Myosin ATPase Reaction Research Articles

Restrictions in ATP diffusion within sarcomeres can provoke ATP-depleted zones impairing exercise capacity in chronic obstructive pulmonary disease

BackgroundChronic obstructive pulmonary disease (COPD) is characterized by the inability of patients to sustain a high level of ventilation resulting in perceived exertional discomfort and limited exercise capacity of leg muscles at average intracellular ATP levels sufficient to support contractility. MethodsMyosin ATPase activity in biopsy samples from healthy and COPD individuals was implemented as a local nucleotide sensor to determine ATP diffusion coefficients within myofibrils. Ergometric parameters clinically measured during maximal exercise tests in both groups were used to define the rates of myosin ATPase reaction and aerobic ATP re-synthesis. The obtained parameters in combination with AK- and CK-catalyzed reactions were implemented to compute the kinetic and steady-state spatial ATP distributions within control and COPD sarcomeres. ResultsThe developed reaction–diffusion model of two-dimensional sarcomeric space identified similar, yet extremely low nucleotide diffusion in normal and COPD myofibrils. The corresponding spatio-temporal ATP distributions, constructed during imposed exercise, predicted in COPD sarcomeres a depletion of ATP in the zones of overlap between actin and myosin filaments along the center axis at average cytosolic ATP levels similar to healthy muscles. ConclusionsATP-depleted zones can induce rigor tension foci impairing muscle contraction and increase a risk for sarcomere damages. Thus, intra-sarcomeric diffusion restrictions at limited aerobic ATP re-synthesis can be an additional risk factor contributing to the muscle contractile deficiency experienced by COPD patients. General significanceThis study demonstrates how restricted substrate mobility within a cellular organelle can provoke an energy imbalance state paradoxically occurring at abounding average metabolic resources.

Abstract P055: Systemic Arterial Hypertension Induces Pulmonary Injury Beyond Protein Degradation and Atrophy of Diaphragm

Background: Systemic arterial hypertension (SAH) is a chronic disease associated with systemic inflammation. Although cardiovascular adaptations resulting from SAH are more evident, little is known about the respiratory alterations. The purinergic receptor P2X7 plays a key role in the immune modulation, beyond to control the vascular tone and the development of inflammation and fibrosis. Aims: Evaluate the effects of SAH on the pulmonary inflammation and remodeling and on the diaphragm, and the involvement of purinergic receptor P2X7 and of ubiquitin-proteasome system (UPS) in this response. Methods: Spontaneously hypertensive rats (SHR) and normotensive Wistar (N) with 18 weeks-of-age were evaluated for: inflammation and remodeling of airways and pulmonary vessels and for P2X7 receptor expression. The morphology and biochemistryin diaphragm muscle for myosin ATPase reaction and ubiquitin-proteasome system (UPS), respectively. Results: The SHR showed a higher wall/lumen of the pulmonary arteries, as well as increased collagen deposition on the wall of these arteries. Increase in P2X7 receptor expression in pulmonary vascular wall (SHR:4.3%±0.7% vs. N:0.3%±0.2%) and bronchial epithelial (SHR:29±0% vs 2.8% N:10.5%±1.3%). The diaphragm was increased cross-sectional area (CSA) of type I fibers (16%) and reduction in CSA of type II (41%), increased the UPS activity and lipid peroxidation. SHR did not change in the analysis of ubiquitinated proteins and misfolded proteins. Conclusion: SAH induces important pulmonary disorders such pulmonary vascular remodeling, with increased expression of the purinergic receptor P2X7 associated with atrophy and protein degradation on diaphragm.

