In this study, whole cell patch clamp recording technique was employed to investigate the effect of Shenmai Injection (SMI) on L-type calcium current of diaphragmatic muscle in rats. The result showed that when the diaphragmatic muscle cell was held at -80 mV and depolarized to +60 mV, 10 microl/ml, 50 microl/ml and 100 microl/ml SMI enhanced the inner peak L-type calcium current from -(6.8 +/- 0.7) pA/pF (n=7) to -(7.3 +/- 0.8) pA/pF (P>0.05, n=7), -(8.6 +/- 1.0) pA/pF (P<0.05, n=7) and -(9.4 +/- 1.2) pA/pF (P<0.05, n=7), respectively, The rates of L-type calcium current were increased by (7.34 +/- 2.37)%, (25.72 +/- 5.94)%, and (38.16 +/- 7.33)%, respectively. However, it had no significant effect on maximal activation potential and reversal potential. Our results suggested that SMI could activate the calcium channel of the diaphragmatic fibers of the rats, increase the influx of Ca2+, and enhance the contractility of diaphragmatic muscles.
Calcium/metabolism ; Calcium Channels, L-Type/*drug effects ; Diaphragm/drug effects ; Diaphragm/*metabolism ; Drug Combinations ; Drugs, Chinese Herbal ; Patch-Clamp Techniques ; Plant Extracts/*pharmacology ; Rats, Wistar

BACKGROUND: Neostigmine, a cholinesterase inhibitor, is known to reverse the neuromuscular blocking action induced by nondepolarizing muscle relaxants at the end of general anesthesia. Some authors, however, reported that neostigmine has the properties of a neuromuscular block in skeletal muscles while others reported that neostigmine caused the smooth muscles such as the diaphragm to relax rather than to contract. The purpose of this study was to evaluate the effect of neostigmine at different doses on the tracheal smooth muscle in rabbits. METHODS: Isolated tracheal ring preparation in rabbits was used. Groups were divided into 7 groups; acetylcholine group (acetylcholine cumulative administered at doses of 10 8, 10 7, 10 6, 10 5, 10 4 and 10 3 M), neostigmine group (neostigmine cumulative administered at doses of 10 8, 10 7, 10 6, 10 5, 10 4 and 10 3 M), acetylcholine 10 6 M + neostigmine group (acetylcholine 10 6 M prior to neostigmine administered at doses of 10 8, 10 7, 10 6, 10 5, 10 4 and 10 3 M), acetylcholine 10 4 M + neostigmine group (acetylcholine 10 4 M prior to neostigmine administered at doses of 10 8, 10 7, 10 6, 10 5, 10 4 and 10 3 M), neostigmine 10 5, 10 4 and 10 3 M groups (neostigmine administered at doses of 10 5, 10 4 and 10 3 M). Smooth muscle contraction was evaluated in isometric tension per gram of tissue. RESULTS: In the acetylcholine group, the contractions increased as the dosage increased (10 8 10 3 M). In the neostigmine group, the contractions increased as the dosage increased (10 8 10 4 M), but at 10 3 M of neostigmine, contractions suddenly decreased. In addition when acetylcholine 10 6 M was given as a pretreatment, there was a sudden decrease in muscle contractions induced by neostigmine at 10 3 M. Also the contractions induced by 10 3 M neostigmine were less than that of 10 4 and 10 5 M. CONCLUSIONS: We concluded that neostigmine caused smooth muscle contraction at low concentrations by blocking acetylcholine metabolism, but at high concentrations, smooth muscle contractions were decreased and this might be due to direct action at the acetylcholine receptor.
Acetylcholine ; Anesthesia, General ; Cholinesterases ; Diaphragm ; Metabolism ; Muscle Contraction ; Muscle, Skeletal ; Muscle, Smooth* ; Neostigmine* ; Neuromuscular Blockade ; Rabbits*

Animals ; Calcium ; metabolism ; Diaphragm ; physiology ; ultrastructure ; Male ; Muscle Contraction ; Rats ; Rats, Sprague-Dawley ; Respiration, Artificial ; Sarcoplasmic Reticulum ; metabolism

