Biomedical subjects

R Quirion

Publications and source records attributed to R Quirion.

At least 19 recordsLinked to original sources

PubMed2000

Stimulation of protein kinase C modulates insulin-like growth factor-1-induced akt activation in PC12 cells.

Activation of protein kinase C (PKC) plays an important role in the negative regulation of receptor signaling, but its effect on insulin-like growth factor-1 (IGF-1) receptor signaling remains unclear. In this study, we characterized the intracellular pathways involved in IGF-1-induced activation of Akt and evaluated the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on the Akt activation by IGF-1. IGF-1 induced a time- and concentration-dependent activation of Akt. The effect of IGF-1 was blocked by the phosphatidylinositide 3-kinase (PI3K) inhibitors LY294002 (50 micrometer) and wortmannin (0.5 micrometer), but not by the MEK inhibitor PD98059 (50 micrometer) or the p70 S6 kinase pathway inhibitor rapamycin (50 nm), suggesting that the stimulation of Akt by IGF-1 is mediated by the PI3K pathway. Interestingly, cotreatment with PMA (400 nm) attenuated IGF-1-induced activation of Akt. The attenuation was blocked completely by the PKC inhibitor GO6983 (0.5 micrometer), but only partially by the MEK inhibitor PD98059 (50 micrometer), indicating that MAPK-dependent and -independent pathways are involved. PMA induced the activation of PKC in PC12 cells, and this induction was blocked by GO6983. These data further support the role of PKC in the effect of PMA. Moreover, PKCdelta is likely involved in the action of PMA on the basis of data obtained using isoform-specific inhibitors such as rottlerin. PMA also decreased IGF-1-induced tyrosine phosphorylation of insulin receptor substrate-1 and its association with PI3K. Taken together, these results suggest, for the first time, that stimulation of PKC modulates IGF-1-induced activation of Akt.

Animals↗

PubMed2000

Dopamine D-5 receptor modulates hippocampal acetylcholine release.

Dopamine is intimately involved in cognitive processes in the brain. Of the several subtypes of dopamine receptors, the possible role of dopamine D1-like receptors in brain functions, especially in learning and memory, has recently generated much interest. However, molecularly the D1-like receptors are comprised of at least two subtypes, namely D-1 and D-5, and it has not been possible to ascertain which of these two receptor classes is responsible for these functions due to the lack of selective ligands. In the present study, utilizing a combined antisense-in vivo dialysis approach, we show that the D-5 subtype is the dopamine D1-like receptor involved in modulating hippocampal acetylcholine (ACh) release, a transmitter implicated in a variety of cognitive processes. This is one of the first evidence for a functional role for the D-5 receptor.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed2000

Olfactory bulbectomy alters NMDA receptor levels in the rat prefrontal cortex.

Olfactory bulbectomized (OBX) rats show a variety of behavioral and biochemical deficits that parallel human depression. We investigated the expression of glutamate receptor subtypes in cortical and subcortical brain regions following bilateral olfactory bulbectomy in adult rats. Quantitative receptor autoradiography using [(125)I]MK-801 (NMDA receptor), [(3)H]AMPA (AMPA receptor), and [(3)H]kainate (kainate receptor) was performed on brain sections at 1-5 weeks following olfactory bulbectomy. Our results show an elevation of NMDA receptors in the medial prefrontal cortex within 1 week following bulbectomy, which persisted up to at least 5 weeks post-bulbectomy. Neither kainate nor AMPA receptors were altered in any brain region examined. The potential significance of these results is discussed in light of experimental findings supporting a role for NMDA receptors in the mechanism of action of antidepressant drugs and the pathophysiology of major depression.

Animals↗

PubMed2000

Behavioral and neurochemical alterations following repeated phencyclidine administration in rats with neonatal ventral hippocampal lesions.

