Restoration of nerve agent inhibited muscle force production in human intercostal muscle strips with HI 6.

An important factor for successful therapy of poisoning with organophosphorus compounds (OP) is the rapid restoration of blocked respiratory muscle function. To achieve this goal, oximes are administered for reactivation of inhibited acetylcholinesterase (AChE). Unfortunately, clinically used oximes, e.g. obidoxime and pralidoxime, are of limited effectiveness in poisoning with different OP nerve agents requiring the search for alternative oximes, e.g. HI 6. In view of substantial species differences regarding reactivation properties of oximes, the effect of HI 6 was investigated with sarin, tabun and soman exposed human intercostal muscle. Muscle force production by indirect field stimulation and the activity of the human muscle AChE was assessed. 30 µM HI 6 resulted in an almost complete recovery of sarin blocked muscle force and in an increase of completely inhibited muscle AChE activity to approx. 30% of control. In soman or tabun exposed human intercostal muscle HI 6 (50 and 100 µM) had no effect on blocked muscle force or on inhibited human muscle AChE activity. In addition, HI 6 up to 1000 µM had no effect on soman blocked muscle force indicating that this oxime has no direct, pharmacological effect in human tissue. These results emphasize that sufficient reactivation of AChE is necessary for a beneficial therapeutic effect on nerve agent blocked neuromuscular transmission.

Pre- and post-treatment effect of physostigmine on soman-inhibited human erythrocyte and muscle acetylcholinesterase in vitro.

Standard treatment of organophosphorus (OP) poisoning includes administration of an antimuscarinic (e.g., atropine) and of an oxime-based reactivator. However, successful oxime treatment in soman poisoning is limited due to rapid aging of phosphylated acetylcholinesterase (AChE). Hence, the inability of standard treatment procedures to counteract the effects of soman poisoning resulted in the search for alternative strategies. Recently, results of an in vivo guinea pig study indicated a therapeutic effect of physostigmine given after soman. The present study was performed to investigate a possible pre- and post-treatment effect of physostigmine on soman-inhibited human AChE given at different time intervals before or after perfusion with soman by using a well-established dynamically working in vitro model for real-time analysis of erythrocyte and muscle AChE. The major findings were that prophylactic physostigmine prevented complete inhibition of AChE by soman and resulted in partial spontaneous recovery of the enzyme by de-carbamylation. Physostigmine given as post-treatment resulted in a time-dependent reduction of the protection from soman inhibition and recovery of AChE. Hence, these date indicate that physostigmine given after soman does not protect AChE from irreversible inhibition by the OP and that the observed therapeutic effect of physostigmine in nerve agent poisoning in vivo is probably due to other factors.

Protection of human muscle acetylcholinesterase from soman by pyridostigmine bromide.

INTRODUCTION: Pretreatment with pyridostigmine bromide (PB) of human intercostal muscle fibers exposed to the irreversible acetylcholinesterase (AChE) inhibitor soman was investigated. METHODS: Muscles were pretreated with 3 × 10(-6) M PB or saline for 20 minutes, then exposed to 10(-7) M soman for 10 minutes. RESULTS: AChE of muscles treated with soman alone was inhibited >95%. In contrast, PB pretreatment of soman-exposed bundles protected 20% of AChE activity. AChE of bundles exposed to PB alone recovered after 4 hours, but bundles exposed to both PB and soman did not. Soman-induced reduction of resting membrane potentials and increment of amplitudes and decay times of miniature endplate potentials (MEPPs) were partially corrected by PB pretreatment. CONCLUSIONS: In vitro pretreatment of human muscles with PB protected up to 20% of muscle AChE and ameliorated some deleterious effects on endplate physiology induced by soman.

Different sensitivity of miniature endplate currents in rat external and internal intercostal muscles to the acetylcholinesterase inhibitor C-547 as compared with diaphragm and extensor digitorum longus.

