Inhibition of muscle carbonic anhydrase slows the Ca2+ transient in rat skeletal muscle fibers

Petra Wetzel, Tanja Kleinke, Simon Papadopoulos, and Gerolf Gros

Zentrum Physiologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A countertransport of H+ is coupled to Ca2+ transport across the sarcoplasmic reticulum (SR) membrane. We propose that SR carbonic anhydrase (CA) accelerates the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reaction so that H+ ions, which are exchanged for Ca2+ ions, are produced or buffered in the SR at sufficient rates. Inhibition of this SR-CA is expected to reduce the rate of H+ fluxes, which then will retard the kinetics of Ca2+ transport. Fura 2 signals and isometric force were simultaneously recorded in fiber bundles of the soleus (SOL) and extensor digitorum longus (EDL) from rats in the absence and presence of the lipophilic CA inhibitors L-645151, chlorzolamide (CLZ), and ethoxzolamide (ETZ), as well as the hydrophilic inhibitor acetazolamide (ACTZ). Fura 2 and force signals were analyzed for time to peak (TTP), 50% decay time (t50), and their amplitudes. L-645151, CLZ, and ETZ significantly increased TTP of fura 2 by 10-25 ms in SOL and by 5-7 ms in EDL and TTP of force by 6-30 ms in both muscles. L-645151 and ETZ significantly prolonged t50 of fura 2 and force by 20-55 and 40-160 ms, respectively, in SOL and EDL. L-645151, CLZ, and ETZ also increased peak force of single twitches and amplitudes of fura fluorescence ratio (R340/380) at an excitation wavelength of 340 to 380 nm. All effects of CA inhibitors on fura 2 and force signals could be reversed. ACTZ did not affect TTP, t50, and amplitudes of fura 2 signals or force. L-645151, CLZ, and ETZ had no effects on myosin-, Ca2+-, and Na+-K+-ATPase activities, nor did they affect the amplitude and half-width of action potentials. We conclude that inhibition of SR-CA by impairing H+ countertransport is responsible for deceleration of intracellular Ca2+ transients and contraction times.

sarcoplasmic reticulum; H+ countertransport; fura 2 transients; single twitches


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AN EXTRACELLULAR, SARCOLEMMAL (SL) carbonic anhydrase (CA), which is GPI anchored, is present in fast- and slow-twitch skeletal muscles, and CAIII occurs in the cytoplasm of slow-twitch muscles (see Ref. 43 for an overview). Several studies have provided evidence for an additional muscle CA bound to the membrane of the sarcoplasmic reticulum (SR). Bruns et al. (3) were the first to report CA activity in isolated SR vesicles from rabbit muscles. By Triton X-114 phase separation experiments, it could be shown that this CA activity originated from a membrane-bound isozyme, rather than from a cytosolic CA. Estimations of inhibition and catalysis constants revealed different properties of the CA of SR vesicle fractions and CA of SL vesicles and confirmed the existence of two membrane-bound CAs in muscle (41). Histochemical studies with the fluorescent CA inhibitor dimethylaminonaphthalene-5-sulfonamide (3, 7) and immunoelectron microscopic studies with ultrathin sections (6) demonstrated an intracellular staining pattern, which is compatible with a CA associated with the SR membrane. In a previous study, we reported that inhibition of this SR-CA leads to significant changes in single twitches of fiber bundles of the soleus (SOL) and extensor digitorum longus (EDL) from rats (45). Inhibition of this enzyme prolonged the rise and relaxation times and slightly increased force production. To investigate whether these changes were mediated by corresponding changes in the intracellular Ca2+ transient, fiber bundles of SOL and EDL were loaded with the ester form of fura 2. We recorded simultaneously intracellular Ca2+ transients by fura 2 fluorescence measurements and isometric single twitches in the absence and presence of the lipophilic and highly membrane-permeable CA inhibitors L-645151, chlorzolamide (CLZ), and ethoxzolamide (ETZ) (2, 22). Our results indicate that the prolonged rise time of twitches is accompanied by a prolonged release of Ca2+ from the SR, the slow muscle relaxation is accompanied by slow kinetics of Ca2+ reuptake, and the increase in peak force is associated with elevated intracellular free Ca2+ concentrations under SR-CA inhibition. A possible role of the SR-CA in the transport of Ca2+ during excitation-contraction coupling is proposed.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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REFERENCES

Preparation of Fiber Bundles

Female Wistar rats (body mass 180-230 g) were killed by an overdose of diethyl ether. The SOL, a slow-twitch muscle, and the EDL, a fast-twitch muscle, were dissected out and kept in oxygenated Krebs-Henseleit solution. From these muscles, fiber bundles were prepared with spring scissors under a Wild M8 microscope (Leica). The bundles consisted of ~15-30 muscle cells.

Preparation of Skinned Fibers

For determination of myosin-ATPase activity, the freshly prepared fiber bundles of SOL and EDL were incubated in a skinning solution overnight at 4°C.

Preparation of SL and SR Vesicles

Four female Wistar rats (body mass 180-230 g) were killed by an overdose of diethyl ether. The white muscles from the hindlimbs were rapidly dissected out and kept in 0.75 M KCl and 5 mM imidazole, pH 7.4, at 4°C. SL and SR vesicles were prepared according to the method of Seiler and Fleischer (33) modified by Wetzel and Gros (40). SR membrane vesicles were obtained from the gradient fraction banding at the 35% sucrose phase. To obtain a purified vesicle fraction highly enriched in SL membranes almost free of contamination with SR vesicles, we further enriched SL membrane fractions obtained from the sucrose gradient centrifugation by centrifugation on a discontinuous dextran density gradient. From this gradient, the pellet was removed, and all other dextran gradient fractions were pooled to give the SL vesicle fraction.

