Carbonic Anhydrase III Research Articles

Carbonic anhydrase III content in various equine muscles

1. 1. In this study, carbonic anhydrase III (CA-III) content in 18 equine muscles was determined by enzyme immunoassay. 2. 2. It was found to differ in several muscles. 3. 3. That in external intercostal muscle, rectus abdominis muscle and splenius muscle from four horses was very high. 4. 4. Although the masseter muscle had only type I fibers, CA-III content was similar to that in mixed-fiber type muscles such as the biceps femoris muscle. 5. 5. It thus appear that equine type I fibers can be further subgrouped.

The differential degradation of two cytosolic proteins as a tool to monitor autophagy in hepatocytes by immunocytochemistry.

The major pathway for cytosolic constituents to enter lysosomes is by autophagy. We used two cytosolic proteins, CuZn superoxide dismutase (SOD) and carbonic anhydrase III (CAIII), as autophagic markers in male rat hepatocytes. We took advantage of the differential presence of the two proteins in autophagic vacuoles because of the high resistance of SOD to lysosomal degradation as compared with CAIII. This allows us to determine the sequence of autophagic vacuole formation. We have double immunogold-labeled SOD and CAIII in cryosections of fasted rat liver and calculated the ratios of SOD over CAIII labeling densities (SOD/CAIII) in autophagic vacuoles (AV), as compared with the cytoplasm. Different classes of AV were defined according to their SOD/CAIII, their morphology, and their additional immunolabeling for the lysosomal markers lgp120 and cathepsin D. Of all AV, 15% exhibited a cytosol-like SOD/CAIII, indicating that degradation had not yet begun. Most of these initial AV (AVi) showed two enveloping membranes. The formation of AVi was prevented by 3-methyladenine, a potent inhibitor of autophagy. Of all AV, 85% showed a SOD/CAIII that exceeded the cytosolic ratio. These single membrane-bound vacuoles were called degradative AV (AVd). Labeling for lysosomal markers allowed the characterization of AV that shared features with both AVi and AVd. These AVi/d had a cytosol-like SOD/CAIII and a double membrane, but showed some labeling for lysosomal markers. Probably these AVi/d represent the recipient compartment for lysosomal components. AVd were positive for cathepsin D and lgp120. We discerned two AVd subclasses. Early AVd with cytosol-like SOD labeling density while CAIII labeling density was consistently lower than in the cytosol. Their size was similar to AVi and AVi/d. Late AVd contained higher SOD concentrations and were mostly larger. Our findings suggest that AV acquire lysosomal constituents by fusion with small nonautophagic structures and that after subsequent elimination of the inner membrane of AVi, degradation starts resulting in the formation of early AVd and late AVd.

Detection of CAIII mRNA in rat skeletal muscle and liver by in situ hybridization.

We carried out a variety of in situ methods of hybridization on rat liver and rat skeletal muscle using 35S-labeled or biotin-labeled rat carbonic anhydrase III (CAIII) cDNA clone. The methods were compared and evaluated. Use of the biotin system produced defined but nonspecific results which were shown not to be due to the biotinylated cDNA probe binding to the mRNA in the muscle sections. This artifact was shown to persist despite various attempts to eliminate it. Alternatively, using 35S-labeled cDNA gave reproducible results which were shown to be consistent with probe binding specifically to mRNA in the muscle section.

Expression of CA III in Rodent Models of Obesity

To achieve a better understanding of the biochemical basis of obesity, we have undertaken comparative analyses of adipose tissue of lean and obese mice. By two-dimensional gel analysis, carbonic anhydrase-III (CA III) has been identified as a major constituent of murine adipose tissue. Quantitative comparisons of CA III protein and mRNA levels indicate that this enzyme is expressed at lower levels in adipose tissue from animals that were either genetically obese or had experimentally induced obesity compared to levels in the corresponding lean controls. This decrease in CA III expression was unique to adipose tissue, since other CA III-containing organs and tissues did not show a change when lean and obese animals were compared. Additionally, levels of CA III in adipose tissue from obese animals responded to acute changes in energy balance of the animal. These results are discussed in light of possible metabolic roles for CA III.