Exposure to chronic phencyclidine (PCP) has been reported to mimic certain aspects of schizophrenia in normal subjects as well as to exacerbate symptoms in schizophrenic patients. Analogous to schizophrenics, adult rats with neonatal ventral hippocampal (VH) lesions have been shown to display enhanced sensitivity to both stress and psychostimulants. In order to examine whether repeated PCP treatment can modulate behavior when administered to neonatal VH-lesioned animals, we examined locomotor activity and immobility time in the forced swimming test (FST) in neonatal VH-lesioned rats following repeated PCP treatment. Receptor autoradiography studies were also performed for dopamine (DA) and N-methyl-D-aspartate (NMDA) receptors to identify neurochemical correlates of the altered behavior in these animals. Though repeated PCP administration resulted in increased levels of locomotor activity and rearing in both VH-lesioned as well as sham rats, the effects were much more enhanced in the lesioned rats compared to sham. However, repeated PCP treatment induced hypolocomotion during the habituation period in both sham and lesioned rats. In the FST paradigm, lesioned rats displayed an altered retention of acquired immobility. Repeated PCP administration increased DA D1-like receptors in the caudate-putamen in lesioned rats and decreased striatal D2-like receptors in both sham and lesioned rats. Moreover, repeated PCP administration in lesioned rats decreased NMDA binding sites in the prefrontal cortex while increasing labelling in the subcortical regions. These results suggest that repeated administration of PCP can qualitatively and quantitatively affect behaviors in neonatal VH-lesioned rats related to abnormal neurodevelopmental processes presumably via prefrontal glutamatergic and subcortical dopaminergic dysfunctions.

Animals↗

PubMed2000

Rediscovering good old friend IGF-I in the new millenium: possible usefulness in Alzheimer's disease and stroke.

Much research has been done over the past two decades on the role of insulin-like growth factors I and II (IGF) in the maintenance of normal body homeostasis, especially in regard to various endocrine functions, growth and aging. For example, IGF-I is a well established promoter of tissue growth and has been used in the clinics for the treatment of growth related disorders, even being abused by athletes to enhance performance in competitions. In contrast, comparatively limited attention has been given to the potential significance of the IGFs in the central nervous system. Over the past few years, we have studied the trophic as well as neuromodulatory roles of the IGFs in the brain. IGF-I and IGF-II are potent modulators of acetylcholine release, IGF-I inhibiting release while IGF-II is a potent stimulant. Moreover, only the internalization of the IGF-I receptor complex was blocked by an inhibitor of phosphotyrosylation. This is in accordance with the differential nature of the IGF-I and IGF-II receptors, the former being a tyrosine kinase receptor while the later is a single transmembrane domain protein bearing binding sites for 6-mannose phosphate containing residues. The activation of IGF-I receptors protected neurons against cell death induced by amyloidogenic derivatives likely by an intracellular mechanism distinct from those involved in the regulation of acetylcholine release and neuronal growth. The stimulation of IGF-I receptors can activate intracellular pathways implicating a PI3/Akt kinase and CREB phosphorylation or modulate the production of free radicals. The effects, particularly those of IGF-I on key markers of the Alzheimer's (AD) brains namely cholinergic dysfunction, neuronal amyloid toxicity, tau phosphorylation and glucose metabolism suggest the potential usefulness of this growth factor in the treatment of neurodegenerative diseases. However, the poor bioavailability, enzymatic stability and brain penetration of IGF-I hamper progress in this regard. The recent development of a small, non-peptidyl mimetic of insulin able to directly activate the insulin receptor [Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M.T., Pelaez, F., Ruby, C., Kendall, R.L., Mao, X., Griffin, P., Calaycay, J., Zierath, J.R., Heck, J. V., Smith, R.G., Moller, D.E., 1999. Science, 284, 974-977] suggests that a similar strategy could be used for IGF-I and the IGF-I receptor leading to the characterization of IGF-I mimics of potential clinical usefulness.

Alzheimer Disease↗

PubMed2000

Acetylcholine induces neuritic outgrowth in rat primary olfactory bulb cultures.

