Immunohistochemical localization of carbonic anhydrase isozyme II in the gustatory epithelium of the adult rat.

The distribution of carbonic anhydrase isozyme II (CA II)-like immunoreactivity (-LI) in the gustatory epithelium was examined in the adult rat. In the circumvallate and foliate papillae, CA II-LI was observed in the cytoplasm of the spindle-shaped taste bud cells, with weak immunoreaction in the surface of the gustatory epithelium. No neuronal elements displayed CA II-LI in these papillae. There was no apparent difference in the distribution pattern between the anterior and posterior portions of the foliate papillae. In immunoelectron microscopy, immunoreaction products for CA II were diffusely distributed in the entire cytoplasm of the taste bud cells having dense round granules at the periphery of the cells. No taste bud cells displaying CA II-LI were detected in the fungiform papillae, but a few thick nerve fibers displayed CA II-LI. In the taste buds of the palatal epithelium, neither taste bud cells nor neuronal elements exhibited CA II-LI. The present results indicate that CA II was localized in the type I cells designated as supporting cells in the taste buds located in the posterior lingual papillae of the adult animal.     

Differential regulation of hepatic carbonic anhydrase isozymes in the streptozotocin-diabetic rat

Most work with the male rat liver carbonic anhydrase isozymes in the past decade has centered on the cytosolic CA III and the mitochondrial CA V. This paper reports that the relative activity of both isozymes is altered in streptozotocin-diabetes. Carbonic anhydrase activity of perfused liver homogenates and disrupted, isolated mitochondria was measured by the mass spectrometric 18O decay technique at 37 degrees C. The contributions of the different isozymes were determined based on intracellular location and sensitivity to acetazolamide inhibition. Diabetes resulted in a twofold increase in the activity of CA V but a halving in the activity of CA III. This is the first time that liver CA V has been shown to be altered by physiological stress. The total carbonic anhydrase activity in the diabetic rat liver was unaltered compared with control rats; however, CA III never accounted for more than 50% of this activity. Since CA isozymes I, II, and IV together account for 30% of the CA activity in control rats and 70% in diabetic rats it is concluded that one or more of these isozymes is subject to regulation in the diabetic male rat. The increase in CA V during diabetes is in accord with this isozyme having an important function in provision of substrate for hepatic gluconeogenesis and ureagenesis.     

Regional differences in androgen thresholds of the epididymis of the castrated rat

The action of graded doses of testosterone and dihydrotestosterone on the maintenance of the functional integrity of the different regions of the epididymis of the castrated rat was investigated. The epididymis required higher amounts of androgens that the accessory glanss for maintenance of its weight and secretory activity. These results are discussed in relation to (a) the androgen differences in the epididymis in its response to androgens and (c) the mode of action of the two androgens.     

Effect of phytoestrogens on gene expression of carbonic anhydrase II in rat uterus and liver

The aim of this study was to characterize carbonic anhydrase II (CA2), as novel estrogen responsive gene, towards its usefulness to elucidate the molecular mechanisms of phytoestrogen action. Effects of estradiol-17beta (E2), and the phytoestrogens genistein (Gen), daidzein (Dai), as well as 8-prenylnaringenin (8PN) on CA2 mRNA expression were investigated in vivo in the uterus and liver of Wistar rats, and in vitro in Fe33 hepatoma cells. Relative amounts of mRNA levels of CA2 were measured by real-time RT-PCR. In vivo CA2 expression in uterus and liver is down-regulated by estrogen in time dependent manner with the most pronounced effect detectable 72 h after treatment. Treatment with Gen results in a slight down-regulation of CA2 expression in the uterus. In liver a response to Gen is detectable only after 7 h, where the expression of the gene is down-regulated to 60%. Treatment with Dai and 8PN for 72 h results in a slight down-regulation of CA2 in both tissues. In contrast in Fe 33 cells CA2 gene expression was up-regulated in response to the treatment with E2 for 7 h. In summary, we could demonstrate that the modulation of CA2 gene expression following treatment with E2 and Gen in rat uterus is comparable to the uterotrophic response of these substances, but with an inverted pattern. Remarkably, of all phytoestrogens 8PN exhibited the strongest uterotrophic response but only induced a very faint decrease of CA2 expression. In addition, we provide the first pieces of evidence that 8PN, like Gen and Dai, cannot be considered as a pure agonist. In conclusion, CA2 shows estrogen sensitivity not only in both tissues studied, but also in many others. Further, it exhibits a differential sensitivity thereby being capable to discriminate between different molecular qualities of phytoestrogens, like demonstrated for Gen and 8PN.     

