Transgenic expression of carbonic anhydrase III in cardiac muscle demonstrates a mechanism to tolerate acidosis.

Carbonic anhydrase III (CAIII) is abundant in liver, adipocytes, and skeletal muscles, but not heart. A cytosolic enzyme that catalyzes conversions between CO2 and HCO3- in the regulation of intracellular pH, its physiological role in myocytes is not fully understood. Mouse skeletal muscles lacking CAIII showed lower intracellular pH during fatigue, suggesting its function in stress tolerance. We created transgenic mice expressing CAIII in cardiomyocytes that lack endogenous CAIII. The transgenic mice showed normal cardiac development and life span under nonstress conditions. Studies of ex vivo working hearts under normal and acidotic conditions demonstrated that the transgenic and wild-type mouse hearts had similar pumping functions under normal pH. At acidotic pH, however, CAIII transgenic mouse hearts showed significantly less decrease in cardiac function than that of wild-type control as shown by higher ventricular pressure development, systolic and diastolic velocities, and stroke volume via elongating the time of diastolic ejection. In addition to the effect of introducing CAIII into cardiomyocytes on maintaining homeostasis to counter acidotic stress, the results demonstrate the role of carbonic anhydrases in maintaining intracellular pH in muscle cells as a potential mechanism to treat heart failure.

Combined proteomic analysis of liver tissue and serum in chronically alcohol-fed rats.

BACKGROUND: Proteomic approaches may provide new insights into pathological conditions associated with alcoholism. The aim of this study was to conduct a proteomic analysis of liver tissue and serum in chronically alcohol-fed rats using agarose 2-dimensional gel electrophoresis (2-DE) and 3-step serum proteome analysis. METHODS: A total of 12 rats were pair-fed nutritionally adequate liquid diet containing ethanol as 36% of the total energy or an isocaloric control diet for 2 months. Rat liver homogenates and cytosol fractions were subjected to agarose 2-DE. Serum samples were subjected to 3-step serum proteome analysis involving immunodepletion of abundant proteins followed by fractionation using reverse-phase high-performance liquid chromatography and 1-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Candidate proteins were digested with trypsin and identified using mass spectrometry. Observed differences in protein expression levels were confirmed using Western blotting. RESULTS: A total of 46 protein spots were found to be differentially expressed in the liver homogenates and cytosol fractions of alcohol-fed rats relative to pair-fed controls. The most notable change was down-regulation of a 29-kDa protein, which was subsequently identified as carbonic anhydrase III (CA III). Down-regulation of this protein in alcohol-fed rats was confirmed by Western blotting. The messenger RNA level of CA III was decreased as well. In rat serum, a total of 41 proteins were differentially expressed. Of these proteins, only betaine-homocysteine methyltransferase (BHMT) was also found to be differentially expressed in the liver. CONCLUSIONS: A combined proteomic analysis of liver tissue and serum in chronically alcohol-fed rats revealed that the expression of CA III is significantly down-regulated in the liver of alcohol-fed rats. Our results also showed that BHMT expression is up-regulated in both the liver and serum of alcohol-fed rats.

Sexual dimorphism in LEC rat liver: suppression of carbonic anhydrase III by copper accumulation during hepatocarcinogenesis.

We examined age-related changes in the protein expression of carbonic anhydrase III (CAIII) in livers of Long-Evans with a cinnamon-like color (LEC) rats using an agouti color (LEA) rats as controls. The levels of the protein of CAIII in the liver of LEC male rats increased before 20 weeks of age, at the stage of acute hepatitis, and were decreased at 54 weeks of age, while those of CAIII in the liver of LEA male rats were highly expressed at all ages. In the normal LEA rats, CAIII showed sexual dimorphism. The level of CAIII in LEA male rat liver relative to female was four times higher. On the other hand, young LEC rat (at 4-12 weeks) showed a higher protein level of CAIII than LEA rats, and then decreased during development of hepatitis. CAIII mRNA also decreased in the LEC rat liver during hepatocarcinogenesis. The level of CAIII in the tumor region was lower than that in the tumor-free region. Immunohistochemical analysis showed that glutathione S-transferase P (GST-P) was positive and CAIII was negative in the precancerous region. The expression of CAIII was suppressed in cancerous lesions in hepatoma-bearing LEC rat liver compared to uninvolved surrounding tissues. These results indicated that suppression of CAIII accompanied hepatocarcinogenesis and it is a secondary consequence of the high copper levels in the liver.

Comparative proteomic analysis reveals a function of the novel death receptor-associated protein BRE in the regulation of prohibitin and p53 expression and proliferation.