The rat olfactory bulb is innervated by basal forebrain cholinergic neurons and is endowed with both nicotinic and muscarinic receptors. The development of this centrifugal cholinergic innervation occurs mainly in early postnatal stages. This developmental time-course and the demonstration that acetylcholine can modulate some aspects of neuronal proliferation, differentiation or death, suggests the possible involvement of cholinergic afferents in the morphogenesis and/or plasticity of the olfactory bulb. The purpose of the present work was to assess whether acetylcholine could modulate neuronal morphogenesis in the olfactory bulb. Toward this aim, we developed a primary culture model of rat olfactory bulbs. Three major cell types were identified on the basis of their morphological and immunocytochemical phenotype: neuronal-shaped cells expressing the neuronal markers neuron specific enolase, microtubule associated protein 2, neural cell adhesion molecule and beta-tubulin III; glial-like cells immunoreactive for glial fibrillary acidic protein and flattened cells immunolabelled with antibodies against beta-tubulin III and nestin, most likely neuronal precursors. After three to six days of treatment with 100-microM carbachol, a cholinergic agonist, significant increase in neuritic length was observed in cultured olfactory bulb neurons. The neurite outgrowth effect of carbachol was abolished by co-treatment with 1 microM alpha-bungarotoxin, an alpha 7 subunit nicotinic receptor antagonist, but was not affected by the addition of 10 microM atropine, a general muscarinic antagonist. The effect of carbachol was also mimicked by the nicotinic agonists, nicotine (100 microM) and epibatidine (10 microM). This pharmacological profile suggested the involvement of nicotinic receptors of the alpha 7-like subtype as confirmed using 125I-alpha-bungarotoxin receptor autoradiography.Taken together, these data argue for a role for nicotinic receptors in neuritic outgrowth in the rat olfactory bulb and provide a cellular support to the previously described effects of acetylcholine on olfactory bulb plasticity in vivo.

Acetylcholine↗

PubMed2000

Decreased levels of neuropeptide Y(5) receptor binding sites in two experimental models of epilepsy.

It has been suggested that the anticonvulsant effects of neuropeptide Y (NPY) could be mediated by the activation of Y(2) and/or Y(5) receptors. NPY Y(1) receptor levels are known to decrease and Y(2) to increase in rat models of epilepsy. By using an autoradiographic approach, we investigated whether epilepsy models (kainic acid and kindling) are also associated with changes in Y(5) receptors. Compared with naive controls, [125I][Leu(31), Pro(34)]PYY/BIBP3226-insensitive (Y(5)) binding sites in the hippocampus (strata oriens and radiatum of CA3 and CA1) and in the neocortex (superficial layers) were unchanged in sham-stimulated rats, but reduced by approximately 50% in kindled rats (seven days after the last stimulus evokes seizure), and further reduced (to approximately -90%) 1h after a kindled seizure. Additionally, Y(5) receptor binding sites in the hippocampus and in the neocortex were unchanged 6h after kainic acid injection, but were highly reduced at 12 and 24h. No changes in Y(5) binding levels were found in the dentate gyrus and the pyramidal cell layer of the hippocampus. The present data suggest that changes in Y(5) receptor levels occur in epilepsy models. These changes may play a role in seizure expression and/or in the maintenance of kindling hyperexcitability.

Animals↗

PubMed2000

Ontogenic profile of the expression of the mu opioid receptor gene in the rat telencephalon and diencephalon: an in situ hybridization study.

The developmental profile of mu (mu) opioid receptor gene expression has been characterized in the embryonic, postnatal and adult rat brain by in situ hybridization histochemistry. By ED12, mu opioid receptor mRNA was detectable in the deep neuroepithelium of the cortical plate. In the developing rat central nervous system (ED13-PD40), transcripts were seen over numerous telencephalic and diencephalic structures, such as the olfactory bulb, caudate-putamen, nucleus accumbens, amygdaloid complex, hippocampal formation, hypothalamus and thalamus. In the vast majority of brain regions examined, the developmental profile of the mu opioid receptor gene expression is similar to that of its translated protein as established using receptor autoradiography. Once a hybridization signal is detected in the prenatal period, it gradually increased to reach maximal levels during the second and third postnatal weeks. By the end of the third postnatal week, mu opioid receptor mRNA levels decreased to reach amounts seen in adulthood. Our study demonstrates that mu opioid receptor gene expression is seen very early on in the embryonic rat brain with transient increases observed during the critical period of neurogenesis, neuronal migration and synaptogenesis, suggesting a role of this opioid receptor subtype in brain developmental processes.

Animals↗

PubMed2000

[(125)I]-GR231118: a high affinity radioligand to investigate neuropeptide Y Y(1) and Y(4) receptors.

