Effects of beta-escin and saponin on the transverse-tubular system and sarcoplasmic reticulum membranes of rat and toad skeletal muscle

Mechanically skinned skeletal muscle fibres from rat and toad were exposed to the permeabilizing agents beta-escin and saponin. The effects of these agents on the sealed transverse tubular system (t-system) and sarcoplasmic reticulum (SR) were examined by looking at changes in the magnitude of the force responses to t-system depolarization, the time course of the fluorescence of fura-2 trapped in the sealed t-system, and changes in the magnitude of caffeine-induced contractures following SR loading with Ca2+ under defined conditions. In the presence of 2 mu g ml(-1) beta-escin and saponin, the response to t-system depolarization was not completely abolished, decreasing to a plateau, and a large proportion of fura-2 remained in the sealed t-system. At 10 mu g ml(-1), both agents abolished the ability of both rat and toad preparations to respond to t-system depolarization after 3 min of exposure, but a significant amount of fura-2 remained in sealed t-tubules even after exposure to 100 mu g ml(-1) beta-escin and saponin for 10 min. beta-Escin took longer than saponin to reduce the t-system depolarizations and fura-2 content of the sealed t-system to a similar level. The ability of the SR to load Ca2+ was reduced to a lower level after treatment with beta-escin than saponin. This direct effect on the SR occurred at much lower concentrations for rat (2 mu g ml(-1) beta-escin and 10 mu g ml(-1) saponin) than toad (10 mu g ml(-1) beta-escin and 150 mu g ml(-1) saponin). The reverse order in sensitivities to beta-escin and saponin of t-system and SR membranes indicates that the mechanisms of action of beta-escin and saponin are different in the two types of membrane. In conclusion, this study shows that: (1) beta-escin has a milder action on the surface membrane than saponin; (2) beta-escin is a more potent modifier of SR function; (3) simple permeabilization of membranes is not sufficient to explain the effects of beta-escin and saponin on muscle membranes; and (4) the t-system network within muscle fibres is not a homogeneous compartment.

Osmotic properties of the sealed tubular system of toad and rat skeletal muscle

A method was developed that allows conversion of changes in maximum Ca2+-dependent fluorescence of a fixed amount of fluo-3 into volume changes of the fluo-3-containing solution. This method was then applied to investigate by confocal microscopy the osmotic properties of the sealed tubular (t-) system of toad and rat mechanically skinned fibers in which a certain amount Of fluo-3 was trapped. When the osmolality of the myoplasmic environment was altered by simple dilution or addition of sucrose within the range 190-638 mosmol kg(-1), the sealed t-system of toad fibers behaved almost like an ideal osmometer, changing its volume inverse proportionally to osmolality However, increasing the osmolality above 638 to 2,550 mosmol kg(-1) caused hardly any change in t-system volume. In myoplasmic solutions made hypotonic to 128 mosmol kg(-1), a loss of Ca2+ from the sealed t-system of toad fibers Occurred, presumably through either stretch-activated cationic channels or store-operated Ca2+ channels. In contrast to the behavior of the t-system in toad fibers, the volume of the sealed t-system of rat fibers changed little (by

Tubular system volume changes in twitch fibres from toad and rat skeletal muscle assessed by confocal microscopy

The volume of the extracellular compartment (tubular system) within intact muscle fibres from cane toad and rat was measured under various conditions using confocal microscopy. Under physiological conditions at rest, the fractional volume of the tubular system (t-sys(Vol)) was 1.38 +/- 0.09% (n = 17),1.41 +/- 0.09% (n = 12) and 0.83 +/- 0.07% (n = 12) of the total fibre volume in the twitch fibres from toad iliofibularis muscle, rat extensor digitorum longus muscle and rat soleus muscle, respectively. In toad muscle fibres, the t-sys(Vol) decreased by 30% when the tubular system was fully depolarized and decreased by 15% when membrane cholesterol was depleted from the tubular system with methyl-beta-cyclodextrin but did not change as the sarcomere length was changed from 1.93 to 3.30 mum. There was also an increase by 30% and a decrease by 25% in t-sys(Vol) when toad fibres were equilibrated in solutions that were 2.5-fold hypertonic and 50% hypotonic, respectively. When the changes in total fibre volume were taken into consideration, the t-sys(Vol) expressed as a percentage of the isotonic fibre volume did actually decrease as tonicity increased, revealing that the tubular system in intact fibres cannot be compressed below 0.9% of the isotonic fibre volume. The results can be explained in terms of forces acting at the level of the tubular wall. These observations have important physiological implications showing that the tubular system is a dynamic membrane structure capable of changing its volume in response to the membrane potential, cholesterol depletion and osmotic stress but not when the sarcomere length is changed in resting muscle.