The SL vesicle fraction displayed high activities in the SL marker enzymes, the ouabain-sensitive Na+-K+-ATPase and the Mg2+-ATPase (13), of 91 ± 7 µmol Pi · mg-1 · h-1 and 1.3 ± 0.2 U/mg, respectively. The activity of the Ca2+-ATPase, a marker enzyme for SR, was 0.8 ± 0.1 U/mg. The SR vesicle fraction was characterized by a high activity of the Ca2+-ATPase of 2.4 ± 0.2 U/mg and by minor activities of the ouabain-sensitive Na+-K+-ATPase of 5 ± 1 µmol Pi · mg-1 · h-1 and the Mg2+-ATPase of 0.09 ± 0.02 U/mg. From the distribution of these marker enzyme activities, we conclude that the SL vesicle fraction is highly enriched in SL membrane vesicles and only slightly contaminated by SR membranes and that the SR vesicle fraction is highly enriched in SR membranes and is contaminated by SL vesicles only to an insignificant degree.

All experiments were carried out in accordance with the guidelines of the Bezirksregierung Hannover.

Simultaneous Recordings of Isometric Single Twitches and Fura 2 Signals

The fiber bundle was transferred to a chamber that was perfused with oxygenated 25 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-5% CO2-buffered Krebs-Henseleit solution and was completely immersed in this solution. One end of the fiber bundle was fixed, and the other end was connected to a force transducer (SensoNor, Friedberg, Germany). The length of the bundle was adjusted to give maximal isometric twitch tension. Single twitches were triggered by direct stimulation of the muscle cells by pulses of 1-ms duration and supramaximal voltage using platinum wires. After an initial phase of 2.5 h, during which peak force and the rise and relaxation times achieved stable values, the fiber bundle was loaded with 0.7 µmol/l fura 2-AM in the presence of 0.0004% Pluronic F-127 (Molecular Probes, Eugene, OR) for 2 h. The Krebs-Henseleit superfusion solution was then completely exchanged to remove the extracellular fura 2-AM. After three to four fura 2 and force signals were recorded as control values within the next 45-60 min, the CA inhibitor was added to the superfusion solution for 60 or 90 min, and the fiber bundle was stimulated every 15 min. Incubation with the CA inhibitor was terminated by a complete exchange of the circulating Krebs-Henseleit solution. Reversibility of inhibitory effects was tested for a further 90 min. The temperature of the superfusion solution was held at 21°C.

Fura 2 was excited by light wavelengths of 340, 360, or 380 nm (ultraviolet filters; Schott, Mainz, Germany). The intensity of the emitted light was measured by a photomultiplier (type HTV R928) attached to a Zeiss fluorescence microscope using a 500- to 530-nm band-pass filter. The time resolution of light intensity data acquisition was 1 ms for each excitation wavelength. Further details concerning the experimental setup have previously been described (42, 44). Recordings at the wavelength of 360 nm, the isosbestic point of the fura 2 excitation spectrum, were used to select an area of the fiber bundle for fura 2 measurements, the light emission of which was not influenced by the movement of the bundle during contraction. The fura 2 signal for one excitation wavelength and the isometric single twitch could be recorded simultaneously with this setup. Figure 1 shows the isometric force recording and the simultaneously recorded fura 2 signal at the excitation wavelength of 380 nm of a SOL fiber. The ratio of fura 2 fluorescence, R340/380 (Fig. 1C), was estimated from separate recordings at 340- and 380-nm excitation wavelengths taken immediately after each other according to the following equation (11): R340/380 = (fura 2 fluorescence at 340 nm - autofluorescence of the fiber bundle at 340 nm)/(fura 2 fluorescence at 380 nm - autofluorescence of the fiber bundle at 380 nm). R340/380 values were determined only when the two force recordings taken with the fluorescence measurements were identical and were both taken from a stable bundle within a few minutes. The force recordings were analyzed for peak force, time to peak (TTP), and time of 50% decay (t50). TTP was defined as the time interval between the point where the traces deviated visibly from the baseline and the peak value. In the case of the fura 2 signals, the former point coincided with the electrical stimulus. The t50 was defined as the time interval between the peak value and the time at which the signal had decayed to 50% of peak amplitude. R340/380 values (Fig. 1C) were analyzed for their value under resting conditions and the peak value after electrical stimulation. The difference between the peak value and the value at rest gives the R340/380 amplitude.


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Fig. 1.   Recordings of single-twitch force, fura 2 fluorescence at 380-nm excitation wavelength, and ratio of fluorescence at 340-nm excitation to that at 380-nm excitation (R340/380) of a soleus (SOL) fiber bundle in the absence and presence of ethoxzolamide (ETZ). A: force recordings of a single twitch of an SOL fiber bundle. B: simultaneously recorded fura 2 signals at an excitation wavelength of 380 nm. C: R340/380 calculated from fura 2 recordings according to Grynkiewicz et al. (11). Resting levels of fura 2 fluorescence between 0 and 0.3 s in B and C were set to 1 to facilitate comparison of curves. Black curves were recorded before and red curves at the end of 45-min exposure to 0.1 mM ETZ. Decay phases of curves in B and C were fitted by a sigmoidal equation with 4 parameters (Sigma Plot 5.0, SPSS, Chicago, IL). Values of 50% decay time (t50) with (red) and without (black) ETZ are indicated by lengths of bars. ETZ caused increases in time to peak (TTP), t50, and peak force of single twitches, which were accompanied by increases in TTP, t50, and amplitudes of fura 2 signals at the 380-nm excitation wavelength and of R340/380.

