Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 431
Filter
Add more filters










Publication year range
1.
Neuroscience ; 134(4): 1377-90, 2005.
Article in English | MEDLINE | ID: mdl-16039802

ABSTRACT

Recent research has shown that neurons in the ventrolateral preoptic nucleus are crucial for sleep by inhibiting wake-promoting systems, but the process that triggers their activation at sleep onset remains to be established. Since evidence indicates that sleep induced by adenosine, an endogenous sleep-promoting substance, requires activation of brain A(2A) receptors, we examined the hypothesis that adenosine could activate ventrolateral preoptic nucleus sleep neurons via A(2A) adenosine receptors in rat brain slices. Following on from our initial in vitro identification of these neurons as uniformly inhibited by noradrenaline and acetylcholine arousal transmitters, we established that the ventrolateral preoptic nucleus comprises two intermingled subtypes of sleep neurons, differing in their firing responses to serotonin, inducing either an inhibition (Type-1 cells) or an excitation (Type-2 cells). Since both cell types contained galanin and expressed glutamic acid decarboxylase-65/67 mRNAs, they potentially correspond to the sleep promoting neurons inhibiting arousal systems. Our pharmacological investigations using A(1) and A(2A) adenosine receptors agonists and antagonists further revealed that only Type-2 neurons were excited by adenosine via a postsynaptic activation of A(2A) adenosine receptors. Hence, the present study is the first demonstration of a direct activation of the sleep neurons by adenosine. Our results further support the cellular and functional heterogeneity of the sleep neurons, which could enable their differential contribution to the regulation of sleep. Adenosine and serotonin progressively accumulate during arousal. We propose that Type-2 neurons, which respond to these homeostatic signals by increasing their firing are involved in sleep induction. In contrast, Type-1 neurons would likely play a role in the consolidation of sleep.


Subject(s)
Adenosine/metabolism , Neurons/cytology , Preoptic Area/cytology , Receptor, Adenosine A2A/metabolism , Sleep/physiology , Adenosine A2 Receptor Agonists , Adenosine A2 Receptor Antagonists , Animals , Neurons/metabolism , Organ Culture Techniques , Patch-Clamp Techniques , Preoptic Area/physiology , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Serotonin/metabolism
2.
Arch Ital Biol ; 142(4): 533-9, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15493554

ABSTRACT

(1) Prostaglandin D2 is essential for the maintenance of the sleep state. (2) The adenosine and A2A receptor system is a link between the humoral and neural mechanisms of sleep-wake regulation. (3) Prostaglandin D2 plays a crucial role in the homeostatic regulation of NREM sleep. Finally, it may not be too far-fetched to say that prostaglandin D2 was most likely the endogenous sleep substance described by Piéron and Ishimori about 100 years ago, and possibly the sleep-inducing factor reported by Professor Jouvet and coworkers some twenty years ago.


Subject(s)
Brain/physiology , Intramolecular Oxidoreductases/genetics , Prostaglandin D2/biosynthesis , Receptors, Immunologic/genetics , Receptors, Prostaglandin/genetics , Sleep/physiology , Animals , Brain/anatomy & histology , Brain/enzymology , Cerebrospinal Fluid/metabolism , Homeostasis/genetics , Humans , Lipocalins , Mice , Prostaglandin D2/genetics , Receptor, Adenosine A2A/genetics , Receptor, Adenosine A2A/metabolism , Sleep/genetics
3.
Neuroscience ; 107(4): 653-63, 2001.
Article in English | MEDLINE | ID: mdl-11720788

ABSTRACT

Considerable evidence indicates that adenosine may be an endogenous somnogen, yet the mechanism through which it promotes sleep is unknown. Adenosine may act via A1 receptors to promote sleep, but an A2a receptor antagonist can block the sleep induced by prostaglandin D(2). We previously reported that prostaglandin D(2) activates sleep-promoting neurons of the ventrolateral preoptic area, and we hypothesized that an A2a receptor agonist also should activate these neurons. Rats were instrumented for sleep recordings, and an injection cannula was placed in the subarachnoid space just anterior to the ventrolateral preoptic area. After an 8-10-day recovery period, the A2a receptor agonist CGS21680 (20 pmol/min) or saline was infused through the injection cannula, and the animals were killed 2 h later. The brains were stained using Fos immunohistochemistry, and the pattern of Fos expression was studied in the entire brain. CGS21680 increased non-rapid eye movement sleep and markedly increased the expression of Fos in the ventrolateral preoptic area and basal leptomeninges, but it reduced Fos expression in wake-active brain regions such as the tuberomammillary nucleus. CGS21680 also induced Fos in the shell and core of the nucleus accumbens and in the lateral subdivision of the central nucleus of the amygdala. To determine whether these effects may have been mediated through A1 receptors, an additional group of rats received subarachnoid infusion of the A1 receptor agonist N(6)-cyclopentyladenosine (2 pmol/min). In contrast to CGS21680, infusion of N(6)-cyclopentyladenosine into the subarachnoid space produced only a small decrease in rapid eye movement sleep, and the pattern of Fos expression induced by N(6)-cyclopentyladenosine was notable only for decreased Fos in regions near the infusion site. These findings suggest that an adenosine A2a receptor agonist may activate cells of the leptomeninges or nucleus accumbens that increase the activity of ventrolateral preoptic area neurons. These ventrolateral preoptic area neurons may then coordinate the inhibition of multiple wake-promoting regions, resulting in sleep.


