Genomic pharmacology: more intracellular sites for drug action.
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Amino Acid Sequence↗
Biomedical subjects
P M Laduron
Publications and source records attributed to P M Laduron.
Neuroprotective actions of riluzole in rodent models of global and focal cerebral ischaemia.
Riluzole (2 amino 6-trifluoromethoxybenzothiazole), when administered at 4 and 8 mg/kg i.p., 0.5, 4.5, 24 and 28 h after the initiation of ischaemia, significantly reduced the prevalence of slow wave, and increased the proportion of higher frequency activity seen in the quantified electrocorticogram (ECoG), during the weeks that followed a 6 min bilateral occlusion of the common carotid arteries in the Mongolian gerbil. In focal ischaemia, provoked in Fischer rats following the occlusion of the middle cerebral artery, administration of riluzole (8 mg/kg) at 30 min and 24.5 h post occlusion significantly reduced the volume of infarcted cortex. These activities of riluzole could be related to its inhibition of sodium channel activity, which in turn inhibits glutamate release.
Animals↗
[3H]RP 62203, a ligand of choice to label in vivo brain 5-HT2 receptors.
There are only a few ligands available for labelling brain receptors simply because in vivo binding requires more severe experimental conditions than in vitro binding. We now describe the in vivo binding properties of [3H]RP 62203, a new potent and selective 5-HT2 antagonist. After intravenous injection into rats, [3H]RP 62203 accumulated predominantly in brain regions containing 5-HT2 receptors, with a frontal cortex/cerebellum ratio of 6 to 7. A good correlation was obtained between the regional distribution of [3H]RP 62203 in the brain and the density of 5-HT2 receptors measured in vitro. In vivo binding of [3H] RP 62203 was saturable in the frontal cortex but not in the cerebellum. The Bmax in the frontal cortex was equal to 42.5 fmol/mg, thus in the same range as was found in vitro. The 5-HT2 selectivity was ascertained by displacement (prevention) experiments; 5-HT2 antagonists or the agonist 2,5-dimethoxy- 4-iodophenylisopropylamine could prevent specific labelling of [3H]RP 62203 only in brain regions containing 5-HT2 receptors. Interestingly, the radioactivity remaining in various brain regions after displacement with pipamperone corresponded exactly to that measured in the cerebellum, with or without pipamperone. In conclusion, [3H]RP 62203 possesses striking properties of in vivo binding which make it a suitable candidate for examining 5-HT2 receptors in human brain by positron emission tomography scanning.
Amphetamines↗
Neurotensin increases tyrosine hydroxylase messenger RNA-positive neurons in substantia nigra after retrograde axonal transport.
In previous studies we have shown that labelled neurotensin injected into the rat striatum was found to be transported retrogradely in dopaminergic neurons through a process which was receptor and microtubule dependent. Now, we show, by in situ hybridization, the consequences of the striatal injection of neurotensin on the gene expression of tyrosine hydroxylase in the substantia nigra. Rats were injected with neurotensin or its fragments in the striatum of one side and with saline or the inactive fragment on the other. The number of nigral cells expressing tyrosine hydroxylase mRNA was found to increase by 40% after injection of neurotensin or its active fragment (neurotensin 8-13). In the same experimental conditions, the inactive fragment (neurotensin 1-8) was without effect. Time-course experiments revealed that the tyrosine hydroxylase mRNA was increased 4 h after neurotensin injection but not at 1 or 16 h. The fact that the increase of mRNA parallels the appearance of labelled neurotensin in the substantia nigra indicates that the changes in the gene expression of tyrosine hydroxylase might be the consequence of the retrograde axonal transport of neurotensin. These results represent the first evidence for the existence of a long-distance retrograde signalling process in which the neuropeptide and presumably its receptor may serve as information molecule between synapses and the cell body.
Animals↗
Distribution of neurokinin B in rat spinal cord and peripheral tissues: comparison with neurokinin A and substance P and effects of neonatal capsaicin treatment.
