Corticosteroids and neuromuscular transmission: Electrophysiological investigation of the effects of prednisolone on normal and anticholinesterase-treated neuromuscular junction

The effect of prednisolone on indirectly stimulated rat muscle twitch was investigated at normal and prostigmine-treated neuromuscular junctions. In vivo, predenisolone up to 150 mg/kg body weight did not affect twitch contraction in normal animals. In neostigmine-pretreated animals, however, doses between 12.5 and 90 mg/kg could entirely abolish the anticholinesterase-induced twitch augmentation. In vitro, prednisolone produced a depressant effect on the twitch of a normal phrenic nerve diaphragm preparation which could amount to 20%. When the preparation was pretreated with neostigmine the augmented twitch could be depressed by 10^–3 to 10^–6 mol/l prednisolone to levels below the untreated control. Part of this effect is owing to a suppression of the neostigmine-induced, stimulus-bound repetitive firing of the motor nerve terminals, but to explain the full effect a further inhibitory action on neuromuscular transmission must be assumed. The latter could be accounted for by a depolarizing interaction of prednisolone and neostigmine on the nerve terminals resulting in conduction block. An action of prednisolone on postsynaptic receptors could also be considered. Such effects of the glucocorticoid might contribute to the exacerbation of muscular weakness occasionally observed in patients with myasthenia gravis at the beginning of steroid therapy.     

Relations between the effect of tetanus toxin on the neuromuscular transmission and histological functional properties of various muscles of the rat

Summary Various doses of tetanus toxin were injected into three hind leg and two fore leg muscles of the rat. The neuromuscular transmission was tested by recording the mass action potential of the muscles elicited by a single electrical stimulus to the motor nerve after strong symptoms of local tetanus had developed. The muscle responses were depressed and blocked at lower toxin doses in the fast tibialis anterior than in the mixed gastrocnemius latemlis, while blocking of the slow soleus required the highest dose. The extensor carpi radialis and the flexor carpi ulnaris muscles showed medium sensitivity. In all five muscles the contraction time was measured and correlated with its individual minimal blocking dose. The more phasic (i.e., the faster) the muscle, the more sensitive its neuromuscular transmission was to tetanus toxin. The proportional distribution of red, white, and intermediate fibres, which are associated with specific end-plate types, was evaluated for the five muscles. The percentage of white fibres in the muscles displayed a very good negative correlation with the blocking dose. The relation between structures of end-plates and effects of tetanus toxin were analysed and it is suggested that the differences in sensitivity to tetanus toxin in the neuromuscular transmission in the five muscles is determined by a differential distribution of endplates with varying sensitivities to this toxin due to structural properties.This study is a part of a doctoral dissertation submitted by one of the authors (H.K.) to the Faculty of Medicine, University of Göttingen. Some of the results were presented at the 48th and 49th Congr. of German Physiol. Soc. (Kretzschmar et al., 1977, 1978) and at the 5th Internat. Conf. on Tetanus (Kretzschmar et al., 1979)     

丹参、生脉注射液对中枢和神经肌接头兴奋传递及肌肉收缩疲劳的影响

目的 :研究复方丹参、生脉注射液对中枢、神经肌接头和肌肉疲劳的影响。方法 :记录蟾蜍坐骨神经干动作电位和腓肠肌收缩曲线 ,测定中枢、神经—肌接头、肌肉出现疲劳的时间。结果 :同对照组比较 ,丹参组和生脉组中枢、神经—肌接头、肌肉出现疲劳的时间都明显延长。差异具有显著性 (P <0 .0 1 )。结论 :复方丹参、生脉注射液具有抗中枢、神经—肌接头和肌疲劳的作用。     

Properties of spontaneous inward currents in rabbit pulmonary artery smooth muscle cells

Spontaneous inward and outward currents were studied with perforated patch recording in freshly dispersed rabbit pulmonary artery smooth muscle cells. With physiological potassium concentrations, spontaneous outward and inward currents were recorded at negative membrane potentials. Ion substitution experiments revealed that the outward and inward currents were respectively potassium and chloride conductance increases. Both conductances were abolished by bath application of caffeine (2–10 mM), which releases calcium from internal stores. The rise time and half-decay time of spontaneous potassium currents were both about 25 ms. The spontaneous chloride current has a rise time of 30 ms and decayed exponentially with a time constant () of 70 ms at –50 mV. The value was increased by depolarization and increased e-fold for a change of 99 mV in membrane potential. In every cell examined when the spontaneous currents occurred as biphasic events, typically between –20 mV and –40 mV, outward currents preceded inward currents in over 90% of these events whereas the inward current always preceded the outward current in caffeine- and noradrenaline-evoked responses. An explanation for these data is that there may be localization of some chloride channels with respect to the caffeine-sensitive calcium store.     

