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Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP and Plp-GFP mice, respectively. Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact. Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species. In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation has proven useful for studies of NMJ development, physiology and pathology. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Homozygous ataxic mice (ax(J)) express reduced levels of the deubiquitinating enzyme Usp14. They develop severe tremors by 2-3 wk of age, followed by hindlimb paralysis, and death by 6-8 wk. While changes in the ubiquitin proteasome system often result in the accumulation of ubiquitin protein aggregates and neuronal loss, these pathological markers are not observed in the ax(J) mice. Instead, defects in neurotransmission were observed in both the central and peripheral nervous systems of ax(J) mice. We have now identified several new alterations in peripheral neurotransmission in the ax(J) mice. Using the two-microelectrode voltage clamp technique on diaphragm muscles of ax(J) mice, we observed that under normal neurotransmitter release conditions ax(J) mice lacked paired-pulse facilitation and exhibited a frequency-dependent increase in rundown of the end plate current at high-frequency stimulation (HFS). Combined electrophysiology and styryl dye staining revealed a significant reduction in quantal content during the initial and plateau portions of the HFS train. In addition, uptake of styryl dyes (FM dye) during HFS demonstrated that the size of the readily releasable vesicle pool was significantly reduced. Destaining rates for styryl dyes suggested that ax(J) neuromuscular junctions are unable to mobilize a sufficient number of vesicles during times of intense activity. These results imply that ax(J) nerve terminals are unable to recruit a sufficient number of vesicles to keep pace with physiological rates of transmitter release. Therefore, ubiquitination of synaptic proteins appears to play an important role in the normal operation of the neurotransmitter release machinery and in regulating the size of pools of synaptic vesicles.

Reactive oxygen species (ROS) are generated as a result of normal cellular metabolism, mainly through the mitochondria and peroxisomes, but their release is enhanced by the activation of oxidant enzymes such as NADPH oxidases or downregulation of endogenous antioxidant enzymes such as manganese-superoxide dismutase (MnSOD) and catalase. Transforming growth factor-β (TGF-β), found to be overexpressed in airway smooth muscle (ASM) from asthmatic and chronic obstructive pulmonary disease patients, may be a pivotal regulator of abnormal ASM cell (ASMC) function in these diseases. An important effect of TGF-β on ASMC inflammatory responses is the induction of IL-6 release. TGF-β also triggers intracellular ROS release in ASMCs by upregulation of NADPH oxidase 4 (Nox4). However, the effect of TGF-β on the expression of key antioxidant enzymes and subsequently on oxidant/antioxidant balance is unknown. Moreover, the role of redox-dependent pathways in the mediation of the proinflammatory effects of TGF-β in ASMCs is unclear. In this study, we show that TGF-β induced the expression of Nox4 while at the same time inhibiting the expression of MnSOD and catalase. This change in oxidant/antioxidant enzymes was accompanied by elevated ROS levels and IL-6 release. Further studies revealed a role for Smad3 and phosphatidyl-inositol kinase-mediated pathways in the induction of oxidant/antioxidant imbalance and IL-6 release. The changes in oxidant/antioxidant enzymes and IL-6 release were reversed by the antioxidants N-acetyl-cysteine (NAC) and ebselen through inhibition of Smad3 phosphorylation, indicating redox-dependent activation of Smad3 by TGF-β. Moreover, these findings suggest a potential role for NAC in preventing TGF-β-mediated pro-oxidant and proinflammatory responses in ASMCs. Knockdown of Nox4 using small interfering RNA partially prevented the inhibition of MnSOD but had no effect on catalase and IL-6 expression. These findings provide novel insights into redox regulation of ASM function by TGF-β.

The importance of the pathological changes in proteoglycans has driven the need to study and design novel chemical tools to control proteoglycan synthesis. Accordingly, we tested the fluorinated analogue of glucosamine (4-fluoro-N-acetyl-glucosamine (4-F-GlcNAc)) on the synthesis of heparan sulfate (HS) and chondroitin sulfate (CS) by murine airway smooth muscle (ASM) cells in the presence of radiolabeled metabolic precursors. Secreted and cell-associated CS and HS were assessed for changes in size by Superose 6 chromatography. Treatment of ASM cells with 4-F-GlcNAc (100 microM) reduced the quantity (by 64.1-76.6%) and decreased the size of HS/CS glycosaminoglycans associated with the cell layer (K(av) shifted from 0.30 to 0.45). The quantity of CS secreted into the medium decreased by 65.7-73.0%, and the size showed a K(av) shift from 0.30 to 0.50. Treatment of ASM cells with 45 microM and 179 microM 4-F-GlcNAc in the presence of a stimulator of CS synthesis, 4-methylumbelliferyl-beta-D-xyloside, reduced the amount of the xyloside-CS chains by 65.4 and 87.0%, respectively. The size of xyloside-CS chains synthesized in the presence of 4-F-GlcNAc were only slightly larger than those with xyloside treatment alone (K(av) of 0.55 compared with that of 0.6). The effects of 4-F-GlcNAc to inhibit CS synthesis were not observed with equimolar concentrations of glucosamine. We propose that 4-F-GlcNAc inhibits CS synthesis by inhibiting 4-epimerization of UDP-GlcNAc to UDP-GalNAc, thereby depleting one of the substrates required, whereas HS elongation is inhibited by truncation when the nonreducing terminus of the growing chain is capped with 4-F-GlcNAc.

