Receptors In Mammalian Brain Research Articles

Neosurugatoxin: CNS acetylcholine receptors and luteinizing hormone secretion in ovariectomized rats

Neosurugatoxin, a neurotoxin isolated from the Japanese ivory shell, inhibits ganglionic nicotinic acetylcholine receptors but not skeletal muscle nicotinic acetylcholine receptors. It has also been reported to inhibit ( 3H) L-nicotine binding to high-affinity agonist acetylcholine receptors in rat brain membrane preparations. In the present study, 10 −5 M neosurugatoxin inhibited the in vitro binding of ( 3H) L-nicotine to the medial habenular nucleus of frozen, coronal sections of rat brain as did 10 −5 M cytisine or nicotine and 10 −4 M dihydro-beta-erythroidine. Neosurugatoxin did not inhibit ( 125I) α-bungarotoxin binding to hypothalamic synaptosomal preparations or to frozen, coronal sections of rat brain. Injection of neosurugatoxin into the third ventricles of ovariectomized rats resulted in a significant decrease in the frequency of pulses of luteinizing hormone (LH) secretion but had no effect on the amplitude of pulses. A low dose (1 μg/injection) of the nicotinic acetylcholine agent cytisine injected into the third ventricle had no significant effect on pulsatile LH secretion. Coadministration of cytisine could block the inhibitory effect of neosurugatoxin on LH secretion. It is suggested that neosurugatoxin is a useful antagonist to study the biological roles of a specific subclass of nicotinic acetylcholine receptors in mammalian brain and reproductive neuroendocrine functions.

Receptor mediation of the discriminative stimulus properties of phencyclidine and sigma-opioid agonists.

Evidence has accumulated that the discriminative stimulus effects of phencyclidine (PCP) may be transduced by a specific receptor in mammalian brain. Two major lines of evidence support this hypothesis. One is the structure-activity correlation that arylcyclohexylamine analogs of PCP have for PCP-like discriminative effects and displacement of [3H]PCP binding. The other, even stronger, evidence is that representatives of some nonarylcyclohexylamine classes of drugs are both generalized from PCP and have activity at PCP-binding sites. These include the psychotomimetic sigma-agonist opioids such as (+)-N-allylnormetazocine (NANM), the 1,3-substituted dioxolanes dexoxadrol and etoxadrol and some benz(f)isoquinolines. Early evidence suggested that there may be a complete overlap in the discriminative effects and receptor systems for PCP and sigma-agonists, and there continues to be evidence to support the commonality of these drug groups. On the other hand, binding studies with radiolabeled sigma agonists have revealed a non-PCP site. The role of this site in the behavioral actions of sigma-agonists is at present unknown. PCP analogs and other PCP-like drugs also can function as reinforcers for self-administration behavior, suggesting that the same cellular mechanisms may be responsible for both discriminative and reinforcing stimulus effects of these drugs. PCP has been shown to block many of the in vitro and in vivo effects of N-methyl-D-aspartate (NMDA), a putative specific agonist for a subtype of excitatory amino acid receptor. Recent evidence that NMDA antagonists are generalized from PCP in rats and pigeons provides evidence that modification of excitatory amino neurotransmission may be a physiological function of the PCP receptor and that this receptor complex may be involved in PCP's discriminative stimulus effects.

Immunohistochemical localization of a neuronal nicotinic acetylcholine receptor in mammalian brain.

A monoclonal antibody generated against purified acetylcholine receptor from Torpedo electric organ was used to immunohistochemically localize a neuronal nicotinic acetylcholine receptor. Regions of the rat brain stained with this antibody paralleled those areas of the brain exhibiting [3H]nicotine binding sites and corresponded to areas in which mRNAs encoding for alpha subunits of the neuronal nicotinic acetylcholine receptor are present. Thus, the anteroventral thalamus, cortex, hippocampus, medial habenula, interpeduncular nucleus, and substantia nigra/ventral tegmental area exhibited significant immunoreactivity. Neurons of the medial habenula and substantia nigra were densely stained, and processes were prominently delineated. Furthermore, in the projection areas of the medial habenula (interpeduncular nucleus and median raphe) axons were strongly immunoreactive and were distributed to distinct subdivisions of the target sites. The present data suggest that there are several discrete neuronal systems in which nicotinic acetylcholine receptors have functional importance. These immunohistochemical studies delineate at the single-cell level the localization within the mammalian central nervous system of certain nicotinic acetylcholine receptors.

