Prefrontal cortex activation during incremental inspiratory loading in healthy participants.

We aimed to investigate whether changes in prefrontal cortex (PFC) oxyhemoglobin (O2Hb) and deoxyhemoglobin (HHb) associates with inspiratory muscle effort during inspiratory threshold loading (ITL) in healthy participants. Participants performed an incremental ITL. Breathing pattern, partial pressure of end-tidal CO2 (PETCO2), mouth pressure and O2Hb and HHb over the right dorsolateral PFC, sternocleidomastoid (SCM), and diaphragm/intercostals (Dia/IC) were monitored. Fourteen healthy participants (8 men; 29 ± 5 years) completed testing. Dyspnea was higher post- than pre-ITL (5 ± 1 vs. 0 ± 1, respectively; P<0.05). PFC O2Hb increased (P < 0.001) and HHb decreased (P = 0.001) at low loads but remained stable with increasing ITL intensities. PFC total hemoglobin increased at task failure compared to rest. SCM HHb increased throughout increasing intensities. SCM and Dia/IC total hemoglobin increased in the at task failure compared to rest. PETCO2 did not change (P = 0.528). PFC is activated early during the ITL but does not show central fatigue at task failure despite greater dyspnea and an imbalance of SCM oxygen demand and delivery.

The Effect of Respiratory Muscle Training on the Pulmonary Function, Lung Ventilation, and Endurance Performance of Young Soccer Players.

This study investigated whether the addition of eight weeks of inspiratory muscle training (IMT) to a regular preseason soccer training program, including incremental endurance training (IET), would change pulmonary function, lung ventilation, and aerobic performance in young soccer players. Sixteen club-level competitive junior soccer players (mean age 17.63 ± 0.48 years, height 182 ± 0.05 cm, body mass 68.88 ± 4.48 kg) participated in the study. Participants were randomly assigned into two groups: experimental (n = 8) and control (n = 8). Both groups performed regular preseason soccer training, including endurance workouts as IET. In addition to this training, the experimental group performed additional IMT for eigght weeks with a commercially available respiratory muscle trainer (Threshold IMT), with a total of 80 inhalations (twice per day, five days per week). Pre- and post-intervention tests of pulmonary function, maximal inspiratory pressure, and the Cooper test were implemented. Eight weeks of IMT had a positive impact on expiratory muscle strength (p = 0.001); however, there was no significant effect on respiratory function parameters. The results also indicate increased efficiency of the inspiratory muscles, contributing to an improvement in aerobic endurance, measured by VO2max estimated from running distance in the cardiorespiratory Cooper test (p < 0.005).

Preliminary study: comparative effects of lung volume therapy between slow and fast deep-breathing techniques on pulmonary function, respiratory muscle strength, oxidative stress, cytokines, 6-minute walking distance, and quality of life in persons with COPD.

BACKGROUND: Lung volume therapy with the Voldyne® device can improve lung volume and has a nonsignificant benefit on respiratory muscle strength via the slow deep-breathing technique (SDBT); whereas respiratory muscle training with a respiratory muscle trainer via the fast deep-breathing technique (FDBT) has produced a significant improvement in people with COPD. Thus, the aim of this study was to compare the efficiency of lung volume therapy with the Voldyne® device with the SDBT and FDBT on pulmonary function, respiratory muscle strength, oxidative stress, cytokines, walking capacity, and quality of life (QoL) in people with COPD. METHODS: A total of 30 COPD patient volunteers with mild (stage I) to moderate (stage II) severity were randomized into two groups: SDBT (n=15) and FDBT (n=15). Pulmonary function (FVC, FEV1, and FEV1/FVC), maximal inspiratory mouth pressure (PImax), oxidative stress status (total antioxidant capacity [TAC], glutathione [GSH], malondialdehyde [MDA], and nitric oxide [NO]), inflammatory cytokines (tumor necrosis factor-alpha [TNF-α] and IL-6), 6-minute walking distance (6MWD), and total clinical COPD questionnaire (CCQ) score were evaluated before and after 4 weeks of training. RESULTS: All the parameters had no statistical difference between the groups before training. The PImax, TAC, IL-6, total QoL score, and 6MWD changed significantly in the SDBT group after the 4-week experiment as compared to those in the pre-experimental period, whereas FVC, FEV1, FEV1%, FEV1/FVC%, PImax, TAC, MDA, NO, TNF-α, IL-6, 6MWD, and total CCQ score changed significantly in the FDBT group as compared to those in the pre-experimental period. The FEV1%, PImax, TNF-α, IL-6, and total CCQ score differed significantly in the FDBT group in the post-experimental period as compared to those in the SDBT group. CONCLUSION: This preliminary study concluded that the application of incentive spirometry with the Voldyne® device via fast deep breathing possibly improved respiratory muscle strength and QoL and reduced inflammatory cytokines, MDA, and NO better than that via slow deep breathing among people with COPD.

