Capsaicin in the periaqueductal gray induces analgesia via metabotropic glutamate receptor-mediated endocannabinoid retrograde disinhibition.

BACKGROUND AND PURPOSE: Capsaicin, an agonist of transient receptor potential vanilloid 1 (TRPV1) channels, is pro-nociceptive in the periphery but is anti-nociceptive when administered into the ventrolateral periaqueductal gray (vlPAG), a midbrain region for initiating descending pain inhibition. Here, we investigated how activation of TRPV1 channels in the vlPAG leads to anti-nociception. EXPERIMENTAL APPROACH: We examined synaptic transmission and neuronal activity using whole-cell recordings in vlPAG slices in vitro and hot-plate nociceptive responses in rats after drug microinjection into the vlPAG in vivo. KEY RESULTS: Capsaicin (1-10 µM) depressed evoked GABAergic inhibitory postsynaptic currents (eIPSCs) in vlPAG slices presynaptically, while increasing miniature excitatory PSC frequency. Capsaicin-induced eIPSC depression was antagonized by cannabinoid CB1 and metabotropic glutamate (mGlu5) receptor antagonists, and prevented by inhibiting diacylglycerol lipase (DAGL), which converts DAG into 2-arachidonoylglycerol (2-AG), an endocannabinoid. Capsaicin induced membrane depolarization in 2/3 neurons recorded but, overall, increased neuronal firings by increasing evoked postsynaptic potentials. Intra-vlPAG capsaicin reduced hot-plate responses in rats, effects blocked by CB1 and mGlu receptor antagonists. Effects of capsaicin were antagonized by SB 366791, a TRPV1 channel antagonist. CONCLUSIONS AND IMPLICATIONS: Capsaicin activated TRPV1s on glutamatergic terminals to release glutamate which activated postsynaptic mGlu5 receptors, yielding 2-AG from DAG by DAGL hydrolysis. 2-AG induces retrograde inhibition (disinhibition) of GABA release via presynaptic CB1 receptors. This disinhibition in the vlPAG leads to anti-nociception by activating the descending pain inhibitory pathway. This is a novel TRPV1 channel-mediated anti-nociceptive mechanism in the brain and a new interaction between vanilloid and endocannabinoid systems.

Plasma calcitonin gene-related peptide in diagnosing and predicting paediatric migraine.

To investigate the role of plasma calcitonin gene-related peptide (CGRP) in paediatric migraine, we prospectively collected 134 blood samples during or between attacks from 66 migraine, 33 non-migraine headache (non-migraine) and 22 non-headache patients, aged 4-18 years. Plasma CGRP concentrations were measured by enzyme-linked immunosorbent assay and disability by Pediatric MIgraine Disability ASsessment (PedMIDAS) questionnaire. Migraineurs had higher plasma CGRP levels than non-migraine patients (P = 0.007). The attack level was higher than the non-attack level in migraine (P = 0.036), but not in non-migraine, patients. This was also revealed in paired comparison (n = 9, P = 0.015 vs. n = 4, P = 0.47). Using a threshold of 55.1 pg/ml, the sensitivity of the attack level in predicting migraine was 0.81, and specificity 0.75. The PedMIDAS score tended to be higher in the high CGRP (> 200 pg/ml, n = 7) group than in the low (< 200 pg/ml, n = 33) group (26.07 vs. 19.32, P = 0.16) using Mann-Whitney test. Plasma CGRP is useful for diagnosis in paediatric migraine.

Nociceptin/orphanin FQ peptide receptors: pharmacology and clinical implications.

