Expression of sarcoplasmic-endoplasmic reticulum Ca-ATPase isoforms in masticatory muscles.

The aim of this study was to characterize the sarcoplasmic-endoplasmic reticulum Ca-ATPase (SERCA) isoforms in rabbit masticatory muscles compared with those in fast-twitch muscle. It was hypothesized that combined expression of the SERCA isoforms in fast- and slow-twitch muscles accounts for lower Ca-ATPase activity. SERCA was isolated by differential centrifugation, the isoforms were determined by ELISA, and the activity of each isoform was measured using a colorimetric method. Activity was tested for significance by anova, and the distribution of isoforms was assessed using the chi-square test (P < 0.05) and correlated to SERCA activity using Spearman's rank correlation. SERCA1 was predominant (90.5%) in fast-twitch muscle, whereas a mixture of SERCA isoforms was found in masticatory muscles: 62-78% was SERCA2, 20-37% was SERCA1, and the SERCA3 content was negligible. Depressor muscles showed a significantly higher content (77.8%) of SERCA2, and elevator muscles showed a higher content (35.4%) of SERCA1. Elevator muscles showed higher expression of SERCA2a (58%), and depressor muscles showed higher expression of SERCA2b (20%). The SERCA1 content was mainly SERCA1a and significantly higher for elevator muscles (33%), whereas depressor muscles showed a higher content of SERCA1b (4%). The SERCA1 content of fast-twitch muscle was mainly SERCA1a (88.5%). It is concluded that the mixture of different SERCA isoforms, along with a substantial content of SERCA2b, in masticatory muscles would support lower Ca-ATPase activity and calcium transport.

Characterization of the sarcoplasmic reticulum Ca-ATPase from rabbit temporalis muscle.

OBJECTIVE: The aim of this work was to isolate the sarcoplasmic reticulum (SR) Ca-ATPase from rabbit temporalis muscle and to determine the optimal conditions for calcium transport and enzymatic activity. DESIGN: SR vesicles were isolated from rabbit temporalis muscle by differential centrifugation, the protein composition analyzed by electrophoresis and compared to fast-twitch muscle membrane suspensions. ELISA was used to determine the sarcoendoplasmic reticulum Ca-ATPase (SERCA) isoform. Ca-ATPase activity was determined by a colorimetric method. Calcium-binding to the Ca-ATPase, calcium uptake, calcium efflux and phosphorylation by P(i) were determined with radioisotopic techniques. RESULTS: Sixty five percent of the total protein concentration of SR membranes suspensions from rabbit temporalis corresponded to SERCA. Of the total SERCA protein, 64% was SERCA 2, 35% was SERCA 1 and less than 1% was SERCA 3. The optimal conditions of the SERCA isolated from rabbit temporalis muscle were: pH 7.2, 5 µM Ca(2+), 100 µM EGTA, 90 µM Mg(2+), 3mM ATP and 100mM KCl and did not differ from fast-twitch skeletal muscle. The temporalis maximal calcium uptake and Ca-ATPase activity were lower but the sensitivity to the specific Ca-ATPase inhibitor thapsigargin was higher. Calcium-binding to the enzyme and calcium efflux were similar while the phosphorylation of the enzyme by P(i) was lower. CONCLUSION: The lower enzymatic activity and calcium transport capability of the Ca-ATPase isolated from rabbit temporalis, and the higher sensitivity to inhibitory drugs are consistent with the presence of a substantial proportion of SERCA 2, which can be expected in other rabbit masticatory muscles.

Effect of articaine on calcium transport in sarcoplasmic reticulum membranes isolated from medial pterygoid muscle.

Local anesthetics used in dentistry have myotoxic effects. Articaine, also known as carticaine, is one of the local anesthetics most widely used in clinical dentistry. The aim of this work was to describe its effect on the sarcoplasmic reticulum Ca-ATPase isolated from medial pterygoid muscle. Ca-ATPase enzymatic activity was determined by a colorimetric method and ATP-dependent calcium uptake with a radioisotopic technique. Articaine inhibited both Ca-ATPase activity and calcium uptake in a concentration-dependent manner. Both inhibitory effects became evident at articaine concentrations lower than those employed in clinical dentistry. Half-maximal inhibitory concentrations (K) were 15.1 +/- 1.8 mM (n = 6) and 25.2 +/- 1.6 mM (n = 6) for enzymatic activity and calcium uptake, respectively. Preincubation of sarcoplasmic reticulum membranes with articaine enhanced Ca-ATPase activity in the absence of calcium ionophore, suggesting an ionophoric-like effect of the local anesthetic. We conclude that the inhibitory effect of articaine on the sarcoplasmic reticulum Ca-ATPase isolated from medial pterygoid muscle is due to a direct interaction of the anesthetic with the enzyme and to the increased membrane permeability to calcium induced by this drug.

