secretory form of enzymatically active recombinant mouse CA XIV, and one against a synthetic peptide corresponding to the 24 C-terminal amino acids in the human enzyme. Immunostaining for CA XIV was found on neuronal membranes and axons in both mouse and human brain. The highest expression was seen on large neuronal bodies and axons in the anterolateral part of pons and medulla oblongata. Other CA XIV-positive sites included the hippocampus, corpus callosum, cerebellar white matter and peduncles, pyramidal tract, and choroid plexus. Mouse brain also showed a positive reaction in the molecular layer of the cerebral cortex and granular cellular layer of the cerebellum. These observations make CA XIV a likely candidate for the extracellular CA postulated to have an important role in modulating excitatory synaptic transmission in brain.

Expression of the membrane-associated carbonic anhydrase isozyme XII in the human kidney and renal tumors.

Carbonic anhydrase isozyme XII (CA XII) is a novel membrane-associated protein with a potential role in von Hippel-Lindau carcinogenesis. Although Northern blotting has revealed positive signal for CA XII in normal human kidney, this is the first study to demonstrate its cellular and subcellular localization along the human nephron and collecting duct. Immunohistochemistry with a polyclonal antibody (PAb) raised against truncated CA XII revealed distinct staining in the basolateral plasma membrane of the epithelial cells in the thick ascending limb of Henle and distal convoluted tubules, and in the principal cells of the collecting ducts. A weak basolateral signal was also detected in the epithelium of the proximal convoluted tubules. In addition to the normal kidney specimens, this immunohistochemical study included 31 renal tumors. CA XII showed moderate or strong plasma membrane-associated expression in most oncocytomas and clear-cell carcinomas. The segmental, cellular, and subcellular distribution of CA XII along the human nephron and collecting duct suggests that it may be one of the key enzymes involved in normal renal physiology, particularly in the regulation of water homeostasis. High expression of CA XII in some renal carcinomas may contribute to its role in von Hippel-Lindau carcinogenesis.

Expression of transmembrane carbonic anhydrase isoenzymes IX and XII in normal human pancreas and pancreatic tumours.

Carbonic anhydrase (CA) IX and XII are transmembrane isoenzymes which are expressed in several epithelia and overexpressed in some carcinomas. They have recently been linked to von Hippel-Lindau gene-mediated carcinogenesis in that both isoenzymes are downregulated by the product of the wild-type von Hippel-Lindau tumour suppressor gene. This paper describes the localisation of CA IX and XII in the normal human pancreas and pancreatic tumours. Both isoenzymes showed positive reaction in the basolateral plasma membrane of the normal acinar and ductal epithelia. The hyperplastic ductal epithelium in tumour specimens generally showed an increased staining for CA IX. Of 29 malignant tumours of exocrine pancreas, 10 showed moderate or strong immunoreaction for CA IX. The signal for CA XII remained weak in most malignant lesions. The present results show that both CA IX and XII are unevenly expressed in the ductal and acinar compartments of the human pancreas. The expression of these isoenzymes in a relatively low number of malignant tumour specimens suggests that they have a limited value in diagnostic evaluation of pancreatic carcinoma. However, the increased expression of CA IX in hyperplastic ductal epithelium may contribute to the pancreatic tumourigenesis.

Nuclear NonO/p54(nrb) protein is a nonclassical carbonic anhydrase.

The growing carbonic anhydrase (CA) gene family includes 11 enzymatically active isozymes in mammals. Each of them has a characteristic cellular and subcellular distribution pattern. In this report, we demonstrate for the first time a nuclear protein with CA activity. A polypeptide recognized by CA II antibodies was purified from several rat tissues using CA inhibitor affinity chromatography. This polypeptide of apparent 66 kDa mass was characterized using amino acid sequencing and CA activity measurements. It appeared to be identical to nonO/p54(nrb), a previously cloned and characterized RNA and DNA binding nuclear factor. Recombinant nonO generated in baculovirus bound to the CA inhibitor affinity chromatography matrix and revealed detectable CA activity (25 units/mg). Hansson's histochemical staining of rat lymph nodes followed by light and electron microscopy showed nuclear CA activity in lymphocytes, suggesting that the nuclear nonO protein is catalytically active in vivo. These results demonstrate that a previously known transcription factor is a novel, nonclassical CA. Through its CA activity, the nonO may function in the maintenance of pH homeostasis in the nucleus.

