ARTÍCULOS ORIGINALES

Inhibitory effect of lidocaine on the sarcoplasmic reticulum Ca²⁺- dependent ATPase from temporalis muscle

 

Gabriel A. Sánchez, Ana C. Casadoumecq, Guillermo L. Alonso, Delia Takara

Biophysics Department, School of Dentistry, University of Buenos Aires, Argentina.

CORRESPONDENCE Dra. Delia Takara Catedra de Biofisica - Facultad de Odontologia M.T. de Alvear 2142 C1122AAH, Buenos Aires, Argentina delia@biofis.odon.uba.ar

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ABSTRACT

Myotoxic effects of local anesthetics on skeletal muscle fibers involve the inhibition of sarcoplasmic reticulum Ca2+-dependent ATPase activity and Ca2+ transport. Lidocaine is a local anesthetic frequently used to relieve the symptoms of trigeminal neuralgia. The aim of this work was to test the inhibitory and/or stimulatory effect of lidocaine on sarcoplasmic reticulum Ca2+-dependent ATPase isolated from rabbit temporalis muscle. Ca2+-dependent ATPase activity was determined by a colorimetric method. Calcium-binding to the Ca2+- dependent ATPase, Ca2+ transport, and phosphorylation of the enzyme by ATP were determined with radioisotopic techniques. Lidocaine inhibited the Ca2+-dependent ATPase activity in a concentration- dependent manner. The preincubation of the sarcoplasmic reticulum membranes with lidocaine enhanced the Ca2+- dependent ATPase activity in the absence of calcium ionophore. Lidocaine also inhibited both Ca2+ uptake and enzyme phosphorylation by ATP but had no effect on Ca2+-binding to the enzyme. We conclude that the effect of lidocaine on the sarcoplasmic reticulum Ca2+-dependent ATPase from temporalis muscle is due to the drug’s direct interaction with the enzyme and the increased permeability of the sarcoplasmic reticulum membrane to Ca.

Key words: Sarcoplasmic reticulum; Ca²⁺-dependent ATPase; Temporal muscle; Local anesthetics; Lidocaine; Calcium transport.

RESUMEN

Efecto inhibitorio de la lidocaína sobre la calcio ATPasa del retículo sarcoplásmico del músculo temporal

La toxicidad de los anestesicos locales sobre las fibras musculares esqueleticas involucra a la inhibicion de la actividad de la calcio ATPasa del reticulo sarcoplasmico y a la inhibicion del transporte del calcio. Tales efectos inhibitorios no han sido aun descriptos en el musculo temporal. La lidocaina es un anestesico local habitualmente usado para aliviar los sintomas de la neuralgia del trigemino por medio de la anestesia infiltrativa de la region temporal. El objetivo del trabajo fue demostrar el efecto inhibitorio y/o activador de la lidocaina sobre la calcio ATPasa del reticulo sarcoplasmico del musculo temporal del conejo. La actividad de la calcio ATPasa se determino empleando un metodo colorimetrico. La union del calcio a la enzima, el transporte del calcio y la fosforilacion de la ATPasa por ATP se determinaron mediante el empleo de tecnicas radioisotopicas. La lidocaina inhibio a la actividad de la calcio ATPasa. El efecto inhibitorio incremento en funcion de la concentracion del anestesico. La preincubacion de las membranas del reticulo sarcoplasmico en lidocaina incremento la actividad de la calcio ATPasa en ausencia de un ionoforo de calcio. Tal resultado avala el efecto permeabilizante del anestesico local sobre las membranas del reticulo sarcoplasmico del musculo temporal. La lidocaina inhibio la captacion del calcio y la fosforilacion de la calcio ATPasa por ATP, pero no evidencio efecto sobre la union del calcio a la enzima. Concluimos que el efecto de la lidocaina sobre la calcio ATPasa del reticulo sarcoplasmico del musculo temporal se debe a la accion directa de la droga sobre la enzima y al incremento inducido de la permeabilidad de la membrana del reticulo sarcoplasmico al Ca.

Palabras clave: Reticulo sarcoplasmico; Ca²⁺-ATPasa; Musculo temporal; Anestesico local; Lidocaina; Transporte de calcio.

