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Characteristics of the Sarcoplasmic Reticulum Ca²⁺-dependent ATPase from Masticatory Muscles

¹ Biophysics Department, Faculty of Dentistry, University of Buenos Aires, MT de Alvear 2142, 1122 Buenos Aires, Argentina; and
² Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina;

^* corresponding author, gabriel{at}biofis.odon.uba.ar

	ABSTRACT

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We compared the sarcoplasmic reticulum (SR) Ca-ATPase from masseter (M) and medial pterygoid (MP) muscles with that from fast muscles (FM) to examine whether its calcium transport capability and enzymatic activity are different. SR vesicles from FM, M, and MP muscles were obtained according to Champeil et al.(1985). Assays for characterization of the enzyme properties were performed. The results showed similar optimal conditions for the Ca-ATPase activity and calcium transport in M, MP, and FM. However, the maximal values of calcium transport, Ca-ATPase activity, and K_i for thapsigargin were significantly lower in the masticatory muscles. These findings are likely related to different Ca-ATPase isoforms. Since the local anesthetics used in dentistry inhibit Ca-ATPase and calcium transport in FM, it will be important for the effects of these drugs on the Ca-ATPase of masticatory muscles to be assessed.

KEY WORDS: SR Ca-ATPase • masticatory muscles • calcium transport • thapsigargin

	INTRODUCTION

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The sarcoplasmic reticulum (SR) Ca-ATPase is a membrane-bound enzyme responsible for active calcium transport from the myoplasm to the SR lumen at the expense of ATP hydrolysis, leading to muscle relaxation. SR membranes can be isolated as sealed vesicles from muscle homogenates, retaining the membrane-bound Ca-ATPase. The vesicles are right-side out—that is, their outer surface corresponds to the SR surface facing the cytoplasm. The vesicles can be isolated by differential centrifugation (Eletr and Inesi, 1972; Okabe et al., 1982; Champeil et al., 1985) and by zonal density gradient centrifugation (Meissner et al., 1973). These preparations have been largely used as an experimental model to study the cation transport and the linked ATPase activity. Most of the studies published until now were devoted to the clarification of several aspects of SR Ca-ATPase, such as structure, modulation of calcium transport, binding and efflux, ATPase activity, phosphorylation of the enzyme by ATP or inorganic phosphate (P_i), and the effects of different drugs (Caravaca et al., 1995; Takara and Alonso, 1996; Takara et al., 2000), toxins (Plenge-Tellechea et al., 1997), and cations (Bishop and Al-Shawi, 1988). In almost all cases, studies were performed on membranes isolated from fast muscles (De Meis, 1981). Few studies reported results on Ca-ATPase activity and calcium transport from SR membranes in masticatory muscles (Okabe et al., 1985).

The masseter and medial pterygoid muscles are jaw-closing muscles involved in a variety of oral functions, including mastication, deglutition, speech, and mandibular positioning. Most of the rabbit masseter consists of slow and fast fibers. Masticatory muscle functions are involved in both finely graded and less precise tasks, such as mandible position (Bredman et al., 1990). The latter tasks do not demand fast response to specific stimuli, and therefore do not require a high SR Ca²⁺ uptake or release capability. In this view, we thought that the Ca-ATPase isolated from masticatory muscles could be a different isoform than that from fast muscles; therefore, we undertook this study to clarify some aspects of the differences between masticatory and fast muscles. Local anesthetics inhibit the SR Ca-ATPase from fast muscles (Suko et al., 1976; Takara et al., 2000). Since these drugs used in dentistry could eventually diffuse into these masticatory muscle fibers, as a result of an incorrect intra-oral anesthetic technique, undesirable side-effects could affect oral functions. The aims of this work were to isolate the SR Ca-ATPase from two main masticatory muscles, masseter and medial pterygoid muscles, with the lowest degree of contaminants and to test the optimal conditions for calcium transport through these membranes. These goals were planned as the initial steps in our study of the effects of local anesthetics on the SR Ca-ATPase from masticatory muscle fibers.

