Increasing evidence suggests that CCN matricellular proteins play important roles in inflammation. One of the major cell types that handle inflammation in the brain is the astrocyte, which, upon activation, dramatically increases its production of cytokines and chemokines. Here, we report that NOV/CCN3, added to primary cultured rat brain astrocytes, markedly increased the expression of CCL2 and CXCL1 chemokines, as indicated by ELISA and RT‐qPCR assays. This effect was selective, as the production of thirteen other cytokines and chemokines was not affected by NOV. NOV expression by astrocytes was demonstrated by immunocytochemistry and Western blot analysis, and astrocyte transfection with NOV small interfering RNA (siRNA) markedly decreased CXCL1 and CCL2 production, indicating that endogenous NOV played a major role in the control of astrocytic chemokine synthesis. NOV was shown to mediate several of its actions through integrins. Here, we observed that siRNAs against integrins β1 and β5 decreased basal and abrogated NOV‐stimulated astrocyte expression of CCL2 and CXCL1, respectively. Using a panel of kinase inhibitors, we demonstrated that NOV action on CCL2 and CXCL1 production involved a Rho/ROCK/JNK/NF‐κB and a Rho/qROCK/p38/NF‐κB pathway, respectively. Thus, distinct integrins and signaling mechanisms are involved in NOV‐induced production of CCL2 and CXCL1 in astrocytes. Finally, astrocytic expression of NOV was detected in rat brain tissue sections, and NOV intracerebral injection increased CCL2 and CXCL1 brain levels in vivo . Altogether, our data shed light on the signaling pathways operated by NOV and strongly suggest that NOV mediates astrocyte activation and, therefore, might play a role in neuroinflammation. © 2010 Wiley‐Liss, Inc.

Uncontrolled or sustained inflammation is the underlying cause of or actively contributes to the progression of many chronic pathologies such as atherosclerosis, arthritis, or neuroinflammatory diseases. Matricellular proteins of the CCN family (CYR61/CTGF/NOV) have emerged as localized multitasking signal integrators. These structurally conserved secreted proteins specifically interact with and signal through various extracellular partners, in particular integrins, which enable them to play crucial roles in various processes including development, angiogenesis, wound healing and diseases such as fibrosis, vascular disease and cancer. In this review, we discuss the possibility that the CCN family members could represent a putative new class of modulators of inflammation. In this context, we focused on their relationship with cytokines and chemokines. In vitro, CCN expression is finely regulated by diverse inflammatory mediators including cytokines (TNFα, IL1β, TGF-β), small factors such as prostaglandins, nitric oxide, histamine and serotonin, and extracellular matrix enzymes. In addition, CCN proteins acting alone or in concert with their specific partners appear to be potent regulators of the production of cytokines and chemokines in a context-dependent manner. Finally, emerging studies suggest a potential role for CCN proteins in chronic inflammatory diseases such as atherosclerosis, rheumatoid arthritis, inflammatory kidney diseases and neuroinflammatory pathologies such as Alzheimer’s disease. CCN members could therefore represent new potential therapeutic targets for drug development against such diseases.

