Clementina Bianchi

A new selective antagonist of the nociceptin receptor

[Phe1Ψ(CH2‐NH)Gly2]NC(1‐13)NH2 has been tested in the electrically stimulated guinea pig ileum and mouse vas deferens, two nociceptin sensitive preparations. The new compound showed per se little or no effect in the two tissues, but it displaced to the right the concentration‐response curves of nociceptin in a concentration‐dependent manner. Schild analyses of the data indicated a competitive type of antagonism and pA2 values of 7.02 and 6.75 in the guinea‐pig ileum and the mouse vas deferens, respectively. At 10 μM [Phe1Ψ(CH2‐NH)Gly2]NC(1‐13)NH2 does not modify either the inhibitory effect of deltorphin I (the selective δ opioid receptor agonist) in the mouse vas deferens or that of dermorphine (the selective μ opioid receptor agonist) in the guinea‐pig ileum. The present findings indicate that [Phe1Ψ(CH2‐NH)Gly2]NC(1‐13)NH2 is a selective antagonist of the nociceptin receptor.

Characterization of [Nphe1]nociceptin(1‐13)NH2, a new selective nociceptin receptor antagonist

Nociceptin (orphanin FQ) is a novel neuropeptide capable of inducing a variety of biological actions via activation of a specific G‐protein coupled receptor. However, the lack of a selective nociceptin receptor antagonist has hampered our understanding of nociceptin actions and the role of this peptide in pathophysiological states. As part of a broader programme of research, geared to the identification and characterization of nociceptin receptor ligands, we report that the novel peptide [Nphe1]nociceptin(1‐13)NH2 acts as the first truly selective and competitive nociceptin receptor antagonist and is devoid of any residual agonist activity. [Nphe1]nociceptin(1‐13)NH2 binds selectively to recombinant nociceptin receptors expressed in Chinese hamster ovary (CHO) cells (pKi 8.4) and competitively antagonizes the inhibitory effects of nociceptin (i) on cyclic AMP accumulation in CHO cells (pA2 6.0) and (ii) on electrically evoked contractions in isolated tissues of the mouse, rat and guinea‐pig with pA2 values ranging from 6.0 to 6.4. [Nphe1]nociceptin(1‐13)NH2 is also active in vivo, where it prevents the pronociceptive and antimorphine actions of intracerebroventricularly applied nociceptin, measured in the mouse tail withdrawal assay. Moreover, [Nphe1]nociceptin(1‐13)NH2 produces per se a dose dependent, naloxone resistant antinociceptive action and, at relatively low doses, potentiates morphine‐induced analgesia. Collectively our data indicate that [Nphe1]nociceptin(1‐13)NH2, acting as a nociceptin receptor antagonist, may be the prototype of a new class of analgesics.

Noradrenaline inhibition of acetylcholine release from guinea-pig brain.

The effect of noradrenaline (NA) on acetylcholine (ACh) release from guinea-pig brain was investigated in superfused cerebral cortex slices and in unrestrained unanaesthetized animals provided with epidural cups. The amine reduced the ACh release from electrically stimulated tissue and its effect was antagonized by phentolamine and phenoxybenzamine, but not by propranolol and spiroperidol. The injection of NA (150 microgram) into the cerebral ventricles caused sedation, E.Co.G. synchronization and reduced ACh outflow from the parietal cortex. This inhibition was counteracted by alpha-blocking agents. A lower dose of NA (50 microgram) did not change the behaviour, but produced a late increase in ACh outflow, prevented by spiroperidol. These results fit well with the hypothesis that NA restrains, via alpha-receptors, the ACh secretion from the nerve endings and indirectly support the view that the amine reduces the firing rate of the corticopetal cholinergic neurones. The late increase in ACh outflow, observed in vivo, may be referred to secondary activation of the dopaminergic neurones, known to enhance the cortical ACh release in this animal species.

Review Article Reciprocaldopamine-glutamatemodulation of release in the basalganglia

Abstract Dopaminergic and glutamatergic transmissions have long been known to interactatmultiple levels in the basal ganglia to modulate motor and cognitive functions. Oneimportantaspect of their interactions is represented by the reciprocal modulation of release. Thistopic hasbeen the object of interest since the late 70s, particularly in the striatum and inmidbraindopaminergic areas (substantia nigra and ventral tegmental area). Analysisofglutamate-dopamine interactions in the control of each others release is complicated by thefactthat both glutamate and dopamine act on multiple receptor subtypes which can exertdifferenteffects. Therefore, glutamatergic modulation of dopamine release has been reviewed byanalyzingthe effects of glutamatergic selective receptor agonists and antagonists in the striatum (bothmotor and limbic portions) and in midbrain dopaminergic areas, as revealed by in vitro (slices,cell cultures, synaptosomes) and in vivo (push-pull, microdialysis and voltammetrytechniques)experimental approaches. The same approach has been followed for dopaminergicmodulation ofglutamate release. The facilitatory nature of glutamate modulating both presynapticand dendriticdopamine release has clearly emerged from in vitro studies. However, evidence ispresented that,at least in the striatum and in the nucleus accumbens of awake rats,glutamate-mediated inhibitoryeffects may also occur. In vitro and in vivo experiments in thestriatum and midbraindopaminergic areas mainly depict dopamine as an inhibitory modulator ofglutamate release.However, in vivo studies reporting dopamine D1 receptor mediated facilitatoryeffects are alsoconsidered. Therefore, the general notion that glutamate and dopamine actoppositely to regulateeach others release, is only partly supported by the available data.Conversely, the nature of theinteraction between the two neurotransmitters seems to varydepending on the experimentalapproach, the brain area considered and the subtype of receptorinvolved.