Effects of a simple prototype respiratory muscle trainer on respiratory muscle strength, quality of life and dyspnea, and oxidative stress in COPD patients: a preliminary study.

BACKGROUND: The aim of this study was to evaluate the efficiency of a simple prototype device for training respiratory muscles in lung function, respiratory muscle strength, walking capacity, quality of life (QOL), dyspnea, and oxidative stress in patients with COPD. METHODS: Thirty COPD patients with moderate severity of the disease were randomized into three groups: control (n=10, 6 males and 4 females), standard training (n=10, 4 males and 6 females), and prototype device (n=10, 5 males and 5 females). Respiratory muscle strength (maximal inspiratory pressure [PImax] and maximal expiratory pressure [PEmax]), lung function (forced vital capacity [FVC], percentage of FVC, forced expiratory volume in 1 second [FEV1], percentage of FEV1 [FEV1%], and FEV1/FVC), 6-minute walking distance (6MWD), QOL, and oxidative stress markers (total antioxidant capacity [TAC]), glutathione (GSH), malondialdehyde (MDA), and nitric oxide (NO) were evaluated before and after 6 weeks of training. Moreover, dyspnea scores were assessed before; during week 2, 4, and 6 of training; and at rest after training. RESULTS: All parameters between the groups had no statistical difference before training, and no statistical change in the control group after week 6. FVC, FEV1/FVC, PImax, PEmax, QOL, MDA, and NO showed significant changes after 6 weeks of training with either the standard or prototype device, compared to pre-training. FEV1, FEV1%, 6MWD, TAC, and GSH data did not change statistically. Furthermore, the results of significant changes in all parameters were not statistically different between training groups using the standard and prototype device. The peak dyspnea scores increased significantly in week 4 and 6 when applying the standard or prototype device, and then lowered significantly at rest after 6 weeks of training, compared to pre-training. CONCLUSION: This study proposes that a simple prototype device can be used clinically in COPD patients as a standard device to train respiratory muscles, improving lung function and QOL, as well as involving MDA and NO levels.

Efectividad de la estimulación eléctrica en los músculos respiratorios / Effectiveness of electrical stimulation of the respiratory muscles

El buen funcionamiento de los músculos respiratorios es de vital importancia para el mantenimiento de un sistema ventilatorio correcto. Una de las técnicas que se viene utilizando de manera novedosa para el tratamiento de las disfunciones respiratorias es la electroterapia. Por ello, el objetivo de esta revisión es evaluar la efectividad de la estimulación eléctrica aplicada en los músculos respiratorios, en cuanto a la mejora en la función pulmonar, en términos de fuerza muscular y capacidades pulmonares. Para ello se ha realizado una búsqueda bibliográfica en las bases de datos MEDLINE, PUBMED, CINHAL y SCOPUS y en S-CIELO de los artículos publicados entre 2009 y 2014. Se analizaron 13 artículos que superaron los criterios de inclusión y exclusión. Se concluye que se necesita una investigación más profunda para sacar conclusiones definitivas, pero la estimulación eléctrica de los músculos respiratorios parece ser una técnica eficaz en la mejora de la función ventilatoria (AU)

Good respiratory muscle function is vital to maintain a correctly functioning respiratory system. Electrotherapy has been used as a novel technique for the treatment of respiratory disorders. The aim of this review was to evaluate the effectiveness of electrical stimulation applied to the respiratory muscles, in terms of improvement in function lung, muscle strength and lung capacity. A literature search was conducted in MEDLINE, PUBMED, CINAHL and SCOPUS and S-CIELO for articles published between 2009 and 2014. Thirteen articles were analysed that met the inclusion and exclusion criteria. Further research is needed to draw definitive conclusions, but electrical stimulation of the respiratory muscles appears to be an effective technique in improving respiratory function (AU)

Cardiorespiratory and Muscle Metabolic Responses During Conventional Versus Motion Sensor-Assisted Strategies for Functional Electrical Stimulation Standing After Spinal Cord Injury.