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INTRODUCTION

The sarcoplasmic reticulum (SR) Ca2+-dependent ATPase is a membrane-bound protein responsible for active Ca2+ accumulation during muscle relaxation. The essential role of this enzyme in skeletal muscles is to keep myoplasmic Ca2+ concentration low^1,2. The ATPase has one high affinity ATP binding site (catalytic site) and two high affinity Ca2+ binding sites (transport sites)^3,4. Lacapere and Guillain⁵ proposed an enzymatic cycle of the Ca2+- dependent ATPase in which the two main enzymatic conformations of the cycle are known as E1 and E2. In the forward direction of the reaction cycle, E1 binds two Ca2+ (Step 1) and it is subsequently phosphorylated by ATP (Step 2). Thus, it drives the movement of Ca2+ from the myoplasm to the SR lumen, allowing the translocation of the cation across the SR membrane (Step 3). In the backward  direction, the ATPase (E2) is phosphorylated by inorganic phosphate (Pi) (Step 4) and the energy derived from the calcium gradient is used by the enzyme to synthesize ATP from ADP and Pi ⁶.
Ca2+-dependent ATPase activity and Ca2+ transport has been previously reported in some masticatory muscles^{7- 9}, but not in temporalis, a main jaw-closing muscle involved in a variety of oral functions. Lidocaine is an amide-type local anesthetic frequently used to relieve the acute symptoms of trigeminal neuralgia through infiltrative anesthesia of the temporal region¹⁰. Moreover, some local anesthetics decrease Ca2+ uptake^11-14 and increase Ca2+ efflux^14-17 through Ca2+-dependent ATPase in fast skeletal muscles, but studies on the Ca2+-dependent ATPase activity measured in the presence of Ca2+ ionophore are lacking¹⁴. The diffusion of local anesthetics into muscle fibers might trigger undesired effects such as the inhibition of the Ca2+ pump and the consequent increase in myoplasmic Ca2+ concentration. Since the sarcoplasmic reticulum Ca2+-dependent ATPase is one of the myoplasm Ca2+-removing systems involved in muscle relaxation, the alteration of the enzyme function by local anesthetics might be responsible for the side effect of these drugs^18,19. Some pathological conditions, such as the sustained muscle contraction could be associated with the action of local anesthetics.
The aim of this work was to determine the inhibitory and/ or stimulatory effect of lidocaine on the sarcoplasmic reticulum Ca2+-dependent ATPase from temporalis muscle. We tested the hypothesis that lidocaine inhibits or stimulates some steps of the sarcoplasmic reticulum Ca2+-dependent enzymatic cycle.

MATERIALS AND METHODS

Membrane Preparation. Temporalis muscles were sampled from adult New Zealand rabbits (6 months old, males, 2 kg) for the isolation of SR membranes as sealed vesicles by centrifugation²⁰. The protein concentration was measured by Lowry et al.²¹. 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, School of Dentistry, University of Buenos Aires.

Ca2+-dependent ATPase Activity. SR membranes (0.1 mg/ml) were incubated at 37oC for 2 min in 50 mM MOPS-Tris buffer (pH 7.2), 10 μM calcimycin (calcium ionophore A23187), 3 mM ATP, 100 mM KCl, 3 mM MgCl2, 0.1 mM CaCl2, 0.1 mM EGTA and lidocaine at various concentrations. Since Ca2+ accumulation inside the vesicles inhibits Ca2+- dependent ATPase activity, calcimycin was added to dissipate the Ca2+ gradient generated by the ATPase. Reactions were stopped with 5% trichloroacetic acid (final concentration). The denatured protein was precipitated by centrifugation and inorganic phosphate was measured in the supernatants²² and taken as an index of the ATPase activity. When indicated, prior to incubations, the membranes (0.5 mg protein/ml) were exposed to 50 mM MOPS-Tris buffer (pH 7.2) and 21 mM lidocaine (concentration for half-maximal inhibition (Ki) of Ca2+-dependent ATPase activity). Later, the media were diluted 1:5 in solutions without lidocaine. The other reagents reached final concentrations as above. Blanks without SR membranes were run in parallel and subtracted from the experimental values.

