Zentrum Physiologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany
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ABSTRACT |
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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
sarcoplasmic reticulum; H+ countertransport; fura 2 transients; single twitches
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INTRODUCTION |
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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.
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METHODS |
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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 · mg1 · 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 HCOFura 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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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 MProtein
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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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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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
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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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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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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 × 10Effects 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 ofLack 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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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 · mg1 · 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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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 =
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DISCUSSION |
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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
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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
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
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 ClEffects 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 withIt 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.
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ACKNOWLEDGEMENTS |
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This research was supported by Deutsche Forschungsgemeinschaft Grant 489/4.
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FOOTNOTES |
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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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