Petra Wetzel

Extracellular carbonic anhydrase activity facilitates lactic acid transport in rat skeletal muscle fibres

1 In skeletal muscle an extracellular sarcolemmal carbonic anhydrase (CA) has been demonstrated. We speculate that this CA accelerates the interstitial CO2/HCO3− buffer system so that H+ ions can be rapidly delivered or buffered in the interstitial fluid. Because > 80 % of the lactate which crosses the sarcolemmal membrane is transported by the H+‐lactate cotransporter, we examined the contributions of extracellular and intracellular CA to lactic acid transport, using ion‐selective microelectrodes for measurements of intracellular pH (pHi) and fibre surface pH (pHs) in rat extensor digitorum longus (EDL) and soleus fibres. 2 Muscle fibres were exposed to 20 mm sodium lactate in the absence and presence of the CA inhibitors benzolamide (BZ), acetazolamide (AZ), chlorzolamide (CZ) and ethoxzolamide (EZ). The initial slopes (dpHs/dt, dpHi/dt) and the amplitudes (ΔpHs, ΔpHi) of pH changes were quantified. From dpHi/dt, ΔpHi and the total buffer factor (BFtot) the lactate fluxes (mm min−1) and intracellular lactate concentrations ([lactate]i) were estimated. 3 BFtot was obtained as the sum of the non‐HCO3− buffer factor (BFnon‐HCO3) and the HCO3− buffer factor (BFHCO3). BFnon‐HCO3 was 35 ± 4 mmΔpH−1 for the EDL (n = 14) and 86 ± 16 mmΔpH−1 for the soleus (n = 14). 4 In soleus, 10 mm cinnamate inhibited lactate influx by 44 % and efflux by 30 %; in EDL, it inhibited lactate influx by 37 % and efflux by 20 %. Cinnamate decreased [lactate]i, in soleus by 36 % and in EDL by 45 %. In soleus, 1 mm DIDS reduced lactate influx by 18 % and efflux by 16 %. In EDL, DIDS lowered the influx by 27 % but had almost no effect on efflux. DIDS reduced [lactate]i by 20 % in soleus and by 26 % in EDL. 5 BZ (0.01 mm) and AZ (0.1 mm), which inhibit only the extracellular sarcolemmal CA, led to a significant increase in dpHs/dt and ΔpHs by about 40 %‐150 % in soleus and EDL. BZ and AZ inhibited the influx and efflux of lactate by 25 %‐50 % and reduced [lactate]i by about 40 %. The membrane‐permeable CA inhibitors CZ (0.5 mm) and EZ (0.1 mm), which inhibit the extracellular as well as the intracellular CAs, exerted no greater effects than the poorly permeable inhibitors BZ and AZ did. 6 In soleus, 10 mm cinnamate inhibited the lactate influx by 47 %. Addition of 0.01 mm BZ led to a further inhibition by only 10 %. BZ alone reduced the influx by 37 %. 7 BZ (0.01 mm) had no influence on the Km value of the lactate transport, but led to a decrease in maximal transport rate (Vmax). In EDL, BZ reduced Vmax by 50 % and in soleus by about 25 %. 8 We conclude that the extracellular sarcolemmal CA plays an important role in lactic acid transport, while internal CA has no effect, a difference most likely attributable to the high internal vs. low extracellular BFnon‐HCO3. The fact that the effects of cinnamate and BZ are not additive indicates that the two inhibitors act at distinct sites on the same transport pathway for lactic acid.

Carbonic anhydrase III is not required in the mouse for normal growth, development, and life span.

ABSTRACT Carbonic anhydrase III is a cytosolic protein which is particularly abundant in skeletal muscle, adipocytes, and liver. The specific activity of this isozyme is quite low, suggesting that its physiological function is not that of hydrating carbon dioxide. To understand the cellular roles of carbonic anhydrase III, we inactivated the Car3 gene. Mice lacking carbonic anhydrase III were viable and fertile and had normal life spans. Carbonic anhydrase III has also been implicated in the response to oxidative stress. We found that mice lacking the protein had the same response to a hyperoxic challenge as did their wild-type siblings. No anatomic alterations were noted in the mice lacking carbonic anhydrase III. They had normal amounts and distribution of fat, despite the fact that carbonic anhydrase III constitutes about 30% of the soluble protein in adipocytes. We conclude that carbonic anhydrase III is dispensable for mice living under standard laboratory husbandry conditions.

Rat skeletal muscle membrane associated carbonic anhydrase is 39-kDa, glycosylated, GPI-anchored CA IV.

