Targeting the cis-dimerization of LINGO-1 with low MW compounds affects its downstream signalling.

BACKGROUND AND PURPOSE: The transmembrane protein LINGO-1 is a negative regulator in the nervous system mainly affecting axonal regeneration, neuronal survival, oligodendrocyte differentiation and myelination. However, the molecular mechanisms regulating its functions are poorly understood. In the present study, we investigated the formation and the role of LINGO-1 cis-dimers in the regulation of its biological activity. EXPERIMENTAL APPROACH: LINGO-1 homodimers were identified in both HEK293 and SH-SY5Y cells using co-immunoprecipitation experiments and BRET saturation analysis. We performed a hypothesis-driven screen for identification of small-molecule protein-protein interaction modulators of LINGO-1 using a BRET-based assay, adapted for screening. The compound identified was further assessed for effects on LINGO-1 downstream signalling pathways using Western blotting analysis and AlphaScreen technology. KEY RESULTS: LINGO-1 was present as homodimers in primary neuronal cultures. LINGO-1 interacted homotypically in cis-orientation and LINGO-1 cis-dimers were formed early during LINGO-1 biosynthesis. A BRET-based assay allowed us to identify phenoxybenzamine as the first conformational modulator of LINGO-1 dimers. In HEK-293 cells, phenoxybenzamine was a positive modulator of LINGO-1 function, increasing the LINGO-1-mediated inhibition of EGF receptor signalling and Erk phosphorylation. CONCLUSIONS AND IMPLICATIONS: Our data suggest that LINGO-1 forms constitutive cis-dimers at the plasma membrane and that low MW compounds affecting the conformational state of these dimers can regulate LINGO-1 downstream signalling pathways. We propose that targeting the LINGO-1 dimerization interface opens a new pharmacological approach to the modulation of its function and provides a new strategy for drug discovery.

Molecular characterization of the family of choline transporter-like proteins and their splice variants.

We show here that the choline transporter-like (CTL) family is more extensive than initially described with five genes in humans and complex alternative splicing. In adult rat tissues, CTL2-4 mRNAs are mainly detected in peripheral tissues, while CTL1 is widely expressed throughout the nervous system. During rat post-natal development, CTL1 is expressed in several subpopulations of neurones and in the white matter, where its spatio-temporal distribution profile recalls that of myelin basic protein, an oligodendrocyte marker. We identified two major rat splice variants of CTL1 (CTL1a and CTL1b) differing in their carboxy-terminal tails with both able to increase choline transport after transfection in neuroblastoma cells. In the developing brain, CTL1a is expressed in both neurones and oligodendroglial cells, whereas CTL1b is restricted to oligodendroglial cells. These findings suggest specific roles for CTL1 splice variants in both neuronal and oligodendrocyte physiology.

Hedgehog interacting protein in the mature brain: membrane-associated and soluble forms.

Hedgehog interacting protein (Hip) is considered as a membrane protein implicated in sequestering the hedgehog (hh) morphogens during embryonic development. Here, we demonstrate that Hip transcription also occurs in cells scattered in discrete brain areas of adult rodents and we identify the presence of membrane-associated and soluble forms of Hip in the mature brain. Moreover, we show that soluble forms of Hip, present in the conditioned medium of HEK293 cells overexpressing Hip, inhibit Sonic hedgehog (Shh)-induced differentiation of C3H10T1/2 cells, a well-characterised response associated with Shh signalling. After transfection in HEK293 cells, Hip partitions with the raft component ganglioside GM1 during density gradient centrifugation. Analysis of tagged Hip constructs reveals that the putative transmembrane domain of Hip is not cleaved suggesting that other mechanisms are implicated in the release of its soluble forms. Taken together, these data are consistent with the involvement of both membrane-associated and soluble Hip in the regulation of Shh signalling in adult neural tissues.

Intrastriatal sonic hedgehog injection increases Patched transcript levels in the adult rat subventricular zone.

