Dopamine receptors – IUPHAR Review 13

The variety of physiological functions controlled by dopamine in the brain and periphery is mediated by the D₁, D₂, D₃, D₄ and D₅ dopamine GPCRs. Drugs acting on dopamine receptors are significant tools for the management of several neuropsychiatric disorders including schizophrenia, bipolar disorder, depression and Parkinson''s disease. Recent investigations of dopamine receptor signalling have shown that dopamine receptors, apart from their canonical action on cAMP-mediated signalling, can regulate a myriad of cellular responses to fine-tune the expression of dopamine-associated behaviours and functions. Such signalling mechanisms may involve alternate G protein coupling or non-G protein mechanisms involving ion channels, receptor tyrosine kinases or proteins such as β-arrestins that are classically involved in GPCR desensitization. Another level of complexity is the growing appreciation of the physiological roles played by dopamine receptor heteromers. Applications of new in vivo techniques have significantly furthered the understanding of the physiological functions played by dopamine receptors. Here we provide an update of the current knowledge regarding the complex biology, signalling, physiology and pharmacology of dopamine receptors.     

α7-Nicotinic receptors and cognition

Nicotinic α7 receptors have been shown in a variety of studies with animal models to play important roles in diverse components of cognitive function, including learning, memory and attention. Mice with α7 receptor knockouts show impairments in memory. Selective α7 agonists significantly improve learning, memory and attention. α7 receptors in limbic structures such as the hippocampus and amygdala have been demonstrated to play critical roles in memory. Blockade of α7 receptors in these areas cause memory impairments. In the brains of people with schizophrenia α7 receptors are impaired. This may be related to pronounced cognitive impairments seen with schizophrenia. There has been a major effort to develop α7 nicotinic agonists for helping to reverse cognitive impairment. These receptors are a promising target for development of therapeutic treatments for a variety of diseases of cognitive impairment including Alzheimer's disease, attention deficit hyperactivity disorder (ADHD) and schizophrenia.     

Nicotine withdrawal and κ-opioid receptors

Rationale  

The synthesis and release of dynorphin are increased in the caudate/putamen (CPU) and nucleus accumbens (NAc) of nicotine-withdrawn mice, suggesting a role in the nicotine abstinence syndrome.     

Protease-activated receptors: sentries for inflammation?

Cell-surface protease-activated receptors (PARs) appear to have evolved to detect extracellular enzymatically active serine proteases such as trypsin and thrombin. The predominant location of PARs on endothelia and epithelia and the discovery of enzymes such as trypsin within these tissues, together with the linkage of PARs to cytoprotective pathways, provide new information on autocrine and paracrine signalling within these critical barriers. In this article, the ways in which the distribution and function of PARs could be harnessed by pharmacologists as novel anti-inflammatory therapeutic strategies are discussed.     

β-Adrenergic receptors in guinea-pig myocardial tissue

Summary Stereospecific binding sites for (–) [³H]-alprenolol, a -adrenergic antagonist, have been identified in guinea-pig myocardial broken cell preparations. The concentration of the sites was 0.3 pmoles per mg of protein and the dissociation constant (at 37°C) 10^–8 M. A close correlation between the ability of various -adrenergic antagonists to compete with tracer alprenolol binding and to block the response of isoprenaline-stimulated myocardial adenylate cyclase has been found. Low affinity sites for the labelled -adrenergic antagonist in contrast to stereospecific sites are heat stable and do not discriminate between the (–) and the (+) forms of the -adrenergic antagonists. Adenylate cyclase in guineapig myocardial tissue is poorly stimulated by isoprenaline or 5-guanylylimidodiphosphate. This is attributed to a high basal activity which could be lowered by a preincubation at 37°C.     

Binding properties of C-truncated delta opioid receptors

目的：研究δ阿片受体Ｃ末端在受体结合配体的亲和力及选择性中的作用．方法：在中国苍鼠卵巢细胞（ＣＨＯ细胞）中分别稳定表达Ｃ末端截短３１个氨基酸残基及野生型δ阿片受体，用受体结合分析法研究表达产物与配体的结合特征．结果：表达得到典型突变受体克隆ＣＨＯＴ及野生型受体克隆ＣＨＯＷ．ＣＨＯＴ结合［３Ｈ］ｄｉｐｒｅｎｏｒｐｈｉｎｅ（Ｄｉｐ）及［３Ｈ］［ＤＡｌａ２，ＤＬｅｕ５］ｅｎｋｅｐｈａｌｉｎ（ＤＡＤＬＥ）的Ｋｄ值与ＣＨＯＷ一致，δ选择性激动剂对二者与［３Ｈ］Ｄｉｐ的结合均有很强的抑制作用，且Ｋｉ相似；而μ及κ选择性激动剂则对二者均无抑制作用．结论：δ阿片受体的Ｃ末端与受体结合配体的亲和力及选择性无关．     

Stoichiometry of δ subunit containing GABAA receptors

Background and Purpose

Although the stoichiometry of the major synaptic αβγ subunit-containing GABA_A receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach.

