Synthesis and pharmacology of 6-substituted benztropines: discovery of novel dopamine uptake inhibitors possessing low binding affinity to the dopamine transporter.

A series of 6alpha- and 6beta-substituted benztropines were synthesized. A marked enantioselectivity was observed for the 6beta-methoxylated benztropines, the (1R)-isomers being more potent than the corresponding (1S) compounds. The racemic 6alpha-methoxy-3-(4',4' '-difluorodiphenylmethoxy)tropane (5 g) was the most potent compound. It has been found that modifications at the 6-position of benztropine might reduce the DAT binding affinity, maintaining otherwise a significant dopamine uptake inhibitory activity. A reinvestigation of the absolute configuration of 6beta-methoxytropinone proved the 6R configuration for the (+)-enantiomer.

Striatal glutamate release evoked in vivo by NMDA is dependent upon ongoing neuronal activity in the substantia nigra, endogenous striatal substance P and dopamine.

The aim of the present microdialysis study was to investigate whether the increase in striatal glutamate levels induced by intrastriatal perfusion with NMDA was dependent on the activation of extrastriatal loops and/or endogenous striatal substance P and dopamine. The NMDA-evoked striatal glutamate release was mediated by selective activation of the NMDA receptor-channel complex and action potential propagation, as it was prevented by local perfusion with dizocilpine and tetrodotoxin, respectively. Tetrodotoxin and bicuculline, perfused distally in the substantia nigra reticulata, prevented the NMDA-evoked striatal glutamate release, suggesting its dependence on ongoing neuronal activity and GABA(A) receptor activation, respectively, in the substantia nigra. The NMDA-evoked glutamate release was also dependent on striatal substance P and dopamine, as it was antagonized by intrastriatal perfusion with selective NK(1) (SR140333), D(1)-like (SCH23390) and D(2)-like (raclopride) receptor antagonists, as well as by striatal dopamine depletion. Furthermore, impairment of dopaminergic transmission unmasked a glutamatergic stimulation by submicromolar NMDA concentrations. We conclude that in vivo the NMDA-evoked striatal glutamate release is mediated by activation of striatofugal GABAergic neurons and requires activation of striatal NK(1) and dopamine receptors. Endogenous striatal dopamine inhibits or potentiates the NMDA action depending on the strength of the excitatory stimulus (i.e. the NMDA concentration).

Early and delayed glutamate effects in rat primary cortical neurons. Changes in the subcellular distribution of protein kinase C isoforms and in intracellular calcium concentration.

Glutamate-induced changes in the subcellular distribution of protein kinase C isoforms and in the intracellular calcium concentration were investigated in rat primary cortical neurons. Western blot analysis of protein kinase C isoforms (alpha, beta1, beta2, gamma, delta, epsilon, zeta and theta), performed 30 min after a 10 min treatment with 30 microM glutamate, revealed a decrease in the total beta1 (-24%) and beta2 (-40%) isoform levels, without any significant change in any of the other isozymes. All conventional isoforms translocated to the membrane compartment, while delta, epsilon, zeta and theta; maintained their initial subcellular distribution. Twenty-four hours after glutamate treatment, the total protein kinase C labelling had increased, particularly the epsilon isoform, which accounted for 34% of the total densitometric signal. At this time, protein kinase C beta1, delta, epsilon and zeta isoforms were mainly detected in the membrane compartment, while gamma and theta; signals were displayed almost solely in the cytosol. Basal intracellular calcium concentration (FURA 2 assay) was concentration-dependently increased (maximum effect +77%) 30 min, but not 24h after a 10 min glutamate (10-100 microM) treatment, while the net increase induced by electrical stimulation (10 Hz, 10s) was consistently reduced (maximum effect -64%). The N-methyl-d-aspartate receptor antagonist, MK-801, 1 microM, prevented glutamate action both 30 min and 24 h after treatment, while non-selective protein kinase C inhibitors, ineffective at 30 min, potentiated it at 24 h. These findings show that protein kinase C isoforms are differently activated and involved in the early and delayed glutamate actions, and that the prevailing effect of their activation is neuroprotective.

Blockade of nociceptin/orphanin FQ transmission in rat substantia nigra reverses haloperidol-induced akinesia and normalizes nigral glutamate release.

