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.

Differential mechanism of the effects of ester-type local anesthetics on sarcoplasmic reticulum Ca-ATPase.

The effect of the local anesthetics procaine and tetracaine on sarcoplasmic reticulum membranes isolated from two masticatory muscles, masseter and medial pterygoid, was tested and compared to fast-twitch muscles. The effects of the anesthetics on Ca-ATPase activity, calcium binding, uptake, and phosphorylation of the enzyme by inorganic phosphate (Pi) were tested with radioisotopic methods. Calcium binding to the Ca-ATPase was non-competitively inhibited, and the enzymatic activity decreased in a concentration-dependent manner. The inhibition of the activity depended on pH, calcium concentration, the presence of the calcium ionophore calcimycin, and the membrane protein concentration. Unlike fast-twitch membranes, the pre-exposure of the masseter and medial pterygoid membranes to the anesthetics enhanced the enzymatic activity in the absence of calcimycin, supporting their permeabilizing effect. Procaine and tetracaine also interfered with the calcium transport capability, decreasing the maximal uptake without modification of the calcium affinity for the ATPase. Besides, the anesthetics inhibited the phosphorylation of the enzyme by Pi in a competitive manner. Tetracaine revealed a higher inhibitory potency on Ca-ATPase compared to procaine, and the inhibitory concentrations were lower than usual clinical doses. It is concluded that procaine and tetracaine not only affect key steps of the Ca-ATPase enzymatic cycle but also exert an indirect effect on membrane permeability to calcium and suggest that the consequent myoplasmic calcium increase induced by the anesthetics might account for myotoxic effects, such as sustained contraction and eventual rigidity of both fast-twitch and masticatory muscles.

The masticatory contractile load induced expression and activation of Akt1/PKBalpha in muscle fibers at the myotendinous junction within muscle-tendon-bone unit.

The cell specific detection of enzyme activation in response to the physiological contractile load within muscle-tendon-bone unit is essential for understanding of the mechanical forces transmission from muscle cells via tendon to the bone. The hypothesis that the physiological mechanical loading regulates activation of Akt1/PKBalpha at Thr308 and at Ser473 in muscle fibers within muscle-tendon-bone unit was tested using quantitative immunohistochemistry, confocal double fluorescence analysis, and immunoblot analysis. In comparison to the staining intensities in peripheral regions of the muscle fibers, Akt1/PKBalpha was detected with a higher staining intensity in muscle fibers at the myotendinous junction (MTJ) areas. In muscle fibers at the MTJ areas, Akt1/PKBalpha is dually phosphorylated at Thr308 and Ser473. The immunohistochemical results were confirmed by immunoblot analysis. We conclude that contractile load generated by masticatory muscles induces local domain-dependent expression of Akt1/PKBalpha as well as activation by dually phosphorylation at Thr308 and Ser473 in muscle fibers at the MTJ areas within muscle-tendon-bone unit.

Local anesthetics inhibit Ca-ATPase in masticatory muscles.

Local anesthetics have myotoxic effects and inhibit Ca-ATPase activity and Ca transport in skeletal muscles. Such effects have not been fully elucidated in masticatory muscles. We tested the hypothesis that local anesthetics increase myoplasmic calcium in masticatory muscles by inhibiting Ca-ATPase at a concentration similar to that of dental cartridges. The effects of lidocaine and bupivacaine on Ca-ATPase from rabbit masseter and medial pterygoid muscles were tested with radioisotopic and colorimetric methods. Bupivacaine had an action similar to that of lidocaine on Ca-ATPase activity, but less effect on calcium transport. The pre-exposure of the membranes to the anesthetics enhanced the Ca-ATPase activity in the absence of calcium ionophore, supporting their permeabilizing effect. The results demonstrate that amide-type anesthetics do not inhibit calcium binding, but do reduce calcium transport and enzyme phosphorylation by ATP, and suggest that the myoplasmic calcium increase induced by lidocaine and bupivacaine might promote masticatory muscle contraction and eventual rigidity.

Characteristics of the sarcoplasmic reticulum Ca2+-dependent ATPase from masticatory muscles.

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.

Histoenzymology and morphometry of the masticatory muscles of tufted capuchin monkey (Cebus apella Linnaeus, 1758).

