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University of Groningen

The 2-aminotetralin system as a structural base for new dopamine- and melatonin-receptor agents

Copinga, Swier

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1994

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Copinga, S. (1994). The 2-aminotetralin system as a structural base for new dopamine- and melatonin- receptor agents. s.n.

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PART I

5,6-(OH)2-PTAT:

A MIXED Dl/D2-RECEPTOR AGONIST

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CHAPTER 1

INTRODUCTION PART I

1.1 DOPAMINE: A NEUROTRANSMITTER IN THE CNS

In the late 1950's dopamine was identified as a normal constituent of the central nervous system (CNS) and it was shown that dopamine might act as a neurotransmitter in the CNS [I]. Since then our knowledge about the organization of dopamine neuronal systems in the CNS and the individual stages of the central dopamine neuro- transmission, like the biosynthesis, the storage, the release, the inactivation, and the interaction with specific dopamine receptors has increased enormously. This chapter deals with: a) the anatomical characterization of central dopamine neuronal systems, b) some basic aspects of central dopamine neurotransmission, c) recent developments in the field of specific dopamine receptors and selective dopamine-receptor agents, and d) the current status of functional dopamine-receptor interactions, and their potential implications for basal ganglia* movement disorders, like Parkinson's disease.

Extensive neuroanatomical tracing studies of dopamine pathways in the CNS have shown that dopamine neurons tend to be organized in highly localized neuronal systems. These can be divided into ascending, descending and local dopamine pathways (for reviews and references, see ref. 2-5).

Nowadays, two major groups of ascending doparnine neurons are defined, the mesostriatal dopamine system and the meso(limbo)cortical dopamine system (Figure 1.1), and one minor group, the mesothalamic doparnine system. The mesostriatal dopamine system can be subdivided into a dorsal component and a ventral component.

The dorsal component consists of dopamine neurons with cell bodies in the substantia nigra pars compacta and, to a limited extent, in the ventral tegmental area and with nerve terminals in the caudate nucleus/putamen complex (striatum), the globus pallidus and the subthalamic nucleus. In contrast, the ventral component is composed of dopamine neurons with cell bodies in the ventral tegmental area and, to a limited extent, in the substantia nigra pars compacta and with nerve terminals in the nucleus accumbens, a limbic region that can be considered as a ventral extension of the striatum.

Within the mesocortical dopamine system, one can, based on the phylogenetical age of

-

* The basal ganglia represent a number of subwrtical nuclei closely arranged in the midbrain. The basal ganglia include the caudate nucleus/putamen complex (striatum), the nucleus accumbens, the substantia nigra, the ventral tegmental area, the globus pallidus, and the subthalamic nucleus (see 1.4.4).

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STRIATUM

SEPTUM 1 HIPPOCAMPUS CINGULATE

PREFRONTAL UBSTANTIA NlGRA

PARS COMPACTA

VENTRAL TEGMENTAL

PERlRHlNAL AREA

ACCUMBENS ENTORHINAL

CORTEX

TUBERCLE GLOBUS SUBTHALAMIC PALLIDUS NUCLEUS

Figure 1.1 Schematic representation of a sagittal section of a rat brain, showing the mesostriatal and meso(limbo)cortical dopamine systems.

the innervated limbic cortical derivative, distinguish mesoallocortical and mesoneo- cortical projections [3,6]. The mesocortical dopamine neurons link the ventral tegmental area and the substantia nigra pars cornpacta with allocortical areas, such as the olfactory tubercle, the septum, the amygdala, the hippocampus and the piriform cortex, and neocortical areas, such as the entorhinal cortex, the perirhinal cortex, the prefrontal cortex, and the cingulate cortex. The mesothalamic dopamine neurons project from the ventral tegmental area to the habenula, an important relay station for circuits in the extrapyramidal system [4].

It is generally accepted that the mesostriatal dopamine system has an activating action on movement. Hence, degeneration of this system, as seen in Parkinson's disease (idiopathic parkinsonism), or a reduction of the dopamine neurotransmission in this system, e.g. as a result of the treatment with dopamine-receptor blocking agents (secondq parkinsonism), leads to the development of rigidity, hypokinesia, and tremors. An excessive activity of the mesostriatal dopamine system has been implicated in hyperkinetic movement disorders, such as Huntington's chorea, a disease characterized by abnormal involuntary movements.

The involvement of the mesocortical dopamine system and the ventral component of the mesostriatal doparnine system in the control of mood and emotional behaviours, like aggressiveness, anxiety and sexuality, is clear, but to what extent is controversial. It has been hypothesized that an overactivity of these systems contributes to the symptomatology of psychotic disorders, such as schizophrenia, whereas reduced functioning of these systems might play a role in the cognitive abnormalities found in patients with Parlunson's disease.

The most important descending dopamine neurons belong to the dienchephalospinal dopamine system. These dopamine neurons originate mainly in the periventricular grey

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of the caudal thalamus and the dorsal hypothalamus and innervate the dorsal grey of the spinal cord as well as the intermediolateral cell column of the spinal cord. This system seems to be involved in the regulation of the activity of preganglionic sympathetic neurons. There are also indications of minor descending dopamine neurons projecting from the midbrain to the locus coeruleus and to the cerebellum.

Of the local dopamine pathways, the hypothalamic tuberoinfundibular and tubero- hypophyseal dopamine systems have received the most attention. The cell bodies of the dopamine neurons, belonging to these systems, are mainly situated in the arcuate and periventricular hypothalamic nuclei. The tuberoinfundibular dopamine neurons innervate mainly the external layer of the median eminence and of the infundibular stalk, whereas the tuberohypophyseal dopamine neurons project predominantly to the intermediate and posterior lobes of the pituitary. Within the median eminence dopamine exerts an inhibitory action on the release of luteinizing hormone releasing hormone (LHRH), thyrotropin releasing hormone (TRH) and somatotropin inhibiting hormone (somatostatine). In addition, dopamine itself is released from the medial palisade zone of the median eminence as the prolactin inhibiting hormone (PIH), which inhibits the secretion of prolactin fiom the anterior lobe of the pituitary. In the intermediate lobe of the pituitary dopamine plays a role in the inhibition of the secretion of melanotropin (a- melanocyte stimulating hormone, a-MSH) and in the posterior lobe of the pituitary in the regulation of the release of vasopressin and oxytocin.

Local dopamine neurons also appear to exist in the olfactory bulb and the retina.

Thus, some periglomerular cells of the olfactory bulb and some innerplexiform cells of the retina utilize dopamine as neurotransmitter, regulating the activity in the olfactory glomeruli and modulating retinal physiology, respectively.

Dopamine is synthesized within the dopamine neurons in a series of enzymatic steps (Chart 1.1) (for reviews and references, see ref. 5,7). The biosynthesis of dopamine starts from the amino acid L-tyrosine, which is taken up from the blood stream across the blood-brain barrier and is accumulated in dopamine neurons by carrier-mediated processes. The first step, the hydroxylation of L-tyrosine to L-3,4- dihydroxyphenylalanine (L-DOPA), is mediated by the specific cytoplasmatic enzyme tyrosine hydroxylase (TH). As TB is the rate-limiting enzyme in the biosynthesis of dopamine, an alteration of its activity changes doparnine levels. Hence, the regulation of its activity is important for the maintenance of constant functional dopamine levels.

