Biomedical subjects

A Beaudet

Publications and source records attributed to A Beaudet.

At least 37 records · Page 2Linked to original sources

PubMed2000

Association of neuropeptide Y Y1 receptors with glutamate-positive and NPY-positive neurons in rat hippocampal cultures.

The hippocampus is particularly enriched with neuropeptide tyrosine (NPY) and NPY receptors including the Y1, Y2 and Y5 subtypes. We have previously reported on the enrichment of cultured rat hippocampal neurons in specific [125I][Leu31, Pro34]PYY/BIBP3226-sensitive (Y1) binding sites and Y1 receptor mRNAs [St-Pierre et al. (1998) Br. J. Pharmacol., 123, p183]. We have now identified which cell types express the Y1 receptor. The majority of Y1 receptors, visualized using either the radiolabeled probe [125I][Leu31,Pro34]PYY or two antibodies directed against distinct domains of the Y1 receptor, was expressed in neurons as revealed by neuron-specific enolase (NSE) immunostaining. One antibody was directed against the second extracelllular loop of the Y1 receptor (amino acids 185-203) whereas the second was directed against the intracellular C-terminal loop (amino acids 355-382). The labelling was evident over both perikarya and processes. Neurons labelled by the various Y1 receptor probes were mostly glutamate-positive as revealed by double immunostaining. Most interestingly, a number of NPY-positive cultured hippocampal neurons were also enriched with the Y1 receptor, suggesting that this subtype may act as an autoreceptor to regulate NPY release in the hippocampus. These results thus provide an anatomical basis for the modulation of glutamate and NPY release by the Y1 receptor in the hippocampus.

Amino Acid Sequence↗

PubMed2000

A highly sensitive quantitative cytosensor technique for the identification of receptor ligands in tissue extracts.

Because G-protein-coupled receptors (GPCRs) constitute excellent putative therapeutic targets, functional characterization of orphan GPCRs through identification of their endogenous ligands has great potential for drug discovery. We propose here a novel single cell-based assay for identification of these ligands. This assay involves (a) fluorescent tagging of the GPCR, (b) expression of the tagged receptor in a heterologous expression system, (c) incubation of the transfected cells with fractions purified from tissue extracts, and (d) imaging of ligand-induced receptor internalization by confocal microscopy coupled to digital image quantification. We tested this approach in CHO cells stably expressing the NT1 neurotensin receptor fused to EGFP (enhanced green fluorescent protein), in which neurotensin promoted internalization of the NT1-EGFP receptor in a dose-dependent fashion (EC(50) = 0.98 nM). Similarly, four of 120 consecutive reversed-phase HPLC fractions of frog brain extracts promoted internalization of the NT1-EGFP receptor. The same four fractions selectively contained neurotensin, an endogenous ligand of the NT1 receptor, as detected by radioimmunoassay and inositol phosphate production. The present internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range that should prove useful for the identification of putative natural and synthetic ligands for GPCRs.

Animals↗

PubMed2000

Intracellular dynamics of sst5 receptors in transfected COS-7 cells: maintenance of cell surface receptors during ligand-induced endocytosis.

Internalization of G protein-coupled receptors is crucial for resensitization of phosphorylation-desensitized receptors, but also for their long term desensitization through sequestration. To elucidate the mechanisms regulating cell surface availability of the somatostatin (SRIF) receptor subtype sst5, we characterized its internalization properties in transfected COS-7 cells using biochemical, confocal microscopic, and electron microscopic techniques. Our results demonstrated rapid and efficient sequestration of specifically bound [125I]Tyr0-D-Trp8-SRIF (up to 45% of bound radioactivity). Combined immunocytochemical detection of sst5 and visualization of a fluorescent SRIF analog by confocal microscopy revealed that whereas the internalized ligand progressively clustered toward the cell center with time, immunoreactive receptors remained predominantly associated with the plasma membrane. The preservation of cell surface receptors was confirmed by binding experiments on whole cells revealing a lack of saturability of [125I]Tyr0-D-Trp8-SRIF binding at 37 C. Binding was rendered saturable by the drug monensin, showing that receptor recycling played a key role in the preservation of cell surface receptors. Electron microscopy demonstrated that in addition to receptor recycling, internalization of receptor-ligand complexes triggered a massive recruitment of sst5 receptor molecules from intracellular stores to the membrane. This combination of recycling and recruitment of spare receptors may protect sst5 from long term down-regulation through sequestration and, therefore, facilitate extended SRIF signaling.

