金褐霉素（Aureofuscin）对神经末梢...

By means of the intracellular recording technique, the effect of aureofuscin (20 micrograms/ml, oversaturation solution) on the ACh release from motor nerve terminals and on muscle cell membrane potential were investigated in phrenic nerve diaphragm preparations of the mice. The results showed that (a) aureofuscin reduced the resting membrane potential of the muscle cell slightly; (b) the frequency of miniature end-plate potentials and the mean quantal content of end-plate potentials increased at first and then recovered approximately to the control level; (c) the depolarization produced by aureofuscin in the muscle cell membrane was reversible and the aureofuscin-invoked facilitation in miniature end-plate potential discharges was Ca(2+)-dependent; and (d) aureofuscin did not block neuromuscular transmission.     

Acetylcholinesterase deficiency contributes to neuromuscular junction dysfunction in type 1 diabetic neuropathy

Diabetic neuropathy is associated with functional and morphological changes of the neuromuscular junction (NMJ) associated with muscle weakness. This study examines the effect of type 1 diabetes on NMJ function. Swiss Webster mice were made diabetic with three interdaily ip injections of streptozotocin (STZ). Mice were severely hyperglycemic within 7 days after the STZ treatment began. Whereas performance of mice on a rotating rod remained normal, the twitch tension response of the isolated extensor digitorum longus to nerve stimulation was reduced significantly at 4 wk after the onset of STZ-induced hyperglycemia. This mechanical alteration was associated with increased amplitude and prolonged duration of miniature end-plate currents (mEPCs). Prolongation of mEPCs was not due to expression of the embryonic acetylcholine receptor but to reduced muscle expression of acetylcholine esterase (AChE). Greater sensitivity of mEPC decay time to the selective butyrylcholinesterase (BChE) inhibitor PEC suggests that muscle attempts to compensate for reduced AChE levels by increasing expression of BChE. These alterations of AChE are attributed to STZ-induced hyperglycemia since similar mEPC prolongation and reduced AChE expression were found for db/db mice. The reduction of muscle end-plate AChE activity early during the onset of STZ-induced hyperglycemia may contribute to endplate pathology and subsequent muscle weakness during diabetes.     

Characterization of the neurotoxic constituents of Conus geographus (L) venom

The crude venom of the marine gastropod Conus geographus (L) has been separated into three lethal constituents and their actions at the mammalian neuromuscular junction examined.Chromatography of the venom of Sephadex G-50 gave one toxic fraction, which was resolved by ion exchange chromatography on SP-Sephadex into three toxic components. These components were individually purified by diafiltration and Sephadex G-15 chromatography to give Toxins I,II and III. Toxins I and II in concentrations greater than 5 ug/ml reduced the amplitude of end-plate potentials and miniature end-plate potentials; Toxin I also blocked the depolarization of muscle fibres produced by carbachol; neither toxin affected the generation of action potentials in muscle fibres. Toxin III in concentrations greater than 5 ug/ml rapidly and reversibly blocked the generation of action potentials in muscle fibres; it had no effect on resting membrane potential nor on the amplitude of epps or mepps. It also slowly blocked the compound action potential recorded from isolated sciatic nerves but this was not reversible in the experiments. The rate at which this toxin blocked action potentials was increased by stimulation of the preparation. It is suggested that Toxin III acts by blocking the inward movement of sodium during activity. Toxin III appeared to be a nonadeca or eicosa peptide possibly having a cystine residue in the N-terminal position.     

Supersensitivity to ACh in muscles after prolonged nerve block.

Sciatic nerves of rats and tibial nerves of rabbits were kept anaesthetized in situ for periods of 3-11 days by applying silastic cuffs containing lidocaine base or marcaine hydrochloride. To insure a more uniform release of the rapidly diffusing lidocaine base, the drug was contained in compartments at some distance from the nerve and the cuffs were covered with polystyrene. The completeness of anaesthesia and the functional state of the nerve were tested by stimulating the exposed nerves proximal and distal to the cuff and by observing the behaviour of the muscle prior to killing the animals. The ACh sensitivity was tested by electrophoretic application of ACh from micropipettes and by recording the results changes of the resting membrane potential in individual muscle fibres. The ACh sensitivity was found to be present in the extrajunctional area of all muscle fibres including those displaying miniature end-plate potentials. These was no difference between the behaviour of muscles from rats and rabbits and between the action of lidocaine base and marcaine hydrochloride. Previous reports on the absence of extrajunctional ACh sensitivity in muscles of rabbits whose nerves had been treated by lidocaine base were explained by a relatively rapid loss of the drug from the usual type of nerve cuffs (more than 70% of the drug lost in one day), permitting a premature recovery of the nerves from anaesthesia.     