The advance of functional genomics revealed the superfamily of G-protein coupled receptors (GPCRs). Hundreds of GPCRs have been cloned but many of them are orphan GPCRs with unidentified ligands. The first identified orphan GPCR is the opioid receptor like orphan receptor, ORL1. It was cloned in 1994 during the identification of opioid receptor subtypes and was de-orphanized in 1995 by the discovery of its endogenous ligand, nociceptin or orphanin FQ (N/OFQ). This receptor was renamed as N/OFQ peptide (NOP) receptor. Several selective ligands acting at NOP receptors or other anti-N/OFQ agents have been reported. These include N/OFQ-derived peptides acting as agonists (cyclo[Cys(10),Cys(14)]N/OFQ, [Arg(14), Lys(15)]N/OFQ, [pX]Phe(4)N/OFQ(1-13)-NH(2), UFP-102, [(pF)Phe(4),Aib(7), Aib(11),Arg(14),Lys(15)]N/OFQ-NH(2)) or antagonists (Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)-NH(2), [Nphe(1)]N/OFQ(1-13)-NH(2), UFP-101, [Nphe(1), (pF)Phe(4),Aib(7),Aib(11),Arg(14),Lys(15)]N/OFQ-NH(2)), hexapeptides, other peptide derivatives (peptide III-BTD, ZP-120, OS-461, OS-462, OS-500), non-peptide agonists (NNC 63-0532, Ro 64-6198, (+)-5a compound, W-212393, 3-(4-piperidinyl)indoles, 3-(4-piperidinyl) pyrrolo[2,3-b]pyridines) and antagonists (TRK-820, J-113397, JTC-801, octahydrobenzimidazol-2-ones, 2-(1,2,4-oxadiazol-5-yl)-1 H-indole, N-benzyl-D-prolines, SB-612111), biostable RNA Spiegelmers specific against N/OFQ, and a functional antagonist, nocistatin. Buprenorphine and naloxone benzoylhydrazone are two opioid receptor ligands showing high affinity for NOP receptors. NOP receptor agonists might be beneficial in the treatment of pain, anxiety, stress-induced anorexia, cough, neurogenic bladder, edema, drug dependence, and, less promising, in cerebral ischemia and epilepsy, while antagonists might be of help in the management of pain, depression, dementia and Parkinsonism. N/OFQ is also involved in cardiovascular, gastrointestinal and immune regulation. Altered plasma levels of N/OFQ have been reported in patients with various pain states, depression and liver diseases. This review summarizes the pharmacological characteristics of, and studies with, the available NOP receptor ligands and their possible clinical implications.

ATP-sensitive K+ channels and cellular actions of morphine in periaqueductal gray slices of neonatal and adult rats.

ATP-sensitive K+ (K(ATP)) channels were reported to be involved in morphine analgesia in vivo. The present study, using patch-clamp technique in brain slices of neonatal (P12-P16) and adult rats, investigated cellular actions of K(ATP) channel ligands and their interactions with morphine in the ventrolateral periaqueductal gray (PAG), a crucial site for morphine analgesia. In neonatal PAG neurons, morphine depressed evoked inhibitory postsynaptic currents (IPSCs) in almost all tested neurons and elicited an inwardly rectifying K+ current in one-third of tested neurons. Glibenclamide (1-10 microM), a K(ATP) channel blocker, did not affect the membrane current or synaptic current per se but also failed to affect the effects of morphine. No outward current was elicited upon using microelectrodes containing ATP-free internal solution. In adult neurons, morphine, at the concentration up to 300 microM, failed to activate K+ current in all 25 neurons tested but depressed IPSCs to a comparable extent as that in neonatal neurons. Glibenclamide also failed to alter the effect of morphine in adult neurons. The openers of K(ATP) channels, lemakalim (10-30 microM) and diazoxide (10-500 microM), unlike morphine, did not increase membrane currents in both neonatal and adult neurons. However, diazoxide induced a glibenclamide-sensitive outward current in hippocampal CA1 neurons. It is concluded that K(ATP) channels display little functional role per se and might not be involved in effects of morphine in the ventrolateral PAG. The correlation between the insensitivity in K+ channel activation and the less antinociceptive response to morphine in adults was discussed.

Differential antagonism by naloxone benzoylhydrazone of the activation of inward rectifying K+ channels by nociceptin and a mu-opioid in rat periaqueductal grey slices.