Photoaffinity labeling of the β-adrenergic receptor in synaptic membranes of rat cerebral cortex and cerebellum

The β-adrenergic receptor polypeptides in synaptic membranes of rat cerebral cortex and cerebellum have been identified using the photoaffinity label[ 125I]iodoazidobenzylpindolol. The major receptor polypeptides possess apparent molecular weights of 62, 000 and 49,000 (cerebral cortex) or 59,000 and 46,000 (cerebellum). Treatment of the membranes with endoglycosidase F caused the receptor polypeptides from both tissues to exhibit lower apparent molecular weight (51,000) on sodium dodecyl sulfate polyacrylamide gels, indicating that β-adrenergic receptors in mammalian brain are glycoproteins as are the receptors in peripheral tissues.

O-258 - Phorbol ester receptors in mammalian brain: characterization, localization and target size

O-258 - Phorbol ester receptors in mammalian brain: characterization, localization and target size

Adenosine A1 receptors in mammalian brain: Species differences in their interactions with agonists and antagonists

AbstractThe binding of the selective ligand [3H]cyclohexyladenosine (CHA) to adenosine A1 receptors was studied in brain membrane from six species; cow, rabbit, rat mouse, human, and guinea pig. Saturation analysis gave evidence for a single binding site in each species. The number of binding sites (2.6–4.7 pmol/mg protein), based on saturation analysis, was virtually identical in each species. The Kd values were, however, different; whereas rat, rabbit, and mouse A1 receptors had similar affinities (Kd=1–2 nM), the binding site in bovine tissue had three to four times greater affinity (Kd=0.59 nM). Adenosine binding sites in human and guinea pig brain had two to three times less affinity, with Kd values 3.7 and 6.6 nM, respectively, than those in rat, rabbit, and mouse brain. Examination of the binding of five agonists, the R and S diastereomers of N6‐phenylisopropropyladenosine (PIA), CHA, 5′N‐ethylcarboxamido adenosine (NECA), and 2‐chloroadenosine, and of five xanthine antagonists, PACPX [1,3,dipropyl‐8‐(2‐amino‐4‐chloro)phenylxanthine], 1,3 diethyl‐8‐phenylxanthine (DPX), 8‐phenyltheophylline (8PT), 8‐parasulfophenyl‐theophylline (8PST), and theophylline, showed that across the species N6‐substituted adenosine analogs showed consistent differences in rank order activity. R‐PIA ≥ CHA >S‐PIA, with the compounds being most active in bovine tissue and least active in guinea pig, showing an 18‐fold difference. In contrast, the 2‐substituted analog, 2‐chloroadenosine was most active in guinea pig and human brain membranes, and least active in rat and bovine brain. The 5′‐substituted analog NECA had similar activity in all species studied. PACPX was the most potent of the xanthines studied, being most active in bovine brain tissue and least active in guinea pig; the difference in activities was 393‐fold. The interaction of the remainder of the xanthines studied was complex. In general, the rank order of potency was PACPX > DPX=8 phenyltheophylline > 8PST >> theophylline. However, in human and rabbit brain 8PT was more active than DPX, and in guinea pig 8PT was twofold less active than DPX. In addition, in human and guinea pig brain, 8PST had comparable activities. These differences in pharmacological profiles may be attributable to differences in the adenosine systems studied as previously noted by Murphy and Snyder [Mol. Pharmacol. 22:250–257, 1982], which may in turn reflect distinct differences in A1 receptors or the presence of different subtypes of adenosine receptor in the various species. While these interspecies studies need to be extended to both A2 receptors and to coupled, i.e., adenylate cyclase, systems, they underline the need for care in choosing the mammalian species in which structure activity relationships are generated. Some caution is necessary in the use of bovine brain tissue in development programs targeted towards therapeutic entities in the adenosine A1 antagonist area.

Two types of receptor for insulin-like growth factors in mammalian brain.