ATP-dependent Calcium Uptake. SR membranes (0.1 mg/ml) were incubated at 37oC for 30 sec in 3 mM ATP, 100 mM KCl, 3 mM MgCl2, 0.1 mM (45Ca)CaCl2 (450 cpm/nmol), 0.1 mM EGTA, 50 mM MOPS-Tris buffer (pH 7.2) and lidocaine at different concentrations. Reactions were stopped by filtration (Millipore filters, 0.45 μm pore size, Bedford, MA, USA). Filters were immediately washed with cold 3 mM LaCl3. The radioactivity retained in the filters was measured in a liquid scintillation counter. Blanks without ATP were run in parallel and subtracted from the experimental values. The effect of lidocaine on ATP-dependent Ca2+ uptake was also determined at different free Ca2+ and ATP concentrations. Lidocaine concentrations for half-maximal inhibition (Ki) of Ca2+-dependent ATPase activity (21 mM) and Ca2+ uptake (= ~ 30 mM) were used. Free Ca2+ concentrations were calculated by Fabiato & Fabiato²³. The Ca2+ transport in a single enzyme turnover at different lidocaine concentrations was measured as described by Davidson & Berman²⁴.

Passive Ca2+-binding to the Enzyme. SR vesicles (0.2 mg/ml) were incubated at room temperature for 30 sec in 50 mM MOPS-Tris buffer (pH 7.2), 0.1 mM EGTA, (45Ca)CaCl2 (450 cpm/nmol) at different concentrations and without or with 30 mM lidocaine. The media were filtered through Millipore filters and the radioactivity retained was measured in a liquid scintillation counter. Blanks without SR membranes were run in parallel and subtracted from the experimental values.

Chemicals and Radioisotopes. Disodium ATP (adenosine triphosphate), calcimycin, bovine seroalbumin, lidocaine, MOPS (3-[n-morpholino]propanesulfonic acid) and Tris (Tris[hydroximethyl]aminomethane) were purchased from Sigma Chemical Co. (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. Mean values of the results are given with the SD. The halfmaximal concentrations of lidocaine that inhibit the Ca2+-dependent ATPase activity or calcium uptake (Ki) are reported with the SEM. The difference in Ki values was tested for its significance by Student’s t test. The level of significance was p<0.05.

RESULTS

Lidocaine inhibited the Ca2+-dependent ATPase activity in a concentration-dependent manner (Fig. 1A, Table 1). Some additional information was obtained by repeating the experiments in the presence of different membrane protein concentrations. The inhibitory effect of lidocaine did not depend on this parameter (data not shown). The Ca2+-dependent ATPase activity varied with the pre-incubation time of the sarcoplasmic reticulum membranes from temporalis muscle with 21 mM lidocaine (Fig. 1B).

Fig. 1. Ca2+- dependent ATPase activity: (A) Effect of increasing lidocaine concentrations on Ca2+- dependent ATPase activity. Error bars indicate SD; n = 4 (independent experiments performed in duplicate). (B) Effect of lidocaine preincubation time on Ca2+- dependent ATPase activity. (○) without calcimycin. (●) with calcimycin. Error bars indicate SD; n = 4 (independent experiments performed in duplicate).

Table 1: Concentrations of lidocaine for half-maximal inhibition (Ki) of Ca2+-dependent ATPase activity and Ca2+ uptake in temporalis muscle.

The Ca2+-dependent ATPase activity appeared inhibited with increased pre-incubation time when measured in the presence of calcimycin, whereas it appeared enhanced when measured in the absence of the Ca2+ ionophore (Fig. 1B). Figure 2A shows that lidocaine inhibited the Ca2+ uptake in a concentration-dependent manner. The Ca2+ uptake decreased upon increasing the lidocaine concentration. The concentration of lidocaine for half-maximal inhibition of Ca2+ uptake (Ki) is shown in Table 1. Figure 2B plots Ca2+ uptake as a function of the extravesicular Ca2+ concentration. The curve in the absence of lidocaine uncovered a sigmoidal profile corresponding to the calcium activation phenomenon. The presence of lidocaine at 21 and 30 mM in the reaction medium decreased the maximal Ca2+ accumulation but did not affect Ca2+ affinity.

Fig. 2: Effect of lidocaine on the ATP-dependent calcium uptake. (A) Under optimal conditions. Error bars indicate SD; n = 4 (independent experiments performed in duplicate). (B) At different Ca2+ concentrations. (●) without lidocaine, (Δ) 21 mM lidoaine, (○) 30 mM lidocaine.