Derivative of 6-methyluracil, selective cholinesterase inhibitor C-547 potentiates miniature endplate currents (MEPCs) in rat external intercostal muscles (external ICM) more effectively than in internal intercostal muscles (internal ICM). Effect of the C-547 on intercostal muscles was compared with those on extensor digitorum longus (EDL) and diaphragm muscles. Half-effective concentrations for tau of MEPC decay arranged in increasing order were as follows: EDL, locomotor muscle, most sensitive = 1.3 nM, external ICM, inspiration muscle = 6.8 nM, diaphragm, main inspiration muscle = 28 nM, internal ICM, expiration muscle = 71 nM. External ICM might therefore be inhibited, similarly as the limb muscles, by nanomolar concentrations of the drug and do not participate in inspiration in the presence of the C-547. Moreover, internal ICM inhibition can hinder the expiration during exercise-induced fast breathing of C-547- treated experimental animals.

Correlation of therapeutic effect of obidoxime and dosing time in the acute intoxication by chlorfenvinphos in rats.

The ability of obidoxime with atropine and diazepam mixture to reactivate acetylcholinesterase inhibited by the organophosphorus compound chlorfenvinphos was compared in the central nervous system and peripheral tissues of rats. The animals were intoxicated with chlorfenvinphos (6 mg/kg, p.o.) and treated immediately, 24 and 48 hrs later with obidoxime (50 mg/kg, i.p.), atropine (10 mg/kg, i.p.), and diazepam (10 mg/kg, i.p.) in a single dose, or in various combinations (with 2-3 drugs) simultaneously. Total tissue acetylcholinesterase activities were monitored at 2, 72, and 168 hrs after intoxication. Enzyme activity was determined using Ellman's colorimetric method. The results of the present study show that obidoxime administered separately and jointly with atropine and diazepam 24 hrs after intoxication was effective on reactivation of chlorfenvinphos-inhibited acetylcholinesterase in the central nervous system and in the peripheral tissues. However, the application of obidoxime alone or in combination with atropine and diazepam 48 hrs after chlorfenvinphos intoxication caused an increased unfavourable effect in rats. The results obtained also indicate an unfavourable interaction of obidoxime with diazepam in the course of chlorfenvinphos poisoning, when antidotes were administered immediately, 24 and 48 hrs after intoxication.

PECAM-1 interacts with nitric oxide synthase in human endothelial cells: implication for flow-induced nitric oxide synthase activation.

OBJECTIVE: We have previously shown that fluid shear stress (FSS) triggers endothelial nitric oxide synthase (eNOS) activity in endothelial cells and that the mechanotransduction mechanisms responsible for activation discriminate between rapid changes in FSS and FSS per se. We hypothesized that the particular sublocalization of eNOS at the cell-cell junction would render it responsive to activation by FSS temporal gradients. METHODS AND RESULTS: In human umbilical vein endothelial cells (HUVECs), immunofluorescence revealed strong eNOS membrane staining at the cell-cell junction colocalizing with platelet/endothelial cell adhesion molecule-1 (PECAM-1). In PECAM-1-/- mouse aorta, eNOS junctional localization seen in the wild type was absent. Similarly, junctional staining was lost in wild-type aorta near intercostal artery branches. eNOS/PECAM-1 association in HUVECs was confirmed by coimmunoprecipitation. When HUVECs were subjected to a 0.5s impulse of 12 dynes/cm2, a transient disruption of the eNOS/PECAM-1 complex was observed, accompanied by an increase in eNOS activity (cGMP production). Ramped flow did not trigger complex dissociation or an increase in cGMP production. In a cell-free system, a direct inhibition of eNOS activity by PECAM-1 is shown. CONCLUSIONS: These results suggest that eNOS is complexed with PECAM-1 at the cell-cell junction and is likely involved in the modulation of eNOS activity by FSS temporal gradients but not by FSS itself.

Induction of diaphragmatic nitric oxide synthase after endotoxin administration in rats: role on diaphragmatic contractile dysfunction.