Measurement of Action Potentials

The microelectrodes were pulled from borosilicate glass tubing with a filament (KBF-112080, ZAK Products, Marktheidenfeld, Germany) and filled with a solution containing 1.5 M KCl and 1.5 M potassium acetate (pH adjusted to 6.6-6.7 with HCl). Two microelectrodes were impaled into the same muscle cell of an SOL fiber bundle: one was used for voltage recording and the other for current injection. The resistance of the microelectrodes for voltage recording varied between 8 and 20 MOmega , and that of the electrodes used for current injection varied between 2 and 5 MOmega . Currents of 40-90 µA were injected into the cell by 1-ms pulses. 2,3-Butanedione monoxime (10 mM) was added to the Krebs-Henseleit solution, while the NaCl concentration was reduced by 10 mM, to minimize the movement of the fiber caused by electrical stimulation.

Protein

Protein contents of SL and SR vesicle fractions, as well as skinned fibers, were measured according to the method of Lowry et al. (18) modified by Peterson (29) using a protein assay kit from Sigma. Myosin was prepared by extraction from skinned fibers by incubation in 0.6 M KCl, 10 mM EGTA, 1 mM tetrasodium diphosphate, 0.1 mM dithiothreitol, 5 mM KH2PO4, 5 mM K2HPO4, and 1 mM phenylmethylsulfonyl fluoride, pH 6.8, and proteins were suspended by an ultrasound scanner.

Myosin-ATPase

Myosin-ATPase of skinned fibers from SOL and EDL was measured in an assay medium containing 50 mM imidazole, pH 7.4, 6 mM KCl, 4 mM MgCl2, 0.5 mM EGTA, 5 mM NaN3, 2.2 mM CaCl2, 4.2 mM Na2ATP, 0.33 mM NADH, 1.2 mM phosphoenolpyruvate, 15 U/ml lactate dehydrogenase, and 14 U/ml pyruvate kinase in a spectrophotometer at 37°C. The formation of ADP by myosin-ATPase activity was coupled to the formation of pyruvate and ATP from phosphoenolpyruvate and ADP. The pyruvate-to-lactate conversion was coupled to the oxidation of NADH, which was continuously monitored by the decrease in absorbance at 340 nm.

Ca2+- and Mg2+-ATPases

Ca2+- and Mg2+-ATPases were measured as described by Seiler and Fleischer (33). Inorganic phosphate was measured according to the method of Ottolenghi (27). The Ca2+-dependent ATPase activity was calculated as the difference between the activity of the total ATPase and the activity of the Mg2+-ATPase.

Na+-K+-ATPase

Na+-K+-ATPase was measured as described by Seiler and Fleischer (33). Ouabain-sensitive Na+-K+-ATPase was calculated as the difference between the total Na+-K+-ATPase and the Na+-K+-ATPase measured in the presence of 1 mM ouabain.

Solutions

Krebs-Henseleit solution was composed of (in mM) 120 NaCl, 3.3 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.3 CaCl2, and 25 NaHCO3. Skinning solution consisted of (in mM) 5 KH2PO4, 3.0 magnesium acetate, 50 creatine phosphate, 5 EGTA, 1 Na2ATP, 1 dithiothreitol, and 0.5% Triton X-100, pH 7.4.

CA Inhibitors

Acetazolamide (ACTZ) and ETZ were purchased from Sigma-Aldrich (Munich, Germany). CLZ and L-645151 were generous gifts of Lederle Laboratories (Pearl River, NY). Stock solutions of ETZ, CLZ, and L-645151 at 10 mM each were prepared in Krebs-Henseleit solution in the presence of 20 mM NaOH. Aliquots of these stock solutions were added to the superfusion solution in 100-fold dilution (20-fold in the case of CLZ), and the amount of added NaOH was compensated by the addition of the equivalent amount of HCl. The final inhibitor concentration in the superfusion solution was always checked photometrically.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of Fura 2 on Contraction Parameters of Single Twitches

The contraction parameters TTP, t50, and peak force were determined before, during, and after the fiber bundles were loaded with fura 2 to test whether they were affected by fura 2 acting as a Ca2+ buffer. Table 1 gives data for SOL and EDL. In SOL, TTP was not affected by fura 2 loading, whereas t50 was reduced from 270 ± 36 ms (control) to 252 ± 11 ms after 120 min of loading with fura 2 and remained stable for a further 2 h. Peak force of SOL significantly decreased from 100% to 88 ± 7% during fura 2 loading, but in the following 2 h the values remained stable. In a separate series of experiments in which SOL fibers were not loaded with fura 2, the TTP and t50 did not change in 8 h, and at a point comparable to that after 120 min of fura 2 loading, force was reduced only to 97 ± 6%, and the difference from 88 ± 7% is significant as estimated by Student's unpaired t-test (P < 0.05). In EDL, fura 2 loading of muscle fibers did not affect TTP and t50 but significantly reduced peak force by 12% (Table 1). When EDL fibers were not loaded with the Ca2+ indicator, force was stable over a comparable period of time at 99 ± 2%, and the difference from 88 ± 11% was significant (P < 0.05, Student's unpaired t-test). Thus fura 2 loading led to a decrease in peak force by ~12% in SOL and EDL and a reduction in t50% by ~7% in SOL. A fluorescent Ca2+ indicator acting as a Ca2+ buffer may reduce the amplitude of the free intracellular Ca2+ concentration and, consequently, peak force and will prolong the decay of free intracellular Ca2+ and, consequently, force relaxation, as shown by Ashley et al. (1) and Noble and Powell (25). Our results indicate that some Ca2+ buffering effect of fura 2 might be responsible for the slight decrease in peak force of twitches, but it did not lead to a prolongation of the kinetics of twitches. All effects of the CA inhibitors on TTP, t50, and the amplitudes of Ca2+ transients and force recordings were compared with control values obtained after fura 2 loading and were tested in terms of reversibility.

