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MUSCLE CELL BIOLOGY AND CELL MOTILITY
1Zentrum Physiologie and 2Zentrum Biochemie, Medizinische Hochschule Hannover, Hannover, Germany; and 3Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri
Submitted 9 February 2007 ; accepted in final form 18 April 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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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 Ca2+-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 50% in CA XIV KO mice does not lead to an impairment of SR function. sarcoplasmic reticulum; sarcolemma; isometric contraction; Ca2+-ATPase; ryanodine receptor
The most recently identified CA isozyme, besides CA XV, is CA XIV. CA XIV is a polypeptide with a molecular mass of 54 kDa (12, 23, 27). It contains an extracellular NH2-terminal catalytic domain, a membrane-spanning segment of 22 amino acids, and a short intracellular COOH-terminal segment with several potential phosphorylation sites (8, 19). CA XIV possesses one N-glycosylation site at Asn195 and a disulfide bond between Cys23 and Cys203 residues (36). The amino acid sequence identity of human CA XIV relative to CA IV, CA IX, and CA XII is 35–43% (19). The CA XIV protein is expressed in the brain on plasma membranes of neuronal bodies and axons (23), in the kidney preferentially on the apical membranes of proximal tubules (12), and in the liver on the plasma membranes of hepatocytes (24). CA XIV is also found in crypts and surface epithelial cuff regions of the colon (24) and in the mouse retina on plasma membranes of retinal pigment epithelium, Müller cells, and astrocytes (20, 21). Recently, we (25) have reported the expression of CA XIV on sarcolemmal (SL) membranes as well as on membranes of the longitudinal sarcoplasmic reticulum (SR) of the mouse heart.
CA XIV mRNA has, among other tissues, been found in skeletal muscle (8, 19). Juel et al. (10) reported a CA XIV polypeptide of 51 kDa in homogenates of human skeletal muscles, and Shah et al. (27) reported a CA XIV protein of 54 kDa in homogenates of mouse skeletal muscles. In the present study, we wanted to investigate 1) whether CA XIV is present in SL and/or SR membranes of skeletal muscles; 2) the contribution of CA XIV activity to the total CA activity found in SL and SR membranes, respectively; and 3) whether CA XIV is a glycosylated protein in the skeletal muscle of mice. Double immunostaining with anti-CA XIV and anti-sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA1) as well as with anti-CA XIV and anti-ryanodine receptor (RyR) antibodies was employed to further characterize the localization of CA XIV along the SR membrane. Isometric contraction measurements using muscle fiber bundles of the fast-twitch extensor digitorum longus (EDL) muscle and of the slow-twitch soleus (SOL) muscle from wild-type (WT) and CA XIV knockout (KO) mice were performed to answer the question of whether CA XIV is involved in excitation-contraction coupling (34). The results of the present study suggest that 50% of the total CA activity of the SR as present in the CA XIV KO mice is sufficient to sustain normal muscle function.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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WT mice and mice deficient for CA XIV were narcotized by diethyl ether. Blood samples were taken from the choroid blood vessels, and, after that, mice were immediately killed by cervical dislocation. Skeletal muscles were rapidly excised and kept in 0.75 M KCl and 5 mM imidazole (pH 7.4) at 4°C. SL membrane vesicles as well as SR membrane fractions were prepared as previously described (33). SR membrane vesicles were obtained from the gradient fraction banding at the 35% sucrose phase, and SL membrane vesicles were obtained by centrifugation on a discontinuous Dextran T10 density gradient. All experiments were carried out in accordance with guidelines of the Bezirksregierung Hannover.
Protein
Protein concentration was measured by a micro-Lowry protein assay (15).
Enzyme Measurements
Na+-K+-ATPase. Na+-K+-ATPase was measured as described by Seiler and Fleischer (26). Inorganic phosphate was measured according to the method of Ottolenghi (22). Ouabain-sensitive Na+-K+-ATPase was calculated as the difference between total Na+-K+-ATPase activity and the activity measured in the presence of 1 mM ouabain.
Ca2+- and Mg2+-ATPases. Ca2+- and Mg2+-ATPases were measured as described by Seiler and Fleischer (26). Total ATPase activity was determined in the presence of 50 µM CaCl2, and Mg2+-ATPase activity was determined in the presence of 1 mM Tris-EGTA. The Ca2+-dependent ATPase activity is the difference between total and Mg2+-dependent ATPase activity.