GR231118 (also known as 1229U91 and GW1229), a purported Y(1) antagonist and Y(4) agonist was radiolabelled using the chloramine T method. [(125)I]-GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [(125)I]-GR231118 binds with very high affinity (K(d) of 0.09 - 0.24 nM) in transfected HEK293 cells with the rat Y(1) and Y(4) receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y(2) or Y(5) receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [(125)I]-GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y(1), but not rat Y(4), receptor cDNA. Autoradiographic studies demonstrated that [(125)I]-GR231118 binding sites were fully inhibited by the Y(1) antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [(125)I]-GR231118/BIBO3304-insensitive binding sites were detected in few areas. These [(125)I]-GR231118/BIBO3304-insensitive binding sites likely represent labelling to the Y(4) receptor subtype. In summary, [(125)I]-GR231118 is a new radiolabelled probe to investigate the Y(1) and Y(4) receptors; its major advantage being its high affinity. Using highly selective Y(1) antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [(125)I]-GR231118 to the Y(1) receptor allowing for the characterization and visualization of the purported Y(4) subtype. British Journal of Pharmacology (2000) 129, 37 - 46

Animals↗

PubMed2000

BIIE0246, a potent and highly selective non-peptide neuropeptide Y Y(2) receptor antagonist.

1. BIIE0246, a newly synthesized non-peptide neuropeptide Y (NPY) Y(2) receptor antagonist, was able to compete with high affinity (8 to 15 nM) for specific [(125)I]PYY(3 - 36) binding sites in HEK293 cells transfected with the rat Y(2) receptor cDNA, and in rat brain and human frontal cortex membrane homogenates. 2. Interestingly, in rat brain homogenates while NPY, C2-NPY and PYY(3 - 36) inhibited all specific [(125)I]PYY(3 - 36) labelling, BIIE0246 failed to compete for all specific binding suggesting that [(125)I]PYY(3 - 36) recognized, in addition to the Y(2) subtype, another population of specific NPY binding sites, most likely the Y(5) receptor. 3. Quantitative receptor autoradiographic data confirmed the presence of [(125)I]PYY(3 - 36)/BIIE0246-sensitive (Y(2)) and-insensitive (Y(5)) binding sites in the rat brain as well as in the marmoset monkey and human hippocampal formation. 4. In the rat vas deferens and dog saphenous vein (two prototypical Y(2) bioassays), BIIE0246 induced parallel shifts to the right of NPY concentration-response curves with pA(2) values of 8.1 and 8.6, respectively. In the rat colon (a Y(2)/Y(4) bioassay), BIIE0246 (1 microM) completely blocked the contraction induced by PYY(3 - 36), but not that of [Leu(31), Pro(34)]NPY (a Y(1), Y(4) and Y(5) agonist) and hPP (a Y(4) and Y(5) agonist). Additionally, BIIE0246 failed to alter the contractile effects of NPY in prototypical Y(1) in vitro bioassays. 5. Taken together, these results demonstrate that BIIE0246 is a highly potent, high affinity antagonist selective for the Y(2) receptor subtype. It should prove most useful to establish further the functional role of the Y(2) receptor in the organism.

Animals↗

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

The Ginkgo biloba extract (EGb 761) protects hippocampal neurons against cell death induced by beta-amyloid.

Substantial evidence suggests that the accumulation of beta-amyloid (Abeta)-derived peptides, and to a lesser extent free radicals, may contribute to the aetiology and/or progression of Alzheimer's disease (AD). Ginkgo biloba extract (EGb 761) is a well-defined plant extract containing two major groups of constituents, i.e. flavonoids and terpenoids. It is viewed as a polyvalent agent with a possible therapeutic use in the treatment of neurodegenerative diseases of multifactorial origin, e.g. AD. We have investigated here the potential effectiveness of EGb 761 against toxicity induced by (Abeta)-derived peptides (Abeta25-35, Abeta1-40 and Abeta1-42) on hippocampal primary cultured cells, this area being severely affected in AD. A co-treatment with EGb 761 concentration-dependently (10-100 microg/mL) protected hippocampal neurons against toxicity induced by Abeta fragments, with a maximal and complete protection at the highest concentration tested. Similar, albeit less potent protective effects were seen with the flavonoid fraction of the extract (CP 205), while the terpenes were ineffective. Most interestingly, EGb 761 (100 microg/mL) was even able to protect (up to 8 h) hippocampal cells from a pre-exposure to Abeta25-35 and Abeta1-40. EGb 761 was also able to both protect and rescue hippocampal cells from toxicity induced by H2O2 (50-150 microM), a major peroxide possibly involved in mediating Abeta toxicity. Moreover, EGb 761 (10-100 microg/mL), and to a lesser extent CP 205 (10-50 microg/mL), completely blocked Abeta-induced events, e.g. reactive oxygen species accumulation and apoptosis. These results suggest that the neuroprotective effects of EGb 761 are partly associated with its antioxidant properties and highlight its possible effectiveness in neurodegenerative diseases, e.g. AD via the inhibition of Abeta-induced toxicity and cell death.

