Identification of presynaptic serotonin autoreceptors using a new ligand: 3H-PAT

Binding studies with appropriate labelled ligands have revealed the existence of two types of serotonin (5-HT) receptor, 5-HT1 and 5-HT2, in the central nervous system of mammals. The 5-HT1 type is characterized by a higher affinity for agonists than for antagonists, whereas the 5-HT2 type binds preferentially to antagonists. However, neither of these receptor types apparently corresponds to the presynaptic autoreceptor controlling 5-HT release. In an attempt to identify the presynaptic autoreceptor directly, we synthesized the tritiated derivative of 8-hydroxy-2-(di-n-propylamino) tetralin (PAT), a new tetralin derivative with potent 5-HT agonist properties and carried out binding studies with rat brain membranes. As we report here, in the hippocampus, the properties of 3H-PAT binding sites correspond closely to those of 5-HT1 sites. In contrast, in the striatum, 3H-PAT binding sites exhibit a subcellular distribution and pharmacological characteristics usually associated with presynaptic autoreceptors. Furthermore, a marked loss of 3H-PAT binding sites occurs in the striatum (but not in the hippocampus) after the selective degeneration of serotoninergic fibres in 5,7-hydroxytryptamine (5,7-HT)-treated rats. Conversely, the sprouting of additional 5-HT terminals in the brain stem of adult rats treated at birth with 5,7-HT is associated with an increased density of 3H-PAT binding sites in this region. 3H-PAT thus seems to be a useful ligand for studying the biochemical and pharmacological characteristics of presynaptic autoreceptors in selected regions of rat brain.     

Localization of D-2 dopamine receptors to intrinsic striatal neurones by quantitative autoradiography

Recent work with positron emission and single photon emission computed tomography has demonstrated the feasibility of studying striatal dopamine receptors in the living human brain. For the proper interpretation of these studies in normal and diseased states, the cellular localization of these receptors must be definitively established. It has been claimed, on the basis of receptor binding studies with tissue homogenates in rats, that 30-50% of striatal D-2 dopamine receptors are located on axons or terminals of the corticostriatal pathway. This finding has been incorporated into major reviews and classifications of dopamine receptors. The recent development of quantitative autoradiographic methods for diffusible ligands has facilitated the study of neurotransmitter receptors in cytoarchitechtonically intact tissue. Because this technique provides the necessary anatomic resolution that is lacking in homogenate binding studies, we have used it to re-examine the localization of striatal dopamine receptors. Here we present evidence that D-2 receptors are located exclusively on kainic acid-sensitive intrinsic neuronal elements in the striatum. We report that discrete cortical ablation does not alter 3H-spiperone binding to rat striatum and thus our results do not support the existence of D-2 dopamine receptors on the terminals of the corticostriatal pathway.     

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.     

Differential regulation of the alpha 2-adrenergic receptor by Na+ and guanine nucleotides

Many hormones interact with receptors which stimulate the enzyme adenylate cyclase. Less well characterized ar those receptors which mediate an inhibition of adenylate cyclase activity. However, guanine nucleotides are clearly important in the regulation of both stimulatory and inhibitory receptors. Monovalent cations, notably Na+, regulate many inhibitory receptor systems but apparently not stimulatory receptors. We investigate here the effects of Na+ and guanine nucleotides on the adenylate cyclase-coupled inhibitory alpha 2-adrenergic receptor of the rabbit platelet. Computer modelling of adrenaline competition curves with 3H-dihydroergocryptine (3H-DHE) indicates that adrenaline induces two distinct affinity states of the alpha 2 receptor--one of higher (alpha 2H) and the other of lower (alpha 2L) affinity. Guanyl-5'-yl-imidodiphosphate (Gpp(NH)p) seems to reduce adrenaline affinity to converting the high-affinity state into the low-affinity form of the receptor. In contrast, Na+ reduces adrenaline affinity at both the high- and low-affinity states of the alpha 2 receptor while preserving receptor heterogeneity. Thus, guanine nucleotides and Na+ differ in the manner by which each reduces agonist affinity for the alpha 2-adrenergic receptor.     