Acute regulation of the activity of TH is achleved through Inhibition by L-DOPA and dopamine and through activation by phosphorylation, which might be triggered by increased levels of intracellular ca2+ following depolarization [8]. The activity of TH can also be affected by released doparnine via synthesis-controlling dopamine autoreceptors (see 1.2.3). Likewise, it is possible to inhibit its activity by pharmaco-

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L-TYR L-DOPA D A

Chart 1.1 Biosynthesis o f dopamine. Abbreviations: AAAD, aromatic L-amino acid decarboxylase;

DA, doparnine; L-DOPA, L-3,4-dihydroxyphenylalanine; TH, tyrosine hydroxylase; L- TYR, L-tyrosine.

logical intervention, e.g. a-methyl-p-tyrosine (AMPT) is a competitive inhibitor of TH.

Due to the fact that TH is completely saturated with L-tyrosine, changes in plasma L- tyrosine levels do not affect the biosynthesis of dopamine.

The second and final step in the pathway to dopamine is the rapid decarboxylation of L-DOPA to dopamine, mediated by the nonspecific cytoplasmatic enzyme aromatic L-amino acid decarboxylase (AAAD). This conversion is the basis for the L-DOPA therapy of Parkinson'disease. AAAD can be mhlbited by drugs such as carbidopa and benserazide. Because these drugs fail to pass the blood-brain barrier, they can be used clinically as peripheral-acting adjuvants in the L-DOPA therapy of Parkinson's disease.

Newly synthesized dopamine is protected from immediate intraneuronal degradation by specific ATP-dependent uptake into membrane-bounded synaptic vesicles of different sizes, where it is stored until required for release (for reviews and references, see ref. 5,7). This uptake and storage can be prevented by the alkaloid reserpine, leading to an irreversible depletion of the neurotransmitter.

Upon the arrival of a depolarizing stimulus at the nerve terminal of a dopamine neuron, dopamine is released by exocytosis from synaptic vesicles into the synaptic cleft (for reviews and references, see ref. 5,7). It is thought that the trigger for the fusion of a synaptic vesicle with the presynaptic membrane is the influx of caZf through voltage-activated ~ a 2 + channels. The release probably takes place at highly specialized regions of the presynaptic membrane, also referred to as active zones [gal. These active zones are often in register with specialized postsynaptic membrane regions with a high concentration of postsynaptic receptors (postsynaptic densities). After being released, dopamine elicits biological responses by interacting with specific dopamine receptors (see 1.2). Besides this release fiom nerve terminals dopamine was also shown to be released from dendrites and/or cell bodies, especially in the substantia nigra [9b-9d].

A major factor determining the synaptic release of doparnine by a dopamine neuron is the impulse flow of that neuron, which depends mainly on the balance between the excitatory and inhibitory inputs to that neuron. Released doparnine can influence the electrical activity of its own neuron in more than one way. First, dopamine released from dendrites, e.g. in the substantia nigra, can inhibit this activity by acting on impulse flow-controlling somadendritic dopamine autoreceptors (see 1.2.3) or on dopamine receptors located on the nerve terminals of neurons afferent to dopamine neurons. Second, dopamine released from nerve terminals, e.g. in the striatum, can inhibit this activity by acting on postsynaptic dopamine receptors (see 1.2.3), which

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activate negative feedback neuronal circuits linking dopamine target cells with cell bodies of dopamine neurons. Although the firing rate is important, the release of dopamine is not solely dependent on this activity. Release-controlling receptors, dopamine autoreceptors (see 1.2.3) as well as receptors for different kinds of neurotransmitters and neuromodulators, present at dopamine nerve terminals modulate negatively or positively the release of doparnine independently of the firing rate of a dopamine neuron. A possible mode of action of these receptors could be the modulation of intracellular ca2+ availability through ~ a 2 + channels. The release of dopamine can be induced pharmacologically by psychostimulants, like amphetamine.

After being released and having fulfilled its action as neurotransmitter, dopamine is inactivated primarily (70 to 80%) by re-uptake into the presynaptic dopamine nerve terminal, where it can be restored in synaptic vesicles or metabolized enzymatically (for reviews and references, see ref. 5,7). The carrier-mediated dopamine re-uptake process is inhibited potently by drugs, like benztropine, nomifensine, amphetamine and cocaine.

However, many of these drugs also enhance the release of dopamine. Cocaine appears most specific in this regard causing little or no release of dopamine. Dopamine that is not taken back into the presynaptic nerve terminal is partly taken up extraneuronally, where it can be metabolized, and is partly removed by passive diffusion from the synaptic cleft.

Metabolism of dopamine, intraneuronally as well as extraneuronally, depends on three enzymes: monoamine oxidase (MAO), aldehyde dehydrogenase (AD) and catechol 0-methyltransferase (COMT), as outlined in Chart 1.2. The combined action

DOPAC

Chart 1.2 Metabolism of dopamine in the brain. Abbreviations: AD, aldehyde dehydrogenase; COMT, catechol 0-methyltransferase; DA, dopamine; DOPAC, 3,4dihydroxyphenylacetic acid;

HVA, homovanillic acid; MAO, monoamine oxidase; 3-MT, 3-methoxytyramine.

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of M A 0 and AD converts dopamine to 3,4-dihydroxyphenylacetic acid (DOPAC), which can be subsequently 0-methylated by COMT to homovanillic acid (HVA). T h s metabolic route accounts approximately for 80% of the enzymatic degradation of dopamine [lo]. Another metabolic route involves the 0-methylation of doparnine to 3- methoxytyramine (3-MT) by COMT, always followed by the conversion of 3-MT to HVA by M A 0 and AD. DOPAC and HVA are removed from the brain in unchanged or in conjugated form.

M A 0 and AD are both membrane-bound enzymes, which are found in the outer layer of mitochondria in neurons and other cells. The enzyme M A 0 exists in two forms, MAO-A and MAO-B, which are homologous proteins with different substrate specificities and sensitivities to inhibitors. However, dopamine is a substrate for both forms. Both forms are present in human brain, although the B form predominates. This finding rationalized the use of selective MAO-B inhibitors, like L-deprenyl and pargyline, as adjuvants in the L-DOPA therapy of Parkinson's disease. COMT is found in the cytoplasm of many cells, including neurons and neuroglial cells.

1.2.1 INTRODUCTION

Nowadays, it is generally accepted that dopamine elicits its biological effects by interacting with specific dopamine receptors (for reviews and references, see ref. 5,11- 20). These receptors, which are more sensitive to dopamine than any other neurotransmitter, are integral membrane glycoproteins, containing a binding site for dopamine, and are located in the lipid membrane of neurons or other cells. Due to the coupling of these glycoproteins to intracellular effector systems through guanine nucleotide-regulatory proteins (G-proteins), activation by dopamine induces intra- cellular changes, which ultimately cause biological effects. Pharmacological agents causing similar biological effects as dopamine by binding at dopamine receptors and subsequently activating them, are called dopamine-receptor agonists (see 1.3.2).