Miniature End-plate Potentials at Mammalian Neuromuscular Junctions Poisoned by Botulinum Toxin

IT is generally accepted that botulinum toxin entirely blocks transmitter release from motor nerve terminals without affecting nerve conduction or the sensitivity of the muscle membrane to acetylcholine. In particular, it has been reported that with both acute and chronic intoxication with type A botulinum, miniature end-plate potentials (m.e.p.p.s.) disappear completely from a muscle at about the time that transmission is blocked^1,2. The action of botulinum toxin has been reinvestigated following acute application of toxin to the rat diaphragm in vitro and chronic paralysis of rat soleus muscle following a single intramuscular injection of toxin; miniature potentials have been observed to persist following blockade of neuromuscular transmission.     

Nicardipine inhibits axon conduction but causes dual changes of acetylcholine release in the mouse motor nerve

The effects of nicardipine, a dihydropyridine Ca2(+)-channel antagonist, on neuromuscular transmission and impulse-evoked release of acetylcholine were compared with those of nifedipine. In the isolated mouse phrenic nerve diaphragm, nicardipine (50 microM), but not nifedipine (100 microM), induced neuromuscular block, fade of tetanic contraction, and dropout or all-or-none block of end-plate potentials. Nicardipine had no significant effect on the resting membrane potential and the amplitude of miniature end-plate potentials but increased the frequency and caused the appearance of large size miniature potentials. The quantal contents of evoked end-plate potentials were increased. In the presence of tubocurarine, however, nicardipine depressed the amplitude of end-plate potentials. The compound nerve action potential was also decreased. It is concluded that nicardipine blocks neuromuscular transmission by acting on Na+ channels and inhibits axonal conduction. Nicardipine appeared to affect the evoked release of acetylcholine by dual mechanisms, i.e., an enhancement presumably by an agonist action on Ca2+ channels, like Bay K 8644 and nifedipine, and inhibition by an effect on Na+ channels, like verapamil and diltiazem. In contrast with its inactivity on the amplitude of miniature end-plate potentials, depolarization of the end plate in response to succinylcholine was greatly depressed. The contractile response of baby chick biventer cervicis muscle to exogenous acetylcholine was noncompetitively antagonized by nicardipine (10 microM), but was unaffected by nifedipine (30 microM). These results may implicate that nicardipine blocks the postsynaptic acetylcholine receptor channel by enhancing receptor desensitization or by a use-dependent effect.     

An ultrastructural study of isolated rat skeletal muscle mitochondria in various metabolic states

Summary An electron microscopic examination was made of isolated rat skeletal muscle mitochondria in various functional states. Shifts in the inner membrane ultrastructure of populations of mitochondria were observed under certain conditions. However, ultrastructural transformations reported by others during rapid changes in biochemical states were not observed in skeletal muscle mitochondria. There does not appear to be a strict correlation between metabolic states and ultrastructural states in isolated rat skeletal muscle mitochondria as has been observed in isolated mitochondria from several other tissue types.     

Effect of prolonged low-frequency stimulation on acetylcholine sensitivity of the postsynaptic membrane during blocking of neuromuscular transmission

Sensitivity of the postsynaptic chemoreceptive membrane of the frog sartorius muscle fiber to acetylcholine was studied during the development of a block to neuromuscular transmission in the course of prolonged indirect low-frequency stimulation. Calculation of the mean amplitude of miniature end-plate potentials, measurement of the input resistance of the electrogenic membrane of the muscle fiber, and application of acetylcholine to the postsynaptic membrane showed that sensitivity of the postsynaptic membrane to mediator is unchanged at the time of onset of the neuromuscular block. A decrease in amplitude of the end-plate potentials during development of fatigue is due to a reduction in their quantum composition, consequent upon negative antidromic influences from the muscle on motor nerve endings, with the participation of chemical agents formed in the muscle during the activity of its contractile system.     