In the present study, highly specific radioimmunoassays were developed and used to measure neurokinin B, neurokinin A and substance P in the rat spinal cord and various peripheral tissues. The results are as follows. (1) Neurokinin B and neurokinin A were distributed all along the rostrocaudal axis of the spinal cord, as is substance P, and were more concentrated in the dorsal than in the ventral region. (2) Substance P was more abundant in the central and peripheral nervous tissues than neurokinin A, while in certain peripheral organs, neurokinin A was more abundant than substance P. In the spinal cord, neurokinin B concentrations were lower than those of the other two tachykinins. (3) In contrast to neurokinin A and substance P, neurokinin B was not detected in any of the peripheral tissues examined. (4) Capsaicin treatment reduced by half neurokinin A and substance P concentrations in the dorsal region of the spinal cord, the dorsal root ganglia and the sciatic nerve, but was without effect on neurokinin B concentrations in the spinal cord. Neurokinin A, like substance P, may therefore have an important function in the transmission of sensory information, particularly in nociceptive transmission from the periphery to the spinal cord and in peripheral neurogenic inflammation. In contrast, since neurokinin B was not found in the sensory neurons, it is not likely to have these functions, but may perhaps control them.
Animals↗
Light and electron microscopic localization of retrogradely transported neurotensin in rat nigrostriatal dopaminergic neurons.
We previously demonstrated the existence of a retrograde axonal transport of radioactivity to the substantia nigra pars compacta following injection of mono-iodinated neurotensin in rat neostriatum. In the present study, the topographical and cellular distribution of this retrogradely transported material was examined by light and electron microscopic autoradiography. Four and a half hours after unilateral injection of [125I]neurotensin in the caudoputamen, retrogradely labelled neuronal cell bodies were detected by light microscopic autoradiography throughout the ipsilateral substantia nigra pars compacta as well as within the ventral tegmental area and retrorubral field. In semithin sections, silver grains were prevalent over the perinuclear cytoplasm of neuronal cell bodies but were also detected over neuronal nuclei. Analysis of electron microscopic autoradiographs revealed that the vast majority (greater than 85%) were associated with neuronal perikarya, unmyelinated and myelinated axons, dendrites and terminals. Within the soma, a significant proportion of silver grains (16% of somatic grains) was detected over the nucleus. However, the majority were identified over the cytoplasm where they often encompassed cytoplasmic organelles, including rough endoplasmic reticulum, mitochondria, Golgi apparatus, lysosomes, and multi-vesicular bodies. In dendrites and axons, a substantial percentage of silver grains (63-89%) was localized over the plasma membrane. A minor proportion (13% of total) of the autoradiographic labelling was detected over myelin sheaths, astrocytes, and oligodendrocytes. The present results are consistent with previous light-microscopic evidence for internalization and retrograde transport of intrastriatal neurotensin within nigrostriatal dopaminergic neurons. They further suggest that retrogradely transported neurotensin may be processed along a variety of intracellular pathways including those mediating degradation in lysosomes and recycling in rough endoplasmic reticulum. The detection of specific autoradiographic labelling in the nucleus supports the concept that neurotensin alone, or complexed to its receptor, might be involved in the regulation of gene expression through direct or indirect interactions with nuclear DNA. Consequently, the retrograde transport of neurotensin in nigrostriatal dopaminergic neurons might provide a vehicle through which events occurring at the level of the axon terminal may initiate long-term biological responses.
Animals↗
RP 62203, a 5-hydroxytryptamine2 antagonist, enhances deep NREM sleep in rats.
RP 62203, a naphtosultam derivative, is an antagonist at the 5-hydroxytryptamine2 (HT2) receptor. The sleep pattern of rats treated orally with RP 62203 was studied at doses ranging from 0.5 to 4 mg/kg. Following RP 62203 administration, the duration of deep nonrapid eye movement (NREM) sleep was found to increase at the expense of wakefulness in a dose-dependent manner from 0.5 mg/kg. The 5-HT2 receptor agonist DOI and the 5-HT1a receptor agonist 8 OH-DPAT induced a dose-related increase in wakefulness; treatment with RP 62203 reversed the enhancement of wakefulness produced by DOI but not that produced by 8 OH-DPAT. These data provide further evidence for the involvement of 5-HT2 receptors in the regulation of NREM sleep in rats. RP 62203 could therefore be of clinical interest in the management of sleep disorders, particularly those developing within a psychiatric context.
Animals↗
Polyclonal antibodies against the rat NK1 receptor: characterization and localization in the spinal cord.
We have developed antibodies against the NK1 receptor and have investigated its cellular distribution. Rabbit polyclonal antibodies were generated against peptide (19-32) of the rat brain NK1 receptor. They were very specific to the NK1 site as shown by ELISA against various epitopes of NK1, NK2 and NK3 receptors and by immunoblotting of proteins from bacteria transfected with rat brain NK1 receptor cDNA and from rat cortex. Determining how immunostained NK1 receptors are distributed in the rat spinal cord made it possible to identify the cellular structures on which NK1 receptors are located and where they form synapses with SP terminals. In the superficial layers of the dorsal horn, the NK1 receptors appeared mainly of dendritic nature and were, like SP, abundant. In the deep layers of the dorsal horn and in the ventral horn, they were associated mostly with cell bodies.