Pharmacological agents inhibiting biological effects of dopamine, by binding at doparnine receptors, but not activating them, are called dopamine-receptor antagonists (see 1.3.3). Dopamine receptors do not appear to constitute a uniform population. They can be differentiated according to anatomical localization, pharmacological, biochemical, and structural properties. They are possibly also functionally different.

During the past twenty years numerous classification schemes have been proposed to describe various subtypes of dopamine receptors.

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1.2.2 BIOCHEMICAL/PHARMACOLOGICAL CLASSIFICATION:

DOPAMINE Dl RECEPTORS W R S U S DOPAMINE

D2

RECEPTORS

Based on biochemical studies the existence of two subtypes of dopamine receptors, designated as D l and D2 receptors by Kebabian and Calne, was suggested at the end of the 1970's [21]. D l receptors were defined as doparnine receptors, which mediate the ability of dopamine to stimulate cyclic adenosine monophosphate (CAMP) formation by activating the enzyme adenylyl cyclase. D2 receptors were considered to be dopamine receptors not coupled to adenylyl cyclase activity. Subsequently, it was shown that stimulation of D2 receptors in the pituitary as well as the striaturn leads to an inhibition of adenylyl cyclase activity [22-241. However, it appeared also that subpopulations of D2 receptors, not linked to CAMP formation, do exist [25,26]. To incorporate this aspect of the D2 receptor function, the biochemical Dl/D2 receptor classification scheme was revised in the mid 1980's (for reviews, see ref. 11,13,27).

A further important development in the 1970's was the identification of specific binding sites for dopamine using radiolabelled dopamine-receptor agents. Although initially, on the basis of in vitro radioligand binding studies, as much as four distinct dopamine binding sites were proposed, the general consensus in the 1980's emerged that there are two dopamine binding sites. These are the D l and D2 binding sites in correspondance with the biochemically classified Dl and D2 receptors. Both can occur in two interconvertible states exhibiting either high or low affinity for dopamine- receptor agonists (for reviews, see ref. 11,12,27,28). The high-affmity states of these two dopamine binding sites appear to be induced by the association of a G-protein to these binding sites [12,29].

The identification of tissues possessing only one of the above-defined dopamine- receptor subtypes made it possible to determine the pharmacological characteristics of these Dl and D2 receptors (for reviews and references, see ref. 11,13,14). The dopamine receptor in the parathyroid gland is considered the prototypical D 1 receptor.

Stimulation of this receptor results in an increase of the CAMP formation and ultimately in the release of parathyroid hormone. In contrast, the dopamine receptor in the anterior and intermediate lobe of the pituitary gland is considered the prototypical D2 receptor.

Stimulation of this receptor inhibits the release of prolactin and a-melanocyte stimulating hormone (a-MSH), respectively. These functional models of the two dopamine-receptor subtypes, in combination with in vitro radioligand binding studies, aided the identification of dopamine-receptor agonists and antagonists interacting selectively with either Dl or D2 receptors (see 1.3). The existence of these selective agents considerably strengthened the postulated Dl/D2-receptor classification.

Recent evidence suggested that D2 receptors may additionally be linked to other signal transduction systems, including the inhibition of phosphoinositide (PI) turnover through the enzyme phospholipase C and the activation of K+ channels 1301. Moreover, recent studies indicated even that in addition to Dl receptors stimulating adenylyl cyclase, D l receptors exist in the CNS, which are positively coupled to the formation of

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inositol phophates [3 1,321. In all these signal transduction systems the linkage of Dl and D2 receptors to its effector system (adenylyl cyclase, phospholipase C or ion channel) is mediated through guanine nucleotide-binding regulatory proteins (G- proteins) (for reviews, see ref. 33-35). Thus, dopamine receptors characterized pharmacologically using selective dopamine-receptor agents as either D l or D2 receptors do not always fit into the biochemical DlID2-receptor classification of the mid 1980's [36].

By using selective Dl- and D2-receptor agents radioactively labelled with

P-

( 3 ~ ) or low-energy photon (1251) emitters in receptor autoradiography, the regional distribution and densities of Dl and D2 receptors in the brains of several mammalian species, including human, were studied [37-421. The main findings of these studies can be summarized as follows. The pattern of regional distribution of these dopamine- receptor subtypes is largely comparable but not totally identical among the studied mammalian species. The highest values of Dl- and D2-receptor densities in the studied species are found in the basal ganglia and associated areas such as the striatum (caudate nucleus/putamen complex), the nucleus accumbens, the olfactory tubercle, the globus pallidus, the amygdala, the islands of Calleja and the substantia nigra pars compacta and pars reticulata. These are the areas considered to be richly innervated by dopamine neurons fiom the midbrain or to contain the cell bodies and dendrites of these dopamine neurons. In all the mammalian species investigated, Dl-receptor densities are much higher than D2-receptor densities and the ratio is almost similar in all of them.

However, the absolute amounts of dopamine receptors varied markedly among the studied species. It appears that during evolution a decrease in dopamine-receptor densities took place.

The exact cellular localization of Dl and D2 receptors in the CNS, in particular in the mesostriatal dopamine system, was also studied extensively by using membrane receptor-binding assays and receptor autoradiography in combination with several kinds of chemical and/or electrolflc lesions. The data obtained from these studies suggested that D2 receptors are not only located on non-dopamine neurons (D2 heteroreceptors) but also on dopamine neurons (D2 autoreceptors), while Dl receptors are solely located on non-dopamine neurons (D 1 heteroreceptors). In other terms, D2 receptors are present pre- and postsynaptically, whereas

Dl

receptors are present exclusively post- synaptically (see 1.2.3). Thus, in the striatum D2 receptors are not only found on the nerve terminals of afferent nigrostriatal dopamine neurons, but also on the cell bodies and the nerve tenninals of intrinsic striatal neurons, especially acetylcholine interneurons, the cell bodies of efferent striatal neurons, such as GABA output neurons, and the nerve terminals of af5erent corticostriatal glutamate neurons [43-491. However, this last finding is not supported by all investigators [50,51]. Striatal D l receptors are mainly localized on the cell bodies of GABA output neurons [52-551. In the substantia nigra the vast majority of the present Dl receptors is located on the nerve terminals of striatonigral GABA neurons, whereas D2 receptors reside predominantly on the cell

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bodies and dendrites of nigrostriatal dopamine neurons [44,48,52-54,56-591. Further studies using, for example, fluorescent analogues of selective Dl and Dpreceptor agents [60-641 may help to answer the questions still existing about the cellular localization of both dopamine-receptor subtypes.

The visualization and quantification of Dl and D2 receptors by receptor autoradio- graphy was not only used to study the regional distribution and cellular localization of both dopamine-receptor subtypes in the CNS; these procedures allowed also the analysis of the influence of several physiological, pathological and pharmacological conditions on the densities of Dl and D2 receptors as well as some aspects of their ontogeny and age-related modulation [59,65-671. However, a major drawback of this receptor imaging technique is that post-mortem brain tissue must be used for the analysis studies. New receptor imaging techniques, such as PET (positron emission tomography) and SPECT (single photon emission computerized tomography) have emerged and analogues of selective D l - and D2-receptor agents, radioactively labelled with radioisotopes applied in these emission computerized tomography techniques, have been developed. This has opened the way to investigate in vivo the regional distributions and densities of Dl and D2 receptors under physiological (aging), pathological (Parkinson's disease, Huntington's chorea and schizophrenia) and clinical pharmacological (antipsychotic treatment with neuroleptics) circumstances [68-731.