Effect of articaine on calcium transport in sarcoplasmic reticulum membranes isolated from medial pterygoid muscle

Local anesthetics used in dentistry have myotoxic effects. Articaine, also known as carticaine, is one of the local anesthetics most widely used in clinical dentistry. The aim of this work was to describe its effect on the sarcoplasmic reticulum Ca-ATPase isolated from medial pterygoid muscle. Ca-ATPase enzymatic activity was determined by a colorimetric method and ATP-dependent calcium uptake with a radioisotopic technique. Articaine inhibited both Ca-ATPase activity and calcium uptake in a concentrationdependent manner. Both inhibitory effects became evident at articaine concentrations lower than those employed in clinical dentistry. Half-maximal inhibitory concentrations (Ki) were 15.1± 1.8 mM (n = 6) and 25.2 ± 1.6 mM (n = 6) for enzymatic activity and calcium uptake, respectively. Preincubation of sarcoplasmic reticulum membranes with articaine enhanced Ca-ATPase activity in the absence of calcium ionophore, suggesting an ionophoriclike effect of the local anesthetic. We conclude that the inhibitory effect of articaine on the sarcoplasmic reticulum Ca-ATPase isolated from medial pterygoid muscle is due to a direct interaction of the anesthetic with the enzyme and to the increased membrane permeability to calcium induced by this drug.

Los anestésicos locales de uso odontológico tienen efectos miotóxicos. La carticaína, también conocida como articaína, es uno de los anestésicos locales más usados en la clínica odontológica actual. El objetivo del trabajo fue describir el efecto de la carticaína sobre la Ca-ATPasa del retículo sarcoplásmico aislada del músculo pterigoideo interno. La actividad enzimática de la bomba de calcio se determinó por un método colorimétrico y se utilizó un método radioisotópico a fin de determinar la captación de calcio dependiente de ATP. La carticaína inhibió la actividad enzimática y la captación de calcio en función de su concentración. Ambos efectos se observaron a concentraciones de carticaína menores a las utilizadas en la clínica. Las concentraciones de carticaína necesarias para inhibir la actividad Ca-ATPásica y la captación de calcio a la mitad de su valor máximo (Ki) fueron 15.1 ± 1.8 mM (n = 6) y 25.2 ± 1.6 mM (n = 6) respectivamente. La preincubación con carticaína de las membranas de retículo sarcoplásmico del músculo pterigoideo interno, en ausencia de ionóforo de calcio, incrementó la actividad de la enzima, evidenciando un efecto ionofórico del anestésico local. Concluimos que el efecto inhibitorio de la carticaína sobre la Ca-ATPasa de retículo sarcoplásmico del músculo pterigoideo interno se debe a la acción directa del anestésico local sobre la enzima y al incremento de la permeabilidad de la membrana del retículo sarcoplásmico al calcio inducido por esta droga.

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

Myotoxic effects of local anesthetics on skeletal musclefibers involve the inhibition ofsarcoplasmic 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 Ca 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.

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

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.

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.

Effect of carticaine on the sarcoplasmic reticulum Ca2+-adenosine triphosphatase. II. Cations dependence.

Ca2+-ATPase is a major intrinsic protein in the sarcoplasmic reticulum (SR) from skeletal muscles. It actively transports Ca2+ from the cytoplasm to the SR lumen, reducing cytoplasmic [Ca2+] to promote muscle relaxation. Carticaine is a local anesthetic widely used in operative dentistry. We previously showed that carticaine inhibits SR Ca2+-ATPase activity and the coupled Ca(2+) uptake by isolated SR vesicles, and increases the rate of Ca2+ efflux from preloaded vesicles. We also found that these effects were antagonized by divalent cations, and concluded that they were mainly due to the direct interaction of carticaine with the Ca2+-ATPase protein. Here we present additional results on the modulation of the above effects of carticaine by Ca2+ and Mg2+. The activating effect of Ca2+ on the ATPase activity is competitively inhibited by carticaine, indicating a decreased Ca2+ binding to the high affinity Ca2+ transport sites. The activating effect of Mg2+ on the phosphorylation of Ca2+-ATPase by orthophosphate is also inhibited by carticaine. The anesthetic does not affect the reaction mechanism of the cations acting as cofactors of ATP in the catalytic site. On the basis of the present and our previous results, we propose a model that describes the effect of carticaine on the Ca2+-ATPase cycle.