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.     

Immunocytochemical localization of carbonic anhydrase isozymes I, II, and III in rat skeletal muscle

Specific antisera were raised against the three carbonic anhydrase (CA) isozymes, CAI, CAII, and CAIII, and were used to determine the fiber distribution of these isozymes in skeletal muscle. Fiber types were determined by ATPase staining, and the CA isozymes were detected using a peroxidase-anti-peroxidase (PAP) technique. All three isozymes were present in type I fibers; CAII and CAIII were exclusive to these fibers, and CAI were also present in some small type 2A fibers.     

Distribution of CAIII in fetal and adult human tissue

Carbonic anhydrase III (CAIII), an enzyme recently shown by conventional electrophoresis to be muscle specific, has been quantitated by “rocket” immunoelectrophoresis. This more sensitive technique has shown that the enzyme is virtually specific to skeletal muscle, where it occurs at a level of 5mg/g, with trace levels in smooth muscle, cardiac muscle, and lung. In man there does not appear to by any correlation between CAIII levels and the proportion of red and white muscle fibers. The fetal development of CAIII has also been examined using immunoelectrophoresis, and the enzyme can be detected at 11 weeks' gestation. The CAIII level rises gradually up to 25 weeks, and there is then a more dramatic increase to reach approximately half adult level at birth.     

Induction of rat muscle carbonic anhydrase by denervation demonstrated with immunofluorescence

Immunofluorescence microscopy of carbonic anhydrase III (CA III) was performed on sections of rat anterior tibialis (AT), extensor digitorum longus (EDL) and soleus after denervation. In contralateral control muscles, CAIII was located only in type I fibres whereas following the operation, CAIII was markedly induced in type II fibers of all the muscles, most strikingly in EDL.     

Quantification of carbonic anhydrase III and myoglobin in different fiber types of human psoas muscle

Summary Carbonic anhydrase (CA III) and myoglobin contents from isolated human muscle fibers were quantified using a sensitive time-resolved fluoroimmunoassay. Human psoas muscle specimens were freeze-dried, and single fibers were dissected out and classified into type I, IIA and IIB by myosin ATPase staining. Fiber typing was further confirmed by SDS-PAGE. CA III and myoglobin were found in all fiber types. Type I fibers contained higher concentrations of CA III and myoglobin than type IIA and IIB fibers. The relative concentrations of CA III in type IIA and IIB fibers were respectively 24% and 10% of that in type I fibers. The relative concentrations of myoglobin in type IIA and IIB fibers were 60% and 28% of that in type I fibers. Anti-CA III immunoblotting results from fiber-specific pooled samples agreed well with quantitative measurements. The results indicate that CA III is a more specific marker than myoglobin for type I fibers.     

Carbonic anhydrase III--a marker of the myoepithelial cells of human salivary glands

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.     

A non-methylated CpG-rich island associated with the human muscle-specific carbonic anhydrase III gene

A cluster of CpG dinucleotides immediately upstream from exon 1 in the muscle-specific carbonic anhydrase III gene (CAIII) resembles the 'HpaII tiny fragment' (HTF) islands characteristic of mammalian 'housekeeping' genes. Since this CAIII gene shows tissue-specific expression we have carried out a detailed examination of methylation status within the CpG cluster using a polyacrylamide gel/electroblot procedure to extend the range of conventional Southern blotting. None of the clustered CpGs are methylated in DNA from muscle or other somatic tissues or in DNA from spermatozoa although flanking CpGs are methylated. Comparison with a candidate HTF island from the more ubiquitously expressed carbonic anhydrase II gene (CAII) shows that the CAII CpG cluster is markedly more CpG-rich than that from the strictly tissue-specific CAIII gene.     

The effects of aging on carbonic anhydrase concentrations in rat liver and skeletal muscle.