Two types of receptor for insulin-like growth factors (IGFs) have been identified on adult rat and human brain plasma membranes by competitive binding assay, affinity labelling, receptor phosphorylation and interaction with antibodies to insulin receptors. The type I IGF receptor consists of two species of subunits: alpha-subunits (mol. wt. approximately 115 000), which bind IGF I and IGF II with almost equal affinity and beta-subunits (mol. wt. approximately 94 000), the phosphorylation of which is stimulated by IGFs. The alpha-subunits of type I IGF receptors in brain and other tissues differ significantly (mol. wt. approximately 115 000 versus 130 000), whereas the beta-subunits are identical (mol. wt. approximately 94 000). The type II IGF receptor in brain is a monomer (mol. wt. approximately 250 000) like that in other tissues. Two antibodies to insulin receptors, B2 and B9, interact with type I but not with type II IGF receptors. B2 is more potent than B9 in inhibiting IGF binding and in immunoprecipitating type I IGF receptors, in contrast to their almost equal effects on insulin receptors. This pattern is characteristic for IGF receptors in other cells. The presence of two types of IGF receptor in mammalian brain suggests a physiological role of IGFs in regulation of nerve cell function and growth. Since IGF II, but not IGF I, is present in human brain, we propose that IGF II interacts with both types of IGF receptor to induce its biological actions.

Insulin receptors in the mammalian central nervous system: binding characteristics and subunit structure

Insulin receptors in rat and human central nervous system have been identified by binding of 125I-insulin on purified synaptic plasma membranes; affinity labelling of receptors by chemical cross-linking 125I-insulin; or phosphorylation of receptors with [gamma-32P]ATP. Brain insulin receptors showed significant differences in their binding characteristics and subunit structure when compared with receptors in other tissues like adipose and liver cells: absence of negatively cooperative interactions; a distinct binding specificity i.e. porcine proinsulin, coypu insulin and insulin-like growth factor I and II showed 2-5 times higher binding affinity in brain than in other cell types; a smaller molecular size of the brain receptor alpha-subunit than in other tissues (Mr approximately 115,000 instead of 130,000). In contrast, the size (Mr approximately 94,000) and function of the insulin receptor beta-subunit kinase was identical with that described in other cells. We conclude, that insulin receptors in mammalian brain represent a receptor subtype which may mediate growth rather than metabolic activity of insulin.

High-affinity stereospecific binding of cyclodiene insecticides and γ-hexachlorocyclohexane to γ-aminobutyric acid receptors of rat brain

The γ-aminobutyric acid (GABA)/benzodiazepine/picrotoxin receptor complex of rat brain was identified by the specific binding of three radiolabeled ligands in the same membrane preparation: [ 3H]flunitrazepam ([ 3H]Flu) for the benzodiazepine receptor site, [ 3H]muscimol for the GABA receptor site, and t-[ 35S]butylbicyclophosphorothionate ([ 35S]TBPS) for the picrotoxinin site. Seven cyclodiene insecticides, γ-hexachlorocyclohexane (γ-BHC), β-BHC, and hexachlorobenzene had no significant effect on the specific binding of [ 3H]Flu or [ 3H]muscimol. But the cyclodienes and γ-BHC were very potent in displacing [ 35S]TBPS binding with IC 50 values ranging from 30 n M for endrin and endosulfan I to 500 n M for aldrin. Inhibition of [ 35S]TBPS binding by endrin was competitive. Binding was stereospecific since the more toxic stereoisomer of three pairs of compounds was more potent with the following IC 50's (in n M): endrin, 30; dieldrin, 100; endosulfan I, 30; endosulfan II, 60; γ-BHC, 150; β-BHC, > 10,000. Also the more toxic dieldrin epoxide and heptachlor epoxide had IC 50's of 100 and 70n M, respectively, compared to 500 and 400 n M for the less toxic aldrin and heptachlor, respectively. The results suggest that the ionic channel of the GABA receptor in mammalian brain is a primary target for the toxic action of γ-BHC and cyclodienes.

The binding of [3H]mepyramine to histamine H1 receptors in monkey brain.