	MATERIALS & METHODS

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Abbreviations
EGTA, ethyleneglycolbis (ß-aminoethyl ether)-N,N'-tetraacetic acid; MOPS, 3-[n-morpholino] propanesulfonic acid; MES, 2[N-morpholino]ethanesulfonic acid; SDS, sodium dodecyl sulfate; SERCA, sarcoplasmic-endoplasmic reticulum Ca-ATPase; Tris, Tris[hydroxymethyl]aminomethane.

Membrane Preparation
From adult New Zealand rabbits (6 mos old, males, 2 kg), several fast-type back (serratus, major and minor dorsal muscles) and leg (anterior and posterior tibialis, extensor and flexor digitorium longus) muscles and masseter and medial pterygoid muscles were sampled for the isolation of SR membranes by various centrifugation techniques. All leg and back muscles were mixed and homogenized for comparison with the two separate masticatory muscles as to Ca-ATPase activity and calcium transport. Masticatory muscles were dissected according to the directions reported by Widmer et al.(1997) with regard to insertion of the muscles and anatomical partitions. The National Institute of Health guidelines for the care and use of laboratory animals were observed. The animal use protocol was reviewed and approved by the Ethics Commission, Faculty of Dentistry, University of Buenos Aires. SR membranes from masticatory muscles were isolated as reported by Champeil et al.(1985). After several centrifugation steps, the membranes were isolated as sealed vesicles and stored (–28°C) in frozen aliquots until use. The protein concentration was measured by the colorimetric procedure of Lowry et al.(1951), with bovine serum albumin as the standard.

Ca-dependent ATPase Activity
The enzymatic activity was assayed in media containing MOPS-Tris or MES-Tris buffer (with variable pH depending on the experiment), calcimycin (calcium ionophore A 23187), ATP, KCl, MgCl₂, CaCl₂, and EGTA at various concentrations as indicated in the Fig. captions. The ionized Ca²⁺ and Mg²⁺ concentrations were calculated as described by Fabiato and Fabiato (1979). The incubations were at 37°C, and the reaction times were adjusted to yield an ATP hydrolysis ranging between 10 and 20%. We began the reactions by adding SR membrane suspensions to the media and stopped them with cold 5% trichloroacetic acid (final concentration). The denatured membranes were precipitated by centrifugation, and P_i was measured in the supernatants according to the method of Baginski et al.(1967). [P_i] was taken as an index of the ATPase activity. Blanks without SR vesicles were run in parallel and subtracted from the experimental values. The Ca²⁺-dependent activity was calculated from the difference between the total and the Ca²⁺-independent activities. The Ca²⁺-independent activity was evaluated in the presence of 1 mmol/L EGTA and no added Ca²⁺, or in the presence of thapsigargin, a specific inhibitor of Ca-ATPase (Sagara and Inesi, 1991).

ATP-dependent Calcium Transport
ATP-dependent calcium transport was determined with a radioisotopic technique. SR vesicles were incubated at 37°C for 30 sec in media containing 3 mmol/L ATP, 100 mmol/L KCl, 3 mmol/L MgCl₂, 0.1 mmol/L (⁴⁵Ca)CaCl₂ (450 cpm/nmol), 0.1 mmol/L EGTA, and 50 mmol/L MOPS-Tris buffer (pH 7.2). Reactions were started by the addition of 0.1 mL of SR vesicles (0.1 mg/mL, final concentration) and stopped by filtration (Millipore filters, 0.45 µm pore size, Bedford, MA, USA). Filters were immediately washed with cold and freshly prepared 3 mmol/L LaCl₃. Calcium uptake by sealed vesicles was taken as an index of the calcium transport capability. Radioactivity retained in the filters was measured in a liquid scintillation counter (Beckman LS 6500). Blanks without ATP were run in parallel and subtracted from the experimental values.