KCl stimulation and ultrastructural responses of tannic acid-stained cholinergic synaptic terminals

The quantal-vesicular hypothesis equates miniature end-plate potentials (MEPPs) with fusions of synaptic vesicles. MEPP production thus predicts vesicle losses, increases in vesicle fusions and increases in terminal plasma membrane. MEPP production and these ultrastructural parameters have been evaluated in the cholinergic presynaptic terminals of skate electric organ following tannic acid saline incubation, known to promote capture and selective staining of dense-core granule fusions, and KCl stimulation, known to elevate MEPP production dramatically in these cholinergic terminals. After pretreatment in tannic acid-elasmobranch saline, KCl stimulation produced MEPPs at 40/s/microm(2)of terminal surface for several minutes with gradual reduction to spontaneous levels by 25-30 min. No loss of vesicles, no vesicle fusions, no expansions of plasma membrane and no tannic acid enhanced staining of vesicles or vacuoles accompanied the generation of 800 MEPPs/microm(3)of terminals having densities of 567 vesicles/microm(3). No ultrastructural footprints were found to support the notion that unnaturally high rates of vesicular exocytosis had occurred.     

Effect of carbachol on miniature end-plate potentials and currents of rat skeletal muscle

Experiments on rats showed that carbachol (10 µM) appreciably quickens miniature end-plate potentials in the soleus muscle kept in medium with potassium chloride concentration increased to 13 mM. Potentiation of spontaneous mediator release also took place when the membrane potential of the muscle fibers was clamped at the level of the potassium equilibrium potential. It is concluded that the presynaptic action of carbachol on synaptic liberation of acetylcholine from motor nerve endings is unconnected with depolarization of the postsynaptic membrane but takes place as a result of the direct effect of the mimetic on motor nerve endings.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 14, No. 2, pp. 185–189, March–April, 1982.     

Electrophysiological analysis of the effect of cortisone on the rat neuromuscular apparatus]

The influence of cortisone (1.5 mg per 100 g of body weight, daily, for 10 days) on the neuro-muscular system was studied in rats in situ. The action potentials of the nerve and muscle were recorded with the extracellular electrodes. The rest potentials (RP) of the muscle fibers and the miniature end-plate potentials (MEPP) were recorded with the intracellular glass microelectrodes. A decrease of the RP and the MEPP amplitude, and an increase of the MEPP frequency and prolongation of the neuromuscular transmission time were revealed in rats given daily doses of cortisone, 1.5 mg/100 g of body weight, during 10 days; reliability of the neuro-muscular transmission (acceleration of the fall of the muscle action potential amplitude during tetanus) proved to decrease under the action of cortisone.     

Intense ultraterminal sprouting from motor nerves and ultrastructural aspects of the neuromuscular junction and non-junctional sarcolemma of the soleus (slow-twitch) muscle in motor endplate disease in the mouse

Motor end-plate disease (med) in the mouse is an hereditary defect of the neuromuscular system, with partial functional denervation and muscle inactivity in late stages of the disease. Motor end-plate disease is characterized by an intense ultraterminal sprouting of the motor nerves from swollen nerve terminal branches in the soleus muscle. At the ultrastructural level, the neuromuscular junctions extend to very wide territories, often outside the original motor end-plate, in regions where the nerve sprouts are in simple apposition to the muscle fiber, with no secondary synaptic folds. The nerve terminals are rich in neurofilaments and poor in synaptic vesicles.Freeze fracture analysis of the pre-synaptic and post-synaptic membrane specializations fails to reveal any important structural alteration which could suggest a defect in acetylcholine release or in muscle membrane excitability. However, the non-junctional sarcolemmal specializations (the so-called ‘square arrays’) arc found with a frequency slightly higher than in normal muscle.The nerve abnormalities at the neuromuscular junction may be either a consequence of muscle inactivity or the morphological expression of some primary nerve abnormality. Further studies of the soleus muscle at early stages of the disease may provide evidence in favor of either possibility.     

Effect of sodium nitroprusside on the mediator release and functional properties of the postsynaptic membrane at the neuromuscular junction]

The effect of nitric oxide donor sodium nitroprusside on the end-plate currents was studied under two-electrode voltage-clamp condition at frog neuro-muscular junction. Sodium nitroprusside (10(-4) M) reduced to the half the amplitude of end-plate currents while did not change miniature end-plate currents indicating the presynaptic nature of end-plate depression. In keeping with such suggestion sodium nitroprusside essentially (to 33%) suppressed the frequency of miniature end-plate currents but did not affect the decay time constant and voltage-dependence of miniature end-plate decay. In contrast to another presynaptic inhibitors sodium nitroprusside rather reduced than increased the presynaptic facilitation and did not change postsynaptic potentials. Thus, nitric oxide is the powerful inhibitor of both evoked and spontaneous transmitter release and did not change postsynaptic potential.     