A novel receptor, the opioid receptor-like orphan receptor (ORL1), is homologous to, but distinct from, classical opioid receptors. Although initially developed as an opioid receptor ligand, naloxone benzoylhydrazone (NalBzOH) is one of the few antagonists at ORL1. The present electrophysiological study of the effects of NalBzOH on the activation of ORL1 and mu-opioid receptors was performed in brain slices of the ventrolateral periaqueductal grey (PAG), a crucial site for opioid-induced supraspinal analgesia. Both orphanin FQ/nociceptin (OFQ/N), an ORL1 agonist, and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), a mu-opioid receptor agonist, activated inwardly rectifying K+ (Kir) channels in the ventrolateral PAG. Of the neurons tested, 96% responded to OFQ/N, but only 65% to DAMGO. NalBzOH (3-30 microM) antagonized the effect of OFQ/N competitively with a pA2 of 5.67. NalBzOH also antagonized, but more potently and non-competitively, the effect of DAMGO. In contrast, NalBzOH did not affect baclofen-induced activation of Kir channels. NalBzOH alone, at concentrations up to 30 microM, had little effect on this inwardly rectifying channel. It is concluded that NalBzOH antagonizes the activation of Kir channels mediated by both ORL1 and mu-opioid receptors in the ventrolateral PAG. It acts not only as a competitive antagonist at ORL1, but also as a more potent and non-competitive antagonist at mu-opioid receptors.

Characterization of synaptic transmission in the ventrolateral periaqueductal gray of rat brain slices.

Synaptic transmission evoked by focal stimulation in the ventrolateral periaqueductal gray was characterized using the whole-cell recording technique in rat brain slices. At resting membrane potential (-62+/-1 mV), focal stimulation (0.05-0.1 ms, 0.03 Hz) usually evoked a 6-cyano-7-nitroquinoxaline-2, 3-dione-sensitive fast excitatory postsynaptic potential and a DL-2-amino-5-phosphonopentanoic acid-sensitive slow excitatory postsynaptic potential with a bicuculline-sensitive inhibitory postsynaptic potential in between. In the presence of kynurenic acid, bicuculline-sensitive inhibitory postsynaptic currents recorded in the voltage-clamp mode displayed a reversal potential of -68+/-3 mV, resembling GABA(A) receptor-mediated inhibitory postsynaptic currents. However, no GABA(B) receptor-mediated inhibitory postsynaptic current was evoked, even at stronger stimulating intensity. 6-Cyano-7-nitroquinoxaline-2,3-dione-sensitive fast excitatory postsynaptic currents were isolated by DL-2-amino-5-phosphonopentanoic acid plus bicuculline and DL-2-amino-5-phosphonopentanoic acid-sensitive slow fast excitatory postsynaptic currents by bicuculline plus 6-cyano-7-nitroquinoxaline-2,3-dione. Both types of excitatory postsynaptic current reversed at potentials near 0 mV. The I-V curve of slow fast excitatory postsynaptic currents or N-methyl-D-aspartate currents displayed a negative slope at potentials more negative than -30 mV in an Mg(2+)-sensitive manner. The control postsynaptic currents reversed at potentials between -50 and -35 mV, inclined to the reversal potential of GABA(A), but not glutamate, receptor channels. It is concluded that, in the ventrolateral periaqueductal gray, focal stimulation elicits both inhibitory and excitatory transmission, while the former is dominant. The inhibitory transmission is mediated by GABA(A) but not GABA(B) receptors. The excitatory transmission is mediated by glutamate acting on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate as well as N-methyl-D-aspartate receptors.

Docosahexaenoic acid inhibits synaptic transmission and epileptiform activity in the rat hippocampus.

Docosahexaenoic acid (DHA) has been suggested to be required for neuronal development and synaptic plasticity. However, in view of the fact that DHA facilitates NMDA responses and blocks K(+) channels, it might predispose the neurons to epileptiform bursting. By using extracellular recording of population spikes in the CA1 region of rat hippocampal slices, we tested this possibility by examining the effect of DHA on the epileptiform activity induced by bicuculline or in Mg(2+)-free medium. When stimuli were delivered to the Schaffer collateral/commissural pathway every 20 or 30 sec, DHA had no significant effect on the epileptiform activity. However, when the frequency of stimulation was increased to 0.2 Hz, DHA attenuated the amplitude of the bursting activity induced by bicuculline to 57.5+/- 10.8% and those induced by Mg(2+)-free ACSF to 65.8+/-13.9% of control. DHA reduced the slope of field excitatory postsynaptic potential (fEPSP) to 77.1+/-7.4% of baseline, without significant effect on the ratio of paired-pulse facilitation (PPF). By intracellular recording of neurons in the stratum pyramidale of rat hippocampal slices, we found that DHA markedly inhibited the repetitive firing of action potentials elicited by depolarizing current pulses but did not affect the initial action potential. Thus, DHA may attenuate epileptic activity mainly through the frequency-dependent blockade of Na(+) channels.