Figure 3A depicts a progressive and saturating increase of Ca2+ accumulation as a function of ATP concentration in the absence of lidocaine. Lidocaine 30 mM inhibited the intravesicular Ca2+ accumulation. It is also observed that increasing ATP concentrations did not relieve the inhibitory effect of lidocaine. Figure 3B shows that lidocaine inhibited the Ca2+ uptake during the first enzymatic cycle in a concentrationdependent manner. Figure 4 illustrates that Ca2+-binding to the enzyme was not modified by lidocaine.

Fig. 3: Effect of lidocaine on the ATP-dependent calcium uptake. (A) At different ATP concentrations. (●) without lidocaine. (○) 30 mM lidocaine. (B) On a single enzyme turnover.

Fig. 4: Effect of lidocaine on Ca2+-binding to the enzyme. (●) without lidocaine. (○) 30 mM lidocaine.

DISCUSSION

The results reported in this work demonstrate that lidocaine inhibits the Ca2+-dependent ATPase activity in SR membranes from temporalis muscle. In addition, the pre-incubation of the SR membranes with lidocaine affects the enzymatic activity. This result demonstrates a dual effect of lidocaine on the Ca2+-dependent ATPase. On the one hand, lidocaine inhibits the optimal Ca2+-dependent ATPase activity measured in the presence of calcimycin. On the other hand, lidocaine increases the SR membrane permeability to Ca2+ when the Ca2+-dependent ATPase activity is measured in the absence of the Ca2+ ionophore. Furthermore, the increased membrane permeability induced by lidocaine precludes the inhibitory effect of the transmembrane Ca2+ gradient increase and the Ca2+-dependent ATPase activity becomes enhanced. This finding points to the ionophoric-like effect of lidocaine. Whether the Ca2+-dependent ATPase is inhibited or activated depends on the experimental conditions. The results here reported are in line with previous studies in which local anesthetics were able to modify the rate of both Ca2+ influx and efflux through either inhibition or activation of the Ca2+-dependent ATPase^12-16. These effects of the local anesthetics on the SR vesicles suggest that these drugs have multiple sites of action. The inhibition of the Ca2+-dependent ATPase by lidocaine did not depend on the protein concentration and it was consistent with a moderate octanol/ water partition coefficient^{25, 26}. Conversely, for diethyl-estilbestrol and ritodrine, the inhibition of the Ca2+-dependent ATPase decreases upon increasing the protein concentration in the reaction medium^{27, 28}. This fact is attributed to drug partitioning into the lipid bilayer. Lidocaine inhibits Ca2+ uptake in SR membranes from temporalis muscle. Our result agrees with previous reports in which local anesthetics were found to decrease Ca2+ uptake and increase Ca2+ efflux in SR membranes from skeletal muscles^{11- 16}.
The inhibitory effect of lidocaine on Ca2+ uptake was reported in fast skeletal muscle¹¹ and recently in masseter muscle9. The results obtained in this work agree with previous reports and suggest that Ca2+ has a protective effect on the ATPase at the required lidocaine concentration, i.e. for a given intravesicular Ca2+ accumulation level, the extravesicular concentration of the cation becomes higher as lidocaine concentration increases. Lidocaine concentration that reduces Ca2+-dependent ATPase activity to one half (Ki) was lower than for Ca2+ uptake. Regarding this point, it must be considered that Ca2+-dependent ATPase activity was measured in the presence of calcimycin while Ca2+ uptake was measured in its absence. We have shown previously²⁹ that the relative distribution of the intermediate species of the enzymatic cycle depends on the presence or absence of calcimycin. Therefore, the apparent affinity of an inhibitory drug will appear increased or decreased depending on the concentration of the target intermediate species. We are reporting that Ki value for lidocaine in temporalis muscle is lower than in other masticatory muscles³⁰. This result indicates a higher affinity of lidocaine for the Ca2+-dependent ATPase in temporalis muscle compared to fast skeletal muscle. Previous assumptions on the presence of a different type of Ca2+-dependent ATPase isoform in masticatory muscles⁸ could account for this. The dependence relation between Ca2+ uptake and ATP concentration here reported shows that Ca2+ uptake increases upon increasing ATP concentration in the presence or absence of lidocaine. However, the maximal Ca2+ uptake is lower in the presence of lidocaine. A similar result was observed for Ca2+ uptake as a function of Ca2+ concentration. Our results demonstrate that the inhibition of lidocaine does not appear to be competitive with respect to Ca2+ and ATP.
The study of partial reactions of the Ca2+-dependent ATPase enzymatic cycle allows the action mechanism of different drugs on this enzyme to be elucidated. The experiments where the enzyme was pre-incubated with 45Ca and later ATP, EGTA and lidocaine were added reflect only the transport of calcium bound to the enzyme, and permitted the exploration of steps 1, 2 and 3 of the enzymatic cycle²⁴. It could be assumed that step 3 would not be involved in the action of lidocaine, since Ca2+ bound to the enzyme is transported even in the presence of lidocaine. We found that Ca2+ transport depends on ATP concentration. However, increasing ATP concentrations does not relieve the inhibitory effect of lidocaine. We cannot discard that interferences in the phosphorylation of the enzyme by ATP would influence Ca2+ transport. It is important to remember that Ca2+ transport and ATP hydrolysis are regulated by limiting steps of the cycle that could be modified depending on the conditions of the incubation medium. Therefore, the inhibitory effect of lidocaine could affect steps 2 and 4. Several authors have reported that local anesthetics have higher affinity for E2 and markedly inhibit the phosphorylation of the enzyme by Pi, step 4 of the reverse cycle^14,15,29. In this work, lidocaine did not affect Ca2+-binding to the Ca2+-dependent ATPase. Since local anesthetics have been reported to be more to likely interact with E2, the inhibitory effect of lidocaine in step 1 was not expected.
We conclude that the effect of lidocaine on the sarcoplasmic reticulum Ca2+-dependent ATPase from temporalis muscle is due to the drug’s direct interaction with the enzyme and the increased sarcoplasmic reticulum membrane’s permeability to Ca. The inhibition of the Ca2+-dependent ATPase could not fully explain lidocaine myotoxicity, but might induce undesired effects such as sustained contraction of the temporalis and masticatory muscles during the treatment of trigeminal neuralgia through infiltrative anesthesia.

