A sleep-promoting role for the Drosophila serotonin receptor 1A

BACKGROUND: Although sleep is an important process essential for life, its regulation is poorly understood. The recently developed Drosophila model for sleep provides a powerful system to genetically and pharmacologically identify molecules that regulate sleep. Serotonin is an important neurotransmitter known to affect many behaviors, but its role in sleep remains controversial. RESULTS: We generated or obtained flies with genetically altered expression of each of three Drosophila serotonin receptor subtypes (d5-HT1A, d5-HT1B, and d5-HT2) and assayed them for baseline sleep phenotypes. The data indicated a sleep-regulating role for the d5-HT1A receptor. d5-HT1A mutant flies had short and fragmented sleep, which was rescued by expressing the receptor in adult mushroom bodies, a structure associated with learning and memory in Drosophila. Neither the d5-HT2 receptor nor the d5-HT1B receptor, which was previously implicated in circadian regulation, had any effect on baseline sleep, indicating that serotonin affects sleep and circadian rhythms through distinct receptors. Elevating serotonin levels, either pharmacologically or genetically, enhanced sleep in wild-type flies. In addition, serotonin promoted sleep in some short-sleep mutants, suggesting that it can compensate for some sleep deficits. CONCLUSIONS: These data show that serotonin promotes baseline sleep in Drosophila. They also link the regulation of sleep behavior by serotonin to a specific receptor in a distinct region of the fly brain.     

The second PGD(2) receptor CRTH2: structure,properties, and functions in leukocytes

Prostaglandin (PG) D(2) plays a broad range of physiological and pathophysiological functions. Until just a few years ago, it was thought that most of the biological actions of PGD(2) are mediated via the classical PGD(2) receptor DP. Recently, we identified a second PGD(2) receptor, chemoattractant receptor-homologous molecule expressed on T helper (Th)2 cells (CRTH2), with different functions relative to DP. Here, we review the recent findings on the structure, tissue distribution, ligand selectivity, signalling pathways, and functions in leukocytes of this receptor. The data suggest that the PGD(2)/CRTH2 system play important roles in allergic inflammation through its stimulatory effects on Th2 cells, eosinophils, and basophils.     

前列腺素D2与睡眠调节

前列腺素D2（PGD2）是目前已知最有潜力的内源性促睡眠物质之一。鼠脑脊液（CSF）中PGD2浓度与睡眠－觉醒周期一致,呈现节律性改变,并且随睡眠剥夺期间嗜睡倾向增加而增加,催化PGH2转变为PGD2的特生酶有两种：Lipocalin型前列腺素D合成酶（L－PGDS）和脾型PGDS。L－PGDS主要在大脑蛛网膜和脉络丛产生,并分泌入CSF。L－PGDS和PGD2在脑室系统、蛛网膜下腔及细胞外间隙中循环,循环中的PGD2可与前脑头端基底腹内侧面的化学感受器中的前列腺素D2受体（DPR）相互作用,通过活化具有腺苷A2a受体的元以产生促进睡眠的信号。PGD2敏感区内DPR的活化可导致腹侧视前区（VLPO）内神经元的活化,其可通过抑制结节乳头核（TMN）而促进睡眠。相反,PGE2在维持大脑清醒状态下起主要作用。PGD2和PGE2的平衡对正常睡眠－觉醒周期的维持十分关键。     

Synthesis and biological activity of trisubstituted adenines as A 2A adenosine receptor antagonists

The discovery of new drugs for the treatment of neurodegenerative disorders, such as Parkinson's disease, has become an attractive field of research. Due to the regulation of D(2) receptor activity by A(2A) adenosine receptor, potent and selective ligands of A(2A) subtype could be useful tools to study neurodegenerative disorders. A series of 2,8-disubstituted-9-ethyladenine derivatives was synthesized and tested in binding affinity assay at human adenosine receptors. New compounds showed good affinity and selectivity at A(2A) receptor versus the other subtypes. The introduction of a bromine atom in 8-position increased the affinity of these compounds, leading to ligands with K(i) in the nanomolar range.     

Molecular mechanisms of sleep-wake regulation: a role of prostaglandin D2

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.     