Pharmacological characterization of the nociceptin receptor, ORL1. Insight from the inward rectifier activation in the periaqueductal gray.

A novel opioid receptor-like orphan receptor (ORL1) was cloned and identified to be homologous to classical opioid receptors but insensitive to traditional opioids. A heptadecapeptide, termed orphanin FQ or nociceptin (OFQ/N), was identified as its endogenous ligand. OFQ/N shares overlapping distribution sites in pain-processing areas and common cellular mechanisms with opioids but exerts diverse effects on nociceptive responses. Of the two reported ORL1 antagonists, [Phe(1)psi(CH(2)-NH)- Gly(2)] nociceptin-(1-13)-NH(2) (Phepsi) and naloxone benzoylhydrazone (NBZ), antagonisms were validated in the activation of inward rectifying K channels induced by OFQ/N, using the patch clamp technique in ventrolateral periaqueductal gray slices. Results showed that Phepsi acted as a partial agonist and NBZ was a weak nonselective antagonist of ORL1. It is comparable with most but not all of the findings from other tissues. Comparing all the reports supports the above inference for these two antagonists. The possible causes for the discrepancy were discussed. A brief review on the putative ORL1 antagonists, acetyl-RYYRIK-NH2, some sigma-ligands and the functional antagonist, nocistatin, is also included. It indicates that a potent and selective ORL1 antagonist is expecting to elucidate the physiological role of OFQ/N.

[Phe1psi(CH2-NH)Gly2]nociceptin-(1 - 13)-NH2 activation of an inward rectifier as a partial agonist of ORL1 receptors in rat periaqueductal gray.

1. [Phe1psi(CH2-NH)Gly2]nociceptin-(1 - 13)-NH2 (Phepsi), a tridecapeptide analogue of orphanin FQ/nociceptin (OFQ/N), was introduced as a competitive antagonist of opioid receptor-like orphan receptor (ORL1) in guinea-pig ileum and mouse vas deferens preparations in vitro but was recently found to act as an agonist in vivo. 2. In the periaqueductal gray, a site enriched with both OFQ/N and ORL1 and involved in OFQ/N-induced hyperalgesia and anti-analgesia, the effects of Phepsi and OFQ/N on the membrane current were studied using whole cell patch clamp recording technique in rat brain slices. 3. OFQ/N (0.01 - 1 microM) activated an inwardly rectifying type of K+ channels in ventrolateral neurons of PAG. Phepsi (0.03 - 1 microM), like OFQ/N, also activated this inward rectifier but had only 30% efficacy of OFQ/N. 4 At maximal effective concentration (1 microM), Phepsi reversed the increment of K+ conductance induced by OFQ/N (300 nM) by 46%. On the other hand, Phepsi also prevented the effect of OFQ/N if pretreated before OFQ/N. 5 It is suggested that Phepsi acts as a partial agonist of ORL1 that mediates the activation of inwardly rectifying K+ channels in ventrolateral neurons of rat periaqueductal gray.

Mechanism underlying increased neuronal activity in the rat ventrolateral periaqueductal grey by a mu-opioid.

1. The overall effect of the mu-opioid receptor agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) on ventrolateral periaqueductal grey (PAG) neurons in brain slices was studied using the whole-cell patch-clamp recording technique. 2. Under current-clamp conditions, DAMGO (1 microM) increased cell firing in many PAG neurons even though the opioid induced hyperpolarization and inhibited excitatory postsynaptic potentials (EPSPs) in these cells. 3. The increase in cell activity by DAMGO was observed in both transverse and horizontal slices. The increase persisted when the membrane potential was re-depolarized to the control level. Thus, different planes of sections or the removal of Na+ channel inactivation could not account for the observation. 4. The GABA antagonist bicuculline caused cell firing, mimicking the excitatory effect of DAMGO. Unlike DAMGO, however, bicuculline depolarized PAG cells. 5. Under voltage-clamp conditions, with the same driving force, the evoked inhibitory postsynaptic currents (IPSCs) in these neurons were 2.3 times larger than the evoked excitatory postsynaptic currents (EPSCs). Furthermore, DAMGO inhibited IPSCs by 60.7% while it inhibited EPSCs by 35.3%. 6. We propose that the overall effect of an opioid depends on the dynamic balance of its excitatory and inhibitory actions. In the PAG, the blockade of the inhibitory drive of GABAergic inputs by DAMGO is large. It overcomes the DAMGO-induced hyperpolarization and inhibition of EPSCs and thus results in the excitation of these neurons.