The morphogen sonic hedgehog (Shh) is implicated in neural tissue patterning and the growth of brain structures during embryogenesis and postnatal development and is also present in the adult brain. Shh signals through interaction with the tumour suppressor Patched (Ptc). This receptor for Shh is associated with Smoothened (Smo), a protein with high homology to the G-protein coupled receptors. However, little is known about the transduction mechanisms implicated in Shh signalling in the adult brain. The study described here shows that injection of aminoterminal myristoylated Shh (myrShhN) into the adult rat striatum robustly increases the levels of Ptc transcripts in selective brain areas including the subventricular zone (SVZ). The adult SVZ contains cell progenitors, which can proliferate and differentiate into new neurons and glia. In the myrShhN injected animals, proliferation and differentiation of these SVZ precursor cells were not affected as demonstrated by BrdU incorporation and immunohistochemistry performed with specific antibodies for nestin (uncommitted neural progenitors), PSA-NCAM (migrating neuroblasts) or GFAP (astrocytes). Together with the presence of Smo expressing cells and amino-terminal Shh (ShhN) protein in SVZ area of untreated animals, the data presented here supports the hypothesis that the Shh pathway may be activated in the adult brain, and that a niche for Shh signalling exists within the adult SVZ.

Co-localization of histamine with GABA but not with galanin in the human tuberomamillary nucleus.

The presence of GABA and galanin in histaminergic neurons was previously reported in the rodent brain but whether such co-localizations also occur in the human brain was not known. We used in situ hybridization histochemistry and immunohistochemistry to study the co-localization of histamine with GABA and galanin in neurons of the tuberomamillary nucleus of adult human posterior hypothalamus. On consecutive formalin-fixed paraffin-embedded sections, co-localization was assessed using the in situ hybridization for L-histidine decarboxylase mRNA and immunocytochemistry for glutamate decarboxylase-67 kDa or galanin in the two profiles of same cell. The pattern of distribution and number of histaminergic neurons identified by in situ hybridization of the synthesizing enzyme gene transcripts were in accordance with data reported for histamine immunoreactivity. The great majority of neurons within the main divisions of the tuberomamillary nucleus containing L-histidine decarboxylase mRNA was also immunoreactive for glutamate decarboxylase-67 kDa. The range of co-localization of the two markers varied from 72% in the lateral part, to 75-87% in the medial part and 83-88% in the ventral part. In contrast, no cell containing L-histidine decarboxylase mRNA was immunoreactive for galanin. We conclude that tuberomamillary neurons in human co-express histamine with GABA but, unlike the neurons in rodents, do not express galanin, indicating that neurotransmitter co-localization patterns differ in the two species.

High expression and anterograde axonal transport of aminoterminal sonic hedgehog in the adult hamster brain.

Sonic hedgehog (SHH) is considered to play an important role in tissue induction and patterning during development, particularly in determining neuronal cell fate in the ventral neural tube and in the embryonic forebrain. SHH precursor is autoproteolytically cleaved to an aminoterminal fragment (SHHN) which retains all known SHH biological activities. Here, we demonstrate the expression of a 22-kDa SHHN immunoreactive peptide in developing and adult hamster brain regions using a rabbit antiserum directed against a mouse SHHN fragment. Interestingly, SHHN was developmentally regulated with the highest expression observed in the adult brain, was resistant to Triton X-100 solubilization at 4 degrees C and partitioned with the raft component ganglioside GM1 during density gradient centrifugation. In rat brain, Shh transcripts were identified by double in situ hybridization in GABAergic neurons located in various basal forebrain nuclei including globus pallidus, ventral pallidum, medial septum-diagonal band complex, magnocellular preoptic nucleus and in cerebellar Purkinje cells as well as in motoneurons of several cranial nerve nuclei and of the spinal cord. We show that radiolabelled SHHN peptides are synthesized in the adult hamster retina and are transported axonally along the optic nerve to the superior colliculus in vivo. Our data indicate that SHHN is associated with cholesterol rich raft-like microdomains and anterogradely transported in the adult brain, and suggest that the roles of this extracellular protein are more diverse than originally thought.