Magnesium is one of the most important factors for regulation of inflammatory response as well as muscle function, and COPD is a multicomponent disease characterized by abnormal inflammatory response of the lungs with systemic muscle dysfunction. Because polymorphonuclear (PMN) cells are significantly represented in the pathogenesis of COPD, concentrations of total (tMg) and ionised magnesium (iMg) were determined in plasma and isolated PMN cells in 46 patients in stable phase of COPD (past smokers, current smokers, and non-smokers), 24 healthy smokers and 37 healthy non-smokers. In the same samples concentrations of total (tCa) and ionised calcium (iCa) were determined, due to the antagonism of magnesium towards calcium. We found decreased biological active iMg in PMN compared to the group of healthy non-smokers (5.42, 1.98-17.31 micromol/10(9) cells vs. 7.50, 3.27-15.15 micromol/10(9) cells, p < 0.05). In the plasma and isolated PMN of the patients the ratio of total calcium/total magnesium (tCa/tMg) was significantly increased (2.89, 2.15-3.86 and 1.19, 0.07-9.87) compared to the group of healthy non-smokers (2.65, 2.19-3.44 and 0.67, 0.14-2.40, p < 0.05) and to the group of healthy smokers (2.58, 2.26-3.24 and 0.66, 0.14-2.85, p < 0.05). In the group of patients the concentration of tCa was significantly increased in all samples compared to the healthy group of non-smokers and healthy smokers. The results of univariant logistic regression analysis for smoking, concentration of tCa and ratio of tCa/tMg in PMN showed high odds ratio for COPD status. These results raise a possibility that intracellular polymorphonuclear value of magnesium could be a distinctive marker for COPD risk disclosure among smokers.

Although 4-hydroxy-2-nonenal (HNE, a product of lipid peroxidation) is a major cause of oxidative damage inside skeletal muscles, the exact proteins modified by HNE are unknown. We used two-dimensional electrophoresis, immunoblotting, and mass spectrometry to identify selective proteins targeted by HNE inside the diaphragm of rats under two conditions: severe sepsis [induced by E. coli lipopolysaccharides (LPS)] and during strenuous muscle contractions elicited by severe inspiratory resistive loading (IRL). Diaphragm HNE-protein adduct formation (detected with a polyclonal antibody) increased significantly after 1 and 3 h of LPS injection with a return to baseline values thereafter. Similarly, HNE-protein adduct formation inside the diaphragm rose significantly after 6 but not 3 h of IRL. Mass spectrometry analysis of HNE-modified proteins revealed enolase 3b, aldolase and triosephosphate isomerase 1, creatine kinase, carbonic anyhdrase III, aconitase 2, dihydrolipoamide dehydrogenase, and electron transfer flavoprotein-beta. Measurements of in vitro enolase activity in the presence of pure HNE revealed that HNE significantly attenuated enolase activity in a dose-dependent fashion, suggesting that HNE-derived modifications have inhibitory effects on enzyme activity. We conclude that lipid peroxidation products may inhibit muscle contractile performance through selective targeting of enzymes involved in glycolysis, energy production as well as CO(2) hydration.