Properties of the vertebrate skeletal muscle tubular system as a sealed compartment

Confocal imaging of impermeant fluorescent dyes trapped in the tubular (t-) system of skeletal muscle fibres of rat and cane toad was used to examine changes in the morphology of the t-system upon mechanical skinning, the time course of dye loss from the sealed t-systern in mechanically skinned fibres and the influence of rapid application and removal of glycerol on the morphology of the sealed t-system. In contrast to intact fibres, which have a t-systern open to the outside, the sealed t-systern of toad mechanically skinned fibres consistently displayed local swellings (vesicles). The occurrence of vesicles in the sealed t-system of rat-skinned fibres was infrequent. Application and removal of 200-400 mM glycerol to the sealed t-system did not produce any obvious changes in its morphology. The dyes fluo-3, fura-2 and Oregon green 488 were lost from the sealed t-system of toad fibres at different rates suggesting that the mechanism of organic anion transport across the tubular wall was not by indiscriminate bulk transport. The rate of fluo-3 and fura-2 loss from the sealed t-system of rat fibres was greater in rat than in toad fibres and could be explained by differences in surface area: volume ratio of the t-system in the two fibre types. Based on the results presented here and on other results from this laboratory, an explanation is given for the formation of numerous vesicles in toad-skinned fibres and lack of vesicle formation in rat-skinned fibres. This explanation can also help with better understanding the mechanism responsible for vacuole formation in intact fibres. (C) 2002 Elsevier Science Ltd. All rights reserved.

Modeling of continuous thermal processing of a non-Newtonian liquid food under diffusive laminar flow in a tubular system

The classic conservative approach for thermal process design can lead to over-processing, especially for laminar flow, when a significant distribution of temperature and of residence time occurs. In order to optimize quality retention, a more comprehensive model is required. A model comprising differential equations for mass and heat transfer is proposed for the simulation of the continuous thermal processing of a non-Newtonian food in a tubular system. The model takes into account the contribution from heating and cooling sections, the heat exchange with the ambient air and effective diffusion associated with non-ideal laminar flow. The study case of soursop juice processing was used to test the model. Various simulations were performed to evaluate the effect of the model assumptions. An expressive difference in the predicted lethality was observed between the classic approach and the proposed model. The main advantage of the model is its flexibility to represent different aspects with a small computational time, making it suitable for process evaluation and design. (C) 2012 Elsevier Ltd. All rights reserved.

Effect of saponin treatment on the sarcoplasmic reticulum of rat, cane toad and crustacean (yabby) skeletal muscle