Multiple subcellular localizations of PCTAIRE-1 in brain.

We developed a selective antibody to a synthetic peptide corresponding to an N-terminal sequence of the PCTAIRE-1 protein. In rodent brain extracts it recognized only the protein doublet characteristic of PCTAIRE-1, and this signal is completely abolished by preincubation of the antibody with the immunopeptide. Immunolabeling experiments done with this PCTAIRE-1-specific antibody reveal that the protein is widely distributed in the rodent brain as are the mRNAs visualized using an antisense riboprobe corresponding to the entire PCTAIRE-1 open reading frame. Two types of PCTAIRE-1 protein localizations were observed: first a diffuse labeling of almost all brain regions, particularly intense in the molecular layer of the cerebellum and the mossy fiber region of the hippocampus, and second a spot-like localization in the nuclei of large neurons such as cerebellar Purkinje cells and pyramidal cells of the hippocampus. Colocalization with the B23 protein allows one to identify these compartments as nucleoli. Our results suggest a nucleolar function of PCTAIRE-1 in large neurons and a role in regions containing important granule cell projections.

Distribution of bone morphogenetic protein and bone morphogenetic protein receptor transcripts in the rodent nervous system and up-regulation of bone morphogenetic protein receptor type II in hippocampal dentate gyrus in a rat model of global cerebral ischemia.

Bone morphogenetic proteins belong to the transforming growth factor-beta superfamily and act through serine/threonine kinase type I and type II receptors such as bone morphogenetic protein receptor type I and type II. In order to further understand the roles that these factors exert in the nervous system, we have examined the expression pattern of seven bone morphogenetic proteins and bone morphogenetic protein receptor type I and II transcripts in the brain and spinal cord of rodent. Whereas bone morphogenetic protein receptor type I expression was low in rat brain, in situ hybridization studies performed with specific digoxigenin-labelled riboprobes revealed the presence of bone morphogenetic protein receptor type II-positive cells throughout the brain, with a notable localization in dopaminergic cells of the substantia nigra. Bone morphogenetic protein receptor type II transcripts were also expressed by large motoneuron-like cells located in the ventral horn of the spinal cord and by sensory neurons of dorsal root ganglia. In addition, we observed a significant up-regulation of bone morphogenetic protein receptor type II in the granule cells of the dentate gyrus 48 h after transient global cerebral ischemia in rat suggesting that modulation of this receptor intervenes during neuronal plasticity or repair that occur upon brain injury. Among the potential ligands for this receptor, bone morphogenetic protein-6 and bone morphogenetic protein-7 were expressed in meninges and the choroid plexus, while bone morphogenetic protein-4-expressing cells were spatially and temporally regulated in myelinated structures during development and in the adult suggesting its expression in oligodendrocytes. These data clearly indicate that besides their roles in bone and embryonic tissues, bone morphogenetic proteins and their receptors may have also important functions in adult neural tissues.

Developmental and adult expression of rat calcium-sensing receptor transcripts in neurons and oligodendrocytes.

The calcium-sensing receptor (CaSR) is a member of a growing family of heptahelical receptors with an unusually large extracellular domain. To further delineate its functions in neurons and glia, we have investigated the expression pattern of CaSR transcripts in the postnatal and adult rat brain, spinal cord and dorsal root ganglia by in situ hybridization. CaSR-expressing cells were spatially and temporally regulated in myelinated structures with a caudo-rostral pattern that paralleled that of myelin basic protein, a marker of myelination, with a downregulation observed in the adult. Double-labelling studies demonstrated that CaSR mRNA colocalizes with myelin basic protein-expressing cells within fibre tracts, suggesting that CaSR is expressed by mature oligodendrocytes. In cultured rat oligodendrocytes, Ca2+ induced stimulation of phosphatidylinositol hydrolysis with an EC50 of 1.4 mM and increased intracellular calcium. NPS R-568 (1 microM), a calcimimetic, significantly stimulates the inositol phosphate response, whereas a less potent stereoisomer, NPS S-568 (1 microM), was without effect. These data suggest that a functional CaSR is expressed in mature oligodendrocytes with a potential role in myelination. CaSR expression was also developmentally regulated in neurons of the orbital cortex and in the CA2 region of the hippocampus, and present in olfactory nuclei, hypothalamic areas and in the area postrema through postnatal days to adulthood. This expression is consistent with a role of CaSR in olfactory or gustatory signal integration, and with the regulation of fluid and mineral homeostasis. CaSR expression in a subpopulation of small cells in dorsal root ganglia suggests additional roles for extracellular Ca2+ in sensory nerves.