Changes in fatty acid transport and transporters are related to the severity of insulin deficiency

We have examined the effects of streptozotocin (STZ)-induced diabetes (moderate and severe) on fatty acid transport and fatty acid transporter (FAT/CD36) and plasma membrane-bound fatty acid binding protein (FABPpm) expression, at the mRNA and protein level, as well as their plasmalemmal localization. These studies have shown that, with STZ-induced diabetes, 1) fatty acid transport across the plasma membrane is increased in heart, skeletal muscle, and adipose tissue and is reduced in liver; 2) changes in fatty acid transport are generally not associated with changes in fatty acid transporter mRNAs, except in the heart; 3) increases in fatty acid transport in heart and skeletal muscle occurred with concomitant increases in plasma membrane FAT/CD36, whereas in contrast, the increase and decrease in fatty acid transport in adipose tissue and liver, respectively, were accompanied by concomitant increments and reductions in plasma membrane FABPpm; and finally, 4) the increases in plasma membrane transporters (FAT/CD36 in heart and skeletal muscle; FABPpm in adipose tissue) were attributable to their increased expression, whereas in liver, the reduced plasma membrane FABPpm appeared to be due to its relocation within the cell in the face of slightly increased expression. Taken together, STZ-induced changes in fatty acid uptake demonstrate a complex and tissue-specific pattern, involving different fatty acid transporters in different tissues, in combination with different underlying mechanisms to alter their surface abundance.     

Tissue-specific depression of mitochondrial proton leak and substrate oxidation in hibernating arctic ground squirrels

A significant proportion of standard metabolic rate is devoted to driving mitochondrial proton leak, and this futile cycle may be a site of metabolic control during hibernation. To determine if the proton leak pathway is decreased during metabolic depression related to hibernation, mitochondria were isolated from liver and skeletal muscle of nonhibernating (active) and hibernating arctic ground squirrels (Spermophilus parryii). At an assay temperature of 37 degrees C, state 3 and state 4 respiration rates and state 4 membrane potential were significantly depressed in liver mitochondria isolated from hibernators. In contrast, state 3 and state 4 respiration rates and membrane potentials were unchanged during hibernation in skeletal muscle mitochondria. The decrease in oxygen consumption of liver mitochondria was achieved by reduced activity of the set of reactions generating the proton gradient but not by a lowered proton permeability. These results suggest that mitochondrial proton conductance is unchanged during hibernation and that the reduced metabolism in hibernators is a partial consequence of tissue-specific depression of substrate oxidation.     

Quantitative investigation of the neuromuscular junction of rat skeletal muscle fibres after double innervation

Summary In normal (untreated) rats the mean length ratio of postsynaptic to presynaptic membrane was 2.7±0.8 for neuromuscular junctions of slow-twitch soleus muscle fibres and 4.2±1.0 for neuromuscular junctions of fast-twitch extensor digitorum longus muscle fibres; this difference was significant (P<0.001). After experimental double innervation by fast and slow muscle nerves for four months, the ratio was (1) 2.9±0.8 for the original slow-twitch fibre end-plate and 2.8±0.8 for the newly established one, both not significantly different from that of the normal slow-twitch fibres; and (2) 2.2±0.5 for the original fast-twitch fibre end-plate and 2.2±0.7 for the newly established one, both significantly smaller than that of the normal fast-twitch fibres (P<0.001). This means that the double innervated slow-twitch muscle fibres retained their original neuromuscular junction type, whereas the doubly-innervated fast-twitch muscle fibres underwent a dramatic transformation of their neuromuscular junction from the fast-muscle to the slow-muscle type. In both doubly innervated fibres, the ultrastructural characteristics of neuromuscular junctions, whether altered or not, were identical at both end-plate regions.     

Role and differential expression of calpastatin mRNA and protein in cultured cardiomyocytes exposed to hypoxic stress