Amino Acid Sequence↗
Striatal injection of neurotensin increases tyrosine hydroxylase mRNA in substantia nigra.
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Animals↗
Retrograde axonal transport of neurotensin in rat dopaminergic neurons.
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Animals↗
Autoradiographic localization of retrogradely transported neurotensin in nigrostriatal neurons.
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Animals↗
[RP 67580, a potent and selective substance P non-peptide antagonist].
The pharmacological properties of 7,7-Diphenyl-2 [1-imino-2 (2-methoxy-phenyl)-ethyl] perhydroisoindol-4-one (3 aR, 7 aR) or RP67580 are described. This compound, derived from a novel chemical family, is a potent and selective substance P (SP) antagonist, in vitro and in vivo. In vitro, it inhibited in a competitive manner (IC50 = 10 nM) 3H-SP binding in rat brain (NK1 receptors). It did not interact with the two other tachykinin receptor sites (NK2 and NK3) nor the other receptor sites tested. Moreover, RP67580 competitively antagonized the contractile activity of SP on guinea-pig ileum (pA2 = 7.16); in contrast, it was inactive in rabbit pulmonary artery and in rat portal vein tissues which contain NK2 and NK3 receptors, respectively. In vivo, in the rat, RP67580 inhibited the plasmatic extravasation induced by administration of SP (ED50 = 0.04 mg/kg i.v.) as well as that induced by antidromic stimulation of a peripheral sensory nerve (ED50 = 0.15 mg/kg i.v.). In mice and rats, RP67580, like morphine, potently blocked the nociceptive effects of phenylbenzoquinone and formalin; its antinociceptive effect does not involve opiate receptors since it was not reversed by naloxone. These results indicate that RP67580 is a particularly valuable tool for investigating the physiological and pathological role of SP.
Analgesics↗
Rapid agonist-induced decrease of neurotensin receptors from the cell surface in rat cultured neurons.
The regulation of neurotensin receptors was studied in vitro in primary cultures of neuronal cells. High affinity receptors for [3H]neurotensin were found in homogenates and at the cell surface of intact neurons cultured from the brain of rat embryos. When intact cells were incubated with 3 nM neurotensin (1-13), a rapid decrease in [3H]neurotensin binding was observed; about 60% of neurotensin receptors disappeared from the cell surface in less than 15 min. This corresponded to a reduction of the Bmax value without a change in the binding affinity. The decrease in neurotensin receptor number was also induced by the active fragment (8-13) of neurotensin but not by its inactive fragment (1-8). It was partially inhibited by bacitracin, at concentrations which are known to interact with receptor internalization, and was not detected when intact cells were incubated at 0-4 degrees with the unlabeled peptide. When intact neurons were incubated with [3H]neurotensin, there was a rapid ligand uptake and the kinetics of endocytosis were similar to those of the cell surface receptor disappearance. Once endocytosed, [3H]neurotensin could not be released (or displaced) from either intact neurons or homogenates, suggesting the sequestration of the labeled peptide in vesicles or other subcellular structures. Therefore, the present results suggest that the rapid agonist-induced decrease in the number of neurotensin receptors from the cell surface corresponds to an internalization process which involves a simultaneous receptor-mediated peptide endocytosis.
Animals↗
Pharmacological properties of a potent and selective nonpeptide substance P antagonist.
We describe here the pharmacological properties of RP 67580 [(3aR,7aR)-7,7-diphenyl-2-[1-imino-2-(2-methoxyphenyl)ethyl] perhydroisoindol-4-one], a nonpeptide antagonist of substance P (SP). In vitro, the compound was found to inhibit in a competitive manner (Ki = 4.16 +/- 0.59 nM) [3H]SP binding to neurokinin receptors type 1 (NK1 receptors) in rat brain membranes. Contractions induced by SP and septide (a selective NK1 agonist) in guinea pig ileum were competitively inhibited by RP 67580 (pA2 = 7.16 and 7.59, respectively). Moreover, RP 67580 displayed the profile of a specific antagonist of NK1 receptors: it was not active on NK2 and NK3 receptors as seen in binding assays and in isolated preparations of rabbit pulmonary artery and rat portal vein. In the rat, low intravenous doses of RP 67580 totally inhibited the plasma extravasation induced by SP in the urinary bladder (ED50 = 0.04 mg/kg i.v.) and by antidromic electrical stimulation of the saphenous nerve in the hind paw skin (ED50 = 0.15 mg/kg i.v.). This compound was also active in two classical analgesic tests in mice: phenylbenzoquinone-induced writhing (ED50 = 0.07 mg/kg s.c.) and the formalin test (ED50 = 3.7 mg/kg s.c.). Its potency was of the same order as that of morphine. Thus we conclude that RP 67580, a SP antagonist, belongs to a class of drugs that may be useful in the management of various clinical pathologies where pain and neurogenic inflammation are involved.