An adenosine A receptor agonist induces sleep by increasing GABA release in the tuberomammillary nucleus to inhibit histaminergic systems in rats

The adenosine A(2A) receptor (A(2A)R) has been demonstrated to play a crucial role in the regulation of the sleep process. However, the molecular mechanism of the A(2A)R-mediated sleep remains to be elucidated. Here we used electroencephalogram and electromyogram recordings coupled with in vivo microdialysis to investigate the effects of an A(2A)R agonist, CGS21680, on sleep and on the release of histamine and GABA in the brain. In freely moving rats, CGS21680 applied to the subarachnoid space underlying the rostral basal forebrain significantly promoted sleep and inhibited histamine release in the frontal cortex. The histamine release was negatively correlated with the amount of non-rapid eye movement sleep (r = - 0.652). In urethane-anesthetized rats, CGS21680 inhibited histamine release in both the frontal cortex and medial pre-optic area in a dose-dependent manner, and increased GABA release specifically in the histaminergic tuberomammillary nucleus but not in the frontal cortex. Moreover, the CGS21680-induced inhibition of histamine release was antagonized by perfusion of the tuberomammillary nucleus with a GABA(A) antagonist, picrotoxin. These results suggest that the A(2A)R agonist induced sleep by inhibiting the histaminergic system through increasing GABA release in the tuberomammillary nucleus.     

G protein-coupled receptor kinase regulates dopamine D3 receptor signaling by modulating the stability of a receptor-filamin-beta-arrestin complex. A case of autoreceptor regulation

In addition to its postsynaptic role, the dopamine D3 receptor (D3R) serves as a presynaptic autoreceptor, where it provides continuous feedback regulation of dopamine release at nerve terminals for processes as diverse as emotional tone and locomotion. D3R signaling ability is supported by an association with filamin (actin-binding protein 280), which localizes the receptor with G proteins in plasma membrane lipid rafts but is not appreciably antagonized in a classical sense by the ligand-mediated activation of G protein-coupled receptor kinases (GRKs) and beta-arrestins. In this study, we investigate GRK-mediated regulation of D3R.filamin complex stability and its effect on D3R.G protein signaling potential. Studies in HEK-293 cells show that in the absence of agonist the D3R immunoprecipitates in a complex containing both filamin A and beta-arrestin2. Moreover, the filamin directly interacts with beta-arrestin2 as assessed by immunoprecipitation and yeast two-hybrid studies. With reductions in basal GRK2/3 activity, an increase in the basal association of filamin A and beta-arrestin2 with D3R is observed. Conversely, increases in the basal GRK2/3 activity result in a reduction in the interaction between the D3R and filamin but a relative increase in the agonist-mediated interaction between beta-arrestin2 and the D3R. Our data suggest that the D3R, filamin A, and beta-arrestin form a signaling complex that is destabilized by agonist- or expression-mediated increases in GRK2/3 activity. These findings provide a novel GRK-based mechanism for regulating D3R signaling potential and provide insight for interpreting D3R autoreceptor behavior.     

Role of dorsal raphe nucleus serotonin 5-HT1A receptor in the regulation of REM sleep

Cholinergic neurons in the laterodorsal (LDT) and the pedunculopontine (PPT) tegmental nuclei act to promote REM sleep (REMS). The predominantly glutamatergic neurons of the REMS-induction region of the medial pontine reticular formation are in turn activated by cholinergic cells, which results in the occurrence of tonic and phasic components of REMS. All these neurons are inhibited by serotonergic (5-HT), noradrenergic, and presumably histaminergic (H2 receptor) and dopaminergic (D2 and D3 receptor) cells. 5-Hydroxytryptamine-containing neurons in the dorsal raphe nucleus (DRN) virtually cease firing when an animal starts REMS, consequently decreasing the release of 5-HT during this state. The activation of GABA(A) receptors is apparently responsible for this phenomenon. Systemic administration of the selective 5-HT1A receptor agonist 8-OHDPAT induces dose-dependent effects; i.e. low doses increase slow wave sleep and reduce waking, whereas large doses increase waking and reduce slow wave sleep and REM sleep. Direct injection of 8-OHDPAT or flesinoxan, another 5-HT1A agonist into the DRN, or microdialysis perfusion of 8-OHDPAT into the DRN significantly increases REMS. On the other hand, infusion of 8-OHDPAT into the LDT selectively inhibits REMS, as does direct administration into the DRN of the 5-HT1A receptor antagonists pindolol or WAY 100635. Thus, presently available evidence indicates that selective activation of the somatodendritic 5-HT1A receptor in the DRN induces an increase of REMS. On the other hand, activation of the postsynaptic 5-HT1A receptor at the level of the PPT/LDT nuclei decreases REMS occurrence.     

Dysregulation of dopamine-dependent mechanisms as a determinant of hypertension: studies in dopamine receptor knockout mice

Dopamine plays an important role in the pathogenesis of hypertension by regulating epithelial sodium transport and by interacting with vasoactive hormones/humoral factors, such as aldosterone, angiotensin, catecholamines, endothelin, oxytocin, prolactin pro-opiomelancortin, reactive oxygen species, renin, and vasopressin. Dopamine receptors are classified into D(1)-like (D(1) and D(5)) and D(2)-like (D(2), D(3), and D(4)) subtypes based on their structure and pharmacology. In recent years, mice deficient in one or more of the five dopamine receptor subtypes have been generated, leading to a better understanding of the physiological role of each of the dopamine receptor subtypes. This review summarizes the results from studies of various dopamine receptor mutant mice on the role of individual dopamine receptor subtypes and their interactions with other G protein-coupled receptors in the regulation of blood pressure.     