Alzheimer Disease↗

PubMed2000

The Ginkgo biloba extract (EGb 761) protects and rescues hippocampal cells against nitric oxide-induced toxicity: involvement of its flavonoid constituents and protein kinase C.

An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. Numerous studies have shown that the Ginkgo biloba extract EGb 761 is a NO scavenger with neuroprotective properties. However, the mechanisms underlying its neuroprotective ability remain to be fully established. Thus, we investigated the effect of different constituents of EGb 761, i.e., flavonoids and terpenoids, against toxicity induced by NO generators on cells of the hippocampus, a brain area particularly susceptible to neurodegenerative damage. Exposure of rat primary mixed hippocampal cell cultures to either sodium nitroprusside (SNP; 100 microM) or 3-morpholinosydnonimine resulted in both a decrease in cell survival and an increase in free radical accumulation. These SNP-induced events were blocked by either EGb 761 (10-100 microg/ml) or its flavonoid fraction CP 205 (25 microg/ml), as well as by inhibitors of protein kinase C (PKC; chelerythrine) and L-type calcium channels (nitrendipine). In contrast, the terpenoid constituents of EGb 761, known as bilobalide and ginkgolide B, as well as inhibitors of phospholipases A [3-[(4-octadecyl)benzoyl]acrylic acid (OBAA)] and C (U-73122), failed to display any significant effects. Moreover, EGb 761 (50 microm) CP 205 (25 microg/ml), and chelerythrine were also able to rescue hippocampal cells preexposed to SNP (up to 1 mM). Finally, EGb 761 (100 microg/ml) was shown to block the activation of PKC induced by SNP (100 microM). These data suggest that the protective and rescuing abilities of EGb 761 are not only attributable to the antioxidant properties of its flavonoid constituents but also via their ability to inhibit NO-stimulated PKC activity.

Alkaloids↗

PubMed2000

A novel receptor for calcitonin gene-related peptide (CGRP) mediates secretion in the rat colon: implications for secretory function in colitis.

The receptor responsible for CGRP-induced ion transport and permeability was examined in tissues from animals treated 7 days previously with trinitrobenzenesulfonic acid to induce colitis or in controls. CGRP caused a concentration-dependent increase in short circuit current (I(sc), EC(50) 21 nM), which was abolished in chloride-free buffer but was not blocked by CGRP(8-37) or tetrodotoxin (TTX). Amylin and adrenomedullin caused only a modest increase in I(sc). The responses to the linear CGRP(2) receptor agonists [Cys(Et)(2,7)] hCGRPalpha and [Cys(Acm)(2,7)] hCGRPalpha were considerably smaller than the response to CGRP. These responses were abolished in chloride-free buffer and were TTX sensitive. Atropine, doxantrazole, and indomethacin did not block the effects of CGRP or the CGRP(2) agonists. The response to [Cys(Et)(2,7)] hCGRPalpha was not affected by prior desensitization of the CGRP receptor and vice versa. Inflamed rats had a similar secretory response to CGRP (I(sc), EC(50) 15 nM) and [Cys(Et)(2,7)] hCGRPalpha as control tissues, while being hyporesponsive to carbachol. CGRP application increased electrical conductance of inflamed preparations. Taken together, these data suggest that CGRP may play an important role in the maintenance of host defense in colitis through an apparently novel CGRP receptor located on the colonic enterocyte.

Animals↗

PubMed2000

Pharmacological characterization of endogenous acetylcholine release from primary septal cultures.