BACKGROUND: Prolongation of the neuromuscular block of mivacurium can occur when there is a genetic deficiency of the enzyme or in the presence of anticholinesterase (AntiChE) which inhibit the activity of the enzyme. The aim of this study was to determine the efficacies of cholinesterase, AntiChE (neostigmine, pyridostigmine), and 4-aminopyridine in reversing mivacurium block, using the phrenic nerve-diaphragm preparation of a rat. METHODS: Forty-eight Sprague-Dawley rats (200~300 g) were anesthetized with peritoneal injection of 2.5% thiopental 5~10 ml. After a stable twitch and train-of-four responses were established for at least 30 minutes in each preparation, incremental dose of mivacurium was added to obtain 90~95% inhibition of control twitch height. The effects of 0.1 and 1.0 u/ml of horse pseudocholinesterase (pChE, Sigma), 0.1 and 1.0 g/ml of neostigmine, 0.2 and 2.0 g/ml of pyridostigmine, and 1.6, 16 g/ml of 4-aminopyridine (P.B.I) on reversal of mivacurium block were tested. The effects of 0.1 g/ml of neostigmine, or 0.2 g/ml of pyridostigmine with and without 0.1 or 1.0 u/ml of pChE following mivacurium were also tested. RESULTS: In reversing mivacurium block, single twitch and TOF ratios were recovered completely with pChE but not with antiChEs or 4-aminopyridine (p<0.05). Second set of experiments showed that antiChE mixed with pChE had a tendency to recover faster (p<0.05). The comparable recovery patterns of pChE 0.1u/ml alone and neostigmine 0.1 g/ml with pChE 0.1u/ml in our study, indicated that neostigmine would prolong the mivacurium block especially in the presence of hereditary or acquired defects of pChE activity. CONCLUSION: The authors conclude that pChE 1.0 u/ml with and without antiChE were equally effective in reversing neuromuscular block of mivacurium. If these results can be extrapolated to human, it is unlikely that mivacurium block is potentiated by antiChE that may slow its metabolism.
4-Aminopyridine* ; Animals ; Cholinesterases ; Diaphragm* ; Horses ; Humans ; Metabolism ; Neostigmine ; Neuromuscular Blockade* ; Pseudocholinesterase* ; Pyridostigmine Bromide ; Rats* ; Rats, Sprague-Dawley ; Thiopental

Background: Neural respiratory drive (NRD) using diaphragm electromyography through an invasive transesophageal multi-electrode catheter can be used as a feasible clinical physiological parameter in patients with chronic obstructive pulmonary disease (COPD) to provide useful information on the treatment response. However, it remains unknown whether the surface diaphragm electromyogram (EMGdi) could be used to identify the deterioration of clinical symptoms and to predict the necessity of hospitalization in acute exacerbation of COPD (AECOPD) patients. Methods: COPD patients visiting the outpatient department due to acute exacerbation were enrolled in this study. All patients who were subjected to EMGdi and classical parameters such as spirometry parameters, arterial blood gas analysis, COPD assessment test (CAT) score, and the modified early warning score (MEWS) in outpatient department, would be treated effectively in the outpatient or inpatient settings according to the Global Initiative for Chronic Obstructive Lung Disease guideline. When the acute exacerbation of the patients was managed, all the examination above would be repeated. Results: We compared the relationships of admission-to-discharge changes (Δ) in the normalized value of the EMGdi, including the change of the percentage of maximal EMGdi (ΔEMGdi%max) and the change of the ratio of minute ventilation to the percentage of maximal EMGdi (ΔVE/EMGdi%max) with the changes of classical parameters. There was a significant positive association between ΔEMGdi%max and ΔCAT, ΔPaCO, and ΔpH. The change (Δ) of EMGdi%max was negatively correlated with ΔPaO/FiOin the course of the treatment of AECOPD. Compared with the classical parameters including forced expiratory volume in 1 s, MEWS, PaO/FiO, the EMGdi%max (odds ratio 1.143, 95% confidence interval 1.004-1.300) has a higher sensitivity when detecting the early exacerbation and enables to predict the admission of hospital in the whole cohort. Conclusions The changes of surface EMGdi parameters had a direct correlation with classical measures in the whole cohort of AECOPD. The measurement of NRD by surface EMGdi represents a practical physiological biomarker, which may be helpful in detecting patients who should be hospitalized timely.
Diaphragm ; physiopathology ; Electromyography ; methods ; Forced Expiratory Volume ; physiology ; Hospitalization ; Humans ; Pulmonary Disease, Chronic Obstructive ; metabolism ; physiopathology ; Spirometry ; Vital Capacity ; physiology

AIMTo detect effect of the different frequency of chronic electrical stimulation (CES) on myofibrillar isoform, myosin heavy chain (MHC) and metabolic enzyme activities.

METHODSThe histochemical method and SDS-polyacrylamide gel electrophoresis were respectively employed.