                              
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Table 1.   TTP, t50, and peak force of single twitches before, during, and after fibers were loaded with fura 2-AM

Effects of ETZ on Force and Fura 2 Signals of a Fiber Bundle From SOL

Figure 1 shows the single twitch, the simultaneously recorded fluorescence signal at 380-nm excitation wavelength, and the calculated R340/380 before the addition of the CA inhibitor ETZ and after 45 min of exposure to 0.1 mM ETZ. When both force signals of Fig. 1A were compared, ETZ increased TTP from 162 to 176 ms, t50 from 343 to 553 ms, and peak force amplitude from 0.95 to 1.07 mN. The increases in TTP and t50 of the single twitches were accompanied by increases in TTP and t50 of the 380-nm signals: TTP was prolonged from 14 to 24 ms and t50 from 105 to 165 ms (Fig. 1B). The increase in peak force was accompanied by an increase in the amplitude of the 380-nm signal from 0.22 to 0.31 (Fig. 1B) and, less markedly, in R340/380 by 0.08 (Fig. 1C).

Effects of CA Inhibitors on TTP of Fura 2 and Force Signals

Figure 2A shows the time courses of TTP of fura 2 and force signals from EDL fiber bundles. L-645151 was added to the Krebs-Henseleit superfusion solution at time 0 and was present for 90 min. L-645151 (0.1 mM) prolonged TTP of fura 2 signals as well as TTP of force. After removal of L-645151, these effects were reversed. To obtain the values of Table 2, control data from -60 to 0 min were combined in one group and given as means ± SD. Data obtained in the presence of CA inhibitor during an exposure time of 15 min to 60 min (CLZ) or to 90 min (L-645151 and ETZ) were combined into one group and given as means ± SD (Table 2). Data of the washout phases from 75 to 150 min (CLZ) and from 105 to 180 min (L-645151 and ETZ) were also combined into one group. Means ± SD of the phases before, during, and after exposure to the CA inhibitor are listed in Table 2. Significance of differences between the control values and the values determined in the period of exposure to the CA inhibitor was estimated by Student's unpaired t-test. Significance of differences between the values in the presence of inhibitor and the values in the washout phase was also estimated. L-645151, CLZ, and ETZ caused significant increases in TTP of fura 2 as well as of force signals (Table 2). All these effects were significantly reversed on removal of the inhibitors.


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Fig. 2.   Time courses of TTP, t50, and amplitudes of fura 2 and force signals of extensor digitorum longus (EDL) fiber bundles. A: time courses of TTP before, during, and after exposure to L-645151 [number of fiber bundles tested (n) = 3]. B: time courses of t50 before, during, and after exposure to L-645151 (n = 3). C: time courses of peak force and of R340/380 amplitude before, during, and after exposure to ETZ (n = 2). open circle , Fura 2 signals at 380-nm excitation wavelength (A and B) and R340/380 amplitudes (C); , simultaneously recorded single-twitch forces. Values are means ± SD. Control values between -60 and 0 min were combined to give 1 group and are shown as means ± SD in Table 2. Values obtained in the presence of L-645151 and ETZ between 15 and 90 min of exposure and values after removal of inhibitors, taken between 105 and 180 min, were combined into groups and are shown as means ± SD in Table 2.


                              
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Table 2.   TTP, t50, and amplitudes of fura 2 and force signals before, during, and after exposure of EDL fiber bundles to CA inhibitors

In SOL, TTP of fura 2 signals and of force signals were also increased by L-645151, CLZ, and ETZ (Table 3; increases in TTP of fura 2 signal by L-645151 and in TTP of force signal by ETZ were not significant). After removal of the CA inhibitors, all effects showed full or partial reversibility.

                              
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Table 3.   TTP, t50, and amplitudes of fura 2 and force signals before, during, and after exposure of SOL fiber bundles to CA inhibitors

Figure 3A shows that TTP of the force signal of SOL bundles is affected by CLZ in a dose-dependent manner. TTP begins to increase at >= 10-4 M CLZ.


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Fig. 3.   Dose-response curves of effect of chlorzolamide (CLZ) on TTP (A) and t50 (B) of force signals from SOL fibers. SOL fiber bundles were exposed to CLZ for 60 min. Single twitches were recorded at 0 min (control value) and after incubation with CLZ. Data at 0 min were set to 100%, and data with inhibitor are expressed as percentage of control. Effects of CLZ reached a plateau at 30 min; therefore, data at 30, 45, and 60 min were combined for each bundle and expressed as means ± SD of 9 measurements from 3 fiber bundles for 2 × 10-5 M CLZ, 12 measurements from 4 fiber bundles for 5 × 10-5 M CLZ, 18 measurements from 6 fiber bundles for 7.5 × 10-5 M CLZ, 24 measurements from 8 fiber bundles for 1 × 10-4 M CLZ, 12 measurements from 3 fiber bundles for 2.2 × 10-4 M CLZ, 24 measurements from 8 fiber bundles for 5 × 10-4 M CLZ, and 9 measurements from 3 fiber bundles for 1 × 10-3 M CLZ.