A detailed investigation of endogenous acetylcholine (ACh) release from primary embryonic septal cultures is described in this study. Applications of veratridine (25 microM) or increasing extracellular concentrations of K(+) (6-100 mM) induced robust increases of endogenous ACh release ( approximately 500-15,000 fmol/well/10 min). Release stimulated with K(+) (25 mM) was sustainable and did not differ significantly over 180 min. ACh release was dependent on extracellular choline and decreased proportionally to choline concentrations (0-10 microM). For example, after 30 min of stimulation with K(+) (25 mM), release in the absence of extracellular choline was approximately 25% of that associated with 10 microM choline. The vesicular transport blocker vesamicol (0-5 microM) almost completely prevented stimulated and basal ACh release at the highest concentration evaluated, which suggests a mostly vesicular mode of release in this model. The M(2)-like muscarinic receptor antagonist AF-DX 384 (0-10 microM) enhanced stimulated ACh release ( approximately 150% at the highest concentration evaluated), whereas the nonspecific muscarinic receptor agonist oxotremorine (0-10 microM) decreased stimulated release (approximately 60% at the highest concentration evaluated), suggesting that functional muscarinic autoreceptors exist in primary embryonic septal cultures. Novel findings concerning ACh release from primary embryonic septal cultures are reported herein, and the demonstration of ACh release gives further credit to the use of these cultures for studying cholinergic system functioning and in relation to physiology and pathology.

Acetylcholine↗

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↗

PubMed2000

Potent and selective tools to investigate neuropeptide Y receptors in the central and peripheral nervous systems: BIB03304 (Y1) and CGP71683A (Y5).

We have evaluated 3 newly developed neuropeptide Y receptor antagonists in various in vitro binding and bioassays: BIBO3304 (Y1), T4[NPY33-36]4 (Y2), and CGP71683A (Y5). In rat brain homogenates, BIBO3304 competes for the same population of [125I][Leu31,Pro34] peptide YY (PYY) binding sites (75%) as BIBP3226, but with a 10 fold greater affinity (IC50 of 0.2 +/- 0.04 nM for BIBO3304 vs. 2.4 +/- 0.07 nM for BIBP3226),while CGP71683A has high affinity for 25% of specific [125I][Leu31,Pro34]PYY binding sites. Both BIBO3304 and CGP71683A (at 1.0 microM) were unable to compete for a significant proportion of specific [125I]PYY3-36/Y2 sites. The purported Y2 antagonist T4[NPY33-36]4 competed against [125I]PYY3-36 binding sites with an affinity of 750 nM. These results were confirmed in HEK 293 cells transfected with either the rat Y1, Y2, Y4, or Y5 receptor cDNA. BIBO3304, but not CGP71683A, competed with high affinity for [125I][Leu31,Pro34]PYY binding sites in HEK 293 cells transfected with the rat Y1 receptor cDNA, whereas the reverse profile was observed upon transfection with the rat Y5 receptor cDNA. Additionally, both molecules were inactive at Y2 and Y4 receptor subtypes expressed in HEK 293 cells. Receptor autoradiographic studies revealed the presence of [125I][Leu31,Pro34]PYY/BIBO3304-insensitive sites in the rat brain as reported previously for BIBP3226. Finally, the selective antagonistic properties of BIBO3304 were demonstrated in a Y1 bioassay (rabbit saphenous vein; pA2 value of 9.04) while being inactive in Y2 (rat vas deferens) and Y4 (rat colon) bioassays. These results confirm the high affinity and selectivity of BIBO3304 and CGP71683A for the Y1 and Y5 receptor subtypes, respectively, while the purported Y2 antagonist, T4[NPY33-36]4 possesses rather low affinity for this receptor.

Animals↗

PubMed1999

Multiple receptors for neuropeptide Y in the hippocampus: putative roles in seizures and cognition.

Neuropeptide Y (NPY) is widely distributed throughout the central nervous system (CNS) and is one of the most conserved peptides in evolution, suggesting an important role in the regulation of basic physiological functions, including learning and memory. In addition, experimental studies have suggested that NPY, together with its receptors, may have a direct implication in several pathological disorders, including epilepsy/seizure. NPY-like immunoreactivity and NPY receptors have been shown to be present throughout the brain, but is concentrated in the hippocampus. The hippocampal formation has been repeatedly implicated in the modulation of cognition, as well as the pathogenesis of seizure. This review will concentrate on the hippocampal distribution of NPY, its receptors and the putative role played by this peptide in seizure, together with the regulation of cognitive function associated with learning and memory.

Animals↗