The recent cloning, functional expression and brain localization of two new potassium channels, TREK and TRAAK, led us to examine whether both channels are present in peripheral nerves and can move along axons by means of axonal transport mechanisms. Using specific antibodies directed against TREK and TRAAK peptides, we found that immunoreactivity for both potassium channels accumulates above and below a ligature in rat sciatic nerves. The process was rapid and bidirectional suggesting that the channels are associated with vesicles. This represents the first report on the axonal transport of potassium channels.

Most of the available drugs act on membrane or nuclear receptors. Membrane receptors desensitize when they undergo an internalization process in response to an excess of agonist. This does not necessarily mean receptor degradation, a process that occurs when endosomes containing internalized receptors fuse with lysosomes, leading to what we call receptor down-regulation. Rather, the internalization of receptors may represent the initial step in retrograde signaling in which the ligand-receptor complex is transported along axons, ultimately leading to longterm effects at the gene level. In neurons, for example, “third messengers” are cytoplasmic proteins which are associated with vesicles containing internalized receptors and which pass into the nucleus. They subsequently serve as transcription factors in the modulation of gene expression. In the future, “third messengers” are expected to be the primary target of new drugs.

Monotherapy is today the normal way to treat diseases. Fortunately for the patients, there are some exceptions to this rule as in cancer,hypertension and tuberculosis but also more recently in aids where a multiple drug approach or the triple therapy is becoming the rule. In fact a very long way has led to the monotherapy concept, starting already at the Renaissance with Paracelse who had reduced his prescription to ten ingredients rather than 40 or 60 as recommended by all the pharmacists of his time. This was one of the reason why the father of pharmacologists was disparaged by his contemporaries. Today monotherapy has been elevated to the rank of dogma and this has been encouraged mostly by the regulatory authorities. Indeed it is more easy to evaluate a drug containing one active substance rather than several molecules which together may produce possible interactions leading to side-effects. However one has to recall that sometimes two compounds may display synergistic effects. As a rule in science the negative aspects of a biological systems are first recognized and these can even mask the more positive effects. The problem of synergy will be discussed later on. Today it is time to evolve from monotherapy to multiple drug therapy. Indeed there are more and more experimental and clinical data showing that combination or multiple drug therapy constitutes a more effective approach in the treament of major diseases. In this paper I would like to discuss some examples to illustrate the notion that multitargetting compounds and multiple drug therapy are made necessary because of nature's diversity and redundancy and the multifactorial nature of most of the diseases. But as we will see there are numerous difficulties to achieve this goal. Nature dictates to us the therapy of the future but mimicking nature is rarely the best way to find innovative drugs. Molecular biology has provided a better understanding of biological processes in showing a greater diversity and redundancy than was previously expected. If asthma is seen as the prototype of multifactorial diseases, it is assumed that other pathologies as schizophrenia for instance appear more to be the consequence of one cause or one gene defect justifying a monotherapy for this disease. However recent data indicate that in many cases more than one gene is involved as in Alzheimer's disease which is associated with genes on at least three different chromosomes. In schizophrenia one needs,of course, neuroleptics with reduced extrapyramidal side-effects.But one needs also drugs that would be able to prevent the increase of dopamine synthesis and release induced by neuroleptics, a fact which is something exactly opposite to what one would expect from an antipsychotic drug. Therefore here again multitargetting compounds and multiple drug therapy are required even if the mulfactorial nature of the disease appears to be much less evident.