Chemicals and Radioisotopes
Disodium ATP, bovine serum albumin, calcimycin, thapsigargin, MOPS, MES, and Tris were purchased from Sigma (St. Louis, MO, USA). All other reagents were of analytical grade. (⁴⁵Ca)CaCl₂ was from New England Nuclear (E.I. Dupont de Nemours, Boston, MA, USA). The radioisotope use protocol was reviewed by the National Commission of Atomic Energy (Argentina).

Data Presentation and Statistical Analysis
The experimental values represent the average of at least four independent experiments performed in duplicate. Mean values of the results are given with the standard deviation (SD). Half-maximal thapsigargin and EGTA concentrations that inhibit the Ca-ATPase activity or calcium transport (K_i) were reported with the standard error of the mean (SEM). Mean values of maximal Ca-dependent ATPase activity and ATP-dependent calcium uptake, and K_i values for thapsigargin were tested for significance by one-way analysis of variance and the Scheffé test. The level of significance used was p < 0.05. Equations were fitted to experimental data as reported by Fraser and Suzuki (1973).

	RESULTS

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Preliminary assays for the characterization of Ca-ATPase were performed, namely, identification on SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) and evaluation of the Ca²⁺-independent activity (basal activity) in the presence of 1 mmol/L EGTA and no added calcium (data not shown). Other tests used to identify the Ca-ATPase, such as the inhibition of Ca-dependent ATPase activity and calcium transport by thapsigargin, were also performed. In preliminary experiments, SR vesicles from masticatory and fast muscles were also obtained according to Eletr and Inesi (1972) and Okabe et al.(1982). Electrophoretic analysis of the preparation obtained by the method of Champeil et al.(1985) revealed the major presence of a protein of 100–110 KDa. This protein, assumed to be Ca-ATPase (MacLennan et al., 1985), was identified on the gel along with the lowest degree of contaminant, such as different glycoproteins, and plasma membranes and T-tubule ATPases (Campbell and MacLennan, 1980). Preliminary assays of the Ca²⁺-independent ATPase activity showed that the procedure described by Champeil et al.(1985) enabled us to obtain SR vesicles with the lowest basal activity for masticatory and fast muscles. Assays of Ca-ATPase activity showed that SR vesicles, from masticatory and fast muscles, with the highest specific activity were obtained through the procedure of Champeil et al.(1985).

The enzymatic activity measured in the absence of EGTA and no added Ca²⁺ (Fig. 1A), in membranes from masticatory muscles, was high (approximately 40% of the maximal activity measured under optimal conditions). Upon addition of EGTA, the activity was inhibited, with K_i = 0.092 ± 0.011 mmol/L (n = 4). The presence of 1 mM EGTA completely reduced the enzymatic activity attributed to contaminant calcium present in the membrane preparations.

View larger version (15K): [in this window] [in a new window]   Figure 1. ATPase activity of SR membranes from masticatory muscles. SR vesicles (0.1 mg/mL) were incubated at 37°C in media containing 50 mmol/L MOPS-Tris (pH 7.2), 3 mmol/L ATP, 3 mmol/L MgCl2, 100 mmol/L KCl, and 10 µmol/L calcimycin. No Ca2+ was added. The reactions and the ATPase activity determinations were performed as indicated in MATERIALS & METHODS. Error bars indicate SD; n = 4. (A) ATPase activity as a function of EGTA concentration. SR vesicles from masseter (•) and medial pterygoid () muscles were incubated in the above medium with EGTA added as indicated in the abscissa. (B) ATPase activity in the presence of thapsigargin for masticatory muscles. SR vesicles from masseter and medial pterygoid muscles were incubated as described above in the presence of 0.1 mmol/L EGTA and with (hatched bars) or without (blank bars) 0.4 µmol/L thapsigargin.

