Effect of glutamate on spontaneous secretion of acetylcholine in the nerve-muscle synapse in rats]

In rats, glutamate was shown to exert no effect on the mean frequency, character of interstimuli distribution, amplitude and temporal parameters of the miniature EPPs. Glutamate suppressed nonquantal release. The glutamate effect depended on its concentration and was abolished by blockade of NMDA receptors, NO-synthase inhibitoin, and NO molecules binding by haemoglobin in extracellular medium. Glutamate seems to modulate the nonquantal acetylcholine secretion by initiation of the NO synthesis in muscle fibres via activation of the NMDA receptors.     

The nerve-muscle synapse of the garter snake

Snake nerve-muscle preparations are well-suited for study of both motor innervation patterns at the systems level and NMJ function at the cellular level. Their small size (～100 myofibers) and thinness (one fiber) allows access to all NMJs in one muscle. Snake NMJs are of three types, two twitch subtypes and a single tonic type. Properties of the NMJs supplied by a particular motor neuron, and of the motor unit fibers they innervate, are precisely regulated by the motor neuron in a manner consistent with the Henneman Size Principle. Unlike its amphibian or mammalian cousins, the snake NMJ comprises ～50 (twitch) or ～20 (tonic) individual one-bouton synapses, similar to synapses found in the central nervous system. Each bouton releases a few quanta per stimulus. Larger fibers, which require more synaptic current to initiate contraction, receive nerve terminals that contain more boutons and express receptor patches with higher sensitivity to transmitter. Quantal analysis suggests that transmitter release sites in one bouton do not behave independently; rather, they may cooperate to reduce fluctuations and enhance reliability. After release, two mechanisms coexist for retrieval and reprocessing of spent vesicles–one involving clathrin-mediated endocytosis, the other macropinocytosis. Unanswered questions include how each mechanism is regulated in a use-dependent manner.     

Effect of delta-aminolevulinic acid on frog nerve-muscle function

Effects of delta-aminolevulinic acid (ALA) on nerve-muscle function $\text{in vitro}$ have been examined using the frog sciatic gastrocnemius preparation. Levels of ALA that did not interfere with nerve conduction did inhibit the muscle's response to nerve stimulation for a period of 50 – 120 min. The amounts of ALA within muscle were declining at 2 hr after topical administration. It is suggested that some of the symptoms in acute attacks of intermittent porphyria may be attributable to effects from ALA.     

L-arginine--an endogenous source of nitric oxide in animal tissues in vivo]

According to EPR data, NG-mononitro-L-arginine (MNA) being intraperitoneally injected to inbred albino mice in the dose of 70-700 mg/kg strongly decreases the formation of mononitrosyl iron complexes (MNIC) with the exogenous ligand, diethyldithiocarbamate (DETC) in liver cells. Simultaneous injections of experimental mice with MNA (70 mg/kg) and L-arginine (700 mg/kg) are unaccompanied by the formation of MNIC-DETC complexes. It is concluded that nitric oxide (NO) which is produced in mouse liver in vivo and which provides for the formation of MNIC complexes with DETC is generated by L-arginine via an enzymatic reaction which is competitively inhibited by MNA. Besides, MNA causes reversible inhibition and augmented synthesis of NO formed in mouse liver after the injection of the exogenous lipopolysaccharide of E. coli.     

Effect of exogenous and endogenous nitric oxide on biofilm formation by Lactobacillus plantarum

Biofilms are a widespread form of occurrence of microorganisms in nature, and understanding the mechanism of regulation of their formation is of unquestionable practical significance for medicine and biotechnology. In the present work, the effect of nitric oxide (NO) on biofilm formation by Lactobacillus plantarum was investigated and the micromolar concentrations of exogenous NO were shown to have a negative effect on this process due to its toxic effect on the cells. However, the decrease in the level of endogenous NO in bacteria in the presence of a nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) impaired the characteristics of the forming biofilms, as was evident from the decrease in their size.     