Event Abstract Back to Event Chemokines are neuromodulators of dopaminergic transmission William Rostene1*, Stephane M. Parsadaniantz1, A. Blancher1, A. Guyon2, JL Nahon2, C. Ewenczyk1, D. Skrzydelski1, P. Kitabgi1 and E. Apartis1 1 Universite Pierre et Marie Curie-Paris 6, Hopital Saint-Antoine, Institut National de la Sante et de la Recherche Medicale (INSERM) U 732, France 2 UMR 6097 CNRS, Institut de Pharmacologie Moleculaire et Cellulaire (IPMC), France Increasing evidence suggest that chemokines act as neuromodulators of brain functions. Thus they may play a key role in human pathological conditions implying central dopaminergic (DA) pathways, such as Parkinson’s disease. This new hypothesis is based on very recent immunohistological data showing that chemokines such as CXCL12 (SDF-1) or CCL2 (MCP-1) and their respective receptors CXCR4/CCR2 are constitutively expressed in DA neurons. We will mainly focus our presentation on CXCL12 (SDF-1). According to this neuroanatomical substratum, we first investigated the physiological role of CXCL12 (SDF-1) on DA system using various in vivo an in vitro approaches in the rat. Patch clamp recording of DA neurons in rat substantia nigra slices revealed that CXCL12 (SDF-1) (0.01-10 nM): (i) increased the amplitude of N-type high voltage-activated calcium currents known to be involved in DA release, (ii) induced a neuronal depolarization and increased action potential frequency and (iii) switched the firing pattern of depolarized DA neurons from a tonic to a burst firing mode. These data suggested that CXCL12 (SDF-1) could increase DA release from neurons. Consistent with this hypothesis, we showed using microdialysis that unilateral intranigral injection of CXCL12 (SDF-1) in freely moving rat increased extracellular concentrations of DA in the ipsilateral dorsal striatum. Such increase in DA release was related to a contralateral circling behaviour. All these effects of CXCL12 (SDF-1) were mediated via CXCR4 as they were all abrogated by administration of a selective CXCR4 antagonist (AMD3100). Using primary cultures of rat mesencephalic neurons, we observed that not only CXCL12 (SDF-1) was able to potentiate K+-induced DA release but also protected DA neurons from MPP+ neurodegeneration. Altogether, these data demonstrate that CXCL12 (SDF-1), via CXCR4, activates nigrostriatal DA transmission. They show that the central functions of chemokines are not restricted, as originally thought, to neuroinflammation, but extend to neuromodulatory actions on well-defined neuronal circuits in pathological and non-pathological conditions. Conference: 3rd Mediterranean Conference of Neuroscience , Alexandria, Egypt, 13 Dec - 16 Dec, 2009. Presentation Type: Oral Presentation Topic: Symposium 17 – Chemokines: new modulators in brain and pituitary functions Citation: Rostene W, Parsadaniantz SM, Blancher A, Guyon A, Nahon J, Ewenczyk C, Skrzydelski D, Kitabgi P and Apartis E (2009). Chemokines are neuromodulators of dopaminergic transmission. Front. Neurosci. Conference Abstract: 3rd Mediterranean Conference of Neuroscience . doi: 10.3389/conf.neuro.01.2009.16.074 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 20 Nov 2009; Published Online: 20 Nov 2009. * Correspondence: William Rostene, Universite Pierre et Marie Curie-Paris 6, Hopital Saint-Antoine, Institut National de la Sante et de la Recherche Medicale (INSERM) U 732, Paris, France, william.rostene@inserm.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers William Rostene Stephane M Parsadaniantz A. Blancher A. Guyon JL Nahon C. Ewenczyk D. Skrzydelski P. Kitabgi E. Apartis Google William Rostene Stephane M Parsadaniantz A. Blancher A. Guyon JL Nahon C. Ewenczyk D. Skrzydelski P. Kitabgi E. Apartis Google Scholar William Rostene Stephane M Parsadaniantz A. Blancher A. Guyon JL Nahon C. Ewenczyk D. Skrzydelski P. Kitabgi E. Apartis PubMed William Rostene Stephane M Parsadaniantz A. Blancher A. Guyon JL Nahon C. Ewenczyk D. Skrzydelski P. Kitabgi E. Apartis Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Neurotensin (NT) is a neuropeptide predominantly expressed in the brain and the gut. The peptide exerts its effects through two G-protein-coupled receptors, the NTS1 and NTS2, and through a single transmembrane domain protein, the NTS3 or sortilin, that belongs to a small family of sorting receptors. In the brain, NT and its receptors are distributed in a regionalized fashion, and the peptide modulates the activity of neuronal systems involved in behavioral responses such as locomotor activity, rewarding, drug seeking, eating, and pain sensation.