Samples of the anterior and posterior regions of the masseter and temporal muscles and of the anterior belly of the digastric muscle of 4 adult male tufted capuchin monkeys (Cebus apella) were removed and stained with HE and submitted to the m-ATPase reaction (with alkaline and acid preincubation) and to the NADH-TR and SDH reactions. The results of the histoenzymologic reactions were similar, except for acid reversal which did not occur in fibers of the fast glycolytic (FG) type in the mandibular locomotor muscles. FG fibers had a larger area and were more frequent in all regions studied. No significant differences in frequency or area of each fiber type were detected, considering the anterior and posterior regions of the masseter and temporal muscles. The frequency of fibers of the fast oxidative glycolytic (FOG) and slow oxidative (SO) types and of FOG area differed significantly between the anterior belly of the digastric muscle and the mandibular locomotor muscle. The predominance of fast twitch (FG and FOG) fibers and the multipenniform and bipenniform internal architecture of the masseter and temporal muscles, respectively, are characteristics that permit the powerful bite typical of tufted capuchin monkeys.

[A histomorphologic and enzyme histochemical study of masticatory muscles affected by distraction osteogenesis of mandible].

OBJECTIVE: The purpose of this study was to study the masticatory muscles affected by distraction osteogenesis of the mandible. METHODS: The distraction osteogenesis (DO) was applied to distract the left mandible of 6 mongrel dogs that were divided into three experimental groups. After different distraction phase and consolidation phase, the masseter and the digastric muscle were taken out. The specimens were stained using hematoxylin/eosin and enzyme histochemistry. Afterwards, the specimens were observed with a light microscope to study the morphologic changes of the muscles. The contents of enzyme in the different groups were measured by VIDAS. RESULTS: The masseter showed consequently atrophy, but the digastric muscle showed a progress of histomorphologic reconstruction, including atrophy and hypertrophy. The changes of the contents of enzyme and histomorphology were identical in the masticatory muscles. CONCLUSION: The digastric muscle parallel to the vector of mandibular distraction adapts the distraction by the way of atrophy, regeneration and hypertrophy. And the contents of enzyme appear to decrease at the beginning, increase afterwards, and return to the normal level finally. But the masseter perpendicular to the vector of mandibular distraction shows consequent atrophy, and the contents of enzyme consequently decrease, which means the metabolism decrease.

Analysis of histochemically and morphometrically in the anterior belly digastric muscle of osteopetrotic (op/op) mice.

The fibers of the anterior belly digastric muscle of mice, fed a granulated diet for various periods, have been studied histochemically and morphometrically. The diameters of the anterior belly digastric fibers in normal mice fed only a granulated diet were smaller than those in mice fed a solid diet. Differences in the succinate dehydrogenase (SDH) activity of muscle fibers between op/op and normal mice gradually appeared in the anterior belly digastric muscle and, by the age of 90 days, under-development of muscle fibers was observed in the mild-belly region of the anterior belly digastric muscle of op/op mice fed a granulated diet. These results indicate mechanical stress in mastication plays an important role in the development of the anterior belly digastric muscle structures.

Histoenzimologia dos músculos da mastigaçäo de ratos (Rattus novergicus) alcoolizados / Histoenzymology of masticatory muscles in alcoholized rats (Rattus novergicus)

A literatura descreve a influência do álcool sobre o tamanho das fibras de contraçäo rápida em vários músculos estriados do corpo. É também de conhecimento geral, que os músculos da mastigaçäo, histoenzimológicamente, apresentam várias diferenças, em relaçäo aos músculos das outras partes do corpos. Näo se constatou na literatura consultada nenhum trabalho sobre a influência do álcool nos músculos da mastigaçäo. Portanto, esse trabalho objetiva evidenciar as características histológicas e histoenzimológicas, a freqüência e diâmetro menor das fibras dos músculo da mastigaçäo do rato, e verificar se o álcool excerce influência sobre as fibras musculares desses músculos. Foram retiradas amostras dos músculos digástrico (ventre anterior), masseter e temporal, de 5 ratos normais e 5 ratos alcoolizados, que foram submetidas às reaçöes de m-ATPase, SDH e coloraçäo de HE, para classificaçäo das fibras musculares. Baseado nos resultados obtidos pode-se concluir que a percentagem e o diâmetro menor dos tipos de fibras é diferente entre os músculos elevadores (masseter e temporal) e depressor (digástrico - ventre anterior) da mandíbula; que nos músculos elevadores da mandíbula as fibras do tipo FG são as predominantes; que o diâmetro menor das fibras do tipo FG é semelhante entre os músculos elevadores da mandíbula, mas maior do que no depressor; que o álcool teve influência sobre o diâmetro menor das fibras de contraçäo rápida (FG e FOG) nos músculos elevadores e depressor da mandíbula, o que näo ocorreu com as fibras de contraçäo lenta (SO)

Developmental changes in enzyme activities and in structural features of rat masticatory muscle mitochondria.