RESULTS(1)There were a significant increase in I myo-fibrillar isoform and I MHC isoform and decrease in II B myofibrillar isoform and II B MHC isoforms in the chronic low frequency electrical stimulation (CLFES) 10 Hz and 20 Hz groups, but opposite results were found in the chronic high frequency electrical stimulation (CHFES) 50 Hz and 100 Hz groups. (2) There were a significant increase in the aerobic-oxidative enzyme activities and capacity, and a concomitant significant drop in glycolysis enzyme activities in CLFES groups, but opposite results were found in CHFES 50 Hz and 100 Hz groups.

CONCLUSIONIt was suggested that there was a significant dependent relation between chronic electrical stimulation frequency and myofibrilla isoforms, myosin heavy chain (MHC) and metabolic enzyme activities.

Adaptation, Physiological ; Animals ; Diaphragm ; enzymology ; metabolism ; physiology ; Electric Stimulation ; Muscle Contraction ; Myosin Heavy Chains ; metabolism ; Nonmuscle Myosin Type IIB ; metabolism ; Protein Isoforms ; Rabbits

OBJECTIVE: To explore the role of endoplasmic reticulum ryanodine receptor 1 (RyR1) expression and phosphorylation in sepsis- induced diaphragm dysfunction. METHODS: Thirty SPF male SD rats were randomized equally into 5 groups, including a sham-operated group, 3 sepsis model groups observed at 6, 12, or 24 h following cecal ligation and perforation (CLP; CLP-6h, CLP-12h, and CLP-24h groups, respectively), and a CLP-24h group with a single intraperitoneal injection of KN- 93 immediately after the operation (CLP-24h+KN-93 group). At the indicated time points, diaphragm samples were collected for measurement of compound muscle action potential (CMAP), fatigue index of the isolated diaphragm and fitted frequencycontraction curves. The protein expression levels of CaMK Ⅱ, RyR1 and P-RyR1 in the diaphragm were detected using Western blotting. RESULTS: In the rat models of sepsis, the amplitude of diaphragm CMAP decreased and its duration increased with time following CLP, and the changes were the most obvious at 24 h and significantly attenuated by KN-93 treatment (P < 0.05). The diaphragm fatigue index increased progressively following CLP (P < 0.05) irrespective of KN- 93 treatment (P>0.05). The frequency-contraction curve of the diaphragm muscle decreased progressively following CLP, and was significantly lower in CLP-24 h group than in CLP-24 h+KN-93 group (P < 0.05). Compared with that in the sham-operated group, RyR1 expression level in the diaphragm was significantly lowered at 24 h (P < 0.05) but not at 6 or 12 following CLP, irrespective of KN-93 treatment; The expression level of P-RyR1 increased gradually with time after CLP, and was significantly lowered by KN-93 treatment at 24 h following CLP (P < 0.05). The expression level of CaMKⅡ increased significantly at 24 h following CLP, and was obviously lowered by KN-93 treatment (P < 0.05). CONCLUSION Sepsis causes diaphragmatic dysfunction by enhancing CaMK Ⅱ expression and RyR1 receptor phosphorylation in the endoplasmic reticulum of the diaphragm.
Rats ; Male ; Animals ; Diaphragm/metabolism* ; Ryanodine Receptor Calcium Release Channel/metabolism* ; Rats, Sprague-Dawley ; Phosphorylation ; Muscle Contraction/physiology* ; Endoplasmic Reticulum ; Sepsis/metabolism*

OBJECTIVETo study the changes in diaphragmatic function and gene expressions of calcium regulatory proteins in diabetic rats and explore the mechanism of diaphragm dysfunction in diabetes mellitus.

METHODSSD rats were randomly divided into normal control group and diabetic (induced by intraperitoneal STZ injection) group. After 4 and 8 weeks, the body weight and diaphragm to body weight ratio were measured, and the activities of succinic dehydrogenase (SDH) in the diaphragm and blood glucose were assayed. The diaphragm contractility was assessed and the alterations of diaphragm ultrastructure were observed. RT-PCR was used to detect the changes in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLB) mRNA expressions in the diaphragm.

RESULTSThe diabetic rats showed a significant weight loss with a lowered diaphragm to body weight ratio (P<0.01) and SDH activity (P<0.01). The peak twitch tension and maximum tetanic tension of the diaphragm were significantly lowered and the time to peak contraction and half relaxation time significantly prolonged (P<0.01) in the diabetic rats, which also exhibited a lowered tetanic force in response to stimulus (P<0.01). Transmission electron microscopy revealed obvious ultrastructural changes of the diaphragm in diabetic rats. RT-PCR showed significantly decreased SERCA and increased PLB mRNA expressions in diabetic rat diaphragm (P<0.01), and these changes intensified with time (P<0.01).