Receptors involved in intercellular communication at the cell surface share the capacity to desensitize through molecular and cellular mechanisms. Cellular desensitization is a rapid and dynamic process whereby membrane receptors internalize in response to an excess of agonists. The internalized receptors may recycle rapidly or undergo down-regulation when following a degradative pathway. However, receptor internalization does not necessarily mean degradation; it also represents the initial step of a retrograde signalling system whereby an "interiorized" message, the ligand-receptor complex, can be transported in contrast to second messengers, along axons or in the cytoplasm leading to long-term effects in the nucleus. Such "third messengers" have to undergo nuclear translocation to serve as transcriptional regulators in the control of gene expression. The "third messengers" are thus cytoplasmic proteins, including the receptor itself, which may be associated with internalized vesicles and released by mechanisms which have not yet been elucidated. They represent already good targets for the development of new drugs, and multi-targeting and synergistic approaches are likely to increase their usefulness.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNew indole derivatives as potent and selective serotonin uptake inhibitors. [Erratum to document cited in CA119(2):8726n]Jean Luc Malleron, Claude Gueremy, Serge Mignani, Jean Francois Peyronel, Alain Truchon, Jean Charles Blanchard, Adam Doble, Pierre Laduron, Odile Piot, and Cite this: J. Med. Chem. 1993, 36, 15, 2242Publication Date (Print):July 1, 1993Publication History Published online1 May 2002Published inissue 1 July 1993https://pubs.acs.org/doi/10.1021/jm00067a024https://doi.org/10.1021/jm00067a024research-articleACS PublicationsRequest reuse permissionsArticle Views116Altmetric-Citations3LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

There are only a few ligands available for labelling brain receptors simplybecause in vivo requires more severe experimental conditions han tin 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 correspond exactly to that measured in the cerebellum, with or without pipamperone. In conclusion, [3H]RP 62203 posseses 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.

Annals of the New York Academy of SciencesVolume 668, Issue 1 p. 323-325 Autoradiographic Localization of Retrogradely Transported Neurotensin in Nigrostriatal Neurons MARIE-NOELLE CASTEL, MARIE-NOELLE CASTEL Rhǒne-Poulenc Rorer, 94400 Vitry-sur-Seine, FranceSearch for more papers by this authorJOHN WOULFE, JOHN WOULFE Neuroanatomy Laboratory, Montreal Neurological Institute, McGill University, Montreal, Quebec, H3A 2B4 CanadaSearch for more papers by this authorXUDONG WANG, XUDONG WANG Neuroanatomy Laboratory, Montreal Neurological Institute, McGill University, Montreal, Quebec, H3A 2B4 CanadaSearch for more papers by this authorPIERRE M. LADURON, PIERRE M. LADURON Rhǒne-Poulenc Rorer, 94400 Vitry-sur-Seine, FranceSearch for more papers by this authorALAIN BEAUDET, Corresponding Author ALAIN BEAUDET Neuroanatomy Laboratory, Montreal Neurological Institute, McGill University, Montreal, Quebec, H3A 2B4 CanadaAddress for correspondence: A. Beaudet, Neuroanatomy Lab, Montreal Neurological Institute, 3801 University St., Montreal, Quebec, H3A 2B4 Canada.Search for more papers by this author MARIE-NOELLE CASTEL, MARIE-NOELLE CASTEL Rhǒne-Poulenc Rorer, 94400 Vitry-sur-Seine, FranceSearch for more papers by this authorJOHN WOULFE, JOHN WOULFE Neuroanatomy Laboratory, Montreal Neurological Institute, McGill University, Montreal, Quebec, H3A 2B4 CanadaSearch for more papers by this authorXUDONG WANG, XUDONG WANG Neuroanatomy Laboratory, Montreal Neurological Institute, McGill University, Montreal, Quebec, H3A 2B4 CanadaSearch for more papers by this authorPIERRE M. LADURON, PIERRE M. LADURON Rhǒne-Poulenc Rorer, 94400 Vitry-sur-Seine, FranceSearch for more papers by this authorALAIN BEAUDET, Corresponding Author ALAIN BEAUDET Neuroanatomy Laboratory, Montreal Neurological Institute, McGill University, Montreal, Quebec, H3A 2B4 CanadaAddress for correspondence: A. Beaudet, Neuroanatomy Lab, Montreal Neurological Institute, 3801 University St., Montreal, Quebec, H3A 2B4 Canada.Search for more papers by this author First published: October 1992 https://doi.org/10.1111/j.1749-6632.1992.tb27364.xCitations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume668, Issue1The Neurobiology of NeurotensinOctober 1992Pages 323-325 RelatedInformation

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 (DTX(i)) 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 DIX(i), 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 DTX(i) 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, the preventive effect of riluzole in this model 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.