However, in vivo dopamine receptor imaging is still limited by the difficulties of quantifying the process of ligand-receptor binding in terms of affinity constants and receptor concentration.

1.2.3 CLASSIFICATION BASED ON ANATOMICAL LOCALIZATION:

PRESYNAPTIC DOPAMINE RECEPTORS (DOPAMINE AUTORECEPTORS)

mmus

POSTSYNAPTIC DOPAMINE RECEPTORS (DOPAMINE HETERORECEPTORS) Based on studies suggesting the involvement of dopamine receptors in the local, negative feedback regulation of the activity of dopamine neurotransmission, it was proposed in the 1970's to divide dopamine receptors on the basis of their anatomical localization in presynaptic dopamine receptors and postsynaptic dopamine receptors. As presynaptic dopamine receptors are located on dopamine neurons, the term dopamine autoreceptors is also used to describe these receptors. Postsynaptic dopamine receptors located on non-dopamine target neurons are also called dopamine heteroreceptors (for reviews and references, see ref. 74-77). Doparnine autoreceptors serve different autoregulatory functions dependent on their cellular distribution over the dopamine neuron. Stimulation of dopamine autoreceptors localized on cell bodies and dendrites gives rise to a decreased firing rate of dopamine neurons. Stimulation of doparnine autoreceptors present at nerve terminals causes an Inhibition of the biosynthesis and the release of dopamine. Dopamine autoreceptors were characterized pharmacologically as being of the D2-receptor subtype [I 1,13,76,77]. Postsynaptic dopamine receptors are

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considered to mediate primarily the physiological and behavioural effects of dopamine and dopamine-receptor agonists (for review, see ref. 13).

In vivo studies revealed that D2 autoreceptors are more sensitive to stimulation by dopamine-receptor agonists than their postsynaptic counterparts (for reviews, see ref.

75-77). This differential responsiveness was explained in several ways. Carlsson and co-workers launched the hypothesis that these two types of D2 receptors do not represent two distinct forms of the D2 receptor but are derived from a homogenous population of D2 receptors existing in different states of adaptation as a result of variations in previous dopamine-receptor agonist (dopamine) occupancy [78-801.

According to this hypothesis, the higher sensitivity of D2 autoreceptors than of postsynaptic D2 receptors to stimulation by dopamine-receptor agonists under physiological conditions results from a lower endogenous tone of dopamine at D2 autoreceptors than at postsynaptic D2 receptors. This is assumed to be caused by the localization of D2 autoreceptors largerly or entirely outside the synaptic cleft, whereas postsynaptic D2 receptors are located inside the synaptic cleft. Another hypothesis, put forward by Meller and colleagues, clarifies the difference in sensitivity of D2 autoreceptors and postsynaptic D2 receptors by the presence or absence of receptor reserve [81-841. According to this hypothesis, the higher sensitivity of D2 autoreceptors with respect to postsynaptic D2 receptors results from the presence of a higher percentage of spare receptors, i.e. a more efficient receptor-effector coupling presynaptically than postsynaptically.

This preferential activation of D2 autoreceptors by dopamine-receptor agonists formed a strong incentive for many research groups to attempt to develop selective D2- autoreceptor agonists. Mzlbition of dopamine neurotransmission by selective activation of D2 autoreceptors seemed to hold the promise of an elegant way in treating schizophrenia, a neuropsychiatric disorder thought to involve an increased dopamine activity in the CNS, without evoking extrapyramidal side effects [85]. In recent years, various agents were introduced which allegedly displayed a selective agonist profile at dopamine autoreceptors (see 1.3.2). On first impression, this would suggest a pharmacological distinction between D2 autoreceptors and postsynaptic D2 receptors.

However, Drukarch and Stoof showed, in their recent review about the existence of selective agents for D2 autoreceptors, that D2 autoreceptors and postsynaptic D2 receptors display similar pharmacological characteristics [77]. Based on these findings they concluded that D2 autoreceptors and postsynaptic D2 receptors are similar if not identical entities. A sound strategy to preferentially inhibit the activity of dopamine neurons would be the development of D2-receptor agonists with a partial agonist profile [86]. The most favourable D2-receptor partial agonists would act presynaptically almost as full agonists and postsynaptically as partial agonists with very low intrinsic efficacy.

This last property is needed to avoid the negative effects of antagonism at postsynaptic D2 receptors. According to the hypothesis of Carlsson and co-workers, the intrinsic efficacy of such D2-receptor partial agonists would be at maximum at those D2 receptors, where the endogenous dopamine occupancy is at minimum [87]. Thus, not

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only at extrasynaptical D2 autoreceptors, but also at postsynaptic D2-receptors under conditions of chronic dopamine deprivation [reserpine; 6-hydroxydopamine (6-OHDA)].

Keeping this in mind, if supersensitivity of dopamine receptors occurs in schizophrenia, the abovementioned D2-receptor partial agonists might worsen the condition by acting almost as full agonists at supersensitive postsynaptic D2 receptors. However, if supersensitivity of dopamine receptors can be refuted in schizophrenia, this type of D2- receptor partial agonists could be of great value in the pharmacotherapy of schizophrenia.

During the last decade much effort was put into the molecular characterization of dopamine receptors (for reviews, see ref. 16-20,88). A major breakthrough in this area of research was the cloning of the rat D2 receptor in 1988 by Bunzow and co-workers via the homology approach [89]. Exploiting the known primary amino acid-sequence homologies among members of the superfamily of G-protein-coupled receptors (for reviews, see ref. 33,90,91), they used the hamster P2-adrenergic receptor coding sequence as a hybridization probe to isolate clones encoding putatively G-protein- coupled receptors from a rat genomic library. One clone containing a partial genomic fragment with significant homology to the P2-adrenergic receptor was used to isolate a full-length cDNA from a rat brain cDNA library. This cDNA encoded a protein of 415 amino acids that, expressed in mouse fibroblast cells, possesses the radioligand binding characteristics of a D2 receptor [89]. To show that this protein was a functional D2 receptor, they demonstrated in sequential studies that it can couple to an inhibitory G- protein (Gi-protein) and that this coupling results in an inhibition of adenylyl cyclase activity and an inhibition of prolactin secretion [92,93]. Moreover, it was shown that this D~receptor, dependent on the cell types used for expression, decreases or increases intracellular ~ a 2 + through activation of K+ channels or phospholipase C, respectively [94]. Subsequently, the human homologue of this rat D2 receptor was cloned and shown to exhibit 96% identity with the rat D2 receptor [95,96].

Hydropathy analysis of the primary amino acid sequence of this mammalian D2 receptor showed the existence of seven hydrophobic stretches of approximately 25 amino acids each, which are surrounded by eight hydrophilic stretches of various length [89,95,96]. The hydrophobic regions are predicted to form seven a-helical trans- membranal (TM) domains with the N-terminus of the protein localized extracellularly and the C-terminus of the protein projected into the cytosol, as shown in Figure 1.2.