Experimental Approach

Using site-directed mutagenesis, we inserted a highly characterized 9′ serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits.

Key Results

Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose–response curves of cells co-expressing WT subunits with their respective L9′S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits.

Conclusions and Implications

Subunit stoichiometry is an important determinant of GABA_A receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ.     

Neuroprotection by donepezil against glutamate excitotoxicity involves stimulation of ��7 nicotinic receptors and internalization of NMDA receptors

BACKGROUND AND PURPOSE

Glutamate excitotoxicity may be involved in ischaemic injury to the CNS and some neurodegenerative diseases, such as Alzheimer''s disease. Donepezil, an acetylcholinesterase (AChE) inhibitor, exerts neuroprotective effects. Here we demonstrated a novel mechanism underlying the neuroprotection induced by donepezil.

EXPERIMENTAL APPROACH

Cell damage in primary rat neuron cultures was quantified by lactate dehydrogenase release. Morphological changes associated with neuroprotective effects of nicotine and AChE inhibitors were assessed by immunostaining. Cell surface levels of the glutamate receptor sub-units, NR1 and NR2A, were analyzed using biotinylation. Immunoblot was used to measure protein levels of cleaved caspase-3, total NR1, total NR2A and phosphorylated NR1. Immunoprecipitation was used to measure association of NR1 with the post-synaptic protein, PSD-95. Intracellular Ca²⁺ concentrations were measured with fura 2-acetoxymethylester. Caspase 3-like activity was measured using enzyme substrate, 7-amino-4-methylcoumarin (AMC)-DEVD.

KEY RESULTS

Levels of NR1, a core subunit of the NMDA receptor, on the cell surface were significantly reduced by donepexzil. In addition, glutamate-mediated Ca²⁺ entry was significantly attenuated by donepezil. Methyllycaconitine, an inhibitor of α7 nicotinic acetylcholine receptors (nAChR), inhibited the donepezil-induced attenuation of glutamate-mediated Ca²⁺ entry. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a phosphatidyl inositol 3-kinase (PI3K) inhibitor, had no effect on attenuation of glutamate-mediated Ca²⁺ entry induced by donepezil.

CONCLUSIONS AND IMPLICATIONS

Decreased glutamate toxicity through down-regulation of NMDA receptors, following stimulation of α7 nAChRs, could be another mechanism underlying neuroprotection by donepezil, in addition to up-regulating the PI3K-Akt cascade or defensive system.     

Estrogen receptors and type 1 metabotropic glutamate receptors are interdependent in protecting cortical neurons against β-amyloid toxicity

We examined the interaction between estrogen receptors (ERs) and type 1 metabotropic glutamate receptors (mGlu1 receptors) in mechanisms of neurodegeneration/neuroprotection using mixed cultures of cortical cells challenged with β-amyloid peptide. Both receptors were present in neurons, whereas only ERα but not mGlu1 receptors were found in astrocytes. Addition of 17β-estradiol (17βE2) protected cultured neurons against amyloid toxicity, and its action was mimicked by the selective ERα agonist, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) as well as by a cell-impermeable bovine serum albumin conjugate of 17βE2. The selective ERβ agonist, diarylpropionitrile (DPN), was only slightly neuroprotective. The mGlu1/5 receptor agonist, 3,5-dihydroxyphenylglycine (DHPG), was also neuroprotective against amyloid toxicity, and its action was abolished by the mGlu1 receptor antagonist, (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone (JNJ 16259685). Neuroprotection by 17βΕ2 or PPT (but not DPN) and DHPG was less than additive, suggesting that ERα and mGlu1 receptors activate the same pathway of cell survival. More important, neuroprotection by 17βΕ2 was abolished not only by the ER antagonist fulvestrant (ICI 182,780) but also by JNJ 16259685, and neuroprotection by DHPG was abolished by ICI 182,780. ERα and mGlu1 receptors were also interdependent in activating the phosphatidylinositol-3-kinase pathway, and pharmacological blockade of this pathway abolished neuroprotection by 17βE2, DHPG, or their combination. These data provide the first evidence that ERα and mGlu1 receptors critically interact in promoting neuroprotection, information that should be taken into account when the impact of estrogen on neurodegeneration associated with central nervous system disorders is examined.     