Transient elevation of spontaneous release at the frog neuromuscular junction following acetylcholine iontophoresis

Miniature end-plate currents (m.e.p.c.s) were recorded from frog neuromuscular junctions using a two-electrode voltage clamp. The m.e.p.c. frequency was temporarily elevated following 10 s iontophoretic applications of acetylcholine (ACh) when the junctions were clamped at 100 mV. This post-ACh burst of quanta was also observed at unclamped junctions. At –100 mV, the intensity of the burst was proportional to the amount of current flowing across the end-plate during ACh iontophoresis, but usually did not reach its peak until the end-plate receptor channels were almost completely closed. Addition of 0.5 M TTX to the Ringer's solution, or total replacement of sodium with quanidine, lithium, or methylamine did not inhibit the burst. No burst was observed in Ca²⁺-free, EGTA solutions, or in Ca²⁺-free solutions containing 2 mM Mn²⁺. Sr²⁺ effectively substituted for Ca²⁺. Addition of 2 mM Co²⁺ or Mn²⁺ to normal Ringer's did not inhibit the burst. Presynaptic muscarinic receptors did not obviously contribute to the burst, since it was not blocked by atropine, nor produced by oxotremorine or pilocarpine. The ACh analogs carbachol and acetyl--methylcholine also produced the burst. The burst was highly dependent on the muscle membrane potential during the period of ACh iontophoresis, becoming more intense at potentials negative to –100 mV and disappearing at –60 mV. The critical importance of the post-synaptic membrane potential suggests that the burst may be due to an action of the muscle end-plate on the motor nerve terminal, possibly by the movement of an anionic substance through open end-plate receptor channels, but this hypothesis does not account for the delay of the burst until near the end of the ACh-induced end-plate current.     

Age changes in neuromuscular junctions of masseter muscle

There is little information regarding the relationship between aging and ensuing morphological changes in the neuromuscular junction (NMJ) of the masticatory muscle. The masseter of C57BL/6J male mice at three different ages (young adult, 6 months; mature adult, 12 months; and old, 30 months) was studied. Morphological measurements were taken from zinc iodide osmium stained NMJ. Camera lucida drawings were superimposed on a computer monitor via a video camera and traced using a digitizer. The data were treated statistically using multivariate analysis of variance (MANOVA). No difference was found between 6 and 12 months of age, but a significant decrease in morphological parameters in NMJ from 30-month-old animals was found when compared with mature adult animals. Nerve terminal areas, perimeters, longitudinal extent lengths, and fiber diameters were reduced by 24%, 21%, 15.5% and 23%, respectively. Nerve terminal branches and incidence of sprouts were significantly increased in older animals. Age changes in the NMJ morphology are probably associated with altered balance between degeneration and regeneration of nerve terminals. This view is supported by increased terminal sprouting in old mice which is indicative of the plasticity or remodelling of NMJ during aging. © 1993 Wiley-Liss Inc.     

Serotonin modulates neuromuscular transmission in frogs

The effect of 5-hydroxytryptamine (serotonin) on neuromuscular transmission in frog skeletal muscle was studied using voltage clamp technique. Serotonin produced no effect on end-plate currents during low frequency electrical stimulation of the motor nerve, but increased the amplitude depression of multiquantal currents during high-frequency stimulation similar to motor commands fired by motoneurons. It was shown that the inhibitory effect of serotonin on neuromuscular transmission is determined by slow potential-dependent block of open ionic channels in the postsynaptic membrane accumulating during rhythmic activation of the synapse.     

Effect of dexamethasone on neuromuscular synapse ultrastructure

Laboratory of Evolutionary Histology, A. N. Severtsov Institute of Evolutionary Morphology and Animal Ecology, Academy of Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 105, No. 4, pp. 491–494, April, 1988.     

Polyploidy of smooth myocytes in coronary arteries

Study of smooth myocytes in coronary arteries of dogs with experimental coarctation of the aorta revealed increased DNA content in myocyte nuclei and increased percentage of binucleated cells. Polyploidy of vascular myocytes did not disappear after correction of the aortal defect.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 11, pp. 589–591, November, 2004     

Demonstration of glutamate-like immunoreactivity at rat neuromuscular junctions by quantitative electron microscopic immunocytochemistry