Several laboratories have reported ligand binding studies using radioactive histamine H1 antagonists to label the H1 receptors in mammalian brain. We have extended these studies to a detailed examination of the binding of [3H]mepyramine to monkey brain and have shown that the distribution is similar to that in man, with specific binding sites being concentrated in the frontal cortex with relatively low binding to the pons and basal ganglia. The binding shows a single saturable component with a KD of about 1 nM and a Hill plot slope close to unity. These observations are the same for all structures tested. Comparison with data from other laboratories suggests that in this species, the histamine receptor is the same as that in peripheral tissues. From Ki values for various ligands and comparison of KD estimates in other species, the receptor seems to be essentially identical to the H1 receptor in central and peripheral tissues of the guinea pig and also to that in human brain. The rat and possibly the dog have minor differences from the monkey in terms of KD values for [3H]mepyramine binding.

Co-localization of GABA receptors and benzodiazepine receptors in the brain shown by monoclonal antibodies.

The most abundant inhibitory neurotransmitter in the central nervous system, gamma-aminobutyric acid (GABA), exerts its main effects via a GABAA receptor that gates a chloride channel in the subsynaptic membrane. These receptors can contain a modulatory unit, the benzodiazepine receptor, through which ligands of different chemical classes can increase or decrease GABAA receptor function. We have now visualized a GABAA receptor in mammalian brain using monoclonal antibodies. The protein complex recognized by the antibodies contained high- and low-affinity binding sites for GABA as well as binding sites for benzodiazepines, indicative of a GABAA receptor functionally associated with benzodiazepine receptors. As the pattern of brain immunoreactivity corresponds to the autoradiographical distribution of benzodiazepine binding sites, most benzodiazepine receptors seem to be part of GABAA receptors. Two constituent proteins were identified immunologically. Because the monoclonal antibodies cross-react with human brain, they provide a means for elucidating those CNS disorders which may be linked to a dysfunction of a GABAA receptor.

Nicotinic receptors in mammalian brain

1. Nicotine has marked effects on CNS function increasing brain excitability and spontaneous activity and also has antinociceptive actions. 2. Agonist radioligands for the nicotinic cholinergic receptor bind with high affinity in a saturable manner. Binding is however, insensitive to the ganglionic blockers, hexamethonium and mecamylamine. This suggests that agonists and antagonists bind to different sites on the receptor or that the nicotinic receptor in brain is different from that found in peripheral tissues. 3. The nicotinic antagonist, dihydro-β-erythroidine binds with high affinity (Kd = 4 nM) to rat brain membranes in a stereospecific, saturable, manner with a regional distribution similar to that seen with radiolabeled acetylcholine. Binding is insensitive to hexamethonium and mecamylamine. 4. It is concluded that the nicotinic recognition sites to which dihydro-β-erythroidine binds are neuromuscular rather than ganglionic in nature.

TRH receptors in fish

The brains of goldfish and other teleosts contain high-affinity binding sites for [ 3H][3-methyl-His 2]thyrotropin-releasing hormone ([ 3H]MeTRH) which closely resemble TRH receptors in mammalian brain and pituitary gland in apparent dissociation constant ( K D = 3–4 n M), in pharmacology for eight TRH analogs, and in exhibiting marked regional differences in the density of binding sites, with highest binding in the cerebrum and lowest in the cerebellum. Fish brain differs from mammalian brain in containing a prominent additional class of much lower affinity [ 3H]MeTRH binding sites ( K D about 15 μ M), of unknown nature, which also exhibit regional differences. Fish pituitary glands contain high-affinity [ 3H]MeTRH binding sites, but insufficient tissue has prevented full characterization. Little or no saturable binding of [ 3H]MeTRH was detected in several goldfish peripheral tissues.

Assessment of the chloroethyl analog of 3-PPP (N-n-propyl-3-(3-hydroxyphenyl)-piperidine), 3-PPP-Cl as an irreversible ligand for central dopaminergic recognition sites

NCA, the chloro analog of the potent dopamine agonist NPA is an irreversible ligand at dopamine receptors in mammalian brain. The chloroethyl analog of the recently described putative dopamine autoreceptor agonist 3-PPP, 3-PPP-Cl, was evaluated for its potential use as an irreversible autoreceptor ligand. N-Chloroethylation of 3-PPP reduced the intrinsic affinity of the agonist seven-fold and, consequently, in contrast to NCA, it was found that 3-PPP-Cl was not a good irreversible ligand at dopamine receptors.