This study investigated the beneficial effects and mechanism of action of the juice of Momordica charantia in streptozotocin (STZ)-induced diabetes mellitus in rats. Diabetes mellitus was associated with significant (p < 0.01) time course reductions in body weight, plasma insulin and the number of insulin positive cells per islet and significant (p < 0.01) time course elevation in blood glucose and osmolarity and systolic blood pressure compared to age-matched healthy controls. Oral intake of M. charantia juice by STZ-induced diabetic rats partially reversed all the diabetes-induced effects measured. Daily oral administration of M. charantia juice to STZ-induced diabetic rates significantly (p < 0.01) reduced the Na+- and K+-dependent absorptions of glucose by the brush border membrane vesicles of the jejunum compared to the responses obtained in STZ-induced diabetic rat. Either insulin (100 MM) or the fruit juice lyophilised extract (5 microg x ml(-1)) can stimulate 14C-D-glucose uptake in L6 myotubes. These effects were completely blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. High concentrations (10-200 microg x ml(-1)) of M. charantia juice extract inhibited 14C-D-glucose uptake in L6 myotubes compared to the control response. The effect of M. charantia treatment was also investigated on myelinated fibre abnormalities in the tibial nerve of STZ-induced diabetic and control rats. The results show that diabetes was associated with significant (p < 0.05) reduction in the mean cross-sectional myelinated nerve fibres, axonal area, myelin area and maximal fibre area compared to end controls. Treatment of STZ-induced diabetic rats with M. charantia juice normalised the structural abnormalities of peripheral nerves. The results indicate that M. charantia can exert marked beneficial effects in diabetic rats, and moreover, it can regulate glucose uptake into jejunum membrane brush border vesicles and stimulate glucose uptake into skeletal muscle cells similar to the response obtained with insulin.     

Release of packets of acetylcholine and synaptic vesicles elicited by brown widow spider venom in frog motor nerve endings poisoned by botulinum toxin

Homogenates of venom glands of brown widow spiders caused the release of packets of acetylcholine, observed as miniature end-plate potentials (mepps), from nerve terminals in sartorius and cutaneous pectoris muscles of the frog, even though the appearance of mepps which is normally induced by depolarization of the presynaptic membrane had been completely blocked by prior treatment with type A botulinum toxin (BotTX). The distribution of the amplitudes of the recorded mepps resembled that of the mepps in normal muscle and was quite different from the heavily skewed (to the left) distribution of the mepps recorded from BotTX-treated muscles. This suggests that the mepps elicited by brown venom glands homogenates in muscles blocked by BotTX were due to the release of a population of vesicles which is unaffected by BotTX and which is also released on hyperpolarization of nerve terminals.     

Depression of glutamate-mediated synaptic transmission by benzyl alcohol.

The data obtained from this study suggest that the nonionizable anesthetic benzyl alcohol has two prominent actions on GABA- and glutamate-mediated synaptic transmission at the lobster neuromuscular junction. They are as follows: (1) depression of the excitatory end-plate potential and the postsynaptic membrane response to applied glutamate, and (2) a hyperpolarization of the postsynaptic resting membrane potential associated with a decrease in effective membrane resistance. No change in amplitude of the inhibitory end-plate potential or inhibitory reversal potential was seen. Excitatory miniature end-plate potential frequency was also unaffected. The depression of excitatory synaptic transmission appears to be due to a decreased responsiveness of the postsynaptic receptor-ionophore complex.     

Expression of basic fibroblast growth factor,protein kinase C and members of the apoptotic pathway in skeletal muscle of streptozotocin-induced diabetic rats

This study investigated the potential mechanisms that may underlie diabetes induced amyoatrophy. Sprague-Dawley rats were either injected intraperiotneally with STZ (test group; N = 8) to induce diabetic-like symptoms (blood glucose level ≥16.65 mmol/L) or with buffer (control group; N = 8). Differences in muscle structure between the STZ-induced diabetic and control groups were evaluated by histochemistry. Protein and mRNA levels of basic FGF (bFGF), bax, bcl-2, and caspase 3 in skeletal muscle were compared between the 2 groups using immunohistochemistry and quantitative PCR, respectively. Serum level of insulin and protein kinase C (PKC) were measured by competitive RIA and ELISA, respectively. Unlike control animals, the skeletal muscle fibers from STZ-induced diabetic animals were broken and pyknotic, the sarcomeric structure disrupted, and mild hyperplasia of interstitial adipose tissues was detected. The serum level of PKC was higher (P = 0.003) and the protein and mRNA levels of bFGF in skeletal muscle were lower (P = 0.001) in STZ-induced diabetic versus control animals. Protein and mRNA levels of the apoptosis promoting genes caspase-3 and bax were higher in skeletal muscle from STZ-induced diabetic rats as compared to control animals (P < 0.001 and P = 0.037, respectively), while mRNA and protein levels of bcl-2, an inhibitor of apoptosis, was lower in STZ-induced diabetic rats versus control animals (P = 0.026). Increasing apoptosis in skeletal muscle from STZ-induced diabetic rats was further demonstrated by TNNEL assay. Our findings suggest that enhanced PKC levels, reduction of bFGF expression, and increased in apoptosis might be associated with the development of diabetes-induced myoatrophy.