Dopamine activation of the arachidonic acid cascade as a basis for D1/D2 receptor synergism

Understanding the actions of the neurotransmitter dopamine in the brain is important in view of its roles in neuropsychiatric illnesses. Dopamine D1 receptors, which stimulate both adenylyl cyclase and phospholipase C, and D2 receptors, which inhibit them, can nevertheless act synergistically to produce many electrophysiological and behavioral responses. Because this functional synergism can occur at the level of single neurons, another, as yet unidentified, signalling pathway activated by dopamine has been hypothesized. We report here that in Chinese hamster ovary (CHO) cells transfected with the D2 receptor complementary DNA, D2 agonists potently enhanced arachidonic acid release, provided that such release has been initiated by stimulating constitutive purinergic receptors or by increasing intracellular Ca2+. In CHO cells expressed D1 receptors, D1 agonists exert no such effect. When D1 and D2 receptors are coexpressed, however, activation of both subtypes results in a marked synergistic potentiation of arachidonic acid release. The numerous actions of arachidonic acid and its metabolites in neuronal signal transduction suggest that facilitation of its release may be implicated in dopaminergic responses, such as feedback inhibition mediated by D2 autoreceptors, and may constitute a molecular basis for D1/D2 receptor synergism.     

Agonist-independent activation of metabotropic glutamate receptors by the intracellular protein Homer.

G-protein-coupled receptors (GPCRs) transduce signals from extracellular transmitters to the inside of the cell by activating G proteins. Mutation and overexpression of these receptors have revealed that they can reach their active state even in the absence of agonist, as a result of a natural shift in the equilibrium between their inactive and active conformations. Such agonist-independent (constitutive) activity has been observed for the glutamate GPCRs (the metabotropic glutamate receptors mGluR1a and mGluR5) when they are overexpressed in heterologous cells. Here we show that in neurons, the constitutive activity of these receptors is controlled by Homer proteins, which bind directly to the receptors' carboxy-terminal intracellular domains. Disruption of this interaction by mutagenesis or antisense strategies, or expression of endogenous Homer1a (H1a), induces constitutive activity in mGluR1a or mGluR5. Our results show that these glutamate GPCRs can be directly activated by intracellular proteins as well as by agonists.     

瓜氨酸生产相关基因簇在谷氨酸棒杆菌中的组成型表达

构建1种组成型载体并将载体应用在表达瓜氨酸相关基因簇argCJBDF上。通过去除pXMJ19诱导型启动子上游阻遏蛋白lacI基因的方法,构建组成型质粒pXMJ19-lacI,并将谷氨酸棒杆菌中合成瓜氨酸途径的基因簇argCJBDF克隆到改造过的组成型载体中,实现瓜氨酸合成相关基因簇argCJBDF在谷氨酸棒杆菌的组成型表达。结果表明:重组菌在30℃摇瓶发酵72 h后,N-乙酰谷氨酸激酶的酶活达到(0.323±0.015)U/mg,瓜氨酸的产量达到4.33 g/L。成功构建的组成型表达载体,实现了外源基因簇argCJBDF在谷氨酸棒杆菌中的组成型表达。     