The aim of this study was to investigate whether the heme oxygenase (HO) pathway could modulate proliferation of airway smooth muscle (ASM) and the mechanism(s) involved in this phenomenon. In cultured human ASM cells, 10% fetal calf serum or 50 ng/ml platelet-derived growth factor AB induced cell proliferation, extracellular and intracellular reactive oxygen species (ROS) production and ERK1/2 phosphorylation. Pharmacological HO-1 induction (by 10 microm hemin or by 20 microm cobalt-protoporphyrin) and HO inhibition (by 25 microm tin-protoporphyrin or by an antisense oligonucleotide), respectively, reduced and enhanced significantly both cell proliferation and ROS production. Neither the carbon monoxide scavenger myoglobin (5-20 microm) nor the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one could reverse ASM proliferation induced by tin-protoporphyrin, making a role of the CO-cGMP pathway in HO-modulated proliferation unlikely. By contrast, bilirubin (1 microm) and the antioxidant N-acetyl-cysteine (1 mm) significantly reduced mitogen-induced cell proliferation, ROS production, and ERK1/2 phosphorylation. Furthermore, both bilirubin and N-acetyl-cysteine and the ERK1/2 inhibitor PD98059 significantly reversed the effects of HO inhibition on ASM proliferation. These results could be relevant to ASM alterations observed in asthma because activation of the HO pathway prevented the increase in bronchial smooth muscle area induced by repeated ovalbumin challenge in immunized guinea pigs, whereas inhibition of HO had the opposite effect. In conclusion, this study provides evidence for an antiproliferative effect of the HO pathway in ASM in vitro and in vivo through a bilirubin-mediated redox modulation of phosphorylation of ERK1/2.

Leukotrienes play a critical role in promoting bronchoconstriction in asthma. The purpose of this study was to examine whether interferon (IFN)-gamma, a cytokine upregulated in asthmatic airways, modulates leukotriene (LT)D4 receptor expression and contractile responses in cultured human airway smooth muscle (HASM) cells. Treatment of HASM cells with IFN-gamma (10 to 1,000 U/ml) stimulated a dose-dependent increase in cell-surface expression of cysteinyl leukotriene receptor 1 (CysLT1) as determined by flow cytometry. CysLT1 messenger RNA (mRNA) levels were also significantly enhanced by IFN-gamma, as demonstrated by reverse transcription-polymerase chain reaction. To determine the functional relevance of increased CysLT1 expression in HASM, cell stiffness responses to LTD4 were measured with magnetic twisting cytometry. IFN-gamma (1,000 U/ml for 24 h) markedly increased LTD4-induced changes in cell stiffness, from 4.6 +/- 1 [mean +/- SEM]% to 24.4 +/- 3.7% (n = 8, p < 0.05). Montelukast, a CysLT1 antagonist, completely inhibited LTD4-induced increases in cell stiffness. IFN-gamma had no effect on the cell stiffness responses to bradykinin, another contractile agonist. Collectively, these data suggest that IFN-gamma increases LTD4 responses in HASM cells by increasing cell-surface expression of CysLT1. Our data suggest that increased levels of IFN-gamma in asthmatic individuals may promote airway hyperresponsiveness and asthma exacerbations by directly modulating contractile responses of HASM.

In this study, we searched for signs of thixotropic behavior in human rib cage respiratory muscles. If rib cage respiratory muscles possess thixotropic properties similar to those seen in other skeletal muscles in animals and humans, we expect resting rib cage circumference would be temporarily changed after deep rib cage inflations or deflations and that these aftereffects would be particularly pronounced in trials that combine conditioning deep inflations or deflations with forceful isometric contractions of the respiratory muscles. We used induction plethysmography to obtain a continuous relative measure of rib cage circumference changes during quiet breathing in 12 healthy subjects. Rib cage position at the end of the expiratory phase (EEP) was used as an index of resting rib cage circumference. Comparisons were made between EEP values of five spontaneous breaths immediately before and after six types of conditioning maneuvers: deep inspiration (DI); deep expiration (DE); DI combined with forceful effort to inspire (FII) or expire (FEI); and DE combined with forceful effort to inspire (FIE) or expire (FEE), both with temporary airway occlusion. The aftereffects of the conditioning maneuvers on EEP values were consistent with the supposition that human respiratory muscles possess thixotropic properties. EEP values were significantly enhanced after all conditioning maneuvers involving DI, and the aftereffects were particularly pronounced in the FII and FEI trials. In contrast, EEP values were reduced after DE maneuvers. The aftereffects were statistically significant for the FEE and FIE, but not DE, trials. It is suggested that respiratory muscle thixotropy may contribute to the pulmonary hyperinflation seen in patients with chronic obstructive pulmonary disease.