This overall membrane topography, initially proposed for rhodopsin, has been suggested for all the G-protein-coupled receptors that have been cloned (for reviews, see ref. 33, 90,91,97). The primary amino acid sequence of this D2 receptor displays a high degree of homology with other G-protein-coupled receptors. The regions of greatest homology are clustered within the putative TM domains (e.g. the rat D2 receptor shares 39% and

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Figure 1.2 Model of the human D2 receptor. The shaded areas represent lipid bilayer membrane.

Potential glycosylation sites are indicated as Y. The hexagonal cysteine residues might form a disulphide bridge. The black amino acid residues indicate putative phosphorylation sites.

The square and triangular amino acid residues are expected to be involved in ligand binding.

The shaded amino acid residues in the third cytoplasmic loop result from the alternative RNA splicing event (taken with permission from ref. 16).

48% of its TM sequences with the hamster P2- and human a2-adrenergic receptor, respectively). Other striking structural features of this D2 receptor, conimon to members of the superfamily of G-protein-coupled receptors, particularly the biogenic (catecho1)arnine receptors, are the following. First, the N-terminus contains consensus sequences for potential asparagine-linked glycosylation sites. This observation agrees well with previous biochemical data, indicating that the D2 receptor is a glycoprotein [88]. Second, within the predicted hydrophobic TM domains a number of acidic (e.g. an aspartate in TM domain I1 and one in the middle of TM domain 111) and hydrophilic (e.g. a cluster of serines in TM domain V) amino acids and several proline residues are present at conserved positions. The acidic and hydrophilic amino acids may play not only an important role in the binding of D2-receptor ligands, especially the aspartate in TM domain 111, but also in the maintaining of a functional conformation of the D2 receptor, especially aspartate in TM domain I1 [16-18,33,88,9 1,98- 10 11. Proline residues, which distort the a-helical stuctures due to their cyclic nature, have been postulated to be involved in the conformational changes, which are induced through the binding of ligands at G-protein-coupled receptors [33,90,91]. Third, two conserved extracellular cysteine residues might form a disulphide bridge that could affect ligand binding [16,18,90,91,98]. Fourth, the predicted size of the C-terminus is rather small and it possesses a conserved cysteine residue, possibly serving to anchor the receptor to the membrane through palmitoylation [16- 19,9 11. Fifth, the cytoplasmic loop between TM domains V and VI, likely to be involved in the interaction of the receptor with a G-

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protein, is quite large. These features of having a large third cytoplasmic loop and a short C-terminus seem to be characteristic of receptors that are coupled to a Gi-protein, and consequently, inhibit adenylyl cyclase activity [33,90,91].

Determination of the regional distribution of D2 receptor mRNA in the rat brain showed that the highest concentrations are found in the striatum, the nucleus accumbens, the olfactory tubercle, the substantia nigra pars compacta, and the ventral tegmental area [102-1081. These are the major projection areas of midbrain dopamine neurons as well as areas containing cell bodies and dendrites of these dopamine neurons. Keeping in mind that mRNA is localized primarily in cell bodies, this regional distribution indicates a postsynaptic as well as a presynaptic role for the D2 receptor.

The cellular localization of D~receptor mRNA in the rat striatum was also investigated, showing that approximately 50% of the medium-sized spiny GABA output neurons express D2-receptor mRNA. Preliminary, it was demonstrated that most of these neurons contain also the neuropeptide enkephalin, and belong to the striatal GABA output neurons projecting to the globus pallidus (in humans the external segment of the globus pallidus) (see 1.4.4) [109,110]. D2-receptor mRNA was also observed in large- sized cells of the rat striatum, the majority of which appear to be acetylcholine interneurons [ I l l ] . In addition, D2-receptor mRNA is present in the pituitary gland [107,108].

Shortly after the initial cloning of the mammalian D2 receptor, it was determined that this D2 receptor exists in two protein isoforms generated from the same D2- receptor gene by alternative RNA splicing [95,112-1201. This gene appears to be atypical as it contains six introns in its coding sequence, whereas those encoding most other G-protein-coupled receptors are intronless in their coding sequence. The two D2- receptor isoforms, termed D2s(hort) and D ~ L ( ~ ~ ~ ) , differ by the insertion of 29 amino acids in the putative third cytoplasmic loop (Figure 1.2). This insertion is encoded by a separate exon. Both two isoforms are present in all tissues where D2 receptors are expressed. Interestingly, the longer isofonn appears to be expressed predominantly, although the exact ratio of the two isoforms can vary significantly. Hence, some kind of functional difference was suggested between these two isoforms, especially concerning G-protein coupling and effector regulation due to the involvement of the putative third cytoplasmic loop. Recently, some evidence emerged in support of this hypothesis [121- 1231. Additionally, radioligand-binding studies revealed that several antipsychotic agents, including clozapine and substituted benzamides (see 1.3.3), interact with a 2- to 5-fold higher affinity at the D2s receptor than at the D ~ L receptor, whereas other dopamine-receptor agents fail to differentiate between the two isofoms [124-1261.

In 1990 four research groups, using different variations of the homology approach, reported the cloning of the classical D 1 receptor [127- 1301. They cloned from either rat or human origin a cDNA andlor gene encoding a protein of 446 amino acid residues in length (rat and human protein: 91% identical primary amino acid sequence) that, expressed in various mammalian cells, possesses the pharmacological hallmarks of a Dl

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receptor. Thus, this protein has the ability to bind selectively Dl-receptor ligands as well as the ability to stimulate adenylyl cyclase activity via coupling to a stimulatory G- protein (Gs-protein). Interestingly, the gene encoding this D l receptor possesses no introns in its coding sequence, like most genes encoding G-protein-coupled receptors.

Hence, there is no possibility to form protein isoforms by alternative RNA splicing.

Hydropathy analysis of the primary amino acid sequence of this D l receptor again revealed the existence of seven stretches of hydrophobic amino acids that may form TM domains. In addition, this Dl receptor possesses all the structural features common to catecholamine receptors, such as potential asparagine-linked glycosylation sites, conserved amino acids in the putative TM domains characteristic of catecholarnine recognition, a conserved extracellular disulphide bridge, and a conserved cysteine residue in the C-terminus, as shown in Figure 1.3. The degree of homology in the putative TM domains between the human Dl receptor and the human D2 receptor is about 45%. That is about the same as between this D l receptor and other catecholamine receptors. In contrast to the D2 receptor, the Dl receptor has a small putative third cytoplasmic loop and a long C-terminus. These features appear to be representative of Gs-protein-coupled receptors.

The highest concentrations of D l receptor mRNA are present in the major dopamine-receptive brain areas, such as the striaturn, the nucleus accurnbens, and the olfactory tubercle [106- 108,127,129-1331. In contrast to D~receptor mRNA, Dl- receptor mRNA is absent in the midbrain dopamine cell groups, i.e. the substantia nigra pars compacta and the ventral tegmental area, suggesting that there are no D l autoreceptors. This mRNA is also absent in the entopeduncular nucleus of the rat, or its

Figure 1.3 Model of the human D l receptor (for description, see Figure 1.2; taken with permission from ref. 16).