Nicotinamide Adenine Dinucleotide Tetrazolium Reductase Identifies Microvasculature Activation in Muscle from Adult Patients with Dermatomyositis

Previous work has suggested involvement of the muscle microvasculature in the pathogenesis of dermatomyositis (DM). Our study evaluates whether standard histochemical reactions can identify microvascular changes in muscle biopsies from patients with DM compared to myopathic and nonmyopathic controls. Muscle biopsies were obtained from 111 patients, including 45 patients with DM. Microvascular quantitation was performed on transversely oriented 1-μm toluidine blue-stained plastic sections. Histoenzymatic procedures included alkaline phosphatase (AP), nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR), succinate dehydrogenase (SDH), cytochrome C oxidase (COX), and myosin ATPase reactions. Capillary density was significantly lower in DM muscle biopsies compared to biopsies from patients with noninflammatory myopathies (NIM; n = 26) and healthy control muscle (n = 27; mean ± SD: 252 ± 114 vs 402 ± 56 and 325 ± 109 capillaries/mm(2), respectively; p values < 0.05). In contrast, a marked increase in the number of capillaries staining with NADH-TR was noted in DM compared to other idiopathic inflammatory myopathies (IIM; n = 13), NIM, and controls (49.8 ± 50.7 vs 8.0 ± 7.1, 6.7 ± 7.2, and 3.6 ± 2.8 capillaries/mm(2); p < 0.05 compared to DM). DM capillaries also demonstrated mildly increased staining with AP compared to controls; however, no increased SDH or COX reactivity was observed. DM muscle capillaries are highly reactive with NADH-TR compared to myopathic and nonmyopathic controls. The lack of staining of DM capillaries with mitochondrial SDH and COX reactions suggests that NADH-TR reactivity may be secondary to activation of the microvascular endoplasmic reticulum, rather than mitochondrial hyperplasia.

Mechanism of ATP loss in nonoxidative contracting muscle

The transition from rest to intense exercise is a challenge to cellular energetics ([11][1], [13][2], [15][3]). The metabolic fuels, i.e., the sources of ATP to sustain muscular contraction, are creatine phosphate and glycogen. Two anaerobic metabolic paths, leading to ATP generation, are catalyzed

Yuji Tonomura: A Pioneer in the Field of Energy Transduction in Muscle Contraction

Late Professor Yuji Tonomura has made a great contribution in the study of energy transduction in muscle contraction. He was the investigator who first proposed that a myosin-phosphate intermediate is produced subsequently to the Michaelis-Menten complex in the pre-steady state of the myosin ATPase reaction and that it is a key intermediate for muscle contraction. Here, his proposed intermediate will be viewed from the prospective of today's understanding of actomyosin ATPase kinetics and in the context of myosin motor domain crystal structures.

Muscle fibre composition and electromyographic features of cervical muscles following prolonged head extension in growing rats.

Soft tissue stretching has been proposed as one of the control factors in craniofacial morphogenesis. However, its mechanism remains unclear. The present study investigated electromyographic (EMG) activity and muscle fibre composition of cervical muscles following prolonged head extension in growing rats. Thirty-six male Wistar rats were divided into two experimental (E1, E2) and one control (C) group at 25 days of age. To induce head extension, the experimental rats were raised in cylindrical cages, which were positioned horizontally for group E1 and tilted upward at 45 degrees for group E2. At 55 days of age, EMG activity was recorded from the anterior digastricus (AD), sternohyoideus (SH), sternomastoideus (SM), longus capitis (LC), and biventer cervicis (BC) muscles in the rest position and passive head extension. EMG activity was analysed on its integrated values (IEMG), and composition of muscle fibres was evaluated by myosin ATPase reaction and fibre cross-sectional areas were calculated. Group E1 showed a higher percentage of type I fibres and lower IEMG during passive head extension in AD. In group E2 there was a higher percentage of type I fibres, a higher IEMG at rest, and a lower IEMG during passive head extension in BC. The experimental groups demonstrated altered proportions of type IIA and IIB fibres in SM and LC. They also showed higher percentages of subtype fibres and reduced cross-section areas of type II fibres in most of the muscles investigated. These findings suggest that head extension affects fibre transition, distribution, cross-section area, and recruitment pattern in cervical muscles.

Regenerated rat skeletal muscle after periodic contusions.