Modulation of phosphatidylinositol turnover on central nicotinic receptors

目的：研究磷脂酰肌醇代谢对中枢烟碱受体功能的调节作用，以分析脑烟碱受体与磷脂酰肌醇代谢之间的关系．方法：在小鼠上观察肌醇磷酸酶抑制剂氯化锂对烟碱诱发惊厥作用的影响．结果：氯化锂２５－１０ｍｍｏｌ·ｋｇ－１预处理后，烟碱诱发小鼠惊厥的量效关系发生变化，在高于半数效量的剂量下，烟碱诱发惊厥的作用显著增强．但氧颤莫林００５－０２０ｍｇ·ｋｇ－１预处理后，烟碱诱发小鼠惊厥的量效关系无显著变化．在小鼠上每日注射一次氯化锂５０ｍｍｏｌ·ｋｇ－１７ｄ后，烟碱诱发惊厥的作用显著减弱，半数效量由０５８增至０９７ｍｇ·ｋｇ－１．结论：磷脂酰肌醇代谢可调节中枢烟碱受体的功能．     

Central cholinergic regulation of respiration： nicotinic receptors

Nicotinic acetylcholine receptors （nAChRs） are expressed in brainstem and spinal cord regions involved in the control of breathing. These receptors mediate central cholinergic regulation of respiration and effects of the exogenous ligand nicotine on respiratory pattern. Activation of α4＊ nAChRs in the preBotzinger Complex （preBotC）, an essential site for normal respiratory rhythm generation in mammals, modulates excitatory glutamatergic neurotransmission and depolarizes preBotC inspiratory neurons, leading to increases in respiratory frequency, nAChRs are also present in motor nuclei innervating respiratory muscles. Activation of post- and/or extra-synaptic α4＊ nAChRs on hypoglossal （XII） motoneurons depolarizes these neurons, potentiating tonic and respiratory-related rhythmic activity. As perinatal nicotine exposure may contribute to the pathogenesis of sudden infant death syndrome （SIDS）, we discuss the effects of perinatal nicotine exposure on development of the cholinergic and other neurotransmitter systems involved in control of breathing. Advances in understanding of the mechanisms underlying central cholinergic/nicotinic modulation of respiration provide a pharmacological basis for exploiting nAChRs as therapeutic targets for neurological disorders related to neural control of breathing such as sleep apnea and SIDS.     

Murine macrophage cell lines contain μ3-opiate receptors

Opiate alkaloid-selective, opioid peptide-insensitive μ₃ receptors are present in three murine macrophage cell lines (J774.2; RAW 264.7; BAC1.2F5). The receptor binds morphine, its active metabolite morphine 6-glucuronide and certain other alkaloids, but not morphine 3-glucuronide or any of the opioid peptides tested. The cell lines thus provide valuable model systems for investigation of μ₃-opiate receptors, previously demonstrated to mediate inhibitory effects of morphine on activation of human peripheral blood macrophages (monocytes).     

High affinity ligands for ‘diazepam-insensitive’ benzodiazepine receptors

Structurally diverse compounds have been shown to possess high affinities for benzodiazepine receptors in their ‘diazepam-sensitive’ (DS) conformations. In contrast, only the imidazobenzodiazepinone Ro 15-4513 has been shown to exhibit a high affinity for the ‘diazepam-insensitive’ (DI) conformation of benzodiazepine receptors. We examined a series of 1,4-diazepines containing one or more annelated ring systems for their affinities at DI and DS benzodiazepine receptors, several 1,4-diazepinone carboxylates including Ro 19-4603, Ro 16-6028 and Ro 15-3505 were found to possess high affinities (K_i ∼ 2.6–20 nM) for DI. Nonetheless, among the ligands examined, Ro 15-4513 was the only substance with a DI/DS potency ratio ∼ I: other substances had ratios ranging from 13 to > 1000. Ligands with high to moderate affinities at Di were previously classified as partial agonists, antagonists, or partial inverse agonists at DS benzodiazepine receptors, but behaved as ‘GABA neutral’ (antagonist) substances at DI. The identification of several additional high affinity ligands at DI benzodiazepine receptors may be helpful in elucidating the pharmacological and physiological importance of these sites.     

Lubiprostone targets prostanoid EP₄ receptors in ovine airways

BACKGROUND AND PURPOSE

Lubiprostone, a prostaglandin E₁ derivative, is reported to activate ClC-2 chloride channels located in the apical membranes of a number of transporting epithelia. Lack of functioning CFTR chloride channels in epithelia is responsible for the genetic disease cystic fibrosis, therefore, surrogate channels that can operate independently of CFTR are of interest. This study explores the target receptor(s) for lubiprostone in airway epithelium.

EXPERIMENTAL APPROACH

All experiments were performed on the ventral tracheal epithelium of sheep. Epithelia were used to measure anion secretion from the apical surface as short circuit current or as fluid secretion from individual airway submucosal glands, using an optical method.