The isoenzymes carbonic anhydrase II (CAII) and III (CAIII) have been measured by radioimmunoassay in the livers of male and female rats aged from 21 to 800 days. No sexual dimorphism at 21 days was found, but from 50 to 400 days both isoenzymes show sexual differences. From 600 days onwards, these differences are less apparent. CAIII concentrations in two 'fast' fibre muscles and one 'slow' fibre muscle have been determined. There is no sexual dimorphism in muscle, but a wide variation between individuals was observed. Fast muscles show maximal CAIII levels at 800 days, whereas in slow muscle the concentration of the isoenzyme is declining at this time.     

Purification and localization of human carbonic anhydrase

Summary Three different isoenzymes of human carbonic anhydrase are now well characterized. Carbonic anhydrase I and II have been known for several years and are located in high amounts in red blood cells as well as in many other tissues.Carbonic anhydrase III, a protein showing CO₂ hydratase and p-nitrophenylphosphatase activity was isolated from skeletal muscle some years ago. Earlier observations based on enzyme activity and radioimmunoassay studies have suggested that this protein is present in greater quantities in red skeletal muscles than in white ones. We have purified CA III from human soleus muscle and using obtained monospecific polyclonal antibody localized this protein in the same muscle fibers which show acid resistant ATPase activity. Using this protein as a marker for type I muscle fibers, fiber classification into type I and II could now be done also from paraffin embedded sections.This study is supported by the Research Council of Physical Education and Sport, Ministry of Education, Finland     

Characterisation of cDNA clones for rat muscle carbonic anhydrase III

cDNA clones for rat muscle carbonic anhydrase III have been isolated from a gt-11 library and sequenced. Comparison with human CAIII cDNA showed about 90% homology to rat. The rat clones were used to estimate mRNA from liver and muscle on Northern blots and showed that the sexual dimorphism of CAIII in rat liver relates to a difference in mRNA levels.     

Carbonic anhydrase III regulates peroxisome proliferator-activated receptor-γ2

Carbonic anhydrase III (CAIII) is an isoenzyme of the CA family. Because of its low specific anhydrase activity, physiological functions in addition to hydrating CO(2) have been proposed. CAIII expression is highly induced in adipogenesis and CAIII is the most abundant protein in adipose tissues. The function of CAIII in both preadipocytes and adipocytes is however unknown. In the present study we demonstrate that adipogenesis is greatly increased in mouse embryonic fibroblasts (MEFs) from CAIII knockout (KO) mice, as demonstrated by a greater than 10-fold increase in the induction of fatty acid-binding protein-4 (FABP4) and increased triglyceride formation in CAIII(-/-) MEFs compared with CAIII(+/+) cells. To address the underlying mechanism, we investigated the expression of the two adipogenic key regulators, peroxisome proliferator-activated receptor-γ2 (PPARγ2) and CCAAT/enhancer binding protein-α. We found a considerable (approximately 1000-fold) increase in the PPARγ2 expression in the CAIII(-/-) MEFs. Furthermore, RNAi-mediated knockdown of endogenous CAIII in NIH 3T3-L1 preadipocytes resulted in a significant increase in the induction of PPARγ2 and FABP4. When both CAIII and PPARγ2 were knocked down, FABP4 was not induced. We conclude that down-regulation of CAIII in preadipocytes enhances adipogenesis and that CAIII is a regulator of adipogenic differentiation which acts at the level of PPARγ2 gene expression.     

Androgen-linked control of rat liver carbonic anhydrase III

The concentration of carbonic anhydrase III (CAIII) in male rat liver was found to be 30 times greater than that in the female. Castration of male rats led to marked reduction in liver CAIII concentrations which could be partially restored to control levels by testosterone replacement. Marked developmental and senescence changes in liver CAIII were also observed in male rats.     

Reduction (dethiolation) of protein mixed-disulfides; distribution and specificity of dethiolating enzymes and N,N'-bis(2-chlorethyl)-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-[3H]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 Km for glutathione for the GSH-dependent dethiolase was 15 microM with Phb as substrate and 10 microM with CAIII. Thus, the GSH-dependent dethiolase is probably not affected by normal changes in the cardiac glutathione content (normally approximately 3 mM). 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 microM 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.     