In the present study we evaluated the morphological aspect and changes in the area and incidence of muscle fiber types of long-term regenerated rat tibialis anterior (TA) muscle previously submitted to periodic contusions. Animals received eight consecutive traumas: one trauma per week, for eight weeks, and were evaluated one (N = 8) and four (N = 9) months after the last contusion. Serial cross-sections were evaluated by toluidine blue staining, acid phosphatase and myosin ATPase reactions. The weight of injured muscles was decreased compared to the contralateral intact one (one month: 0.77 +/- 0.15 vs 0.91 +/- 0.09 g, P = 0.03; four months: 0.79 +/- 0.14 vs 1.02 +/- 0.07 g, P = 0.0007, respectively) and showed abundant presence of split fibers and fibers with centralized nuclei, mainly in the deep portion. Damaged muscles presented a higher incidence of undifferentiated fibers when compared to the intact one (one month: 3.4 +/- 2.1 vs 0.5 +/- 0.3%, P = 0.006; four months: 2.3 +/- 1.6 vs 0.3 +/- 0.3%, P = 0.007, respectively). Injured TA evaluated one month later showed a decreased area of muscle fibers when compared to the intact one (P = 0.003). Thus, we conclude that: a) muscle fibers were damaged mainly in the deep portion, probably because they were compressed against the tibia; b) periodic contusions in the TA muscle did not change the percentage of type I and II muscle fibers; c) periodically injured TA muscles took four months to reach a muscle fiber area similar to that of the intact muscle.

Myosin light-chain domain rotates upon muscle activation but not ATP hydrolysis.

We have studied the correlation between myosin structure, myosin biochemistry, and muscle force. Two distinct orientations of the myosin light-chain domain were previously resolved using electron paramagnetic resonance (EPR) spectroscopy of spin-labeled regulatory light chains in scallop muscle fibers. In the present study, we measured isometric force during EPR spectral acquisition, in order to define how these two light-chain domain orientations are coupled to force and the myosin ATPase cycle. When muscle fibers are partially activated with increasing amounts of calcium, the distribution between the two light-chain domain orientations shifts toward the one associated with strong actin binding. This shift in distribution is linearly related to the increase in force, suggesting that rotation of the light-chain domain is coupled to strong actin binding. However, when nucleotide analogues are used to trap myosin in the pre- and posthydrolysis states of its ATPase cycle in relaxed muscle, there is no change in the distribution between light-chain domain orientations, showing that the rotation of the light-chain domain is not directly coupled to the ATP hydrolysis step. Instead, it is likely that in relaxed muscle the myosin thick filament stabilizes two light-chain domain orientations that are independent of the nucleotide analogue bound at the active site. We conclude that a large and distinct rotation of the light-chain domain of myosin is responsible for force generation and is coupled to strong actin binding but is not coupled to a specific step in the myosin ATPase reaction.

Analysis of stress in the active site of myosin accompanied by conformational changes in transient state intermediate complexes using photoaffinity labeling and 19F‐NMR spectroscopy

Myosin forms stable ternary complexes with ADP and the phosphate analogues, fluoroaluminate (Al F4-), fluoroberyllate (BeFn) or orthovanadate (Vi); these ternary complexes mimic transient intermediates in the myosin ATPase cycle. Moreover, we previously demonstrated that these complexes may mimic different myosin ATPase reaction intermediates corresponding to separate steps in the cross-bridge cycle [Maruta, S., Henry, G. D., Sykes, B. D. & Ikebe, M. (1993) J. Biol. Chem. 268, 7093-7100]. Park et al. suggested that the changing conformation of ATP during hydrolysis stresses the active site of myosin subfragment-1 (S-1) through protein-nucleotide contacts at the gamma-phosphate and nucleotide base, and the stress-induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure [Park, S., Ajtai, K. & Burghardt, T. P. (1997) Biochemistry 36, 3368-3372]. In the present study, the photoactive ADP analogue, 3'-O-(N-methylanthraniloyl)-2-azido-ADP (Mant-2-N3-ADP), and the 19F-labeled ADP analogue, 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate, were employed to examine conformational differences in protein-nucleotide contact in the ATP-binding site that may correlate with energy transduction. Mant-2-N3-ADP was trapped within the active site of skeletal and smooth muscle myosin in the presence of AlF4-, BeFn or Vi. For both skeletal and smooth muscle myosins, trapped Mant-2-N3-ADP was covalently linked to the 25-kDa N-terminal fragment of S-1 of both myosin/Mant-2-N3-ADP/AlF4- and BeFn complexes, presumably at Trp130. However, the efficiency of the incorporation was much higher for skeletal than for smooth muscle myosin suggesting that the conformations of the adenine-binding pockets of the two myosins are somewhat different. Although the amount of Mant-2-N3-ADP trapped in the presence of AlF4- and BeFn was the same for both myosins, the efficiency of photolabeling skeletal muscle myosin was approximately two times higher for BeFn complex than for AlF4- complex. The 19F-NMR spectra of the bound 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate in the ternary complexes formed in the presence of AlF4-, BeFn or Vi showed small but distinguishable differences. Taken together, these results indicate that there is some variation in the protein-nucleotide contacts at the nucleotide base among the ternary complexes studied, and these differences mimic separate steps occurring transiently during the contractile cycle.