Nitric oxide (NO), a free radical that is negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by an NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to examine iNOS induction in the rat diaphragm after Escherichia Coli LPS inoculation (1.6 mg/kg i.p.), and its involvement in diaphragmatic contractile dysfunction. Inducible NOS protein and activity could be detected in the diaphragm as early as 6 h after LPS inoculation. 6 and 12 h after LPS, iNOS was expressed in inflammatory cells infiltrating the perivascular spaces of the diaphragm, whereas 12 and 24 h after LPS it was expressed in skeletal muscle fibers. Inducible NOS was also expressed in the left ventricular myocardium, whereas no expression was observed in the abdominal, intercostal, and peripheral skeletal muscles. Diaphragmatic force was significantly decreased 12 and 24 h after LPS. This decrease was prevented by inhibition of iNOS induction by dexamethasone or by inhibition of iNOS activity by N(G)-methyl-L-arginine. We conclude that iNOS was induced in the diaphragm after E. Coli LPS inoculation in rats, being involved in the decreased muscular force.

Accumulation in fetal muscle and localization to the neuromuscular junction of cAMP-dependent protein kinase A regulatory and catalytic subunits RI alpha and C alpha.

Using probes specific for cAMP-dependent protein kinase, we have analyzed by in situ hybridization the patterns of expression of regulatory and catalytic subunits in mouse embryos and in adult muscle. RI alpha transcripts are distributed in muscle fibers exactly as acetylcholinesterase, showing that this RNA is localized at the neuromuscular junction. The transcript levels increase upon denervation of the muscle, but the RNA remains localized, indicating a regulation pattern similar to that of the epsilon subunit of nicotinic acetylcholine receptor. RI alpha transcripts have accumulated in the muscle by day 12 of mouse embryogenesis, and localization is established by day 14, at about the time of formation of junctions. This localization is maintained throughout development and in the adult. Immunocytochemical analysis has demonstrated that RI alpha protein is also localized. In addition, RI alpha recruits C alpha protein to the junction, providing at this site the potential for local responsiveness to cAMP. PKA could be implicated in the establishment and/or maintenance of the unique pattern of gene expression occurring at the junction, or in the modulation of synaptic activity via protein phosphorylation. Embryonic skeletal muscle shows a high level of C alpha transcripts and protein throughout the fiber; the transcripts are already present by day 12 of embryogenesis, and their elevated level is maintained only through fetal life. In the adult, the C alpha hybridization signal of muscle is weak and homogeneous.

Differences in diaphragm fiber types in SIDS infants.

The diaphragm is a vital respiratory muscle in the sleeping infant. Any changes in diaphragm fiber type number or size could represent either a primary developmental delay or a secondary reaction to increased workload, and could give a clue as to the pathogenesis of sudden infant death syndrome (SIDS). We therefore quantitated by point counting on ATPase histochemistry the numbers and areas of type 1 and 2 fibers in the diaphragm, external intercostal and psoas muscles of 37 SIDS and 20 control infants. The amount of slow, fast and fetal myosin in the diaphragm and psoas muscles was measured by electrophoresis to check the ATPase quantitation. There were fewer type 1 fibers in SIDS (median 30.0%) compared with control (median 40.0%) infants (p < 0.02), whereas the diameter of type 1 fibers in SIDS (median 33.9 microns) was larger than in control (median 30.3 microns) infants (p < 0.007). The total cross-sectional area occupied by type 1 and 2 fibers was similar in both groups. No changes were found in the external intercostal or psoas. The amount of slow and fast myosins correlated well with type 1 and type 2 fibers, respectively. The finding of fewer type 1 (fatigue-resistant) fibers of large diameter in SIDS diaphragms suggests that differences in muscle fiber types may predispose these infants to diaphragm fatigue and respiratory failure.

Effect of spaceflight on oxidative and antioxidant enzyme activity in rat diaphragm and intercostal muscles.