Possible regulatory function of acetylcholine receptor in maintenance of retinotectal synapses.

alpha-Neurotoxins bind to cholinergic receptor, block transmission, and induce sprouting of retinal terminals in the toad tectum. New connections retain an orderliness that suggests a selective affinity between presynaptic terminals. The results suggest that postsynaptic cells exert a control, associated with receptors, on the growth of presynaptic terminals and on the maintenance of their synaptic connections.     

A dopamine- and cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions

Several mammalian neurotransmitter candidates, for example, serotonin, dopamine and noradrenaline, may exert some of their synaptic effects by regulating protein phosphorylation systems. Comparison of the regional distribution of brain phosphoproteins with neurotransmitter systems may help to identify the specific phosphoproteins involved in the functions of particular neurotransmitters. Here we report the association of one such phosphoprotein with the dopamine pathways in brain. This protein, of apparent molecular weight (MW) 32,000 (32K), seems to be present only in nervous tissue. Its regional distribution within the brain is very similar to the pattern of dopamine-containing nerve terminals; more specifically, the protein appears to be enriched in those dopaminoceptive neurones which possess D-1 receptors (dopamine receptors coupled to adenylate cyclase). The state of phosphorylation of the protein in these dopaminoceptive neurones can be regulated by both dopamine and cyclic AMP. These results suggest that the phosphoprotein may mediate certain of the trans-synaptic effects of dopamine acting on dopaminoceptive neurones.     

Cloning of the gene and cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin

The adenylate cyclase system, which consists of a catalytic moiety and regulatory guanine nucleotide-binding proteins, provides the effector mechanism for the intracellular actions of many hormones and drugs. The tissue specificity of the system is determined by the particular receptors that a cell expresses. Of the many receptors known to modulate adenylate cyclase activity, the best characterized and one of the most pharmacologically important is the beta-adrenergic receptor (beta AR). The pharmacologically distinguishable subtypes of the beta-adrenergic receptor, beta 1 and beta 2 receptors, stimulate adenylate cyclase on binding specific catecholamines. Recently, the avian erythrocyte beta 1, the amphibian erythrocyte beta 2 and the mammalian lung beta 2 receptors have been purified to homogeneity and demonstrated to retain binding activity in detergent-solubilized form. Moreover, the beta-adrenergic receptor has been reconstituted with the other components of the adenylate cyclase system in vitro, thus making this hormone receptor particularly attractive for studies of the mechanism of receptor action. This situation is in contrast to that for the receptors for growth factors and insulin, where the primary biochemical effectors of receptor action are unknown. Here, we report the cloning of the gene and cDNA for the mammalian beta 2AR. Analysis of the amino-acid sequence predicted for the beta AR indicates significant amino-acid homology with bovine rhodopsin and suggests that, like rhodopsin, beta AR possesses multiple membrane-spanning regions.     

Pertussis toxin reverses adenosine inhibition of neuronal glutamate release

Adenosine and its analogues are potent inhibitors of synaptic activity in the central and peripheral nervous system. In the central nervous system (CNS), this appears to arise primarily by inhibition of presynaptic release of transmitters, including glutamate, which is possibly the major excitatory transmitter in the brain. In addition, postsynaptic effects of adenosine have been reported which would also serve to reduce neurotransmission. The mechanism by which adenosine inhibits CNS neurotransmission is unknown, although it appears to exert its effect via an A1 receptor which in some systems is negatively coupled to adenylate cyclase. In an attempt to elucidate the mechanism of inhibition, we have examined the effect of pertussis toxin (PTX) on the ability of the stable adenosine analogue (-)phenylisopropyladenosine (PIA) to inhibit glutamate release from cerebellar neurones maintained in primary culture. PTX, by ADP-ribosylating the nucleotide-binding protein Ni, prevents coupling of inhibitory receptors such as the A1 receptor to adenylate cyclase. As reported here, we found that PTX, as well as preventing inhibition of adenylate cyclase by PIA, also converts the PIA-induced inhibition of glutamate release to a stimulation. Our results suggest strongly that purinergic inhibitory modulation of transmitter release occurs by inhibition of adenylate cyclase.     