Postnatal development of the masseter muscles in the Japanese field vole Microtus montebelli, with special attention to differentiation of the fast-twitch oxidative fiber.

Postnatal development and differentiation of the masseter muscles consisting only of fast-twitch oxidative (FO) fibers in the adult Japanese field vole Microtus montebelli were studied using histochemical and electron microscopic techniques. The masseter muscles were composed of myotubes and muscle fibers at day 0 (birth day). Most muscle cells showed the strong reaction for myosin ATPase after both alkaline and acid preincubations. For NADH-dehydrogenase (NADH-DH), small granular diformazan deposits were recognized in the sarcoplasm. Afterwards, the masseter muscles consisted of myofibers and satellite cells at day 5. For myosin ATPase, weakly-reactive fibers after acid preincubation (fast-twitch fibers) increased in number. For NADH-DH, granular diformazan deposits in all the myofibers increased in size. Since all the myofibers had numerous sarcoplasmic reticula, and they reacted strongly after alkaline preincubation and weakly after acid preincubation for myosin ATPase at day 10 when the young start to take solid food, it seems that the masseter muscles become contractive fast. At day 15 (before weaning), all the myofibers showed the adult-like strong reaction for NADH-DH and had numerous well-developed mitochondria, thus they acquired the ability of the fast and sustained contraction. It is accordingly considered that the masseter muscles of the vole mature in a short time after birth because of adaptation for herbivorous food habit.

Histochemical properties of intrinsic laryngeal muscles in cats

As one means in elucidating the contractile properties of the intrinsic laryngeal muscles, we examined the histochemical properties of the posterior cricoarytenoid (PCA), thyroarytenoid (TA), lateral cricoarytenoid (LCA), arytenoid (A) cricothyroid (CT) muscles in cats using the reaction for myosin ATPase following acid and alkali preincubation. The muscle fiber compositions of the intrinsic laryngeal muscles differed not only across the muscles but also across the muscle fascicles within a single muscle. The relative frequency of type-1 fibers was the smallest (9%) in the TA and the largest (45%) in the CT, and that of type-2A fibers was 39–48% in each muscle. The relative frequency of type-2B fibers was the smallest (9%) in the CT and the largest (42–45%) in the TA, LCA and A, and that of type-2C fibers was less than 1.0% in each muscle. Across the muscle fascicles, the TA was the most heterogeneous in muscle fiber distribution. The mediocaudal part of the TA was mainly composed of the muscle fascicles with type-1 fibers, while the laterocaudal and rostral parts of the TA were mostly composed of the muscle fascicles without type-1 fibers. The PCA was the most homogeneous in muscle fiber distribution. Our results demonstrate that the contractile properties of the intrinsic laryngeal muscles differ across the muscles and across the muscle fascicles within a single muscle and suggest that the delicate laryngeal movements are established by the coordinated activities of the intrinsic laryngeal muscle fibers with different contractile properties.