Animals↗

PubMed2000

Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor.

The neuropeptide neurotensin (NT) is known to be internalized in a receptor-mediated fashion into its target cells. To gain insight into the mechanisms underlying this process, we monitored in parallel the migration of the NT1 neurotensin receptor subtype and a fluorescent analog of NT (fluo-NT) in COS-7 cells transfected with a tagged NT1 construct. Fluo-NT internalization was prevented by hypertonic sucrose, potassium depletion and cytosol acidification, demonstrating that it proceeded via clathrin-coated pits. Within 0-30 minutes, fluo-NT accumulated together with its receptor in Acridine Orange-positive, acidic organelles. These organelles concentrated transferrin and immunostained positively for rab 5A, therefore they were early endosomes. After 30-45 minutes, the ligand and its receptor no longer colocalized. Fluo-NT was first found in rab 7-positive late endosomes and later in a nonacidic juxtanuclear compartment identified as the Trans-Golgi Network (TGN) by virtue of its staining for syntaxin 6. This juxtanuclear compartment also stained positively for rab 7 and for the TGN/pericentriolar recycling endosome marker rab 11, suggesting that the ligand could have been recruited to the TGN from either late or recycling endosomes. By that time, internalized receptors were detected in Lamp-1-immunoreactive lysosomes. These results demonstrate that neurotensin/NT1 receptor complexes follow a recycling cycle that is unique among the G protein-coupled receptors studied to date, and provide the first evidence for the targeting of a nonendogenous protein from endosomes to the TGN.

Animals↗

PubMed2000

Sub-population of cultured hippocampal astrocytes expresses neuropeptide Y Y(1) receptors.

The expression and pharmacological characterization of neuropeptide Y (NPY) receptors of the Y(1) subtype on cultured hippocampal neurons was reported using radioreceptor assays and immunohistochemical approaches (St-Pierre et al., 1998). The present study aimed to establish the presence of NPY Y(1) receptors on cultured hippocampal astrocytes using similar strategies. Immunocytochemical experiments were carried out using three antisera directed against distinct domains (amino acids sequence 185-203, 198-213 and 355-382) of the Y(1) receptor. Double-labeling experiments and confocal microscopy with these Y(1) receptor antisera demonstrated their recognition of the same sub-population (20%) of GFAP-positive astrocytes in culture. The immunostaining seen with all three Y(1) receptor antisera took the form of large irregular clusters distributed throughout cell bodies and processes. Further experiments using radioactive ligands confirmed the presence of NPY receptors on cultured hippocampal astrocytes. Emulsion receptor autoradiography using a newly developed ligand, [(125)I]GR231118 in the presence of PYY, hPP or BIBP3226 (1 microM), pharmacologically established the Y(1) nature of these receptors. Specific [(125)I]GR231118 binding was competed by PYY and the selective Y(1) antagonist BIBP3226 but not by hPP (a Y(4)/Y(5) agonist). Similar autoradiographic labeling patterns were obtained using [(125)I][Leu(31).Pro(34)]PYY (a Y(1)/Y(4)/Y(5) agonist) whereas [(125)I]PYY(3-36) (a Y(2)/Y(5) agonist) failed to generate any specific signal. Hence, rat cultured hippocampal astrocytes express a typical Y(1) receptor without evidence for the presence of Y(2), Y(4) or Y(5) subtypes. These data suggest a preferential regulation by NPY, acting via the Y(1) receptors, of astrocytic function.

Amino Acid Sequence↗

PubMed1999

Immunohistochemical distribution of the somatostatin receptor subtype 5 in the adult rat brain: predominant expression in the basal forebrain.