CA. CA was measured as described by Bruns et al. (5). The total CA activity of membrane vesicle fractions was determined in the presence of 0.1% Triton X-100. In the presence of 0.1% Triton X-100, CA was inhibited either by 1:80 diluted anti-CA XIV serum or by 0.1 mM dorzolamide (DZ).
Western Blot Analysis
SL and SR membrane proteins were separated on 10% SDS-polyacrylamide gels under reducing conditions. SL membranes contained 3 µg protein, and SR membranes contained 86 µg protein. Western blot analysis was performed as previously described (31). The polypeptide for CA XIV was characterized using primary rabbit anti-mouse CA XIV antibody at a dilution of 1:3,000 and secondary goat anti-rabbit IgG antibody conjugated with peroxidase (1 ng/ml, Calbiochem). Blots were developed using a luminol/peroxide buffer kit from Pierce Biotechnology (Supersignal West Femto, Rockford, IL). Low-molecular-mass standard proteins were from Bio-Rad Laboratories (Hercules, CA). Endoglycosidase F (Endo F) treatment was performed as described by Zhu and Sly (38), and endoglycosidase H (Endo H) treatment was performed as described by Waheed et al. (32).
SL-WT and SR-WT bands of 54 kDa were also quantitatively analyzed to estimate the distribution of CA XIV protein between SL and SR membranes. Therefore, the amounts of SL-WT and SR-WT protein (6.5 µg and 72 µg, respectively) were chosen so that the samples applied to both membrane fractions contained the same amount of CA enzyme units, namely, 0.033 enzyme units. The quantitative analysis of CA XIV protein bands was performed with ImageMaster 1D software (Pharmacia Biotechnology, Uppsala, Sweden).
Immunofluorescence Staining
Isolated muscle fibers of the EDL from WT and CA XIV KO mice were fixed with 3% paraformaldehyde and 100% methanol and permeabilized in 0.1% Triton X-100 for 5 min as previously described (18). Fibers were incubated with primary antibodies for 30 min. The primary rabbit anti-mouse CA XIV antibody was diluted 1:400, and the primary goat anti-mouse SERCA1, goat anti-mouse RyR, and goat anti-mouse monocarboxylate transporter-4 (MCT-4) antibodies were diluted 1:200. An incubation with FITC-labeled anti-rabbit IgG secondary antibody and with TRITC-labeled anti-goat IgG secondary antibody was performed for a further 30 min. The subcellular localization of CA XIV, SERCA1, RyR, and MCT-4 was examined by confocal laser scanning microscopy (CLSM; Leica, Wetzlar, Germany) and analyzed with Image Span software (Leica).
Blood Parameters
The acid-base status of blood samples from WT and CA XIV KO mice was analyzed by a blood gas and electrolyte system (ABL 505, Radiometer Medical A/S, Copenhagen, Denmark). The values of pH, PCO2, PO2, and base excess (BE) as well as plasma HCO3– concentrations at PCO2 = 40 mmHg (= standard – HCO3–) were determined.
Measurements of Isometric Contractions
EDL and SOL muscles were dissected from euthanized WT and CA XIV KO mice, respectively. Small intact muscle fiber bundles were prepared from tendon to tendon consisting of 50 muscle cells as previously described (35). Muscle bundles were mounted in a chamber with one tendon fixed and the other tendon attached to a force transducer (type AE 801, SensoNor, Horton, Norway). Muscle fiber bundles were directly stimulated using platinum wires and a stimulator (type SD 9, Grass, West Warwick, RI). Isometric contraction signals were displayed on a storage oscilloscope (Nicolet 3091, Madison, WI). Fiber bundles were superfused with Krebs-Henseleit solution containing (in mM) 120 NaCl, 3.3 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.3 CaCl2, and 25 NaHCO3. The Krebs-Henseleit solution was equilibrated with 95% O2-5% CO2 to give a pH of 7.4. In the case of CA inhibition experiments, 0.5 mM DZ or 0.1 mM benzolamide (BZ) was added to the Krebs-Henseleit solution. The temperature of the solution was held at 25.0 ± 0.2°C. The length of the fiber bundles was adjusted to maximize isometric single twitch force. Single twitches were elicited by stimulation with pulses of 1-ms duration and supramaximal voltage. Tetani of EDL fiber bundles were elicited by a 0.4-s train of 1-ms pulses at 100 Hz and tetani of SOL fiber bundles by a 1.5-s train of 1-ms pulses at 50 Hz. During the fatigue test, EDL fibers were stimulated with 0.2-s tetani at 55 Hz repeated every 12.5 s for 40 min, and SOL fibers were stimulated with 1.2-s tetani at 15 Hz repeated every 5 s for 40 min. Single twitches were analyzed for time to peak (TTP), 50% relaxation time (T50%), and peak force. TTP was defined as the time interval between deviation from the initial baseline and peak force. T50% was defined as the time interval between peak force and 50% decay of force. Tetani were analyzed for maximum tetanic force and T50%. All forces are given as force normalized for cross-sectional area (mN/mm2) and were corrected for the varying size of the fiber bundles.