Dopaminergic neurons of the substantia nigra constitutively express the CXCR4 receptor for the chemokine stromal-cell-derived factor 1 alpha (CXCL12) but, to date, no direct effect of CXCR4 activation by CXCL12 on membrane conductance of dopaminergic neurons has been demonstrated. We tested the effects of CXCL12 on whole-cell currents of dopaminergic neurons recorded in patch clamp in substantia nigra slices and showed that CXCL12 (0.01-10 nM) increased the amplitude of total high-voltage-activated (HVA) Ca currents through CXCR4 activation. This effect was reversibly reduced by pi-conotoxin-GVIA, suggesting that CXCL12 acted on N-type Ca currents, known to be involved in dopamine (DA) release. We therefore investigated the effects of CXCL12 on DA release from cultured dopaminergic neurons from the rat mesencephalon. In basal conditions, CXCL12 alone had no effect on DA release. When neurons were depolarized with KCl (20 mM), and thus when HVA Ca currents were activated, low CXCL12 concentrations (1-50 nM) increased DA release via CXCR4 stimulation. These data strongly suggest that the chemokine CXCL12 can act directly as a neuromodulator of dopaminergic neuronal electrical activity through the modulation of HVA currents.

Physical insults including but not limited to nerve damage, inflammation, visceral pathologies and cancer generate long lasting pain commonly referred as chronic pain. Recently, members of the chemokine family and their receptors emerged as key modulators in nociceptive influx transmission in neuropathic and inflammatory chronic pain models. To this day, rodents defective in specific chemokine receptors have provided evidence of the implication of chemokine in pain sensitivity. In addition, up-regulation of chemokines and their receptors at multiple levels in the central nervous (CNS) and peripheral (PNS) systems is associated in the development of chronic pain. Indeed, we point out the fact that chemokines are synthesized and released by both neuronal and non-neuronal cells and act as neuromodulators. Even if their functional roles in the CNS remain largely unknown, chemokines participate in the glial activation and modulation of neuronal excitability as well as neurotransmitter release. This review focuses on three chemokines (i.e. CCL2, CXCL12, CX3CL1) recently identified as important mediators of the initiation and maintenance of pain hypersensitivity, thus broadening the panel of new strategies for the management of chronic pain.

La chimiokine SDF-1 participe à la physiopathologie de certaines maladies neurodégénératives. Son fragment SDF-1 (5-67), toxique in vivo sur le striatum de souris, pourrait être impliqué dans celle de la maladie de Parkinson. Les effets neurotoxiques potentiels du SDF-1 (5-67) sur les neurones dopaminergiques sont inconnus. Nous étudions l'effet in vitro du SDF-1 (5-67) sur la survie des neurones dopaminergiques de rat, seul ou en présence de MPP +. Nous avons travaillé sur un modèle de cultures primaires de neurones mésencéphaliques de rat. Le SDF-1 (5-67) a été appliqué à 100 nM pendant deux jours seul et avec du MPP + (1 μM), ainsi qu'à 1000 nM seul pendant quatre jours. La survie des neurones dopaminergiques (DA) a été évaluée à l'aide de paramètres structurels en immunocytochimie (nombre de cellules immunoréactives pour la tyrosine hydroxylase (TH +), taille des corps cellulaires), et fonctionnels avec la recapture de la [3H]DA. L'application de SDF-1 (5-67) à 100 nM n'a pas modifié la survie neuronale DA. L'application de SDF-1 (5-67) à 1 000 nM a provoqué une baisse significative du nombre de cellules TH +, une hausse également significative de la recapture de la [3H]DA par neurone et de la taille des corps cellulaires des neurones DA. L'application de SDF-1 (5-67) à 100 nM conjointement au MPP + a provoqué une amélioration de la survie cellulaire et une moindre baisse de la recapture de la [3H]DA par rapport aux cellules traitées par MPP + seul. À 1 000 nM, le SDF-1 (5-67) induit une mort neuronale DA avec une augmentation concomitante et paradoxale des autres paramètres DA. On peut postuler que : (1) il existe 2 sous-populations de neurones DA dont une entre en apoptose, (2) les neurones DA survivants sont hyperstimulés selon un mode compensatoire par un mécanisme à définir. Par ailleurs il semble exister un effet neuroprotecteur à 100 nM vis-à-vis de la toxicité du MPP +. Nos résultats suggèrent que le SDF-1 (5-67) agit sur la fonction, la trophicité et la survie neuronale DA. S'ils sont confirmés, cela pourrait constituer une nouvelle approche physiopathologique de la maladie de Parkinson.