Mdmx as an essential regulator of p53 activity

The murine double minute 2 (Mdm2) is a critical negative regulator of the p53 tumor suppressor. Almost 10 years ago, a search for new p53-interactors revealed the existence of an Mdm2-structurally related protein, Mdmx (or Mdm4). Since then a large body of biochemical data has accumulated on the functions of Mdmx, often leading to conflicting molecular models. Nevertheless, virtually all these data pointed toward a critical role for Mdmx in the regulation of the p53-Mdm2 network. A view that was recently confirmed by genetic studies. This review is a summary of our current understanding of this molecule, its structure and biological functions, as well as its relationship to its known binding partners.     

Dual hypocretin receptor antagonism is more effective for sleep promotion than antagonism of either receptor alone

The hypocretin (orexin) system is involved in sleep/wake regulation, and antagonists of both hypocretin receptor type 1 (HCRTR1) and/or HCRTR2 are considered to be potential hypnotic medications. It is currently unclear whether blockade of either or both receptors is more effective for promoting sleep with minimal side effects. Accordingly, we compared the properties of selective HCRTR1 (SB-408124 and SB-334867) and HCRTR2 (EMPA) antagonists with that of the dual HCRTR1/R2 antagonist almorexant in the rat. All 4 antagonists bound to their respective receptors with high affinity and selectivity in vitro. Since in vivo pharmacokinetic experiments revealed poor brain penetration for SB-408124, SB-334867 was selected for subsequent in vivo studies. When injected in the mid-active phase, SB-334867 produced small increases in rapid-eye-movement (REM) and non-REM (NR) sleep. EMPA produced a significant increase in NR only at the highest dose studied. In contrast, almorexant decreased NR latency and increased both NR and REM proportionally throughout the subsequent 6 h without rebound wakefulness. The increased NR was due to a greater number of NR bouts; NR bout duration was unchanged. At the highest dose tested (100 mg/kg), almorexant fragmented sleep architecture by increasing the number of waking and REM bouts. No evidence of cataplexy was observed. HCRTR1 occupancy by almorexant declined 4-6 h post-administration while HCRTR2 occupancy was still elevated after 12 h, revealing a complex relationship between occupancy of HCRT receptors and sleep promotion. We conclude that dual HCRTR1/R2 blockade is more effective in promoting sleep than blockade of either HCRTR alone. In contrast to GABA receptor agonists which induce sleep by generalized inhibition, HCRTR antagonists seem to facilitate sleep by reducing waking "drive".     

Receptor crosstalk: haloperidol treatment enhances A2A adenosine receptor functioning in a transfected cell model

A(2A) adenosine receptors are considered an excellent target for drug development in several neurological and psychiatric disorders. It is noteworthy that the responses evoked by A(2A) adenosine receptors are regulated by D(2) dopamine receptor ligands. These two receptors are co-expressed at the level of the basal ganglia and interact to form functional heterodimers. In this context, possible changes in A(2A) adenosine receptor functional responses caused by the chronic blockade/activation of D(2) dopamine receptors should be considered to optimise the therapeutic effectiveness of dopaminergic agents and to reduce any possible side effects. In the present paper, we investigated the regulation of A(2A) adenosine receptors induced by antipsychotic drugs, commonly acting as D(2) dopamine receptor antagonists, in a cellular model co-expressing both A(2A) and D(2) receptors. Our data suggest that the treatment of cells with the classical antipsychotic haloperidol increased both the affinity and responsiveness of the A(2A) receptor and also affected the degree of A(2A)-D(2) receptor heterodimerisation. In contrast, an atypical antipsychotic, clozapine, had no effect on A(2A) adenosine receptor parameters, suggesting that the two classes of drugs have different effects on adenosine-dopamine receptor interaction. Modifications to A(2A) adenosine receptors may play a significant role in determining cerebral adenosine effects during the chronic administration of antipsychotics in psychiatric diseases and may account for the efficacy of A(2A) adenosine receptor ligands in pathologies associated with dopaminergic system dysfunction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11302-010-9201-z) contains supplementary material, which is available to authorized users.     