Epigallocatechin gallate preserves endothelial function by reducing the endogenous nitric oxide synthase inhibitor level

Asymmetric dimethylarginine (ADMA), the endogenous nitric oxide synthase inhibitor, is thought to be a key factor contributing to endothelial dysfunction. Tea catechins can cause an endothelium-dependent vasorelaxation. The present study examined the effect of epigallocatechin gallate (EGCG), the major component of tea catechins, on endothelial dysfunction induced by native low density lipoprotein (LDL) in rats and oxidized LDL (ox-LDL) in cultured endothelial cells, and whether the protective effect of EGCG is related to reduction of ADMA level. A single injection of LDL (4 mg x kg(-1), i.v.) markedly reduced endothelium-dependent relaxation and the serum nitrite/nitrate (NO) level, and increased serum concentrations of ADMA, malondialdehyde (MDA), and tumor necrosis factor-alpha (TNF-alpha). EGCG (10 or 50 mg x kg(-1), i.p.) significantly attenuated the inhibition of vasodilator response to acetylcholine and the decreased serum nitrite/nitrate level, and reduced the elevated levels of ADMA, MDA, and TNF-alpha. Exposure of endothelial cells to ox-LDL (100 microg x mL(-1)) for 24 h markedly increased the medium levels of lactate dehydrogenase (LDH), ADMA, TNF-alpha, and MDA, and decreased the level of nitrite/nitrate in the medium and the activity of dimethylarginine dimethylaminohydrolase (DDAH) in the endothelial cells. EGCG (10 and 100 microg x mL(-1)) significantly decreased the levels of LDH, ADMA, TNF-alpha, and MDA, and increased the level of nitrite/nitrate and the activity of DDAH. These results suggest that EGCG protects endothelial dysfunction induced by native LDL in vivo or by ox-LDL in endothelial cells, and the protective effect of EGCG on the endothelium is related to decrease in ADMA level via increasing of DDAH activity.     

Potentiating effect of allatostatin on transmitter quantal secretion in the mouse nerve-muscle synapse

Action of allatostatin on the spontaneous and evoked quantal acetylcholine secretion was studied for the first time in the mouse nerve-muscle synapse. End plate miniature potentials (EPMP) and miniature currents (EPMC) as well as end plate single evoked currents (SEP) were recorded in mouse semidiaphragm. Allatostatin (1 nm–1 μM) produced a dose-dependent increase of the EPMP amplitude (that reached 209% of control at 1 μM of peptide), but without affecting statistically significantly the EPMP frequency and membrane potential of muscle fibers throughout the entire range of its concentrations. The potentiating action of 1 μM peptide on the EPMP amplitude was accompanied by a rise of time of the EPMP ascent and semidrop (by 17 and 13%, respectively). Allatostatin (1 μM) caused a twofold rise of EPMC amplitude, but the time parameters of miniature postsynaptic currents did not change statistically significantly. Amplitude of SEP also increased more than twice under effect of 1 μM peptide, but the SEP quantal composition remained at the control level. On the background of allatostatin there were revealed no rise of the postsynaptic membrane input resistance (on the contrary, it decreased by 25%) and no changes of the EPMC potential-dependent amplitude and of the droptime constant that characterize cholinoreceptor conductivity. The potentiating allatostatin effect on EPMP amplitude was prevented by vesamicol (1 μM), a blocker of transport of acetylcholine into synaptic vesicles. Preliminary treatment of the nerve-muscle preparation with the inhibitor of protein kinase A (PKA) H-89 (50 nM) prevented the allatostatin-evoked EPMC amplitude increment. The obtained data allow us to suggest that allatostatin in the mouse nerve-muscle synapse acts at the presynaptic level by producing an increase of the acetylcholine quantum size due to an intraterminal cascade of reaction with participation of PKA.     

Effect of cardiac pacing on forearm vascular responses and nitric oxide function

We examined the hypothesis that changes in heart rate at rest influence bioactivity of nitric oxide (NO) in humans by examining forearm blood flow responses during cardiac pacing in six subjects. Peak forearm and mean forearm blood flows across the cardiac cycle were continuously recorded at baseline and during pacing, with the use of high-resolution brachial artery ultrasound and Doppler flow velocity measurement. The brachial artery was cannulated to allow continuous infusion of saline or N(G)-monomethyl-L-arginine (L-NMMA). As heart rate increased, no changes in pulse pressure and mean or peak blood flow were evident. L-NMMA had no effect on brachial artery diameter, velocity, or flows compared with saline infusion. These results contrast with our recent findings that exercise involving the lower body, associated with increases in heart rate and pulse pressure, also increased forearm blood flow, the latter response being diminished by L-NMMA. These data suggest that changes in blood pressure, rather than pulse frequency, may be the stimulus for shear stress-mediated NO release in vivo.     