Motor nerve terminals and adjacent structures in the extensor digitorum longus and soleus muscles of young adult rats were examined for their content of glutamate by means of quantitative, electron microscopic immunocytochemistry employing colloidal gold particles as markers. The level of glutamate immunoreactivity was stronger in the extensor digitorum longus terminals than in the soleus terminals. In both muscles the glutamate immunolabelling was stronger in the nerve terminals than in the synaptic clefts and the postsynaptic tissue separating the secondary clefts, but the differences were larger in the extensor digitorum longus than in the soleus muscle. The myofibrils of the soleus muscle were more densely labelled than those in the extensor digitorum longus muscle: The level of immunoreactivity was high in the Schwann cells of both muscles. By comparing the labelling intensity of motor nerve terminals with that of muscle fibres and hippocampal mossy fibres (compartments that have been analysed previously with respect to their glutamate content), the mean concentration of fixed glutamate in the extensor digitorum terminals was estimated to be in the range of 10–20 mmol/l. An association of glutamate immunoreactivity with synaptic vesicles was demonstrated in the most strongly labelled terminals. Whether these epitopes were localized in the interior of the vesicles or at their external surface could not be resolved with the present technique. These data indicate that motor nerve terminals contain glutamate, and that the enrichment of this amino acid is more pronounced in the terminals of the extensor digitorum longus muscle (a fast muscle) than in those of the soleus muscle (a slow muscle). A possible modulatory or trophic role of glutamate in the mammalian neuromuscular junction should be considered.     

Elements of molecular machinery of GABAergic signaling in the vertebrate cholinergic neuromuscular junction

It is generally accepted that gamma-aminobutyric acid (GABA) is a signaling molecule abundant in central synapses. In a number of studies though, it has been shown that GABA signaling functions in the peripheral nervous system as well, in particular, in the synapses of sympathetic ganglia. However, there exists no firm evidence on the presence of GABAergic signaling cascade in the intercellular junctions of the somatic nerve system.By the use of immunohistochemistry methods, in the synaptic area of cholinergic neuromuscular contact in rat diaphragm, we have detected glutamate decarboxylase, the enzyme involved in synthesis of GABA, molecules of GABA, and also GAT-2, a protein responsible for transmembrane transport of GABA. Earlier we have also shown that metabotropic GABA_B receptors have overlapping localization in the same compartment. Moreover, activation of GABA_B receptors affects the intensity of acetylcholine release. These data taken together, allows us to suggest that in the mammalian cholinergic neuromuscular junction, GABA is synthesized and performs certain synaptic signaling function.     

Inhibitory actions of adenosine differ between ear and mesenteric arteries in the rabbit

In isolated ear and mesenteric arteries of rabbit, adenosine inhibited nerve-mediated contractions to a similar extent. However, the amplitude of the excitatory junction potentials evoked by perivascular nerve stimulation was increased by adenosine in the ear artery and decreased in the mesenteric artery. Outflows of noradrenaline and its metabolite, 3,4-dihydroxyphenylglycol, evoked by perivascular nerve stimulation were increased and decreased by adenosine in the ear and mesenteric arteries, respectively. Adenosine hyperpolarized the smooth muscle cells, by increasing potassium conductance of the membrane, with no relation to the endothelial cells. The hyperpolarizing action of adenosine was stronger in the ear artery than in the mesenteric artery. The inhibition of the nerve-mediated contraction by adenosine may be mainly due to postjunctional events in the ear artery and prejunctional events in the mesenteric artery.     

Neuromuscular transmission in hypoxemic patients with chronic obstructive pulmonary disease

Many studies have focused on the systemic effects of chronic obstructive pulmonary disease (COPD), but none has examined neuromuscular junction transmission (NMT). We evaluated NMT dysfunction using single-fiber electromyography (SFEMG) in patients with COPD. Twenty patients with COPD and 20 age-matched healthy controls were included in the study. All patients and controls underwent SFEMG. Abnormal NMT was found in seven of 20 patients (35%), but in none of the control subjects. The COPD patients were subgrouped according to the presence of hypoxemia. The patients with normoxemia were classified as Group 1, and the patients with hypoxemia were classified as Group 2. Abnormal NMT was found in six patients in Group 2 and in one in Group 1. While there was significant difference in terms of abnormal NMT between Group 2 and the controls, there was none between Group 1 and the controls. Our results show that NMT abnormalities can be present in hypoxemic patients with COPD.     

