Mark W. Hamblin

Behavioral and radioligand binding evidence for irreversible dopamine receptor blockade by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an irreversible alpha adrenergic antagonist, also acts as a potent and longlasting in vivo antagonist of D-2 dopamine receptors. Rats given EEDQ 3-10 mg/kg i.p. exhibit catalepsy and greatly reduced apomorphine-induced stereotypy, behavioral effects associated with D-2 dopamine receptor blockade. These effects are apparent up to 4 days after drug administration, with scores returning to control level by day 7. In vitro receptor binding assays of striatal membrane preparations from these animals using the radioligand 3H-spiroperidol directly demonstrate that EEDQ is a potent D-2 dopamine receptor antagonist, revealing the apparent basis of the behavioral effects of EEDQ. This antagonism proceeds via a reduction in D-2 receptor Bmax, with no change in the observed KD for 3H-spiroperidol, and is resistant to extensive washing of the membrane preparation after in vivo EEDQ exposure. These observations suggest that EEDQ inhibition of D-2 receptors is irreversible. Administration of behaviorally active doses of EEDQ effect a reduction of 50-85% in D-2 receptor number. Recovery of this loss roughly parallels recovery of normal catalepsy and apomorphine stereotypy scores. These doses of EEDQ also reduce binding of 3H-flupentixol to D-1 and 3H-dopamine to D-3 type dopaminergic binding sites, putative dopamine receptors with no known behavioral correlates. Recovery of D-1 and D-3 binding also occurs with a similar timecourse. Because of the apparent covalent nature of its interaction with dopamine receptors and because of its activity after peripheral administration, EEDQ may prove useful in the study of the function and turnover of dopamine receptors.

3H-dopamine binding to rat striatal D-2 and D-3 sites: enhancement by magnesium and inhibition by guanine nucleotides and sodium.

Previous studies have demonstrated high affinity 3H-dopamine binding sites on mammalian striatal membranes. These putative dopamine receptors of unknown physiological significance have been termed D-3 sites. Such studies have failed, however, to demonstrate high affinity 3H-dopamine binding to D-2 sites, which can be labeled by 3H-butyrophenones, and which represent the putative dopamine receptors most strongly implicated in the behavioral correlates of dopaminergic CNS activity. We now know that preincubation of membrane homogenates with Mg++ and inclusion of Mg (1-10mM) or other divalent metal cations during binding allows high affinity D-2 specific 3H-dopamine binding in rat striatal membranes, and that these ions also increase the Bmax of D-3 specific 3H-dopamine binding. GTP, GDP, and GppNHp can completely abolish all D-2 specific 3H-dopamine binding, while only a magnesium-dependent portion of D-3 sites appears to be GTP sensitive. These data are consistent with the hypothesis that the striatal D-2 receptor exists in two agonist affinity states whose interconversion is effected by guanine nucleotides and divalent metal cations. The GTP sensitive/magnesium dependent nature of 3H-dopamine binding to so-called D-3 sites suggests that some such sites may in fact represent a high agonist-affinity state of the D-1 adenylate cyclase stimulating dopamine receptor also found in this tissue.

Ascorbic acid enables reversible dopamine receptor 3H-agonist binding

Abstract The effects of ascorbic acid on dopaminergic 3 H-agonist receptor binding were studied in membrane homogenates of bovine anterior pituitary and caudate, and rat striatum. In all tissues virtually no stereospecific binding (defined using luM (+)butaclamol) of the 3 H-agonists N-propylnorapomorphine (NPA), apomorphine, or dopamine could be demonstrated in the absence of ascorbic acid. Although levels of total 3 H-agonist binding were three to five times greater in the absence than in the presence of 0.1% ascorbic acid, the increased binding was entirely non-stereospecific. Greater amounts of dopamine-inhibitable 3 H-NPA binding could be demonstrated in the absence of 0.1% ascorbic acid, but this measure of “specific binding” was demonstrated not to represent dopamine receptor binding since several other catecholamines and catechol were equipotent with dopamine and more potent than the dopamine agonist (±)amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) in inhibiting this binding. High levels of dopamine-displaceable 3 H-agonist binding were detected in fresh and boiled homogenates of cerebellum, an area of brain which receives no dopaminergic innervation, further demonstrating the non-specific nature of 3 H-agonist binding in the absence of ascorbic acid. These studies emphasize that under typical assay conditions ascorbic acid is required in order to demonstrate reversible and specific 3 H-agonist binding to dopamine receptors.

Dopamine receptors in the central nervous system

Publisher Summary Dopamine (DA) is an important central nervous system (CNS) pituitary modulator of prolactin, β -endorphin, and α -MSH secretion. This chapter discusses central DA receptors, the pharmacological characteristics and anatomical localizations of the several distinct DA-receptor subtypes delineated through radioligand binding and biochemical studies, the functions of these receptors, and actions of dopaminergic agonists and antagonists. Although, several DA receptors are thought to function in the CNS, there is a great deal yet to be understood regarding their biochemical, physiological, and behavioral roles. Radioligand-binding studies have proved to be very useful in determining the mechanisms involved in the regulation and alteration of neuronal activity; however, future investigations may exploit this technique for the study of neuronal and neurochemical plasticity. Regulation of neurotransmitter and hormone-receptor binding should be examined during the aging process, because if receptor regulation is progressively disturbed, it may be amenable to pharmacological therapy.

CNS Dopamine Receptors

The past 20 years has seen our appreciation of the function of dopamine in the brain elevated from that of a precursor for other catecholamines, principally norepinephrine, to a neurotransmitter in its own right. The association of disturbances of dopaminergic neurotransmission with neurological and psychiatric disorders has further emphasized the crucial role of this neurotransmitter in normal brain functioning and stimulated much basic research into dopaminergic neurotransmission. Dopaminergic agonists with the ability to cross the blood-brain barrier now have a firmly established role in the treatment of Parkinson’s disease, and may be of value in the therapy of tardive dyskinesia. Dopaminergic antagonists have a longer history in the treatment of schizophrenia, Huntington’s disease, and Gilles de la Tourette’s syndrome. This pharmacological arsenal, available because of the pharmaceutical industries’ search for better therapeutic agents, also provides the major tools for experimental approaches. Both agonists and antagonists are available from diverse structural groups. Some exist as stereo or geometric isomers which differ markedly in their ability to interact with dopaminergic systems.

Radioligand Binding Studies of Agonist Interactions with Dopamine Receptors

Since 1975, the elegantly simple radioligand binding technique has allowed direct examination of neurotransmitter and drug interactions with dopamine receptors. The simplification obtained through elimination of factors such as alteration of neurotransmitter synthesis or other regulators of dopamine’s second messenger systems is the chief advantage of this approach to the study of receptor biochemistry and pharmacology. This simplification, however, also presents a major challenge—to demonstrate that the binding sites identified in vitro have functional relevance in the physiological milieu. It is a task of utmost importance, and often of considerable difficulty, to demonstrate that receptor binding sites can be clearly associated with some biological function. Although problems remain, this correspondence between binding sites and their function, on both the behavioral and biochemical level, is steadily being established for the dopamine receptors.