Carbonic anhydrase isozyme distribution and characterization in metabolic fiber types of the dorsal levator muscle of the blue crab, Callinectes sapidus

Carbonic anhydrase (CA) distribution and characterization were examined in white and light pink fibers of the dorsal levator muscle of the blue crab. White fibers were structurally and metabolically characterized as fast twitch glycolytic, while the light pink fibers were fast oxidative. All subcellular fractions of both fiber types had significant levels of CA activity; cytoplasmic and microsomal activity was significantly higher in light pink vs white fibers. Cytoplasmic CA from both fiber types was highly sensitive to the inhibitors acetazolamide and chlorzolamide, with Ki values of approximately 2 and 0.4 nM, respectively. Further analysis confirmed that cytoplasmic CA from both fiber types was kinetically similar to the high turnover Type II isoform. It appears that the evolution of the CA Type III isoform, found in vertebrate red muscle, did not occur with the differentiation of metabolic fiber types in crustaceans. Membrane-associated CA, which was also kinetically similar to the Type II isoform, was 20-fold higher in light pink fibers, suggesting a physiological role in facilitated CO2 efflux from the muscle fiber during periods of prolonged maximal activity.     

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.     

Hypophysectomy abolishes sexual dimorphism of liver carbonic anhydrase III

Hypophysectomy was found to have no effect on the concentration of carbonic anhydrase III (CAIII) in male rat liver, whereas in the female, CAIII was elevated 10-fold, to male levels.     

Forced MyHCIIB expression following targeted genetic manipulation of conditionally immortalized muscle precursor cells

The ability to carry out gene targeting in somatic stem cells while maintaining their stem cell characteristics would have important implications for gene therapy and for the analysis of gene function. Using mouse myoblasts, we have explored this possibility by attempting to alter the promoter of a myosin heavy chain gene (MyHCIIB) characteristic of physiologically "fast" muscle so as to force its unscheduled expression in physiologically "slow" muscle fibers. Conditionally immortalized muscle precursor cells were transfected with a gene targeting construct designed to replace the MyHCIIB promoter with that for the carbonic anhydrase III gene (CAIII), which is highly expressed in slow muscle. A potentially targeted clone was isolated and differentiated in culture to form myotubes which expressed MyHCIIB. Cells from the same clone were injected into both slow and fast muscle of host mice, where they contributed to fiber formation. In slow muscle, the fibers derived from this clone did not express MyHCIIB; this may reflect an instability of the targeted MyHCIIB locus and/or a failure of the hybrid promoter to function in slow fibers in vivo. Nonetheless, we have demonstrated that a "promoter knock-in" gene targeting procedure can be used to generate unique MyHCIIB-expressing myotubes in culture and that conditionally immortalized myoblasts can be subjected to extensive passaging and genetic manipulation without losing their ability to form fibers in culture and in vivo.     

Nucleotide sequence and derived amino acid sequence of a cDNA encoding human muscle carbonic anhydrase

We report the nucleotide (nt) sequence of a full length cDNA clone, pCA15, which encodes the human muscle-specific carbonic anhydrase, CAIII. pCA15 identifies a 1.7-kb mRNA, which is present at high levels in skeletal muscle, at much lower levels in cardiac and smooth muscle and which appears to be developmentally regulated. The CAIII mRNA is distinguished by a 887-nt long 3'-untranslated region, containing two AAUAAA signal sequences and is longer than either of the mRNAs encoding the erythrocyte CAs, CAI and CAII, which each have relatively shorter 3'-untranslated regions, 360 and 670 nt long, respectively. The derived amino acid (aa) sequence for human CAIII shows 85% homology with ox CAIII, 62% homology with human CAII and 54% with human CAI when simple pairwise aa comparisons are made. We describe an allelic variation at a TaqI restriction site for CAIII which occurs at high frequency in the European population.