Somatostatin exerts its actions by means of a family of G protein-coupled receptors, five of which have so far been cloned. Whereas mRNAs for receptor subtypes sst(1)-sst(4) have been unequivocally localized in the brain, the data concerning the fifth subtype, sst(5), are contradictory. Moreover, whereas sst(1) and sst(2A) receptor proteins have been localized by immunohistochemistry, the distribution of sst(3)-sst(5) receptor proteins and/or subtype-specific binding remains to be determined in the central nervous system. In the present study, we investigated the distribution of immunoreactive sst(5) in adult rat brain and pituitary and demonstrated the presence of this receptor protein in the central nervous system by using an affinity-purified antibody generated against the C-terminus of the receptor. The specificity of the antibody for sst(5) was established by immunoblotting experiments on membranes prepared from cells transfected with cDNA encoding different somatotropin release inhibiting (SRIF) receptor subtypes as well as from anterior pituitary. In both systems, the antibody specifically recognized a band at approximately 50 kDa molecular mass, corresponding well to the reported size of the cloned receptor (48 kDa). Immunofluorescence in COS-7 cells transfected with individual SRIF receptor subtypes as well as in sections of rat pituitary demonstrated the antibody's applicability to the immunohistochemical detection of sst(5) receptors. In rat brain sections, sst(5) immunoreactivity was predominantly associated with neuronal perikarya and primary dendrites. Immunolabeling was most prominent in the olfactory tubercle, islands of Calleja, diagonal band of Broca, substantia innominata, and magnocellular preoptic nucleus of the basal forebrain as well as in the reticular nucleus of the thalamus. Other, less intensely labeled areas included the cerebral cortex, hippocampus, amygdala, preoptic area as well as the lateroanterior nucleus of the hypothalamus. The present findings provide the first characterization of the anatomic distribution of sst(5) receptors in the rat brain. They demonstrate a prominent expression of these receptors in the basal forebrain, suggesting that they may be involved in the mediation of somatostatin effects on the sleep-wake cycle through their association with cortically projecting subcortical systems.

Animals↗

PubMed1999

Receptor-mediated internalization is critical for the inhibition of the expression of growth hormone by somatostatin in the pituitary cell line AtT-20.

The inhibitory effect of the neuropeptide somatostatin on the expression of growth hormone was measured by quantitative polymerase chain reaction in the pituitary cell line AtT-20. We demonstrate that this effect is dependent on the internalization of somatostatin-receptor complexes and that it is totally independent from the peptide-induced inhibition of adenylate cyclase. Indeed, the inhibitory effect of the peptide on growth hormone mRNA levels was totally insensitive to pertussis toxin treatment but was totally abolished under conditions which block somatostatin receptor internalization. Comparative confocal microscopic imaging of fluorescent somatostatin sequestration and fluorescence immunolabeling of sst1, sst2A, and sst5 receptors suggests that sst2A is most probably responsible of the inhibitory effect of somatostatin on growth hormone expression.

Animals↗

PubMed1999

Immunohistochemical distribution of the prohormone convertase PC5-A in rat brain.

Prohormone convertase 5 is an endoprotease of the kexin/subtilisin-like family, which has been postulated to play a role in the proteolytic maturation of a variety of pro-peptides in the mammalian brain. In order to gain insight into the functional role of prohormone convertase 5 in the central nervous system, the regional, cellular and subcellular distributions of the enzyme were investigated by immunohistochemistry in rat brain using an N-terminal-directed specific antibody shown previously to recognize both the mature and unprocessed forms of the enzyme. Throughout the brain, prohormone convertase 5 immunoreactivity was concentrated within nerve cell bodies and proximal dendrites. No prohormone convertase 5 immunoreactivity was associated with astrocytes, as confirmed by the absence of prohormone convertase 5 immunolabeling in cells immunopositive for the glial protein S-100alpha. Within neurons, prohormone convertase 5 immunoreactivity was concentrated within the Golgi apparatus, as revealed immunohistochemically within the same sections using antibodies against the medial cisternae protein MG-160. It was also present within small vesicular-like elements distributed throughout the cytoplasm of perikarya and dendrites, but not of axons, as confirmed by its lack of co-localization with the synaptic terminal marker Dynamin-1. These results suggest that prohormone convertase 5 is active within early compartments of the neuronal regulated secretory pathway and that it is unlikely to be released with its processed substrates. At the regional level, prohormone convertase 5-immunoreactive perikarya were distributed extensively throughout the forebrain. The most numerous and intensely labeled were detected in the olfactory bulb, cerebral cortex, globus pallidus, endopeduncular and subthalamic nuclei, septum, diagonal band of Broca, magnocellular and medial preoptic areas, supraoptic and arcuate nuclei of the hypothalamus, and anterodorsal, laterodorsal, paraventricular and reticular nuclei of the thalamus. Moderate to dense neuronal labeling was also evident in the olfactory tubercle, caudate-putamen, claustrum, bed nucleus of the stria terminalis, substantia innominata, hippocampus, amygdala, and remaining thalamic and hypothalamic nuclei. This widespread distribution suggests that prohormone convertase 5 is involved in the processing of a variety of neuropeptide and/or neurotrophin precursors in mammalian brain.