We recently showed that pharmacological blockade of nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors located in the substantia nigra stimulates the nigrostriatal dopaminergic pathway and motor behavior (Marti et al. J. Neurosci. 2004, 24, 6659-6666). To investigate whether such motor-stimulating action was dependent on functional dopaminergic transmission, the selective NOP receptor peptide antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101) was microinjected into the substantia nigra reticulata of rats made cataleptic by systemic haloperidol administration. UFP-101 reduced haloperidol-induced akinesia as measured by immobility time in the bar test. UFP-101 also induced contralateral turning in cataleptic rats. To investigate the mechanisms involved in the anti-akinetic action of UFP-101, nigral glutamate release was monitored by microdialysis technique. The anti-akinetic action of UFP-101 correlated with normalization of nigral glutamate release, previously elevated by haloperidol injection. We conclude that endogenous N/OFQ in the substantia nigra sustains akinesia generated by impaired DA transmission and subthalamic nucleus overactivation. NOP receptor antagonists may be beneficial in the symptomatic therapy of parkinsonism, via normalization of subthalamonigral glutamatergic transmission.

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.

Pharmacological profile of nociceptin/orphanin FQ receptors regulating 5-hydroxytryptamine release in the mouse neocortex.

A synaptosomal preparation was employed to pharmacologically characterize the role of presynaptic nociceptin/orphanin FQ (N/OFQ) receptors (NOP receptors) in the regulation of 5-hydroxytryptamine release in the Swiss mouse neocortex. In the present study, the NOP receptor ligands N/OFQ, Ac-RYYRWK-NH(2) and [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)-NH(2) inhibited the K(+)-induced [(3)H]-5-HT overflow with similar maximal effects ( approximately -35%) but different potencies (pEC(50) of 8.56, 8.35 and 7.23, respectively). The novel agonist [Arg(14),Lys(15)]N/OFQ also inhibited [(3)H]-5-HT overflow, but the concentration-response curve was biphasic and the efficacy higher ( approximately -45%). Receptor selectivity of NOP receptor agonists was demonstrated by showing that synaptosomes from NOP receptor knockout mice were unresponsive to N/OFQ, [Arg(14),Lys(15)]N/OFQ and [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)-NH(2) but maintained full responsiveness to endomorphin-1. Moreover, the inhibitory effect of N/OFQ was prevented by peptide ([Nphe(1)]N/OFQ(1-13)-NH(2) and UFP-101) and nonpeptide (J-113397 and JTC-801) NOP receptor selective antagonists. Desensitization occurred under perfusion with high (3 and 10 microm) N/OFQ concentrations. This phenomenon was prevented by the protein kinase C inhibitor, bisindolylmaleimide. Moreover, N/OFQ-induced desensitization did not affect mu opioid receptor responsiveness. Finally, it was observed in a similar preparation of rat cerebrocortical synaptosomes, although it was induced by higher N/OFQ concentrations than that used in the mouse. Together, these findings indicate that presynaptic NOP receptors inhibit 5-hydroxytryptamine release in the mouse neocortex. Based on present and previous studies, we conclude that NOP receptors in the mouse are subtly different from the homologous receptor population in the rat, strengthening the view that there exist species differences in the pharmacology of central NOP receptors.

Nociceptin inhibition of acetylcholine efflux from different brain areas.

The effects of nociceptin on [3H]choline [3H](Ch) efflux from electrically-stimulated rat cortical, hippocampal and caudatal slices as well as from KCl-depolarized synaptosomes and tetrodotoxin-pretreated slices have been studied. The inhibition of electrically evoked [3H]Ch efflux by nociceptin (0.03-3 microM) was moderate (max -33%), more evident in the neocortex than in the hippocampus and was prevented by [Nphe1]NC(1-13)NH(2) 10 microM. This effect was absent in the caudate nucleus, in cortical synaptosomes and in tetrodotoxin-pretreated cortical slices. These data point to a distinct localization of NOP receptors in the different brain areas and to a prevailing inhibitory control by nociceptin on the cortical cholinergic input at pre-terminal level. However, the reported impairment of neocortical and hippocampal function by nociceptin may be referred to the inhibition not only of the cholinergic signal but also of other transmitters such as glutamate.