Analgesics↗
Are 5-HT2 antagonists endowed with anxiolytic properties in rodents?
The precise role of serotonin (5-HT) in anxiety remains unclear. We report here on the effects of RP 62203, a new 5-HT2 antagonist, and ritanserin in different animal models of anxiety. In the elevated plus-maze in mice, RP 62203 increased dose-dependently the percentage of entries onto, and time spent on open arms, over the dose range 0.25-4 mg.kg-1 p.o. By contrast, ritanserin was ineffective up to the dose of 4 mg.kg-1 p.o. In addition, both compounds were tested against the anxiogenic compound FG 7142 (20 mg.kg-1, i.p.) in the plus-maze test in mice and via electrocorticographic recordings (ECoG) in rats. The anxiolytic effect of RP 62203 is antagonized by FG 7142 at a dose devoid of anxiogenic properties. A similar interaction between RP 62203 and FG 7142 is observed in ECoG studies. In contrast, ritanserin seemed to potentiate the anxiogenic and awakening activities of FG 7142. These results demonstrate that RP 62203, a selective 5-HT2 antagonist, possesses anxiolytic properties in rodents suggesting that 5-HT2 receptors are involved in the control of anxiety.
Animals↗
Reduction of the amount of neurotensin retrogradely transported in dopaminergic neurons of senescent rats.
It is now clearly established that fast anterograde axonal transport can be altered during ageing, both in the central and the peripheral nervous systems, but no information is yet available concerning the modifications of fast retrograde axonal transport during senescence. In the present paper, we report the changes occurring in the retrograde axonal transport of neurotensin in dopaminergic neurons of old rats. When iodinated neurotensin was injected into the striatum, a diminution of approximately 50% in the amount of the labelling measured in the ipsilateral substantia nigra was observed in senescent rats by comparison with young adult rats. Nevertheless, the rate of neurotensin transport was not modified. Our results clearly indicate that less neurotensin is transported from the nerve terminals towards the cell bodies in senescent rats which may have possible consequences for dopaminergic neurons.
Animals↗
Riluzole prevents hyperexcitability produced by the mast cell degranulating peptide and dendrotoxin I in the rat.
Using electroencephalographic (EEG) recordings in freely moving rats and extracellular neuronal firing-rate recordings in hippocampal slices, we examined the effects of riluzole (RP 54274), a compound with anti-glutamate properties, against the convulsive seizures and the cellular hyperexcitability produced by the mast-cell degranulating peptide (MCD), dendrotoxin I (DTXi) and 4-aminopyridine (4-AP). I.c.v. administration of riluzole (10 nmol) prevented the seizures induced by MCD, and to a lesser extent those due to DIXi, whilst leaving 4-AP seizures unaffected. This effect was also present after oral administration of the compound (4 mg kg-1) and lasted for approximately 6 h. Electrophysiological recordings in vitro confirmed that riluzole dose dependently and reversibly abolished the sustained increase in firing rate induced by both MCD and DTXi in the hippocampus. These results indicate that the anti-epileptic spectrum of riluzole in this model has similarities with, but is not identical to, that of classical potassium channel openers, and differs from that of calcium channel blockers or other glutamate antagonists such as D(-)-2-amino-5-phosphono-valeric acid. However, since MCD releases glutamate, the preventive effect of riluzole in this model may involve direct or indirect interaction with glutamatergic processes.
4-Aminopyridine↗
Identification of intact neurotensin in the substantia nigra after its retrograde axonal transport in dopaminergic neurons.
Two hours after the injection of (3-[125I]iodotyrosyl3) neurotensin into the striatum, a labeling was observed in the ipsilateral substantia nigra. In the present study, we demonstrated by HPLC that this radioactivity corresponded to intact neurotensin and to degradation products of this peptide. This finding provides the first clearcut evidence that a neuropeptide can be internalized and retrogradely transported in brain neurons. Therefore, the fact that intact neurotensin can be seen to exist over a long period of time in the cell body suggests that the retrograde transport process could perhaps be involved in the long-term effects of neuropeptides.
Animals↗