1. Mechanically skinned fibres from skeletal muscles of the rat, toad and yabby were used to investigate the effect of saponin treatment on sarcoplasmic reticulum (SR) Ca2+ loading properties. The SR was loaded submaximally under control conditions before and after treatment with saponin and SR Ca2+ was released with caffeine. 2. Treatment with 10 mu g ml(-1) saponin greatly reduced the SR Ca2+ loading ability of skinned fibres from the extensor digitorum longus muscle of the rat with a rate constant of 0.24 min(-1). Saponin concentrations up to 150 mu g ml(-1) and increased exposure time up to 30 min did not further reduce the SR Ca2+ loading ability of the SR, which indicates that the inhibitory action of 10-150 mu g ml(-1) saponin is not dose dependent. The effect of saponin was also not dependent on the state of polarization of the transverse-tubular system. 3. Treatment with saponin at concentrations up to 100 mu g ml(-1) for 30 min did not affect the Ca2+ loading ability of SR in skinned skeletal muscle fibres from the twitch portion of the toad iliofibularis muscle but SR Ca2+ loading ability decreased markedly with a time constant of 0.22 min(-1) in the presence of 150 mu g ml(-1) saponin. 4. The saponin dependent increase in permeability could be reversed in both rat and toad fibres by short treatment with 6 mu M Ruthenium Red, a potent SR Ca2+ channel blocker, suggesting that saponin does affect the SR Ca2+ channel properties in mammalian and anuran skeletal muscle. 5. Treatment of skinned fibres of long sarcomere length (> 6 mu m) from the claw muscle of the yabby (a freshwater decapod crustacean) with 10 mu g ml(-1) saponin for 30 min abolished the ability of the SR to load Ca2+, indicating that saponin affects differently the SR from skeletal muscles of mammals, anurans and crustaceans. 6. is concluded that at relatively low concentrations, saponin causes inhibition of the skeletal SR Ca2+ loading ability in a species dependent manner, probably by increasing the Ca2+ loss through SR Ca2+ release channels.

Histoenzymological and ultrastructural changes in lateral muscle fibers of Oreochromis niloticus (Teleostei: Cichlidae) after local injection of veratrine

The effects of veratrine have been investigated in mammalian, amphibian, and crustacean muscle, but not in fish. In this work, the action of veratrine was studied in the lateral muscle of the freshwater teleost Oreochromis niloticus after intramuscular injection. Histoenzymological typing and electron microscopy of muscle fibers before and 15, 30, and 60 min after veratrine injection (10 ng/kg fish) were used to indirectly assess the morphological changes and the oxidative and m-ATPase activities. In some cases, muscles were pretreated with tetrodotoxin to determine whether the ultrastructural changes were the result of Na+ channel activation by veratrine. Veratrine altered the metabolism of fibers mainly after 30 min. Oxidative fibers showed decreased NADH-TR activity, whereas that of glycolytic and oxidative-glycolytic type fibers increased. There was no change in the m-ATPase activity of the three fiber types, except at 60 min postveratrine, when a novel fiber type, which showed no reversal after acidic and alkaline preincubations, appeared. Ultrastructural damage involved sarcomeres, myofibrils, and mitochondria, but the T-tubules remained intact. Pretreatment with tetrodotoxin (1 ng/ml) prevented the ultrastructural changes caused by veratrine. These results show that in fish skeletal muscle veratrine produces some effects that are not seen in mammalian muscle.

γ-Aminobutyric acid type B receptors are expressed and functional in mammalian cardiomyocytes

γ-Hydroxybutyrate (GHB), an anesthetic adjuvant analog of γ-aminobutyrate (GABA), depresses cell excitability in hippocampal neurons by inducing hyperpolarization through the activation of a prominent inwardly rectifying K+ (Kir3) conductance. These GABA type B (GABAB)-like effects are clearly shown at high concentrations of GHB corresponding to blood levels usually reached during anesthesia and are mimicked by the GABAB agonist baclofen. Recent studies of native GABAB receptors (GABABRs) have favored the concept that GHB is also a selective agonist. Furthermore, cloning has demonstrated that GABABRs assemble heteromeric complexes from the GABABR1 and GABABR2 subtypes and that these assemblies are activated by GHB. The surprisingly high tissue content, together with anti-ischemic and protective effects of GHB in the heart, raises the question of a possible influence of GABAB agonists on excitable cardiac cells. In the present study, we provide electrophysiological evidence that GHB activates an inwardly rectifying K+ current in rat ventricular myocytes. This effect is mimicked by baclofen, reversibly inhibited by GABAB antagonists, and prevented by pertussis toxin pretreatment. Both GABABR1 and GABABR2 are detected in cardiomyocytes by Western blotting and are shown to coimmunoprecipitate. Laser scanning confocal microscopy discloses an even distribution of the two receptors in the sarcolemma and along the transverse tubular system. Hence, we conclude that GABABRs are distributed not only in neuronal tissues but also in the heart, where they can be activated and induce electrophysiological alterations through G-protein-coupled inward rectifier potassium channels.