Structures of murine carbonic anhydrase IV and human carbonic anhydrase II complexed with brinzolamide: molecular basis of isozyme-drug discrimination.

Carbonic anhydrase IV (CAIV) is a membrane-associated enzyme anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage. We have determined the 2.8-angstroms resolution crystal structure of a truncated, soluble form of recombinant murine CAIV. We have also determined the structure of its complex with a drug used for glaucoma therapy, the sulfonamide inhibitor brinzolamide (Azopt). The overall structure of murine CAIV is generally similar to that of human CAIV; however, some local structural differences are found in the active site resulting from amino acid sequence differences in the "130's segment" and the residue-63 loop (these may affect the nearby catalytic proton shuttle, His-64). Similar to human CAIV, the C-terminus of murine CAIV is surrounded by a substantial electropositive surface potential that may stabilize the interaction with the phospholipid membrane. Binding interactions observed for brinzolamide rationalize the generally weaker affinity of inhibitors used in glaucoma therapy toward CAIV compared with CAII.     

Expression of carbonic anhydrase IV in mouse placenta

Carbonic anhydrase (CA) facilitates acid-base transport in several tissues. Acidosis upregulates membrane-bound SDS-resistant hydratase activity in various tissues and CA IV mRNA in rabbit kidney. This study was designed to assess whether the expression of membrane-bound CA IV isozyme in mouse placenta is regulated developmentally and by maternal ammonium chloride loading at the end of pregnancy. For this purpose we used Northern blot analysis, Western blots of microsomal membranes, and immunocytochemistry. The expression of CA IV mRNA on Northern blots tripled from day 11 to day 15 and then remained stable until the end of pregnancy. Expression of CA IV immunoreactive protein on Western blot tripled from day 11 to day 15 and decreased almost to baseline by day 19. Strong staining for CA IV was detected by immunocytochemistry in labyrinthine trophoblast, in the endodermal layer of the yolk sac (both intra- and extraplacental) and in the uterine epithelium. Weak staining was observed in most fetal endothelial cells at 11 days but not later in gestation. Maternal acidosis did not upregulate the expression of CA IV mRNA or CA IV immunoreactive protein. Thus CA IV expression in mouse placenta is developmentally regulated. Maternal acidosis during the last quarter of pregnancy does not upregulate CA IV mRNA or CA IV immunoreactive protein.     

Effects of fluid shear stress on mRNA expression of carbonic anhydrase II in polarized rat osteoclasts

The present study was designed to determine the effects of fluid shear stress on the mRNA expression of carbonic anhydrase II (CAII) in polarized rat osteoclasts. Cellular morphology of the polarized osteoclasts generated by a mechanical anatomical technique was examined by tartrate-resistant acid phosphatase (TRAP) staining and the osteoclastic resorption of dentine slices. The polarized osteoclasts were then stress-loaded by using a flow shear stress device newly developed by the osteoclast research group (patent number 200420034438; China), at 9 dyne/cm(2) for various time periods [0 (control group), 15, 30, 60, and 120 min], or at various stress levels [0 (control), 0.9, 2.9, 8.7, and 26.3 dyne/cm(2)] for 30 min. The mRNA expression of CAII was quantified using real-time fluorescent quantitative PCR (RT-PCR) and the data were analyzed with SPSS 12.0 software. The polarized osteoclasts were larger than regular monocytes (about 30 microm diameter) with irregular configuration, and the majority of polarized osteoclasts appeared to be spherical and had approximately 2-20 nuclei. The TRAP positive polarized osteoclasts showed asymmetrical red staining in the cytoplasm, and had many filaments and vacuoles. These cells formed resorptive pits in dentine slices. The levels of CAII mRNA expression were shown to be time-dependent, with the E+5 copy numbers being 7.88+/-0.09, 11.14+/-0.12, 15.83+/-0.18, 1.94+/-0.02, and 1.37+/-0.01 in cells treated at 9 dyne/cm(2) for 0, 15, 30, 60 and 120 min, respectively (P < 0.05). The levels of CAII mRNA expression (E+5 copy numbers) in cells treated with the stress levels of 0, 0.9, 2.9, 8.7 and 26.3 dyne/cm(2) were 7.97+/-0.201, 11.26+/-0.688, 15.94+/-0.201, 31.88+/-1.496, and 45.08+/-2.639, respectively (P < 0.05). These results indicate that there is a relationship between the fluid shear stress and the mRNA expression of CAII in polarized rat osteoclasts.     