Effects of CA Inhibitors on t50 of Fura 2 and Force Signals

Figure 2B shows the time courses of t50 of fura 2 and force signals from EDL fibers. L-645151 led to a distinct increase in t50 of fura 2 and of force signals. The observation that in EDL the absolute values of t50 of fura 2 signals are higher than those of force signals has been discussed in a previous study (42). Table 2 gives the data for EDL before, during, and after exposure to the inhibitors. All three membrane-permeable inhibitors caused a significant prolongation of t50, which was significantly reversed on their removal. Table 3 summarizes the effects of L-645151, CLZ, and ETZ in SOL fibers. In SOL, as in EDL, t50 values of fura 2 and of force signals were prolonged by the inhibitors (prolongation of t50 of fura 2 signals by L-645151 and, not significantly, by ETZ), and their effects were reversible. Figure 3B illustrates that t50 of the force signal of SOL also varies with CLZ concentration in a dose-dependent manner, reaching a plateau at ~5 × 10-4 M CLZ.

Effects of CA Inhibitors on Peak Force of Single Twitches and R340/380

In Fig. 2C, peak forces are given as percentages. Because it was not possible to prepare fiber bundles of identical size, the values of force varied: in SOL between 1.5 and 3.5 mN and in EDL between 2 and 6 mN. Therefore, the mean value derived from the values of the control phase of -60 to 0 min (Fig. 2C) has been set at 100%, and the values of peak force during and after exposure to the CA inhibitor have been expressed as a percentage of this mean value. ETZ at 0.1 mM increased peak force and amplitude of R340/380 (Fig. 2C). The three lipophilic and membrane-permeable CA inhibitors, L-645151, CLZ, and ETZ, significantly increased peak force of twitches: in EDL by 10-80% and in SOL by 10-30% (Tables 2 and 3). To determine whether these increases in peak force were accompanied by an increase in Ca2+ release from the SR, R340/380 values were calculated and analyzed for their peak values and amplitudes. In the case of L-645151, this was not possible, because this inhibitor displayed an autofluorescence at 340- and 380-nm excitation wavelengths. In EDL, CLZ led to a small increase in peak force by 9%, which was not paralleled by an increase in the amplitude of R340/380 (Table 2). ETZ caused a significant increase in peak force (~40%), which was accompanied by a significant increase in the amplitudes of R340/380 from 0.5 ± 0.1 to 0.8 ± 0.2 (Table 2, Fig. 2C). In SOL, CLZ increased peak force of single twitches by ~20% and the amplitudes of R340/380 from 0.99 ± 0.30 to 1.27 ± 0.04 (Table 3). ETZ led to an increase in peak force by 12%, which coincided with a slight, not significant, increase in the amplitudes of R340/380 (Table 3). In some, but not all, cases, the increases in peak force were accompanied by increased amplitudes of R340/380. It appears possible that, in the presence of permeable inhibitors, more Ca2+ is released on muscle activation. The values of R340/380 under resting conditions were not influenced by the CA inhibitors.

Lack of Effects of ACTZ on Fura 2 and Force Signals of SOL Fibers

ACTZ is a hydrophilic and, therefore, poorly membrane-permeable CA inhibitor (22) that predominantly inhibits the extracellular SL-CA and affects the intracellular CAs only to a minor degree. In SOL, ACTZ at 1 mM, which is 10 times higher than the concentration of L-645151 and ETZ and 2 times higher than that of CLZ, did not lead to a prolongation of TTP in fura 2 or in force signals (Fig. 4A). The t50 values of fura 2 signals were not affected by ACTZ (Fig. 4B). The t50 of force was only slightly increased by ~10-20 ms by ACTZ (Fig. 4B), whereas L-645151 and ETZ increased t50 of force by ~140 and 70 ms, respectively (Table 3). Neither the amplitudes of R340/380 nor the values of peak force were increased by ACTZ (Fig. 4C, Table 3).


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Fig. 4.   Time courses of TTP and t50 of fura 2 and force signals and of amplitudes of R340/380 and peak force of SOL fiber bundles in the absence and presence of acetazolamide (ACTZ). A: TTP values of fura 2 signals (open circle ) and single twitches (). B: t50 values of fura 2 signals (open circle ) and single twitches (). C: amplitudes of R340/380 (open circle ) and peak force of single twitches (). Values are means ± SD; 4 SOL fiber bundles were tested.

Lack of Effects of CA Inhibitors on Myosin-, Ca2+-, and Ouabain-Sensitive Na+-K+-ATPase Activities

Myosin-ATPase. Myosin-ATPase activities were measured in skinned fibers. First, the Ca2+-activated myosin-ATPase activity was monitored for 5-10 min, then the inhibitor was added to the assay medium, and the activity was measured for a further 5-10 min. Because of the different size of fibers, the absolute activities varied between 0.9 and 4.7 µmol ADP · mg-1 · h-1. Therefore, the myosin-ATPase activities in the absence of CA inhibitor were set at 100% (i.e., control values), and the activities in the presence of the inhibitor were expressed as percentage of controls. The myosin-ATPase activities of SOL and EDL were not significantly affected by L-645151, CLZ, or ETZ (Table 4).