1. Previous studies in dogs have demonstrated that the maximum change in airway pressure (DeltaPao) produced by a particular respiratory muscle is the product of three factors, namely the mass of the muscle, the maximal active muscle tension per unit cross-sectional area ( approximately 3.0 kg cm-2), and the fractional change in muscle length per unit volume increase of the relaxed chest wall (i.e. the muscle's mechanical advantage). In the present studies, we have used this principle to infer the DeltaPao values generated by the parasternal intercostal and triangularis sterni muscles in man. 2. The mass of the muscles and the direction of the muscle fibres relative to the sternum were first assessed in six cadavers. Seven healthy individuals were then placed in a computed tomographic scanner to determine the orientation of the costal cartilages relative to the sternum and their rotation during passive inflation to total lung capacity. The fractional changes in length of the muscles during inflation, their mechanical advantages, and their DeltaPao values were then calculated. 3. Passive inflation induced shortening of the parasternal intercostals in all interspaces and lengthening of the triangularis sterni. The fractional shortening of the parasternal intercostals decreased gradually from 7.7 % in the second interspace to 2.0 % in the fifth, whereas the fractional lengthening of the triangularis sterni increased progressively from 5.9 to 13.8 %. These rostrocaudal gradients were well accounted for by the more caudal orientation of the cartilages of the lower ribs. 4. Since these fractional changes in length corresponded to a maximal inflation, the inspiratory mechanical advantage of the parasternal intercostals was only 2.2-0. 6 % l-1, and the expiratory mechanical advantage of the triangularis sterni was only 1.6-3.8 % l-1. In addition, whatever the interspace, parasternal and triangularis muscle mass was 3-5 and 1-3 g, respectively. As a result, the magnitude of the DeltaPao values generated by a maximal contraction of the parasternal intercostals or triangularis sterni in all interspaces would be only 1-3 cmH2O. 5. These studies therefore confirm that the parasternal intercostals in man have an inspiratory action on the lung whereas the triangularis sterni has an expiratory action. However, these studies also establish the important fact that the pressure-generating ability of both muscles is substantially smaller than in the dog.

Potential relationships between the structure of the diaphragm and external intercostals and several indices of respiratory muscle function, lung function and nutrition in 27 patients (61+/-10 yrs of age) subjected to thoracotomy as a result of a lung neoplasm have been investigated. Prior to surgery the nutritional status of the patients was assessed and lung function (spirometry, lung volumes, transfer factor of the lungs for carbon monoxide, arterial blood gases) and respiratory muscle function (maximal inspiratory pressure (MIP) and diaphragmatic function were measured). Biopsies of the diaphragm (and external intercostals) were obtained during surgery. On average, patients showed mild airflow limitation (forced expiratory volume in one second (FEV1), 70+/-14% of predicted value, FEV1/forced vital capacity (FVC), 70+/-9%) with some air trapping (residual volume (RV), 139+/-50% pred) and normal gas exchange (arterial oxygen tension (Pa,O2), 11.3+/-1.33 kPa (85+/-10 mmHg)) and arterial carbon dioxide tension (Pa,CO2) 5.4+/-0.5 kPa (40.6+/-4 mmHg). MIP was 77+/-25% pred; maximal transdiaphragmatic pressure was 90+/-27 cmH2O. Most morphometric measurements of the diaphragm and external intercostals were within the range of values reported previously in other skeletal muscles. The size of the fibres of these two respiratory muscles was positively related (p<0.05) to MIP (% pred). There were no significant relationships between the structure of both muscles and nutritional status or any index of lung function. In conclusion, in the population studied, the fibre size of the diaphragm and external intercostals appears to relate to their ability to generate force.

Immunohistochemical screening for monoclonal antibodies prepared by immunization of mice with a rat osteoblastic cell population led to identification of one antibody that reacted against a small population of cells present in the soft connective tissue compartment of 21 days fetal rat calvaria. The morphology of the cells and the immunohistochemical staining characteristics (a distinct intracellular granular pattern) suggested that the antibody might be reacting specifically against mast cells. We used combined histochemistry and immunohistochemistry to further characterize this antibody, designated RCJ102. Cryosections containing calvaria bone, soft connective tissues and skin were prepared from the top of the head of 21 days fetal rats, and from adult rats cryosections of lung, muscle, adipose tissue and small intestine were prepared. Some sections were labelled by indirect immunofluorescence with RCJ102; corresponding sections were labelled histochemically with toluidine blue. There was a direct correspondence between mast cells identified histochemically and cells labelling with RCJ102 in all tissues except intestine, in which the mast cell detectable by histochemistry were not labelled by RCJ102. These results suggest that the RCJ102 antibody will be a valuable new reagent for further elucidation of the heterogeneity described between connective tissue and intestinal mucosal mast cells.

Interest in the adaptive strategies of respiratory muscles in response to exercise training has grown in recent years. Animal studies have clearly demonstrated that regular endurance exercise training results in small but significant increases in oxidative and antioxidant enzyme activities in both inspiratory and expiratory muscles. Further, exercise training has been shown to promote a shift in the fast myosin heavy chain isoforms (e.g. from type IIb to IId) within the costal diaphragm of endurance-trained rodents. Human studies using numerous respiratory muscle training programmes have shown that respiratory muscle training results in an increased work capacity of the ventilatory musculature. However, the issue of whatever respiratory muscle training improves whole body endurance capacity remains controversial. Although some authors have reported that ventilatory muscle training results in improved whole body exercise, many investigators argue that respiratory muscle performance does not limit high intensity exercise tolerance or influence maximum oxygen table uptake (VO2max). The explanation for the divergent findings is unclear but may be due to variance in the exercise tasks used to evaluate exercise endurance. This is an interesting area for future research.