Characterization of the major DNA adducts in the liver of rats chronically exposed to tamoxifen for 18 months.

Our previous study has shown that chronic exposure to tamoxifen (TAM) induced formation of high levels of DNA adducts in the liver, the target tissue of TAM-induced carcinogenesis in rats. One of the major DNA adducts (spot 1), as detected by 32P-postlabeling, accounted for 53% of the total adducts. To characterize this major adduct, the current study has compared spot 1 with two previously identified TAM-DNA adducts, i.e. alpha-TAM-N2-deoxyguanine (alpha-TAM-N2-dG) and alpha-N-desmethyl TAM-N2-deoxyguanine (alpha-N-dmTAM-N2-dG) by various rechromatography methods. It was found that spot 1 was further resolved into two fractions during rechromatography analysis, one fraction co-migrated with the alpha-TAM-N2-dG and the other fraction co-migrated with the alpha-N-dmTAM-N2-dG. These findings have demonstrated that chronic exposure to tamoxifen induced the same major DNA adducts, i.e. alpha-TAM-N2-dG and alpha-N-dmTAM-N2-dG as those detected in acutely exposed rats.

Carbonic anhydrase inhibitor suppresses invasion of renal cancer cells in vitro.

Acidification of the extracellular milieu of malignant tumors is reported to increase the invasive behavior of cancer cells. In normal tissues, production of acid is catalyzed by carbonic anhydrases (CAs), some of which are known to be overexpressed in certain cancers. To investigate the functional role of CA activity in such cancer cells, we analyzed the effect of acetazolamide, a potent CA inhibitor, on the invasive capacity of four renal carcinoma cell lines (Caki-1, Caki-2, ACHN, and A-498). We found that 10 microM acetazolamide inhibited the relative invasion rate of these cell lines between 18-74%. The Caki-2 and ACHN cell lines displayed the highest responsiveness, and their responses clearly depended on the acetazolamide concentration in the culture medium. Immunocytochemical and Western blotting results identified the presence of CA isoenzyme II in the cytoplasm of all four cell lines and CA XII on the plasma membrane in three of four cell lines. Because acetazolamide alone reduced invasiveness of these cancer cells in vitro, we conclude that the CAs overexpressed in these renal cancer cells contribute to invasiveness, at least in vitro, and suggest that CA inhibitors may also reduce invasiveness in other tumors that overexpress one or more CAs.

Expression of a novel transmembrane carbonic anhydrase isozyme XII in normal human gut and colorectal tumors.

Carbonic anhydrase isozyme XII is a recently discovered member of the alpha-carbonic anhydrase gene family with a suggested role in von Hippel-Lindau gene-mediated carcinogenesis. Increased expression of its mRNA has been observed in renal and lung carcinomas. This paper presents the localization of CA XII in the normal human gut and in colorectal tumors. Immunohistochemistry performed using a polyclonal antibody raised against truncated CA XII revealed prominent polarized staining for CA XII in the basolateral plasma membrane of the enterocytes of the normal large intestine, the reaction being most intense in the surface epithelial cuff region. Most colorectal tumors displayed abnormal expression of CA XII; the most dramatic change was observed in the deep parts of the adenomatous mucosa, where the positive immunoreaction clearly increased along with the grade of dysplasia. Adenomas with severe dysplasia and carcinomas showed an equal, diffuse staining pattern. The results indicate region-specific regulation of CA XII expression along the cranial-caudal axis of the human gut, whereas its diffuse expression in the most malignant tumors seems to correlate with their biological behavior.

Dna adducts, mutations, and cancer 2000.