This is a case series study with the objective of comparing two motion sensor automated strategies to avert knee buckle during functional electrical stimulation (FES)-standing against a conventional hand-controlled (HC) FES approach. The research was conducted in a clinical exercise laboratory gymnasium at the University of Sydney, Australia. The automated strategies, Aut-A and Aut-B, applied fixed and variable changes of neurostimulation, respectively, in quadriceps amplitude to precisely control knee extension during standing. HC was an "on-demand" increase of stimulation amplitude to maintain stance. Finally, maximal FES amplitude (MA) was used as a control condition, whereby knee buckle was prevented by maximal isometric muscle recruitment. Four AIS-A paraplegics undertook 4 days of testing each, and each assessment day comprised three FES standing trials using the same strategy. Cardiorespiratory responses were recorded, and quadriceps muscle oxygenation was quantified using near-infrared spectroscopy. For all subjects, the longest standing times were observed during Aut-A, followed by Aut-B, and then HC and MA. The standing times of the automated strategies were superior to HC by 9-64%. Apart from a lower heart rates during standing (P = 0.034), the automation of knee extension did not promote different cardiorespiratory responses compared with HC. The standing times during MA were significantly shorter than during the automated or "on-demand" strategies (by 80-250%). In fact, the higher isometric-evoked quadriceps contraction during MA resulted in a greater oxygen demand (P < 0.0001) and wider arteriovenous oxygen extraction (P = 0.08) when compared with the other strategies. In conclusion, even though increased standing times were demonstrated using automated control of knee extension, physiological benefits compared with HC were not evident.

GH responses to two consecutive bouts of respiratory muscle endurance training in healthy adults.

Repetition of voluntary exercise bouts and of different pharmacological GH-releasing stimuli at 2-h intervals is associated with a complete abolishment of GH responsiveness. By contrast, a different pattern is observed after repeated neuromuscular electrostimulation, which is characterized by preservation of GH responsiveness. Aim of the study was to evaluate GH responses to repeated bouts of respiratory muscle endurance training (RMET) by mean of a specific commercially available device (Spiro Tiger®). Eight healthy men underwent an incremental progressive RMET protocol of 11 daily sessions. Blood samplings for GH, cortisol and lactate (LA) determinations were collected during the 12th session, which was composed of two consecutive bouts of RMET (of identical intensity and duration: 1 min at a respiration rate of 28 acts/min, 5 min at 32 acts/min, 5 min at 34 acts/min, 4 min at 36 acts/min) at a 2 h interval. Baseline GH levels (mean: 0.9±0.4 ng/ml) significantly (p<0.01) increased after the first bout of RMET (peak: 15.7±4.0 ng/ml). The administration of the second bout of RMET resulted in a significantly lower (p<0.05) GH increase (peak: 3.9±0.8 ng/ml) in comparison with the first one. Baseline LA levels (mean: 1.2±0.1 mmol/l) significantly increased (p<0.001) after the first bout of RMET (peak: 2.3±0.2 mmol/l). The administration of the second RMET bout resulted in a comparable LA increase (from a basal value of 1.2±0.1 mmol/l up to a peak of 2.0±0.1 mmol/l, p<0.001). The first bout of RMET caused a significant increase of cortisol levels (p<0.01), starting from a basal mean value of 142.9±9.4 ng/ml up to a peak of 188.8±10.3 ng/ml. By contrast, the second bout of RMET did not induce any significant change of cortisol levels (basal: 149.1±9.0 ng/ml, peak: 168.5±5.1 ng/ml). In conclusion, a single bout of RMET is capable of stimulating GH and cortisol secretions and LA production. When a second bout is repeated after 2 h, there is a blunting of GH and cortisol responses with a preservation of LA release. Further studies are needed to schedule long-term RMET protocols capable of persistently stimulating GH-IGF-I release and to maximally enhance the ergogenic and metabolic benefits of this intervention either in normal subjects (e.g. athletes) or patients with an impairment of motor capabilities requested to perform normal daily activities (i.e. severely obese and elderly people).

Inhibition of polyadenylation reduces inflammatory gene induction.