We recently demonstrated that dopaminergic (DA) neurons of the rat substantia nigra constitutively expressed CXCR4, receptor for the chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 (SDF-1). To check the physiological relevance of such anatomical observation, in vitro and in vivo approaches were used. Patch clamp recording of DA neurons in rat substantia nigra slices revealed that SDF-1 (10 nmol/L) induced: (i) a depolarization and increased action potential frequency; and (ii) switched the firing pattern of depolarized DA neurons from a tonic to a burst firing mode. This suggests that SDF-1 could increase DA release from neurons. Consistent with this hypothesis, unilateral intranigral injection of SDF-1 (50 ng) in freely moving rat decreased DA content and increased extracellular concentrations of DA and metabolites in the ipsilateral dorsal striatum, as shown using microdialysis. Furthermore, intranigral SDF-1 injection induced a contralateral circling behavior. These effects of SDF-1 were mediated via CXCR4 as they were abrogated by administration of a selective CXCR4 antagonist. Altogether, these data demonstrate that SDF-1, via CXCR4, activates nigrostriatal DA transmission. They show that the central functions of chemokines are not restricted, as originally thought, to neuroinflammation, but extend to neuromodulatory actions on well-defined neuronal circuits in non-pathological conditions.

Stromal cell-derived factor 1alpha (SDF-1alpha), a chemoattractant for leucocytes and neurons, and its receptor, CXCR4 are expressed in subsets of neurons of specific brain areas. In rat lateral hypothalamic area (LHA) we show, using immunocytochemistry, that CXCR4 is localized within melanin-concentrating hormone (MCH)-expressing neurons, mainly involved in feeding behaviour regulation. We investigated whether SDF-1alpha may control MCH neuronal activity. Patch-clamp recordings in rat LHA slices revealed multiple effects of SDF-1alpha on the membrane potential of MCH neurons, indirect through glutamate/GABA release and direct through GIRK current activation. Moreover, SDF-1alpha at 0.1-1 nM decreased peak and discharge frequency of action potential evoked by current pulses. These effects were further confirmed in voltage-clamp experiments, SDF-1alpha depressing both potassium and sodium currents. At 10 nM, however, SDF-1alpha increased peak and discharge frequency of action potential evoked by current pulses. Using a specific CXCR4 antagonist, we demonstrated that only the depressing effect on AP discharge was mediated through CXCR4 while the opposite effect was indirect. Together, our studies reveal for the first time a direct effect of SDF-1alpha on voltage-dependent membrane currents of neurons in brain slices and suggest that this chemokine may regulate MCH neuron activity.

In the CNS, immune-like competent cells (microglia and astrocytes) were first described as potential sites of chemokine synthesis, but more recent evidence has indicated that neurones might also express chemokines and their receptors. The aim of the present work was to investigate further, both in vivo and in vitro, CC Chemokine Family Receptor 2 (CCR2) expression and functionality in rat spinal cord neurones. First, we demonstrated by RT-PCR and western blot analysis that CCR2 mRNA and protein were present in spinal extracts. Furthermore, we showed by immunolabelling that CCR2 was exclusively expressed by neurones in spinal sections of healthy rat. Finally, to test the functionality of CCR2, we used primary cultures of rat spinal neurones. In this model, similar to what was observed in vivo, CCR2 mRNA and protein were expressed by neurones. Cultured neurones stimulated with Monocyte Chemoattractant Protein-1 (MCP-1)/CCL2, the best characterized CCR2 agonist, showed activation of the Akt pathway. Finally, patch-clamp recording of cultured spinal neurones was used to investigate whether MCP-1/CCL2 could modulate their electrophysiological properties. MCP-1 alone did not affect the electrical properties of spinal neurones, but potently and efficiently inhibited GABA(A)-mediated GABAergic responses in these neurones. These data constitute the first demonstration of a modulatory role of MCP-1 on GABAergic neurotransmission and contribute to our understanding of the roles of CCR2 and MCP-1/CCL2 in spinal cord physiology, in particular with respect to nociceptive transmission, as well as the implication of this chemokine in neuronal adaptation or dysfunction during neuropathy.