Differential responsiveness of rat striatal nerve endings to the mitochondrial toxin 3-nitropropionic acid: implications for Huntington's disease.

Rat striatal synaptosomes and slices were used to investigate the responsiveness of different populations of nerve terminals to 3-nitropropionic acid (3-NP), a suicide inhibitor of the mitochondrial enzyme succinate dehydrogenase, and to elucidate the ionic mechanisms involved. 3-NP (0.3-3 mm) stimulated spontaneous gamma-aminobutyric acid (GABA), glutamate and [3H]-dopamine efflux but left unchanged acetylcholine efflux from synaptosomes. This effect was associated with a >70% inhibition of succinate dehydrogenase, as measured in the whole synaptosomal population. The facilitation was not dependent on extracellular Ca2+ but relied on voltage-dependent Na+ channel opening, because it was prevented by tetrodotoxin and riluzole. 3-NP also elevated spontaneous glutamate efflux from slices but in a tetrodotoxin-insensitive way. To investigate whether energy depletion could change the responsiveness of nerve endings to a depolarizing stimulus, synaptosomes were pretreated with 3-NP and challenged with pulses of KCl evoking 'quasi-physiological' neurotransmitter release. 3-NP potentiated the K+-evoked GABA, glutamate and [3H]-dopamine release but inhibited the K+-evoked acetylcholine release. The 3-NP induced potentiation of GABA release was Ca2+-dependent and prevented by tetrodotoxin and riluzole whereas the 3-NP-induced inhibition of acetylcholine release was tetrodotoxin- and riluzole-insensitive but reversed by glipizide, an ATP-dependent K+ channel inhibitor. We conclude that the responsiveness of striatal nerve endings to 3-NP relies on activation of different ionic conductances, and suggest that the selective survival of striatal cholinergic interneurons following chronic 3-NP treatment (as in models of Huntington's disease) may rely on the opening of ATP-dependent K+ channels, which counteracts the fall in membrane potential as a result of mitochondrial impairment.

Plasticity of glutamatergic control of striatal acetylcholine release in experimental parkinsonism: opposite changes at group-II metabotropic and NMDA receptors.

To investigate whether adaptive changes of glutamatergic transmission underlie dysfunction of the cholinergic system in experimental parkinsonism, the effects of group-II metabotropic glutamate and NMDA receptor ligands on acetylcholine release was studied in striatal slices and synaptosomes obtained from naive rats, 6-hydroxydopamine hemi-lesioned rats and 6-hydroxydopamine hemi-lesioned rats chronically treated with levodopa (L-DOPA) plus benserazide (non-dyskinetic). Group-II metabotropic glutamate receptor agonists LY354740, DCG-IV and L-CCG-I inhibited the electrically-evoked endogenous acetylcholine release from slices, while NMDA facilitated it. LY354740 also inhibited K+-evoked acetylcholine release from synaptosomes. LY354740-induced inhibition was prevented by the group-II metabotropic glutamate receptor antagonist LY341495. In hemi-parkinsonian rats, sensitivity towards LY354740 was reduced while that to NMDA was enhanced in the lesioned (denervated) compared with unlesioned striatum. Moreover, dizocilpine inhibited acetylcholine release in the lesioned compared with unlesioned striatum. Chronic treatment with L-DOPA normalized sensitivity towards glutamatergic agonists. We conclude that striatal dopamine denervation results in plastic changes at group-II metabotropic glutamate and NMDA receptors that may shift glutamatergic control of acetylcholine release towards facilitation. From a clinical perspective, L-DOPA and NMDA antagonists appear effective in counteracting overactivity of striatal cholinergic interneurones associated with Parkinson's disease.

Pharmacological profiles of presynaptic nociceptin/orphanin FQ receptors modulating 5-hydroxytryptamine and noradrenaline release in the rat neocortex.