CAIII a marker for early myogenesis: Analysis of expression in cultured myogenic cells

Carbonic anhydrase III (CAIII) is an abundant muscle protein characteristic of adult type-1, slow-twitch, muscle fibers. We demonstrate that CAIII is not confined to mature muscle but is also expressed in cultured myogenic cells that were originally derived from adult and fetal limb muscle (G8 and C2C12) and by azacytidine treatment of 10T1/2 fibroblasts (23A2). Transcripts may accumulate in these cells to levels that correspond to 6.5% of that found in mature muscle. CAIII is expressed in mononucleate myoblasts and is abundant in those that preferentially fuse to form myotubes, and these findings contrast with those for many other muscle genes whose transcripts only accumulate on or after terminal differentiation. Preliminary promoter-function assays by transfection shows that 2.8 kb of sequence flanking the 5' end of the human CAIII gene efficiently promotes transcription of the bacterial chloramphenicol acetyltransferase gene in myogenic cells. However, none of the sequences within this region are sufficient to confer muscle-specific expression. Removal of sequences 5' to -715 bp leads to a major loss of transcriptional activity of the CAIII promoter. These results imply that the proximal CAIII promoter, which includes a putative CArG box and four potential MyoD binding sites, is not adequate for either myoblast-specific or maximal transcription.

Reduction (dethiolation) of protein mixed-disulfides; distribution and specificity of dethiolating enzymes and N,N′-bis(2-chloroethyl)- N-nitrosourea inhibition of an NADPH-dependent cardiac dethiolase

The S-thiolated proteins phosphorylase b (Phb) and carbonic anhydrase III (CAIII) were prepared with [ 3H]glutathione in a reaction initiated with diamide. These substrates were used to measure the rate of reduction (dethiolation) of protein mixed-disulfides by enzymes with properties similar to those of thioredoxin and glutaredoxin. This enzyme activity is termed a dethiolase since the identities of the enzymes are still unknown. The dethiolation of either S-[ 3H]glutathiolated Phb or S-[ 3 H]glutathiolated CAIII was employed in tissue assays and for study of two partially purified dethiolases from cardiac tissue. NADPH-dependent dethiolase activity was most abundant except in rat liver and muscle. Total dethiolase activity was approximately 10-fold higher in neutrophils, 3T3-L1 cells, and Escherichia coli than in other sources. Rat skeletal muscle had 3- to 4-fold higher dethiolase activity than rat heart or liver. These data indicate that protein dethiolase activity is ubiquitous and that normal expression of the two dethiolase activities varies considerably. A partially purified cardiac NADPH-dependent dethiolase acted on Phb approximately 1.5 times faster than CAIII, and a glutathione (GSH)-dependent dethiolase acted on Phb 3 times faster than CAIII. The K m for glutathione for the GSH-dependent dethiolase was 15 μ m with Phb as substrate and 10 μ m with CAIII. Thus, the GSH-dependent dethiolase is probably not affected by normal changes in the cardiac glutathione content (normally approximately 3 m m). Partially purified cardiac NADPH-dependent dethiolase was inactivated by BCNU ( N,N′-bis(2-chloroethyl)- N-nitrosourea) and the GSH-dependent dethiolase was unaffected under similar conditions. In a soluble extract from bovine heart, 200 μ m BCNU inhibited NADPH-dependent dethiolase by more than 60% but did not affect GSH-dependent activity. These results demonstrate that BCNU is a selective inhibitor of the NADPH-dependent dethiolase.

Efficacy of hemoperfusion in myasthenia patients monitored by serum laser correlation spectroscopy

Carbonic anhydrase III (CAIII) is the isoenzyme purified from the human skeletal slow muscle immunohistochemically revealed in smooth muscle cells of uterus, myoepithelial cells of salivary, lactiferous and prostatic glands of man. Immunohistochemical determination of CAIII is a useful approach to correct the identification of myoepithelial cells in human salivary glands.

Distributions of Extracellular Matrix and Carbonic Anhydrase-III during Bovine Palatine Ridge Development.

The present study describes histological alterations and immunohistochemical distributions of extracellular matrices (ECMs) and the carbonic anhydrase isozyme-III (CA-III) during the period of bovine palatine ridge formation. Morphogenesis of bovine palatine ridges was preceded by epidermal placodes and the mesenchymal condensation (MC). During the early stages of less than 44 cm crown rump length (CRL), fibronectin (FN) was distributed densely in the MC. Strong reactions against type I collagen (C-1) were detected outer to the FN positive site. In the stages of more than 44 cm CRL, FN and C-1 were distributed diffusely in subepithelial mesenchyme. Laminin (LN) and type IV collagen were distributed in the epithelial and endothelial basement membranes (BMs) in all of the stages examined, except in the stage of 7 cm CRL, where LN was not detected only in the BM just beneath the epidermal placode. CA-III was detected in basal epithelial cells except for palatine ridge rudiments in the stages of more than 21 cm CRL. It is suggested that the expressions of LN and CA-III might play a role in the spatial determination of rudiments of bovine fetal palatine ridges.