These experiments examined the metabolic properties of the canine respiratory muscles. Because the costal diaphragm (COD), crural diaphragm (CRD), parasternal intercostals (PI), triangularis sterni (TS), and transversus abdominis (TA) are active during quite breathing in the dog, we hypothesized that these muscles would have different metabolic profiles (i.e., higher oxidative and antioxidant enzyme activities) compared with ventilatory muscles recruited only at increased ventilatory requirements [e.g., scalene (SC) and external oblique (EO)] and locomotor muscles [e.g., deltoid (DEL)]. To test this hypothesis, muscle samples were removed from six healthy adult dogs and analyzed to determine the activities of citrate synthase (CS), phosphofructokinase (PFK), 3-hydroxyacyl-CoA dehydrogenase (HADH), and superoxide dismutase (SOD). The activities of these enzymes were interpreted as relative measures of metabolic capacities, and enzyme activity ratios were considered as representing relationships between different metabolic pathways. Analysis revealed that CS and HADH activities were significantly higher (P < 0.05) in the PI, COD, CRD, and TS compared with those in all other muscles. Muscles with the lowest CS, HADH, and SOD activities (i.e., SC, TA, EO, DEL) generally had the highest PFK activities, Furthermore, the PFK/CS ratio was significantly lower in the PI, COD, CRD, and TS compared with that in all other muscles studied. These data support the notion that the canine PI, COD, CRD, and TS are metabolically different from other key ventilatory muscles.

In this study the effects of administration of cortisone acetate (100 mg kg-1 body weight subcutaneously for 11 days) on distribution and cross-sectional area of different fibre types of rat skeletal muscles were investigated. Diaphragm, parasternal intercostal (PI), extensor digitorum longus (EDL) and soleus muscles were examined in cortisone treated animals (CA) in comparison with ad libitum controls (CTRL) and pair-fed (PF) controls. Four fibre types (I or slow and IIA, IIX, IIB or fast) were identified on the basis of their myosin heavy chain composition using a set of monoclonal antibodies. In CA rats the reduction of cross-sectional area was above 30% in IIX fibres of diaphragm, IIB fibres of PI and in all fast fibres of EDL. In all muscles slow fibres were spared from atrophy. Significant variations in fibre type distribution were found in the muscles of CA rats when compared to CTRL. The percentage of IIB fibres decreased in EDL, PI and diaphragm. This decrease was accompanied by an increase in the percentage of IIA fibres in the same muscles. No changes in the percentage of slow fibres and of fast IIX fibres were observed in EDL, PI and diaphragm of CA rats in comparison with CTRL. In soleus of CA rats the proportion of IIA fibres was lower than in CTRL. In EDL of PF rats atrophy of IIA fibres and changes in fibre type distribution were similar to those observed in CA rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Calponin and caldesmon are two proteins considered to play a regulatory role in smooth muscle contraction, which have never previously been found to be expressed in subcultured cells. In the present study, immunocytochemistry and immunoblotting were performed to identify these proteins in smooth muscle cells (SMC) from human bronchi. It was found that human airway SMC, kept in a non-proliferative state, continued to express caldesmon and calponin at least until the 8th passage. The expression of alpha-smooth muscle actin studied under the same conditions was also shown to be preserved in subcultured bronchial SMC.

This study investigated the histochemical and morphometric properties of fibres in laryngeal, hyoid, tongue and pharyngeal muscles which contribute in maintaining patency of the upper airway. Muscle specimens from adult female goats were stained for nicotinamide adenine dinucleotide dehydrogenase-tetrazolium reductase and myosin adenosine triphosphatase activities, and the composition and size of the fibre types determined. These muscles contained types 1, 2A, 2B and 2C fibres with type 2 fibres predominating and the fibres possessed oxidative enzyme activity suggesting fast contraction speed and yet moderate resistance to fatigue. Abductor laryngeal muscles contained more type 1 fibres than the adductors. Among pharyngeal muscles fibre size and type 1 fibre composition increased progressively from the hyopharyngeus caudally. Upper airway muscles contained relatively small fibres (range of mean diameter: 25.7 to 46.1 microns) with the pharyngeal and lingualis proprius muscles containing the smallest fibres. These properties might influence the response of upper airway muscles to neuromuscular blocking drugs.