Annexin A1 is a biomarker of T-tubular repair in skeletal muscle of nonmyopathic patients undergoing statin therapy

Skeletal muscle complaints are a common consequence of cholesterol-lowering therapy. Transverse tubular (T-tubular) vacuolations occur in patients having statin-associated myopathy and, to a lesser extent, in statin-treated patients without myopathy. We have investigated quantitative changes in T-tubular morphology and looked for early indicators of T-tubular membrane repair in skeletal muscle biopsy samples from patients receiving cholesterol-lowering therapy who do not have myopathic side effects. Gene expression and protein levels of incipient membrane repair proteins were monitored in patients who tolerated statin treatment without myopathy and in statin-naive subjects. In addition, morphometry of the T-tubular system was performed. Only the gene expression for annexin A1 was up-regulated, whereas the expression of other repair genes remained unchanged. However, annexin A1 and dysferlin protein levels were significantly increased. In statin-treated patients, the volume fraction of the T-tubular system was significantly increased, but the volume fraction of the sarcoplasmic reticulum remained unchanged. A complex surface structure in combination with high mechanical loads makes skeletal muscle plasma membranes susceptible to injury. Ca(2+)-dependent membrane repair proteins such as dysferlin and annexin A1 are deployed at T-tubular sites. The up-regulation of annexin A1 gene expression and protein points to this protein as a biomarker for T-tubular repair.

Identification of the coupling between skeletal muscle store-operated Ca2+ entry and the inositol trisphosphate receptor

Examination of store-operated Ca2+ entry (SOC) in single, mechanically skinned skeletal muscle cells by confocal microscopy shows that the inositol 1,4,5-trisphosphate (IP3) receptor acts as a sarcoplasmic reticulum [Ca2+] sensor and mediates SOC by physical coupling without playing a key role in Ca2+ release from internal stores, as is the case with various cell types in which SOC was investigated previously. The results have broad implications for understanding the mechanism of SOC that is essential for cell function in general and muscle function in particular. Moreover, the study ascribes an important role to the IN receptors in skeletal muscle, the role of which with respect to Ca2+ homeostasis was ill defined until now.

Características morfológicas da região epididimária do pombo doméstico (Columba livia, L.)

A região epididimária do pombo doméstico compreende a parte principal da rede testicular, os dúctulos eferentes do testículo e o ducto epididimário. Os canais epiteliais da rede testicular são contínuos com o segmento extratesticular da rede, o qual, por sua vez, é seqüente com os dúctulos eferentes proximais e distais e, finalmente, o ducto epididimário se forma em continuidade aos eferentes distais. O epitélio de revestimento deste sistema tubular extratesticular é cúbico simples na rede testicular e pseudo-estratificado colunar nos outros ductos da região epididimária, com células ciliadas e não-ciliadas presentes principalmente nos dúctulos eferentes. As características ultra-estruturais das células epiteliais dos túbulos da região epididimária do pombo, com base comparativa, permitiram inferir que a reabsorção de fluido seminífero parece ser a função principal dessas células, embora outros papéis citofisiológicos foram também propostos, tais como: endocitose adsorptiva, ciliogênese, e possivelmente secreção apócrina nas células não-ciliadas escuras.

Alterações histopatológicas dos rins de macacos prego, Cebusapella (Linnaeus 1758) após exposição crônica a baixas doses de metilmercúrio