Subject(s)
Adenosine/analogs & derivatives , Neurons/metabolism , Preoptic Area/cytology , Proto-Oncogene Proteins c-fos/biosynthesis , Purinergic P1 Receptor Agonists , Sleep/drug effects , Adenosine/pharmacology , Animals , Antihypertensive Agents/pharmacology , Brain Chemistry/drug effects , Male , Neurons/chemistry , Phenethylamines/pharmacology , Preoptic Area/physiology , Proto-Oncogene Proteins c-fos/analysis , Rats , Rats, Sprague-Dawley , Receptor, Adenosine A2A , Specific Pathogen-Free Organisms , Subarachnoid Space , Wakefulness/drug effects
4.
Proc Natl Acad Sci U S A ; 98(20): 11674-9, 2001 Sep 25.
Article in English | MEDLINE | ID: mdl-11562489

ABSTRACT

Infusion of prostaglandin (PG) D(2) into the lateral ventricle of the brain induced an increase in the amount of non-rapid eye movement sleep in wild-type (WT) mice but not in mice deficient in the PGD receptor (DP). Immunofluorescence staining of WT mouse brain revealed that DP immunoreactivity was dominantly localized in the leptomeninges (LM) of the basal forebrain but that PGD synthase immunoreactivity was widely distributed in the LM of the entire brain. Electron microscopic observation indicated that DP-immunoreactive particles were predominantly located on the plasma membranes of arachnoid trabecular cells of the LM. The region with the highest DP immunoreactivity was clearly defined as bilateral wings in the LM of the basal forebrain located lateral to the optic chiasm in the proximity of the ventrolateral preoptic area, one of the putative sleep centers, and the tuberomammillary nucleus, one of the putative wake centers. The LM of this region contained DP mRNA 70-fold higher than that in the cortex as judged from the results of quantitative reverse transcription-PCR. PGD(2) infusion into the subarachnoid space of this region increased the extracellular adenosine level more than 2-fold in WT mice but not in the DP-deficient mice. These results indicate that DPs in the arachnoid trabecular cells of the basal forebrain mediate an increase in the extracellular adenosine level and sleep induction by PGD(2).


Subject(s)
Receptors, Calcitriol/genetics , Sleep/physiology , Adenosine/metabolism , Amino Acid Sequence , Anesthesia, General , Animals , Arachnoid/physiology , Base Sequence , DNA Primers , Electroencephalography , Electromyography , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Intramolecular Oxidoreductases/analysis , Kinetics , Lipocalins , Medulla Oblongata/physiology , Mice , Mice, Knockout , Molecular Sequence Data , Neocortex/physiology , Pentobarbital/pharmacology , Perfusion , Polymerase Chain Reaction , Prostaglandin D2/pharmacology , RNA/genetics , RNA/isolation & purification , RNA, Messenger/analysis , RNA, Messenger/genetics , Receptors, Calcitriol/analysis , Receptors, Calcitriol/chemistry , Sleep Stages/physiology , Sleep, REM/physiology , Subarachnoid Space/drug effects , Subarachnoid Space/metabolism
5.
Proc Natl Acad Sci U S A ; 98(17): 9965-70, 2001 Aug 14.
Article in English | MEDLINE | ID: mdl-11493714

ABSTRACT

Orexin neurons are exclusively localized in the lateral hypothalamic area and project their fibers to the entire central nervous system, including the histaminergic tuberomammillary nucleus (TMN). Dysfunction of the orexin system results in the sleep disorder narcolepsy, but the role of orexin in physiological sleep-wake regulation and the mechanisms involved remain to be elucidated. Here we provide several lines of evidence that orexin A induces wakefulness by means of the TMN and histamine H(1) receptor (H1R). Perfusion of orexin A (5 and 25 pmol/min) for 1 hr into the TMN of rats through a microdialysis probe promptly increased wakefulness for 2 hr after starting the perfusion by 2.5- and 4-fold, respectively, concomitant with a reduction in rapid eye movement (REM) and non-REM sleep. Microdialysis studies showed that application of orexin A to the TMN increased histamine release from both the medial preoptic area and the frontal cortex by approximately 2-fold over the baseline for 80 to 160 min in a dose-dependent manner. Furthermore, infusion of orexin A (1.5 pmol/min) for 6 hr into the lateral ventricle of mice produced a significant increase in wakefulness during the 8 hr after starting infusion to the same level as the wakefulness observed during the active period in wild-type mice, but not at all in H1R gene knockout mice. These findings strongly indicate that the arousal effect of orexin A depends on the activation of histaminergic neurotransmission mediated by H1R.