Adenosine receptors in the mammalian central nervous system

1. 1. Adenosine by interaction with discrete extracellular recognition sites can modulate cyclic AMP formation and cell firing in the mammalian CNS. 2. 2. The effects of adenosine on cyclic AMP formation are mediated through two extracellular recognition sites: a high affinity (Kd = 10 −9 M) site designated A-1, activation of which results in an inhibition of adenylate cyclase activity and a lower affinity site (Kd = 10 −6 M) designated A-2, activation of which stimulates adenylate cyclase activity. Stable radiolabeled analogs of adenosine have been used to label A-1 receptors in mammalian brain. 3. 3. Adenosine and its stable analogs are potent inhibitors of neurotransmitter release. 4. 4. In addition to being phosphodiesterase inhibitors, the alkylxanthines are also adenosine antagonists, stimulating neurotransmitter release and increasing cell firing by antagonism of the effects of endogenous adenosine. These effects have been attributed to the presence of an inhibitory purinergic tone. 5. 5. Adenosine and related purines have been implicated in the mode of action of several centrally active drugs including anxiolytics, antidepressants and analgesics. 6. 6. Future progress in understanding the potential physiological role of adenosine in the mammalian CNS will depend on the availability of more potent and specific adenosine antagonists, ligands specific for the A-2 receptor, and a better understanding of the factors that regulate adenosine availability.

Characterization of a putative nicotinic acetylcholine receptor in mammalian brain

The preponderance of evidence suggests that neurons respond electrophysio-logically to ACh and ACh antagonists in a manner consistent with the existence of AChRs, and at least some of these AChRs are nicotinic in nature. The binding of neurotoxins to brain tissue generally occurs in areas where other cholinergic markers exist. The biochemical properties of binding sites are remarkably similar to muscle tissues in size, the presence of a sulf hydryl group that alters ligand affinities, pharmacology, and glycoprotein nature. The dissimilarities, however, are also unmistakable: the association and dissociation rates of toxin binding are different, the inability of Butx to block electrophysiological activity in some tissues, the ratio of toxin sites blocked by MBTA and the apparent lack of antigenic similarity between peripheral nAChRs and brain. In at least one area the relationship to other cholinergic markers has been questioned. It is not likely that brain possesses non-functional sites with such similarity to nAChRs, but it is possible that some sites are non-synaptic and possibly non-specific (see ref. 74 for a discussion of some of these topics). We believe it is possible, however, that the dissimilarities between toxin binding sites in brain and muscle tissue reflect a structural difference between nAChRs. It is likely that toxins bind to nAChRs but do not prevent ACh binding to all sites. Since ACh has apparently not undergone evolutionary change, it is likely that evolutionary changes in cholinergic functionring are reflected in receptor specialization 36. The neuromuscular synapse evolved for a highly specialized function requiring fast, excitatory post-synaptic depolarization. The muscle nAChR probably evolved from a primitive, possibly less specific, form of a molecule sensitive to acetylcholine. Although little is known regarding phylogenetic changes in receptor sites, evidence has recently been presented that the four Torpedo subunits are not immunologically distinct and may have evolved from a common precursor 116. While some CNS neurons may have evolved with similar needs, other CNS neuronal groups might necessitate both the possibility of excitation and inhibition. Other synapses may have retained simpler evolutionary traits of mixed nicotinic-muscarinic responsiveness. There is, also, evidence for the interaction of ACh with neuromodulators and other putative neurotransmitters. It is presently not clear whether the characteristics of a receptor are similar when it is pre-synaptic, non-synaptic or inhibitory, in contrast to the peripheral excitatory post-synaptic nAChR. Future studies must determine if currently used neurotoxins bind to a functional CNS nAChR site in any of these cases. We must also find a ligand that specifically binds to and blocks nAChRs that are not affected by Butx. Only then will we be able to address the central questions. Specifically, do Butx and similar neurotoxins bind to an nAChR?, and are there nAChRs which are not bound by currently known neurotoxins?

