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.

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.

[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.

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.

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.

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.

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

Neuromuscular block by verapamil and diltiazem and inhibition of acetylcholine release.

The effects of Ca2+-channel antagonists, verapamil, diltiazem and nifedipine, on the neuromuscular transmission were studied in the isolated mouse phrenic nerve-diaphragm preparations. All 3 drugs increased the twitch response evoked by direct single stimulation at 10-100 microM. The neuromuscular transmission at 0.1 Hz was blocked by verapamil and diltiazem, but not by nifedipine, only at very high concentrations (greater than or equal to 100 microM). In the time course of block, no endplate potential (e.p.p.) could be recorded, whenever the junction failed to elicit an action potential, suggesting that the block is due to an axonal conduction failure. Conduction block became apparent in both axon and muscle at low concentrations (greater than 10 microM) of verapamil and diltiazem at 100 Hz. When the safety margin of neuromuscular transmission was reduced by tubocurarine or low Ca2+ plus high Mg2+, verapamil and diltiazem, but not nifedipine, reduced the single twitch response to nerve stimulation at concentrations that did not cause axon conduction block. The inhibition was dependent on the frequency of nerve stimulation, enhanced by low-Ca2+ and antagonized by high-Ca2+. Verapamil (50 microM) inhibited the mean amplitude of the median size miniature e.p.p. by only 8%, whereas it increased the frequency by 4-5-fold and the proportion of both small and giant miniature e.p.p.s. The e.p.p. amplitude was inhibited by verapamil by about 67% in low-Ca2+ media and by about 38% in normal Tyrode. Similar but somewhat lesser effect was obtained with diltiazem. It is concluded that verapamil and diltiazem, but not nifedipine, inhibit the transmitter release.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

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.

A selective inhibitor of cAMP-specific phosphodiesterase, Ro 20-1724, has no effect on the quantal release of acetylcholine from the mouse phrenic nerve.

The indirect twitch response of the mouse isolated phrenic nerve-diaphragm preparation, partially paralysed by tubocurarine, was restored only by about 10% by Ro 20-1724 at 2 to 280 microM. The solvent vehicle, dimethylsulphoxide, also showed the same effect to a similar extent. Intracellular recordings with glass microelectrodes revealed that Ro 20-1724 (40 microM) affected neither the resting membrane potential, the amplitude and frequency of miniature endplate potentials nor the amplitude of nerve-impulse evoked endplate potentials recorded in curarized preparations. The result indicates that Ro 20-1724 at a concentration four times the IC50 of phosphodiesterase inhibition has no effect on the quantal release of acetylcholine from a mammalian motor nerve and suggests that cAMP has no modulatory effect on the transmitter release.

A comparison of the antagonisms by neostigmine and diaminopyridine against the neuromuscular block caused by cobrotoxin and (+)-tubocurarine.

Cobrotoxin was about 11-fold more potent than (+)-tubocurarine on a weight basis in blocking neuromuscular transmission in mouse isolated phrenic nerve-diaphragm preparations. Neostigmine and diaminopyridine increased the concentrations of cobrotoxin for 70% inhibition of indirect contraction by 290 and 320%, and increased those of (+)-tubocurarine by 180 and 230%, respectively. More than additive increases were obtained when neostigmine and diaminopyridine were used simultaneously. Cobrotoxin, however, was only 6-fold more toxic than (+)-tubocurarine after intraperitoneal injection in mice. The lethal dose of (+)-tubocurarine was increased by 80% when both antidotes were used together, but only by 15-20% when used alone. In contrast, the lethality of cobrotoxin was not decreased by these drugs. Unexpectedly, the time to death after treatment with cobrotoxin was shortened when mice were pretreated with these antidotes.

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.

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.

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.

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.

Effect of clonidine on neuromuscular transmission and the nicotinic acetylcholine receptor.

The effects of clonidine on neuromuscular transmission were investigated in the mouse phrenic nerve-diaphragms and chicken biventer cervicis. Clonidine inhibited the indirect twitch response dose-dependently and reversibly without an effect on the direct response of the muscles to electrical stimulation and KCl. This effect was antagonized effectively by diaminopyridine but not by yohimbine, phentolamine or physostigmine. The quantal content was not affected although the amplitudes of end-plate potential (epp) and spontaneous miniature epp (mepp) were markedly depressed. Clonidine also decreased the slope of the ACh dose-response curve and maximal response in denervated mouse diaphragms as well as the carbachol response in the chinck muscle. In the latter, ACh response was not depressed by clonidine probably because of its inherent anticholinesterase activity. Clonidine facilitated the fading of ACh-contracture either in mouse or chick muscle. It is concluded that clonidine impairs the neuromuscular transmission by a noncompetitive blockade of ACh receptors, most likely affecting the ACh channel but not the recognition site of the ACh receptor. Its inhibitory effect is not mediated by alpha 2-adrenoceptor, suggesting that there is no alpha 2-adrenoceptor on the motor nerve terminal to modulate the transmitter release.