Carbonic anhydrase III in equine tissues and sera determined by a highly sensitive enzyme‐immunoassay

A sensitive sandwich enzyme immunoassay (EIA) for measuring equine carbonic anhydrase III (CA-III) was established using a microplate as a solid-phase and peroxidase as a labelling enzyme. The assay can detect concentrations as low as 5 ng/ml using 20 microliters of sample sera. Within-run coefficients of variation obtained using standard equine CA-III were less than 5 per cent. CA-III levels in equine serum ranged from 5 to 50 ng/ml (n = 370), and apparently abnormal levels of CA-III from 100 to 1900 ng/ml (n = 27) were observed. The concentrations of immunoreactive CA-III in the extracts of various equine tissues were also determined; it was present at high concentrations in skeletal muscle and liver and to a much lesser extent in the thymus. Other tissues contained much smaller amounts.

Carbonic Anhydrase III in the Salivary Glands and Kidney of the Japanes Monkey (Macaca fuscata)

Carbonic anhydrase III (CA-III) was found in muscles of the Japanese monkey by the double immunodiffusion test and western blotting using antiserum raised against equine CA-III. Immunocytochemical localization of CA-III in the salivary glands and kidney of the monkey was studied using an avidin-biotinylated glucose oxidase complex. CA-III was found mainly in the striated duct and interlobular duct cells of the parotid glands. In the submandibular glands, striated duct, interlobular duct, and excretory duct cells were strongly stained for CA-III. In the kidney of the monkey, CA-III was localized mainly in the dark cells of the collecting duct at the medulla and in the epithelial cells of thick limb of Henle's loop.

Chronic stimulation-induced effects point to a coordinated expression of carbonic anhydrase III and slow myosin heavy chain in skeletal muscle

Chronic low-frequency Stimulation of rat fast-twitch muscle induces 3.7-fold elevations in cytochrome c oxidase activity, but remains without effect on carbonic anhydrase III (CAIII) mRNA and protein. This is in contrast with the situation in the rabbit where chronic stimulation elicits more than 10-fold elevations in CAIII activity and mRNA content which coincide with an enhanced expression of the slow myosin heavy chain (HCI). Since chronic stimulation of rat muscle does not enhance the expression of HCI, we conclude that CAIII is expressed in parallel with HCI and, therefore, is present only in type I and C fibers.

The episodic secretory pattern of growth hormone regulates liver carbonic anhydrase III. Studies in normal and mutant growth-hormone-deficient dwarf rats

Carbonic anhydrase III (CAIII) occurs in male rat liver at concentrations twenty times those in the female, and is sensitive to the pattern of growth hormone (GH) release. Males release GH episodically and have high concentrations of CAIII; females produce GH in a more continuous fashion and have lower CAIII levels. In normal female rats, the endogenous GH secretory pattern was masculinized, either by regular injections of GH-releasing factor (GRF) or by intermittent infusions of somatostatin (90 min on/90 min off). Both treatments induced regular GH pulses and stimulated growth, but only intermittent somatostatin infusions raised CAIII levels (controls, 1.5 +/- 0.5; somatostatin-treated, 9.0 +/- 2.9 micrograms/mg; means +/- S.D.). GRF pulses (4 micrograms every 4 h) did not however raise CAIII levels (controls 1.8 +/- 0.5; GRF-treated 1.4 +/- 0.4 micrograms/mg). Surprisingly, hepatic CAIII is also sexually dimorphic (males, 18.8 +/- 3; females, 2.22 +/- 0.4 micrograms/mg) in a GH-deficient dwarf rat strain which has low plasma GH levels without 3-hourly GH peaks. Intermittent somatostatin infusions in female dwarf rats partially masculinized hepatic CAIII, an effect reduced by co-infusion with GRF. This CAIII response was not secondary to growth induction, since neither somatostatin nor GRF stimulated growth in dwarf rats, and pulses of exogenous GH stimulated growth in female dwarfs without masculinizing CAIII levels. Furthermore, continuous GH infusion in male dwarf rats partially feminized hepatic CAIII levels (to 9.1 +/- 2.4 micrograms/mg), whereas infusions of insulin-like growth factor-1, which induced the same body weight gain, did not affect hepatic CAIII (20.8 +/- 6 micrograms/mg). These results show that hepatic CAIII expression is highly sensitive to the endogenous GH secretory pattern, independent of growth. They also implicate the low basal GH levels between pulses, rather than the peak GH levels, as the primary determinant of the sexually dimorphic hepatic CAIII expression in the rat.

Presence of carbonic anhydrase III in erythrocyte.

It is well known that there is present in hemolyzed serum a factor that interferes with the radioimmunoassay for detection of skeletal muscle carbonic anhydrase III (CA-III) in serum. This inhibitory factor cross-reacts with muscle anti-CA-III antibody and its level increases in parallel with the degree of hemolysis. In order to identify this factor, we purified the factor from a hemolysate of human erythrocytes, characterized it, compared it with known carbonic anhydrase isozymes from erythrocytes and with CA-III from skeletal muscle. We concluded that the factor was biochemically and immunochemically identical to CA-III from skeletal muscle. Calculation indicated the factor to represent about 1% of the total enzymes in human erythrocytes.