Cerebellar GABAA receptor selective for a behavioural alcohol antagonist

Benzodiazepines are widely prescribed anxiolytics and anticonvulsants which bind with high affinity to sites on the GABAA receptor/Cl- channel complex and potentiate the effect of the neurotransmitter GABA (gamma-aminobutyric acid). The heterogeneity of benzodiazepine recognition sites in the central nervous system was revealed by studies showing different classes of GABAA receptor subunits (classes alpha, beta and gamma) and variant subunits in these classes, particularly in the alpha-class. Expression of recombinant subunits produces functional receptors; when certain alpha-variants are coexpressed with beta- and gamma-subunits the resulting receptors have pharmacological properties characteristic of GABAA-benzodiazepine type I or type II receptors. The alpha-variants are differentially expressed in the central nervous system and can be photoaffinity-labelled with benzodiazepines. Here we report a novel alpha-subunit (alpha 6) of cerebellar granule cells. We show that recombinant receptors composed of alpha 6, beta 2 and gamma 2 subunits bind with high affinity to the GABA agonist [3H]muscimol and the benzodiazepine [3H]Ro15-4513 but not the other benzodiazepines or beta-carboniles. The same distinctive pharmacology is observed with GABAA receptors from rat cerebellum immunoprecipitated by an antiserum specific for the alpha 6 subunit. We conclude that this alpha-subunit is part of a cerebellar receptor subtype, selective for Ro15-4513, an antagonist of alcohol-induced motor incoordination and ataxia.     

Multiple-conductance channels activated by excitatory amino acids in cerebellar neurons

In the mammalian central nervous system amino acids such as L-glutamate and L-aspartate are thought to act as fast synaptic transmitters. It has been suggested that at least three pharmacologically-distinguishable types of glutamate receptor occur in central neurons and that these are selectively activated by the glutamate analogues N-methyl-D-aspartate (NMDA), quisqualate and kainate. These three receptor types would be expected to open ion channels with different conductances. Hence if agonists produce similar channel conductances this would suggest they are acting on the same receptor. Another possibility is suggested by experiments on spinal neurons, where GABA (gamma-amino butyric acid) and glycine appear to open different sub-conductance levels of one class of channel while acting on different receptors. By analogy, several types of glutamate receptor could also be linked to a single type of channel with several sub-conductance states. We have examined these possibilities in cerebellar neurons by analysing the single-channel currents activated by L-glutamate, L-aspartate, NMDA, quisqualate and kainate in excised membrane patches. All of these agonists are capable of opening channels with at least five different conductance levels, the largest being about 45-50 pS. NMDA predominantly activated conductance levels above 30 pS while quisqualate and kainate mainly activated ones below 20 pS. The presence of clear transitions between levels favours the idea that the five main levels are all sub-states of the same type of channel.     

Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine

Responses to the excitatory amino acid N-methyl-D-aspartate (NMDA) are markedly potentiated by nanomolar concentrations of glycine. This is due to the action of glycine at a novel strychnine-resistant binding site with an anatomical distribution identical to that for NMDA receptors, suggesting that the NMDA receptor channel complex contains at least two classes of amino-acid recognition site. Antagonists at the glycine-binding site associated with NMDA receptors act as potent non-competitive antagonists, but do not alter the mean open time or conductance, as estimated by fluctuation analysis. The mechanisms by which glycine acts on NMDA receptors are unknown, but single-channel recording experiments show an increase in opening frequency with no change in mean open time or conductance, suggesting that glycine could regulate transitions to states that are intermediate between binding of NMDA receptor agonists and ion-channel gating. It has been suggested that glycine acts as a co-agonist at the NMDA receptor, and that responses to NMDA cannot be obtained in the complete absence of glycine, but in these experiments the response to NMDA was measured at equilibrium, and it is unlikely that sufficient temporal resolution was achieved to detect rapid alterations in receptor gating. Using a fast perfusion system we find that glycine regulates desensitization at NMDA receptors; this has a major effect on the response to NMDA measured at equilibrium, as would occur with slower applications of agonist. Reduction of NMDA receptor desensitization by glycine provides an example of a novel mechanism for regulation of ion-channel activity.     

