Carbonic anhydrase activity in mammalian skeletal and cardiac muscle.
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ABSTRACT:
SUBMITTER: Moynihan JB
PROVIDER: S-EPMC1183806 | biostudies-other | 1977 Dec
REPOSITORIES: biostudies-other
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Project description:We have studied the distribution of carbonic anhydrases (CA) in several skeletal muscles of the hindlimb of rabbits and rats and in cardiac muscle of the rabbit. To remove erythrocyte CA, hindlimbs and hearts were thoroughly perfused with dextran solution, and the effectiveness of the perfusion was in most cases assessed by determining the contamination of the muscles with radioisotopes that had been used to label the erythrocytes before the perfusion was started. We observed three forms of CA: (1) cytosolic (sulphonamide-resistant) CA III; (2) a cytosolic sulphonamide-sensitive CA, probably isoenzyme II; (3) a membrane-bound form that was extracted from the particulate fraction using Triton X-100. These CA isoforms were distributed as follows. (1) CA III is located in the cytoplasm of slow, oxidative skeletal muscles and is absent from or low in fast skeletal and cardiac muscle; this holds for rabbits and rats and is identical with the pattern previously described for several other species. (2) The cytosolic sulphonamide-sensitive CA is present in fast rabbit muscles and absent from slow muscles of this species. In contrast, all skeletal muscles of the rat studied here lack, or possess only very low, activity of this isoenzyme. (3) The membrane-bound form of CA is present in all rabbit muscles studied; its activity appears somewhat higher in fast than in slow skeletal muscles. (4) Cardiac muscle constitutes an exception among all striated muscles of the rabbit as it possesses no form of cytosolic CA but a high activity of the membrane-bound form.
| S-EPMC1130903 | biostudies-other
Project description:We investigated the activity of carbonic anhydrase in blood-free perfused white skeletal muscles of the rabbit. Carbonic anhydrase activities were measured in supernatants and in Triton extracts of the particulate fractions of white-skeletal-muscle homogenate by using a rapid-reaction stopped-flow apparatus equipped with a pH electrode. An average carbonic anhydrase concentration of about 0.5 microM was determined for white skeletal muscle. This concentration is about 1% of that inside the erythrocyte. Some 85% of the muscle enzyme was found in the homogenate supernatant, and only 15% appeared to be associated with membranes and organelles. White-skeletal-muscle carbonic anhydrase was characterized in terms of its Michaelis constant and catalytic-centre activity (turnover number) for CO2 and its inhibition constant towards ethoxzolamide. These properties were identical with those of the rabbit erythrocyte carbonic anhydrase C, suggesting that a type-C enzyme is present in white skeletal muscle. Affinity chromatography of muscle supernatant and of lysed erythrocytes showed that, whereas rabbit erythrocytes contain about equal amounts of carbonic anhydrase isoenzymes B and C, the B isoenzyme is practically absent from white skeletal muscle. Similarly, ethoxzolamide-inhibition curves suggested that white skeletal muscle contains no carbonic anhydrase A. It is concluded that white skeletal muscle contains essentially one carbonic anhydrase isoenzyme, the C form, most of which is probably of cytosolic origin.
| S-EPMC1158521 | biostudies-other
Project description: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.
| S-EPMC1148849 | biostudies-other
Project description:Metastatic tumors are often hypoxic exhibiting a decrease in extracellular pH (~6.5) due to a metabolic transition described by the Warburg Effect. This shift in tumor cell metabolism alters the tumor milieu inducing tumor cell proliferation, angiogenesis, cell motility, invasiveness, and often resistance to common anti-cancer treatments; hence hindering treatment of aggressive cancers. As a result, tumors exhibiting this phenotype are directly associated with poor prognosis and decreased survival rates in cancer patients. A key component to this tumor microenvironment is carbonic anhydrase IX (CA IX). Knockdown of CA IX expression or inhibition of its activity has been shown to reduce primary tumor growth, tumor proliferation, and also decrease tumor resistance to conventional anti-cancer therapies. As such several approaches have been taken to target CA IX in tumors via small-molecule, anti-body, and RNAi delivery systems. Here we will review recent developments that have exploited these approaches and provide our thoughts for future directions of CA IX targeting for the treatment of cancer.