Cordycepin (3' deoxyadenosine) has long been used in the study of in vitro assembled polyadenylation complexes, because it terminates the poly(A) tail and arrests the cleavage complex. It is derived from caterpillar fungi, which are highly prized in Chinese traditional medicine. Here we show that cordycepin specifically inhibits the induction of inflammatory mRNAs by cytokines in human airway smooth muscle cells without affecting the expression of control mRNAs. Cordycepin treatment results in shorter poly(A) tails, and a reduction in the efficiency of mRNA cleavage and transcription termination is observed, indicating that the effects of cordycepin on 3' processing in cells are similar to those described in in vitro reactions. For the CCL2 and CXCL1 mRNAs, the effects of cordycepin are post-transcriptional, with the mRNA disappearing during or immediately after nuclear export. In contrast, although the recruitment of RNA polymerase II to the IL8 promoter is also unaffected, the levels of nascent transcript are reduced, indicating a defect in transcription elongation. We show that a reporter construct with 3' sequences from a histone gene is unaffected by cordycepin, while CXCL1 sequences confer cordycepin sensitivity to the reporter, demonstrating that polyadenylation is indeed required for the effect of cordycepin on gene expression. In addition, treatment with another polyadenyation inhibitor and knockdown of poly(A) polymerase α also specifically reduced the induction of inflammatory mRNAs. These data demonstrate that there are differences in the 3' processing of inflammatory and housekeeping genes and identify polyadenylation as a novel target for anti-inflammatory drugs.

Inspiratory muscle training abolishes the blood lactate increase associated with volitional hyperpnoea superimposed on exercise and accelerates lactate and oxygen uptake kinetics at the onset of exercise.

We examined the effects of inspiratory muscle training (IMT) upon volitional hyperpnoea-mediated increases in blood lactate ([lac(-)](B)) during cycling at maximal lactate steady state (MLSS) power, and blood lactate and oxygen uptake kinetics at the onset of exercise. Twenty males formed either an IMT (n = 10) or control group (n = 10). Prior to and following a 6-week intervention, two 30 min trials were performed at MLSS (207 ± 28 W), determined using repeated 30 min constant power trials. The first was a reference trial, whereas during the second trial, from 20 to 28 min, participants mimicked the breathing pattern commensurate with 90% of the maximal incremental exercise test minute ventilation ([Formula: see text]). Prior to the intervention, the MLSS [lac(-)](B) was 3.7 ± 1.8 and 3.9 ± 1.6 mmol L(-1) in the IMT and control groups, respectively. During volitional hyperpnoea, [Formula: see text] increased from 79.9 ± 9.5 and 76.3 ± 15.4 L min(-1) at 20 min to 137.8 ± 15.2 and 135.0 ± 19.7 L min(-1) in IMT and control groups, respectively; [lac(-)](B) concurrently increased by 1.0 ± 0.6 (+27%) and 0.9 ± 0.7 mmol L(-1) (+25%), respectively (P < 0.05). Following the intervention, maximal inspiratory mouth pressure increased 19% in the IMT group only (P < 0.01). Following IMT only, the increase in [lac(-)](B) during volitional hyperpnoea was abolished (P < 0.05). In addition, the blood lactate (-28%) and phase II oxygen uptake (-31%) kinetics time constants at the onset of exercise and the MLSS [lac(-)](B) (-15%) were reduced (P < 0.05). We attribute these changes to an IMT-mediated increase in the oxidative and/or lactate transport capacity of the inspiratory muscles.

Evaluation of a respiratory muscle biofeedback procedure-effects on heart rate and dyspnea.

Patients with respiratory diseases or anxiety frequently complain about dyspnea, which may be partly related to chronic tension of respiratory muscles and/or dynamic hyperinflation. In two experiments we tested a biofeedback technique that recorded electromyographic (EMG) activity from a bipolar surface electrode placement over the right external intercostal muscles with visual signal feedback. Healthy participants were tested in their ability to alter the signal. Heart rate was measured continuously throughout training trials. In the second experiment, dyspnea was rated on a modified Borg scale after each trial. Participants were able to increase their EMG activity considerably while heart rate and dyspnea increased substantially. Changes in EMG activity were achieved mostly by manipulating accessory muscle tension and/or altering breathing pattern. Thus, the technique is capable of altering respiratory muscle tension and associated dyspnea. Further studies may test the procedure as a relaxation technique in patients with respiratory disease or anxiety.