Ligand binding to the beta-adrenergic receptor involves its rhodopsin-like core

Recently the genes for several hormone receptors that interact with guanine nucleotide binding proteins (G proteins) have been cloned, including the hamster beta 2-adrenergic receptor (beta 2AR), a human beta AR, the turkey erythrocyte beta AR and the porcine muscarinic acetylcholine receptor (MAR). All these receptors share some amino-acid homology with rhodopsin, particularly in 7 hydrophobic stretches of residues that are believed to represent transmembrane helices. To determine whether differences in ligand specificity result from the divergence in the sequences of the hydrophilic regions of these receptors, we have expressed in mammalian cells genes for the wild-type hamster and human beta AR proteins, and a series of deletion mutant genes of the hamster beta 2AR. The pharmacology of the expressed receptors indicates that most of the hydrophilic residues are not directly involved in the binding of agonists or antagonists to the receptor. In addition, we have identified a mutant receptor that has high agonist affinity but does not couple to adenylate cyclase.     

Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1.

Dopamine receptors belong to a superfamily of receptors that exert their biological effects through guanine nucleotide-binding (G) proteins. Two main dopamine receptor subtypes have been identified, D1 and D2, which differ in their pharmacological and biochemical characteristics. D1 stimulates adenylyl cyclase activity, whereas D2 inhibits it. Both receptors are primary targets for drugs used to treat many psychomotor diseases, including Parkinson's disease and schizophrenia. Whereas the dopamine D1 receptor has been cloned, biochemical and behavioural data indicate that dopamine D1-like receptors exist which either are not linked to adenylyl cyclase or display different pharmacological activities. We report here the cloning of a gene encoding a 477-amino-acid protein with strong homology to the cloned D1 receptor. The receptor, called D5, binds drugs with a pharmacological profile similar to that of the cloned D1 receptor, but displays a 10-fold higher affinity for the endogenous agonist, dopamine. As with D1, the dopamine D5 receptor stimulates adenylyl cyclase activity. Northern blot and in situ hybridization analyses reveal that the receptor is neuron-specific, localized primarily within limbic regions of the brain; no messenger RNA was detected in kidney, liver, heart or parathyroid gland. The existence of a dopamine D1-like receptor with these characteristics had not been predicted and may represent an alternative pathway for dopamine-mediated events and regulation of D2 receptor activity.     

Movement of 'gating charge' is coupled to ligand binding in a G-protein-coupled receptor

Activation by agonist binding of G-protein-coupled receptors (GPCRs) controls most signal transduction processes. Although these receptors span the cell membrane, they are not considered to be voltage sensitive. Recently it was shown that both the activity of GPCRs and their affinity towards agonists are regulated by membrane potential. However, it remains unclear whether GPCRs intrinsically respond to changes in membrane potential. Here we show that two prototypical GPCRs, the m2 and m1 muscarinic receptors (m2R and m1R), display charge-movement-associated currents analogous to 'gating currents' of voltage-gated channels. The gating charge-voltage relationship of m2R correlates well with the voltage dependence of the affinity of the receptor for acetylcholine. The loop that couples m2R and m1R to their G protein has a crucial function in coupling voltage sensing to agonist-binding affinity. Our data strongly indicate that GPCRs serve as sensors for both transmembrane potential and external chemical signals.     

Expression of functional interleukin-2 receptors in human light chain/Tac transgenic mice

The growth of mature T lymphocytes is regulated by interaction between interleukin-2 (IL-2) and its receptor. Three distinct binding sites for IL-2, namely low- (Kd 10 nM), intermediate- (Kd 100 pM) and high- (Kd 10 pM) affinity sites, have been found on human and primate T lymphocytes. Chemical crosslinking of labelled IL-2 to human T cells shows that two polypeptide chains, p55 (L chain) and p75 (H chain), bind IL-2 with low and intermediate affinities respectively. The high-affinity binding was shown to arise from ternary complex formation of IL-2, L and H chains. Construction of mutants of the L-chain complementary DNA indicated that the L chain is not directly involved in growth signal transduction. Nevertheless, expression of the IL-2 receptor L chain is tightly regulated by antigen or mitogen stimulation. To investigate the L chain function, we have produced transgenic mice using human L-chain cDNA of the IL-2 receptor under the control of a constitutive promoter. Studies on the L-chain transgenic mice showed that functionally active IL-2 receptors with high affinity were expressed on unstimulated spleen and thymus cells. The results indicate that the H chain of the IL-2 receptor is constitutively expressed in T cells.     