The Ca-ATPase activity of membranes from masticatory and fast muscles also depended on pH (Fig. 2). The enzymatic activity showed a maximum at 7.2, being inhibited at lower or higher pH values. This behavior was observed in masticatory and fast muscles. However, a higher maximal activity value (pH 7.2) was found in fast muscles.

View larger version (19K): [in this window] [in a new window]   Figure 2. Dependence of Ca-ATPase activity on pH. SR vesicles (0.1 mg/mL) from masseter (•), medial pterygoid (), and fast muscles () were incubated at 37°C in media containing 50 mmol/L MOPS-Tris (pH 6.5–8.5) or MES-Tris (pH 5.0–6.4), 3 mmol/L ATP, 0.1 mmol/L CaCl2, 0.1 mmol/L EGTA, 3 mmol/L MgCl2, 100 mmol/L KCl, and 10 µmol/L calcimycin. The reactions and the Ca-ATPase activity determinations were performed as indicated in MATERIALS & METHODS. Bars, SD; n = 5.

View larger version (28K): [in this window] [in a new window]   Figure 3. Ca-ATPase activity as a function of the substrate and cofactor concentrations. SR vesicles (0.1 mg/mL) from masseter (•), medial pterygoid (), and fast muscles () were incubated at 37°C in a basic medium containing MOPS-Tris (pH 7.2), ATP, MgCl2, KCl, CaCl2, EGTA, and calcimycin modified as follows. (A) Ca-ATPase activity as a function of [Ca2+]. SR vesicles were incubated in the above basic medium except for the addition of various free calcium and EGTA concentrations to render concentrations as indicated in the abscissa. (B) Ca-ATPase activity as a function of [Mg2+]. SR vesicles were incubated in the above basic medium except for the addition of various free magnesium concentrations as indicated in the abscissa. (C) Ca-ATPase activity as a function of [KCl]. SR vesicles were incubated in the above basic medium except for the addition of various KCl concentrations as indicated in the abscissa. (D) Ca-ATPase activity as a function of [ATP]. SR vesicles were incubated in the above basic medium except for the addition of various ATP concentrations as indicated in the abscissa. (A-D) Reactants at constant concentration within each Fig. were as in Fig. 2. The reactions and the Ca-ATPase activity determinations were performed as indicated in MATERIALS & METHODS. Error bars indicate SD; n = 4.

View larger version (15K): [in this window] [in a new window]   Figure 4. Inhibition of Ca-ATPase by thapsigargin. SR vesicles (0.1 mg/mL) from masseter (•), medial pterygoid (), and fast muscles () were incubated at 37°C in media containing 50 mmol/L MOPS-Tris (pH 7.2), 3 mmol/L ATP, 3 mmol/L MgCl2, 100 mmol/L KCl, 0.1 mmol/L CaCl2, 0.1 mmol/L EGTA, and thapsigargin as indicated in the abscissa. (A) Inhibition of Ca-ATPase activity. SR vesicles were incubated in the above medium with 10 µmol/L calcimycin added. The reactions and the Ca-ATPase activity determinations were performed as indicated in MATERIALS & METHODS. Bars, SD; n = 5. (B) Inhibition of ATP-dependent calcium uptake. SR vesicles were incubated in the above medium with the addition of tracer amounts of (45Ca)CaCl2. The reactions were performed as indicated in MATERIALS & METHODS. Bars, SD; n = 4.

	DISCUSSION

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The masticatory muscle preparations contain sufficient contaminant calcium concentration to activate the enzyme, although not optimally (Fig. 1A). The addition of 1 mmol/L EGTA reduced the enzymatic activity due to contaminant calcium, as previously reported (Alonso et al., 1998), indicating the calcium-dependence of the ATPase. The ATPase activity measured in the presence of 0.1 mmol/L EGTA was inhibited by thapsigargin (Fig. 1B). This fact also indicates that ATP hydrolysis is catalyzed by a SR Ca-ATPase, since the basal Ca- or Mg-dependent ATPase from T-tubules (Malouf and Meissner, 1979; Hidalgo et al., 1983), usually present in various amounts in SR membrane fractions, was not inhibited by thapsigargin.