Animals↗

PubMed1999

Pharmacological, molecular and functional characterization of glial neurotensin receptors.

The pharmacological properties, molecular identity and physiopathological regulation of neurotensin receptors expressed by central astrocytes were investigated in primary glial cultures and sections from the adult rat brain. Binding experiments carried out on astrocytes in culture revealed the presence of a single apparent class of neurotensin binding sites. These sites bound [125]neurotensin with an affinity (6 nM) comparable to that of the recently cloned NT2 low-affinity receptor expressed in transfected cells. The glial receptor was sensitive to the antihistamine, levocabastine, but less so than the NT2 site expressed in heterologous expression systems, suggesting the presence of an additional site or a differential coupling of the NT2 receptor in glia. Reverse transcription-polymerase chain reaction experiments demonstrated that both NT2 and NT3 neurotensin receptor sub-types were in fact expressed by cortical glial cells in culture. Confocal microscopic visualization of specifically bound fluorescent neurotensin indicated that this expression concerned only a sub-population of astrocytes in culture, in conformity with earlier reports of a heterogeneous expression of neuropeptides and their receptors by glial cells. To further investigate the functionality of NT2 receptors expressed in astrocytes, dual immunohistochemical labeling of glial fibrillary acidic protein and in situ hybridization of NT2 messenger RNA was performed on sections of normal and lesioned rat brain. In sections from normal brain, only a small subset of immunolabeled astrocytes hybridized NT2 messenger RNA. By contrast, in sections of stab-wounded rat brains, there was a marked increase in the number of NT2-hybridizing astrocytes in the surround of the lesion. Furthermore, NT2 expression within immunopositive reactive astrocytes was significantly enhanced as compared to immunolabeled glial cells in the brain of control animals. These results indicate that NT2 receptor expression is up-regulated during astrocytic reaction, suggesting that NT2 receptors may play a role in regulating glial response to injury.

Animals↗

PubMed1999

Sexually dimorphic expression of sst1 and sst2 somatostatin receptor subtypes in the arcuate nucleus and anterior pituitary of adult rats.

The pattern of growth hormone (GH) secretion and rate of somatic growth are markedly sexually dimorphic, but the underlying neuroendocrine mechanisms are far from clear. In the present study, we tested the hypothesis that the sexual dimorphism of GH secretion may be due to gender-related differences in the transduction of somatostatin's actions in brain and/or pituitary. To accomplish this, we compared the distributional pattern and level of expression of two somatostatin receptor subtypes, sst1 and sst2, in the brain and pituitary of adult male and female rats by in-situ hybridization using 35S-labelled antisense riboprobes. In the brain, the hybridization pattern and labelling density of sst1 and sst2 mRNA-expressing cells, as revealed by computer-assisted image analysis, in areas including the cerebral cortex, medial habenula (MHb) and ventromedial hypothalamic nucleus (VMN), were similar in male and female rats. In contrast, there was a marked sex-related difference in sst1 expression in the arcuate nucleus of the hypothalamus; both the number and labelling density of sst1 mRNA-expressing cells were two- to threefold greater in males than in females and this significant increase was homogenous throughout the rostrocaudal extent of the nucleus. No gender-related differences in arcuate sst2 mRNA levels were found. At the level of the anterior pituitary, the labelling density of sst2 mRNA in males was significantly higher than that of females. No sex-related difference in pituitary sst1 mRNA was observed. These results demonstrate a sexual dimorphism in the expression of two somatostatin receptor subtypes, sst1 and sst2, at the level of the arcuate nucleus and anterior pituitary, respectively. Such dimorphism suggests a differential involvement of sst1 and sst2 in GH regulation with respect to gender, and may imply roles for sst2 and sst1 in transducing somatostatin's actions on pituitary somatotrophs and GH-releasing hormone-containing arcuate neurones, respectively, to generate the lower basal and higher GH pulse levels characteristic of the male rat.