前列腺素D2及其受体与哺乳动物生殖

前列腺素D2(PGD2)是前列腺素(PGs)家族成员之一,广泛分布于各种哺乳动物组织中,并发挥多种生理功能。PGD,可以促进睡眠、诱导过敏反应、抑制血小板凝集及松弛平滑肌等,并且在生殖系统中起重要作用。机体中存在生化和免疫功能截然不同的两类前列腺素D合成酶(PGDs)：脑型PGDS(L-PGDS)和生血型PGDS(hPGDS)。生殖系统中,L-PGDS主要存在于雄性生殖道,可能在睾丸发育、精子发生、精子成熟以及血一睾和血一附睾屏障等方面发挥重要作用。hPGDS在妊娠时期的子宫内膜和胚胎滋养层中表达,由其产生的PGD2可能通过DP和CRTH2两种受体来维持妊娠。此外,PGD2还可能与女性不孕以及精子在雌性生殖道内的运输等有关。     

1,2,4-Benzothiadiazine derivatives as alpha1 and 5-HT1A receptor ligands

A series of new 1,2,4-benzothiadiazine derivatives with an arylpiperazine mojety linked at position 3 of the heterocyclic ring were synthesized and assessed for their pharmacological profiles at alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B) and alpha(1D)) by functional experiments and by in vitro binding studies at human cloned 5-HT(1A) receptor. Compound 1 was identified as a novel alpha(1D) antagonist (pK(b)alpha(1D)=7.59; alpha(1D)/alpha(1A)>389; alpha(1D)/alpha(1B)=135) with high selectivity over 5-HT(1A) receptor (5-HT(1A)/alpha(1D)<0.01), while compound 6, a 3,4-dihydro-derivative, was characterized as a novel 5-HT(1A) receptor ligand, highly selective over alpha(1D)-adrenoceptor subtype (pK(i)5-HT(1A)=8.04; 5-HT(1A)/alpha(1D)=1096). Further pharmacological studies demonstrated that 6 is a partial agonist at 5-HT(1A) receptor (E(max)=23, pD(2)=6.92).     

Epigenetic mechanisms regulate the prostaglandin E receptor 2 in breast cancer

The increase in local oestrogen production seen in oestrogen receptor positive (ER+) breast cancers is driven by increased activity of the aromatase enzyme. CYP19A1, the encoding gene for aromatase, is often overexpressed in the oestrogen-producing cells of the breast adipose fibroblasts (BAFs) surrounding an ER+ tumour, and the molecular processes underlying this upregulation is important in the development of breast-specific aromatase inhibitors for breast cancer therapy. Prostaglandin E2 (PGE2), a factor secreted by tumours, is known to stimulate CYP19A1 expression in human BAFs. The hormonal regulation of this process has been examined; however, what is less well understood is the emerging role of epigenetic mechanisms and how they modulate PGE2 signalling. This present study characterises the epigenetic processes underlying expression of the prostanoid receptor EP2 in the context of ER+ breast cancer. Sodium bisulphite sequencing of CpG methylation within the promoter region of EP2 revealed that an inverse correlation existed between methylation levels and relative EP2 expression in breast cancer cell lines MDA-MB-231, MCF7 and MCF10A but not in HS578t and T47D. Inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5aza) and histone deacetylation with Trichostatin A (TSA) resulted in upregulation of EP2 mRNA in all cell lines with varying influences of each epigenetic process observed. Expression of EP2 was detected in human BAFs despite a natively methylated promoter, and this expression was further increased upon 5aza treatment. An examination of 3 triple negative, 3 ductal carcinoma in situ and 3 invasive ductal carcinoma samples revealed that there was no change in EP2 promoter methylation status between normal and cancer associated stroma, despite observed differences in relative mRNA levels. Although EP2 methylation status is inversely correlated to expression levels in established breast cancer cell lines, we could not identify that such a correlation existed in tumour-associated stroma cells.     

Cyclooxygenase-2-derived prostaglandin D(2) is an early anti-inflammatory signal in experimental colitis

The ability of nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors to exacerbate inflammatory bowel disease suggests that prostaglandins are important anti-inflammatory mediators in this context. Prostaglandin D(2) has been suggested to exert anti-inflammatory effects. We investigated the possibility that prostaglandin D(2) derived from cyclooxygenase-2 plays an important role in downregulating colonic inflammation in rats. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. At various times thereafter (from 1 h to 7 days), colonic prostaglandin synthesis and myeloperoxidase activity (index of granulocyte infiltration) were measured. Prostaglandin D(2) synthesis was elevated >4-fold above controls within 1-3 h of induction of colitis, preceding significant granulocyte infiltration. Treatment with a selective cyclooxygenase-2 inhibitor abolished the increase in prostaglandin D(2) synthesis and caused a doubling of granulocyte infiltration. Colonic granulocyte infiltration was significantly reduced by administration of prostaglandin D(2) or a DP receptor agonist (BW-245C). These results demonstrate that induction of colitis results in a rapid increase in prostaglandin D(2) synthesis via cyclooxygenase-2. Prostaglandin D(2) downregulates granulocyte infiltration into the colonic mucosa, probably through the DP receptor.