Relative increase in areas of muscle fibre types in horses during growth

SummaryThe relative growth of muscle fibre types as distinguished by the myosin ATPase reaction at pH 9.5 in m. semitendinosus, m. diaphragma and m. pectoralis transversus of growing Thoroughbreds and other horses ranging in age from perinatal life to adulthood was investigated.The growth rate of the area of myosin ATPase high‐reacting fibres in m. semitendinosus of both types of horse and in m. pectoralis transversus of Thoroughbreds was greater than that of low reacting fibres. Myosin ATPase low reacting fibres increased in area faster than high reacting fibres in the diaphragm of both types of horse and m. pectoralis transversus of other horses. These differential growth rates result in myosin ATPase high reacting fibres being larger than myosin ATPase low reacting fibres in m. semitendinosus and m. pectoralis transversus in adults of both types however, low‐reacting fibres are larger than high reacting fibres in m. diaphragma of adults of both types. These results suggest that the greater relative muscle mass of the proximal limbs of Thoroughbreds and associated greater mean fibre area associated are with relatively faster growing high reacting fibres in their muscles than those of other horses.

Postnatal Morphological and Histochemical Type Differentiation of the Porcine Muscle Fibres

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Morphological and Histochemical Type Differentiation of Muscle Fibres in Newborn Pigs: Its Relationship to Topical Myopathies

K a man I.: Morphological and Histochemical Type Differentiation of Muscle Fibres in Newborn Pigs: Its Relationship to Topical Myopathies. Acta vet. Bmo 1995,64: 35-47. Seven topographically and functionally different muscles of neonates of the wild pig, primitive domestic breed and ten modem breeds at various degrees of selection and their hybrids were examined in order to ascertain the degree of muscle fibre differentiation in neonates of the pig as a species. Using myosin ATPase reactions after acid or alkaline preincubation, the author was able to differentiate muscle fibres of type I and II (MF I, MF II), and an intermediary type IIC, with its transformation subtypes mild/strong, strong/strong and strong/mild (MS, SS and SM) in muscle fibres of type I. In some cases, a differentiation ofMF II into MF IIA and MF lIB was also observed. SDH detection and PAS reaction failed to differentiate MF in neonatal piglets reliably, or did not differentiate them at all. Although the degree of MF differentiation in neonatal piglets was mainly determined by their breed, it was also related to their birth weight, MF type and its relationship to the blood bed. Muscle fibres were fully morphologically and adequately histochemically differentiated in the neonatal . wild pig, primitive domestic pig, and in the less selected breeds of domestic pigs. Modern medium-selected meat breeds exhibited a myofibrillat deficiency of individual MF at an extent of a physiological vatiation. Myofibrillat hypoplasia (MFH) at various levels of severity was chatacteristic of pigs intensively selected for an extreme muscle growth. MFH affected primarily extrafusal anaerobic MF II and muscle groups with a predominance ofMF II, for which those pigs have unilaterally been selected. Myofibrillat hypoplasia did not affect intrafusal MF. Neonatal pig, wild pig, muscles, muscle fibre differentiation, classification, myofibrillar hypoplasia

Myocardial ischemic contracture. Metabolites affect rigor tension development and stiffness.