Animals↗

PubMed1999

Confocal microscopy reveals thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) in the classical secretory pathway.

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) and neurolysin (EC 3.4.24.16; EP24.16) are closely related enzymes involved in the metabolic inactivation of bioactive peptides. Both of these enzymes were previously shown to be secreted from a variety of cell types, although their primary sequence lacks a signal peptide. To investigate the mechanisms responsible for this secretion, we examined by confocal microscopy the subcellular localization of these two enzymes in the neuroendocrine cell line AtT20. Both EP24.15 and EP24.16 were found by immunohistochemistry to be abundantly expressed in AtT20 cells. Western blotting experiments confirmed that the immunoreactivity detected in the soma of these cells corresponded to previously cloned isoforms of the enzymes. At the subcellular level, both enzymes colocalized extensively with the integral trans-Golgi network protein, syntaxin-6, in the juxtanuclear region. In addition, both EP24.15 and EP24.16 were found within small vesicular organelles distributed throughout the cell body. Some, but not all, of these organelles also stained positively for ACTH. These results demonstrate that both EP24.15 and EP24.16 are present within the classical secretory pathway. Their colocalization with ACTH further suggests that they may be targeted to the regulated secretory pathway, even in the absence of a signal peptide.

Animals↗

PubMed1998

Correlative ultrastructural distribution of neurotensin receptor proteins and binding sites in the rat substantia nigra.

Neurotensin (NT) produces various stimulatory effects on dopaminergic neurons of the rat substantia nigra. To gain insight into the subcellular substrate for these effects, we compared by electron microscopy the distribution of immunoreactive high-affinity NT receptor proteins (NTRH) with that of high-affinity 125I-NT binding sites in this region of rat brain. Quantitative analysis showed a predominant association of immunogold and radioautographic labels with somata and dendrites of presumptive dopaminergic neurons, and a more modest localization in myelinated and unmyelinated axons and astrocytic leaflets. The distributions of immunoreactive NTRH and 125I-NT binding sites along somatodendritic plasma membranes were highly correlated and homogeneous, suggesting that membrane-targeted NTRH proteins were functional and predominantly extrasynaptic. Abundant immunocytochemically and radioautographically labeled receptors were also detected inside perikarya and dendrites. Within perikarya, these were found in comparable proportions over membranes of smooth endoplasmic reticulum and Golgi apparatus, suggesting that newly synthesized receptor proteins already possess the molecular and conformational properties required for effective ligand binding. By contrast, dendrites showed a proportionally higher concentration of immunolabeled than radiolabeled intracellular receptors. A fraction of these immunoreactive receptors were found in endosomes, suggesting that they had undergone ligand-induced internalization and were under a molecular conformation and/or in a physical location that precluded their recognition by and/or access to exogenous ligand. Our results provide the first evidence that electron microscopic immunocytochemistry of the NT receptor identifies sites for both the binding and trafficking of NT in the substantia nigra.

Animals↗

PubMed1998

PC5-A-mediated processing of pro-neurotensin in early compartments of the regulated secretory pathway of PC5-transfected PC12 cells.

Among the members of the proprotein convertase (PC) family, PC1 and PC2 have well established roles as prohormone convertases. Another good candidate for this role is PC5-A that has been shown to be present in the regulated secretory pathway of certain neuroendocrine tissues, but evidence that it can process prohormones is lacking. To determine whether PC5-A could function as a prohormone convertase and to compare its cleavage specificity with that of PC1 and PC2, we stably transfected the rat pheochromocytoma PC12 cell line with PC5-A and analyzed the biosynthesis and subcellular localization of the enzyme, as well as its ability to process pro-neurotensin/neuromedin N (pro-NT/NN) into active peptides. Our data showed that in transfected PC12 cells, PC5-A was converted from its 126-kDa precursor form into a 117-kDa mature form and, to a lesser extent, into a C-terminally truncated 65-kDa form of the 117-kDa product. Metabolic and immunochemical studies showed that PC5-A was sorted to early compartments of the regulated secretory pathway where it colocalized with immunoreactive NT. Furthermore, pro-NT/NN was processed in these compartments according to a pattern that differed from that previously described in PC1- and PC2-transfected PC12 cells. This pattern resembled that previously reported for pro-NT/NN processing in the adrenal medulla, a tissue known to express high levels of PC5-A. Altogether, these data demonstrate for the first time the ability of PC5-A to function as a prohormone convertase in the regulated secretory pathway and suggest a role for this enzyme in the physiological processing of pro-NT/NN.