Subject(s)
Arousal/drug effects , Carrier Proteins/pharmacology , Histamine/physiology , Hypothalamic Area, Lateral/drug effects , Hypothalamus/physiology , Intracellular Signaling Peptides and Proteins , Nerve Tissue Proteins/drug effects , Neuropeptides/pharmacology , Receptors, Histamine H1/drug effects , Sleep/drug effects , Wakefulness/drug effects , Animals , Electroencephalography , Electromyography , Frontal Lobe/physiology , Hypothalamic Area, Lateral/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microdialysis , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Orexin Receptors , Orexins , Preoptic Area/physiology , Rats , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled , Receptors, Histamine H1/deficiency , Receptors, Histamine H1/genetics , Receptors, Histamine H1/physiology , Receptors, Neuropeptide
6.
J Exp Med ; 192(9): 1327-38, 2000 Nov 06.
Article in English | MEDLINE | ID: mdl-11067881

ABSTRACT

Members of the genus Trypanosoma cause African trypanosomiasis in humans and animals in Africa. Infection of mammals by African trypanosomes is characterized by an upregulation of prostaglandin (PG) production in the plasma and cerebrospinal fluid. These metabolites of arachidonic acid (AA) may, in part, be responsible for symptoms such as fever, headache, immunosuppression, deep muscle hyperaesthesia, miscarriage, ovarian dysfunction, sleepiness, and other symptoms observed in patients with chronic African trypanosomiasis. Here, we show that the protozoan parasite T. brucei is involved in PG production and that it produces PGs enzymatically from AA and its metabolite, PGH(2). Among all PGs synthesized, PGF(2alpha) was the major prostanoid produced by trypanosome lysates. We have purified a novel T. brucei PGF(2alpha) synthase (TbPGFS) and cloned its cDNA. Phylogenetic analysis and molecular properties revealed that TbPGFS is completely distinct from mammalian PGF synthases. We also found that TbPGFS mRNA expression and TbPGFS activity were high in the early logarithmic growth phase and low during the stationary phase. The characterization of TbPGFS and its gene in T. brucei provides a basis for the molecular analysis of the role of parasite-derived PGF(2alpha) in the physiology of the parasite and the pathogenesis of African trypanosomiasis.


Subject(s)
Dinoprost/biosynthesis , Prostaglandin-Endoperoxide Synthases/isolation & purification , Prostaglandin-Endoperoxide Synthases/metabolism , Trypanosoma brucei brucei/enzymology , Amino Acid Sequence , Animals , Arachidonic Acid/metabolism , Cell Extracts , Cells, Cultured , Cloning, Molecular , Dinoprost/metabolism , Dinoprostone/biosynthesis , Dinoprostone/metabolism , Gas Chromatography-Mass Spectrometry , Kinetics , Molecular Sequence Data , Multigene Family , Phylogeny , Prostaglandin D2/biosynthesis , Prostaglandin D2/metabolism , Prostaglandin H2 , Prostaglandin-Endoperoxide Synthases/chemistry , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandins H/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Analysis, Protein , Substrate Specificity , Trypanosoma brucei brucei/cytology , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/metabolism
7.
Biochim Biophys Acta ; 1482(1-2): 259-71, 2000 Oct 18.
Article in English | MEDLINE | ID: mdl-11058767

ABSTRACT

Lipocalin-type prostaglandin (PG) D synthase (PGDS) catalyzes the isomerization of PGH(2), a common precursor of various prostanoids, to produce PGD(2), a potent endogenous somnogen and nociceptive modulator, in the presence of sulfhydryl compounds. PGDS is an N-glycosylated monomeric protein with an M(r) of 20000-31000 depending on the size of the glycosyl moiety. PGDS is localized in the central nervous system and male genital organs of various mammals and in the human heart and is secreted into the cerebrospinal fluid, seminal plasma, and plasma, respectively, as beta-trace. The PGDS concentrations in these body fluids are useful for the diagnosis of several neurological disorders, dysfunction of sperm formation, and cardiovascular and renal diseases. The cDNA and gene for PGDS have been isolated from several animal species, and the tissue distribution and cellular localization have also been determined. This enzyme is considered to be a dual functional protein; i.e. it acts as a PGD(2)-producing enzyme and also as a lipophilic ligand-binding protein, because the enzyme binds biliverdin, bilirubin (K(d)=30 nM), retinaldehyde, retinoic acid (K(d)=80 nM) with high affinities. X-ray crystallographic analyses revealed that PGDS possesses a beta-barrel structure with a hydrophobic pocket in which an active thiol, Cys(65), the active center for the catalytic reaction, was located facing to the inside of the pocket. Gene-knockout and transgenic mice for PGDS were generated and found to have abnormalities in the regulation of nociception and sleep.


Subject(s)
Intramolecular Oxidoreductases/chemistry , Intramolecular Oxidoreductases/genetics , Amino Acid Sequence , Animals , Biomarkers/analysis , Chromosome Mapping , DNA, Complementary/analysis , Humans , Intramolecular Oxidoreductases/metabolism , Intramolecular Oxidoreductases/physiology , Lipocalins , Mice , Mice, Knockout , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Substrate Specificity , Tissue Distribution
8.
J Comp Neurol ; 428(1): 62-78, 2000 Dec 04.
Article in English | MEDLINE | ID: mdl-11058225