                              
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Table 4.   Activities of myosin-, Ca2+-, and Na+-K+- ATPases in the absence and presence of CA inhibitors

Ca2+-ATPase. Ca2+-ATPase activity was determined in SR vesicles of white hindlimb muscles from rat. Different concentrations of the Ca2+ ionophore A-23187 were tested, and 5 µg/ml gave the maximum activation of the Ca2+-ATPase. Because the activities were dependent on the lot number of the ionophore used in the assay and more than one lot number of the ionophore was needed for all determinations, it was necessary that Ca2+-ATPase activities be given as percentages so that they could be compared with activity values that were determined with different lot numbers of ionophore. The mean value of the total ATPase activity was set at 100%, and the ATPase activities in the absence of Ca2+ were expressed as percentage of this mean value. The Ca2+-ATPase activity was calculated as the difference between the total and the basal Mg2+-dependent ATPase activities. Total and basal ATPase activities were measured in the absence and presence of CA inhibitors. Table 4 shows the Ca2+-ATPase activities determined in the absence of CA inhibitor (i.e., control values) and the activities determined in the presence of L-645151, CLZ, and ETZ. None of these inhibitors significantly affected the Ca2+-ATPase in a stimulatory or an inhibitory manner.

Ouabain-sensitive Na+-K+-ATPase. The ouabain-sensitive Na+-K+-ATPase activity of white muscle SL vesicles was not affected by the CA inhibitors L-645151, CLZ, and ETZ (Table 4).

Lack of Effects of CLZ and ETZ on the Action Potentials of SOL Fibers

Action potentials (APs) were measured in single muscle cells of SOL at room temperature. APs were evoked every 3-5 min over a 40- to 100-min period by current injection into a single cell. Fifty-two APs of four muscle cells were measured in the absence of a CA inhibitor and used as controls. Seventy APs of six muscle cells were elicited in the presence of 0.1 mM ETZ and 50 APs of four muscle cells in the presence of 0.22 mM CLZ. Each AP signal was analyzed for its amplitude and its half-width. The time courses of AP amplitudes under control and inhibitory conditions are shown in Fig. 5. The linear regression calculation of AP amplitude vs. time gave the intercept a of the linear regression line with the y-axis and the regression coefficient m. Under control conditions, a = 78 mV and m = -13 mV/h. In the presence of ETZ, the linear regression line is given by a = 88 mV and m = -7.9 mV/h and in the presence of CLZ by a = 88 mV and m = -15 mV/h (Fig. 5). In the case of the half-width of AP signals (not shown), the linear regression line of control is given by a = 2.34 ms and m = 0.82 ms/h. In the presence of ETZ, a = 1.98 ms and m = 0.44 ms/h, and in the presence of CLZ a = 2.10 ms and m = 0.56 ms/h. The linear regression lines obtained in the presence of ETZ or CLZ are not significantly different from control.


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Fig. 5.   Time courses of amplitudes of action potentials stimulated in single SOL fibers in the absence of ETZ and CLZ (), with extracellular 0.1 mM ETZ (open circle ), and with extracellular 0.22 mM CLZ (black-down-triangle ).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hypothesis on the Role of SR-CA for Countertransport of H+ During Ca2+ Release and Reuptake by the SR

Studies of Somlyo et al. (35), Pape et al. (28), and Kamp et al. (14) postulated or showed a transport of H+ into the SR during Ca2+ release from the SR. The influx of H+ can occur via the extremely high H+ permeability of the SR membrane (9, 24, 26). An H+ ejection coupled to Ca2+ uptake by SR vesicles has been demonstrated by several authors (4, 15, 21, 23, 38, 39). In line with this, it has been reported that the Ca2+-ATPase directly effects an exchange of H+ for Ca2+ (17, 39, 46-48). All these studies have provided unambiguous evidence for a vectorial transport of H+ associated with the Ca2+ transport across the SR membrane. In addition, fluxes of K+, Mg2+, and Cl- contribute to counterbalance the charge transfer by the Ca2+ (23, 35). The protons required for these proton fluxes may be generated by the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> buffer system. This system can serve as an H+ source and sink that produces the required H+ on one side of the SR membrane and buffers the transported H+ on the other side of the SR membrane (Fig. 6). However, the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> system can fulfill this task only when it is catalyzed by CA, because the half-time of the uncatalyzed reaction of ~7 s is far too slow in view of the fast kinetics of Ca2+ fluxes. Ca2+ release lasts ~20-50 ms, and Ca2+ reuptake lasts ~200-300 ms (Fig. 2, Tables 2 and 3) (44). The need for catalysis may be illustrated by the following rough calculation. Let us consider a Ca2+ release lasting for 30 ms and causing a depletion of intra-SR Ca2+ concentration by 2 mM (12). Ca2+ then moves across the SR membrane at a rate of
<FR><NU>d[Ca<SUP>2+</SUP>]</NU><DE>d<IT>t</IT></DE></FR> = <FR><NU>0.002 mol</NU><DE>l × 0.03 s</DE></FR> = 0.07 mol · l<SUP>−1</SUP> · s<SUP>−1</SUP> (1)
where [Ca2+] is Ca2+ concentration and l represents 1 liter of intra-SR volume. If protons move into the SR during the Ca2+ release, they will have to be rapidly buffered inside the SR. We estimate the rate of H+ buffering by the uncatalyzed dehydration reaction of carbonic acid by assuming an intra-SR pH of 7, an HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration of 10 mM, and a reaction velocity constant (kd) of 130,000 l · mol-1 · s-1 as derived from a CO2 hydration velocity constant of 0.1 s-1 and pK'1 of 6.1. For further simplification, we neglect the backreaction, which yields an overestimate of the possible rate of H+ buffering, (d[H+]/dt)
<FR><NU>d[H<SUP>+</SUP>]</NU><DE>d<IT>t</IT></DE></FR><IT>=k</IT><SUB>d</SUB> × [H<SUP>+</SUP>] × [HCO<SUP>−</SUP><SUB>3</SUB>] (2)