The pattern of glycogen utilization was used to determine whether various muscle fiber types in the rat diaphragm are differentially susceptible to neuromuscular transmission failure. Muscle segments from the midcostal region were repetitively stimulated directly or via the phrenic nerve at 10 or 75 Hz. Muscle fiber types were classified histochemically as type I, IIa, or IIb. The amount of muscle fiber glycogen depletion with direct stimulation depended on stimulation rate (75 Hz > 10 Hz) and fiber type (IIb > IIa > I). However, with nerve stimulation, muscle fiber glycogen depletion did not display the same dependency on stimulation rate (10 Hz > 75 Hz), although with stimulation at 10 Hz, the same rank order of fiber depletion was observed (IIb > IIa > I). This rank order of depletion was reversed (I > IIa > IIb) during repetitive stimulation of the nerve at 75 Hz. By intermittently stimulating the muscle directly during continuous nerve stimulation, we determined that neuromuscular transmission failure contributed significantly to the force decline after 2 min of stimulation at 75 Hz but relatively little to the force decline after 2 min of stimulation at 10 Hz. A significantly greater fraction of the force decline could be attributed to neuromuscular transmission failure with repetitive bouts of stimulation at 10 Hz. We conclude that neuromuscular transmission failure causes a significant portion of the force decline after 8 min of stimulation at 10 and 75 Hz, that all diaphragm fiber types are susceptible to neuromuscular transmission failure, but that type IIb fibers are particularly susceptible at higher frequencies of stimulation.

The effects of long-term (24- to 28-wk) continuous respiratory resistive loading on diaphragm mass, contractility, fatigue, and fiber types were studied in male rats. Increased respiratory resistance was produced by extratracheal banding, and results were compared with sham-operated pair-fed controls. At the time the animals were killed, banded tracheal segment internal diameter was reduced by 57% of control values. Diaphragm surface area and muscle mass (normalized for body mass) increased by 19% of control values. Isometric diaphragm contractility and fatigue resistance indexes were measured using an in vitro diaphragm costal strip preparation at 37 degrees C. Twitch and tetanic stimulations were evoked using direct stimulation. Compared with controls, baseline tensions (normalized for diaphragm cross-sectional area) were significantly decreased at low frequencies. Fatigue resistance (endurance) indexes were significantly increased at all frequencies. These findings were consistent with observed increases in number and cross-sectional area of type I (low-tension high-endurance) fibers. We conclude that the diaphragm adapts to chronic long-term resistive loads by sacrificing peak tensions for an increase in endurance capacity.

This study was designed to determine the histochemical properties, size and composition of fibres in the diaphragm, intercostal and abdominal muscles of goats to clarify whether reported similarities in respiratory muscle physiology between goats and humans have a structural basis. Serial sections (10 microns) of muscular tissue from adult female goats were stained for myosin adenosine triphosphatase and reduced nicotinamide adenine dinucleotide dehydrogenase-tetrazolium reductase activities; the fibres were classified into type I, IIA and IIB; and their mean diameter and composition were determined. Abdominal and intercostal muscles contained types I, IIA and IIB fibres in the ratio 1:1:1, and the mean diameter of the fibres ranged from 49.2 to 62.2 microns. In contrast, the diaphragm contained 58.9% type I and 41.1% type II fibres, and the latter could not be differentiated into types IIA and IIB. Diaphragmatic fibres were also smaller (36.9-40.9 microns). These findings contrast with those in humans, where the diaphragm, intercostal and abdominal muscles contain > 50% type I fibres and have fibres of identical diameter. The differences in fibre characteristics between the diaphragm, intercostal and abdominal muscles of goats and the differences between goats and humans need to be taken into consideration in interpreting the results from studies in respiratory muscle physiology.

Recent evidence suggests that cellular sodium regulation may be abnormal in muscular dystrophy. We have measured intracellular sodium concentration (Nai) in muscles of mdx mice (a model of Duchenne muscular dystrophy) using two techniques. Nai in isolated diaphragm (measured using a microelectrode) was 13.0 +/- 0.3 mM and 23.5 +/- 0.7 mM (mean +/- SE) in the control and mdx mice respectively. Nai in gastrocnemius muscle (calculated from extra- and intracellular volumes using serum and whole-muscle sodium concentrations) was 13 +/- 3 mM and 24 +/- 2 mM (mean +/- SE) in control and mdx, respectively. We argue that this abnormality in mdx tissues could reflect a reduced flux through the Na/K ATPase, although a contribution from increased Na leak cannot be ruled out. We also discuss possible consequences of an increased Nai: for example, raised Nai may lead to defective cell volume control in Duchenne dystrophy and the mdx mouse.