ABSTRACT

We applied high-resolution laser-scanning microscopy, electron microscopy, and non-radioactive in situ hybridization histochemistry to determine the cellular and intracellular localization of lipocalin-type prostaglandin D synthase, the major brain-derived protein component of cerebrospinal fluid, and its mRNA in leptomeninges, choroid plexus, and parenchyma of the adult rat brain. Both immunoreactivity and mRNA for prostaglandin D synthase were located in arachnoid barrier cells, arachnoid trabecular cells, and arachnoid pia mater cells. Furthermore, meningeal macrophages and perivascular microglial cells, identified by use of ED2 antibody, were immunopositive for prostaglandin D synthase. In the arachnoid trabecular cells, the immunoreactivity for prostaglandin D synthase was located in the nuclear envelope, Golgi apparatus, and secretory vesicles, indicating the active production and secretion of prostaglandin D synthase. In the meningeal macrophages, prostaglandin D synthase was not found around the nucleus but in lysosomes in the cytoplasm, pointing to an uptake of the protein from the cerebrospinal fluid. Furthermore, the existence of meningeal cyclooxygenase (COX) -1 and COX-2 was investigated by Western blot, Northern blot, and reverse transcriptase-polymerase chain reaction (RT-PCR), and the colocalization of COX-2 and prostaglandin D synthase was demonstrated in virtually all cells of the leptomeninges, choroid plexus epithelial cells, and perivascular microglial cells, suggesting that these cells synthesize prostaglandin D(2) actively. Alternatively, oligodendrocytes showed prostaglandin D synthase immunoreactivity without detectable COX-2. The localization of lipocalin-type prostaglandin D synthase in meningeal cells and its colocalization with COX-2 provide evidence for its function as a prostaglandin D(2)-producing enzyme.


Subject(s)
Central Nervous System/enzymology , Intramolecular Oxidoreductases/metabolism , Meninges/enzymology , Rats, Sprague-Dawley/metabolism , Animals , Arachnoid/metabolism , Arachnoid/ultrastructure , Brain/metabolism , Brain/ultrastructure , Central Nervous System/ultrastructure , Choroid Plexus/metabolism , Choroid Plexus/ultrastructure , Cyclooxygenase 1 , Cyclooxygenase 2 , Intramolecular Oxidoreductases/genetics , Isoenzymes/genetics , Isoenzymes/metabolism , Lipocalins , Male , Membrane Proteins , Meninges/ultrastructure , Pia Mater/metabolism , Pia Mater/ultrastructure , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandin-Endoperoxide Synthases/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley/anatomy & histology
9.
Neurochem Res ; 25(5): 733-8, 2000 May.
Article in English | MEDLINE | ID: mdl-10905636

ABSTRACT

Cytosolic prostaglandin (PG) E synthase was purified from human brain cortex. The N-terminal amino acid sequence, PMTLGYXNIRGL, was identical to that of the human mu-class glutathione transferase (GST) M2 subunit. Complementary DNAs for human GSTM2, GSTM3, and GSTM4 subunits were cloned, and recombinant proteins were expressed as homodimers in Escherichia coli. The recombinant GSTM2-2 and 3-3 catalyzed the conversion of PGH2 to PGE2 at the rates of 282 and 923 nmol/min/mg of protein, respectively, at the optimal pH of 8, whereas GSTM4-4 was inactive; although all three enzymes showed GST activity. The PGE synthase activity depended on thiols, such as glutathione, dithiothreitol, 2-mercaptoethanol, or L-cysteine. Michaelis-Menten constants and turnover numbers for PGH2 were 141 microM and 10.8 min(-1) for GSTM2-2 and 1.5 mM and 130 min(-1) for GSTM3-3, respectively. GSTM2-2 and 3-3 may play crucial roles in temperature regulation, nociception, and sleep-wake regulation by producing PGE2 in the brain.


Subject(s)
Cerebral Cortex/enzymology , Glutathione Transferase/metabolism , Intramolecular Oxidoreductases/metabolism , Amino Acid Sequence , Cloning, Molecular , Cytosol/enzymology , Glutathione Transferase/chemistry , Glutathione Transferase/genetics , Humans , Intramolecular Oxidoreductases/chemistry , Intramolecular Oxidoreductases/genetics , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , Peptide Fragments/chemistry , Prostaglandin-E Synthases , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate Specificity , Sulfhydryl Compounds/metabolism
10.
J Biol Chem ; 275(40): 31239-44, 2000 Oct 06.
Article in English | MEDLINE | ID: mdl-10871602

ABSTRACT

Hematopoietic prostaglandin (PG) D synthase (PGDS) is the first identified vertebrate ortholog in the Sigma class of the glutathione S-transferase (GST) family and catalyzes both isomerization of PGH(2) to PGD(2) and conjugation of glutathione to 1-chloro-2, 4-dinitrobenzene. We introduced site-directed mutations of Tyr(8), Arg(14), Trp(104), Lys(112), Tyr(152), Cys(156), Lys(198), and Leu(199), which are presumed to participate in catalysis or PGH(2) substrate binding based on the crystallographic structure. Mutants were analyzed in terms of structure, GST and PGDS activities, and activation of the glutathione thiol group. Of all the mutants, only Y8F, W104I, K112E, and L199F showed minor but substantial differences in their far-UV circular dichroism spectra from the wild-type enzyme. Y8F, R14K/E, and W104I were completely inactive. C156L/Y selectively lost only PGDS activity. K112E reduced GST activity slightly and PGDS activity markedly, whereas K198E caused a selective decrease in PGDS activity and K(m) for glutathione and PGH(2) in the PGDS reaction. No significant changes were observed in the catalytic activities of Y152F and L199F, although their K(m) for glutathione was increased. Using 5,5'-dithiobis(2-nitrobenzoic acid) as an SH-selective agent, we found that only Y8F and R14E/K did not accelerate the reactivity of the glutathione thiol group under the low reactivity condition of pH 5.0. These results indicate that Lys(112), Cys(156), and Lys(198) are involved in the binding of PGH(2); Trp(104) is critical for structural integrity of the catalytic center for GST and PGDS activities; and Tyr(8) and Arg(14) are essential for activation of the thiol group of glutathione.


