RESUMO
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.
Assuntos
Anidrases Carbônicas/metabolismo , Ácido Láctico/metabolismo , Músculo Esquelético/metabolismo , Animais , Transporte Biológico , Anidrases Carbônicas/genética , Eletrodos , Eletrofisiologia , Concentração de Íons de Hidrogênio , Imuno-Histoquímica/métodos , Ácido Láctico/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/genética , MúsculosRESUMO
The subcellular localization of carbonic anhydrase (CA) IV and CA IX in mouse skeletal muscle fibers has been studied immunohistochemically by confocal laser scanning microscopy. CA IV has been found to be located on the plasma membrane as well as on the sarcoplasmic reticulum (SR) membrane. CA IX is not localized in the plasma membrane but in the region of the t-tubular (TT)/terminal SR membrane. CA IV contributes 20% and CA IX 60% to the total CA activity of SR membrane vesicles isolated from mouse skeletal muscles. Our aim was to examine whether SR CA IV and TT/SR CA IX affect muscle contraction. Isolated fiber bundles of fast-twitch extensor digitorum longus and slow-twitch soleus muscle from mouse were investigated for isometric twitch and tetanic contractions and by a fatigue test. The muscle functions of CA IV knockout (KO) fibers and of CA IX KO fibers do not differ from the function of wild-type (WT) fibers. Muscle function of CA IV/XIV double KO mice unexpectedly shows a decrease in rise and relaxation time and in force of single twitches. In contrast, the CA inhibitor dorzolamide, whether applied to WT or to double KO muscle fibers, leads to a significant increase in rise time and force of twitches. It is concluded that the function of mouse skeletal muscle fibers expressing three membrane-associated CAs, IV, IX, and XIV, is not affected by the lack of one isoform but is possibly affected by the lack of all three CAs, as indicated by the inhibition studies.
Assuntos
Anidrase Carbônica IV/metabolismo , Anidrases Carbônicas/metabolismo , Músculo Esquelético/enzimologia , Animais , Anidrase Carbônica IV/genética , Anidrase Carbônica IX , Anidrases Carbônicas/genética , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/genética , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Microscopia Confocal , Contração Muscular/genética , Fibras Musculares de Contração Rápida/enzimologia , Fibras Musculares de Contração Rápida/ultraestrutura , Fibras Musculares de Contração Lenta/enzimologia , Fibras Musculares de Contração Lenta/ultraestrutura , Músculo Esquelético/ultraestrutura , Sarcolema/enzimologia , Sarcolema/ultraestrutura , Retículo Sarcoplasmático/enzimologia , Retículo Sarcoplasmático/ultraestrutura , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Fatores de Tempo , Vesículas Transportadoras/enzimologia , Vesículas Transportadoras/ultraestruturaRESUMO
The expression of carbonic anhydrase (CA) XIV was investigated in mouse skeletal muscles. Sarcoplasmic reticulum (SR) and sarcolemmal (SL) membrane fractions were isolated from wild-type (WT) and CA XIV knockout (KO) mice. The CA XIV protein of 54 kDa was present in SR and SL membrane fractions as shown by Western blot analysis. CA activity measurements of WT and KO membrane fractions showed that CA XIV accounts for approximately 50% and 66% of the total CA activities determined in the SR and SL fractions, respectively. This indicates the presence of at least one other membrane-associated CA isoform in these membranes, e.g., CA IV, CA IX, or CA XII. Muscle fibers of the extensor digitorum longus (EDL) muscle were immunostained with anti-CA XIV/FITC and anti-sarco(endo)plasmic reticulum Ca(2+)-ATPase 1/TRITC, with anti-CA XIV/FITC and anti-ryanodine receptor/TRITC, or with anti-CA XIV/FITC and anti-monocarboxylate transporter-4/TRITC. CA XIV was expressed in the plasma membrane and in the longitudinal SR but not in the terminal SR. Isometric contraction measurements of single twitches and tetani and a fatigue protocol applied to fiber bundles of the fast-twitch EDL and of the slow-twitch soleus muscle from WT and KO mice showed that the lack of SR membrane-associated CA XIV did not affect maximum force, rise and relaxation times, and fatigue behavior. Thus, it is concluded that a reduction of the total SR CA activity by approximately 50% in CA XIV KO mice does not lead to an impairment of SR function.