Chinese herb constituent beta-eudesmol alleviated the electroshock seizures in mice and electrographic seizures in rat hippocampal slices.

We have found that beta-eudesmol, a sesquiterpenol constituent of Chinese herb antagonized organophosphate-induced lethal toxicity by reversing the neuromuscular failure and reducing the occurrence of convulsions. Its possible antiepileptic action was further explored in electroshock seizure mice in vivo and in high potassium treated rat hippocampal slices in vitro. At a dose with little effect on the motor activity, beta-eudesmol prevented the convulsions and lethality induced by maximal electroshock but not those by pentylenetetrazol or picrotoxin. At subeffective doses, beta-eudesmol and phenytoin showed additive effect in preventing electroshock seizures. Extracellular recording of field potentials in CA1 pyramidal layer of hippocampal slices showed that beta-eudesmol reduced the high potassium (8.5 microM)-induced electrographic seizure activity. The potential of beta-eudesmol to serve as an antiepileptic or a conjuvant in phenytoin therapy is suggested.

Enhancement by benzodiazepines of the inhibitory effect of adenosine on skeletal neuromuscular transmission.

1. Interactions of benzodiazepines with adenosine on the neuromuscular transmission were studied in mouse diaphragm preparations. 2. In tubocurarine (0.6-0.8 microM)-partially paralyzed preparations, diazepam (35 microM) and Ro 5-4864 (3-30 microM), a peripheral type benzodiazepine receptor agonist, potentiated the inhibitory effect of adenosine on indirect twitch responses. 3. The central type receptor agonist, clonazepam did not affect the inhibitory effect of adenosine. 4. The peripheral benzodiazepine receptor antagonist, PK11195 (1-10 microM) attenuated the adenosine inhibition and antagonized the potentiation by Ro 5-4864. 5. Ro 5-4864 failed to enhance further the inhibitory effect of adenosine in the presence of dipyridamole, an adenosine uptake inhibitor that also potentiated adenosine inhibition. 6. Neither Ro 5-4864 nor PK 11195 affected the inhibition produced by a stable adenosine analogue, 2-chloroadenosine, which is not a substrate for the adenosine uptake system. 7. Ro 5-4864 did not affect endplate potentials (e.p.ps) in the absence of adenosine, but reduced the amplitude of e.p.ps in the presence of adenosine without affecting miniature e.p.ps. 8. It is suggested that benzodiazepines potentiate the adenosine-effected presynaptic inhibition of neuromuscular transmission by an inhibition of adenosine uptake through activation of peripheral type benzodiazepine receptors.

beta-Eudesmol as an antidote for intoxication from organophosphorus anticholinesterase agents.

beta-Eudesmol, a sesquiterpenol present in Chinese herbs, improved the tetanic contraction impaired by diisopropylfluorophosphate in isolated mouse diaphragm preparations by an inhibition of the regenerative acetylcholine release. The antagonism was enhanced when a small concentration of obidoxime was present. Neither enzyme reactivation nor curare-like action was evident. beta-Eudesmol (300 mg/kg, i.p.) elevated the LD50 of diisopropylfluorophosphate (s.c.) in control mice from 4.2 to 6.4 mg/kg and in mice pretreated with atropine from 7.8 to 10.6 mg/kg. In mice pretreated with atropine and obidoxime, beta-eudesmol showed a greater synergistic effect, increasing the LD50 from 281 to more than 800 mg/kg. beta-Eudesmol also markedly alleviated diisopropylfluorophosphate-induced muscle fasciculation, tremor and convulsion and prolonged the time to death. It is proposed that beta-eudesmol may be added to the standard antidotal regimen (atropine plus obidoxime) for treating organophosphate intoxication.