Subject(s)
Hematopoietic Stem Cells/enzymology , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/metabolism , Amino Acids/metabolism , Animals , Arginine/chemistry , Arginine/metabolism , Catalysis , Circular Dichroism , Crystallography, X-Ray , DNA Primers , Dinitrobenzenes/pharmacology , Escherichia coli/metabolism , Glutathione/metabolism , Glutathione Transferase/metabolism , Immunoblotting , Immunohistochemistry , Kinetics , Lipocalins , Models, Molecular , Mutagenesis, Site-Directed , Plasmids/metabolism , Polymerase Chain Reaction , Rats , Recombinant Proteins/metabolism , Time Factors , Tyrosine/chemistry , Tyrosine/metabolism , Ultraviolet Rays
11.
Eur J Biochem ; 267(11): 3315-22, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10824118

ABSTRACT

Hematopoietic prostaglandin D synthase (H-PGDS) is the key enzyme for the production of the D and J series of prostanoids, and the first recognized vertebrate homolog of sigma-class glutathione S-transferase (GST). We isolated the genes and cDNAs for human and mouse H-PGDSs. The human and mouse cDNAs contained a coding region corresponding to 199 amino-acid residues with calculated molecular masses of 23 343 and 23 226, respectively. Both H-PGDS proteins recombinantly expressed in Escherichia coli showed bifunctional activities for PGDS and GST, and had almost the same catalytic properties as the rat enzyme. Northern analyses demonstrated that the H-PGDS genes were expressed in a highly species-specific manner. Whereas the human gene was widely distributed, in contrast, the mouse gene was detected only in samples from oviduct and skin. By fluorescence in situ hybridization, the chromosomal localization of the human and mouse H-PGDS genes were mapped to 4q21-22 and 3D-E, respectively. The human and mouse H-PGDS genes spanned approximately 41 and 28 kb, respectively, and consisted of six exons divided by five introns. The exon/intron boundaries of both genes were completely identical to those of the sigma-class GST subfamily, although the amino-acid sequences of the latter were only 17.0-21.5% identical to those of either H-PGDS. These findings suggest that the H-PGDS genes evolved from the same ancestral gene as the members of the sigma-class GST family.


Subject(s)
Chromosomes, Human, Pair 4/genetics , Crystallins/genetics , Evolution, Molecular , Glutathione Transferase/genetics , Hematopoietic System/enzymology , Intramolecular Oxidoreductases/genetics , Amino Acid Sequence , Animals , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Cells, Cultured , Chickens/genetics , Chickens/metabolism , Chromosome Mapping , DNA, Complementary/genetics , Decapodiformes/enzymology , Decapodiformes/genetics , Helminth Proteins/genetics , Humans , In Situ Hybridization, Fluorescence , Lipocalins , Lymphocytes/ultrastructure , Mice , Molecular Sequence Data , Organ Specificity , RNA, Messenger/biosynthesis , Rats , Recombinant Fusion Proteins/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity
12.
Proc Natl Acad Sci U S A ; 97(9): 4903-7, 2000 Apr 25.
Article in English | MEDLINE | ID: mdl-10781097

ABSTRACT

To examine the function of prostaglandin (PG) D synthase (PGDS) gene, as well as endogenously produced PGD(2) in sleep regulation in vivo, we generated transgenic (TG) mice that overexpress human PGDS gene to study their sleep behavior. Although no difference was observed in the sleep/wake patterns between wild-type and TG mice, a striking time-dependent increase in non-rapid eye movement (NREM), but not in rapid eye movement (REM), sleep was observed in two independent lines of TG mice after stimulation by tail clipping. Concomitantly, the spontaneous locomotor activity of TG animals was drastically decreased in response to the tail clip. Induction of NREM sleep in TG mice was positively correlated with the PGD(2) production in the brain. Sleep, locomotion, and PGD(2) content were essentially unchanged in wild-type mice after tail clipping. The results with TG mice demonstrate the involvement of the PGDS gene in the regulation of NREM sleep.