An electric lobe suppressor for a yeast choline transport mutation belongs to a new family of transporter-like proteins.

Choline is an important metabolite in all cells due to the major contribution of phosphatidylcholine to the production of membranes, but it takes on an added role in cholinergic neurons where it participates in the synthesis of the neurotransmitter acetylcholine. We have cloned a suppressor for a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation. The full-length clone encodes a protein with 10 putative transmembrane domains, two of which contain transporter-like motifs, and whose expression increased high-affinity choline uptake in mutant yeast. The gene was called CTL1 for its choline transporter-like properties. The homologous rat gene, rCTL1, was isolated and found to be highly expressed as a 3. 5-kb transcript in the spinal cord and brain and as a 5-kb transcript in the colon. In situ hybridization showed strong expression of rCTL1 in motor neurons and oligodendrocytes and to a lesser extent in various neuronal populations throughout the rat brain. High levels of rCTL1 were also identified in the mucosal cell layer of the colon. Although the sequence of the CTL1 gene shows clear homology with a single gene in Caenorhabditis elegans, several homologous genes are found in mammals (CTL2-4). These results establish a new family of genes for transporter-like proteins in eukaryotes and suggest that one of its members, CTL1, is involved in supplying choline to certain cell types, including a specific subset of cholinergic neurons.

Changes in histamine H3 receptor responsiveness in mouse brain.

Changes in various histamine (HA) H3 receptor-mediated responses and H3 receptor binding in brain were investigated in mice receiving single or repeated administration of ciproxifan, a potent brain-penetrating and selective H3 receptor antagonist. Blockade of the H3 autoreceptor was nearly as effective in enhancing levels of tele-methylhistamine (t-MeHA), a major HA metabolite, in brain areas when ciproxifan was administered once either at 7 a.m. or 8 p.m., in spite of the large differences of basal levels at these two phases of the circadian cycle. Blockade after a single ciproxifan administration was, however, followed by a transient decrease in striatal t-MeHA levels, possibly reflecting rapid development of autoreceptor hypersensitivity. Following a 5-day administration of ciproxifan and a 2-day drug-free period, basal t-MeHA levels were significantly decreased (approximately -20%) in three brain areas, and the ED50 values of the drug to enhance t-MeHA levels were increased by 5-15 times without significant change in maximal response, indicating that H3 autoreceptor hypersensitivity had developed. However, in synaptosomes from the cerebral cortex of these animals, the H3 receptor-mediated inhibition of K+-induced [3H]HA release was not significantly modified. Subchronic administration of ciproxifan for 10 days also resulted in an increased binding of [125I]iodoproxyfan to the H3 receptor of striatal and hypothalamic membranes by 40-54%. Hypersensitivity at H3 somatodendritic autoreceptors and at heteroreceptors attributable to an increased number of HA binding sites could account for the various changes observed in this study.

Discrete localizations of hedgehog signalling components in the developing and adult rat nervous system.