Obidoxime Antagonizes the Neuromuscular Failure Induced by Neostigmine and Diisopropyl Fluorophosphate via Different Mechanisms.

The efficacies and mechanisms of obidoxime in antagonizing the neuromuscular failure induced by neostigmine and diisopropyl fluorophosphate (DFP) were studied in mouse phrenic nerve/diaphragm preparations. Obidoxime antagonized neostigmine-induced tetanic fade (EC(50): 300 &mgr;M) by inhibiting the regenerative and sustained depolarization during repetitive stimulation. The antagonism was associated with a depression and shortening of single endplate potentials (EPPs) and miniature EPPs (MEPPs). In contrast, the neuromuscular failure induced irreversibly after treatment with DFP and followed by washout was restored by obidoxime at concentrations (EC(50): 0.6 &mgr;M) 500-fold lower than that against neostigmine. The regenerative depolarization was abolished with no depression of single EPPs and MEPPs, and the antagonistic action persisted after washout of obidoxime. The EC(50) of obidoxime was proportionately increased in the presence of increasing concentrations of DFP. Nevertheless, the EC(50) against DFP, at a concentration (30 &mgr;M) 15-fold in excess of that which caused tetanic fade, was still 10-fold lower than that which antagonized neostigmine. In both cases, the amplitudes of train EPPs were increased. It is concluded that obidoxime antagonizes neostigmine-induced neuromuscular failure by a curare-like action but antagonizes DFP by an enzyme reactivation. Copyright 1994 S. Karger AG, Basel

Pharmacological relevance of peripheral type benzodiazepine receptors on motor nerve and skeletal muscle.

1. Effects of agonists and antagonists of peripheral and central benzodiazepine receptors (pBZR and cBZR) on neuromuscular transmission were studied in mouse isolated phrenic nerve-diaphragm preparations. 2. Ro5-4864, a pBZR agonist, had no effect on the neuromuscular transmission but increased muscle contractility and antagonized the tetanic fade induced by neostigmine. 3. Ro5-4864 inhibited the regenerative tonic endplate depolarization caused by repetitive stimulation in the presence of neostigmine without affecting the amplitude and decay time of miniature and evoked single endplate potentials. 4. All the effects of Ro5-4864 were shared by PK11195, a pBZR antagonist, but not by clonazepam and flumazenil, a cBZR agonist and antagonist, respectively. 5. It is suggested that peripheral type benzodiazepine receptors modulate presynaptic function and muscle contraction.

Improvement by diazepam of neuromuscular transmission blocked by anticholinesterase agents in mouse diaphragms.

Effects of diazepam on the neuromuscular transmission blocked by neostigmine were studied in isolated mouse phrenic nerve-diaphragm preparations. Diazepam, in the absence of neostigmine, had no significant effect on the neuromuscular transmission at concentrations lower than 100 microM, except to enhance muscle contractility. Single and train pulses-evoked endplate potentials (e.p.p.s) and miniature e.p.p.s (m.e.p.p.s) were also unaffected. At concentrations of 175 microM or higher, diazepam caused an axonal conduction block. However, neostigmine-induced twitch potentiation, spontaneous fasciculation and tetanic fade were antagonized by diazepam at 3.5-35 microM. Diazepam did not decrease the amplitude of neostigmine-augmented e.p.p.s and m.e.p.p.s but slightly reduced their decay time. The incidence of regenerative depolarization of endplates induced by repetitive stimulation in the presence of neostigmine, was decreased from 92% to 35% junctions and the duration shortened from 650 ms to 230 ms. The amplitude of train e.p.p.s was increased. It is suggested that diazepam reverses the muscle paralysis induced by anticholinesterase agents by inhibiting the regenerative release of acetylcholine.