Subject(s)
Intramolecular Oxidoreductases/genetics , Sleep Stages/genetics , Sleep, REM/genetics , Animals , Biological Clocks , Circadian Rhythm , Gene Expression Regulation, Enzymologic , Humans , Intramolecular Oxidoreductases/metabolism , Lipocalins , Male , Mice , Mice, Inbred Strains , Mice, Transgenic , Motor Activity , Sleep Stages/physiology , Sleep, REM/physiology
13.
Philos Trans R Soc Lond B Biol Sci ; 355(1394): 275-80, 2000 Feb 29.
Article in English | MEDLINE | ID: mdl-10724461

ABSTRACT

Prostaglandin (PG) D2 is a major prostanoid in the brains of rats and other mammals, including humans. When PGD synthase (PGDS), the enzyme that produces PGD2 in the brain, was inhibited by the intracerebroventricular infusion of its selective inhibitors, i.e. tetravalent selenium compounds, the amount of sleep decreased both time and dose dependently. The amount of sleep of transgenic mice, in which the human PGDS gene had been incorporated, increased several fold under appropriate conditions. These data indicate that PGDS is a key enzyme in sleep regulation. In situ hybridization, immunoperoxidase staining and direct enzyme activity determination of tissue samples revealed that PGDS is hardly detectable in the brain parenchyma but is localized in the membrane systems surrounding the brain, namely, the arachnoid membrane and choroid plexus, from which it is secreted into the cerebrospinal fluid (CSF) to become beta-trace, a major protein component of the CSF. PGD2 exerts its somnogenic activity by binding to PGD2 receptors exclusively localized at the ventrorostral surface of the basal forebrain. When PGD2 was infused into the subarachnoid space below the rostral basal forebrain, striking expression of proto-oncogene Fos immunoreactivity (FosIR) was observed in the ventrolateral preoptic area (VLPO), a putative sleep centre, concurrent with sleep induction. Fos expression in the VLPO was positively correlated with the preceding amount of sleep and negatively correlated with Fos expression in the tuberomammillary nucleus (TMN), a putative wake centre. These observations suggest that PGD2 may induce sleep via leptomeningeal PGD2 receptors with subsequent activation of the VLPO neurons and downregulation of the wake neurons in the TMN area. Adenosine may be involved in the signal transduction associated with PGD2.


Subject(s)
Brain Chemistry/physiology , Prostaglandin D2/physiology , Sleep/physiology , Wakefulness/physiology , Animals , Humans , Proto-Oncogene Mas
14.
J Sleep Res ; 9(1): 81-7, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10733693

ABSTRACT

We studied the effect of sleep excess on the sleep-wakefulness pattern of rats. Subarachnoid infusion of prostaglandin D2 or the adenosine A2a receptor agonist CGS21680 effectively induced slow wave sleep (SWS) for the first 12 h of the night-time period, whereas they did not induce sleep during the following 24 h of infusion. An increase in the amount of wakefulness was seen during the last 12 h of prostaglandin D2 infusion. The amounts of wakefulness strongly increased during the following 36-h recovery period. Rebound wakefulness was extraordinarily strong after the cessation of CGS21680 infusion, reaching almost complete insomnia during the night-time. Treatment of animals with prostaglandin D2 overnight, following by treatment with CGS21680 on the next night, resulted in the strongest induction of wakefulness rebound. During the rebound period, the amount of wakefulness reached up to 50 min per hour in the daytime. Rebound of wakefulness depended on the amounts of preceding SWS induced by infusion of prostaglandin D2 for 6 or 12 h and of CGS21680 for 12 h. The larger the amount of SWS, the larger the amount of the following rebound of wakefulness. Rebounds of wakefulness occurred as a result of decrease in SWS amounts, whereas paradoxical sleep amounts did not change. Desensitization of adenosine A2a receptors and accumulation of prostaglandin E2 may be involved in the production of strong wakefulness rebound following relatively long treatments (more than 12 h) with prostaglandin D2 or CGS21680.


Subject(s)
Adenosine/analogs & derivatives , Phenethylamines/pharmacology , Purinergic P1 Receptor Agonists , Receptors, Immunologic , Receptors, Prostaglandin/drug effects , Receptors, Purinergic P1/drug effects , Sleep, REM/physiology , Wakefulness/drug effects , Adenosine/pharmacology , Animals , Male , Rats , Rats, Sprague-Dawley , Receptor, Adenosine A2A , Time Factors
16.
Vitam Horm ; 58: 89-120, 2000.
Article in English | MEDLINE | ID: mdl-10668396

ABSTRACT

Prostaglandin (PG) D synthase catalyzes the isomerization of PGH2, a common precursor of various prostanoids, to produce PGD2 in the presence of sulfhydryl compounds. PGD2 induces sleep, regulates nociception, inhibits platelet aggregation, acts as an allergic mediator, and is further converted to 9 alpha, 11 beta-PGF2 or the J series of prostanoids, such as PGJ2, delta 12-PGJ2, and 15-deoxy-delta 12,14-PGJ2. We have purified two distinct types of PGD synthase; one is the lipocalin-type enzyme and the other is the hematopoietic enzyme. We isolated the cDNA and the gene for each enzyme and determined the tissue distribution profile and the cellular localization in several animal species. Lipocalin-type PGD synthase is localized in the central nervous system and male genital organs of various mammals and the human heart and is secreted into cerebrospinal fluid, seminal plasma, and plasma, respectively. The human enzyme was identified as beta-trace, which is a major protein in human cerebrospinal fluid. This enzyme is considered to be a dual-function protein; it acts as a PGD2-producing enzyme and also as a lipophilic ligand-binding protein, because the enzyme binds retinoids, thyroids, and bile pigments, with high affinities. Hematopoietic PGD synthase is widely distributed in the peripheral tissues and localized in the antigen-presenting cells, mast cells, and megakaryocytes. The hematopoietic enzyme is the first recognized vertebrate homolog of the sigma class of glutathione S-transferase. X-ray crystallographic analyses and generation of gene-knockout and transgenic mice for each enzyme have been performed.