Evidence for the presence of carbonic anhydrase III in the myoid cells of the bovine thymus

An immunohistochemical study was carried out to detect the localization of carbonic anhydrase III (CA-III) in the bovine thymus. It was found that the CA-III activity was localized in the cells forming small clusters dispersed in the medullary region. By ultrastructural observation, these cells were identified as myoid cells.

Induction of carbonic anhydrase III mRNA and protein by denervation of rat muscle

Carbonic anhydrase III (CAIII) protein and mRNA amounts in fast- and slow-twitch rat muscles were examined after resection of the sciatic nerve. Striking changes occur in the fast-twitch anterior tibialis (AT) and extensor digitorum longus (EDL) muscles, where CAIII protein and mRNA are increased several-fold 16 days after denervation. The data suggest that these changes are regulated in part by changes in gene transcription and that they perhaps signal a fast-to-slow fibre type transition in these denervated muscles. AT and EDL show some differences in the effects of denervation, which are suggestive of variation in the timing of denervation-induced responses and/or the CAIII protein/mRNA turnover rates in the two muscles.

Changes in skeletal muscle gene transcription induced by chronic stimulation.

The mRNA products of four genes, carbonic anhydrase III (CAIII), fast myosin heavy chain (MHCf), actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were assayed by Northern and slot-blot analysis in rabbit tibialis anterior and extensor digitorum longus muscles that were subjected to electrical stimulation for periods of up to 21 days. Marked changes in mRNA levels were seen for all four genes. The mRNA for CAIII, which is specific for type 1 fibers, rose significantly, whereas the MHCf mRNA fell markedly in the stimulated muscles. Changes in GAPDH mRNA were consistent with a reduced dependence on anaerobic glycolysis as an energy source. Actin mRNA levels were noticeably depressed in the early stages of stimulation. Thus for several classes of muscle protein, the response to chronic low-frequency stimulation appears to involve changes at the level of gene transcription.

Comparative immunochemical studies of carbonic anhydrase III in horses and other mammalian species

1. 1. Carbonic anhydrase III (CA-III) from different mammalian species (horse, cow, dog, cat, rat and rabbit) has been analyzed by the immunodiffusion technique with anti-equine CA-III serum. 2. 2. Immunodiffusion demonstrated the absence of cross-reactivity between isozyme CA-I, CA-II, and CA-III. 3. 3. Cross-reactions were observed between the CA-III from all the species examined except the rabbit. 4. 4. Molecular weights and isoelectric points of CA-III from different species were determined by Western blotting.

Immunocytochemical localisation of carbonic anhydrase isozyme III in equine skeletal muscle.

The location of carbonic anhydrase III (CA-III) in frozen sections of biopsies of Thoroughbred horse skeletal muscle was studied. Fibre types were determined by ATP-ase and succinate dehydrogenase staining. CA-III isozyme was detected using a peroxidase conjugated anti-CA-III antibody. CA-III was found to be localised in slow twitch oxidative fibres (ST), but was also present in fast twitch oxidative (FTH) fibres in small amounts. Fast twitch glycolytic (FT) fibres were stained lightly compared with control sections. The concentrations of CA-III in muscle and liver were 70 micrograms/mg protein and 4 micrograms/mg protein, respectively. CA-I and CA-II were not found in muscle extracts by the double immunodiffusion method.

Thyroidectomy significantly alters carbonic anhydrase III concentration and fiber distribution in rat muscle.

Thyroidectomy has a dramatic effect on rat muscle, greatly increasing the number of Type I fibers and the concentrations of carbonic anhydrase III (CAIII) in the muscle. Carbonic anhydrase III is not confined to the Type I fibers, as was previously believed, but also occurs in fibers that exhibit a level of ATPase staining less than that of 2A fibers but greater than 2B. These fibers are rare in normal muscle but become numerous after thyroidectomy, when they stain heavily for CAIII.

Serum creatine kinase isoenzymes in duchenne muscular dystrophy determined by sensitive enzyme immunoassay methods

Serum levels of creatine kinase (CK) isoenzymes (MM, MB, and BB) were measured by sensitive enzyme immunoassay (EIA) methods in 50 patients with Duchenne muscular dystrophy (DMD) and in 39 controls. MM, MB, and BB levels in DMD patients were higher than in controls, and these three levels decreased with advancing age of DMD patients. Serum MB levels showed a good correlation with serum levels of carbonic anhydrase III (CA-III), but correlated poorly with electrocardiographic findings, suggesting that the main origin of serum MB in DMD is skeletal muscle rather than cardiac muscle.