ACKNOWLEDGEMENT

This work was supported by grant UBACyT O 802 from the University of Buenos Aires, Argentina.

REFERENCES

1. Inesi G. Mechanism of calcium transport. Annu Rev Physiol 1985;47:573-601.         [ Links ]

2. Melzer W, Hermann-Frank A, Luttgau HC. The role of Ca2+ ions in excitation-contraction coupling of skeletal muscle fibres. Biochim Biophys Acta 1995;1241:59-116.         [ Links ]

3. Clarke DM, Loo TW, Inesi G, MacLennan DH. Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+- ATPase. Nature 1989;339:476-478.         [ Links ]

4. Toyoshima C. How Ca2+-ATPase pumps ions across the sarcoplasmic reticulum membranes. Biochim Biophys Acta 2009;1793:941-946.         [ Links ]

5. Lacapere J, Guillain F. Reaction mechanism of Ca2+-ATPase of sarcoplasmic reticulum. Equilibrium and transient study of phosphorylation with Ca.ATP as substrate. J Biol Chem 1990; 265:18762-18768.         [ Links ]

6. De Meis L. The sarcoplasmic reticulum. Transport and energy transduction. Transport in the life Sciences, Bittar E ed. (Vol 2) 1981, John Wiley & Sons, New York.         [ Links ]

7. Okabe E, Kohono H, Kato Y, Odajima C, Ito H. Characterization of the effect of pH on the excitation-contration coupling system of canine masseter muscle. Jpn. J Pharmacol 1985;37:277-283.         [ Links ]

8. Sanchez GA, Takara D, Toma AF, Alonso GL. Characteristics of the sarcoplasmic reticulum Ca2+-dependent ATPase from masticatory muscles. J. Dent. Res 2004;83:557-561.         [ Links ]

9. Sanchez GA, Takara D, Alonso GL. Local anesthetics inhibit Ca-ATPase in masticatory muscles. J. Dent. Res 2010; 89:372-377.         [ Links ]

10. Sindrup SH, Jensen TS. Pharmacotherapy of trigeminal neuralgia. Clin J Pain 2002;18:22-27        [ Links ]

11. Suko J, Winkler F, Scharinger B, Hellman G. Aspect of the mechanism of action of local anesthetics on the sarcoplasmic reticulum of skeletal muscle. Biochim Biophys Acta 1976;441:571-586.         [ Links ]