O mercúrio representa um grande risco ambiental e ocupacional constituindo um problema para a saúde humana na região Amazônica. Muito embora estudos tenham demonstrado que o mercúrio compromete vários tecidos e órgãos, os rins constituem-se órgãos-alvo para a toxicidade do metal. Dessa forma, o objetivo desse trabalho foi investigar os efeitos de uma exposição crônica a baixas doses de metilmercúrio sobre o parênquima renal de macacos Cebusapella, machos, adultos, expostos durante 120 dias consecutivos com doses diárias via oral, de 1,5 μg na dieta. As concentrações de mercúrio total no sangue dos animais foram monitoradas a cada 30 dias usando espectrofotômetro de absorção atômica a vapor frio (Hg 201), comparando ao grupo controle. O método utilizado para análise histopatológica foi a inclusão em parafina com coloração pela Hematoxilina e Eosina, Tricrômico de CAB e PAS. As investigações imuno-histoquímicas compreenderam as reações para a detecção de actina para musculo liso (IA4), actina muscular (HHF35) e pancitoqueratina (AE1 e AE2). Os resultados obtidos demonstraram que o tratamento com mercúrio causou diferença significativa (P<0,001) entre os grupos exposto e controle. Quanto aos níveis de Hg total, foram observadas alterações histopatológicas com características de hidropsia nos Túbulos Proximais, um achado comum na exposição ao metilmercúrio em outras espécies, sem alterações significativas nas concentrações de creatinina e ureia. O teste de correlação de Person demonstrou uma forte relação negativa entre a concentração de mercúrio e a perda de massa corporal dos animais (P<0,0001). Outro achado importante foi a diminuição do número de células mesangiais, o que sugere que o metilmercúrio executou a sua nefrotoxicidade atingindo não somente o sistema tubular renal, como também as células do mesangio glomerular, fazendo-se necessário um maior aporte de estudos experimentais para esclarecer qual o nível de alerta da concentração de mercúrio é capaz de desencadear mecanismos de agressão e injúria renal em indivíduos expostos ao metilmercúrio.

The effects of PTK787/ZK222584, an inhibitor of VEGFR and PDGFR? pathways, on intussusceptive angiogenesis and glomerular recovery from Thy1.1 nephritis

The aim of our study was to investigate the phenomenon of intussusceptive angiogenesis with a focus on its molecular regulation by vascular endothelial growth factor receptor (VEGFR)/platelet-derived growth factor receptor β (PDGFRβ) pathways and biological significance for glomerular recovery after acute injury. Glomerular healing by intussusception was examined in a particular setting of Thy1.1 nephritis, where the lysis of mesangial cells results in an initial collapse and successive rebuilding of glomerular capillary structure. Restoration of capillary structure after induction of Thy1.1 nephritis occurred by intussusceptive angiogenesis resulting in i) rapid expansion of the capillary plexus with reinstatement of the glomerular filtration surface and ii) restoration of the archetypical glomerular vascular pattern. Glomerular capillaries of nephritic rats after combined VEGFR2 and PDGFRβ inhibition by PTK787/ZK222584 (PTK/ZK) were tortuous and irregular. However, the onset of intussusceptive angiogenesis was influenced only after long-term PTK/ZK treatment, providing an important insight into differential molecular regulation between sprouting and intussusceptive angiogenesis. PTK/ZK treatment abolished α-smooth muscle actin and tensin expression by injured mesangial cells, impaired glomerular filtration of microspheres, and led to the reduction of glomerular volume and the presence of multiple hemorrhages detectable in the tubular system. Collectively, treatment of nephritic patients with PTK/ZK compound is not recommended.

Statin therapy induces ultrastructural damage in skeletal muscle in patients without myalgia

Muscle pain and weakness are frequent complaints in patients receiving 3-hydroxymethylglutaryl coenzymeA (HMG CoA) reductase inhibitors (statins). Many patients with myalgia have creatine kinase levels that are either normal or only marginally elevated, and no obvious structural defects have been reported in patients with myalgia only. To investigate further the mechanism that mediates statin-induced skeletal muscle damage, skeletal muscle biopsies from statin-treated and non-statin-treated patients were examined using both electron microscopy and biochemical approaches. The present paper reports clear evidence of skeletal muscle damage in statin-treated patients, despite their being asymptomatic. Though the degree of overall damage is slight, it has a characteristic pattern that includes breakdown of the T-tubular system and subsarcolemmal rupture. These characteristic structural abnormalities observed in the statin-treated patients were reproduced by extraction of cholesterol from skeletal muscle fibres in vitro. These findings support the hypothesis that statin-induced cholesterol lowering per se contributes to myocyte damage and suggest further that it is the specific lipid/protein organization of the skeletal muscle cell itself that renders it particularly vulnerable.