Myocardial ischemia is characterized by a decrease in phosphocreatine (PCr) and Mg(2+)-ATP contents as well as an accumulation of myosin ATPase reaction products (inorganic phosphate [P(i)], protons, and Mg(2+)-ADP). The possibility that these metabolites play a role in rigor tension development was checked in rat ventricular Triton X-100-skinned fibers. Rigor tension was induced by stepwise decreasing [Mg(2+)-ATP] in the presence or in the absence of 12 mmol/L PCr. To mimic the diastolic ionic environment of the myofibrils, [free Ca2+] was set at 100 nmol/L (pCa 7); [free Mg2+], at 1 mmol/L; and ionic strength, at 160 mmol/L. In control conditions (pH 7.1, with no added P(i) or Mg(2+)-ADP), the pMg(2+)-ATP for half-maximal rigor tension (pMg(2+)-ATP50) was 5.07 +/- 0.03 in the presence of PCr. After withdrawal of PCr, the pMg2+)-ATP50 value was shifted toward higher Mg(2+)-ATP values (3.57 +/- 0.03). Addition of 20 mmol/L P(i) shifted the pMg(2+)-ATP50 to 3.71 +/- 0.04 (P < .05) in the absence of PCr and in the opposite direction to 4.98 +/- 0.02 (P < .01) in the presence of PCr. Acidic pH (6.6) strongly increased pMg(2+)-ATP50 in both the absence (3.90 +/- 0.03, P < .001) and presence (5.44 +/- 0.02, P < .001) of PCr. Conversely, Mg(2+)-ADP (250 mumol/L) decreased pMg(2+)-ATP50 to 3.26 +/- 0.06 (P < .001) in the absence of PCr; at pMg(2+)-ATP 4, no rigor tension was observed until PCr concentration was decreased to < 2 mmol/L. At acidic pH, maximal rigor tension was lower by 29% compared with control conditions, whereas in the presence of Mg(2+)-ADP, maximal rigor tension developed to 143% of the control value; P(i) had no effect. The tension-to-stiffness (measured by the quick length-change technique) ratio was lower in rigor (no PCr and pMg(2+)-ATP 6) than during Ca2+ activation in the presence of both PCr and ATP. Compared with control rigor conditions, this parameter was unchanged by Mg(2+)-ADP and decreased by acidic pH, suggesting a proton-induced decrease in the amount of force per crossbridge. In addition to their known effects on active tension, Mg(2+)-ADP and protons affect rigor tension and influence ischemic contracture development. It is concluded that ischemic contracture and increased myocardial stiffness may be mediated by a decreased PCr and local Mg(2+)-ADP accumulation. This emphasizes the importance of myofibrillar creatine kinase activity in preventing ischemic contracture.

Skeletal muscle characteristics in 2 year-old race-trained thoroughbred horses

Skeletal muscle samples were obtained by needle biopsy from two depths of the m. gluteus medius of 50, young race-trained thoroughbred racehorses. Histochemical and biochemical characteristics of the muscle samples were analysed. Fibres were classified as type I, type lia or type IIb on the basis of the pH dependent lability of the myosin ATPase reaction. The activities of citrate synthase (CS) and glycogen phosphorylase (Phos) were determined. Muscle fibre composition varied markedly between deep and superficial muscle samples and this was reflected in differences in the activities of citrate synthase (CS) and phosphorylase (Phos). CS activity was greater in samples taken from a depth of 90 mm (deep) than those taken from a depth of 40mm (superficial: 122 ± 19 compared with 88 ± 16mol/g dry muscle/min at 25°C). Phos activity was greater in superficial samples (137 ± 20) compared with deep samples (117 ± 21). Regression analysis was used to estimate the enzyme activities in the different fibre types. No significant correlations were observed between histochemical and biochemical measures and subsequent racing performance.

Glycosylation pattern and enzyme activities in atrophic, angulated skeletal muscle fibres from ageing rats.

In this study enzyme activities and lectin binding patterns in skeletal muscle from very old rats were investigated in order to evaluate changes in enzyme activity or carbohydrate expression in senile muscle. Activities for adenosine triphosphatase (ATPase), succinic dehydrogenase, non-specific esterase and the binding pattern for 31 lectins were investigated in the soleus muscles from very old (36 months) and young (3 months) rats. In ageing muscles atrophic, angulated muscle fibres are frequent. In cryostat sections these fibres were mostly but not always type II defined by the myosin ATPase reaction; few showed a strong esterase activity. Some showed strong activity for succinic dehydrogenase while others were weakly reacting. A number of lectins strongly bound to the sarcoplasm in angulated fibres while the binding to normal fibres in both old and young rat muscle was much weaker or even absent. Preferential binding to the ageing, angulated fibres was seen with Aleuria aurentia, Galantus nivalis, Caragana abborecens, Triticum vulgaris, Maackia amurensis, Sambucus nigra, Phaseolus vulgaris erythroagglutinin, and Phaseolus coccineus. Samples of homogenized and centrifuged muscles were run by electrophoresis and the gels blotted to nitrocellulose paper. Subsequent lectin staining of the blots detected that two glycoproteins with molecular weights around 25,000 and 21,000 daltons were present in old muscle, but not in young. Aberrant or elevated expression of sarcoplasmic glycoconjugates is involved in ageing muscle atrophy.