There are limited data regarding changes in oxidative and antioxidant enzymes induced by simulated or actual weightlessness, and any additional information would provide insight into potential mechanisms involving other changes observed in muscles from animals previously flown in space. Thus, the NASA Biospecimen Sharing Program was an opportunity to collect valuable information. Oxidative and antioxidant enzyme levels, as well as lipid perioxidation, were measured in respiratory muscles from rats flown on board Space Shuttle mission STS-54. The results indicated that there was an increasing trend in citrate synthase activity in the flight diaphragm when compared to ground based controls, and there were no significant changes observed in the intercostal muscles for any of the parameters. However, lipid peroxidation was significantly (p<0.05) decreased in the flight diaphragm. These results indicate that 6 day exposure to microgravity may have a different effect on oxidative and antioxidant activity in rat respiratory muscles when compared to data from previous 14 day hindlimb suspension studies.

Metabolic characteristics of primary inspiratory and expiratory muscles in the dog.

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.

Structure of parasternal intercostal muscles in the adult hamster: topographic effects.

The parasternal intercostals are primary inspiratory muscles like the costal and crural diaphragm. However, the structure of the rib cage and its impedance to inspiration and expiration varies regionally. We questioned whether topographic differences in rib cage structure and impedance were associated with regional differences in parasternal intercostal muscle structure. Therefore, we examined the size and percentage of histochemically stained fibers in the parasternal intercostal muscles in the first, second, third, fourth, and sixth interspaces in the hamster. We observed a rostrocaudal gradient in the percentage and size of slow oxidative (SO), fast oxidative-glycolytic, and fast glycolytic (FG) fibers in the parasternal intercostal muscles. In particular, the percentage of SO decreased while the percentage of FG increased in a rostrocaudal direction in the first through sixth interspaces. In addition, the size of SO and FG fibers increased from the first to sixth interspace. Furthermore, changes in the size and percent of the three fiber types produced, in a rostrocaudal direction, significant reductions in the relative mass of the parasternal intercostal muscle made up of SO fibers and increases in the mass of fast fibers. We speculate that topographical differences in the size and percentage of fast and slow twitch fibers in the parasternal intercostal are likely to alter force-generating capacity of the parasternal muscles in a rostrocaudal direction and likely reflect regional differences in muscle load and/or activity.

Rostrocaudal variation of fiber type composition in rat intercostal muscles.

We used the histochemical stain for ATPase to compare the fiber-type composition of rat internal and external intercostal muscles from thoracic (T) segments 2-5, 8, and 11. At each level, type II fibers were more numerous than type I fibers, type II B fibers were more numerous than II A fibers, and type I fibers were more numerous in external than in internal intercostals. However, fiber type composition varied from segment to segment. For example, the proportion of type II A fibers increased in a rostrocaudal gradient in internal but not external intercostals, and type I fibers were more prevalent at rostral and caudal than at intermediate levels in both internal and external intercostals. These results provide a basis for interpreting previous physiological and molecular studies which have compared intercostal muscles from different segmental levels.

Responses of intercostal muscle biopsies from normal subjects and patients with myasthenia gravis.

In order to evaluate the mechanisms of weakness in muscles of patients with myasthenia gravis (MG), intercostal muscle biopsies were obtained from 9 normal subjects and 6 MG patients, and the compound muscle action potential (AP) and tension responses to nerve and muscle stimulation, and contracture responses on exposure to caffeine, were monitored in vitro. In normal muscle, on stimulation of the nerve or muscle at 30 to 100 Hz, the AP responses showed decrement in amplitude, one-third of which was attributable to failure of neuromuscular transmission and two-thirds to failure of muscle membrane excitation. On stimulation at 1 to 5 Hz, the AP responses showed very little decrement, while the contractile responses showed significant fade in tension, due to failure of E-C coupling or contractility. In muscle from patients with generalized MG, stimulation of the nerve at all frequencies (1 to 100 Hz) caused much greater decrement in APs and fade in tension responses than in normal muscle, due mainly to failure of neuromuscular transmission. However, at 100 Hz, 40% of the decrement in APs was due to failure of muscle membrane excitation, and at 1 to 5 Hz, 40% of the fade in tension was due to failure of E-C coupling or contractility, as in normal muscle. On direct stimulation the contraction and half-relaxation times were slower and the tetanic tension was smaller than in normal muscle, especially in the MG patient with thymoma. Caffeine-induced contractures were smaller in MG muscle than in normal muscle. These results indicate that while the weakness of MG muscle is due mainly to failure of neuromuscular transmission, it is also partly due to reduced E-C coupling or contractility.