The functional ability of a muscle is closely related to the activities of the mitochondria, which are energy-producing organelles in muscle cells. The development of the mammalian masticatory muscle progresses dramatically when feeding behavior changes from suckling to mastication, but it is unclear how the energy-producing systems of the mitochondria change. In this paper, the development of rat masticatory muscle mitochondria was investigated in terms of enzyme activities of the mitochondrial respiratory chain and the structural and numerical development of mitochondria, especially regarding the change in feeding behavior from suckling to mastication. Using isolated mitochondria from the masticatory muscle, we measured succinate dehydrogenase, NADH dehydrogenase, succinate-O2 oxidoreductase, and NADH-O2 oxidoreductase. These were found to be increased in the 15-day postnatal rat compared with the 0- to 10-day postnatal rat. The structural development of mitochondria was gradual in the 0- to 15-day postnatal rat. However, a notable increase was found in the cross-sectional area of mitochondria between 10 and 15 days postnatally. The number of mitochondria per muscle fiber was apparently constant during the same period. We demonstrated that the change in feeding behavior was well-correlated with an increase in mitochondrial enzyme activity, also supported by the early structural development of mitochondria.

The histochemistry of reactive masticatory muscle hypertrophy.

There are two types of hypertrophy of the muscles of mastication in man: reactive hypertrophy, the more common form; and nonreactive enlargements-myositic, genetic, myopathic, and idiopathic. Reactive hypertrophy develops when the masticatory muscle workload is increased by local bone and dental disorders; such triggers are not powerful but act over long periods, thus demanding increased endurance. Exercise for endurance has a greater effect on the muscles of mastication than it has on the large muscles of the limbs; these react solely by stimulating the oxidative metabolism of type 1 fibers, whereas masticatory muscle reacts structurally by hypertrophy and progressive type 1 fiber predominance. Eventually enzyme instability of type 1 fibers and end stage atrophy of type 2 fibers may appear. Unexpectedly, lesions have also been found in control masticatory muscle, in particular type 2 fiber specific atrophy like that seen in long-standing acquired autoimmune myasthenia gravis. It is suggested that the adverse lesions in hypertrophied and control masticatory muscle are the consequence of post-activation fatigue.

Muscle-specific enzyme activity patterns of the capillary bed of human oro-facial, masticatory and limb muscles.

Enzyme-histochemical methods were used to analyse the activities of alkaline phosphatase (AP), dipeptidylpeptidase IV (DPP IV) and adenosine triphosphatase (ATPase) in capillaries of four different human oro-facial muscles, the major and minor zygomatic, the orbicularis oris and buccinator, one masticatory, the masseter and two limb muscles, the biceps brachii and first dorsal interosseus muscles. In all muscles, except for the orbicularis oris, the majority of the capillaries lacked enzyme activity. Therefore, none of these enzymes seems to be reliable as a general marker for human muscle capillaries. In general, the capillaries of the limb muscles and the major and minor zygomatic and the buccinator, were similar in their staining pattern for AP and ATPase, but differed in DPP IV staining. The orbicularis oris muscle differed from the other muscles by showing the largest proportion of capillaries with AP and ATPase activity. The masseter muscle had the largest proportion of capillaries stained for DPP IV. The muscle specific differences in enzyme activity of the capillaries are in agreement with our previous findings of specific differences between limb, oro-facial and masticatory muscles with respect to capillary supply and composition of fibre types and myosins. The results reflect functional specialization of the capillary bed of human muscles.

Differentiation of histochemical properties of masticatory muscles in Bovidae and Cervidae (Artiodactyla).

We analyzed the masticatory muscles (masseter, temporal, medial pterygoid and lateral pterygoid muscles) of Bovidae and Cervidae (Artiodactyla) for the histochemical characteristics of their fiber types. Analysis of muscle fiber types in the present study was based on the staining reaction for SDH, Sudan black B, alpha-GPDH, and myosin-ATPase after alkaline preincubations. Histochemical properties were found to contribute to masticatory function, including a slow-twitch fatigue resistant activity derived from the high percentage of red fibers, in spite of the differences in the distributions of fiber types in three portions (superficial, medial and profound portions) of each masticatory muscle. These results indicate a correlation between the histochemical profiles of individual masticatory muscles in these species and their functions during jaw movements.

[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.

Myosin isoform transitions during development of extra-ocular and masticatory muscles in the fetal rat.