Lung volume influences the mechanical action of the primary inspiratory and expiratory muscles by affecting their precontraction length, alignment with the rib cage, and mechanical coupling to agonistic and antagonistic muscles. We have previously shown that the canine pectoral muscles exert an expiratory action on the rib cage when the forelimbs are at the torso's side and an inspiratory action when the forelimbs are held elevated. To determine the effect of lung volume on intrathoracic pressure changes produced by the canine pectoral muscles, we performed isolated bilateral supramaximal electrical stimulation of the deep pectoral and superficial pectoralis (descending and transverse heads) muscles in 15 adult supine anesthetized dogs during hyperventilation-induced apnea. Lung volume was altered by application of a negative or positive pressure (+/- 30 cmH2O) to the airway. In all animals, selective electrical stimulation of the descending, transverse, and deep pectoral muscles with the forelimbs held elevated produced negative intrathoracic pressure changes (i.e., an inspiratory action). Moreover, with the forelimbs elevated, increasing lung volume decreased both pectoral muscle fiber precontraction length and the negative intrathoracic pressure changes generated by contraction of each of these muscles. Conversely, with the forelimbs along the torso, increasing lung volume lengthened pectoral muscle precontraction length and augmented the positive intrathoracic pressure changes produced by muscle contraction (i.e., an expiratory action). These results indicate that lung volume significantly affects the length of the canine pectoral muscles and their mechanical actions on the rib cage.

To determine the potential range of diaphragm sarcomere lengths in situ and the effect of changes in sarcomere length on capillary and fiber geometry, rat diaphragms were perfusion fixed in situ with glutaraldehyde at different airway pressures and during electrical stimulation. The lengths of thick (1.517 +/- 0.007 microns) and thin (1.194 +/- 0.048 microns) filaments were not different from those established for rat limb muscle. Morphometric techniques were used to determine fiber cross-sectional area, sarcomere length, capillary orientation, and capillary length and surface area per fiber volume. All measurements were referenced to sarcomere length, which averaged 2.88 +/- 0.08 microns at -20 to -25 cmH2O airway pressure (residual volume) and 2.32 +/- 0.05 microns at +20 to +26 cmH2O airway pressure (total lung capacity). The contribution of capillary tortuosity and branching to total capillary length was dependent on sarcomere length and varied from 5 to 22%, consistent with that shown previously for mammalian limb muscles over this range of sarcomere lengths. Capillary length per fiber volume [Jv(c,f)] was significantly greater at residual volume (3,761 +/- 193 mm-2) than at total lung capacity (3,142 +/- 118 mm-2) and correlated with sarcomere length [l; r = 0.628, Jv(c,f) = 876l + 1,156, P less than 0.01; n = 18]. We conclude that the diaphragm is unusual in that the apparent in situ minimal sarcomere length is greater than 2.0 microns.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the effect of aminophylline (0.1-1 mM) on the contraction threshold (CT) of rat diaphragm fibers (25 degrees C). The CT was measured by direct visualization (x200) of the fiber under current-clamp conditions. The main findings are the following: 1) Aminophylline lowers the CT, in a dose-dependent manner, toward more negative values of the resting membrane potential (Vm). 2) Dibutyryl adenosine 3',5'-cyclic monophosphate (2 mM) shifts the CT, although this change is smaller than in the presence of xanthine. 3) Tetracaine (1 mM), a drug that diminishes Ca release from the sarcoplasmic reticulum, reduces the shift induced by 1 mM aminophylline; this is partially overcome by increasing aminophylline concentration to 5 mM. 4) Hyperpolarization of the fibers shifts the CT to more negative Vm. We suggest that the displacement in the CT to more negative Vm plays an important role in the potentiating effect of aminophylline. This could be the result of an enhancement of Ca release from the sarcoplasmic reticulum.

The mechanisms of neuromuscular blockade by hypaconitine and aconitine were investigated electrophysiologically in isolated phrenic nerve-diaphragm muscles of mice. Hypaconitine (0.08-2 microM) and aconitine (0.3-2 microM) depressed the nerve-evoked twitch tension, without affecting the contraction evoked by stimulation of the muscle. At the concentrations of hypaconitine (up to 5 microM) and aconitine (up to 2 microM) that depressed the nerve-evoked twitch tension, the resting membrane potential of the muscle cells was unchanged. Hypaconitine (0.1-2 microM) and aconitine (2 microM) blocked the end-plate potential (epp), without affecting the amplitude of the miniature epp (mepp). The quantal content of end-plate potentials was decreased by these agents in parallel with the decrement in amplitude. The nerve compound action potential was inhibited by hypaconitine (5 microM) and aconitine (2-10 microM), as well as by 1 microM tetrodotoxin (TTX). When the nerve compound action potential was completely blocked by 2 microM aconitine, the muscle action potential was unaffected, although 1 microM TTX suppressed both potentials to the same degree. These results indicate the neuromuscular blockade produced by hypaconitine and aconitine were caused by reducing the evoked quantal release. The mechanism of this effect was attributed mainly to blocking of the nerve compound action potential.