Sonic hedgehog (Shh), a morphogen molecule implicated in embryonic tissue patterning, displays inductive, proliferative, neurotrophic and neuroprotective activities on various neural cells. Shh might exert its biological functions through binding to patched (Ptc) associated with smoothened (Smo), leading to downstream activation of target genes such as the transcription factor Gli1. We have performed a detailed localization of cells expressing transcripts of Shh, Ptc, Smo and Gli1 in brain and spinal cord of the adult rat as well as in the developing cerebellum. In the adult, Shh-positive cells were mainly observed in forebrain structures, in the Purkinje cells of the cerebellum and in motor neurons. Ptc-positive cells were frequently observed in brain areas devoid of any Shh transcripts, except in the median eminence or the facial nucleus, suggesting local Shh signalling. Interestingly, Smo transcripts were predominantly present within circumventricular organs, in granular cells of the dentate gyrus and in neurons of the reticular thalamic nucleus. The presence of Shh, Ptc and Smo transcripts in hypothalamic areas may indicate a role of Shh signalling in the modulation of neuroendocrine functions. The expression pattern of these three genes as well as of Gli1, and their developmental regulation in the cerebellum, suggest a possible role for Hedgehog signalling in the control of various cell populations within the cerebellum, particularly in granule cell proliferation and/or differentiation that might be impaired in proliferative states such as medulloblastomas.

Atypical neuroleptics enhance histamine turnover in brain via 5-Hydroxytryptamine2A receptor blockade.

Clozapine and olanzapine behave as weak H3-receptor antagonists in vitro with Ki values around 1 and 50 microM, respectively. Despite these modest apparent affinities, both compounds given orally to mice, nearly doubled steady-state tele-methylhistamine levels in brain, with ED50 values as low as 1 and 3 mg/kg, respectively, an effect comparable to those of potent H3-receptor antagonists. This effect corresponded to an enhancement of histamine turnover rate from 45 to 73 ng/g/h as measured in the case of olanzapine using the pargyline test. Other antipsychotics displaying, such as clozapine and olanzapine, high 5-hydroxytryptamine (5-HT)2A receptor antagonist potency, i.e., risperidone, thioridazine, seroquel, and iloperidone, also enhanced markedly tele-methylhistamine levels. This effect was 1) additive with that of a pure H3-receptor antagonist, ciproxifan, 2) mimicked by a 5-HT2A receptor antagonist, ketanserin, 3) reversed by a 5-HT2A receptor agonist, DOI, 4) not shared by antipsychotics with low affinity for the 5-HT2A receptor, i.e., haloperidol, sulpiride, raclopride, or remoxipride that, on the contrary, tended to reduce tele-methylhistamine levels. We conclude that in contrast to "typical" antipsychotics, "atypical" antipsychotics stimulate histamine neuron activity via blockade of the 5-HT2A receptor in vivo. This effect does not appear to account for their reduced extrapyramidal side-effects but may underlie their pro-cognitive properties.

Characterization of 5-ht6 receptor and expression of 5-ht6 mRNA in the rat brain during ontogenetic development.

We have determined the pharmacological characteristics of the rat 5-ht6 receptor stably expressed in CHO cells. Moreover, using RT-PCR experiments the in vivo expression of the gene encoding this receptor was studied in rat at various embryonic days (ED) starting from ED10 to birth (PN0) and at post-natal days (PN) up to PN36. The pharmacological analysis of the [3H]5-HT binding in stably transfected CHO cells expressing rat 5-ht6 receptors revealed the presence of a single class of high affinity saturable binding sites for 5-HT corresponding to an affinity constant: Kd=27.2+/-3.4 nM. This receptor also exhibited a high affinity for a number of typical and atypical antipsychotics, tricyclic antidepressant drugs and ergot alkaloïds. In stably transfected CHO cells, serotonin elicited a potent stimulation of adenylyl cyclase activity which was blocked by antipsychotic and antidepressant drugs. These results confirm the hypothesis that 5-ht6 receptors may correspond to an important target for atypical antipsychotics and reveal an original pharmacological profile for this receptor. The study of the ontogeny of the 5-ht6 mRNA in rat developing brain showed that 5-ht6 mRNA were first detectable with a high level on ED12, slighly decreased up to ED17 and then remained stable at high level until the adult age. The ontogenetic pattern of 5-ht6 mRNA expression appeared to correlate with the occurence of the first cell bodies of serotonergic neurons; the early expression of 5-ht6 mRNA and the fact that this receptor is positively coupled to the production of cAMP may suggest a role for 5-ht6 receptor in the early growth process involving the serotonergic system.