Antibodies and Sulfonamides
Polyclonal rabbit anti-mouse CA XIV antibody was as previously described (23). Goat anti-mouse SERCA 1 (sc-8093) antibody, goat anti-mouse RyR (sc-8170) antibody, and goat anti-mouse MCT-4 (sc-14934) antibody as well as all secondary anti-rabbit IgG and anti-goat IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). DZ was a kind gift from Merck (Rahway, NJ), and BZ was a kind gift from Lederle Laboratories (Pearl River, NY).
CA XIV KO Mice
The CA XIV KO mouse has been characterized previously (27).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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As shown in Table 1, SL membrane fractions of WT as well as of CA XIV KO skeletal muscles were characterized by high activities of ouabain-sensitive Na+-K+-ATPase and Mg2+-ATPase (9). Both enzymes are marker enzymes for SL membranes. Activities of Ca2+-ATPase, which is a marker enzyme for SR membranes, were very low in SL-WT and SL-KO fractions, respectively, and constituted only 2–10% compared with activities found in SR membrane fractions. On the other hand, SR-WT and SR-KO membrane fractions displayed only low activities of Na+-K+-ATPase and Mg2+-ATPase, 1.5–6% of ATPase activities of SL membrane fractions. However, SR-WT and SR-KO membrane fractions were characterized by high activities of Ca2+-ATPase (Table 1). Thus, SL-WT and SL-KO fractions were highly enriched in SL membranes and slightly contaminated with SR membrane vesicles, and SR-WT and SR-KO fractions were highly enriched in SR membranes and showed a minor contamination with SL membrane vesicles.
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Western blot analysis was performed to investigate whether SR and/or SL membranes contain CA XIV. Figure 1 shows an immunoreactive polypeptide of 54 kDa after an incubation of polyvinylidene difluoride membranes with anti-mouse CA XIV serum. CA XIV protein has a molecular mass of 54 kDa. CA XIV was present in SR-WT membranes as well as in SL-WT membranes, whereas CA XIV protein was not detectable in SR-KO and SL-KO membrane fractions (Fig. 1). Treatment of SR and SL membrane vesicles with Endo F revealed that this treatment reduces the molecular mass of CA XIV by 3,000 Da, indicating that CA XIV protein of the mouse contains one N-linked oligosaccharide (Fig. 2). However, treatment of SL with Endo H resulted in a fraction of Endo H-resistant and sensitive N-linked oligosaccharides, suggesting that CA XIV protein of mouse SL membranes contains both a fully processed complex and an unprocessed, high-mannose oligosaccharide (30). To estimate the distribution of CA XIV protein between SR and SL membranes, equal amounts of CA enzyme units of SR-WT and SL-WT fractions were applied. The optical density of the 54-kDa polypeptide band of SL-WT determined by imaging of the gels was set to 100%. The density of the 54-kDa band from SR-WT accounted for 15.3 ± 5.6% of the density of the SL-WT band (n = 8 immunoblots; blots not shown).
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Figure 3A shows CA activities of SL-WT and SL-KO membrane fractions. Total CA activity of the SL-WT fraction, determined in the presence of 0.1% Triton X-100, was 6.3 ± 1.9 U·ml·mg–1. The anti-mouse CA XIV antibody inhibits the CA XIV isozyme, and a serum dilution of 1:80 resulted in a maximal inhibitory effect. Preincubation of SL-WT vesicles with the anti-CA XIV antibody for 30 min at 37°C in the presence of 0.1% Triton X-100 reduced the CA activity to 1.5 ± 0.6 U·ml·mg–1. The remaining activity could be completely inhibited by 0.1 mM DZ, a sulfonamide that inhibits every CA isoform (Fig. 3A). SL-KO membrane vesicles exhibited a total CA activity of 2.0 ± 0.8 U·ml·mg–1. This activity was significantly lower compared with the total CA activity of SL-WT (unpaired t-test, P < 0.01) and similar to the activity of SL-WT determined in the presence of anti-CA XIV serum. Preincubation of SL-KO vesicles with anti-CA XIV serum had no significant effect on the CA activity, but 0.1 mM DZ led to an almost complete inhibition of CA activity of SL-KO vesicles.