The brain and reproductive organ expressed (BRE) gene encodes a highly conserved stress-modulating protein. To gain further insight into the function of this gene, we used comparative proteomics to investigate the protein profiles of C2C12 and D122 cells resulting from small interfering RNA (siRNA)-mediated silencing as well as overexpression of BRE. Silencing of BRE in C2C12 cells, using siRNA, resulted in up-regulated Akt-3 and carbonic anhydrase III expression, while the 26S proteasome regulatory subunit S14 and prohibitin were down-regulated. Prohibitin is a potential tumour suppressor gene, which can directly interact with p53. We found that cell proliferation was significantly increased after knockdown of BRE, concomitant with reduced p53 and prohibitin expression. In contrast, we observed decreased proliferation and up-regulation of p53 and prohibitin when BRE was overexpressed in the D122 cell line. In total, five proteins were found to be up-regulated after BRE over-expression. The majority of these proteins can target or crosstalk with NF-kappaB, which plays a central role in regulating cell proliferation, differentiation and survival. Our results establish a crucial role for BRE in the regulation of key proteins of the cellular stress-response machinery and provide an explanation for the multifunctional nature of BRE.

Suppression of carbonic anhydrase III mRNA level by an aryl hydrocarbon receptor ligand in primary cultured hepatocytes of rat.

The effect of an aryl hydrocarbon receptor (AhR) ligand on the carbonic anhydrase III (CAIII) mRNA level was studied using primary cultured hepatocytes of rats. CAIII gene which is highly suppressible by dioxins in vivo, was also suppressible in primary cultured hepatocytes of rats by an AhR ligand, 3-methylchlanthrene (3MC). The suppression of CAIII by 3MC was observed in a dose-dependent fashion. The suppression was marked at 10 microM MC. It is likely that AhR is involved in the suppression of the CAIII gene. The transcriptional regulation region of rat CAIII gene was cloned by polymerase chain reaction on the basis of the similarity to the mouse and human CAIII genes. A 1.5 kb section upstream of rat CAIII was sequenced and the transcription initiation site of this gene was mapped to 58 bases upstream of the initiation codon. A xenobiotic responsive element (XRE)-like sequence was found at -555 to -549 bp of the transcription initiation site. The location of XRE-like element was conserved between rats and mice those CAIIIs in liver were shown as dioxins-suppressible. Although the roles of the XRE have not been clarified, these results suggest that the AhR ligands could elicit the suppressive effect on hepatic CAIII and the effect on the factors from extrahepatic tissues is not required for the suppression.

The mouse liver content of carbonic anhydrase III and glutathione S-tranferases A3 and P1 depend on dietary supply of methionine and cysteine.

The contents of glutathione S-transferase (GST) subunits, carbonic anhydrase III (CAIII), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a 230 kDa protein are affected by protein deprivation in mouse liver. In order to know if particular amino acids control these contents, the effects of feeding for 5 days with diets containing different amino acids were examined. After an exploration using SDS-PAGE analysis, the action of selected diets was further examined by distinct techniques. The 230 kDa protein was identified as fatty acid synthase (FAS) by both mass spectrometry and amino acid sequence analyses. Dietary tests showed that: (1) a protein-free diet (PFD) increased the content of glutathione S-transferases P1 and M1, and glyceraldehyde-3-phosphate dehydrogenase, while the content of glutathione S-transferase A3, fatty acid synthase and carbonic anhydrase III decreased; (2) a protein-free diet having either methionine or cysteine preserved the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anydrase III; (3) a protein-free diet having threonine preserved partially the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anhydrase III; (4) a protein-free diet having methionine, threonine and cysteine prevented in part the loss of fatty acid synthase; and (5) the glyceraldehyde-3-phosphate dehydrogenase content was controlled by increased carbohydrate level and/or by lower amino acid content of diets, but not by any specific amino acid. These data indicate that methionine and cysteine exert a main role on the control of liver glutathione S-transferases A3 and P1, and carbonic anhydrase III. Thus, they emerge necessary to prevent unsafe alterations of liver metabolism caused by protein deprivation.

Androgen-linked control of carbonic anhydrase III expression occurs in rat perivenous hepatocytes; an immunocytochemical study.

Carbonic anhydrase (CA) isozymes CAII and CAIII were assayed by a radioimmunosorbent technique in liver cytosolic fractions and in isolated hepatocytes of adult male and female rats. Male livers contained 0.16 mg of CAII and 57 mg of CAIII per g cytosolic protein. Corresponding values for female livers were 0.34 mg CAII and 4 mg CAIII. Similar values and differences between CAII and III were found in isolated hepatocytes. Neonatal and adult castration of males reduced the CAIII levels to those of the females. Treatment with testosterone for three weeks restored the copulatory behaviour in the males castrated at adult age, but restored only partially the levels of CAIII. No significant effects of the endocrine manipulations were seen on CAII. Oophorectomy, with or without testosterone substitution, had no significant effect on CAII and CAIII levels in female rats. Immunohistochemistry and histochemistry showed that the regulation of CAIII is confined to perivenous hepatocytes. CAIII can therefore serve as a useful marker in the separation of these cells. CAIII appears to belong to the proteins and enzymes of the rat liver, known to be regulated via the hypothalamo-pituitary-liver axis. It may be used as a model of gene regulation in perivenous hepatocytes.