The late fetal development of rat extra-ocular and masticatory muscles was examined by myosin immunohistochemistry. The pattern of slow and neonatal myosin isoform expression in primary and secondary myotubes in these muscles was generally similar to that seen by others in limb muscles. We observed a consistent difference between the Sprague-Dawley and Wistar rats in the degree of maturity reached by all muscles studied at a particular age. In both strains, extra-ocular muscles were also about one day in advance of the masticatory muscles. Thus, secondary myotubes were first seen at E17 in Wistar extraocular muscles, at E18 in Sprague-Dawley extra-ocular muscles and Wistar masticatory muscles, and at E19 in Sprague-Dawley masticatory muscles. There was a strikingly early and complete type differentiation of primary myotubes in extraocular muscles, and tonic myosin first appeared before birth in presumptive extrafusal tonic fibres in the orbital layer of the oculorotatory muscles. Throughout the late fetal period, retractor bulbi was composed of fast myotubes only, but these myotubes were not arranged in classical clusters. In the masticatory muscles at E17/E18 some slow primary myotubes started to express tonic myosin, and these presumptive spindle bag2 fibres were located only in regions of the muscles known to contain spindles in the adult. Presumptive bag1 fibres appeared about a day later (initially without tonic myosin), and in the region of the spindle cluster in anterior deep masseter extrafusal secondary myotube production appeared to be suppressed.

[Myoglobin content and beta-hydroxyacyl-CoA-dehydrogenase activity of different muscles of cattle and calf]. / Myoglobingehalt und beta-hydroxyacyl-CoA-Dehydrogenase-Aktivität verschiedener Muskeln von Rind und Kalb.

The heart, tongue, jowl, diaphragm and tail as well as shoulder, top round, the longissimus dorsi muscle of slaughtered cattle and the diaphragms of calf were examined with respect to their myoglobin content and beta-hydroxyacyl-CoA-dehydrogenase (HADH) activity. According to Gottesmann and Hamm [1] the product of these two values, the so-called MH value, can serve as the differentiation between the diaphragm and "normal cross striated skeletal muscles". Like the diaphragm, heart, tongue and jowl of cattle show higher MH values than those of "normal beef". Muscles in the tail have the same MH values as those of normal beef muscles. There are no essential differences in the MH values of various cross-striated muscle types of cows and calves. Muscles of cattle show a slightly higher myoglobin content, whereas the HADH activity is lower than in veal.

Structure and function of masticatory muscles in a case of muscular dystrophy.

Histologic examination of muscle biopsies and functional examination comprising electromyography and force measurements in a 19-yr-old boy with muscular dystrophy showed different wasting patterns of mandibular elevator and depressor muscles. Pronounced histopathologic changes were present in the masseter muscle, whereas pathologic findings in the anterior digastric muscle were limited to increased number of cells in slightly enlarged interfiber connective tissue. The masticatory pattern was distorted, and strength of mandibular elevator muscles was less than one third of the norm, whereas depressor strength corresponded more to reference values. This difference of muscular wasting might be caused by protective enzymes in the digastric muscle and/or functionally induced damage of the masseter. As affection from muscular dystrophy may vary greatly between the masticatory muscles, structural and functional examination should be used routinely to clarify prognosis before initiation of treatment procedures.

Variation in histochemical enzyme profile and diameter along human masseter intrafusal muscle fibers.

The histochemical enzyme profile of human masseter intrafusal muscle fibers was analyzed in consecutive serial cross sections along the individual fibers. Two hundred intrafusal fibers in 21 muscle spindles were classified. On the basis of equatorial nucleation, myosin ATPase-staining reactions after alkaline and acid preincubations and diameter, four different populations or types of intrafusal fiber were identified: large-diameter alkaline-stable and acid-stable fibers, bag2; two types of fiber with intermediate-diameter, alkaline-labile and acid-labile fibers corresponding to bag1 and alkaline-labile and acid-stable fibers designated as AS-bag1; and small-diameter alkaline-stable and acid-stable (pH 4.6)-acid-labile (pH 4.3) fibers called chain fibers. Regional variability in staining and diameter along the individual fibers was noted. In general, intrafusal fibers showed stronger oxidative reactions than did extrafusal fibers. The enzyme profile of the human masseter intrafusal fibers differed from that of extrafusal fibers in jaw, limb, and trunk muscles and also from that reported for spindles in limb and trunk muscles in man. The result suggests unique properties of human jaw muscle spindles and the jaw motor system.

Non-specific esterases and esterproteases in masticatory muscles from the muscular dystrophic mouse.

With the aid of histochemical and electrophoretic techniques activities for esterase and esterprotease were investigated in the digastric and masseter muscles from normal and dystrophic mice. The substrates used were alpha-naphthyl acetate and N-acetyl-L-alanine alpha-naphthyl ester. According to the microscopic observations of the dystrophic muscles the histopathological changes in the masseter muscle were much more pronounced than in the digastric muscle. The connective tissue surrounding the myofibers of the dystrophic masseter contained a large number of cells with pronounced enzyme activity. Among them were mast cells that were strongly stained for esterprotease. The connective tissue of the dystrophic digastricus was much less infiltrated with cellular elements reacting for esterprotease. In zymograms the normal digastricus, the dystrophic masseter and the dystrophic digastricus showed a strong activity for certain isoenzymes that were absent or weakly expressed in the normal masseter.