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human equivalent the internal segment of the globus pallidus, and in the substantia nigra pars reticulata, despite the hgh densities of Dl-receptor binding in these two structurally related brain areas, which are considered together as the major output structure of the striatum. This lack of correspondence between the distributions of D 1- receptor binding and Dl-receptor mRNA can be explained by the fact that receptor binding is localized on both cell bodies and fibers, while mRNA is thought to be primarily localized in cell bodies. Thus, e.g. the D l receptors in the substantia nigra pars reticulata are most likely synthesized in the cell bodies of striatonigral neurons and thereafter transported to their nerve terminals. Within the rat striatum, about 50% of the medium-sized spiny GABA output neurons express Dl-receptor mRNA [109].

Preliminary evidence indicates that these neurons project to the entopeduncular nucleus as well as the substantia nigra pars reticulata, and contain as neuropeptides substance P and dynorphin (see 1.4.4) [109, 1341. Thus, mRNA-distribution studies suggest the respective expression of D l and D2 receptors in striatonigral and striatopallidal neurons [109,135]. However, this distribution is not exclusive, i.e. a subset of striatal output neurons (20 to 40%) appears to express Dl as well as D2 receptors [107,135].

Additionally, a D2 receptor-related dopamine receptor was cloned, which was termed the D3 receptor [136-1391. The rat homologue of this D3 receptor contains 446 amino acids and shares 75% and 41% of its putative TM sequences with the rat D ~ L and the rat D l receptor, respectively. Surprisingly, the human homologue has 46 fewer amino acids in its putative third cytoplasmic loop. Excluding this deletion, the human D3 receptor is 88% homologous with the rat protein. This mammalian D3 receptor is encoded by a gene containing five introns in its coding sequence and whose organization is very similar to that of the D2 receptor (four introns in strictly similar positions). However, this organization does not allow the existence of Dyreceptor isoforms similar to the D ~ s - and D~L-receptor isoforms. By contrast, two shorter RNA splice variants were described which seem to be nonfunctional [140,141]. The proposed membrane topography of the D3-receptor is also highly similar to that of the D2 receptor. It contains a large third cytoplasmic loop, a short C-terminus that ends with a cysteine residue, and within the TM domains conserved amino acid residues characteristic of catecholamine recognition. Thus, genetically and structurally the D3 receptor is closely related to the D2 receptor.

Regional analysis of D3 receptor mRNA in the brain indicated that it is much less abundant and more narrowly distributed than D2-receptor rnRNA [108,136,142,143]. In general, D3 receptor mRNA is expressed predominantly in limbic areas including the olfactory tubercle-islands of Calleja complex, the nucleus accumbens, the bed nucleus of the stria terminalis and the mammillary nuclei of the hypothalamus. This suggests a major participation of D3 receptors in dopamine transmission in limbic areas known to be associated with cognitive, emotional and endocrine functions. Importantly, D3- receptor mRNA is also expressed at a low level in dopamine neurons of the midbrain dopamine cell groups, i. e. the substantia nigra pars compacta and the ventral tegmental

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area. Hence, the D3 receptor may play a role as dopamine autoreceptor in addition to the D2 receptor. Such a role is consistent with its pharmacological profile (see below).

Interestingly, Dyreceptor mRNA is almost undetectable in the pituitary gland, the prototypical localization of the D2 receptor.

Determination of the pharmacological profile of the D3 receptor, expressed in Chinese hamster ovary (CHO) cells, revealed that it poorly recognizes selective Dl- receptor agents, whereas it binds with good aff~nities selective D2-receptor agents [136,144]. However, like dopamine itself, several so-called selective D2-receptor agonists, such as quinpirole and quinerolane (see 1.3.2), display higher aff~nities at the D3 receptor than at the D2 receptor. In addition, 7-hydroxy-2-(N,N-di-n-propy1amino)- tetralin (7-OH-DPAT) (see 1.3.2), a proposed selective agent for D2 autoreceptors, was identified as a more selective D3-receptor agent of high affinity [145]. This observation in combination with the fact that dopamine-receptor agonists act preferentially at dopamine autoreceptors suggests that some functions attributed to dopamine- autoreceptor stimulation involve actually the D3 receptor. In agreement, (+)-AJ 76 and (+)-UH 232, classified before as preferential D2-autoreceptor antagonists (see 1.3.3), exhibit a limited D3-receptor selectivity (about 4-fold with respect to the D2-receptor) [136,144,146]. Other dopamine-receptor antagonists examined were between 2- and 30- fold more selective for the D2 receptor than for the D3 receptor. Thus, the D3-receptor pharmacology is similar but not identical to that of the D2 receptor.

Despite its structural similarity to the D2 receptor including the features characteristic of inhibitory linkage to adenylyl cyclase activity, the D3 receptor expressed in CHO cells was shown to lack the ability to affect adenylyl cyclase activity [136]. In addition, the in this manner expressed D3 receptor failed to influence other G- protein-dependent signal transduction mechanisms, such as the phospholipase A2larachidonic acid system, known to be enhanced by the CHO cell-expressed D2 receptor after initial stimulation by intracellular ~ a 2 + [147,148]. To explain this discrepancy it was suggested that t h ~ s type of cells lacks the appropriate G-protein involved in Dyreceptor signalling or that the D3 receptor couples to a still unidentified signal transduction system.

Recently, two more doparnine receptors were cloned, i.e. another D2 receptor- related one, termed the Dq receptor [149,150], and a Dl receptor-related one, termed the Dg, Dip, or D ~ B receptor [151-1541.

The human Dq receptor appears to comprise a protein of 387 amino acids in length with a proposed membrane topography very similar to that of the D2 receptor [149].

However, its putative third cytoplasmic loop is quite short compared to the equivalent loop of the D2 receptor. In its putative TM domains, the human Dq receptor is approximately 55% identical to the human D2 receptor as well as the human D3 receptor (42% identical to the human D l receptor) and contains the amino acids thought to be necessary for catecholamine recognition [19,149]. The coding sequence of the human Dq-receptor gene is interrupted by three introns, equivalently positioned as three

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of those in the D2-receptor gene. Moreover, genetic polymorphism has generated, very likely by internal duplication events, allelic forms of the human Dq-receptor gene [I%].

The human Dq-receptor variants encoded by these alleles differ in the number of sequence repeats, 16 amino acids in length, contained in their putative third cytoplasmic loop. Variants harboring two (the original cloned human Dq receptor), three, four, five, and seven of those sequence repeats have been identified. These repeats are absent in the rat Dq receptor [19,150]. Altogether, it appears that the Dq receptor is related to the D2 receptor, yet more distantly than the D3 receptor.

When expressed, the human Dq-receptor displays similar or lower affinities for dopamine-receptor agents than the human D ~ L receptor [149]. Unexpectedly, the human Dq receptor binds clozapine (see 1.3.3), an antipsychotic agent without extrapyramidal side-effects, with a 10-fold higher affinity than the predominantly expressed long isoform of the D2 receptor [149]. As with D3-receptor mRNA, Dq- receptor mRNA is less abundantly expressed than D2-receptor mRNA. The highest levels of Dq-receptor mRNA are found in the frontal cortex, midbrain areas, the amygdala, and the medulla, areas associated with psychotic etiologies, while a very low level of this mRNA is observed in the striaturn, the site of motor control [149,156,157].