Regional distribution of Sonic Hedgehog, patched, and smoothened mRNA in the adult rat brain.

In vertebrates, Sonic Hedgehog (Shh), Desert Hedgehog (Dhh), and Indian Hedgehog (Ihh) genes encode a family of morphogen proteins that are implicated in a wide range of signaling activities, particularly during embryonic development. These secreted proteins are proposed to mediate their effects on target cells by interacting with their putative receptor, Patched (Ptc), and with a seven-pass transmembrane protein, Smoothened (Smo). However, the roles that these signaling molecules may play in adult tissues, particularly in brain, are not yet clearly defined. Therefore, we investigated the expression of these genes in adult rat tissues. Northern blot analysis revealed expression of Shh, Dhh, and Ihh genes in peripheral tissues, whereas Shh transcript was also identified in brain. It is interesting that northern blot analysis with probes derived from the mouse Ptc and Smo genes revealed the expression of a 7.9-kb and a 3.7-kb transcript, respectively, in all brain tissues examined. In situ hybridization experiments using specific digoxigenin-labeled riboprobes showed expression of Ptc and Smo transcripts in discrete brain areas. Shh-positive cells were observed in restricted regions of the brain. Within the cerebellum, Shh, Ptc, and Smo transcripts were colocalized in the Purkinje cell layer. These data suggest that, besides its roles in determining cell fate and patterning during embryogenesis, the hedgehog signaling pathway may have also important roles in the adult brain.

Detailed mapping of the histamine H2 receptor and its gene transcripts in guinea-pig brain.

Autoradiographic studies of the distribution of the histamine H2 receptor and its messenger RNAs were performed on serial frontal and a few sagittal sections of guinea-pig brain using [(125)I]iodoaminopotentidine for radioligand binding and a 33P-labelled complementary RNA probe for in situ hybridization, respectively. Both probes were validated by assessing non-specific labelling using non-radioactive competing H2 receptor ligands and a sense probe for binding sites and gene transcripts, respectively. In some areas, e.g., cerebral cortex, hippocampal complex or cerebellum, such studies were completed by identification of neurons expressing the H2 receptor messenger RNAs on emulsion-dipped sections. Nissl-stained sections from comparable levels were used to localize brain structures. In many brain areas, the distribution of the H2 receptor and its messenger RNAs appeared to parallel that known for histaminergic axons. For instance. high levels of both H2 receptor markers were detected in striatal and limbic areas known to receive abundant histaminergic projections. In contrast, in septum, hypothalamic, pontine and several thalamic nuclei, a comparatively low density of both H2 receptor markers was detected, suggesting that histamine actions in these areas are mediated by H1 and/or H3 receptors. Generally, the distribution of H2 receptor messenger RNA correlates well with that of [(125)I]iodoaminopotentidine binding sites, although some differences were observed. In a few regions (e.g., substantia nigra, locus coeruleus) high or moderate densities of binding sites were accompanied by a much more restricted expression of H2 receptor transcripts. Conversely, the mammillary region and the pontine nucleus exhibited higher levels of hybridization than of binding sites. In hippocampus, cerebral and cerebellar cortex there was a selective localization of the H2 receptor messenger RNA in the granule cells of dentate gyrus, pyramidal cells of the Ammon's horn and cerebral cortex, and Purkinje cells of cerebellum, whereas [(125)I]iodoaminopotentidine binding sites were located in layers where the dendritic trees of these messenger RNA-expressing neurons extend. The same discrepancy between messenger RNAs and binding sites suggests that striatonigral endings are endowed with the H2 receptor. The histamine H1 and H2 receptors both appear to be present in several brain areas, in some cases in a way suggesting their potential co-expression by the same neuronal populations, e.g., in granule and pyramidal cells in the hippocampal formation. This co-expression accounts for synergic responses, e.g., on cAMP generation, previously observed upon co-stimulation of both receptor subtypes. The widespread distribution of the H2 receptor, namely in thalamic nuclei or in telencephalic areas such as most layers of the cerebral cortex, together with its excitatory role previously established in electrophysiological studies, support its alleged function in mediating the histamine-driven control of arousal mechanisms. In addition, the detection of H2 receptor expression in brainstem areas from which other monoaminergic pathways involved in the control of states of sleep and wakefulness emanate, e.g., several raphe nuclei, locus coeruleus or substantia innominata, suggests possible interrelationships between all of these systems with highly divergent projections to the thalamus and telencephalon. The present mapping of the H2 receptor and its gene transcripts should facilitate neurochemical, neurophysiological and behavioural studies aimed at clarifying the role of histaminergic systems in brain.