Histochemical localization of carbonic anhydrase in the testis and epididymis of the boar.

The testis and epididymis of sexually mature, fertile boars were studied for localization of carbonic anhydrase (CA) using a cobalt precipitation technique. In the testis, cytoplasmic CA was found in the Sertoli cells and in the capillaries surrounding the seminiferous tubules. The epididymal duct was divided into initial, middle and terminal segments, and regional differences in CA activity were observed. The cell membranes of the basal cells were stained in the initial and middle segments. Strong cytoplasmic CA staining was present only in the apical cells in the initial and middle segments. The basolateral cell membranes were stained in the principal cells of the terminal segment and the ductus deferens. As a rule the capillaries surrounding the epididymal duct were unstained. The enzyme, specifically localized in regions of the male genitalia acting as sperm reservoirs, might be related to the quiescence of the stored spermatozoa by influencing the acid-base status of the epididymal fluid.     

Histochemical localization of carbonic anhydrase in the testis and epididymis of the rabbit.

The distribution of carbonic anhydrase (CA) was studied in the testis and epididymis of mature, male rabbits using a cobalt precipitation method. CA was found only in the endothelium of the capillaries in the testis. The epididymal duct was divided into initial, middle and terminal segments. Strong cytoplasmic CA was present in the apical cells in the initial and middle segments. Vacuoles with CA staining in the membranes were found in the principal cells in the middle segment. Intensely stained basal cells were present in the terminal segment. In the last part of the terminal segment and the first of the ductus deferens the basolateral cell membranes were also stained. The function of the enzyme is discussed especially in relation to acidification of the epididymal fluid and facilitation of CO2 diffusion.     

Transport activity of the high-affinity monocarboxylate transporter MCT2 is enhanced by extracellular carbonic anhydrase IV but not by intracellular carbonic anhydrase II

The ubiquitous enzyme carbonic anhydrase isoform II (CAII) has been shown to enhance transport activity of the proton-coupled monocarboxylate transporters MCT1 and MCT4 in a non-catalytic manner. In this study, we investigated the role of cytosolic CAII and of the extracellular, membrane-bound CA isoform IV (CAIV) on the lactate transport activity of the high-affinity monocarboxylate transporter MCT2, heterologously expressed in Xenopus oocytes. In contrast to MCT1 and MCT4, transport activity of MCT2 was not altered by CAII. However, coexpression of CAIV with MCT2 resulted in a significant increase in MCT2 transport activity when the transporter was coexpressed with its associated ancillary protein GP70 (embigin). The CAIV-mediated augmentation of MCT2 activity was independent of the catalytic activity of the enzyme, as application of the CA-inhibitor ethoxyzolamide or coexpressing the catalytically inactive mutant CAIV-V165Y did not suppress CAIV-mediated augmentation of MCT2 transport activity. Furthermore, exchange of His-88, mediating an intramolecular H(+)-shuttle in CAIV, to alanine resulted only in a slight decrease in CAIV-mediated augmentation of MCT2 activity. The data suggest that extracellular membrane-bound CAIV, but not cytosolic CAII, augments transport activity of MCT2 in a non-catalytic manner, possibly by facilitating a proton pathway other than His-88.     