Subject(s)
Intramolecular Oxidoreductases/chemistry , Intramolecular Oxidoreductases/metabolism , Animals , Hematopoiesis , Humans , Intramolecular Oxidoreductases/genetics , Lipocalins , Male , Models, Molecular , Organ Specificity , Prostaglandin D2/metabolism , Prostaglandin D2/pharmacology
17.
Biochem Biophys Res Commun ; 266(3): 641-6, 1999 Dec 29.
Article in English | MEDLINE | ID: mdl-10603301

ABSTRACT

The characterization of unfolding of mouse recombinant lipocalin-type prostaglandin D synthase (L-PGDS) by guanidine hydrochloride (GdnHCl) was carried out. In the presence of low concentrations of GdnHCl (up to 0.75 M), enhancement of the enzyme activity was observed. However, above a 1 M concentration of GdnHCl, the enzyme activity was reduced in a concentration-dependent manner. The maximum enzyme activity induced by GdnHCl was approximately 1. 5-fold compared with the activity under physiological conditions without GdnHCl. The ellipticity in circular dichroism (CD) spectrum of the L-PGDS at 218 nm, reflecting the beta-sheet content, was decreased by GdnHCl (up to 0.75 M), and the minimum ellipticity was observed at 0.5 M GdnHCl. The fluorescence quenching of the intrinsic tryptophan of L-PGDS due to the binding of bilirubin in the presence or absence of GdnHCl was measured. The K(d) values obtained in the presence and absence of 0.5 M GdnHCl were 447 and 115 nM, respectively, indicating lower affinity of the L-PGDS for bilirubin with GdnHCl than without it. Further, an NMR study revealed that the reorganization of hydrogen-bond network in the L-PGDS was observed in the presence of 0.5 M GdnHCl. These results, taken together, indicate that the enzyme activity of L-PGDS is enhanced by the conformational change, especially by the change in the secondary structure.


Subject(s)
Guanidine/chemistry , Intramolecular Oxidoreductases/metabolism , Animals , Bilirubin/chemistry , Circular Dichroism , Enzyme Activation , Fluorescence , Intramolecular Oxidoreductases/chemistry , Lipocalins , Magnetic Resonance Spectroscopy , Mice , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
18.
J Sleep Res ; 8 Suppl 1: 60-4, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10389108

ABSTRACT

Prostaglandin (PG) D2 is the major prostanoid in the mammalian brain, and is the endogenous sleep-promoting substance in mice, rats, and monkeys, and probably in humans as well. When PGD synthase (PGDS), the enzyme responsible for the biosynthesis of PGD2 in the brain, was inhibited in vivo by its selective inhibitors, tetravalent selenium compounds, both slow-wave sleep and rapid-eye-movement sleep were reduced almost completely but reversibly, indicating that PGDS is a key enzyme in sleep regulation. Experiments with transgenic mice also support this contention. In situ hybridization, immunoperoxidase staining, and direct enzyme assay of tissue samples revealed that PGDS is mainly, if not exclusively, localized in the arachnoid membrane and choroid plexus, from which it is secreted into the cerebrospinal fluid to become beta-trace protein. PGD2 exerts its somnogenic activity by binding with PGD2 receptors, exclusively localized at the ventro-rostral surface of the basal forebrain. CGS21680, an adenosine A2a agonist, mimicked the somnogenic activity of PGD2 when applied to the PGD2-sensitive zone. This effect was dose-dependently and selectively abolished by the prior i.p. application of the adenosine A2a antagonist KF17837. Furthermore, the somnogenic activity of PGD2 was also dose-dependently and selectively attenuated by KF17837, indicating the possibility that the sleep induction by PGD2 may be mediated by adenosine through A2a receptors under these conditions. When PGD2 was infused into the subarachnoid space below the rostral basal forebrain, concurrent with sleep induction, striking expression of Fos immunoreactivity was observed in the ventrolateral preoptic area. Fos expression in the ventrolateral preoptic area was positively correlated with the preceding amount of sleep and negatively correlated with Fos expression in the tuberomammillary nucleus. PGD2 also increased Fos IR in the basal leptomeninges and several regions implicated in autonomic regulation. These observations suggest that PGD2 may induce sleep via leptomeningeal PGD2 receptors with subsequent activation of the ventrolateral preoptic area neurons.