Axoplasmic transport of muscarinic receptors

The reality of axoplasmic transport is widely accepted; various neutrotransmitters, enzymes, labelled proteins and peptides are known to move rapidly along the axons of different nerve fibres. In the terminals of sympathetic nerves, noradrenaline release is controlled by various regulatory mechanisms which imply the occurrence of presynaptic receptors. In this regard, there is considerable indirect physiological evidence for the existence of muscarinic cholinergic receptors in the sympathetic nerve endings; the stimulation by acetylcholine of such presynaptic receptors elicits an inhibitory effect on noradrenaline release. We not provide direct biochemical evidence for the occurrence in dog splenic nerve of muscarinic receptors which seem to move along the axon as suggested by their rapid accumulation on either side of a ligature.     

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.     

Fundamental difference between the molecular interactions of agonists and antagonists with the beta-adrenergic receptor.

Antagonist binding to the beta-adrenergic receptor is largely entropy driven, with only a small enthalpy component. The binding of agonists, on the other hand, is associated with a large decrease in enthalpy which permits a highly unfavourable decrease in entropy. The thermodynamic differences between the binding of agonists and antagonists may provide new insights into the molecular basis for hormone stimulation of adenylate cyclase activity.     

Abolition of the expression of inhibitory guanine nucleotide regulatory protein Gi activity in diabetes

Many cell-surface receptors for hormones appear to exert their effects on target cells by interacting with specific guanine nucleotide binding regulatory proteins (G-proteins) which couple receptors to their second-messenger signal generation systems. A common intracellular second messenger, which is used by many hormones, is cyclic AMP. This is produced by adenylate cyclase, whose activity is controlled by two G-proteins, Gs which mediates stimulatory effects and Gi inhibitory effects on adenylate cyclase activity. In liver, the hormone glucagon increases intracellular cAMP concentrations by activating adenylate cyclase by a Gs-mediated process. This effect of glucagon is antagonised by the hormone insulin, although the molecular mechanism by which insulin elicits its actions is obscure. However, insulin receptors exhibit a tyrosyl kinase activity and appear to interact with G-proteins, perhaps by causing phosphorylation of them. In type I diabetes, circulating insulin levels are abnormally low, giving rise to gross perturbations of metabolism as well as to a variety of complications such as ionic disturbances, neuropathies of the nervous system, respiratory and cardiovascular aberrations and predisposition to infection. We show here that experimentally-induced type I diabetes leads to the loss of expression of Gi in rat liver. As it has been suggested that Gi may couple receptors to K+-channels as well as mediating the inhibition of adenylate cyclase, aberrations in the control of expression of this key regulatory protein in type I diabetes may be expected to lead to pleiotropic effects.     

Protection of substantia nigra from MPP+ neurotoxicity by N-methyl-D-aspartate antagonists.

Intake of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) leads to symptoms of Parkinson's disease and produces degeneration of nigrostriatal dopaminergic neurons in humans, giving rise to the hypothesis that this disorder may be caused by endogenous or environmental toxins. Excitation mediated by dicarboxylic amino acids such as L-glutamate or L-aspartate, has been claimed to be involved in pathogenesis of neurodegenerative disorders. We therefore sought to determine whether antagonists active at the NMDA or quisqualate subtypes of L-glutamate receptors prevent toxicity of either MPP+ (1-methyl-4-phenyl-pyridinium ion, the active metabolite of MPTP) or the selective dopaminergic neurotoxin 6-OHDA in the rat substantia nigra pars compacta. We report here that certain selective NMDA antagonists (AP7, CPP, MK-801), but not the preferential quisqualate antagonists CNQX and NBQX, provided short-term (up to 24 h) protection against MPP+ toxicity when coadministered into the substantia nigra. Systemic administration of CPP or MK-801 also offered temporary protection for up to 4 h against MPP+ toxicity. Repeated systemic administration of either compound prolonged protection against MPP+ challenge. Repeated administration for at least 24 h also led to permanent protection, still evident 7 days after intranigral administration of MPP+.     