The monocyte chemoattractant protein‐1 (MCP‐1/CCL2) and its receptor CCR2 are key modulators of immune functions. In the nervous system, MCP‐1/CCL2 is implicated in neuroinflammatory pathologies. However, cerebral functions of MCP‐1/CCL2 under normal conditions are still unclear. In this study, using reverse transcriptase‐polymerase chain reaction (RT‐PCR) and specific rat MCP‐1 enzyme‐linked immunosorbent assay (ELISA) approaches, we observed that MCP‐1/CCL2 mRNA and protein were expressed in different punched regions of the normal rat central nervous system. Immunohistochemical studies further revealed that this chemokine is constitutively expressed not only in astrocytes but also in neurons, in discrete neuroanatomical regions. Neuronal expression of MCP‐1/CCL2 is mainly found in the cerebral cortex, globus pallidus, hippocampus, paraventricular and supraoptic hypothalamic nuclei, lateral hypothalamus, substantia nigra, facial nuclei, motor and spinal trigeminal nuclei, and gigantocellular reticular nucleus and in Purkinje cells in the cerebellum. Moreover, we obtained the first evidence that MCP‐1/CCL2 is constitutively expressed in cholinergic neurons, notably in the magnocellular preoptic and oculomotor nuclei, and in dopaminergic neurons of the substantia nigra pars compacta. In addition, in the lateral hypothalamic area, MCP‐1/CCL2 co‐localized with melanin‐concentrating hormone‐expressing neurons. Interestingly, we demonstrate a co‐localization of MCP‐1/CCL2 with vasopressin in magnocellular neuronal cell bodies and processes in the supraoptic and paraventricular hypothalamic nuclei, as well as in processes in the internal layer of the median eminence and in the posterior pituitary. Taken together, our data suggest that MCP‐1/CCL2 could act as a modulator of neuronal activity and neuroendocrine functions. J. Comp. Neurol. 489:275–292, 2005. © 2005 Wiley‐Liss, Inc.

Chemokines and their receptors are well described in the immune system, where they promote cell migration and activation. In the central nervous system, chemokine has been implicated in neuroinflammatory processes. However, an increasing number of evidence suggests that they have regulatory functions in the normal nervous system, where they could participate in cell communication. In this work, using a semiquantitative immunohistochemistry approach, we provide the first neuroanatomical mapping of constitutive neuronal CCR2 localization. Neuronal expression of CCR2 was observed in the anterior olfactory nucleus, cerebral cortex, hippocampal formation, caudate putamen, globus pallidus, supraoptic and paraventricular hypothalamic nuclei, amygdala, substantia nigra, ventral tegmental area, and in the brainstem and cerebellum. These data are largely in accordance with results obtained using quantitative autoradiography with [(125)I]MCP-1/CCL2 and RT-PCR CCR2 mRNA analysis. Furthermore, using dual fluorescent immunohistochemistry we studied the chemical phenotype of labeled neurons and demonstrated the coexistence of CCR2 with classical neurotransmitters. Indeed, localization of CCR2 immunostaining is observed in dopaminergic neurons in the substantia nigra pars compacta and in the ventral tegmental area as well as in cholinergic neurons in the substantia innominata and caudate putamen. Finally, we show that the preferential CCR2 ligand, MCP-1/CCL2, elicits Ca(2+) transients in primary cultured neurons from various rat brain regions including the cortex, hippocampus, hypothalamus, and mesencephalon. In conclusion, the constitutive neuronal CCR2 expression in selective brain structures suggests that this receptor could be involved in neuronal communication and possibly associated with cholinergic and dopaminergic neurotransmission and related disorders.