Amino Acid Sequence↗

PubMed1998

Regional and cellular distribution of low affinity neurotensin receptor mRNA in adult and developing mouse brain.

Levocabastine-sensitive neurotensin receptor (NTRL) mRNAs were localized by in situ hybridization in adult and developing mouse brain. NTRL hybridization signal was widely distributed throughout the neuraxis. The highest concentrations of NTRL mRNA were detected in the olfactory system, olfactory tubercle, cerebral and cerebellar cortices, hippocampal formation, and selective hypothalamic nuclei. Moderate to dense hybridization signal was also observed in association with a variety of auditory, visual, and somatosensory relay nuclei, suggesting that the NTRL might be involved in a widespread modulation of primary afferent pathways. Finally a high expression of NTRL was evident in brainstem structures implicated in descending antinociceptive influences (e.g., the periaqueductal gray, nucleus raphe magnus, gigantocellular reticular nucleus, pars alpha, and lateral paragigantocellular nucleus) consistent with the proposed mediation of NT-induced analgesia by the NTRL. Although most of the regions found here to express NTRL mRNA were previously reported to be devoid of mRNA encoding the high affinity NT receptor (NTRH), a few areas (e.g., the anterior olfactory nucleus, medial septum, diagonal band of Broca, reticular thalamic nucleus, suprachiasmatic hypothalamic nucleus, and pontine nucleus) were enriched in both receptor subtypes, suggesting a possible coexpression of these receptors by the same cells. Ontogenic studies revealed that in the mouse brain, NTRL mRNA was detected only from postnatal day 14 and did not reach adulthood concentrations before day 30. In cerebral cortex, the developmental increase in NTRL expression was correlated over time with the decrease in NTRH expression previously documented in the rat, suggesting a progressive takeover of the latter by the former for transduction of the effects of NT in this structure.

Animals↗

PubMed1998

Origin of the neurotensinergic innervation of the rat basal forebrain studied by retrograde transport of cholera toxin.

Basal forebrain cholinergic neurons project to the hippocampus and cerebral cortex where they play an important role in cortical activation, attention, and memory. These neurons have been shown to express functional neurotensin receptors and to receive a dense neurotensinergic innervation. In the present study, we investigated the origin of this innervation by combining retrograde transport of cholera toxin with immunohistochemical detection of neurotensin. After injection of cholera toxin in the anterior substantia innominata and diagonal band of Broca, retrogradely labelled cells were widely distributed throughout forebrain limbic structures. Only a small proportion of these cells, located (by decreasing order of frequency) in the lateral septum, medial preoptic area, rostral hypothalamus, nucleus accumbens, and rostral basal forebrain, were dually labelled for neurotensin. After injection of cholera toxin in the posterior substantia innominata and magnocellular preoptic nucleus, retrogradely labelled cells were detected throughout the limbic forebrain and pontomesencephalic tegmentum. Here again, only a small proportion of these cells, located (by decreasing order of frequency) in the nucleus accumbens, lateral septum, rostral basal forebrain, hypothalamus, bed nucleus of the stria terminalis, supramammilliary nucleus, ventral tegmental area, and raphe complex co-localized neurotensin. In view of the burst generating properties of neurotensin on basal forebrain cholinergic neurons, our results suggest that neurotensin projections may be part of the septo-hippocampo-septal loop regulating hippocampal theta activity. More caudally, neurotensin axons originating from the lateral hypothalamus and pontomesencephalic tegmentum may contribute to the contingent of ascending reticular formation fibers involved in the regulation of the sleep-wake cycle.

Animals↗

PubMed1998

Interrelationships between somatostatin sst2A receptors and somatostatin-containing axons in rat brain: evidence for regulation of cell surface receptors by endogenous somatostatin.