Glutamate antagonists prevent morphine withdrawal in mice and guinea pigs

The effects of excitatory amino acid antagonists on increased cortical acetylcholine release and behavioral hyperactivity induced by naloxone in morphine tolerant guinea pigs and mice were studied. The results show that the N-methyl-D-aspartic acid (NMDA) antagonist MK-801 (0.1-1 mg/kg, i.p.) injected 30 min before naloxone (3 mg/kg, s.c.) dose-dependently prevented the neurochemical and behavioral signs of morphine withdrawal in guinea pigs and mice. The non-selective antagonist glutamic acid diethylester only at 100 mg/kg i.p. reduced the naloxone-induced increase of cortical acetylcholine release without affecting the behavioral changes. These findings indicate that the activation of excitatory amino acid receptors, mainly the NMDA receptors, plays a relevant role in the expression of opiate abstinence.

The mouse vas deferens: a pharmacological preparation sensitive to nociceptin

The newly discovered neuropeptide, nociceptin (alias orphanin FQ), was tested for its potential direct effects, as well as for its ability to modify the electrically evoked contractions in several isolated organs suspended in vitro. The electrically stimulated mouse vas deferens is a sensitive preparation on which nociceptin exerts an inhibitory effect which is not affected by naloxone. The mouse vas deferens is therefore proposed as a bioassay for nociceptin and related compounds.

Pharmacological characterization of the nociceptin receptor mediating hyperalgesia in the mouse tail withdrawal assay

1 The newly discovered neuropeptide nociceptin (NC) has recently been reported to be the endogenous ligand of the opioid‐like orphan receptor. Despite its structural similarity to opioids, when injected intracerebroventricularly (i.c.v.) in the mouse, NC exerts a direct hyperalgesic effect and reverses opioid‐induced analgesia. In the present investigation, these two effects of NC were evaluated under the same experimental conditions; in addition, a pharmacological characterization of the receptor mediating these central effects of NC was attempted. 2 NC caused a dose dependent (0.1–10 nmol/mouse), naloxone‐insensitive reduction of tail withdrawal latency with a maximal effect of about 50% of the reaction time observed in saline injected mice. In the same range of doses, NC inhibited morphine (1 nmol/mouse) induced analgesia. 3 The effects of the natural peptide were mimicked by NCNH2 and NC(1–13)NH2 (all tested at 1 nmol/mouse) while 1 nmol NC(1–9)NH2 was found to be inactive either in reducing tail withdrawal latency or in preventing morphine analgesia. 4 [Phe1ψ(CH2‐NH)Gly2]NC(1–13)NH2 ([F/G]NC(1–13)NH2), which has been shown to antagonize NC effects in the mouse vas deferens, acted as an agonist, mimicking NC effects in both the experimental paradigms. In addition, when NC and [F/G]NC(1–13)NH2 were given together, their effects were additive. 5 These results demonstrate that both the direct hyperalgesic action and the anti‐morphine effect of NC can be studied under the same experimental conditions in the mouse tail withdrawal assay. Moreover, the pharmacological characterization of the NC functional site responsible for these actions compared with the peripherally active site, indicates the existence of important differences between peripheral and central NC receptors.

Facilitation of gaba release by neurotensin is associated with a reduction of dopamine release in rat nucleus accumbens

The main aim of the present study was to investigate the effects of local perfusion with the tridecapeptide neurotensin on extracellular GABA and dopamine levels in the nucleus accumbens of the halothane-anaesthetized rat, using in vivo microdialysis. In an initial set of characterization studies we examined the Na+ dependence of neurotransmitter release by local perfusion with ouabain, veratridine and tetrodotoxin. Local perfusion with the Na+ ATPase inhibitor ouabain (10 microM) or the Na+ channel agonist veratridine (20 microM) perfused into the nucleus accumbens increased both extracellular GABA and dopamine levels. The Na+ channel antagonist tetrodotoxin (1 microM) consistently decreased (24% of basal) dopamine levels, while even at 10 microM it did not affect GABA. However, tetrodotoxin (10 microM) abolished the veratridine-induced increase in both GABA and dopamine, demonstrating that Na(+)-dependent neuronal activity is involved in this release mechanism. In a second set of experiments a hypothesis for a functional link between neurotensin, dopamine and GABA in the medial nucleus accumbens was tested. Towards this aim, the effects of local perfusion with a high 1 microM concentration of neurotensin into the nucleus accumbens increased both GABA (210% of basal value) and dopamine (145% of basal) release. However, a low (10 nM) concentration of neurotensin again increased GABA release (160% of basal), but decreased that of dopamine (75% of basal value). Furthermore, the local perfusion with the GABAA receptor antagonist bicuculline abolished the neurotensin (10 nM) induced inhibition of dopamine release without affecting the increase in GABA release. These findings suggest that neurotensin modulates both GABA and dopamine neurotransmission in the nucleus accumbens.(ABSTRACT TRUNCATED AT 250 WORDS)