The main achievements in the DNA adduct field in the 1990s have been technical innovations of methods for specific adducts reaching sensitivities required for low levels encountered in humans. Over 20 specific adducts or closely related groups of adducts have been determined in humans. The sources of the DNA-binding agents are endogenous and exogenous or both. In some of these studies adduct levels have been correlated to metabolic or DNA repair genotypes. An example of DNA adduct studies in human target tissue is taken on UV photoproducts in skin in situ. Adduct-induced mutations, specific mutation spectra, and their relationship to cancer are discussed. The quantitative adduct techniques will enable comparisons of endogenous and exogenous adduct levels and will give important clues to the etiology of human cancer. Furthermore, adducts will provide an intermediary tool for genotyping studies, both for metabolic enzyme and for DNA repair system genotypes. As the common polymorphisms are likely to cause at most moderate increases in the risk of cancer, the intermediary adduct endpoint is a necessary proof of causal relationships. The present and future biomonitoring studies will cover many endpoints to link the mechanistic steps from DNA adducts to cancer via mutations and modulating host susceptibility factors.

Identification of carbonic anhydrase XII as the membrane isozyme expressed in the normal human endometrial epithelium.

Although previous studies demonstrated carbonic anhydrase (CA) activity in the human endometrium, the CA isozyme(s) responsible for this activity has not been established. In this report, we provide the first evidence that the CA isozyme XII, a recently identified transmembrane isozyme that is expressed in normal kidney and greatly overexpressed in some renal cancers, is present in endometrium. We show by immunohistochemistry that CA XII is expressed in the basolateral plasma membrane of epithelial cells of normal human endometrium. Expression of CA XII in uterus was confirmed by Northern blotting. Detergent-solubilized CA XII was isolated from human endometrium by inhibitor affinity chromatography and characterized by isoelectric focusing and Western blot as a polypeptide with a pI of 6.3. The high expression of CA XII in the endometrial epithelium suggests that it may be functionally linked to the pH-dependent events in spermatozoa that precede fertilization. Its basolateral location and extracellular active site could also allow it to influence the morphological changes in endometrium that occur during the menstrual cycle.

Regional expression and androgen regulation of carbonic anhydrase IV and II in the adult rat epididymis.

Carbonic anhydrase (CA) is implicated in the acidification of epididymal fluid and thereby in the regulation of sperm maturation and motility. Among the CA isoenzymes, CA IV and II have been shown to be present in the rat epididymal duct epithelium. In the present study, we examined the expression and androgen regulation of CA IV and II mRNAs along the epididymal duct. Northern blot analysis revealed the presence of CA II mRNA in all regions of the epididymis with the strongest signal in the corpus region, while CA IV mRNA was expressed predominantly in the corpus epididymidis. Three days after bilateral castration, CA IV and II mRNAs were decreased by 80-90% in the corpus epididymidis. Testosterone (T) replacement maintained the expression of CA mRNAs at 50-60% of the control levels, indicating that circulating androgens alone are not sufficient to recover the CA expression in the corpus region. However, unilateral castration did not affect the mRNA levels of CA IV and II, suggesting that factors in testicular fluid do not play a major role in the regulation of CA expression in the corpus epididymidis. Immunoblot analysis showed that CA IV protein levels decreased 3 days after castration, while T administration maintained the protein expression virtually at the precastration levels. These data demonstrate that mRNAs for CA IV and II are predominantly expressed in the corpus region of the rat epididymis and can be regulated by androgens in that region. The present data suggest that the regulation of CA expression in the corpus epididymidis by androgens contributes to the known androgen effects on epididymal acidification.

Salivary carbonic anhydrase isoenzyme VI.

The carbonic anhydrases (CAs) participate in the maintenance of pH homeostasis in various tissues and biological fluids of the human body by catalysing the reversible reaction CO2 + H2O HCO3- + H+ (Davenport & Fisher, 1938; Davenport, 1939; Maren, 1967). Carbonic anhydrase isoenzyme VI (CA VI) is the only secretory isoenzyme of the mammalian CA gene family. It is exclusively expressed in the serous acinar cells of the parotid and submandibular glands, from where it is secreted into the saliva. In this review, we will discuss recent advances in research focused on the physiological role of salivary CA VI in the oral cavity and upper alimentary canal.

Primary structure of a sperm cell anion exchanger and its messenger ribonucleic acid expression during spermatogenesis.