Anaerobic metabolism of inspiratory muscles in COPD.

OBJECTIVE: The purpose of this study was to determine if the respiratory muscles of patients with COPD could be made to function anaerobically, as evidenced by an increase in arterial blood lactate concentration ([lactate](a)) during specific loading of the inspiratory muscles and, if so, the effect of a programme of high-intensity inspiratory muscle training on this function. METHODS: In seven patients with COPD (FEV(1) = 33 +/- 14% of predicted), measurements of [lactate](a) were made each minute during progressive inspiratory threshold loading to voluntary exhaustion. These tests were performed before and after an 8-week programme of specific high-intensity inspiratory muscle training, combined with general whole-body exercise training. RESULTS: During inspiratory muscle loading small increases in [lactate](a) (0.83 +/- 0.32 mM) were observed in two subjects before training, and in five subjects after training (0.69 +/- 0.57 mM). [Lactate](a) only increased when the inspiratory work rate exceeded 6.9 cm H(2)O L/min per kilogram of body weight, and when baseline maximum inspiratory pressure exceeded 65 cm H(2)O. CONCLUSIONS: The results of this study demonstrated that it is possible for COPD patients to increase inspiratory muscle work rate to a level requiring a major energy contribution from anaerobic glycolytic metabolism. This was only seen when inspiratory muscle strength and endurance were sufficient to allow it. Some patients who failed to demonstrate an increase in [lactate](a) at baseline did so after a programme of high-intensity inspiratory muscle training.

Electrical and mechanical properties and mode of innervation in scorpionfish sound-producing muscle fibres.

To obtain information about the neural mechanism underlying sound production in teleost fish, we studied the electrical and mechanical properties and mode of innervation in the swimbladder muscle (SBM) fibres of scorpionfish Sebastiscus marmoratus. Action potentials of the SBM fibres in response to direct electrical stimulation neither exhibited overshoot nor propagated along the fibre. Stimulation of the motor nerve, however, uniformly evoked action potentials along the fibre. When neuromuscular transmission was blocked by curare, motor nerve stimulation uniformly evoked endplate potentials along the fibre. These results indicate that action potentials propagate along the nerve branches but not along the SBM fibre membrane. In accordance with the above results, histochemical studies showed that motor nerve branches run along the SBM fibres to form many endplates with cholinesterase activity, indicating multiterminal innervation. The SBM consisted of about 600 fibres, while its motor nerve contained about 100 axons, giving an innervation ratio of about 1:6. Like mammalian fast muscle fibres, the SBM fibres exhibited a low succinic dehydrogenase activity and a high ATPase activity. These results are discussed in connection with the function of the SBM fibres in producing sound.

Effects of nandrolone decanoate on respiratory and peripheral muscles in male and female rats.

Thirty male and 18 female adult rats received weekly an intramuscular injection of either saline (control; C), 1.5 mg/kg (low-dose; LD) nandrolone decanoate or 7.5 mg/kg (high-dose; HD) nandrolone decanoate during 5 wk. Compared with respective C, growth rate was stunted in male HD rats from 2 wk of treatment on, whereas it was enhanced in female LD and HD rats after 1 wk. Mass of all muscles studied varied proportionally to body weight, except for the gastrocnemius (males: 0.49 +/- 0.04 vs. C: 0.52 +/- 0.03%, not significant; females: 0.17 +/- 0.01 vs. C: 0.15 +/- 0.01%, P < 0.05). In vitro contractile and fatigue properties of the diaphragm remained unchanged, except for a decrease in twitch kinetics (time to peak tension: C, 21 +/- 2; LD, 19 +/- 1; HD, 19 +/- 2 ms, P < 0.05; half-relaxation time: C, 26 +/- 5, LD, 25 +/- 5, HD, 23 +/- 3 ms, P < 0.01). Histochemistry of the diaphragm and the gastrocnemius revealed a significant increase in type IIx/b dimensions. In the gastrocnemius, type I fiber dimensions also increased. A pair-fed study, including another 24 female rats, showed that the changes in oral food intake only partly accounted for the observed anabolic effects.

Probable mechanism of hypoglycemic activity of bassic acid, a natural product isolated from Bumelia sartorum.