Using an antipeptide antibody, we reported previously on the distribution of the somatostatin sst2A receptor subtype in rat brain. Depending on the region, immunolabeled receptors were either confined to neuronal perikarya and dendrites or distributed diffusely in tissue. To investigate the functional significance of these distribution patterns, we examined the regional and cellular relationships between somatostatin axons and sst2A receptors in the rat CNS, using double-labeling immunocytochemistry. Light and confocal microscopy revealed a significant correlation (p < 0.02) between the distribution of somatodendritic sst2A receptor immunoreactivity and that of somatostatin terminal fields, both quantitatively and qualitatively. Furthermore, in regions of somatodendritic labeling, a subpopulation of sst2A-immunoreactive cells was also immunopositive for somatostatin, suggesting that a subset of sst2A receptors consists of autoreceptors. By contrast, in regions displaying diffuse sst2A labeling only moderate to low densities of somatostatin terminals were observed, and no significant relationship was found between terminal density and receptor immunoreactivity. At the electron microscopic level, areas expressing somatodendritic sst2A labeling were found by immunogold cytochemistry to display low proportions of membrane-associated, as compared with intracellular, receptors. Conversely, in regions displaying diffuse sst2A receptor labeling, receptors were predominantly associated with neuronal plasma membranes, a finding consistent with the high density of sst2 binding sites previously visualized in these areas by autoradiography. Double-labeling studies demonstrated that in the former but not in the latter regions, sst2A-immunoreactive somata and dendrites were heavily contacted by somatostatin axon terminals. Taken together, these results suggest that the low incidence of membrane-associated receptors observed in regions of somatodendritic sst2A labeling may be caused by downregulation of cell surface receptors by endogenous somatostatin, possibly through ligand-induced receptor internalization.

Animals↗

PubMed1998

Sensory presynaptic and widespread somatodendritic immunolocalization of central ionotropic P2X ATP receptors.

Recent evidence suggests that extracellular ATP plays a neurotransmitter role in the central nervous system. Its fast ionotropic effects are exerted through a family of P2X ATP-gated channels expressed in brain and spinal cord. To determine the physiological significance of central ATP receptors, we have investigated the localization of a major neuronal P2X receptor at the cellular and subcellular levels using affinity-purified antibodies directed against the C-terminal domain of P2X4 subunit. Subunit-specific anti-P2X4 antibodies detected a single band of 57,000 +/- 3000 mol. wt in transfected HEK-293 cells and in homogenates from adult rat brain. The strongest expression of central P2X receptors was observed in the olfactory bulb, lateral septum, cerebellum and spinal cord. P2X4 immunoreactivity was also evident in widespread areas including the cerebral cortex, hippocampus, thalamus and brainstem. In all regions examined, P2X receptors were associated with perikarya and dendrites where they were concentrated at the level of afferent synaptic junctions, confirming a direct involvement of postsynaptic ATP-gated channels in fast excitatory purinergic transmission. Moreover, P2X4-containing purinoceptors were localized in axon terminals in the olfactory bulb and in the substantia gelatinosa of nucleus caudalis of the medulla and dorsal horn of the spinal cord, demonstrating an important selective presynaptic role of ATP in the modulation of neurotransmitter release in central sensory systems.

Adenosine Triphosphate↗

PubMed1998

Preferential expression of sst2A over sst2B somatostatin receptor splice variant in rat brain and pituitary.

The expression of sst2A and sst2B somatostatin (SRIH) receptor splice variants was examined by polymerase chain reaction (PCR) in rat brain and pituitary. Two sets of primers chosen to recognize either both sst2A and sst2B or only the sst2B transcript yielded bands of the size predicted from the cloned sst2A and sst2B sequences in both mouse and rat tissue extracts. Accordingly, experiments carried out on mouse brain extracts and on AtT-20 pituitary cells yielded sst2A/sst2B expression ratios comparable to those previously published. As in the mouse, rat pituitary extracts contained both sst2A and sst2B transcripts. By contrast, various cerebral regions in which both sst2A and sst2B forms were detected in the mouse contained only sst2A form in rat brain, with the exception of the cerebral cortex which also showed weak sst2B expression. No sst2B mRNA was detected in the arcuate nucleus/median eminence complex, indicating that the sst2A form is the one that is associated with growth-hormone-releasing-hormone-containing neurons documented to express sst2 receptors in this region. Finally, only sst2A transcripts were detected in rat astrocytes in culture, suggesting that this form of the receptor is responsible for sst2-mediated effects of SRIH on these cells.

Animals↗