| S-EPMC6272707 | biostudies-literature
Project description:cea05-01_carbonic-anhydrase - carbonic anhydrase (1 or 2) simple or double mutants - Identification of genes differentially expressed in leaves of single CA1 and CA2 T-DNA insertional mutants and in the corresponding double mutant vs wild type - CA1 (At3g01500) and CA2 (At5g14740) T-DNA insertional mutant lines, the double (CA1+CA2) mutant and wild type Arabidopsis seeds were sown in soil in a phytotron. Leaves were harvested 40 days later for RNA extraction
2012-03-25 | GSE36625 | GEO
Project description:cea05-01_carbonic-anhydrase - carbonic anhydrase (1 or 2) simple or double mutants - Identification of genes differentially expressed in leaves of single CA1 and CA2 T-DNA insertional mutants and in the corresponding double mutant vs wild type - CA1 (At3g01500) and CA2 (At5g14740) T-DNA insertional mutant lines, the double (CA1+CA2) mutant and wild type Arabidopsis seeds were sown in soil in a phytotron. Leaves were harvested 40 days later for RNA extraction 6 dye-swap - gene knock out
2012-03-25 | E-GEOD-36625 | biostudies-arrayexpress
Project description:Carbonic anhydrases (CAs) have been linked to tumor progression, particularly membrane-bound CA isoform IX (CA IX). The role of CA IX in the context of breast cancer is to regulate the pH of the tumor microenvironment. In contrast to CA IX, expression of CA XII, specifically in breast cancer, is associated with better outcome despite performing the same catalytic function. In this study, we have structurally modeled the orientation of bound ureido-substituted benzene sulfonamides (USBs) within the active site of CA XII, in comparison to CA IX and cytosolic off-target CA II, to understand isoform specific inhibition. This has identified specific residues within the CA active site, which differ between isoforms that are important for inhibitor binding and isoform specificity. The ability of these sulfonamides to block CA IX activity in breast cancer cells is less effective than their ability to block activity of the recombinant protein (by one to two orders of magnitude depending on the inhibitor). The same is true for CA XII activity but now they are two to three orders of magnitude less effective. Thus, there is significantly greater specificity for CA IX activity over CA XII. While the inhibitors block cell growth, without inducing cell death, this again occurs at two orders of magnitude above the Ki values for inhibition of CA IX and CA XII activity in their respective cell types. Surprisingly, the USBs inhibited cell growth even in cells where CA IX and CA XII expression was ablated. Despite the potential for these sulfonamides as chemotherapeutic agents, these data suggest that we reconsider the role of CA activity on growth potentiation.
| S-EPMC6242694 | biostudies-literature
Project description:Carbonic anhydrase enzymes (CAs) catalyse the reversible hydration of CO2 to H+ and HCO3- ions. This catalysis is proposed to be harnessed by acid/base transporters, to facilitate their transmembrane flux activity, either through direct protein-protein binding (a 'transport metabolon') or local functional interaction. Flux facilitation has previously been investigated by heterologous co-expression of relevant proteins in host cell lines/oocytes. Here, we examine the influence of intrinsic CA activity on membrane HCO3- or H+ transport via the native acid-extruding proteins, Na+ -HCO3- cotransport (NBC) and Na+ / H+ exchange (NHE), expressed in enzymically isolated mammalian ventricular myocytes. Effects of intracellular and extracellular (exofacial) CA (CAi and CAe) are distinguished using membrane-permeant and -impermeant pharmacological CA inhibitors, while measuring transporter activity in the intact cell using pH and Na+ fluorophores. We find that NBC, but not NHE flux is enhanced by catalytic CA activity, with facilitation being confined to CAi activity alone. Results are quantitatively consistent with a model where CAi catalyses local H+ ion delivery to the NBC protein, assisting the subsequent (uncatalysed) protonation and removal of imported HCO3- ions. In well-superfused myocytes, exofacial CA activity is superfluous, most likely because extracellular CO2/HCO3- buffer is clamped at equilibrium. The CAi insensitivity of NHE flux suggests that, in the native cell, intrinsic mobile buffer-shuttles supply sufficient intracellular H+ ions to this transporter, while intrinsic buffer access to NBC proteins is restricted. Our results demonstrate a selective CA facilitation of acid/base transporters in the ventricular myocyte, implying a specific role for the intracellular enzyme in HCO3- transport, and hence pHi regulation in the heart.
| S-EPMC3948559 | biostudies-literature
Project description:One of the four discrete isoenzymes of carbonic anhydrase hitherto characterized, CA III, has the lowest turnover rate and the greatest resistance to inhibition by sulphonamides. Streptozotocin-induced diabetes mellitus resulted in a reduction in acetazolamide-resistant activity of carbonic anhydrase in the liver, but not in tonic skeletal muscle, of adult male rats. The hepatic activity declined with apparent first-order kinetics [calculated rate constant (k) 0.089 day-1] to a minimum of approx. 6% of control values; the reduction in activity was moderated by administration of insulin.
| S-EPMC1149739 | biostudies-other
Project description:Ischemic stroke is a leading cause of death and disability worldwide. The only pharmacological treatment available to date for cerebral ischemia is tissue plasminogen activator (t-PA) and the search for successful therapeutic strategies still remains a major challenge. The loss of cerebral blood flow leads to reduced oxygen and glucose supply and a subsequent switch to the glycolytic pathway, which leads to tissue acidification. Carbonic anhydrase (CA, EC 4.2.1.1) is the enzyme responsible for converting carbon dioxide into a protons and bicarbonate, thus contributing to pH regulation and metabolism, with many CA isoforms present in the brain. Recently, numerous studies have shed light on several classes of carbonic anhydrase inhibitor (CAI) as possible new pharmacological agents for the management of brain ischemia. In the present review we summarized pharmacological, preclinical and clinical findings regarding the role of CAIs in strokes and we discuss their potential protective mechanisms.
| S-EPMC8126098 | biostudies-literature