Subject(s)
Prostaglandin D2/genetics , Sleep, REM/genetics , Adenosine/antagonists & inhibitors , Animals , Brain/enzymology , DNA, Complementary/genetics , Genes, fos/genetics , Mice , Mice, Transgenic , Prostaglandin D2/biosynthesis , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandin-Endoperoxide Synthases/metabolism , RNA, Messenger/genetics , Rats , Receptors, Purinergic P1/drug effects , Wakefulness/genetics
19.
Biochemistry ; 38(25): 8006-13, 1999 Jun 22.
Article in English | MEDLINE | ID: mdl-10387044

ABSTRACT

Lipocalin-type prostaglandin D synthase is a major protein of the cerebrospinal fluid and was originally known as beta-trace. We investigated the binding ability of prostaglandin D synthase toward bile pigments, thyroid hormones, steroid hormones, and fatty acids in this present study. We found that the recombinant enzyme binds bile pigments and thyroid hormones, resulting in quenching of the intrinsic tryptophan fluorescence, the appearance of induced circular dichroism of the lipophilic ligands, and a red shift of the absorption spectra of bilirubin and biliverdin. The binding of prostaglandin D synthase to lipophilic ligands was also demonstrated by the resonant mirror technique and surface plasmon resonance detection. The dissociation constants were calculated to be 33 nM, 37 nM, 660 nM, 820 nM, and 2.08 microM for biliverdin, bilirubin, L-thyroxine, 3,3',5'-triiodo-L-thyronine, and 3,3', 5-triiodo-L-thyronine, respectively. Biliverdin and bilirubin underwent a shift in their absorption peaks from 375 to 380 nm and from 439 to 446 nm, respectively, after binding to prostaglandin D synthase. Bilirubin bound to the enzyme showed a bisignate CD spectrum with a (-) Cotton effect at 422 nm and a (+) Cotton effect at 472 nm, indicating a right-handed chirality. The ligands also inhibited prostaglandin D synthase activity noncompetitively in a concentration-dependent manner, with IC50 values between 3.9 and 10. 9 microM. Epididymal retinoic acid-binding protein and beta-lactoglobulin, two other lipocalin proteins that bind retinoids such as prostaglandin D synthase, did not show any significant interaction with bile pigments or thyroid hormones. These results show that prostaglandin D synthase binds small lipophilic ligands with a specificity distinct from that of other lipocalins.


Subject(s)
Bilirubin/chemistry , Biliverdine/chemistry , Carrier Proteins/chemistry , Intramolecular Oxidoreductases/chemistry , Neoplasm Proteins , Nerve Tissue Proteins , Thyroid Hormones/chemistry , Animals , Beta-Globulins/chemistry , Beta-Globulins/metabolism , Bilirubin/metabolism , Biliverdine/metabolism , Carrier Proteins/metabolism , Enzyme Inhibitors/chemistry , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Intramolecular Oxidoreductases/antagonists & inhibitors , Intramolecular Oxidoreductases/metabolism , Lactoglobulins/chemistry , Ligands , Lipocalins , Models, Molecular , Myelin P2 Protein/chemistry , Myelin P2 Protein/metabolism , Rats , Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/metabolism , Spectrometry, Fluorescence , Thyroid Hormones/metabolism
20.
Eur J Neurosci ; 11(5): 1587-97, 1999 May.
Article in English | MEDLINE | ID: mdl-10215911

ABSTRACT

The present study has demonstrated that the sleep-promoting potency of 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido adenosine (CGS21680), a selective agonist for the adenosine A2A receptor, varies depending on the location of the administration. CGS21680 was continuously administered to rats through a chronically implanted cannula for 6 h during their active phase. The tip of the cannula was located in the subarachnoid space or the brain ventricle neighbouring the established brain areas implicated in the regulation of sleep-wake phenomena, i.e. rostral basal forebrain, medial preoptic area, lateral preoptic area, posterior hypothalamus, and dorsal tegmentum of the pons and medulla. At an infusion rate of 2.0 pmol/min, the magnitude of increase in non-rapid eye movement sleep varied from 14 min (a 15% increase) to 96 min (a 103% increase), and those of rapid eye movement sleep varied from 6 min (a 40% increase) to 28 min (a 264% increase) from the respective baseline values. The largest increases in both types of sleep occurred when CGS21680 was administered to the subarachnoid space underlying the rostral basal forebrain. These findings were interpreted to mean that the major, if not the only, site responsible for the CGS21680-inducing sleep was located in or near the rostral basal forebrain. This interpretation was supported by the findings that the administration of CGS21680 to the rostral basal forebrain produced predominant expression of Fos within the shell of the nucleus accumbens and the medial portion of the olfactory tubercle, and that the microdialysis perfusion of CGS21680 into the shell of the nucleus accumbens also exhibited a sleep-promoting effect.


Subject(s)
Adenosine/analogs & derivatives , Antihypertensive Agents/pharmacology , Phenethylamines/pharmacology , Receptors, Purinergic P1/physiology , Sleep, REM/drug effects , Sleep, REM/physiology , Adenosine/pharmacology , Animals , Body Temperature , Brain Chemistry/drug effects , Brain Chemistry/physiology , Electroencephalography , Electromyography , Male , Microdialysis , Neurons/chemistry , Neurons/physiology , Nucleus Accumbens/cytology , Nucleus Accumbens/drug effects , Nucleus Accumbens/physiology , Proto-Oncogene Proteins c-fos/analysis , Purinergic P1 Receptor Agonists , Rats , Rats, Sprague-Dawley , Receptor, Adenosine A2A
SELECTION OF CITATIONS
SEARCH DETAIL
...