Effect of nitric oxide synthase inhibitor on diaphragmatic function after resistive loading

We studied the effect of a nitric oxide synthase inhibitor, Nomega-Nitro-L-arginine-methyl-ester (L-NAME), on in vitro diphragmatic function both at rest (control) or after inspiratory resistive loading (IRL). Sprague-Dawley rats were anesthetized, instrumented, and then the following experimental groups: (1) controls; (2) L-NAME (100 mg/kg/body weight intravenously alone); (3) IRL alone; and (4) L-NAME + IRL. The IRL protocol consisted of applying a variable resistor to the inspiratory limb of a two-way valve at 70% of maximal airway pressure until apnea. After the experiment, the animals were sacrificed and diaphragmatic strips were obtained for activity of constitutive nitric oxide synthase (cNOS) and measurements of in vitro contractile properties: tetanic (Po) and twitch tensions (Pt). cNOS activity was significantly decreased in the L-NAME and L-NAME + IRL groups (P < or = 0.05) as compared with control and IRL groups. L-NAME alone did not affect Po or Pt. However, in both IRL groups, with and without was a significant decrease in Po and Pt. This reduction was comparable in both groups. In summary, our data showed that L-NAME resulted in a significant decrease cNOS activity, but in vitro contractility was impaired.     

Role of airway endogenous nitric oxide on lung function during and after exercise in mild asthma.

We hypothesized that nitric oxide (NO), a known mild bronchodilator that can be released by several cell types within pulmonary airways, might protect airways during exercise in asthmatic subjects. We studied 17 individuals with documented exercise-induced asthma (screening exercise evaluation) on 2 study days: after treatment with inhaled NO synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA; 2 ml of 25 mg/ml mist) and after treatment with saline vehicle. Pulmonary resistance (Rl, esophageal manometry) rose and forced expiratory volume in 1 s fell more after l-NMMA compared with saline treatment, suggesting a bronchoprotective role for NO at baseline. The rise in Rl seen after l-NMMA treatment was nearly completely reversed early in exercise, suggesting a non-NO-mediated bronchodilation. A slow rise in Rl during constant-load exercise and dramatic increase in Rl after exercise were the same on the 2 treatment days, indicating little role for NO in regulating airway function during and after exercise. We conclude that endogenous NO plays little role in regulating airway function during and after exercise in subjects with mild asthma.     

Role of endogenous nitric oxide on PAF-induced vascular and respiratory effects

The role of endogenous nitric oxide (NO) on vascular and respiratory smooth muscle basal tone was evaluated in six anaesthetized, paralysed, mechanically ventilated pigs. The involvement of endogenous NO in PAF-induced shock and airway hyperresponsiveness was also studied. PAF (50 ng/kg, i.v.) was administered before and after pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.v.), an NO synthesis inhibitor. PAF was also administered to three of these pigs after indomethacin infusion (3 mg/kg, i.v.). In normal pigs, L-NAME increased systemic and pulmonary vascular resistances, caused pulmonary hypertension and reduced cardiac output and stroke volume. The pulmonary vascular responses were correlated with the increase in static and dynamic lung elastances, without changing lung resistance. Inhibition of NO synthesis enhanced the PAF-dependent increase in total, intrinsic and viscoelastic lung resistances, without affecting lung elastances or cardiac activity. The systemic hypotensive effect of PAF was not abolished by pretreatment with L-NAME or indomethacin. This indicates that systemic hypotension is not correlated with the release of endogenous NO or prostacyclines. Indomethacin completely abolished the PAF-dependent respiratory effects.     

Effect of endogenous nitric oxide on energy metabolism of rat heart mitochondria during ischemia and reperfusion

The effects of ischemia and postischemic reperfusion on the functions of the heart and its mitochondria were studied with special attention to the effect of nitric oxide (NO) by treatment of rat hearts with the nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) or its noninhibitory isomer N^G-nitro-D-arginine methyl ester (D-NAME). NO generated during reperfusion caused increase in coronary flow (CF), but had no effect on the left ventricular pressure (LVP) or heart rate (HR). The ATP level of the heart decreased during ischemia and was not completely restored by introduction of oxygen during reperfusion due to damage of complexes I and II of the respiratory chain of mitochondria by NO. Inhibition of the respiratory chain resulted in generation of hydrogen peroxide, and NO and NO-derived species generated after production of NO caused further damage of various proteins in mitochondria, such as complexes I and II of the respiratory chain and pyruvate dehydrogenase (PDH). These results suggested that NO generated on reperfusion was the primary cause of mitochondrial dysfunction by damage of complexes I and II of the respiratory chain, with consequent increase of CF in the heart.     