1 The pharmacological profiles of presynaptic nociceptin/orphanin FQ (N/OFQ) peptide receptors (NOP) modulating 5-hydroxytryptamine (5-HT) and noradrenaline (NE) release in the rat neocortex were characterized in a preparation of superfused synaptosomes challenged with 10 mM KCl. 2 N/OFQ concentration-dependently inhibited K(+)-evoked [(3)H]-5-HT and [(3)H]-NE overflow with similar potency (pEC(50) approximately 7.9 and approximately 7.7, respectively) and efficacy (maximal inhibition approximately 40%). 3 N/OFQ (0.1 micro M) inhibition of [(3)H]-5-HT and [(3)H]-NE overflow was antagonized by selective NOP receptor antagonists of peptide ([Nphe(1)]N/OFQ(1-13)NH(2) and UFP-101; 10 and 1 microM, respectively) and non-peptide (J-113397 and JTC-801; both 0.1 microM) nature. Antagonists were routinely applied 3 min before N/OFQ. However, a 21 min pre-application time was necessary for J-113397 and JTC-801 to prevent N/OFQ inhibition of [(3)H]-NE overflow. 4 The NOP receptor ligand [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) ([F/G]N/OFQ(1-13)NH(2); 3 microM) did not affect K(+)-evoked [(3)H]-NE but inhibited K(+)-evoked [(3)H]-5-HT overflow in a UFP-101 sensitive manner. [F/G]N/OFQ(1-13)NH(2) antagonized N/OFQ actions on both neurotransmitters. 5 The time-dependency of JTC-801 action was studied in CHO cells expressing human NOP receptors. N/OFQ inhibited forskolin-stimulated cAMP accumulation and JTC-801, tested at different concentrations (0.1-10 microM) and pre-incubation times (0, 40 and 90 min), antagonized this effect in a time-dependent manner. The Schild-type analysis excluded a competitive type of antagonism. 6 We conclude that presynaptic NO receptors inhibiting 5-HT and NE release in the rat neocortex have similar pharmacological profiles. Nevertheless, they can be differentiated pharmacologically on the basis of responsiveness to [F/G]N/OFQ(1-13)NH(2) and time-dependent sensitivity towards non-peptide antagonists.

Dual effects of 5-HT4 receptor activation on GABA release from guinea pig hippocampal slices.

The effects of BIMU-8, a 5-HT4 receptor agonist, were studied on GABA release in guinea pig hippocampal slices. BIMU-8 did not modify GABA outflow at rest but did display a complex action in electrically stimulated slices: at low concentrations it increased, and at higher concentrations inhibited, GABA release. These responses were competitively counteracted by GR 125487, a selective 5-HT4 receptor antagonist. The dual effects of BIMU-8 are consistent with its indirect cholinergic action since the M1 and M3 antagonist, 4-DAMP, prevented BIMU-8-elicited GABA facilitation, whereas the M2 antagonist AFDX-116 cancelled GABA inhibition. These results provide evidence that serotonin exerts a complex modulation on the GABAergic system, via 5-HT4 receptors, and suggest that the amine releases acetylcholine which, in turn, bidirectionally modulates GABA release.

Striatal dopamine-NMDA receptor interactions in the modulation of glutamate release in the substantia nigra pars reticulata in vivo: opposite role for D1 and D2 receptors.

Dual probe microdialysis was employed in conscious rats to investigate whether endogenous dopamine is involved in the stimulation of glutamate release in the substantia nigra pars reticulata following striatal NMDA receptor activation. Intrastriatal perfusion with NMDA (1 and 10 microm) facilitated nigral glutamate release (dizocilpine- and tetrodotoxin-sensitive). The D2 dopamine receptor antagonist raclopride increased spontaneous nigral glutamate release and caused a leftward shift in the NMDA sensitivity, lowering NMDA effective concentrations to submicromolar levels. Conversely, the D1 antagonist SCH23390 prevented the effect of NMDA (1 microm) and caused a rightward shift in the NMDA sensitivity. It was tested whether the antagonist effects were due to dopamine receptor blockade or increased tone on D1/D2 receptors. SCH23390 prevented the raclopride-induced enhancement of spontaneous but not NMDA-evoked glutamate release while raclopride left unchanged the SCH23390-induced inhibition. The physiopathological relevance of the dopaminergic modulation was strengthened by perfusing NMDA in the dopamine-depleted striatum of hemiparkinsonian rats. Nigral glutamate responsiveness to NMDA was enhanced as with raclopride. We conclude that endogenous striatal dopamine regulates both spontaneous and NMDA-induced nigral glutamate release via an opposite control mediated by D1 facilitatory and D2 inhibitory receptors. Alterations of this control may subserve the motor symptoms of Parkinson's disease.