= 1.3 × 10<SUP>5</SUP> <FR><NU>l</NU><DE>mol × s</DE></FR> × 10<SUP>−7</SUP> <FR><NU>mol</NU><DE>l</DE></FR> × 10<SUP>−2</SUP> <FR><NU>mol</NU><DE>l</DE></FR>

= 0.00013 mol · l<SUP>−1</SUP> · s<SUP>−1</SUP>
where [H+] is H+ concentration.


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Fig. 6.   A possible role of the sarcoplasmic reticulum (SR) carbonic anhydrase (CA) for H+ countertransport coupled to Ca2+ release and Ca2+ reuptake by SR. During release of Ca2+ from the SR, H+ will be transported into the SR, probably via the high proton permeability of the SR membrane. Inside the SR, H+ are rapidly buffered by the dehydration reaction catalyzed by SR-CA. On the cytoplasmic surface of the SR, SR-CA may support the fast delivery of H+ by catalyzing the CO2 hydration reaction. During the reuptake of Ca2+, H+ are transported out of the SR by the Ca2+-ATPase. Inside the SR, SR-CA supports fast generation of H+; on the cytoplasmic side, it supports the fast buffering of the transported H+. Inhibition of this CA is expected to cause a deceleration of H+ fluxes, which is expected to impair the Ca2+ transport rates.

We conclude from this consideration that the uncatalyzed dehydration reaction can buffer protons at <= 1/500th of the rate of Ca2+ efflux. Thus, even if equal fluxes of H+ and Ca2+ were required, which would balance 50% of the charges transferred by Ca2+, the carbonic acid dehydration reaction will have to be accelerated by a factor of 500. Indeed, Bruns et al. (3) showed that the SR-CA accelerates the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reaction ~1,000-fold, which reduces the half time from 7 s to ~7 ms.

From this hypothesis, it is predicted that inhibition of the SR-CA will impair the fast production of H+ on the cytoplasmic side of the SR membrane and impair buffering inside the SR of H+ that have moved into the SR during Ca2+ release. This will slow the H+ fluxes into the SR during Ca2+ release, slow the kinetics of Ca2+ release, and prolong the rise time of twitches. On the other hand, during Ca2+ uptake, inhibition of SR CA will impair the fast production of H+ on the intraluminal side of the SR membrane. This will reduce the rate of H+ fluxes that move out of the SR during Ca2+ reuptake, reduce the rate of Ca2+ reuptake, and prolong relaxation of twitches.

The presence of the CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> system may be especially important inside the SR because of the small SR volume compared with the volume of the cytoplasm (5) and because of the absence of major nonbicarbonate buffer systems inside the SR. This is in accordance with the observed distribution of CA activity across the SR membrane. By mass spectrometric measurements (10, 43), about two-thirds of the total CA activity of SR vesicles was found to be intravesicular, and only one-third of the CA activity was located on the outside, i.e., the cytoplasmic side, of the SR vesicles.

Fura 2 and Force Signals are Not Influenced by Possible Effects of CA Inhibitors on Myosin-, Ca2+-, and Na+-K+-ATPases and on AP

To test whether the changes in TTP, t50, and peak values of fura 2 and force signals were caused by the inhibition of SR-CA as postulated by our hypothesis or whether they were influenced by side effects of the CA inhibitors, the effects of CA inhibitors on the activities of different ATPases and on the AP were investigated; e.g., an inhibition of the myosin-ATPase by the CA inhibitors could have led to a prolongation of TTP of twitches or a possible stimulation to an increase in force production. However, neither L-645151 nor CLZ or ETZ affected the activities of the myosin-ATPase (Table 4). An inhibition of the Ca2+-ATPase would cause a prolongation of t50 of fura 2 signals and, consequently, a prolongation of t50 of force. However, none of the CA inhibitors inhibited the Ca2+-ATPase (Table 4). If the CA inhibitors affected the resting potential via changes in Na+-K+-ATPase activity and the AP, the activation of ryanodine receptors (RyR) might be altered. An increased AP might lead to a longer-lasting release of Ca2+ and a greater amount of released Ca2+. This could result in an increase in TTP of fura 2 signals and greater amplitudes of R340/380 values. L-645151, CLZ, and ETZ did not affect the Na+-K+-ATPase and did not significantly alter APs (Table 4, Fig. 5). We cannot rule out a direct effect of sulfonamides on the RyR, but they exhibit an effect on t50 that is qualitatively similar to the effect on TTP, although Ca2+-ATPase is not affected. Therefore, we conclude that the changes in TTP, t50, and peak values of fura 2 and force signals are very likely caused by the inhibition of the SR-CA, rather than by any side effect of the CA inhibitors. This conclusion is strengthened by the dose-dependent effect of CLZ on TTP and t50.