Aluminum fluoride interactions with troponin C

The increasing interest in the metal ion aluminum fluoride and beryllium fluoride complexes as phosphate analogs in the myosin ATPase reaction and in muscle fiber studies prompted the examination of their interactions with the regulatory system of troponin and tropomyosin. In this work, the effects of these metal ion analogs on the spectral properties of the Ca(2+)-binding subunit of troponin, troponin C (TnC), were examined. In contrast to beryllium fluoride which did not change the spectral properties of TnC, aluminum fluoride binding induced an increase in both the alpha-helicity and the tyrosine fluorescence of TnC and exposed a hydrophobic region on this protein for fluorescent probe binding. Aluminum fluoride also reduced the Ca2+ and/or Mg(2+)-induced changes on TnC. These results indicate a direct interaction of aluminum fluoride with TnC and merit consideration in designing muscle fiber experiments with this phosphate analog.

Fiber differentiation of the human laryngeal muscles using the inhibition reactivation myofibrillar ATPase technique.

The aim of the present study was to further subdivide the type II fibers of the human thyroarytenoid and posterior cricoarytenoid muscles by means of a modified myosin ATPase reaction. In order to understand the functioning of these highly strained muscles better, it is important to know the respective percentage of fatigue-resistant type IIA fibers and fatigable type IIB fibers. The material comprised the larynges of seven laryngectomized males aged between 45 and 70 years and four laryngectomized females aged between 39 and 72 years. After having been frozen in nitrogen, 10-microns-thick sections were cut from the laryngeal muscles in a cryostat. The pH-lability of the enzyme that can be utilized in a classical myosin ATPase reaction permits a differentiation between fiber types I, IIA and IIB. Evidently, this is not possible with every human muscle. The fiber types IIA and IIB of the thyroarytenoid and the posterior cricoarytenoid muscles could be clearly distinguished by means of the inhibition reactivation myofibrillar ATPase technique. Using this method, the myosin ATPase enzyme was initially inhibited by hydroxymercuribenzoate and subsequently reactivated by cysteine. Regarding the incidence of type I and IIA fibers, there was a statistically significant difference between the thyroarytenoid and the posterior cricoarytenoid muscles. The type IIA fiber content was statistically significantly higher in the arytenoid muscle than in the posterior cricoarytenoid muscle. The percentage of type IIB fibers was low, not only in the thyroarytenoid muscle and the posterior cricoarytenoid muscle but also in the other laryngeal muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of the carnosine content of different fibre types in the middle gluteal muscle of the thoroughbred horse.

1. Skeletal muscle samples were obtained by needle biopsy from one of two depths of the m. gluteus medius in a group of twenty race-trained thoroughbred horses. 2. The content of carnosine was determined in each muscle sample, part of which was used for histochemical analysis. Fibres were classified as type I, type IIA or type IIB on the basis of the pH dependent lability of the myosin ATPase reaction. 3. Muscle samples with a higher type II fibre section area (FSA) have a higher carnosine content than those with a higher type I FSA. 4. Multiple linear regression analysis was used to estimate the mean carnosine content of individual fibre types. The results estimated a mean carnosine content in type I fibres of 54 mmol (kg dry muscle (DM))-1, in type IIA fibres 85 mmol (kg DM)-1 and in type IIB fibres 180 mmol (kg DM)-1. 5. Based on the estimated values of single fibre carnosine content, there was close concordance between the estimated and the measured carnosine content of mixed fibre samples. 6. It would appear from this and other studies that carnosine has an important role as a physico-chemical buffer in equine middle gluteal muscle and that this is greatest in type IIB fibres, where it may account for up to 50% of physico-chemical buffering of H+ produced by muscle in the pH range 7.1-6.5.