Background: This study was designed to determine whether presynaptic receptor blockade could be differentiated from postsynaptic receptor blockade by examining the effect of increasing frequencies of indirect stimulation on partial twitch depression in vitro rat phrenic nerve hemidiaphragm preparations. Methods: After isolating rat phrenic nerve hemidiaphragm preparation, T200/T1 ratio (twitch height of the 200th stimuli divided by that of the 1st stimuli) at frequencies of 0.2, 0.5, 1.0, and 2.0 Hz using a drug concentration which provided approximately 20% twitch depression at 0.1 Hz was calculated. To compare T200/T1 ratios with TOF ratios, 2.0 Hz TOF response was measured immediately after 200th stimuli at either frequency of stimulation. Results: Hexamethonium caused a marked decrease in T200/T1 ratio at 0.5~2.0 Hz of stimulation, whereas alpha-bungarotoxin caused no change in T200/T1 ratios at up to 2.0 Hz of stimulation. The T200/T1 ratios produced by d-tubocurarine, vecuronium, mivacurium, and rocuronium located intermediate between alpha-bungarotoxin and hexamethonium, however significant differences among four drugs were found at 2.0 Hz. The propensity for decrease in T200/T1 ratios at 2.0 Hz might differ from this study: hexamethonium >d-tubocurarine >rocuronium >mivacurium = vecuronium >alpha-bungarotoxin. T200/T1 ratios at 2.0 Hz were not different from TOF ratios. Conclusions: When the observed effects in this study were provided with result of alpha-bungarotoxin acting predominantly at postsynaptic receptors and hexamethonium acting predominantly at presynaptic receptors, the effects of nondepolarizing muscle relaxants at each binding site could be differentiated by examining the T200/T1 ratios at 2.0 Hz.
Animals ; Binding Sites ; Bungarotoxins ; Depression* ; Hexamethonium ; Phrenic Nerve* ; Rats* ; Receptors, Presynaptic ; Tubocurarine ; Vecuronium Bromide

BACKGROUND: Interactions of neuromuscular blocking agents are antagonistic in a combination of depolarizing and nondepolarizing agents, additive in a combination of relative two compounds or synergistic in a combination of different two nondepolarizing agents. However, the interactions of neuromuscular blocking agents with a different site of action from each other have not been studied clearly. This study was designed to examine the interaction between hexamethonium and lidocaine, alpha-bungarotoxin or decamethonium with markedly different pre and postsynaptic sites of action. METHODS: Square wave, 0.1 Hz supramaximal stimuli or 2 Hz, 0.2 ms train of four (TOF) stimuli, was applied to the rat phrenic nerve-hemidiaphragm preparation, and the twitch height response was recorded mechanomyographically. The cumulative concentration effect and TOF ratio at each point of twitch depression after hexamethonium, lidocaine, alpha-bungarotoxin or decamethonium given were measured. The EC50 and EC95 of hexamethonium, lidocaine, alpha-bungarotoxin and decamethonium were calculated using an inhibitory sigmoid Emax model. In the experiment of each combination of two drugs, three points of the isobole for hexamethonium-lidocaine, hexamethonium-alpha-bungarotoxin and hexamethonium-decamethonium were established using ratios of 1 : 3, 1 : 1 and 3 : 1 of their EC50. Points on the line of theoretical additivity and 95% confidence intervals were calculated according to Tallarida et al. TOF ratios were observed at 75, 50 and 25% of the control twitch height value during each combination ratio of their EC50. RESULTS: Significant deviations of points on the isobole from the line of additivity to the left were found at all EC50 ratios of hexamethonium-lidocaine (P < 0.05 respectively), that to the right was found at all EC50 ratios of a hexamethonium-alpha-bungarotoxin and hexamethonium-decamethonium (P < 0.05 respectively). The magnitude of TOF fade depended upon the mixed ratios for their EC50. CONCLUSIONS: The interaction was found to be synergistic in the combination of hexamethonium- lidocaine, and antagonistic in the combination of hexamethonium-alpha-bungarotoxin and hexamethonium- decamethonium.
Animals ; Bungarotoxins* ; Colon, Sigmoid ; Depression ; Hexamethonium* ; Lidocaine* ; Neuromuscular Blocking Agents ; Rats