Thus, clozapine's lack of extrapyramidal side effects may be a reflection of Dq-receptor localization in the CNS. Recently, however, it was shown that clozapine binds with reasonable aff~nity at the human D2s receptor [126]. This suggests that clozapine is not as selective at the Dq receptor as reported before. Hence, it seems premature to designate the Dq receptor as the only target mediating the antipsycotic action of clozapine [158]. Nevertheless, it was reported very recently that in schizophrenic patients, even "drug-free" patients, the Dq receptor density is elevated 6-fold in comparison to normal subjects [159]. If this observation can be confiied, it can play an important role on the way to a better understanding of schizophrenia.

Using the human Dl-receptor coding sequence as a hybridization probe a human D 1 -receptor-related dopamine receptor, termed the Dg (or D 1

P)

receptor, was cloned [151-1531. This Dg receptor, 477 amino acids in length, displays all the structural and topographical features common to the Dl receptor. Especially, a small putative third cytoplasmic loop and a long C-terminus are characteristic. Within the TM domains the level of homology between these two dopamine receptors is very high, approximately 80%. Moreover, similar to the Dl-receptor gene, the D5-receptor gene lacks introns in its coding sequence. Interestingly, the human genome appears to contain two genes which share almost 100% homology with the Dg-receptor gene. However, due to insertions and deletions resulting in in-frame stop codons, both genes are incapable of encoding functional dopamine receptors, and consequently, are genuine Dg-receptor pseudogenes [152,153,160].

When expressed, the human Dg receptor binds dopamine-receptor agents with a pharmacological profile similar to that of the Dl receptor, but it displays a 10-fold higher affinity for dopamine. Additionally, stimulation of the expressed human Dg

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receptor gives rise to an increase in CAMP formation, proving the coupling of this doparnine receptor to a Gs-protein, analogous to the D 1 receptor [15 1- 153,16 11.

However, the hgher affmity of dopamine at the human D-j receptor is not associated with a higher potency for the stimulation of adenylyl cyclase activity [161].

Recently, the rat homologue of the human Dg receptor, termed the D ~ B receptor, was characterized [154]. This rat Dg receptor, although 95% homologous within the putative TM domains to the human D-j receptor, does not display a pronounced hlgher affinity for dopamine than the rat D l receptor. Determination of the regional distribution of D5-receptor mRNA in rat brain revealed that this distribution is very distinct fiom that of the D l receptor: little expression is found in the striaturn, the nucleus accumbens and the olfactory tubercle, areas in which Dl-receptor mRNA is abundant. In contrast, high levels of Dg-receptor mRNA are expressed in the hypothalamic lateral marnmillary nuclei, the thalamic parafascicular nucleus and several hippocampal layers [154,162]. This restricted distribution suggests that the D5 receptor may have a function quite dissimilar fiom other dopamine receptors, e.g. in the modulation of the thalamic processing of painful stimuli [162].

Based on the molecular biological properties, such as gene organization, proposed overall membrane topography, and percentage identical amino acids within the putative TM domains, as well as the present pharmacological profiles of the up-to-now cloned dopamine receptors, as shown in Table 1.1, two main subfamilies of dopamine receptors can be distinguished, i.e. doparnine "Dl-like" and "D2-like" receptors. These subfamilies comprise currently the D l and Dg receptor and the D2, D3, and Dq receptor, respectively. A major advantage of this differentiation is the maintenance of consistency with the classical biochemicaVpharmacological classification scheme (see 1.2.2).

Consequently, Sibley and Monsma proposed a hierarchical nomenclature for the dopamine-receptor family [17]. They took the well-defined Dl- and D ~ r e c e p t o r subtypes for which prototypical receptors have been cloned (see above) as starting-point for their nomenclature. Thus, in their opinion, if the pharmacological profile of a cloned dopamine receptor is highly similar and the amino acid-sequence homology within the putative TM domains is greater than 50% compared with one of these prototypical receptors, then it should be designated as a member of that subfamily using an A,B,C etc. nomenclature. Using this method, they designated the Dl and D-j receptor as the D ~ A and DIB receptor, and the D2, D3, and Dq receptor as the D ~ A , D ~ B , and D2c receptor, respectively. If a novel dopamine receptor is cloned that does not meet these criteria, then it will constitute the prototypical member of a new subfamily of dopaminereceptors, with designation D3, Dq etc. However, reviewing the percentage of identical transmembranal amino acids between the Dq receptor on the one hand and the D2 and D3 receptor on the other hand (Table 1.1), maybe it is better to insert one additional hierarchical step in the molecular biologicaVpharmacological classification of dopamine receptors, as shown in Figure 1.4.

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Table 1.1 Properties of cloned dopamine receptors. a Based on ref. 19. b See text. See 1.3. Binding of dopamine is guanyl nucleotide-sensitive. e See ref. 17. f see Figure 1.4.

GENE

human chromosome 5q 1 lq 3q 1 1~ 4~

introns in coding sequence 0 6 5 3 0

selective gene expression

--

altern.splicing -- human polyrnorph, human

" 2 ~ ~ ~ 2 ~ (D4.2,D4.4,D4.7) pseudogenes PROTEIN

amino acids : human 446 414/443 400 387 477

rat 446 4 15/444 446 385 475

third cytoplasmic loop small large large large small

C-terminal tail long short short short long

sequence I) 1 100 47 45 42 82

identity (%) D2 100 77 5 1 44

in human D3 100 54 40

TM domainsa: D4 100 45

"5 100

mRNA

highest brain densitiesb striatum striatum olfacttuber. medulla h~ppocampus n.accumb. n.accumb. islands of Call. amygdala rnammil.nuc1.

01fact.tuber. 01fact.tuber. n.accumb. frontal cortex parafasc.nuc1.

amygdala subst.nigra mammil .nucl. midbrain ventr.tegm.

PHARMACOLOGY

characteristic agentsC SCH 23390 spiperone spiperone spiperone SCEI 23390 SKF 38393 remoxipride 7-OH-DPAT clozapine SKF 38393

AJ 76,UH 232 PD 128907 BIOCHEMISTRY

G-protein couplingd Yes Yes ? Yes Yes

adenylyl cyclase stimulation inhibition ? ? stimulation

NOMENCLATURE

two-step hierarchicale D l ~ D2A D 2 ~ " 2 ~ D l ~

three-step hierarchicalf D 1~~ D2Aa D2Ap D2Ba " l ~ p

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classification criteria:

+ gene organization and membrane topography

+ transmembranal

- - -

homology

?

+ ~ndrv~dual varratlon and pharmacologrcal profile

Dl, D1,p

'

D,& DMp ? ,D, ? ?

( 4 ) (D5) (02) (D3) (D4)

Figure 1.4 A possible molecular biologicaVpharmawlogica1 classification of dopamine receptors Although unexpected a few years ago, the multiplicity of the dopamine receptor family is not totally surprising in view of the complexity of the other receptor families which are part of the superfamily of G-protein-coupled receptors. How many more dopamine receptors will be found is unknown. However, it seems likely that still not all dopamine receptors have been cloned, because the presently cloned dopamine receptors do not exhibit all the properties previously attributed to dopamine receptors [17,36].

