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.Public Library of Scienc

Schematic representation of the cooperation of the MCT4 and the three membrane-bound CAs in lactic acid transport across the sarcolemma.

<p>About half of lactic acid transport (in this scheme efflux) occurs via the surface membrane, supported by the buffering action of CA XIV and CA IV. The other half occurs via the T tubular membrane and is supported by the buffering action of CA IX. CA IX and half of the total sarcolemmal MCT4 are colocalized in the T tubule. The removal of lactic acid from the T tubules occurs by outward diffusion of lactate, while the H⁺ are transported out by an inward diffusion of HCO₃⁻ in combination with an outward diffusion of CO₂, a CO₂- HCO₃⁻ shuttle. This removal mechanism operates effectively in spite of the long diffusion distance from the T tubule interior to the extracellular space due to very large concentration gradients of lactate and HCO₃⁻ that can build up along the T tubule. These gradients and the mobility of protons are much smaller in the sarcoplasm of the fiber.</p

Lactate influxes and amplitudes of surface pH transients.

<p>Influxes (a) and amplitudes (b) are shown for WT fibers and for fibers lacking CA IV, CA XIV or both. On the right, both figures show the effects of the extracellular CA inhibitor benzolamide.</p

Lactate influx and efflux measurements in EDL fibers from various knockout mice.

<p>a) Lactate influxes in WT, CA IV ko, CA XIV ko and CA IX ko fibers. b) Lactate effluxes from the same combination of fibers as in 4a. c) Comparisons of lactate influxes in WT vs. CA IX ko fibers (left), in CA IV-CA XIV double ko vs. CA IV-CA IX-CA XIV triple ko (middle), and WT vs. triple ko, both in the presence of the membrane-permeable CA inhibitor ethoxzolamide (right). Columns for lactate influxes in WT, CA IV ko, CA XIV ko, and CA IV/CA XIV double ko are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015137#pone-0015137-g003" target="_blank">Fig. 3</a>. Stars indicate significant differences from WT (* P<0.05; ** P<0.01). #(s) indicates a significant difference between the CA IX ko and the triple ko fluxes (P<0.05), &(ns) indicates that there is no significant difference between double ko and triple ko fluxes.</p

Lactic acid fluxes in mouse EDL fibers.

<p>a) Schematic representation of the mechanisms of H⁺ production and H⁺ buffering associated with lactic acid influx and efflux in skeletal muscle. Fluxes can be quantitated by following the changes in intracellular pH. The changes in surface pH illustrate the associated processes of proton consumption or production on the surface membrane. b) pH_S and pH_I during exposure to and after subsequent withdrawal of lactate in the bathing solution in a WT EDL fiber. c) pH_S and pH_I during the same maneuver as in 2b in a CA IV-CA XIV double ko mouse EDL fiber. Lactate fluxes are decreased in comparison to 2b and pH_S transients are greatly enhanced. Note that pH_S curves in b and c are shifted on the pH ordinate by + 0.3 units to improve visibility. The standard bathing solution is Krebs-Henseleit solution, pH 7.4, at room temperature and equilibrated with 5% CO₂/95%O₂.</p

Immunocytochemical CLSM images from fibers of mouse EDL muscle.

<p>a) Simultaneous exposure to antibodies against CA IV, CA IX or CA XIV and MCT4. The microscope was focussed on the plane of the fiber surface membrane. CA IV, basal homogeneous membrane staining and enrichment at entrances to T tubuli. CA IX, absence from the surface membrane. CA XIV, homogeneous surface membrane staining. b) Simultaneous staining with antibodies against CA IV, CA IX or CA XIV and RyR. The microscope was focussed on a plane inside the cell exhibiting triads. CA IV, staining of entire SR. CA IX, staining of triads (T tubules) but not SR. CA XIV, staining of light SR but not the triads (terminal cisternae of the SR and T tubules). c) Simultaneous staining with antibody against CA IX and RyR or MCT4. In the upper lane, the microscope was focussed on a plane inside the fiber free of triads; nevertheless, CA IX staining exihibits the same staining pattern as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015137#pone-0015137-g001" target="_blank">Fig. 1b</a>, middle lane, indicating it is associated with T tubules. In the middle lane a plane is shown, in which RyR are fully, or partially or not at all visible, causing an incomplete RyR pattern compared to that seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015137#pone-0015137-g001" target="_blank">Fig. 1b</a>. Nevertheless, the staining pattern of CAIX is as complete and regular as in the lane above. This confirms that CAIX staining is associated with T tubules but not necessarily with RyR. In the lowest lane it is seen that CA IX and MCT4 are perfectly colocalized in T tubules.</p