Localization of carbonic anhydrase in the rat lung

Summary The localization of carbonic anhydrase in the rat lung has been demonstrated, at light and electron microscopic levels, by the cobalt bicarbonate histochemical method of Hansson. Focal deposits of the cobalt sulfide reaction product were found not only in the capillary endothelium of the alveolar walls, but also in the small and large alveolar cells. The histochemical reaction was abolished by two potent inhibitors, acetazolamide (10^–5 to 10^–6 M) and KCNO (5×10^–3 to 10×10^–3 M). Physiological assay with Maren's method indicated that values for carbonic anhydrase activity in rat lung are 4.4±0.8 UA/mg of protein, 25.0±5.5 UA/mg of nitrogen, and 369±86 UA/g of wet weight. In addition, it was calculated that after fixation in glutaraldehyde-formaldehyde-picric acid about 9% activity is retained.     

Cell-specific expression of mitochondrial carbonic anhydrase in the human and rat gastrointestinal tract.

Mitochondrial carbonic anhydrase V (CA V) in liver provides HCO3- to pyruvate carboxylase for the first step in gluconeogenesis and HCO3- to carbamyl phosphate synthetase I for the first step in ureagenesis. Because carbamyl phosphate synthetase I and ornithine transcarbamylase are also expressed in enterocytes, we tested the hypothesis that CA V is expressed in the gastrointestinal tract in addition to liver. Polyclonal rabbit antisera were raised against a polypeptide of 17 C-terminal amino acids of human CA V and against purified recombinant mouse isozyme and were used in Western blotting and immunoperoxidase staining of human and rat tissues. Immunohistochemistry showed that CA V is expressed cell-specifically in the alimentary canal mucosa from stomach to rectum. Immunoreactions for CA V were detected in the parietal cells and gastrin-producing G-cells of the stomach and in intestinal enterocytes. Western blotting of human and rat gastrointestinal tissues with isozyme-specific antibodies showed positive signals for CA V with the expected molecular mass. The findings in human tissues paralleled those in rat. The cell-specific pattern of CA V expression suggests a role for CA V in alimentary canal physiology. We propose that mitochondrial CA V participates in the detoxification of ammonia produced in the gastrointestinal tract by providing bicarbonate to carbamyl phosphate synthetase I. (J Histochem Cytochem 47:517-524, 1999)     

Novel carbonic anhydrase isozymes I, II and IV activators incorporating sulfonyl-histamino moieties.

Sulfonylamido(ureido) derivatives of histamine were synthesized by an original procedure in order to obtain tight-binding activators of the zinc enzyme carbonic anhydrase (CA), exploiting the binding energy of the alkyl/arylsulfonyl moieties with amino acid residues at the entrance of the active site. In contrast to the lead molecule, histamine, the new derivatives possessed higher affinity for three different CA isozymes, as evidenced by compairing the affinity constants of these compounds for isozyme CA II.     

Engineering the hydrophobic pocket of carbonic anhydrase II

Wild-type and mutant human carbonic anhydrases II, where mutations have been made in the hydrophobic pocket of the active site, have been studied by X-ray crystallographic methods. Specifically, mutations at Val-143 (the base of the pocket) lead to significant changes in catalytic activity and protein structure. The obliteration of a well-defined pocket in the Val-143----Phe and Val-143----Tyr mutants results in significantly diminished enzyme activity [(5 x 10(4))-fold and (3 x 10(5))-fold, respectively]; however, the activity of the Val-143----His mutant is diminished less (10(2)-fold), and deepening the pocket in the Val-143----Gly mutant results in only a 2-fold decrease in activity [Fierke et al., 1991 (preceding paper in this issue)]. These results indicate that the hydrophobic pocket is important for substrate association with the enzyme, but there are probably several catalytically acceptable substrate trajectories through this region of the enzyme structure. Additionally, each mutant protein exhibits long-range (ca. 10-15 A) compensatory structural changes which accommodate the Val-143 substitution. As such, the genetic-structural approach represented in this work serves as a three-dimensional paradigm for the redesign of specificity pockets in other protein catalysts.     

Region- and cell-specific differences in the distribution of carbonic anhydrases II, III, XII, and XIV in the adult rat epididymis.