Differential response of enzyme activities in rat diaphragm and intercostal muscles to exercise training.

To determine whether respiratory muscles undergo alterations in enzyme activities of energy metabolism as a result of increased mechanical activity, adult male Wistar rats were subjected to a prolonged endurance training program. Analysis off maximal enzyme activity patterns in the diaphragm following 15 weeks of extreme training (final running duration: 210 min per day, 27 m.min-1 at 15 degrees grade, indicated significant reductions in the marker enzymes of the citric acid cycle (citrate synthase), glycolysis (pyruvate kinase, PK; lactate dehydrogenase, LDH), ketone body utilization (3-keto acid: CoA transferase) and transamination (glutamate pyruvate transaminase, GPT). No changes were found for the enzymes of glycogenolysis (phosphorylase, PHOSPH), glycolysis (glyceraldehyde phosphate dehydrogenase, GAPDH), glucose phosphorylation (hexokinase, HK) and beta-oxidation (3-hydroxyacyl: CoA dehydrogenase, HAD) following training. In contrast, in the external intercostal muscle, increases in the range of 57-77% were noted for the enzymes CS and HAD, whereas in the internal intercostal muscles no training induced alteration was evident for these enzymes. For both the intercostal muscles, a consistent trend was noted towards a reduction in all of the glycolytic enzymes investigated, however, significantly lower values were recorded for only PK and LDH in the internal intercostals. GPT was increased in the internal intercostal muscles. These findings indicate that the response pattern observed in the enzyme activities studied following training are to some degree specific to the respiratory muscle investigated.

Metabolic enzymatic activities in the intercostal and serratus muscles and in the latissimus dorsi of middle-aged normal men and patients with moderate obstructive pulmonary disease.

The glycolytic and oxidative enzyme activities (lactate dehydrogenase (LDH), hexokinase (HK), citrate synthase (CS) and 3-hydroxyacyl-CoA-dehydrogenase (HAD] were measured in the fifth internal and external intercostal muscles, in the vertical and horizontal parts of the serratus, an accessory inspiratory muscle, and in a non-respiratory muscle, the latissimus dorsi (LD) of twenty middle-aged men: nine subjects with normal lung function and eleven patients with moderate chronic obstructive pulmonary disease (COPD). In the normal subjects the enzyme activities of the respiratory muscles were similar to those of the LD, and there were no differences between the internal and the external intercostal muscles. In the COPD patients the metabolic activities of HK, CS and HAD were higher in both intercostals than in LD. Furthermore, there was a significant increase in these enzymatic activities as compared to the intercostals of the normal subjects. These data support the hypothesis that the internal and external intercostal muscles play a more important role in COPD patients than in normal subjects. They are consistent with the hypothesis that COPD has an endurance training effect on both intercostal muscles which could compensate for diaphragmatic disuse.

Fibre types, capillary supply and enzyme activities in human intercostal muscles.

The relative occurrence of slow twitch (ST) and fast twitch (FTa and FTb) fibres, fibre size, capillary supply, and glycolytic and oxidative enzyme activities in external (EXT) and internal (INT) intercostal muscles was determined on biopsies obtained during thoracotomy from the mid-axillary line in six patients with normal lung function. EXT and INT showed a similar occurrence of ST fibres. Both FTa and FTb fibres were seen in EXT while only FTa fibres were seen in INT. The size of the three fibre types in EXT was similar. In INT the fibre size was larger for FTa than for ST (P less than 0.05). INT appeared to have a larger number of capillaries per fibre than EXT (P less than 0.05). A positive correlation was demonstrated between variables measured during spirometry and fibre size in INT only. No difference in glycolytic and oxidative enzyme activities between EXT and INT was observed. The histochemical characteristics suggest that, in the mid-axillary line, INT are more used than EXT, though this conclusion did not appear to be confirmed by the enzyme activities determined.