BACKGROUND: alpha-Bungarotoxin, decamethonium or lidocaine has a neuromuscular blocking effect. The aim of this study was to evaluate the pharmacodynamic properties of these drugs at the neuromuscular junction and the reversal effects of antagonists in vitro. METHODS: The effects of evoked twitch tension response have been studied on the isolated phrenic nerve hemidiaphragm preparation of the rat, using a single twitch (0.1 Hz) and the train of four (TOF; 2 Hz for 2 s) stimulation. The cumulative concentration effect and TOF ratio at each point of twitch depression after alpha-bungarotoxin, decamethonium or lidocaine were measured mechanomyographically. The EC50 and EC95 of alpha-bungarotoxin, decamethonium or lidocaine were calculated using an inhibitory sigmoid Emax model. The reversal effects of various doses of neostigmine, pyridostigmine or 4-aminopyridine (4-AP) to the partial neuromuscula r block produced by EC50 of alpha-bungarotoxin, decamethonium or lidocaine were determined. RESULTS: The EC50 and EC95 of alpha-bungarotoxin, decamethonium or lidocaine were 0.179 and 0.320 microgram/ml, 17.07 and 26.84 microgram/ml or 76.80 and 105.70 microgram/ml. TOF fade was produced by alpha-bungarotoxin or decamethonium but not by lidocaine. Neostigmine or pyridostigmine did not reverse the partial neuromuscular block induced by alpha-bungarotoxin, decamethonium or lidocaine. However, 4-AP produced a dose-dependent recovery of the twitch response (P < 0.05). CONCLUSIONS: alpha-Bungarotoxin, decamethonium or lidocaine produced different degree of TOF fade, and it means that this may be due to different site of action of these drugs. 4-AP reversed effectively the partial neuromuscular block induced by alpha-bungarotoxin, decamethonium or lidocaine, whereas neostigmine or pyridostigmine did not.
4-Aminopyridine ; Animals ; Bungarotoxins* ; Colon, Sigmoid ; Depression ; Lidocaine* ; Neostigmine ; Neuromuscular Blockade* ; Neuromuscular Junction ; Phrenic Nerve ; Pyridostigmine Bromide ; Rats*

BACKGROUND: This study was performed to evaluate the presynaptic effects of depolarizing neuromuscular blocking drugs by using slow and fast frequencies of indirect stimulation on partial twitch depression in vitro. METHODS: A rat phrenic nerve hemidiaphragm was dissected and was mounted in an organ bath containing an oxygenated Krebs solution. The phrenic nerve was stimulated supramaximally and the twitch response (0.1 Hz) was stabilized for at least 30 minutes. T200/T1 ratio (twitch height of the 200th stimuli divided by that of the first stimuli) at frequencies of 0.2, 0.5, 1.0, and 2.0 Hz using a drug concentration which provided approximately 20% twitch depression at 0.1 Hz was calculated. To compare T200/T1 ratios with TOF ratios, a 2.0 Hz TOF response was measured immediately after the 200th stimuli at either frequency of stimulation. RESULTS: T200/T1 ratios produced by succinylcholine (SCC) and decamethonium (C10) were located between alpha-bungarotoxin (ABX) and hexamethonium (C6), however, significant differences among the four drugs were found at 2.0 Hz. The propensity for a decrease in T200/T1 ratios at 2.0 Hz might differ from this study: C6 > C10 > SCC > ABX. T200/T1 ratios at 2.0 Hz were not different from TOF ratios. CONCLUSIONS: It is concluded that small doses of C10 have a greater presynaptic activity than that of SCC, when the observed effects in this study were compared with the result of ABX acting predominantly at postsynaptic receptors and C6 acting predominantly at presynaptic receptors.
Animals ; Baths ; Bungarotoxins ; Depression ; Hexamethonium ; Neuromuscular Blockade* ; Neuromuscular Blocking Agents* ; Oxygen ; Phrenic Nerve ; Rats ; Receptors, Presynaptic ; Succinylcholine