Antagonism by beta-eudesmol of neostigmine-induced neuromuscular failure in mouse diaphragms.

beta-Eudesmol, a sesquiterpenol extracted from a Chinese herb, Atractylodes lancea, at 10-80 microM, did not affect muscle action potentials, miniature and evoked endplate potentials and acetylcholine-induced depolarization in the presence or absence of neostigmine in mouse phrenic nerve-diaphragms. However, the tetanic fade, muscle fasciculation and twitch potentiation induced by neostigmine were effectively antagonized by 20 microM beta-eudesmol. When trains of pulses were applied to the nerve in the presence of neostigmine, beta-eudesmol reduced the incidence of explosive depolarization of the endplate from 95% to 35-67% of junctions, and shortened the duration when it occurred. Moreover, both the maximal and steady-state depolarizations during repetitive stimulation were reduced while the amplitudes of steady-state endplate potentials were increased. The results suggest that beta-eudesmol antagonized neostigmine-induced neuromuscular failure mainly by a presynaptic action to depress the regenerative release of acetylcholine during repetitive stimulation. The mechanism of antagonism is obviously not tubocurarine-like and it is unrelated to desensitization of acetylcholine channels.

Mechanisms of the synergistic interactions between organic calcium channel antagonists and various neuromuscular blocking agents.

The effects of Mn2+, neomycin and four organic Ca2(+)-channel antagonists (OCA): nicardipine, nifedipine, diltiazem and verapamil on the neuromuscular blocking activities of tubocurarine, succinylcholine (SCh), decamethonium and neomycin were studied in isolated mouse phrenic nerve-diaphragm preparations. The effective concentration of SCh for 50% inhibition (IC50) of single indirect twitch responses were reduced markedly by more than 3-fold when the preparations were pretreated with OCA at 10 microM; the latter alone did not appreciably affect the indirect twitch response or the amplitude of miniature endplate potentials. The neuromuscular blocking effect of decamethonium was also enhanced synergistically by OCA to a similar extent. On the other hand, under the comparable condition. the combined uses of OCA plus tubocurarine or neomycin, neomycin plus tubocurarine or SCh, and Mn2+ plus tubocurarine, SCh or neomycin all resulted in insignificant potentiation. These results suggest that OCA have a specific effect to enhance the agonist effect of depolarizing agents on nicotinic acetylcholine receptors. Nicardipine at 2 microM non-competitively inhibited depolarizations of endplates elicited by SCh and decamethonium and abolished them completely at 10 microM nicardipine. The IC50's in inhibiting endplate potentials and miniature endplate potentials by SCh and decamethonium were also reduced 2 to 3.5-fold by nicardipine. It is inferred that OCA are endowed with a unique capability to allosterically affect the postsynaptic nicotinic acetylcholine receptor, promoting its desensitization liability, hence synergistic interaction with depolarizing agents. Presynaptic effects of OCA are probably not involved.

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.

Selective antagonism to succinylcholine-induced depolarization by alpha-bungarotoxin with respect to the mode of action of depolarizing agents.

1. The interactions of alpha-bungarotoxin or tubocurarine with the neuromuscular block and endplate depolarization induced by succinylcholine (SCh) in the phrenic nerve-diaphragm preparation of mice were studied in order to elucidate the role of depolarization by SCh in the neuromuscular blockade. 2. The SCh concentrations required to depress the indirect twitch response by 20% and the evoked endplate potential in cut muscle preparations by 80% were 10 microm and 6 microM, respectively, while only 2 microM SCh was needed to induce maximal endplate depolarization from -80 mV to about -60 mV. 3. SCh blocked the neuromuscular transmission synergistically with either alpha-bungarotoxin or tubocurarine. There was an initial partial reversal of the neuromuscular inhibition caused by tubocurarine, but not that by alpha-bungarotoxin. 4. alpha-Bungarotoxin (0.025 microM) antagonized SCh (10 microM)-induced depolarization more effectively than it depressed miniature endplate potentials and the antagonism was insurmountable by increasing SCh concentration. By contrast, tubocurarine preferentially depressed miniature endplate potentials and antagonized SCh-depolarization competitively. 5. The above difference was attributed to the irreversible nature of alpha-bungarotoxin binding to acetylcholine receptors, to the slow diffusion of the toxin molecule into the synaptic cleft and thus to the more rapid binding with perijunctional receptors compared with junctional ones. 6. It is concluded that the sustained depolarization of the endplate by SCh results largely from an action on the perijunctional receptor in mice and, unlike cats, the neuromuscular block by SCh is not due to the depolarization per se but rather to a direct attenuation of endplate potential.