The possible anxiolytic activity of riluzole, a drug which interferes with glutamic acid neurotransmission, was studied in rats using operant conflict procedures. In both “anxiolytic” and “anxiogenic” procedures, riluzole alone did not possess any anticonflict or proconflict effect at doses of 2 and 4 mg/kg PO. Riluzole over the same dose-range was able to antagonize the well known proconflict effect of the β-carboline derivative FG 7142, an inverse agonist at the GABA-benzodiazepine-chloride ionophore receptor complex. This effect could be related to the possible interaction of riluzole with glutamic acid neurotransmission, since it has been demonstrated previously that β-carbolines such as DMCM and β-CCM were able to deplete the levels of aspartic and glutamic acids in rodent cortex, perhaps by enhancing release of amino acid neurotransmitters. If one subscribes to the hypothesis that the anxiety induced by β-carboline derivatives is related to depression, riluzole might be of value in the treatment of anxiety related to depression.

An iodinated azido derivative of ketanserin, 7-azido-8-[125I]iodoketanserin ( [125I]AZIK), has been used to label the monoamine transporter of bovine chromaffin granule membranes by the technique of photoaffinity labeling. In the dark, this derivative was found to bind reversibly to the membranes, with an equilibrium dissociation constant estimated to be 6 nM at 0 degrees C. As for ketanserin, binding occurred at the tetrabenazine site: (i) [125I]AZIK was displaced efficiently from its binding site by tetrabenazine, ketanserin, and 7-azidoketanserin, whereas serotonin, which is a substrate for the transporter but has a low affinity for tetrabenazine binding site, was a poor displacer; pipamperone and pyrilamine, two antagonists of respectively serotonin S2 and histamine H1 receptors, were inactive. (ii) 7-Azidoketanserin was a competitive inhibitor of [3H]dihydrotetrabenazine binding, and it inhibited the ATP-dependent uptake of serotonin by chromaffin granule ghosts. Irradiation of [125I]AZIK with long-wavelength UV light, followed by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels and autoradiography, revealed irreversible labeling of a membrane component with an apparent molecular weight of 73,000. Tetrabenazine inhibited the labeling of this 73-kDa band in a manner parallel to the binding of [125I]AZIK in the dark. Such a labeling is totally compatible with previous results obtained through photolabeling with a tetrabenazine derivative or by target size analysis. Moreover, preliminary experiments showed that [125I]AZIK can label the tetrabenazine binding sites of various sources including rat striatum, rabbit platelets, human pheochromocytoma, and human adrenal medulla. Therefore, this molecule appears to be an excellent probe to label the monoamine transporter of different amine storage vesicles even without purification.

The subcellular localization of neurotensin-receptor sites (NT2 sites) and neurotensin-acceptor sites (NT1 sites) was studied in rat caudate-putamen by isopycnic centrifugation in sucrose density gradients. [3H]Neurotensin binding to NT2 sites occurred as a major peak at higher sucrose densities, colocalized with [3H]dopamine uptake, and as a small peak at a lower density; whereas binding to NT1 sites occurred as a single large peak at an intermediate density. 6-Hydroxydopamine lesions of the median forebrain bundle resulted in a total loss of NT2 sites in the caudate-putamen but did not affect NT2 sites in the nucleus accumbens and the olfactory tubercle. NT1 sites were not affected. Kainic acid injections into the rat caudate-putamen led to a partial decrease of NT1 sites in this region 5 days later. After a few weeks they returned to normal. Therefore NT2 sites are probably associated with presynaptic nigrostriatal dopaminergic terminals in the caudate-putamen but not in the nucleus accumbens and the olfactory tubercle. A possible association of NT1 sites with glial cells is suggested.