Airway smooth muscle cells of bovine trachealis muscle from immature (2 weeks old), developing (5 months old) and mature animals (greater than 5 years old) have resting membrane potentials of -63.5 +/- 0.4 mV, -62.2 +/- 0.8 mV and -60.3 +/- 0.7 mV, respectively. These resting potentials were not significantly different. The contribution of the electrogenic sodium pump to the resting membrane potential of airway smooth muscle cells decreases significantly with age being 38.3% +/- 0.9, 30.6% +/- 0.8 and 21.3% +/- 0.9 in tissues from immature, developing and mature cows, respectively. The contribution of the electrogenic sodium pump to resting membrane potential was not influenced by the degree of stretch (applied load) of the airway smooth muscle cells. However, resting membrane potential was reduced with increasing tone especially when loads between 5 to 10 g were applied. The densities of Na+/K+ pump sites, as measured by [3H] ouabain binding, decreased significantly as a consequence of maturation and were 4.12 +/- 5.23, 3.08 +/- 0.26 and 1.94 +/- 0.38 pmoles/mg protein ouabain binding in tissues from immature, developing and mature animals respectively. The affinity of the pump sites for the cardiac glycoside, ouabain, did not change during maturation. We conclude that maturation alters both the number of Na+/K+ pump sites and the contribution of the electrogenic sodium pump to the membrane potential of airway smooth muscle cells. These age related changes may contribute to the reduced airway reactivity to both bronchoconstrictor and bronchodilator agents previously observed both in vitro and in vivo.

The motor neurons for the accessory muscles of respiration, pectoralis, trapezius, external oblique, and the rectus abdominis were studied in the spinal cord. The objective was to determine if the localization and morphology of the motor neurons for these muscles bear any distinct relationship to the specialized function of these muscles, serving both as supportive skeletal muscles and as accessory respiratory muscles. In addition, it was of interest to know if the inspiratory role of the pectoralis and trapezius muscles and the expiratory role of the external oblique and rectus abdominis are related to the spatial organization of the motor neurons; this knowledge may be important in the discrimination of influences from afferent connections. The motor neurons for these muscles were retrogradely labeled with true blue and were compared with the triceps motor neurons. All neurons occurred ipsilateral and most labeling occurred in C6-7. The motor neurons for the accessory muscles were mainly confined to the ventrolateral tip of the ventral gray matter. The triceps neurons were dorsolateral to the respiratory related neurons in C6-7. Within the confines of the ventrolateral area, the majority of neurons for the pectoralis were localized medial to ventromedial, those for the trapezius were ventrolateral, and those for the external oblique were in the extreme ventrolateral to ventral sections of C7. No neurons were observed in C2 to T2 for the rectus abdominis. A second neuronal column occurred medioventrally in the ventral gray of C4-6 for the trapezius, and is distinct and separated from the C6-7 cell column.(ABSTRACT TRUNCATED AT 250 WORDS)

Patterns of muscle activity during lung ventilation, patterns of innervation and some contractile properties were measured in the hypaxial muscles of green iguanas. Electromyography shows that only four hypaxial muscles are involved in breathing. Expiration is produced by two deep hypaxial muscles, the transversalis and the retrahentes costarum. Inspiration is produced by the external and internal intercostal muscles. Although the two intercostal muscles are the main agonists of inspiration, neither is involved in expiration. This conflicts with the widely held notion that the different fibre orientations of the two intercostal muscles determine their ventilatory action. Several observations indicate that ventilation is produced by slow (i.e. nontwitch) fibres of these four muscles. First, electromyographic (EMG) activity recorded from these muscles during ventilation has an unusually low range of frequencies (less than 100 Hz). Such low-frequency signals have been suggested to be characteristic of muscle fibres that do not propagate action potentials (i.e. slow fibres). Second, during inspiration, EMG activity is restricted to he medical sides of the two intercostal muscles. Muscle fibres from this region have multiple motor endplates and exhibit tonic contraction when immersed in saline solutions of high potassium content. Like the intercostals, the transversalis and retrahentes costarum muscles also contain fibres with multiple motor endplates. Thus, although breathing is a phasic activity, it is produced by tonic (i.e. slow) muscle fibres. The intercostal muscles are also involved in postural and locomotor movements of the trunk. However, such movements employ twitch as well as slow fibres of the intercostal muscles.