CONCLUSIONDiabetes can cause impairment of diaphragmatic ultrastructure, mitochondrial injuries, and lowered SDH activity and ATP production. Decreased SERCA and increased PLB mRNA expressions in diabetes result in reduced Ca(2+) uptake by the diaphragm sarcoplasmic reticulum to induce diaphragm dysfunction.

Animals ; Body Weight ; Calcium ; metabolism ; Calcium-Binding Proteins ; metabolism ; Diabetes Mellitus, Experimental ; metabolism ; physiopathology ; Diaphragm ; metabolism ; physiopathology ; Glucose ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Succinate Dehydrogenase ; metabolism

In this study, whole cell patch clamp recording technique was employed to investigate the effect of Shenmai Injection (SMI) on L-type calcium current of diaphragmatic muscle in rats. The result showed that when the diaphragmatic muscle cell was held at -80 mV and depolarized to +60 mV, 10 microl/ml, 50 microl/ml and 100 microl/ml SMI enhanced the inner peak L-type calcium current from -(6.8 +/- 0.7) pA/pF (n=7) to -(7.3 +/- 0.8) pA/pF (P>0.05, n=7), -(8.6 +/- 1.0) pA/pF (P<0.05, n=7) and -(9.4 +/- 1.2) pA/pF (P<0.05, n=7), respectively, The rates of L-type calcium current were increased by (7.34 +/- 2.37)%, (25.72 +/- 5.94)%, and (38.16 +/- 7.33)%, respectively. However, it had no significant effect on maximal activation potential and reversal potential. Our results suggested that SMI could activate the calcium channel of the diaphragmatic fibers of the rats, increase the influx of Ca2+, and enhance the contractility of diaphragmatic muscles.
Animals ; Calcium ; metabolism ; Calcium Channels, L-Type ; drug effects ; Diaphragm ; drug effects ; metabolism ; Drug Combinations ; Drugs, Chinese Herbal ; Male ; Patch-Clamp Techniques ; Plant Extracts ; pharmacology ; Rats ; Rats, Wistar

OBJECTIVETo study the changes of inducible nitric oxide synthase (iNOS) activity and apoptosis-related genes Bcl-2, Bax and caspase-3 mRNA expressions in endotoxemia-induced rat diaphragm injury and analyze the related apoptosis mechanism.

METHODSThirty-two male SD rats were randomly divided into 4 groups (n = 8): control group (saline 0.5 ml ip), endotoxin 24 h, 48 h and 96 h group (endotoxin 12 mg/kg ip, animals were killed either 24, 48 or 96 h after injections). Body weight were measured, the ratio between diaphragm weight and body weight, activities of constitutive nitric oxide syntheses (cNOS), iNOS and succinate dehydrogenase (SDH) were also measured. The expressions of Bcl-2, Bax and caspase-3 mRNA were detected by RT-PCR analysis.

RESULTSEndotoxin induced significant reductions in diaphragm mass in endotoxin 96 h group (P < 0.05). Endotoxin increased diaphragm cNOS or iNOS activities, and they were significantly higher in endotoxin 96 h group than those in endotoxin 24 h and 48 h groups, diaphragm SDH activity was reduced, and it was lower in endotoxin 96 h group than that in endotoxin 24 h and 48 h groups (P < 0.01). Endotoxin significantly increased Bax and caspase-3 mRNA expressions, and they were higher in endotoxin 48 h and 96 h groups than those in endotoxin 24 h group (P < 0.01). Endotoxin significantly reduced Bcl-2 mRNA expression and the ratio of Bcl-2/Bax, and they were lower in endotoxin 48 h and 96 h groups than those in endotoxin 24 h group (P < 0.01).

CONCLUSIONiNOS is activated in endotoxemia-induced rat diaphragm injury. It damages mitochondria, upregulates Bax expression and downregulates Bcl-2 expression, then induces caspase-3 related apoptotic pathway. These changes may cause diaphragm injury and atrophy.

Animals ; Apoptosis ; Caspase 3 ; metabolism ; Diaphragm ; metabolism ; physiopathology ; Endotoxemia ; metabolism ; Gene Expression ; Male ; Nitric Oxide Synthase Type II ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; metabolism