Proton inhibition of N-methyl-D-aspartate receptors in cerebellar neurons.

Mammalian neurons contain at least three types of excitatory amino-acid receptors, selectively activated by N-methyl-D-aspartate (NMDA) or aspartate, (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate ((S)-AMPA) and kainate. An important aspect of NMDA receptors is their regulation by a variety of factors such as glycine, Mg2+ and Zn2+ that are present in vivo. We show here that NMDA receptor responses are selectively inhibited by protons, with a 50% inhibitory concentration (IC50) that is close to physiological pH, implying that NMDA receptors are not fully active under normal conditions. (S)-AMPA and kainate responses remain unchanged at similar pH levels. Proton inhibition is voltage-insensitive and does not result either from fast channel block, a change in channel conductance, or an increase in the 50% excitatory concentration (EC50) of aspartate/NMDA or glycine. Instead, protons seem to decrease markedly the opening frequency of 30-50 pS NMDA channels, and reduce the relative proportion of longer bursts. This feature of NMDA receptors could be relevant to neurotoxic activation of NMDA receptors during ischaemia, as well as to seizure generation, as extracellular proton changes occur during both of these pathological situations. Furthermore, these results may have implications for normal NMDA receptor function as transient changes in extracellular protons occur during synaptic transmission.     

组胺能与γ-氨基丁酸能神经传递在间位核神经元信息加工中的作用(英文)

小脑间位核(interpositus nucleus,IN)主要接受γ-氨基丁酸(GABA)能纤维支配,同时接受组胺能纤维的调节.本研究在小脑脑片上研究了GABA和组胺对单个IN神经元电活动的共同作用.持续灌流组胺或同时施加组胺和GABA,81.2%(69/85)神经元,GABA及其激动剂的效应都被组胺削弱(持续灌流n=33;同时施加n=36).这种削弱效应能够被组胺H2受体阻断剂ranitidine(n=10)和PKA抑制剂H-89阻断(n=8),fors-kolin模拟组胺的效应(n=9).结果表明组胺和GABA对IN神经元的电活动具有交互调节作用:通过激活H2受体偶联的G-protein-AC-PKA信号通路,磷酸化GABAB和GABAA受体,降低受体功能.推测受体间的对话的工作模式,可能是整个大脑神经元活动的某些药理作用和生理活动调节的基础;如果对话紊乱,可能导致大脑功能障碍.     

Auto-inhibition of brain histamine release mediated by a novel class (H3) of histamine receptor

Although histaminergic neurones have not yet been histochemically visualized, there is little doubt that histamine (HA) has a neurotransmitter role in the invertebrate and mammalian central nervous system. For example, a combination of biochemical, electrophysiological and lesion studies in rats have shown that histamine is synthesized in and released from a discrete set of neurones ascending through the lateral hypothalamic area and widely projecting in the telencephalon. Histamine acts on target cells in mammalian brain via stimulation of two classes of receptor (H1 and H2) previously characterized in peripheral organs and probably uses Ca2+ and cyclic AMP, respectively, as second messengers. It is well established that several neurotransmitters affect neuronal activity in the central nervous system through stimulation not only of postsynaptic receptors, but also of receptors located presynaptically which often display distinct pharmacological specificity and by which they may control their own release. Such 'autoreceptors' have been demonstrated (or postulated) in the case of noradrenaline, dopamine, serotonin, acetylcholine and gamma-aminobutyric acid (GABA) neurones but have never been demonstrated for histamine. We show here that histamine inhibits its own release from depolarized slices of rat cerebral cortex, an action apparently mediated by a class of receptor (H3) pharmacologically distinct from those previously characterized, that is, the H1 and H2 receptors.     