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Subcellular Localization of CA XIV
Freshly isolated EDL muscle fiber bundles of WT mice were immunostained using anti-CA XIV/FITC and anti-SERCA1/TRITC. The intracellular staining was examined by CLSM. The CA XIV staining was apparent from the green fluorescence shown in Fig. 4A, and the SERCA 1 staining was apparent from the red fluorescence shown in Fig. 4B. The merge of both fluorescence signals showed a highly expressed colocalization of CA XIV and SERCA1 in the same SR membrane regions (Fig. 4C). Figure 4, D–F, shows double immunostaining with anti-CA XIV/FITC and anti-RyR/TRITC. The merge image, shown in Fig. 4F, clearly showed that the fluorescent sites of CA XIV and RyR do not overlap. Because of the positive colocalization with SERCA1 and the negative colocalization with RyR, it is concluded that CA XIV is preferentially localized in the region of the longitudinal SR and not in the region of the terminal SR. Double immunostaining of EDL fibers from CA XIV KO mice with anti-CA XIV/FITC and anti-RyR/TRITC resulted in a negative control (Fig. 4, G–I). Double immunostaining of EDL fibers from WT mice with preimmune serum/FITC and anti-RyR/TRITC also resulted in a negative control (data not shown).
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Parameters of the acid-base status of blood samples from the retroorbital sinus are shown in Table 2. The values of pH, PCO2, and PO2 indicate that venous blood was obtained. This blood might be slightly affected by a reduced ventilation of the narcotized animals during withdrawal of the blood samples, as judged by PCO2 values >50 mmHg and PO2 values <40 mmHg. However, it is obvious that in WT mice as well as in CA XIV KO mice, values of BE and standard – HCO3– concentrations were normal (Table 2). This excludes a renal acidosis, which might be expected to occur in KO mice since the lack of renal CA XIV might lead to a renal loss of HCO3– in these KO mice.
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To investigate whether skeletal muscles deficient for CA XIV exhibit altered muscle function, isometric contractions of fast-twitch EDL muscle fibers as well as of slow-twitch SOL fibers from WT and CA XIV KO mice were studied. The kinetics of force development and relaxation were investigated by single twitches, the maximum force by tetani, and the fatigue behavior by a fatigue protocol. Figure 6, A and B, shows typical recordings of a single twitch and of a tetanus, respectively, from an EDL fiber bundle. A typical recording of a tetanus as applied during the fatigue test is shown in Fig. 6C.
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Figure 8 shows the values of maximum tetanic forces (left y-axis) and of T50% of tetani (right y-axis). Compared with the tetanic forces of EDL-WT fibers, tetanic forces of EDL-KO, EDL-WT + DZ, and EDL-WT + BZ fiber bundles were not significantly different. It should be noted that in the presence of DZ, EDL-WT fibers exhibited the greatest value of maximum tetanic force. In SOL fibers, the tetanic force of KO fibers was slightly greater than the tetanic force of WT fibers, but the difference was not significant. DZ, but not BZ, significantly increased values of tetanic force in SOL-WT fibers. T50% of tetani from WT, KO, and WT + BZ fibers were comparable; this held for EDL as well as for SOL muscles. T50% of EDL-WT and of SOL-WT fibers were significantly prolonged by DZ.