Glycogenolysis induced by serotonin in brain: identification of a new class of receptor

Serotonin-containing neurones in brain have been proposed to have a role in the control of physiological mechanisms such as sleep, thermoregulation, pain perception and endocrine secretions as well as in the physiopathology of migraine or depressive illness. One difficulty in testing these possibilities lies in the scarcity of pharmacological agents able to interact selectively with the probably multiple classes of serotonin receptors in the central nervous system. Development of such agents would be facilitated by simple in vitro models in which biological responses to serotonin in mammalian brain could be quantified. Thus a serotonin-sensitive adenylate cyclase has been characterized in rat brain, but the response to serotonin is weak in newborn and practically absent in adult animals. In addition, two pharmacologically distinct classes of serotoninergic binding site have been identified using 3H-serotonin and 3H-spiperone as ligands, but their identification as receptors remains to be established. More recently, serotonin has been shown to stimulate phosphorylation of a neuronal protein in slices from the facial motor nucleus, although the receptors mediating this action were not characterized. We now report that serotonin stimulates glycogen hydrolysis in slices of cerebral cortex, that this action is mediated by a novel class of receptors and that tricyclic antidepressants are among the best competitive antagonists of the indolamine.     

Pure beta-adrenergic receptor: the single polypeptide confers catecholamine responsiveness to adenylate cyclase

The beta-adrenergic receptor binding subunits from frog erythrocytes, hamster lung and guinea pig lung have been purified to apparent homogeneity and in all cases reside on a single polypeptide. Insertion of the pure receptors into phospholipid vesicles and subsequent fusion of these vesicles with a receptor-deficient cell conveys beta-adrenergic responsiveness to the adenylate cyclase system of the acceptor cell. Such responsiveness is linearly dependent on the amount of receptor used in the fusion experiments and is independent of the receptor source. Moreover, this responsiveness displays appropriate beta-adrenergic specificity. These results indicate that the beta-adrenergic receptor polypeptide contains both the ligand binding site and the site responsible for mediating stimulation of adenylate cyclase activity, presumably via interaction with the guanine nucleotide regulatory protein.     

Stimulation of specific GTP binding and hydrolysis activities in lymphocyte membrane by interleukin-2

Interleukin-2 (IL-2) is a polypeptide growth factor which stimulates the proliferation and differentiation of T lymphocytes. The receptor for IL-2 is expressed on activated T lymphocytes, cloned IL-2 dependent cells and several other cell types. Analysis of the primary structure and of immune-precipitated receptor suggests that this molecule has no intrinsic signal transduction function, unlike other growth factors. IL-2 interaction with a high affinity receptor has been shown, however, to activate the calcium/phospholipid-dependent protein kinase C (PK-C) presumably via phosphoinositide hydrolysis. Members of a family of closely related guanine nucleotide binding proteins (G proteins) regulate a diverse group of metabolic events. Two of them, Gs and Gi, stimulate and inhibit adenylate cyclase activity respectively, and other G proteins are involved in diverse signal transduction system. Another member, Go, has no known function and activation of phospholipase C has been attributed to the action of an unidentified G protein, Gp. Since it has been observed that IL-2 inhibits the catalytic activity of adenylate cyclase and that agents such as PGE2 which stimulate adenylate cyclase activity inhibit the lymphoproliferative response to IL-2, association of GTP binding proteins with IL-2 signal transduction was investigated. In this report we describe for the first time the participation of a GTP binding protein in the action of a polypeptide growth factor, interleukin-2.     

青霉素惊厥与脑中GABA_A和GABA_B受体亲和力的关系

脑室微量注射青霉素（１１．９ｍｇ·ｍｌ－１，１５μｌ）制作小白鼠惊厥模型；并以同位素示踪法研究大脑皮层、小脑、海马、下丘脑四个脑区ＧＡＢＡＡ和ＧＡＢＡＢ受体亲和力的变化。结果显示，青霉素惊厥时大脑皮层和小脑ＧＡＢＡＡ受体亲和力显著减弱，而海马、下丘脑ＧＡＢＡＡ受体亲和力无变化；青霉素惊厥使四个脑区中ＧＡＢＡＢ受体均显著下降。提示，除了海马和下丘脑的ＧＡＢＡＡ受体以外，四个脑区的ＧＡＢＡＡ和ＧＡＢＡＢ受体均参与了青霉素的致惊厥过程。青霉素可能通过竞争内源性ＧＡＢＡ与ＧＡＢＡＡ和ＧＡＢＡＢ受体的结合，阻断了ＧＡＢＡ介导的突触前和突触后抑制效应并增加了兴奋性递质的释放，显示了惊厥效应。     