The retrograde transport of receptor-bound opiate was markedly enhanced in the vagus nerves of rats housed for 25 days in an atmosphere of ethanol vapor. This increase disappeared after 8 days of withdrawal as did the animal preference for ethanol. In contrast, the axonal transport of muscarinic receptors in sciatic nerves was slightly reduced. This provides more evidence for the idea that chronic alcoholization and morphine addiction share a common denominator and that the retrograde transport of receptor-bound signal molecules represents a link between the synapses and the cell-body of neurones.

The monoamine carrier of noradrenergic synaptic vesicles was assayed in the rat sciatic nerve by in vitro binding of [3H]dihydrotetrabenazine. The number of binding sites increased with time on both sides of a ligature, indicating fast anterograde and retrograde axoplasmic transports of comparable amplitude. Anterogradely and retrogradely transported binding sites had similar pharmacological properties, which were characteristic of binding to the monoamine carrier embedded within a lipid bilayer. Contrary to the secreted synaptic vesicle matrix constituents, the intrinsic monoamine carrier assayed by [3H]dihydrotetrabenazine binding is a marker adapted to retrograde transport studies.

There is an increasing amount of evidence indicating that presynaptic receptors move antero- and retrogradely along axons of neurones through axoplasmic transport mechanisms. The main features of this dynamic process will be presented: it is bidirectional, fast, microtubule dependent and associated with vesicles which are presumably recycled in the cell body. Receptor axonal transport is impaired or enhanced in certain pathological conditions. The implications of this process and its possible involvement in long-term memory will be discussed.

7-Azido-8-[125I]ketanserin ([125I]AZIK) was characterized as a potent photoaffinity probe for serotonin-S2 receptors. In reversible binding experiments, [125I]AZIK bound with high affinity (Kd = 0.69 nM) to rat frontal cortex membranes. When incubation with [125I]AZIK was followed by UV irradiation, the binding was found to be irreversible. Protection experiments with various drugs demonstrated the serotonin-S2 nature of the photoaffinity labelling. SDS-polyacrylamide gel electrophoresis of the photolabelled membranes allowed one to identify the serotonin-S2 receptor ligand binding site as a single polypeptide with a molecular mass of approx. 67500 Da. [125I]AZIK will be a valuable tool for the elucidation of the serotonin-S2 receptor structure.

Two distinct neurotensin binding sites have been identified in rat brain: the NT1-acceptor site (levocabastine-sensitive) and the NT2-receptor site. In rat forebrain, NT2-receptors were present at birth, revealed a maximal level (13.8 fmol/mg tissue) on day 10 of postnatal life but a much lower plateau (3.0 fmol/mg tissue) in adult rats. NT1-acceptors were not detected before day 10 and became maximal at day 30. It is suggested that the loss of NT2-receptor sites during the postnatal development may be the expression of the regression of a transient redundancy of neuronal connections.

Monoiodo-[125I-Tyr3]neurotensin (NT) bound to a high affinity, low capacity binding component and a lower affinity, high capacity component in rat brain synaptic membranes. The antihistamine H1 agent levocabastine, which bears no structural relationship to NT, selectively and totally inhibited NT binding to its low affinity binding sites. The IC50 for levocabastine was 7 nM. Lowering the temperature of the binding assay from 25 to 4 degrees C markedly reduced the affinity of the high affinity NT binding site but did not affect the ability of levocabastine to discriminate between high and low affinity NT binding sites in rat brain membranes and tissue sections. Radioautographic studies of [125I-Tyr3]NT binding to rat brain tissue sections in the absence and presence of levocabastine revealed markedly different regional distributions of the two NT binding components. The levocabastine-sensitive NT binding site was present in membranes from rat and mouse brain but absent from rabbit brain membranes and from human brain tissue sections. It was also absent from mouse neuroblastoma N1E115 and human colonic adenocarcinoma HT29 cell membranes, two cell lines which have previously been shown to possess NT receptors functionally coupled to intracellular second messenger-generating systems. These findings are discussed in the light of the known properties of the high and low affinity NT binding sites in rat brain.