Inhibition of SL CA by ACTZ Does Not Affect Fura 2 and Force Signals

ACTZ is a hydrophilic and, therefore, poorly membrane-permeable CA inhibitor (22) that predominantly inhibits the extracellular SL-CA but not the SR-CA and the CAIII in SOL. ACTZ did not prolong TTP of fura 2 and force signals or t50 of fura 2 signals and prolonged only to a small degree t50 of force (Fig. 4, Table 3). ACTZ exerted no effects on the amplitudes of R340/380 and had no effect on peak force. From these results, we conclude that inhibition of the SL-CA cannot be responsible for the effects on TTP, t50, and amplitudes of fluorescence ratios and force signals caused by L-645151, CLZ, and ETZ. These three CA inhibitors are rather lipophilic and, therefore, highly membrane permeable (2, 22) and inhibit the extracellular SL-CA as well as the SR-CA and the CAIII of SOL. Because they exerted their effects in SOL as well as in EDL, which has no CAIII but the same two membrane-bound CA forms as SOL, CAIII can be excluded as the enzyme responsible for these changes. Therefore, we conclude that the inhibitory effects of L-645151, CLZ, and ETZ are very likely caused by inhibition of the SR-CA in SOL as well as in EDL.

Effects of Membrane-Permeable CA Inhibitors on TTP are Consistent With the Proposed Function of SR-CA

The prolongations of TTP of fura 2 as well as of force signals by L-645151, CLZ, and ETZ are in full agreement with the model of Fig. 6: CA inhibition reduces the rates of H+ influx, Ca2+ release, and force development. Dettbarn and Palade (8) observed that counterions of Ca2+ can markedly affect the rate of Ca2+ release. They reported that the rate of Ca2+ release from SR vesicles was reduced by 75-90% when the countertransport of K+ and Cl- was prevented by the replacement of K+ by choline and the replacement of Cl- by gluconate, respectively. However, their experiments were conducted in the absence of CO2. Conversely, when CA is inhibited in the present experiments, the impairment of H+ influx may partly be compensated by increased fluxes of the other balancing ions K+, Mg2+, and Cl-. This may be the reason for the moderate effect of SR-CA inhibition on TTP.

Effects of Membrane-Permeable CA Inhibitors on t50 are Consistent With the Proposed Function of SR-CA

The t50 values of fura 2 and force signals were significantly prolonged by the highly membrane-permeable CA inhibitors L-645151, CLZ, and ETZ. Again, this is consistent with the prediction from the hypothesis of Fig. 6. Analogously, a stimulating effect of countermovements of K+ on the rates of Ca2+ uptake has been reported by several authors (17, 39, 48, 49). Another result, which is consistent with the proposed function of the SR-CA, has been reported by Levy et al. (17), who found that increasing intravesicular buffer capacities by increasing intravesicular concentrations of PIPES increased the rates of Ca2+ uptake. In conclusion, reducing the rates of H+ fluxes coupled to Ca2+ uptake by inhibition of SR CA may be responsible for the slow kinetics of Ca2+ reuptake. The latter probably causes the slowdown in muscle relaxation.

Effects of Inhibition of SR CA on Peak Force and Amplitude of R340/380

The CA inhibitors caused significant increases in peak force of single twitches (Tables 2 and 3). The decrease in peak force reported for CLZ in a previous study (44) is only seen with >= 1 mM CLZ and is not seen with the other lipophilic inhibitors ETZ and L-645151. In the present study, we used CLZ at <1 mM only.

It is possible, as indicated by the increased amplitudes of R340/380 seen in some cases (Tables 2 and 3), that the increase in force was induced by a somewhat greater amount of Ca2+ released from the SR in the presence of inhibitor. It may be speculated that, in the presence of CA inhibitors, local pH disequilibria cause an increased open probability of the RyR (16, 19, 20, 30, 34) and, thus, an enhanced release of Ca2+. There is an alternative explanation for an increase in peak force under CA inhibition that does not involve an increase in the amount of Ca2+ released from the SR. Because of the prolonged Ca2+ transient (Fig. 1), the contractile apparatus is exposed to elevated intracellular free Ca2+ concentrations for a longer period of time than under control conditions (Fig. 1, B and C). This should lead to an increase in force generation (31, 36). Thus, by mechanisms different from those effecting the slowdown of Ca2+ transients, inhibition of the SR-CA appears to lead to an increase in force generation by exposing the contractile apparatus to intracellular Ca2+ concentrations that are either higher or last longer than under control conditions. In conclusion, although the observed increases in peak force are not predicted by our hypothesis and may be secondary effects, the described prolongations of TTP and t50 are in excellent agreement with the proposed function of SR-CA.


    ACKNOWLEDGEMENTS

This research was supported by Deutsche Forschungsgemeinschaft Grant 489/4.


    FOOTNOTES

Address for reprint requests and other correspondence: P. Wetzel, Zentrum Physiologie, Medizinische Hochschule Hannover, Carl-Neuberg Str. 1, 30625 Hannover, Germany (E-mail: Wetzel.Petra{at}MH-Hannover.de).

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.

10.1152/ajpcell.00106.2002

Received 7 March 2002; accepted in final form 20 May 2002.


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METHODS
RESULTS
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Am J Physiol Cell Physiol 283(4):C1242-C1253
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