BACKGROUND: beta-Bungarotoxin irreversibly changes the presynaptic membrane, hexamethonium acts on the presynaptic nicotinic receptor, and verapamil blocks the ion channels on the presynaptic membrane. The effect of these drugs on twitch height and train of four (TOF) ratio were investigated, as well as the reversal effects of neostigmine, pyridostigmine or 4-aminopyridine (4-AP) on the partial neuromuscular blockade induced by these drugs. METHODS: Square wave, 0.1 Hz supramaximal stimuli or 2 Hz, 0.2 ms train of four stimuli, was applied to the phrenic nerve-hemidiaphragm preparation of the rat, and the twitch height response was recorded mechanomyographically. The cumulative concentration effects and TOF ratios at each point of twitch depression after beta-bungarotoxin, hexamethonium or verapamil were measured. TOF ratios were observed at 75, 50 and 25% of the control twitch height value during observation of the concentration effect. The EC50 and EC95 of beta-bungarotoxin, hexamethonium or verapamil were calculated using an inhibitory sigmoid Emax model. The reversal effect of some doses of neostigmine, pyridostigmine or 4-aminopyridine to the partial neuromuscular block produced by EC50 of beta- bungarotoxin, hexamethonium or verapamil was determined. RESULTS: The EC50 and EC95 of beta-bungarotoxin, hexamethonium and verapamil were 0.0695 and 0.1160 microgram/ml, 1267.0 and 2033.5 microgram/ml and 29.45 and 37.99 microgram/ml respectively. TOF fade was marked with hexamethonium or verapamil but small with beta-bungarotoxin. Neostigmine or pyridostigmine did not reverse the partial neuromuscular block induced by beta-bungarotoxin, hexamethonium or verapamil. However, 4-AP produced a dose-dependent recovery of the twitch response (P < 0.05). CONCLUSIONS: beta-Bungarotoxin, hexamethonium and verapamil produced different degree of TOF fade, and this may be due to different sites of action of these drugs. 4-AP reversed effectively the partialneuromuscular block induced by beta-bungarotoxin, hexamethonium and verapamil, whereas, neostigmine and pyridostigmine did not.
4-Aminopyridine ; Animals ; Bungarotoxins* ; Colon, Sigmoid ; Depression ; Hexamethonium* ; Ion Channels ; Membranes ; Neostigmine ; Neuromuscular Blockade* ; Pyridostigmine Bromide ; Rats* ; Receptors, Nicotinic ; Verapamil*

Nicotine enhances the function of learning and memory, but the underlying mechanism still remains unclear. Hippocampal long-term potentiation (LTP) is assumed to be a cellular mechanism of learning and memory. Our previous experiments showed that with the single pulses evoking 80% of the maximal population spike (PS) amplitude, nicotine (10 μmol/L) induced LTP-like response in the hippocampal CA1 region. In the present study, the nicotinic acetylcholine receptor (nAChR) subtypes and relevant neurotransmitter releases involved in LTP-like response induced by nicotine were investigated by extracellularly recording the PS in the pyramidal cell layer in the hippocampal CA1 region in vitro. LTP-like response induced by nicotine was blocked by mecamylamine (1 μmol/L) or κ-bungarotoxin (0.1 μmol/L), but not by dihydro-β-erythtroidine (DHβE, 10 μmol/L). Moreover, it was inhibited by propranolol (10 μmol/L), but not by phentolamine (10 μmol/L) or atropine (10 μmol/L). The results suggest that noradrenaline release secondary to the activation of κ-bungarotoxin-sensitive nAChRs participates in LTP-like response induced by nicotine in the hippocampal CA1 region.
Animals ; Bungarotoxins ; CA1 Region, Hippocampal ; physiology ; Long-Term Potentiation ; drug effects ; Nicotine ; pharmacology ; Norepinephrine ; secretion ; Receptors, Nicotinic ; metabolism

OBJECTIVE: It has been well known that alcohol can modulate several ligand-gated ion channel and voltage-gated ion channels. But the roles of alcohol in the autonomic neurons still remain unclear. In this study, thus we characterized the neuronal acetylcholine receptor (nnAChRs) and investigated the modulation of nnAChRs by ethanol (EtOH). METHODS: We used whole-cells which were acutely dissociated male rat major pelvic ganglion (MPG) neurons, and used gramicidin perforated patch clamp techniques. RESULTS: MPG neurons can be classified on the basis of the response of the soma membrane to depolarizing current pulses ; either tonic or phasic neurons. Sympathetic neurons expressing T-type Ca(2+) channels showed tonic firing pattern, while parasympathetic neurons lacking T-type Ca(2+) channels phasic firing to depolarizing current pulses. When hyperpolarizing currents were injected, sympathetic neurons produced post-anodal rebound spikes, while parasympathetic neurons were silent. Under current clamp mode, Acetylcholine (ACh) evoked significant membrane depolarization and produced subsequently marked membrane hyperporization. Under whole-cell mode, application of ACh-induced inward currents held at holding potentials below 0 mV and reversal potential was close to 0 mV, an equilibrium potential of nonselective cation channel. The ACh-activated current was blocked by methyllycaconitine (MLA ; 10 micrometer), hexamethonium (100 micrometer) and alpha-bungarotoxin (alpha-BuTx ; 100 nM), nAChRs antagonists. EtOH (40 mM) potentiated ACh-induced depolarization and hyperpolarization. EtOH also increased both alpha-BuTx-sensitive and -insensitive ACh-activated currents. Futhermore, EtOH potentiated 5-HT-activated current but had a little effect on GABA-activated current. CONCLUSION: These results suggest that EtOH modulates nnAChRs and 5-HT receptors in MPG neurons.
Acetylcholine ; Animals ; Bungarotoxins ; Carisoprodol ; Ethanol ; Fires ; Ganglia, Autonomic ; Ganglion Cysts ; Gramicidin ; Hexamethonium ; Humans ; Ion Channels ; Male ; Membranes ; Neurons ; Patch-Clamp Techniques ; Rats ; Receptors, Nicotinic* ; Receptors, Serotonin