Effect of armin on the ultrastructure of the neuromuscular synapse]

Alterations induced by the cholinesterase inhibitor armin (5.10(-7) g/ml) in the ultrastructure of motor nerve endings of the rat phrenic diaphragmal preparations at rest or electric stimulation of the nerve were studied. It was shown that armin at rest induced ultrastructural lesions in the endings similar to those in the control preparations during nerve stimulation. Electric stimulation did not produce additional changes in the ultrastructure of the neuromuscular junction under armin action. It is suggested that the disorder of the nerve ending function may be of importance in the mechanism of the blocking action of armin on the neuromuscular transmission.     

脑啡肽增强胶质细胞的神经营养作用与NO生成减少有关

本文在ＳＤ大鼠大脑皮层胶质细胞神经元共培养模式上,以神经元存活、突起生长、生长相关蛋白４３（ｇｒｏｗｔｈａｓｏｃｉａｔｅｄｐｒｏｔｅｉｎ４３,ＧＡＰ４３）ｍＲＮＡ的表达为指标,观察了脑啡肽对胶质细胞神经营养作用的影响,并对其机理作了初步探讨。结果表明,经脑啡肽处理的胶质细胞能使神经元的存活计数增加２８％（Ｐ＜００５）,单个神经元突起总长度增加１１％（Ｐ＜００５）,最长突起长度增加１６％（Ｐ＜００５）,ＧＡＰ４３ｍＲＮＡ的表达增加２６％（Ｐ＜００５）。然后又观察了脑啡肽（１０－６～１０－１２ｍｏｌ／Ｌ）对培养胶质细胞生成一氧化氮（ＮＯ）的影响。结果表明,浓度为１０－８,１０－１０ｍｏｌ／Ｌ的脑啡肽能明显抑制其生成（Ｐ＜００５）。结果提示,脑啡肽可能增强胶质细胞的神经营养作用,其机制之一可能是通过抑制胶质细胞ＮＯ的生成。     

Vasorelaxing effects of Caesalpinia sappan involvement of endogenous nitric oxide

Methanolic extract and two purified compounds (brazilin and hematoxylin) from Caesalpinia sappan were examined for their relaxant effects in isolated rat thoracic aorta. The methanolic extract significantly and dose-dependently relaxed the alpha1-receptor agonist phenylephrine-precontracted aortic rings, without affecting passive tension of these vessels. Removal of the vascular endothelium, inhibition of nitric oxide (NO) synthase with 0.1 mM Nomega-nitro-L-arginine and of cGMP biosynthesis with 10 microM methylene blue abolished the vasorelaxant effects of the herbal extract at doses up to 30 microg/ml. Similar vasorelaxant effects were observed with brazilin and hematoxylin. Therefore, these results suggest that brazilin and hematoxylin may be responsible for the vascular relaxant effects of C. sappan, via endogenous NO and subsequent cGMP formation. The vascular relaxant effects of the plant may contribute to its therapeutic actions.     

Effect of age on bone mineral density and the serum concentration of endogenous nitric oxide synthase inhibitors in rats

Results of previous studies have indicated that bone mineral density (BMD) is decreased in aged animals and elderly humans, and that treatment with nitric oxide (NO) donors prevents bone loss. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, can inhibit NO synthesis. In the study reported here, we examined age-related changes in the serum content of ADMA and in BMD in various skeletal regions. The BMD in the lumbar part of the spine, the femur, and the tibia in 12-month-old rats was markedly increased, compared with that in 6-month-old rats, and the BMD in 20-month-old rats was decreased, compared with that in 12-month-old rats. Serum concentration of ADMA in 20-month-old rats was significantly increased, compared with that in 6- or 12-month-old rats. A similar age-related change in the concentration of lipid peroxide also was seen in the three age groups. These results suggest that the increased amount of endogenous ADMA may be associated with an age-related decrease in BMD in rats.