Histochemical and biochemical correlates of ventilatory muscle fatigue in emphysematous hamsters.

Histochemical and biochemical characteristics of the ventilatory muscles were evaluated in control and elastase-induced emphysematous hamsters. The emphysematous group was divided into sedentary and endurance-trained groups. Endurance training consisted of treadmill running, 1 h a day, 7 d a week. The experimental period lasted 24 wk. Histochemically, the diaphragm from the sedentary emphysematous hamsters revealed a selective fast fiber atrophy which was prevented by endurance training. Training also led to a hypertrophy of the slow, high oxidative fibers. The external intercostals from both emphysematous groups revealed an increased proportion of fast oxidative fibers at the expense of a decreased number of fast glycolytic fibers. However, the fast fibers in both emphysematous groups were significantly atrophied as compared with controls. The internal intercostals revealed no adaptive changes in either size or proportion distribution of the various fiber types. Biochemically, the diaphragm of the emphysematous animals had a significantly improved oxidative potential as measured by citrate synthase, and a reduced glycolytic capacity as indicated by phosphofructokinase activity, compared with controls. The magnitudes of the biochemical changes were similar in both emphysematous groups and were consistent for diaphragmatic samples taken from the costal and crural segments. The combined internal and external intercostals also underwent significant biochemical increases in their oxidative capacity. In addition, training of the emphysematous group led to an increased glycolytic potential of the intercostals.

Development of histochemical and functional properties of baboon respiratory muscles.

We assessed morphological, histochemical, and physiological characteristics of respiratory muscles of a non-human primate, Papio cynocephalus, from midgestation through adult life. Samples were taken of diaphragm muscles for histochemical analysis, electron microscopy, and assessment of contractile properties and fatigability. Histochemical analyses were also performed on samples of intercostal muscles. Initially, developing fibers are type IIc but differentiate into types I and IIa fibers by term. We observed no IIb fibers in respiratory muscles of premature baboons. Beginning late in gestation, muscle fibers grew rapidly. After term, IIb fibers were found, and fiber size ranked by increasing mean fiber area became types I, IIa, and IIb. After term, we rarely observed type IIc fibers. In electron micrographs we observed large numbers of interfibrillar mitochondria in all muscle fibers of premature baboons but not in all IIb fibers of adults. Histochemical observations were supported by contractile properties. Muscles of premature baboons had significantly longer contraction and relaxation times than adult muscles. Muscles from premature baboons were more resistant to fatigue than those of adult baboons. We conclude that the fibers of respiratory muscles are high in oxidative capacity and are resistant to fatigue during gestation. Fatigue of the respiratory muscle fibers secondary to low oxidative capacity is not a likely cause of respiratory distress in premature baboons.

Changes in histochemical profile of rat respiratory muscles in hypo- and hyperthyroidism.

Rat respiratory muscles underwent considerable changes in histochemical fibre type profile in response to hypo- and hyperthyroidism. Hypothyroidism increased the proportion of type 1 slow oxidative fibres in diaphragm and to a lesser extent in intercostal muscles. Hyperthyroidism resulted in a decreased proportion of type 1 fibres in both diaphragm and intercostals. These changes were broadly comparable to those reported previously in rat limb muscles. In normal rat respiratory muscles, the type 1 fibres were characterized by very high levels of beta-hydroxybutyrate dehydrogenase which was thought to contribute to the fatigue-resistance of these muscles. The type 2B fast glycolytic fibres, and to a lesser extent type 2A fast oxidative fibres, contained high levels of mitochondrial alpha-glycerophosphate dehydrogenase, an enzyme known to be specifically affected in dysthyroid states. The implications of the observed changes in fibre type profile with respect to the oxidative metabolism of rat respiratory muscles are discussed.