Direct and indirect inhibition by nociceptin/orphanin FQ on noradrenaline release from rodent cerebral cortex in vitro.

1 The modulation exerted by nociceptin/orphanin FQ (NC) on noradrenaline (NE) release in rodent cerebral cortex slices and synaptosomes was studied. 2 Rat, mouse and guinea-pig cortical slices and synaptosomes were preincubated with 0.1 micro M [(3)H]-NE and superfused. NE release was evoked by 2 min of electrical (3 Hz) stimulation in slices and by 1 min pulse of 10 mM KCl in synaptosomes. 3 In rat cortical slices, 0.01-3 micro M NC reduced the evoked [(3)H]-NE efflux (E(max)-54%), with a bell-shaped concentration-response curve, which regained its monotonic nature in the presence of either 0.1 micro M naloxone (NX) or 30 micro M bicuculline. In synaptosomes, the NC effect curve was sygmoidal in shape and reached a plateau at 1 micro M concentration. 4 In the rat, both 1 micro M [Phe(1)psi(CH(2)-NH)Gly(2)]NC(1-13)NH(2) and 10 micro M [Nphe(1)]NC(1-13)NH(2) (NPhe) antagonised NC-induced inhibition, without per se modifying [(3)H]-NE efflux. The effects of 0.3-1 micro M NC concentrations were partially prevented by 1 micro M NX; 1 micro M D-Phe-Cys-Thr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) was also an effective antagonist, but 0.1 micro M norbinaltorphimine was not. 5 In the mouse cerebral cortex, NC-induced inhibition of NE release (pEC(50) 6.87, E(max)-61%, in the slices) was prevented by Nphe but was NX-insensitive. In guinea-pig cortical slices, NC effect (pEC(50) 6.22, E(max)-38%) was prevented by Nphe, but was NX-insensitive. 6 These findings demonstrate that NC inhibits NE release from rodent cerebral cortex via presynaptically located ORL(1) receptors. In the rat, micro opioid and GABA(A) receptors are involved as well.

Evaluation of the Dmt-Tic pharmacophore: conversion of a potent delta-opioid receptor antagonist into a potent delta agonist and ligands with mixed properties.

Analogues of the 2',6'-dimethyl-L-tyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) pharmacophore were prepared to test the hypothesis that a "spacer" and a third aromatic center in opioid peptides are required to convert a delta-antagonist into ligands with delta-agonist or with mixed delta-antagonist/mu-agonist properties. Potent delta-agonists and bifunctional compounds with high delta- and mu-opioid receptor affinities were obtained by varying the spacer length [none, NH-CH(2), NH-CH(2)-CH(2), Gly-NH-CH(2)] and C-terminal aromatic nucleus [1H-benzimidazole-2-yl, phenyl (Ph) and benzyl groups]. C-terminal modification primarily affected mu-opioid receptor affinities, which increased maximally 1700-fold relative to the prototype delta-antagonist H-Dmt-Tic-NH(2) and differentially modified bioactivity. In the absence of a spacer (1), the analogue exhibited dual delta-agonism (pEC(50), 7.28) and delta-antagonism (pA(2), 7.90). H-Dmt-Tic-NH-CH(2)-1H-benzimidazole-2-yl (Bid) (2) became a highly potent delta-agonist (pEC(50), 9.90), slightly greater than deltorphin C (pEC(50), 9.56), with mu-agonism (pE(50), 7.57), while H-Dmt-Tic-Gly-NH-CH(2)-Bid (4) retained potent delta-antagonism (pA(2), 9.0) but with an order of magnitude less mu-agonism. Similarly, H-Dmt-Tic-Gly-NH-Ph (5) had nearly equivalent high delta-agonism (pEC(50), 8.52) and mu-agonism (pEC(50), 8.59), while H-Dmt-Tic-Gly-NH-CH(2)-Ph (6) whose spacer was longer by a single methylene group exhibited potent delta-antagonism (pA(2), 9.25) and very high mu-agonism (pEC(50), 8.57). These data confirm that the distance between the Dmt-Tic pharmacophore and a third aromatic nucleus is an important criterion in converting Dmt-Tic from a highly potent delta-antagonist into a potent delta-agonist or into ligands with mixed delta- and mu-opioid properties.