5-HT1A agonists increase and 5-HT3 agonists decrease acetylcholine efflux from the cerebral cortex of freely-moving guinea-pigs

1 The influence of 5‐hydroxytryptamine1A (5‐HT1A), 5‐HT2 and 5‐HT3 agonists and antagonists on acetylcholine (ACh) release from the cerebral cortex was studied in freely moving guinea‐pigs. 2 8‐Hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT, 0.01–1 mg kg−1, s.c.) caused the 5‐HT syndrome and dose‐dependently increased ACh release. Ru 24969 (1–10 mg kg−1, s.c.) shared the same effects, but it was less potent. (−)‐Propranolol (5 mg kg−1) and metitepine (2 mg kg−1) prevented these behavioural and neurochemical responses. 3 (±)‐1(4‐Iodo‐2,5‐dimethoxyphenyl)2‐aminopropane (DOI) up to 2 mg kg−1 did not modify ACh release and ketanserin (0.5 mg kg−1) was ineffective on 5‐HT‐induced changes of ACh outflow. 4 2‐Methyl‐5‐HT (500 μg, i.c.v.) and 5‐HT (500 μg, i.c.v.) plus metitepine (2 mg kg−1, s.c.) inhibited the gross behaviour and ACh release. ICS 205–930 (0.5 mg kg−1) prevented these responses. 5 2‐Methyl‐5‐HT, up to 10 μmoll−1, and 8‐OH‐DPAT, up to 0.1 μmoll−1, (like 5‐HT) did not change [3H]‐choline efflux from cerebral cortex slices. 6 These results suggest that exogenous 5‐HT and related selective agonists modulate guinea‐pig cortical cholinergic structures through 5‐HT1A and 5‐HT3 receptors. The stimulation of 5‐HT1A autoreceptors may lead to disinhibition of the cholinergic cells, tonically inhibited by the tryptaminergic control. Conversely, the stimulation of 5‐HT3 receptors inhibits ACh release, possibly through an interneurone. No direct 5‐HT modulation of the cholinergic nerve endings was found.

Blockade of Nociceptin/Orphanin FQ Receptor Signaling in Rat Substantia Nigra Pars Reticulata Stimulates Nigrostriatal Dopaminergic Transmission and Motor Behavior

A multidisciplinary approach was followed to investigate whether the opioid-like peptide nociceptin/orphanin FQ (N/OFQ) regulates the nigrostriatal dopaminergic pathway and motor behavior. Nigrostriatal dopaminergic cells, which express N/OFQ peptide (NOP) receptors, are located in the substantia nigra pars compacta and extend their dendrites in the substantia nigra pars reticulata, thereby modulating the basal ganglia output neurons. In vitro electrophysiological recordings demonstrated that N/OFQ hyperpolarized the dopaminergic cells of the substantia nigra pars compacta and inhibited their firing activity. In vivo dual-probe microdialysis showed that N/OFQ perfused in the substantia nigra pars reticulata reduced dopamine release in the ipsilateral striatum, whereas UFP-101 ([Nphe1,Arg14,Lys15]N/OFQ(1-13)-NH2) (a selective NOP receptor peptide antagonist) stimulated it. N/OFQ microinjected in the substantia nigra pars reticulata impaired rat performance on a rotarod apparatus, whereas UFP-101 enhanced it. Electromyography revealed that N/OFQ and UFP-101 oppositely affected muscle tone, inducing relaxation and contraction of triceps, respectively. The selective NOP receptor nonpeptide antagonist J-113397 (1-[3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H benzimidazol-2-one), either injected intranigrally or given systemically, also elevated striatal dopamine release and facilitated motor activity, confirming that these effects were caused by blockade of endogenous N/OFQ signaling. The inhibitory role played by endogenous N/OFQ on motor activity was additionally strengthened by the finding that mice lacking the NOP receptor gene outperformed wild-type mice on the rotarod. We conclude that NOP receptors in the substantia nigra pars reticulata, activated by endogenous N/OFQ, drive a physiologically inhibitory control on motor behavior, possibly via modulation of the nigrostriatal dopaminergic pathway.