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.

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.

[Evaluation of a histochemical method specific for myosin ATPase activity in the masticatory muscles of the rat]. / Mise au point d'une méthode histochimique spécifique de l'activité myosine ATPase dans les muscles manducateurs du rat.

A biological study of masticatory muscle behaviour (Divry and Westphal, 1991) suggested the analysis of physiologic correlates (biologic parameters related to a behavioural event) such as histochemical reactions of muscular fibres studied by M-ATPase and SDH activities. For such investigations, routine methods are needed. In the present study, a modification of the original method of Tunell and Hart (1977) was used, in which three features of the original alkaline preincubation method (composition, incubation time and pH) were modified. These allowed a single step differentiation of the various fibre types found in rat masticatory muscles, for which the classical technics gave only a weak contrast, not suitable for image analysis. Acid preincubation was also tested but failed to give new information. By combining this modified technic with SDH staining (Nachlas, 1957) a classification of fibres into 12 theoretical types was proposed.

Postnatal development of the mouse temporal muscle and effects of a fine-grained diet on the muscle spindle.

The postnatal development of the mouse temporal muscle and effects of a fine-grained diet on muscle spindles were investigated. On d 25 after birth, 3 types of extrafusal muscle fibers could be distinguished on the basis of SDH activity, but not on the basis of their diameters. And in 50-d-old mice, 3 types of the muscle fibers had different diameters each other. In intrafusal muscle fibers, the postnatal changes of the SDH activity were similar to those of extrafusal muscle fibers, but the diameter distribution showed unimodal statistical distribution from 10th to 50th postnatal day. On the other hand, in control 180-d-old mice, 80 percent of muscle spindles in the temporal muscle had complete annulospiral endings, but the ratio of muscle spindles with complete annulospiral endings was decreased significantly in mice fed a fine-grained diet. The diet also decreased the diameter of the endings significantly. These results suggest that the main changes in the morphological and functional properties of the mouse temporal muscle occur in during and early after weaning period, and that normal mastication is needed to maintain the homeostatic condition of the mouse temporal muscle.

Adaptations in the temporalis muscles of rabbits after masseter muscle removal.

Masseter muscles were surgically removed in six young female rabbits so that we could study adaptations of the superficial temporalis muscles (ST) to increased functional requirements. Eight weeks following surgery, we used morphological measurements, histochemistry, contractile properties in situ, and occlusal force in vivo to compare the muscles in the experimental animals and six control rabbits. Analysis of the results demonstrated a decrease in fatigability of ST after masseter myectomy. Incisal occlusal force decreased by 65% during the first two weeks, and no recovery was observed during the following six weeks. At eight weeks post-surgery, the mass, twitch tensions, and tetanic tensions of ST were not significantly different from those of the controls. An increase in the percent of the cross-sectional area composed of fast fatigue-resistant fibers, a slower time-to-peak twitch tension, and a decrease in fatigability suggest an increase in oxidative metabolism. Analysis of these results suggests that muscles used for highly repetitious activities with submaximal loadings adapt to increased functional requirements by increasing fatigue-resistant properties.

Histochemical enzyme profile of the masseter, temporal and lateral pterygoid muscles of the European hedgehog (Erinaceus europeaus).

In man, there are large differences in histochemical fibre-type composition, distribution and size between jaw and trunk muscles, probably related to the special functions of the human stomatognathic system. In the hedgehog, the influence of alkaline and acid pre-incubations on the reaction for myofibrillar ATPase was different from that in man, suggesting a different myosin structure; the fibre composition was different also. The masseter, the superficial portion of the temporal and the lateral pterygoid muscles all showed a homogeneous fibre type profile with almost 100 per cent alkali-stable fibres. In two animals, the deep temporal muscles showed an apparent heterogeneous fibre pattern with 81 per cent alkali-stable fibres, 4 per cent alkali-labile fibres and 15 per cent ATPase-intermediate fibres; in one animal 87 per cent alkali-stable fibres and 13 per cent ATPase-intermediate fibres. There was no difference in cross-sectional area between the three fibre types within each muscle, but the fibres of the lateral pterygoid were smaller than the alkali-stable and the alkali-labile fibres of the masseter and temporal muscles. The limb and trunk muscles showed reactions for myofibrillar ATPase similar to the jaw muscles, but had a heterogeneous fibre-type profile. There was no significant difference in cross-sectional fibre area between the jaw and the limb muscles. Thus the jaw and limb muscles of the hedgehog have similar fibre types and about equal fibre size.

Further histochemical studies on masticatory muscles.

This paper describes a histochemical and histographic analysis of the masticatory muscles obtained from 78 early autopsy samples from subjects from 4 days to 87 years old. Five groups of muscles have been stuied: the temporalis, the medial and lateral pterygoid, the superficial bundle of the masseter and the mylohyoideus. All adult muscles have consistently shown a markedly increased number oftype II fibres and a disparity in the size of the two main fibre types, the average diameter of type II fibres being about half that of type I fibres. Fibres of intermediate size and stain were observed with myofibrillar ATPase at pH 9.40. A negative relation between the percentage of type II fibres and intermediate fibres was found, but not between the percentage of type I fibres and of intermediate fibres. Another negative relation was found between the number and the size of type II fibres, again not present in type I nor in intermediate fibres. In children, from 6 days old, an increased number of type II fibres and a definite disparity in the size of the two main fibre types were found. Intermediate fibres were present on the 17th day. Up to the age of 13 years, their diameter was greater than that of type I fibres. The analysis of the distribution and size modifications of the various fibre types seems to indicate a progressive adaptation of the masticatory muscles. This adaptation of the fibres to the successive reactions and to the various movements of the masticatory system is then discussed.