The stromal cell-derived factor-1 (SDF-1)/CXCL12 and its receptor CXCR4 are key modulators of immune functions. In the nervous system, SDF-1/CXCL12 is crucial for neuronal guidance in developing brain, intercellular communication and the neuropathogenesis of acquired immunodeficiency syndrome. However, cerebral functions of SDF-1/CXCL12 in adult brain are poorly understood. The understanding of its role in the adult brain needs a detailed neuroanatomical mapping of SDF-1/CXCL12. By dual immunohistochemistry we demonstrate that this chemokine is constitutively expressed not only in astrocytes and microglia but also in neurons, in discrete neuroanatomical regions. Indeed, neuronal expression of SDF-1/CXCL12 is mainly found in cerebral cortex, substantia innominata, globus pallidus, hippocampus, paraventricular and supraoptic hypothalamic nuclei, lateral hypothalamus, substantia nigra and oculomotor nuclei. Moreover, we provide the first evidence that SDF-1/CXCL12 is constitutively expressed in cholinergic neurons in the medial septum and substantia innominata and in dopaminergic neurons in substantia nigra pars compacta and the ventral tegmental area. Interestingly we also show, for the first time, a selective co-localization of SDF-1/CXCL12 with vasopressin-expressing neurons in the supraoptic and paraventricular hypothalamic nuclei. In addition, in the lateral hypothalamic area, SDF-1/CXCL12 was found to be located on melanin concentrating hormone-expressing neurons. Altogether, these original data suggest that SDF-1/CXCL12 could be a modulatory neuropeptide regulating both central cholinergic and dopaminergic systems. In addition, a key role for SDF-1/CXCL12 in neuroendocrine regulation of vasopressin-expressing neurons represents an exciting new field of research.

1 The neurotensin receptor 1, NTS1, is a G protein-coupled receptor with seven transmembrane domains (TM) that mediates most of the known effects of the neuropeptide. Our previous studies have pointed to extracellular loop 3 and adjacent TM7 as being potentially involved in agonist-induced activation of the NTS1.2 Here we investigated residues in these domains that might be involved in transconformational activation of the rat NTS1. Single amino acid mutated receptors were expressed in COS cells and inositol phosphate (IP) and cyclic AMP productions were studied.3 The F358A mutation in TM7 resulted in a time- and receptor concentration-dependent increase in spontaneous IP production. At expression levels of 12 pmol mg(-1), agonist-independent IP production was increased 10 fold over basal for the F358A mutant receptor whereas the wild type NTS1 exhibited virtually no spontaneous activity at expression levels of 7.5 pmol mg(-1).4 Neurotensin remained agonist on the F358A mutant receptor with a maximal effect that amounted to greater than twice basal IP levels. SR 48692 was inverse agonist at the mutant receptor, reversing IP production almost back to the levels measured in wild type NTS1-transfected cells.5 Cyclic AMP production was not constitutively activated with the F358A mutant receptor but was stimulated by neurotensin with the same concentration dependence as that observed with the wild type NTS1.6 This is the first report, to our knowledge, of a constitutively active mutant of the NTS1. The data are consistent with TM7 being involved in the transconformational changes that lead to agonistinduced coupling of the NTS1 to Gq.

Accumulating evidence supports a role of chemokines and their receptors in brain function. Up to now scarce evidence has been given of the neuroanatomical distribution of chemokine receptors. Although it is widely accepted that chemokine receptors are present on glial cells, especially in pathological conditions, it remains unclear whether they are constitutively present in normal rat brain and whether neurons have the potential to express such chemokine receptors. CXCR4, a G protein-coupled receptor for the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) was reported to have possible implications in brain development and AIDS-related dementia. By dual immunohistochemistry on brain sections, we clearly demonstrate that CXCR4 is constitutively expressed in adult rat brain, in glial cells (astrocytes, microglia but not oligodendrocytes) as well as in neurons. Neuronal expression of CXCR4 is mainly found in cerebral cortex, caudate putamen, globus pallidus, substantia innominata, supraoptic and paraventricular hypothalamic nuclei, ventromedial thalamic nucleus and substantia nigra. Using confocal microscopy, a differential distribution of CXCR4 in neuronal perikarya and dendrites can be observed according to the brain structure. Furthermore, this work demonstrates for the first time the coexistence of a chemokine receptor with classical neurotransmitters. A localization of CXCR4 is thus observed in neuronal cell bodies expressing choline acetyltransferase-immunoreactivity in the caudate putamen and substantia innominata, as well as in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. In conclusion, the constitutive neuronal CXCR4 expression suggests that SDF-1/CXCL12 could be involved in neuronal communication and possibly linked up with cholinergic and dopaminergic neurotransmission and related disorders.