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60%. As shown in Fig. 9B, the decrease in fractional tetanic forces of SOL-KO fiber bundles was comparable to the decrease seen in tetanic forces of SOL-WT fibers. After 40 min, fractional tetanic forces declined by 70%. Also, in the presence of 0.5 mM DZ, fractional tetanic forces decreased by 60% in EDL-WT fibers and by 70% in SOL-WT fibers (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Juel et al. (10) and Shah et al. (27) studied homogenates of human and mouse skeletal muscles, respectively, by Western blot analysis for CA XIV. Muscle homogenates contain a mixture of SL, SR, and mitochondrial membranes. Here, Western blot analysis using isolated SL and SR membrane vesicle fractions clearly demonstrated that CA XIV protein was associated with SL as well as SR membranes. Since the SR-WT membrane fraction exhibited minor activities of Na+-K+-ATPase and Mg2+-ATPase amounting to 1.5–6% and a low specific CA activity of 6% compared with activities of the SL-WT fraction, one might speculate that the SR-WT fraction was slightly contaminated with SL membrane vesicles and consequently does not contain an intrinsic CA. However, double immunostaining with anti-CA XIV/FITC and anti-SERCA1/TRITC gave clear evidence for CA XIV being colocalized with SERCA1 along the longitudinal SR membrane. The negative controls using preimmune serum in WT fibers and anti-CA XIV serum in CA XIV KO fibers confirmed this result. Double immunostaining with anti-CA XIV/FITC and anti-MCT-4/TRITC confirmed the expression of CA XIV in the SL membrane. Thus, CA XIV is not only expressed in SR and SL membranes of the mouse heart (25) but also in SR and SL membranes of mouse skeletal muscles.
CA XIV Is a Glycoprotein
Mori et al. (19) and Fujikawa-Adachi et al. (8) reported that CA XIV possesses an N-glycosylation motif in its CA domain. The X-ray crystallographic data reported by Whittington et al. (36) are consistent with an N-glycosylation of Asn195 within the CA XIV structure. Treatment of SL and SR membrane fractions with Endo F or Endo H reduced the molecular mass of CA XIV from 54 to 51 kDa (Fig. 2), indicating that CA XIV contains one N-linked complex or high-mannose oligosaccharide chain.
Distribution of Activities and Protein Contents of CA XIV Between SL and SR Membranes
The comparison of total CA activities between SL-WT and SL-KO fractions revealed that CA XIV contributes 66% to the total SL-CA activity (Fig. 3A). The comparison of total CA activities between SR-WT and SR-KO fractions showed that CA XIV constitutes 50% of the total SR-CA activity (Fig. 3B). This is qualitatively in line with the observation that inhibitory anti-CA XIV antibody did not lead to a complete inhibition of CA activities of SL-WT and SR-WT fractions (Fig. 3, A and B). Only the CA inhibitor DZ, which does not distinguish among the different CA isozymes, caused an almost-complete CA inhibition of both membrane fractions. From these data, it can be concluded that CA XIV is not the only CA isoform present in SL and SR membranes of skeletal muscle. Other putative candidates are CA IV, CA IX, and CA XII. Previous biochemical and immunohistochemical studies have provided evidence for CA IV being associated with SL and SR membranes of rat skeletal muscles (6, 31) and for CA IV, CA IX, and CA XIV being associated with mouse cardiac muscle membranes (25).
When the same amounts of CA enzyme units were applied for Western blot analysis, the SR-WT fraction contained only 15.3 ± 5.6% of CA XIV protein of that found in the SL-WT fraction. In combination with the finding that CA XIV accounts for 66% of the total SL-CA activity and for 50% of the total SR-CA activity, this would indicate that CA XIV exhibits an about two times higher specific activity in the SR membrane than it does in the SL membrane. Therefore, we speculate that the activity of CA XIV in SL and SR membranes may be regulated, possibly by phosphorylation/dephosphorylation of the COOH terminus, by oxidation/reduction of the disulfide linkage between Cys23 and Cys203, or by processing of oligosaccharides (30).
Acid-Base Status of WT and CA XIV KO Mice
Mori et al. (19) and Kaunisto et al. (12) demonstrated that CA XIV is expressed in the proximal tubules of mouse and rat kidneys. Since >90% of HCO3– reabsorption occurs in the proximal tubules, it might be expected that CA XIV KO mice suffer from renal acidosis due to a renal loss of HCO3–. However, this assumption can be excluded as shown by the data in Table 2. It is possible that other CA isoforms such as CA II, CA IV, and CA XII compensate the lack of CA XIV in the kidneys of CA XIV-deficient mice. A mixed respiratory and metabolic acidosis occurs in CA II-deficient mice (4, 14). Beekley et al. (2) recently reported that EDL and SOL muscle fibers of CA II-deficient mice exhibit an adaptation of their contractile properties to the acidosis. To exclude a similar situation in CA XIV KO mice, it seemed necessary to study their acid-base status. The results (Table 2) showed that at least metabolic acidosis is not present in these animals.