Two distinct serotonin receptors: regional variations in receptor binding in mammalian brain

Two distinct serotonin receptors in mammalian brain are labeled respectively with [ 3H]serotonin (5-HT 1) and [ 3H]spiperone (5-HT 2). In general, agonists display highest affinities for 5-HT 1 while antagonists prefer 5-HT 2 sites. To conduct regional studies of 5-HT receptors, we estimated 5-HT 2 sites with [ 3H]spiperone, using the 5-HT 2 specific antagonist cinanserin to displace binding to 5-HT 2 but not dopamine receptors and sulpiride to displace [ 3H]spiperone from dopamine but not 5-HT receptors. About 15% of cerebral cortical [ 3H]spiperone binding appears to involve dopamine sites and the remainder involves 5-HT 2 receptors. In the corpus striatum about 80% of [ 3H]spiperone binding labels dopamine receptors and the rest involves 5-HT 2 sites. [ 3H]mianserin binds about equally to 5-HT 2 sites are studies selectively by displacing histamine H 1-receptor binding with the H 1-antihistamine triprolidine. [ 3H]LSD labels both 5-HT 1 and 5-HT 2 receptors. Its binding to 5-HT 1 sites is displaced selectively with 5-HT, while its binding to 5-HT 2 receptors is displaced with cinanserin. [ 3H]5-HT labels only 5-HT 1 receptors. The regional distribution of the two 5-HT receptors is similar in rat, guinea pig and bovine brain. However, the regional patterns of 5-HT 1 and 5-HT 2 receptors differ considerably in all 3 species. The hippocampus is quite high in 5-HT 1 receptors but low in 5-HT 2 sites. The cerebellum contains the lowest levels of both 5-HT 1 and 5-HT 2 receptors. In bovine brain, most areas contain similar numbers of 5-HT 1 and 5-HT 2 receptors. However, the substantia nigra, the richest 5-HT 1 area inbovine brain, possesses 10 times more 5-HT 1 than 5-HT 2 sites.

3H-apomorphine labels both dopamine postsynaptic receptors and autoreceptors.

Evidence points to the existence of multiple classes of dopamine (DA) receptor in mammalian brain which can be distinguished by their pharmacological specificities and localizations, and by the actions they mediate. Among them, dopaminergic autoreceptors are regulatory receptors presumed to be present in the membrane of the DA neurones themselves, and believed to mediate an inhibition of these neurones' activity, either at nerve endings or on cell bodies. However, the pharmacology of autoreceptors remains to be established because attempts to characterize autoreceptors by 3H-ligand binding techniques have produced controversial data. Thus Seeman and co-workers stated that 3H-apomorphine selectively labels autoreceptors, whereas Creese et al. concluded that this ligand selectively labels postsynaptic DA receptors. In addition, large differences in the capacity and drug specificity of 3H-apomorphine receptor sites in rat striatum have been reported. We demonstrate here that 3H-apomorphine labels two classes of DA receptor, distinguishable using domperidone, a selective DA antagonist. Lesion studies indicate that they correspond to a certain class of postsynaptic receptor and to autoreceptors, respectively. Each of these classes displays a clearly distinct pharmacological specificity for antipsychotics and DA agonists.

GABA receptors in bovine cerebral blood vessels: Binding studies with [ 3H]muscimol

[ 3H]Muscimol, a potent GABA agonist used to label GABA receptor sites in brain and invertebrate striated muscle, was found to bind specifically to sites in a crude membrane fraction prepared from bovine cerebral blood vessels. Specific [ 3H]muscimol binding was saturable of high affinity ( K d = 41 nM), and was selectively inhibited by GABA, specific GABA agonists, and the antagonist bicuculline with potencies similar to what has been found for GABA receptors in mammalian brain. GABA and several GABA agonists including muscimol have been reported to dilate isolated cerebral arteries, but not peripheral blood vessels. The pharmacology of the [ 3H]muscimol binding site correlated well with that of the vasodilatory response. No significant specific [ 3H]muscimol binding was detected in aorta and mesenteric arteries. The characteristics of the cerebrovascular muscimol binding site thus are indicative of a physiologically relevant GABA receptor associated with cerebral blood vessels. These findings suggest a direct role for GABA in cerebral vascular function.

Animal and molecular pharmacology of mixed agonist‐antagonist analgesic drugs.

1 The pharmacology of analgesic drugs with mixed agonist-antagonist action is reviewed in the light of the theory of drug action at single and multiple receptors. 2 Evidence for a heterogeneous population of opiate receptors in mammalian brain and in isolated tissue preparations is discussed. 3 The unusual pharmacological and pharmacokinetic profiles of the oripavine derivative buprenorphine are discussed in relation to the action of the drug at a molecular level.