We employed RT-PCR followed by light microscope immunocytochemistry on St. Marie's- and Bouin's-fixed tissues to define the distribution of carbonic anhydrase (CA) isoforms in the male reproductive tract. The data revealed that CA II, III, IV, XII, and XIV were expressed in rat epididymis. Whereas CA III was found in principal cells of all epididymal regions, CA II was localized in narrow cells of the initial segment and principal cells of all regions. CA XII expression was most intense in the corpus and proximal cauda regions, where it appeared over the basolateral plasma membranes of principal cells. Narrow cells of the initial segment also revealed intense reactions, as did basal cells of the corpus and proximal cauda regions. Principal cells of the initial segment and proximal caput regions showed diffuse apical cytosolic reactions and occasional basolateral staining for CA XIV, whereas principal cells of distal regions showed more diffuse cytosolic reactions highlighting both apical and basal regions of the cell, with basal cells also being reactive. These data suggest subtle differences in cell type and subcellular- and region-specific distributions for CAs in their role of fine-tuning pH in the lumen, cell cytosol, and intervening intercellular spaces of the epididymis.     

Investigation of piperazines as human carbonic anhydrase I,II, IV and VII activators

Four human (h) carbonic anhydrase isoforms (CA, EC 4.2.1.1), hCA I, II, IV, and VII, were investigated for their activation profile with piperazines belonging to various classes, such as N-aryl-, N-alkyl-, N-acyl-piperazines as well as 2,4-disubstituted derivatives. As the activation mechanism involves participation of the activator in the proton shuttling between the zinc-coordinated water molecule and the external milieu, these derivatives possessing diverse basicity and different scaffolds were appropriate for being investigated as CA activators (CAAs). Most of these derivatives showed CA activating properties against hCA I, II, and VII (cytosolic isoforms) but were devoid of activity against the membrane-associated hCA IV. For hCA I, the K_As were in the range of 32.6–131?µM; for hCA II of 16.2–116?µM, and for hCA VII of 17.1–131?µM. The structure-activity relationship was intricate and not easy to rationalize, but the most effective activators were 1-(2-piperidinyl)-piperazine (K_A of 16.2?µM for hCA II), 2-benzyl-piperazine (K_A of 17.1?µM for hCA VII), and 1-(3-benzylpiperazin-1-yl)propan-1-one (K_A of 32.6?µM for hCA I). As CAAs may have interesting pharmacologic applications in cognition and for artificial tissue engineering, investigation of new classes of activators may be crucial for this relatively new research field.     

The distribution of carbonic anhydrase II in human, pig and rat oesophageal epithelium

Carbonic anhydrase II (CA II) is present in human oesophageal epithelial cells and probably involved in protecting the mucosa against acidic gastric refluxate. If this is the case, then it is likely that the enzyme will be more concentrated at or near the gastro-oesophageal junction. To answer this question, and determine whether CA II is present and similarly distributed in other species, we also examined the oesophageal epithelium of the rat and pig. In the rat, CA II was largely absent from the oesophageal epithelium, but present in the stratified squamous epithelium of the gastric forestomach as an approximately 2 mm-long collar around the entrance to the corpus, a site that roughly corresponds to the gastro-oesophageal junction in other animals. The enzyme was present mainly in basal and prickle cells. In upper and middle pig oesophagus, CA II was largely confined to basal cells and isolated groups of stratified superficial prickle cells. CA II-containing epithelial cells were highly concentrated in the thickened epithelium at the gastro-oesophageal junction (about four-times thicker than upper or middle). Reactive cells were present throughout the depth of the epithelium, but noticeably more concentrated in the basal and superficial prickle cell layers. CA II was also prominent in the most superficial cell layers in islands of the oesophageal mucosa within the gastric cardia. In man, CA II was confined largely to the basal half of the epithelium in the upper and middle regions of oesophagus. The distribution of CA II at the gastro-oesophageal junction took different forms. In general, there were more CA II-reactive cells at or closer to the lumen. The superficial prickle cell layers tended to exhibit more CA II than the deeper layers, with basal and epibasal cells containing little or no enzyme. In other regions of the same specimens, CA II-containing cells were present from the basal to the most luminal layers. If CA II in oesophageal epithelial cells in the region of the gastro-oesophageal junction (or in the case of the rat the forestomach/corpus junction) is important in the defence against refluxate, then it is in a vulnerable site, since bile salts are potent inhibitors of the enzyme. The action of bile salts on CA II may be an important factor in the initiation of oesophageal disease.