Expression of 5-HT7 receptor mRNA in rat brain during postnatal development.

The present study is the first one to demonstrate the expression of 5-HT7 receptor mRNA by in situ hybridization during postnatal development. No quantitative developmental changes in the 5-HT7 gene expression was observed in neocortex, pyramidal layers of CA1 and CA2, dentate gyrus, most of thalamic nuclei, mammillary region, superior colliculus and central gray. However, in retrosplenial cortex, subiculum and medial habenula an increase of labeling is observed between postnatal days (PN) PN15 and PN21. Striatum showed a transient expression during the first stages of development to be undetectable in adults. CA3 pyramidal cell layer, intramediodorsal thalamic nucleus and lateral habenula displayed a high mRNA expression at PN5 and PN8 which decreased throughout development but it was still present in adults. A possible non-neurotransmitter trophic function of 5-HT mediated through 5-HT7 receptors could be suggested.

Binding of histamine H3-receptor antagonists to hematopoietic progenitor cells. Evidence for a histamine transporter unrelated to histamine H3 receptors.

Hematopoietic progenitor cells can take up histamine or release IL-3-induced histamine through a bi-directional transport system that is blocked by H3-receptor antagonists. In the present study we demonstrate a correlation between the affinity of various H3-receptor antagonists and their potency as inhibitors of histamine uptake. All compounds that blocked histamine uptake also inhibited IL-3-induced histamine release. Yet, classical H3 receptors are not involved in this biological activity, since highly specific histamine H3-receptor agonists neither alter histamine uptake nor affect the release of endogenous histamine synthesized in response to IL-3. Furthermore, the inhibitory effect of H3-receptor antagonists on histamine uptake was not reversed by the agonists. Unlike H3-receptor antagonists, the agonists did not displace the binding of the labeled antagonist iodoproxyfan.

Inhibition of histamine versus acetylcholine metabolism as a mechanism of tacrine activity.

Following tacrine administration i.p. to mice, the histamine N-methyltransferase activity of brain homogenates was more potently inhibited than the acetylcholinesterase activity (ID50 of 5.3 mg/kg vs. 13.6 mg/kg). The formation of the metabolite, tele-methylhistamine, in brain of mice treated with an histamine H3 receptor antagonist was abolished by tacrine with an ID50 as low as 1.2 +/- 0.4 mg/kg. The participation of histamine in the actions of tacrine and the relevance of histamine H3 receptor antagonists in Alzheimer's disease are suggested.

Prodynorphin mRNA expression in the rat dentate gyrus after cerebral ischemia.

The beneficial effects of exogenous kappa receptor agonists in preventing neuronal damage elicited by brain ischemia suggest a role for endogenous dynorphins. In agreement prodynorphin (PDYN) gene expression in granule cells of the dentate gyrus detected by in situ hybridization was drastically but transiently decreased 18-32 h after four-vessel cerebral ischemia for 20 min in rats. We propose that decreased dynorphin synthesis and release could contribute to the delayed neuronal death of hippocampal pyramidal cells in this model.