Glutamate-induced long-term potentiation of the frequency of miniature synaptic currents in cultured hippocampal neurons.

Glutamate application at synapses between hippocampal neurons in culture produces long-term potentiation of the frequency of spontaneous miniature synaptic currents, together with long-term potentiation of evoked synaptic currents. The mini frequency potentiation is initiated postsynaptically and requires activity of NMDA receptors. Although the frequency of unitary quantal responses increases strongly, their amplitude remains little changed with potentiation. Tests of postsynaptic responsiveness rule out recruitment of latent glutamate receptor clusters. Thus, postsynaptic induction can lead to enhancement of presynaptic transmitter release. The sustained potentiation of mini frequency is expressed even in the absence of Ca2+ entry into presynaptic terminals.     

Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development

How environmental cues regulate adult stem cell and cancer cell activity through surface receptors is poorly understood. Angiopoietin-like proteins (ANGPTLs), a family of seven secreted glycoproteins, are known to support the activity of haematopoietic stem cells (HSCs) in vitro and in vivo. ANGPTLs also have important roles in lipid metabolism, angiogenesis and inflammation, but were considered 'orphan ligands' because no receptors were identified. Here we show that the immune-inhibitory receptor human leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse orthologue paired immunoglobulin-like receptor (PIRB) are receptors for several ANGPTLs. LILRB2 and PIRB are expressed on human and mouse HSCs, respectively, and the binding of ANGPTLs to these receptors supported ex vivo expansion of HSCs. In mouse transplantation acute myeloid leukaemia models, a deficiency in intracellular signalling of PIRB resulted in increased differentiation of leukaemia cells, revealing that PIRB supports leukaemia development. Our study indicates an unexpected functional significance of classical immune-inhibitory receptors in maintenance of stemness of normal adult stem cells and in support of cancer development.     

Multiple conductance channels in type-2 cerebellar astrocytes activated by excitatory amino acids

L-GLUTAMATE and L-aspartate are thought to have a widespread function as synaptic transmitters in the mammalian central nervous system and there are at least three types of neuronal glutamate receptors, which can be activated by the selective agonists N-methyl-D-aspartate (NMDA), quisqualate and kainate. Recent experiments indicate that glutamate receptors also occur in astrocytes. We have used patch-clamp methods to determine whether one type of macroglial cell, the type-2 astrocyte, possesses glutamate receptors, as previously proposed from neurochemical studies. We find that glutamate and related amino acids can evoke whole-cell and single-channel currents in type-2 astrocytes from rat cerebellum. Although these cells are found mainly in white matter, where neurotransmission does not occur, their processes are closely associated with axons at nodes of Ranvier, suggesting that such receptors are involved in neuronal-glial signalling at the node. Our experiments show that glial cells possess quisqualate- and kainate-receptor channels but lack receptors for NMDA. Interestingly, these glutamate channels exhibit multiple conductance levels that are similar in amplitude to the neuronal glutamate channels.     

5-HT3 receptors mediate inhibition of acetylcholine release in cortical tissue

The release of cerebral acetylcholine from terminals in the cerebral cortex has been shown to be regulated by 5-hydroxytryptamine (5-HT) but it is not known which subtype of the 5-HT receptor is involved. 5-HT receptor agonists increase acetylcholine levels in vivo, indicating a reduced turnover, and reduce release of acetylcholine from striatal slices in vitro. Depleting 5-HT by inhibiting synthesis or by destroying the neurons containing 5-HT potentiates acetylcholine release, and increases acetylcholine turnover in the cerebral cortex and hippocampus. Selective antagonists for the 5-HT3 receptor subtypes which seem to have effects on mood and activity may exert their effect through the regulation of acetylcholine release in the cortex and limbic system. Radioligand binding studies show a high density of 5-HT3 receptors in the cholinergic-rich entorhinal cortex and we provide evidence that a reduction in cortical cholinergic function can be effected in vitro by 5-HT3 receptors.