Proneurotensin/neuromedin N (pro-NT/NN) is the common precursor of two biologically active related peptides, neuromedin N (NN) and neurotensin (NT). It undergoes a tissue-specific processing leading to the formation in some tissues and cancer cell lines of large peptides ending with the NT (large NT) or NN (large NN) sequence. In this study, we prepared and purified high amounts of recombinant large NT and large NN using the Drosophila S2 cell expression system. The binding and pharmacological properties of recombinant large peptides were characterized and compared to those of NT and NN using either COS cells transfected with the human subtype-1 NT receptor (hNTS1) or the human colon adenocarcinoma HT29 cell line that endogenously expresses hNTS1. Furthermore, the metabolic stability of the large peptides, when exposed to HT29 cells, was compared to that of NT and NN. Both large NT and large NN were able to bind to and activate hNTS1 with potencies that were approximately 10 times lower than that of their small counterpart. In addition, the large forms proved to be far less sensitive to degradation than the small peptides. Taken together, these data suggest that the large forms might represent endogenous, long-lasting activators of hNTS1 in a number of physiopathological situations.

The neurotensin receptor 1, NTS1, is a G protein-coupled receptor. We have shown previously that the NTS1 receptor-binding site of the peptide agonist involved residues in extracellular loop 3 and at the extracellular junction of transmembrane domains 4 and 6. Here, we investigated by site-directed mutagenesis residues in extracellular loop 3 that might be involved in agonist-induced activation of the rat NTS1 (rNTS1) receptor. Wild type and mutated receptors were expressed in COS (African green monkey kidney fibroblasts) cells. Labeled agonist and antagonist binding as well as inositol phosphate and cAMP productions were studied. Compared to the wild type NTS1 receptor, the W339A, F344A, H348A and Y349A mutant receptors exhibited (i) decreased proportion of high over low affinity agonist binding sites, (ii) increased sensitivity of high affinity agonist binding to GTP gamma S, and (iii) impaired G protein coupling of high affinity agonist-receptor complexes. The data are consistent with the C-terminal part of extracellular loop 3 being essential for allowing high affinity agonist-NTS1 receptor complexes to couple to G proteins.

Two G protein-coupled neurotensin (NT) receptors, termed NTR1 and NTR2, have been identified so far. In contrast to the NTR1, which has been extensively studied, little is known about the pharmacological and biological properties of the NTR2. In the course of characterizing NT analogs that exhibited binding selectivity for the NTR2, we discovered that this receptor constitutively activated inositol phosphate (IP) production. Here, we report on the constitutive activity of the human NTR2 (hNTR2) transfected in COS cells and on compounds that exhibit agonism, inverse agonism, and neutral antagonism at this receptor. IP levels increased linearly with time, whereas they remained constant in mock-transfected cells. Furthermore, IP production was proportional to the amount of hNTR2 present at the cell membrane. SR 48692, a nonpeptide antagonist of the NTR1, stimulated IP production, whereas levocabastine, a nonpeptide histamine H1 antagonist that binds the NTR2 but not the NTR1, behaved as a weak partial inverse agonist. NT analogs modified at position 11 of the NT molecule, in particular by the introduction of bulky aromatic D amino acids, exhibited binding selectivity at the hNTR2 and also behaved as partial inverse agonists, reversing constitutive IP production up to 50%. Finally, NT barely affected constitutive IP production but antagonized the effects of both agonist and inverse agonist compounds, thus behaving as a neutral antagonist. The unique pharmacological profile of the hNTR2 is discussed in the light of its sequence similarity with the NTR1 and the known binding site topology of NT and SR 48692 in the NTR1.