CA XIV and Isometric Muscle Contractions
Isometric contractions were performed to investigate whether muscle function is affected by the deficiency of CA XIV, especially of SR-CA XIV. Previous studies on rat EDL and SOL fiber bundles have shown that the membrane-permeable CA inhibitors chlorzolamide (CLZ) and L-645,151 led to an increase in peak force, TTP, and T50% of single twitches, whereas the less membrane-permeable CA inhibitors BZ and acetazolamide had no effect (34, 35). All four inhibitors inhibited extracellularly active CAs, which are bound to the SL membrane, but only the membrane-permeable inhibitors CLZ and L-645,151 were able to inhibit SR-CAs and thus affected the time course of single twitches. It has been postulated that SR-CAs are necessary for catalyzing the intracellular CO2/HCO3– buffer system to provide a fast supply of H+, which enters the SR during Ca2+ release by the RyR (7, 11, 17, 28) and a fast buffering of H+ that leaves the SR during Ca2+ uptake by SERCA (13, 16, 37). As previously described (34, 35), inhibition of SR-CAs slows down the H+ transport, which consequentially reduces the rates of Ca2+ release and uptake, which will prolong the rise (TTP) and relaxation (T50%) of twitches. As shown in Figs. 7 and 8, the membrane-permeable CA inhibitor DZ caused an increase in peak force, TTP, and T50% of single twitches as well as of T50% of tetani in EDL and SOL fiber bundles. In contrast, BZ, a membrane-impermeable CA inhibitor, had no effect on twitches and tetani (Figs. 7 and 8). This demonstrates that not only in rats but also in mice, the inhibition of SL-CAs by DZ and BZ does not influence isometric contractions, but it is the inhibition of SR-CAs by DZ that affects muscle contraction. The activity of CA XIV accounts for 50% of the total CA activity found in the SR (Fig. 3B). Does a reduction of the SR-CA activity by 50% affect single twitches and tetani in the same way as full CA inhibition by DZ does? Values of peak force, TTP, and T50% of twitches as well as tetanic forces and T50% of tetani from EDL-KO fiber bundles, being deficient for CA XIV, were comparable with those of EDL-WT fibers (Figs. 7 and 8). Also, fatigue curves of EDL-KO and EDL-WT fiber bundles were nearly identical (Fig. 9A). Thus, it is concluded that in the EDL muscle of mice, a reduction of SR-CA activity by one-half due to CA XIV KO is not sufficient to evoke the effects seen with DZ. It has been shown that CA XIV belongs to the group of sulfonamide-sensitive CAs characterized by Ki values in the nanomolar range, e.g., the Ki of CA XIV for BZ is 0.2 ± 0.02 µM (27). As shown in Fig. 3, 0.1 mM DZ led to an almost total CA inhibition in SL and SR membranes. Thus, it can be assumed that 0.5 mM DZ in the superfusing solution caused a nearly total inhibition of SR-CAs in the contraction experiments and that only inhibition by >>50% will have an impact on single twitches and tetani. It was seen that in the SOL muscle, the parameters of single twitches and tetani from KO fibers were not significantly different from those of WT fibers (Figs. 7 and 8), but it should be noted that the values of force, TTP, and T50% for KO fibers ranged between values of SOL-WT fibers and of SOL-WT fibers in the presence of DZ. This might be taken as an indication that possibly CA XIV is more important in the SR of slow-twitch muscle fibers than in the SR of fast-twitch fibers of mice.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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  R. J. Scheibe, K. Mundhenk, T. Becker, J. Hallerdei, A. Waheed, G. N. Shah, W. S. Sly, G. Gros, and P. Wetzel Carbonic anhydrases IV and IX: subcellular localization and functional role in mouse skeletal muscle Am J Physiol Cell Physiol, February 1, 2008; 294(2): C402 - C412. [Abstract] [Full Text] [PDF]
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, EDL-WT (n = 8 bundles); 
, EDL-KO (n = 8 bundles). B: SOL fiber bundles were stimulated by 1.2-s tetani at 15 Hz repeated every 5 s. 
, SOL-WT (n = 12 bundles); 
, SOL-KO (n = 11 bundles). The amplitude of the first tetanus at time 0 was set to 1.0. The amplitudes of the following tetani were determined every minute, and their amplitudes are expressed as fractional tetanic forces of 1.0. All values are means ± SD. The significance of differences between the mean values of WT and KO fibers determined at the same point of time during the fatigue protocol was calculated by an unpaired t-test. Every calculation was not significant.