The optimal pH of the enzyme was 7.2 (Fig. 2). The enzymatic activity was inhibited when the proton concentration was increased, as previously described for canine masseter muscle preparations (Okabe et al., 1985), and also when the proton concentration was decreased. In this work, significantly different maximum activity values were found between fast muscles and masticatory muscles (F = 32.21, p < 0.01). The maximal activity values were not significantly different between masseter and medial pterygoid membranes [t₍₈₎ = 0.86, p = 0.56].

The maximal Ca-ATPase activity was reached at 10 µmol/L Ca²⁺ (Fig. 3A) and was inhibited at higher concentrations, as reported for the Ca-ATPase isolated from fast muscles (De Meis, 1981). This inhibition by Ca²⁺ is presumably due to the replacement of ATP.Mg by ATP.Ca as substrate. The phosphoenzyme formed has a very low turnover, and the steady-state ATPase activity decreases (Yamada and Ikemoto, 1980; Lacapere and Guillain, 1990). Ca-ATPase activity is a function of the ATP concentration (Fig. 3D), increasing to a saturation level of 3 mmol/L. The Ca-ATPase activity mean values obtained in the presence of 3 and 5 mmol/L were not significantly different, either in masticatory muscles [t₍₆₎ = 1.45, p = 0.20] or in fast muscle vesicles [t₍₆₎ = 1.21, p = 0.28]. Similar behavior was observed when the KCl concentration was increased (Fig. 3C). Ca-ATPase activity increased when [Mg²⁺] was increased until it reached a maximal value (Fig. 3B) and was then inhibited at higher concentrations, as described for the Ca-ATPase isolated from other muscle tissues (Takara and Alonso, 1996).

The maximal specific enzymatic activity value obtained under optimal conditions for masticatory muscles was lower than that obtained in fast muscles, suggesting that the Ca-ATPase isolated from masticatory muscles could be a different isoform from that from fast muscles, SERCA 1a (Burks et al., 1989), possibly SERCA 2a, or a mixture of SERCA 2 and SERCA 1. The SERCA 2a isoform is present in slow muscle fibers, whose functions do not involve precise tasks. Although more specific tests, such as antigen-antibody reactions, should be conducted to determine the isoform type, the previous suggestion could be supported as well by the fact that significantly lower K_i values were obtained for thapsigargin in masticatory muscles (F = 45.93, p < 0.001). In addition, the lower enzymatic activity observed in masticatory muscles when compared with fast muscles could be attributed to a lower ATP hydrolysis rate, supporting the preceding assumptions, since a higher ATPase activity has been described in SERCA 1a (Martonosi and Pikula, 2003).

The maximal ATP-dependent calcium uptake value recorded for masticatory muscle SR membranes was significantly lower than that observed in fast muscles (F = 40.09, p < 0.001). The ATP-dependent calcium uptake was also inhibited by thapsigargin, with significantly different K_i values also in masticatory and fast muscles. These facts reinforce our previous assumptions regarding the suggestion that SR Ca-ATPase from masticatory muscles is a different isoform from that present in fast muscles. In addition to this assumption, it could also be speculated that K_i values for other drugs of infiltrative dental use could also be lower in masticatory muscles. Local anesthetics used in dentistry have been demonstrated to inhibit the SR Ca-ATPase activity and calcium transport in fast muscles (Suko et al., 1976; Takara et al., 2000). Experiments with local anesthetics in SR vesicles from masticatory muscles are being performed in our laboratory.

	ACKNOWLEDGMENTS

 
This work was supported by a grant from the University of Buenos Aires, Argentina (#OD 011).

Received August 11, 2003; Last revision March 25, 2004; Accepted May 5, 2004

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