KEY RESULTS

The EP₄ antagonists L-161982 and {"type":"entrez-nucleotide","attrs":{"text":"GW627368","term_id":"290498219","term_text":"GW627368"}}GW627368 inhibited short circuit current responses to lubiprostone, while EP_1,2&3 receptor antagonists were without effect. Similarly, lubiprostone induced secretion in airway submucosal glands was inhibited by L-161982. L-161982 effectively competed with lubiprostone with a K_d value of 0.058 µM, close to its value for binding to human EP₄ receptors (0.024 µM). The selective EP₄ agonist L-902688 and lubiprostone behaved similarly with respect to EP₄ receptor antagonists. Results of experiments with H89, a protein kinase A inhibitor, were consistent with lubiprostone acting through a G_s-protein coupled EP₄ receptor/cAMP cascade.

CONCLUSIONS AND IMPLICATIONS

Lubiprostone-induced short-circuit currents and submucosal gland secretions were inhibited by selective EP₄ receptor antagonists. The results suggest EP₄ receptor activation by lubiprostone triggers cAMP production necessary for CFTR activation and the secretory responses, a possibility precluded in CF tissues.     

β-endorphin-sensitive opioid receptors in the rat tail artery

Summary Isolated tail arteries of rats were perfused and field-stimulated every 2 min with 2 pulses at 1 Hz. Different opioid peptides depressed the contractile responses to stimulation; their concentration-response curves showed a maximum at about 40% inhibition. The rank order of potency of the peptides was -endorphin (IC₅₀ = 97 nmol/1) BAM-22P > FK-33824 > DAGO > [d-Ala²,d-Leu⁵]-enkephalin metorphamide > dynorphin A-(1-13) [Met⁵]enkephalin. All these substances have in common a certain activity at opioid -receptors, although the enkephalins are preferential -, and the dynorphins preferential -agonists. However, the selective -agonist [d-Pen²,l-Pen⁵]enkephalin was ineffective at up to 10 mol/l, and the -agonists ethylketocyclazocine and U-50488 acted only at concentrations higher than 3 mol/l. Whereas the effects of -endorphin, DAGO and [d-Ala²,d-Leu⁵]enkephalin could be reduced by the -preferential antagonist naloxone, the effects of ethylketocyclazocine and U-50488 were not changed. The -selective antagonist ICI 174864 did not influence the action of [d-Ala²,d-Leu⁵]enkephalin. Naloxone in a concentration (1 mol/l) which nearly abolished the effect of DAGO 3 mol/l, slightly enhanced responses to stimulation. Neither -endorphin nor DAGO influenced vasoconstriction evoked by the application of noradrenaline or adenosine triphosphate; U-50488 reduced it. In arteries preincubated with [³H]noradrenaline DAGO depressed, whereas naloxone enhanced the tritium overflow and vasoconstriction evoked by field stimulation (0.4 Hz, 24 pulses every 14 min). In addition, naloxone antagonized the effect of DAGO. We suggest that the axon terminals of postganglionic sympathetic neurones in the rat tail artery possess -endorphin-sensitive opioid receptors of the -type. The activation of these receptors by exogenous or endogenous opioids inhibits the release of the neuroeffector transmitter.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 325) Send offprint requests to P. Illes at the above address     

Herpesviral Fcgamma receptors: culprits attenuating antiviral IgG?

Production of IgG in response to virus infection is central to antiviral immune effector functions and a hallmark of B cell memory. Antiviral antibodies (Abs) recognising viral glycoproteins or protein antigen displayed on the surface of virions or virus-infected cells are crucial in rendering the virus noninfectious and in eliminating viruses or infected cells, either acting alone or in conjunction with complement. In many instances, passive transfer of Abs is sufficient to protect from viral infection. Herpesviruses (HV) are equipped with a large array of immunomodulatory functions which increase the efficiency of infection by dampening the antiviral immunity. Members of the alpha- and beta-subfamily of the Herpesviridae are distinct in encoding transmembrane glycoproteins which selectively bind IgG via its Fc domain. The Fc-binding proteins constitute viral Fcgamma receptors (vFcgammaRs) which are expressed on the cell surface of infected cells. Moreover, vFcgammaRs are abundantly incorporated into the envelope of virions. Despite their molecular and structural heterogeneity, the vFcgammaRs generally interfere with IgG-mediated effector functions like antibody (Ab)-dependent cellular cytolysis, complement activation and neutralisation of infectivity of virions. vFcgammaRs may thus contribute to the limited therapeutic potency of antiherpesviral IgG in clinical settings. A detailed molecular understanding of vFcgammaRs opens up the possibility to design recombinant IgG molecules resisting vFcgammaRs. Engineering IgG with a better antiviral efficiency represents a new therapeutic option against herpesviral diseases.