Molecular heterogeneity of benzodiazepine receptors

Benzodiazepines exhibit reversible, stereospecific high affinity binding to mammalian brain membranes, and the respective binding sites for 3H-flunitrazepam represent pharmacologically and clinically relevant receptors for benzodiazepines. Recently it has been demonstrated that reversibly bound 3H-flunitrazepam becomes irreversibly attached to a specific membrane protein with apparent molecular weight of 50,000 when incubations are performed in the presence of UV light. Irreversible binding of 3H-flunitrazepam to this protein had pharmacological properties similar to reversible benzodiazepine receptor binding, indicating that 3H-flunitrazepam is a photoaffinity label for the benzodiazepine receptor. Using irreversible binding of 3H-flunitrazepam and subsequent electrophoretic separation of the labelled proteins in SDS-gels followed by fluorography, we found that in hippocampus and several other brain regions at least two different types of benzodiazepine receptors exist. Each seems to be associated with a gamma-aminobutyric acid (GABA) receptor.     

Identification and distribution of 5-HT3 receptors in rat brain using radioligand binding

Functional serotonin (5-hydroxytryptamine, 5-HT) receptors have been divided into three subtypes: 5-HT1-like, 5-HT2 and 5-HT3 (ref. 1). Brain binding sites have been identified for both the 5-HT1 and 5-HT2 subtypes. Receptors of the 5-HT3 type have been characterized on isolated peripheral tissue models such as the rat vagus nerve, guinea-pig ileum and isolated rabbit heart. Using these models, selective 5-HT3 receptor antagonists such as MDL 72222 (ref. 5), ICS 205-930 (ref. 6), GR38032F (ref. 7) and BRL 43694 (ref. 8) have been developed. Recently, GR38032F, MDL 72222 and ICS 205-930 have been shown to have behavioural effects in rodents and primates that undoubtedly reflect an action in the central nervous system (refs 9-11 and unpublished observations), suggesting the existence of 5-HT3 receptors in the brain. Here we report direct evidence for the existence of 5-HT3 receptors in rat brain tissue and their distribution, based on high affinity binding of the potent 5-HT3 receptor antagonist 3H-GR65630 to homogenates of rat entorhinal cortex. Selective 5-HT3 receptor antagonists and agonists inhibited binding of 3H-GR65630 with high affinities which correlated well with their actions on the rat isolated vagus nerve. Binding was differentially distributed throughout the brain with high concentrations in cortical and limbic areas.     

Opioids block long-term potentiation of inhibitory synapses

Excitatory brain synapses are strengthened or weakened in response to specific patterns of synaptic activation, and these changes in synaptic strength are thought to underlie persistent pathologies such as drug addiction, as well as learning. In contrast, there are few examples of synaptic plasticity of inhibitory GABA (gamma-aminobutyric acid)-releasing synapses. Here we report long-term potentiation of GABA(A)-mediated synaptic transmission (LTP(GABA)) onto dopamine neurons of the rat brain ventral tegmental area, a region required for the development of drug addiction. This novel form of LTP is heterosynaptic, requiring postsynaptic NMDA (N-methyl-d-aspartate) receptor activation at glutamate synapses, but resulting from increased GABA release at neighbouring inhibitory nerve terminals. NMDA receptor activation produces nitric oxide, a retrograde signal released from the postsynaptic dopamine neuron. Nitric oxide initiates LTP(GABA) by activating guanylate cyclase in GABA-releasing nerve terminals. Exposure to morphine both in vitro and in vivo prevents LTP(GABA). Whereas brief treatment with morphine in vitro blocks LTP(GABA) by inhibiting presynaptic glutamate release, in vivo exposure to morphine persistently interrupts signalling from nitric oxide to guanylate cyclase. These neuroadaptations to opioid drugs might contribute to early stages of addiction, and may potentially be exploited therapeutically using drugs targeting GABA(A) receptors.