Since Willis described 'fatigable weakness' in 1672, most physicians consider it as a kind of hysteria due to the inconsistent fluctuation of symptoms. Erb presented three cases of 'bulbal palsy' in the 1870s, and Oppenheim and Hopper considered myasthenia gravis as a disease similar to curare poisoning and as a disease induced by attack of the motor centers by intrinsic toxins, respectively. In 1903, Elliot suggested that a 'chemical substance' mediates the nerve impulses at synapse. However, it was not until 1921 that this was demonstrated by Loewi, who provided evidence from the famous two-frog-hearts experiment. Dale later revealed the substance to be acetylcholine, and he also suggested that myasthenia gravis is due to a problem with the motor end plate. In 1934, Walker was prompted by the resemblance between myasthenia gravis and curare poisoning to apply physostigmine, a curare-poisoning antidote, to a patient, which produced a dramatic result. Since then the use of anticholinesterase inhibitors has been adopted for standard therapeutic modality. Some prominent surgeons have also applied thymectomy as a surgical modality. The most recent focus of myasthenia gravis has been immunological. In 1960, Simpson proposed the autoimmune hypothesis, and Chang et al. showed that snake venom contained a selective antagonist of the nicotinic acetylcholine receptor, alpha-bungarotoxin. The immunization of rabbits with acetylcholine receptor purified from the electrical organs of electric eels by Patrick et al. induced myasthenic symptoms and signs, and these were reversed by acetylcholinesterase inhibitors. The role of the autoimmune system has led to the introduction of an immunosuppressive modality and plasma exchange to the field of clinical neurology.
Acetylcholine ; Action Potentials ; Bungarotoxins ; Cholinesterase Inhibitors ; Curare ; Electrophorus ; History of Medicine ; Humans ; Hysteria ; Immunization ; Motor Endplate ; Myasthenia Gravis ; Physostigmine ; Plasma Exchange ; Rabbits ; Receptors, Nicotinic ; Snake Venoms ; Synapses ; Thymectomy

The effects of presynaptic nicotinic acetylcholine receptors (nAChRs) on excitatory synaptic transmission in CA1 pyramidal neurons of the rat hippocampus were examined by blind whole-cell patch clamp recording from hippocampal slice preparations. Local application of the nAChRs agonist dimethylphenyl-piperazinium iodide (DMPP) did not induce a postsynaptic current response in CA1 pyramidal cells. However, DMPP enhanced the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC) in these cells in a dose-dependent manner. This enhancement was blocked by the selective nicotinic alpha-7 receptor antagonist alpha-bungarotoxin, but not by the antagonist mecamylamine, hexamethonium or dihydro-beta-erythroidine. The frequency of miniature excitatory postsynaptic current (mEPSC) in CA1 pyramidal neurons was also increased by application of DMPP, indicating a presynaptic site of action of the agonist. Taken together, these results suggest that activation of presynaptic nAChRs in CA1 pyramidal neurons, which contain alpha-7 subunits, potentiates presynaptic glutamate release and consequently modulate excitatory synaptic transmission in the hippocampus.
Animals ; Bungarotoxins ; physiology ; Dimethylphenylpiperazinium Iodide ; pharmacology ; Glutamic Acid ; pharmacology ; Hippocampus ; physiology ; Male ; Neurons ; physiology ; Nicotinic Agonists ; pharmacology ; Pacemaker, Artificial ; Patch-Clamp Techniques ; Rats ; Rats, Wistar ; Receptors, Nicotinic ; physiology ; Receptors, Presynaptic ; physiology ; Synapses ; physiology ; Synaptic Transmission ; alpha7 Nicotinic Acetylcholine Receptor