Oxygen-dependent mechanisms in cerebral autoregulation

Autoregulatory adjustments in the caliber of cerebral arterioles were studied in anesthetized cats equipped with cranial windows for the direct observation of the pial microcirculation. Increased venous pressure caused slight, but consistent, arteriolar dilation, at normal and at reduced arterial blood pressure and irrespective of whether or not intracranial pressure was kept constant or allowed to increase. Arterial hypotension caused arteriolar dilation which was inhibited partially by perfusion of the space under the cranial window with artificial CSF equilibrated with high concentrations of oxygen. This vasodilation was inhibited to a greater extent by perfusion of the space under the cranial window with fluorocarbon FC-80, equilibrated with high concentrations of oxygen. CSF or fluorocarbon equilibrated with nitrogen did not influence the vasodilation in response to arterial hypotension. The response to increased venous pressure was converted to vasoconstriction when fluorocarbon equilibrated with high concentrations of oxygen was flowing under the cranial window. The vasodilation in response to arterial hypotension was inhibited by topical application of adenosine deaminase. The results show that both metabolic and myogenic mechanisms play a role in cerebral arteriolar autoregulation. Under normal conditions, the metabolic mechanisms predominate. The presence of the myogenic mechanisms may be unmasked by preventing the operation of the metabolic mechanisms. The major metabolic mechanism seems to be dependent on changes in PO₂ within the brain with secondary release of adenosine.     

A method for the isolation of cells from arteries of various sizes

Summary This report describes a technique for the isolation of smooth muscle cells (SMC) from arteries of different sizes. SMC from dog carotid arteries were dissociated by collagenase-elastase perfusion under constant pressure. The freshly isolated cells were spindle shaped with an average length of 118.0 + 8.0 μm. A distinct basal lamina, ovoid nucleus, and both thick and thin filaments were observable by transmission electron microscopy. Scanning electron microscopy of the cells revealed a continuous plasma membrane and several fingerlike processes. A chamber used to study the contractile properties of isolated cells is also described. Studies of the contractile properties of SMC isolated by this technique found that cells exposed to calcium (2.5 mM) or potassium (50 mM) or both contracted by 25 to 35%. Thus, SMC isolated by the collagenase-elastase perfusion technique described here possess morphologic and contractile characteristics typical of SMC. This research was supported by Veterans Administration medical research funds.     

Formation of ectopic neuromuscular junctions in adult rats

The formation of ectopic neuromuscular junctions between a transplanted foreign nerve (the superficial fibular nerve) and the denervated soleus muscle was examined in adult rats. Formation of new junctions was induced by denervating the soleus muscle by cutting the tibial nerve. Junctional transmission began 3 days after denervation when denervation was done 3 weeks after transplantation of the foreign nerve, and progressively later when denervation was done 8, 16 and 24 weeks after transplantation. The rate at which the transmission, once started, acquired mature characteristics was approximately the same in each case. Initially, spontaneous m. e.p. p.s were infrequent, long lasting with a skewed amplitude distribution. The e. p.p. s evoked by stimulation of the foreign nerve were then commonly below threshold for eliciting action potentials and were occasionally no larger than the size of the spontaneous m. e.p. p.s. M.e. p.p. characteristics became normal in the 2nd week after transmission had started. Fully effective evoked transmission with every innervated fibre responding with overshooting action potential occurred 1–3 months after onset of transmission.     

The effects of pH on the conductance change evoked by iontophoresis in the frog neuromuscular junction

Summary The amplitude of the electrophoretically evoked end-plate potential increases with changing the pH of the bathing solution from 9.4 to 5.4 at room temperature. This change is not observed at lower temperature. The underlying current (e.p.c. _I ) is slightly decreasing at room temperature by lowering the pH. The relationship between the amplitude of the e.p.c. _I and membrane potential is highly non-linear at pH 9.4, while it is quite linear at pH 5.4. The time course of the e.p.c. _I is changed neither by different pH, nor by different membrane potential. The data suggest that during the e.p.c. _I , the mediator (ACh), the receptor (R) and the mediator-receptor complex are in equilibrium: the amplitude of the e.p.c. _I will thus depend on the affinity constant of the reversible reaction between ACh and R. It is concluded that by decreasing the pH, the affinity constant is decreased.     

A study of excitatory neuromuscular transmission in the bovine trachea

1. The excitatory innervation of bovine tracheal smooth muscle has been studied with the sucrose-gap apparatus.2. Single 2 ms electrical stimuli applied to the whole tissue excited intrinsic nerves, and produced a small transient depolarization of the smooth muscle, the excitatory junction potential (e.j.p.). The e.j.p. caused a twitch-type contraction; twitches and e.j.p.s summated during repetitive stimulation but facilitation was not observed, and action potentials were never elicited.3. The effects of electrical stimulation could be abolished by atropine (5 x 10(-7) mol/l) and augmented by neostigmine (4 x 10(-6) mol/l), and were mimicked by exogenous acetylcholine (1.0 mug/ml).4. With the electron microscope, the density of innervation was found to be low (one axon per ninety smooth muscle cells). Axons were found in small groups in the clefts between bundles of cells, but no axons penetrated within the muscle bundles. Naked axon varicosities containing agranular vesicles were seen, but no axon approached within 200 nm of a smooth muscle cell.5. It is difficult to reconcile the sparsity of innervation with the dependence of the tissue on nerve excitation to initiate activity.