12. Nash-Adler P, Louis CF, Fudyma G, Katz A. The modification of unidirectional calcium fluxes by dibucaine in sarcoplasmic reticulum vesicles from rabbit fast skeletal muscle. Mol Pharmacol. 1980;17:61-65.         [ Links ]

13. Shoshan-Barmatz V. ATP-dependent interaction of propranolol and local anesthetic with sarcoplasmic reticulum. Stimulation of Ca2+ efflux. Biochem. J 1988; 256:733-739.         [ Links ]

14. Takara D, Sanchez GA, Alonso GL. Effect of carticaine on the sarcoplasmic reticulum Ca2+-dependent adenosine triphosphatase. Naunyn Schmiedebergs Arch Pharmacol 2000;362:497-503.         [ Links ]

15. Wolosker H, Pacheco AGF, De Meis L. Local anesthetics induce fast Ca2+ efflux through a nonenergized state of the sarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 1992; 267:5785-5789.         [ Links ]

16. Volpe P, Palade P, Costello B, Mitchell RD, Fleisches S. Spontaneous calcium release from sarcoplasmic reticulum. Effect of local anesthetics. J Biol Chem 1983;258:12434-12442.         [ Links ]

17. Escudero B, Gutierrez-Merino C. Effects of local anesthetics on the passive permeability of sarcoplasmic reticulum vesicles to Ca2+ and Mg2+. Biochim Biophys Acta 1987; 902:374-384.         [ Links ]

18. Zink W, Seif C, Bohl JR, Hacke N, Braun PM, Sinner B. The acute myotoxic effects of bupivacaine and ropivacaine after continuous peripheral nerve blockdes. Anesth Analg 2003;97:1173-1179.         [ Links ]

19. Zink W, Goetz Missler MS, Barbara S, Eike M, Rainer HA, Bernhard M. Differential effects of bupivacaine and ropivacaine enantiomers on intracellular Ca2+ regulation in murine skeletal muscle fibers. Anesthesiology 2005;102:793-798.         [ Links ]

20. Champeil P, Guillain F, Venien C, Gingold MP. Interaction of magnesium and inorganic phosphate with calciumdeprived sarcoplasmic reticulum adenosinetriphosphatase as reflected by organic solvent induced perturbation. Biochemistry 1985;24:69-81.         [ Links ]

21. Lowry OH, Rosembrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275.         [ Links ]

22. Baginski ES, Foa PP, Zak B. Microdetermination of inorganic phosphate, phospholipids, and total phosphate in biologic materials. Clin Chim Acta 1967;13:326-332.         [ Links ]

23. Fabiato A, Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75:463-505.         [ Links ]

24. Davidson GA, Berman MC. Calcium dependence during single-cycle catalysis of the sarcoplasmic reticulum ATPase. J Biol Chem 1988;263:11786-11791.         [ Links ]

25. Leo A, Hansch C, Elkins D. Hydrophobic parameter: measurement and calculation. Methods Enzymol 1991;202: 544-591.         [ Links ]

26. Courtney KR. Structure-activity relations for frequencydependent sodium channel block in nerve by local anesthetics. J Pharmacol Exp Ther 1980;213:114-119.         [ Links ]

27. Caravaca F, Soler F, Gomez-Fernandez JC, Fernandez-Belda F. Drug action of ritodrine on the sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle. Arch Biochem Biophys 1995;318:97-104.         [ Links ]

28. Martinez-Azorin F, Teruel JA, Fernandez-Belda F, Gomez-Fernandez J.C. Effect of diethyl-estilbestrol and related compounds on Ca2+-transporting of the sarcoplasmic reticulum. J Biol Chem. 1992;267:11923- 11929.         [ Links ]

29. Takara D, Sanchez GA, Toma AF, Bonazzola P, Alonso GL. Effect of carticaine on the sarcoplasmic reticulum Ca2+-adenosine triphosphatase. II. Cations dependence. Naunyn Schmiedebergs Arch Pharmacol 2005;371:375- 382.         [ Links ]

30. Sanchez G, Takara D, Toma A, Alonso GL. Inhibitory effect of local anesthetics on the sarcoplasmic reticulum Ca- ATPase from masseter and medial pterigoyd muscles. J Dent Res 2003;82:3.         [ Links ]