Bassic acid, an unsaturated triterpene acid isolated from an ethanol extract of Bumelia sartorum rootbark, elicited significant hypoglycemic activity in alloxan-diabetic rats and altered the pattern of glucose tolerance in these animals. In addition, bassic acid treatment increased significantly the glucose uptake process and glycogen synthesis in isolated rat diaphragm. Bassic acid treatment increased plasma insulin levels significantly in alloxan-diabetic rats. It is suggested that the hypoglycemia activity of bassic acid may be mediated through enhanced secretion of insulin from the pancreatic beta-cells.

Target-flow inspiratory muscle training: breathing patterns and metabolic costs.

In target-flow inspiratory muscle training (TF-IMT), the generated inspiratory mouth pressure and the duration of the inspiration and expiration are standardized to given an adequate training stimulus to the inspiratory muscles. The acute effects of TF-IMT on the efficiency of breathing were studied in a group of 12 COPD patients with a ventilatory limitation of their exercise capacity (mean age 58, mean FEV1 46.2% of predicted) and in 15 normal subjects (mean age 30). Also, the effect of a 10 week period of TF-IMT on the maximal inspiratory mouth pressure (PImax) in the COPD patients was measured. After an unloaded baseline period, the subjects started to inspire through a target-flow device during 15 min, followed by a recovery phase of 5 min. During TF-IMT minute ventilation (VE) decreased only in the COPD group. The ventilatory equivalent for O2 (VE/VO2) and the dead space to tidal volume ratio (VD/VT) decreased in both groups. During recovery, VE, VE/VO2 and VD/VT remained below baseline values in the COPD group, but not in the control group. PCO2 and lactate concentrations did not change during TF-IMT. After the 10 week training period, PImax [means) (SD] increased from 5.7(2.2) to 8.2(2.7) kPa (p less than 0.05). The results indicate that with standardized TF-IMT, the inspiratory muscles can be trained effectively in COPD patients with a ventilatory limitation. The persistence of the decrease in VE, VE/VO2 and VD/VT after a training session may be an additional beneficial effect of TF-IMT.

Studies on contractures induced in mouse diaphragm by caffeine and cupric and selenite ions.

Mouse diaphragm contractures induced by Cu2+, caffeine and selenite were studied comparatively. Both Cu2+- and caffeine-contractures were produced rapidly and relaxed spontaneously; the selenite-contracture occurred after a latent period of about 45 min and lasted for more than 3 hr. All contractures were myogenic, since neither d-tubocurarine nor tetrodotoxin prevented them. The susceptibility of these contractures to the depletion and replenishment of Ca2+ differed: the Cu2+-contracture increased proportionally with rising extracellular Ca2+ concentrations ranging from 2.5 to 12.5 mM and were abolished by 5 mM EGTA. Caffeine- and selenite-contractures were not affected by changes in extracellular Ca2+ concentration. The caffeine-contracture was abolished by EGTA in high concentration (30 mM) and the selenite-contracture was inhibited by 50 mM EGTA. After removal of Ca2+ with 5 mM EGTA, followed by replacement with 2.5 mM Ca2+ for 1 min, the Cu2(+)-contracture was fully restored. Caffeine- and selenite-contractures were restored only after a longer period (10-20 min) of re-exposure to Ca2+. These findings suggest that the Cu2(+)-contracture is dependent on external Ca2+ and probably caused by an increasing Ca2+ entry through sarcolemma. Caffeine- and selenite-contractures apparently result from internal Ca2+ release by sarcoplasmic reticulum. Substitution of either Sr2+ or Co2+ for Ca2+ fully supports the Cu2(+)-contracture. 45Ca2+ uptake and calcium content of the diaphragm were markedly increased by Cu2+ but not by selenite. Furthermore, the Cu2(+)-contracture was inhibited by exposing the outer membrane to trypsin, phospholipase C or saponin. The selenite-contracture was inhibited only by trypsin. The caffeine-contracture was unaffected by these treatments. These results support the notion that the Cu2(+)-contracture is induced by an increased entry of Ca2+ through the outer membrane. Cu2(+)-, caffeine- and selenite-contractures were respectively abolished, potentiated and unaffected by chronic denervation of the diaphragm. This and the other findings provide evidence that Cu2(+)-, caffeine- and selenite-contractures are induced in mouse diaphragm muscle via different sites of action.