Importantly, this dopamine receptor multiplicity may provide the opportunity to develop dopamine-receptor agents as new therapeutic agents, which have fewer side-effects due to a better dopamine-receptor subtype selectivity.

Since the discovery that dopamine fulfills its function as central neurotransmitter through the interaction with specific dopamine receptors, many pharmacological agents have been identified which interfere directly with this event (for reviews and references, see ref. 11,13,15,163-169). From a functional point of view, these dopamine-receptor agents can be divided into dopamine-receptor agonists and dopamine-receptor antagonists (see 1.2.1). However, this division is complicated by the existence of dopamine-receptor mixed agonists/antagonists, also referred to as dopamine-receptor partial agonists. Beside this division, dopamine-receptor agents can also be classified on the basis of their selectivity for the proposed subtypes of dopamine receptors (see 1.2).

In particular, the identification of selective dopamine-receptor agents for the two classical biochemical subtypes of dopamine receptors (see 1.2.2), was a landmark in the research on doparnine-receptor agents. In the near future the recently identified

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molecular biological multiplicity of dopamine receptors (see 1.2.4) will very llkely have a major impact on this area of research. Up-to-now, however, although some knowledge about the selectivity of dopamine-receptor agents for the five currently cloned dopamine receptors has been gathered, not many very selective dopamine-receptor agents were identified for the newly discovered subtypes of dopamine receptors.

Due to the abundance of dopamine-receptor agents developed, the following sections on dopamine-receptor (partial) agonists and antagonists do not have the intention of a review, but merely to highlight some particularly interesting compounds.

Additionally, one should bear in mind that the next sections are based on data mostly retrieved before the recent expansion of the dopamine-receptor family. Thus, if selectivity is brought up, it is about selectivity for the classical subtypes of dopamine receptors. If this is not the case, it will be stated explicitly.

1.3.2 DOPAMINE-RECEPTOR AGONISTS AND PARTIAL AGONISTS

One of the prototypical dopamine-receptor agonists is (6aR)-(-)-apomorphine (la).

In fact, h s semi-synthetic compound was the first compound shown to mimic potently the action of dopamine [170]. (6aR)-(-)-Apomorphine (la), which bears the dopamine moiety in its a-rotameric conformation,* is generally considered as a mixed DllD2- receptor agonist. However, close examination of its pharmacological effects showed that this compound acts as a full agonist at the D2 receptor, while it behaves as a partial agonist at the D l receptor [171]. Additional studies showed that its affinity at the D2 receptor is several times higher than at the Dl receptor 11721. Originally, the optical antipode (lb) was shown to be inactive as a dopamine-receptor agonist [173]. Later studies revealed that this compound acts as a weak antagonist at the D l receptor as well as the D2 receptor [171]. In contrast, both enantiomers of isoapomorphine (2a,b),

* In 1975 Cannon proposed that dopamine might interact with its receptors in the two possible conformational extremes of the trans coplanar form, which he designated as the a- and P-rotameric conformations. In both rotamers the ethylamine side chain displays coplanarity with the aromatic ring and possesses an extended conformation. In the a-rotamer the meta-hydroxyl group is projected over the ethylamine side chain, whereas in the p-rotamer the meta-hydroxyl group is directed away from the ethylamine side chain [ I 5,163-1651.

OH a-rotamer

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Chart 1.3 Chemical structures of (6aR)-(-)-apomorphine (la), (6aS)-(+)-apomorphine (lb), (6aR)- (-)-isoapomorphine ( 2 4 , and (6aS)-(+)-isoapomorphine (2b).

containing the dopamine moiety in its p-rotameric conformation, are inactive as dopamine-receptor agonists or antagonists [174,175].

SAR-studies on analogues of (R)-(-)-apomorphine (la) revealed that (R)-(-)-N-n- propylnorapomorphine (3), (R)-(-)- 1 l-hydroxyaporphine (4) and (R)-(-)- 1 1 -hydroxy- N-n-propylnoraporphine (5) are potent dopamine-receptor agonists. The last two com-

Chart 1.4 Chemical structures of (R)-(-)-apomorphine analogues (3-5)

pounds contain only a meta-hydroxyl group.

In

contrast to (R)-(-)-apomorphine (la), (R)-(-)-N-n-propylnorapomorphme (3) acts as a full agonist at both dopamine-receptor subtypes [171]. Moreover, both N-propyl analogues (3,s) possess more than a 10-fold higher a f f i t y at the D2 receptor than (R)-(-)-apomorphine (la), while the affinity at the D l receptor stays almost the same [172,176]. In this respect, several 2-substituted analogues of (R)-(-)-N-n-propylnorapomorphine (3), such as (R)-(-)-2-hydroxy-N-n- propylnorapomorphine (6) and (R)-(-)-2-fluoro-N-n-propylnorapomorphine (7), are highly selective D2-receptor agonists [177,178]. Particularly, (R)-(-)-2-fluoro-N-n- propylnorapomorphine (7) has the highest D2-receptor affinity and D2/D1-receptor selectivity of any agent yet described [179]. Recently, it was also shown that (R)-(-)- I 1 -hydroxyaporphine (4) acts as an antagonist at the D 1 receptor, while it acts as an agonist at the D2 receptor. In fact, all of the (R)-(-)-1 l-monohydroxyaporphmes studied, such as the 10-bromo analogue (8), behave as Dl-receptor antagonists [180].

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Chart 1.5 Chemical structures of (R)-(-)-2-hydroxy-N-n-propylnorapomorphie (6), (R)-(-)-2-fluoro- N-n-propylnorapomorphine (7), and (R)-10-bromo-1 1-hydroxyaporphine (8).

Another prototypical dopamine-receptor agonist is (25)-(-)-5,6-dihydroxy-2-(N,N- di-n-propy1amino)tetralin (9). It is not only a chiral congener of the fkagment of (6aR)- (-)-apomorphine (la), believed to be responsible for its dopamine-like activity, but also a cyclic, semi-rigid analogue of dopamine in its a-rotameric conformation [ 15,164,165, 167,1691. This optical-active 2-aminotetralin derivative acts as a very potent mixed DlKI2-receptor agonist with full intrinsic efficacy at both subtypes of dopamine receptors. The (2R)-(+)-enantiomer is much less active as dopamine-receptor agonist. In sharp contrast, the (2R)-(+)-enantiomer of the isomeric, dopamine P-rotamer-containing 6,7-dihydroxy-2-(N,N-di-n-propy1amino)tetralin (lo), which displays also prominent dopamine-receptor agonism, is more active than its (28-(-)-optical antipode.

Chart 1.6 Chemical structures of (29-(-)-5,6dihydroxy-2-(N,Ndi-n-propy1o)rain (9), (2R)- (+)-6,7-dihydroxy-2-(N,N-di-n-propyl- (lo), (29-(-)-5 -hydroxy-2-(N,N-di-n- propy1amino)tetralin (ll), and ( 2 R ) - ( + ) - 7 - h y d r o x y - 2 - ( N , N - d i - n - p r o p y l ~ l i n (12).

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