Chloride/bicarbonate (Cl-/HCO(3)-) exchangers are a family of proteins (anion exchanger [AE] gene family) that regulate many vital cellular processes such as intracellular pH, cell volume, and Cl- concentration. They may also be involved in the regulation of sperm cell motility and acrosome reaction during fertilization, as these two phenomena are bicarbonate dependent, and we have previously shown that a polypeptide immunologically related to erythrocyte band 3 is expressed in mammalian sperm cells. We have now identified this putative sperm cell anion exchanger as the AE2 isoform of this gene family. First, we determined its complete primary structure from the human testis lambda gt 11 cDNA library. The cloned sequence was found to consist of 3896 base pairs (bp) with an open reading frame of 3726 bp, and to be almost identical to the previously published human genomic AE2 sequence. Only four amino acid disparities were found between these two sequences. Second, our in situ hybridization analyses showed that AE2 mRNA is expressed in developing sperm cells, indicating that the cloned sequence corresponds to the sperm cell AE. Our reverse transcription-polymerase chain reaction analyses suggested further that the expression of AE2 mRNA was variable to some extent during the epithelial cell cycle. Strongest expression was observed at stages VII-XIV except for stage X, i.e., when major structural and morphological changes take place. These results suggest that the full-length AE2 isoform regulates HCO(3)- transport in mature sperm cells and thus their motility in vivo.

A low concentration of carbonic anhydrase isoenzyme VI in whole saliva is associated with caries prevalence.

Carbonic anhydrases maintain pH homeostasis in various tissues of the human body by catalyzing the reversible reaction CO2 + H2O <=> HCO3- + H+. Carbonic anhydrase isoenzyme VI (CA VI) is secreted into human saliva by the serous acinar cells of the parotid and submandibular glands. Although it represents about 3% of the total protein in stimulated parotid saliva, its exact physiological significance in the saliva has not been established. In the present study, saliva samples were collected under strictly controlled conditions from young, healthy men and assayed for CA VI concentrations using a specific time-resolved immunofluorometric assay. Salivary secretion rate, pH, buffering capacity, alpha-amylase activity levels, lactobacillus and Streptococcus mutans counts were also determined, and the results were correlated with the dental status of the subjects. Salivary CA VI concentration, pH and buffering capacity values correlated negatively with the numbers of decayed, missing and filled teeth (DMFT index). The correlations between salivary CA VI concentration and DMFT index were most significant in subjects with poor oral hygiene. No correlation was found between salivary CA VI concentration and lactobacillus or Streptococcus mutans counts. As predicted, salivary lactobacillus and Streptococcus mutans counts showed a close positive correlation with the DMFT index. In contrast, no significant correlation was seen between salivary secretion rate or amylase activity and the DMFT index. The present results indicate that low salivary CA VI concentrations are associated with increased caries prevalence, particularly in subjects with neglected oral hygiene.

Salivary carbonic anhydrase isoenzyme VI is located in the human enamel pellicle.

Salivary carbonic anhydrase (CA VI) appears to protect teeth from caries via mechanisms other than direct regulation of salivary pH and buffering capacity. To elucidate whether CA VI acts in the local microenvironment of the tooth surface, we studied the location and activity of the enzyme in the human enamel pellicle. The study was performed using a specific rabbit antiserum to human CA VI in conjunction with immunostaining and immunoblot techniques. CA activity was demonstrated using a histochemical staining method. CA VI immunostaining of extracted teeth having in vivo formed pellicle showed that the enzyme is present in the enamel pellicle. Immunostaining for salivary alpha-amylase, which is known to be present in the pellicle, showed a similar staining pattern. The presence of CA VI in the enamel pellicle was confirmed by immunoblotting of in vivo formed pellicle proteins. In vitro studies showed that CA VI binds to polished enamel surfaces from both saliva and solutions of purified enzyme. The intensity of the CA VI immunostaining on the enamel surface was dependent on the concentration of the applied enzyme. The histochemical staining of in vitro formed enamel pellicle confirmed that the bound enzyme retains its enzymatic activity. The presence of active CA VI in the human enamel pellicle suggests that it may accelerate the removal of acid by functioning locally in the pellicle layer on dental surfaces.