Sarcolemmal membrane vesicle preparations from white and red muscles of rat were found to contain a carbonic anhydrase which was indistinguishable from carbonic anhydrase IV from rat lung. This isozyme appears to account for all of the carbonic anhydrase activity in the sarcolemmal vesicle preparations. Digestion of 39-kDa CA IV with endoglycosidase F reduced the Mr to 36 kDa, suggesting that it contains one N-linked oligosaccharide. Treatment of sarcolemmal vesicles with phosphatidylinositol-specific phospholipase C released all of the activity, indicating that the enzyme is anchored to membranes by a phosphatidylinositol-glycan linkage. White muscle sarcoplasmic reticulum vesicles also contain a small amount of 39-kDa CA IV-type enzyme. A 52-kDa polypeptide in sarcoplasmic reticulum membranes cross-reacts with anti-human CA II and anti-rat CA II antisera, but does not bind to the sulfonamide affinity column. This cross-reacting polypeptide has no detectable CA activity.

Expression of Membrane-bound Carbonic Anhydrases IV, IX, and XIV in the Mouse Heart

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-α-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.

T Tubules and Surface Membranes Provide Equally Effective Pathways of Carbonic Anhydrase-Facilitated Lactic Acid Transport in Skeletal Muscle

We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL) by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA) of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV-CAIX-CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.

Carbonic anhydrase inhibition and calcium transients in soleus fibers.

We simultaneously measured cytoplasmic Ca2+ transients using Fura‐2 and isometric force in rat soleus fiber bundles. In the presence of the carbonic anhydrase inhibitor, chlorzolamide, we observed a decreased amplitude and retarded decay of the Ca2+ signal. This corresponded with a decreased isometric force and a retarded muscle relaxation. We conclude that muscle carbonic anhydrase participates in excitation‐contraction coupling, possibly by rapidly providing protons that are exchanged for Ca2+ across the sarcoplasmic reticulum membrane.

Sarcolemmal carbonic anhydrase in red and white rabbit skeletal muscle

Sarcolemmal vesicles of white and red skeletal muscles of the rabbit were prepared by consecutive density gradient centrifugations in sucrose and dextran according to Seiler and Fleischer (1982, J. Biol. Chem. 257, 13,862-13,871). White and red muscle membrane fractions enriched in sarcolemma were characterized by high ouabain-sensitive Na+, K(+)-ATPase, by high Mg2(+)-ATPase activity, and by a high cholesterol content. Ca2(+)-ATPase activity, a marker enzyme for sarcoplasmic reticulum, was not detectable in the highly purified white and red muscle sarcolemmal fractions. White and red muscle sarcolemmal fractions exhibited no significant differences with regard to Na+, K(+)-ATPase, Mg2(+)-ATPase, and cholesterol. Specific activity of carbonic anhydrase in white muscle sarcolemmal fractions was 38 U.ml/mg and was 17.6 U.ml/mg in red muscle sarcolemma. Inhibition properties of sarcolemmal carbonic anhydrase were analyzed for acetazolamide, chlorzolamide, and cyanate. White muscle sarcolemmal carbonic anhydrase is characterized by inhibition constants, KI, toward acetazolamide of 4.6 X 10(-8) M, toward chlorzolamide of 0.75 X 10(-8) M, and toward cyanate of 1.3 X 10(-4) M. Red muscle sarcolemmal carbonic anhydrase is characterized by KI values toward acetazolamide of 8.1 X 10(-8) M, toward chlorzolamide of 6.3 X 10(-8) M, and toward cyanate of 0.81 X 10(-4) M. In contrast to the high specific carbonic anhydrase activities in sarcolemma, carbonic anhydrase activity in sarcoplasmic reticulum from white muscle varied between values of only 0.7 and 3.3 U.ml/mg. Carbonic anhydrase of red muscle sarcoplasmic reticulum ranged from 2.4 to 3.7 U.ml/mg.

Carbonic anhydrases in striated muscle

The purpose of this chapter is to review the localization and function of various carbonic anhydrase (CA) isoforms in skeletal muscle. We will concentrate on those forms, for which it has been possible to develop clear functional concepts during the last few years. The isozymes, the presence of which has been established for mammalian skeletal muscle, are the following: (1) A membrane-bound carbonic anhydrase associated with the sarcoplasmic reticulum (2) A membrane-bound carbonic anhydrase (CA IV) bound to the exterior surface of the sarcolemma (GPI-anchored) (3) A cytosolic form, CA III, that essentially occurs in the slow, type I fibers (4) Another cytosolic form, CA II, whose presence has been demonstrated for fast or type IIb/IIa fibers in rabbit muscles, but which interestingly is absent in fast rat muscles.