Progress in NeurobiologyVol. 34, pp. 387 to 400, 1990 Printed in Great Britain.All rights reserved

0301-0082/90/'$0.00+ 0.50 ~ 1990PergamonPress plc

DOPAMINE UPTAKE: A REVIEW OF PROGRESS IN THE LAST DECADE A. S. HORN* University Centre for Pharmacy, 2, Antonius Deusinglaan, University of Groningen, Groningen, The Netherlands

(Received 6 November 1989)

CONTENTS I. Introduction 2. Effects of various drugs on [3H]DA uptake 2.1. Cocaine 2.2. MPTP (l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine) 2.3. Psychotomimetics 2.4. Antidepressants 2.5. Miscellaneous drugs 2.6. Up-regulation of the DA transporter by drugs 3. Effects of metallic ions on [3H]DA uptake 3.1. Lead 3.2. Miscellaneous metals 4. Effects of hypoxia and ischemia on [~H]DA uptake 5. Radioligand binding studies and DA uptake sites 5. I. [3H]Cecaine 5.2. GBR derivatives 5.2.1. [3H]GBR 12935 5.2.2. [3H]GBR 12783 5.3. Miscellaneous radioligands 6. The stability of in vitro preparations used to study [3H]DA uptake 7. The uptake of [~H]DA into the median eminence and posterior pituitary 8. Studies on the uptake of [3H]DA into miscellaneous tissues and preparations 9. Possible relationships between DA uptake and release I0. In vivo studies on DA reuptake in animals II. DA uptake inhibitors and behaviour 12. [3H]DA uptake into human blood platelets 13. The DA uptake system in the human brain 14. The molecular nature of the DA transport system 15. Conclusions and future prospectives References

1. INTRODUCTION The first review articles devoted solely to the properties of the dopamine (DA) uptake system were published some years ago (Horn, 1976, 1978). The fact that the last review (Horn, 1979) appeared 10 years ago means that a survey o f some recent trends is long overdue. However, due to limitations of space only certain highlights of the recent literature can be covered. This article is therefore by no means an exhaustive review of the literature of DA uptake over the last decade. The general interest in DA uptake has increased in recent years and this is not surprising bearing in mind the fact that this neuronal transport system is probably the most important mechanism for the inacti*Deceased 2 January 1990.

387 387 387 388 389 389 389 389 390 390 390 390 390 390 391 391 391 391 391 392 392 392 393 393 394 394 395 395 396

vation of the synaptic effects of DA (Horn, 1979). Early work on the anatomical, biochemical and pharmacological properties of DA uptake and its inhibition have been dealt with in the above mentioned reviews. A brief summary of some of these properties is shown in Table I. 2. EFFECTS OF VARIOUS DRUGS ON [31-1IDA UPTAKE 2. I. COCAINE It is well established that cocaine (Fig. I) inhibits DA uptake and due to its status as a popular drug of abuse much effort has been devoted to elucidating its precise neurochemical mode of action (Dackis and Gold, 1985). Ritz et al. 0957) have shown that the potencies of cocaine-like drugs in self=administration 387

A.S.

HOR~

388

TABLE [. CHARACTERISTICSOF THE DOPAMINE TRANSPORTER

Kinetic parameters--uptake K~ (rat striatum) = I/~,, = Km (human caudate)= Vm,. = Tissue/medium =

1.3 × 10- st 25.3 pmol 100~g protein. 2 min 1.85 × 10-' ~t 38.47 pmol mg protein/10 rain 170 (actual gradient may 1000: 1)

Na + dependent Kinetic parameters--binding [3H]GBR 12935 (rat striatum) KD = B~x = {}H]GBR 12935 (human caudate) Ko B,~ = Na + dependent Highest density of binding, corpus striatum (rat) Radioligands available

0.82 n,~t 5.54 pmol/mg protein 3.4 nM 2.7 pmol/mg protein

[3H]GBR 12783, GBR 12935, cocaine, methylphenidate, mazindol and nomifensine

Potent and selective uptake inhibitors

GBR 12909, GBR 13069

Biochemical composition

Glycoprotein?

studies correlate with their potencies in inhibiting [3H]mazindol binding to the DA transport system in rat striatum. They propose that the cocaine receptor related to drug abuse is associated with DA uptake inhibition. Similar conclusions have been reached by Calligaro and Eldefrawi 0988) who investigated [~H]cocaine binding to striatal membranes. They found an excellent correlation between IC~o values for the inhibition of [3H]cocaine binding to rat striatal membranes and [3H]DA uptake into striatai synaptosomes. They suggest that the cocaine receptor may be an allosteric protein with mazindol and cocaine binding to overlapping sites, while Na + and DA are allosteric modulators which stabilize a lower affinity state for cocaine.

Me .~ N~.

2.2. MPTP ( i -METHYL-4-PHENYL-1,2,3,6-TETi~HYDROPYRIDINE) MPTP (Fig. 2) is a neurotoxin which has been shown to cause a Parkinson, like syndrome i n m a n and monkeys characterized by an extensive ~ selective degeneration of the nigro-striatal DA neuronal system (for reviews se¢ Langston and Irwin (1986) and Kinemuchi et al. (1987). Extensive research by various groups has shown that oxidative metabolism of MPTP by monoamine oxidase (MAO) B with the formation o f the toxic metabolite 1-methyl4-phenylpyridin¢ (MPP +) (Fig. 2) is an essential step in the neurotoxic process. This is thought to occur mainly outside the dopamin-

_COOMe

o Cocaine

0 ~CH Benztrol~ine

X X . H , Y . "CH2"CH- CH',Z =H.GBR 12783 X . F , Y . "{CH2)3-,Z .H.GBR 12908 X . H,Y. "{CH2}3".Z . H,GBR 12935 X.F.Y.-CH2-CH,.CH-,Z=H

GBR13069

X . F , Y . "(CH2)3",Z . F GBR 13098

~'Ma NH 2

Nomifenline CI Mazindol

FiG. I. Chemical structures of some DA uptake inhibitors.

DOP~tN~ Ui*t'AI¢~

0

[~ ~ M e

NH2

O

~~'~"-O

H

I,,,,.Mo Amphetamine

Amphonelic acid

COOMe

Melhylpheni(:lale

Me

Me

MPTP

MPP •

Fro. 2. Chemical structures of compounds interacting with the DA transporter. ergic neuron (Kinemachi et al., 1987). Several groups have shown that [~H]MPP + can De accumulated selectively to high levels within DA neurons via the dopaminergic uptake system (Javitch and Snyder, 1985; Javitch et al., 1985; Chiba et al., 1985). The Km and Vm,x values are similar to those found for DA itself. It is thought that MPP + brings about its neurotoxic effects once inside the neuron by an action on the mitochondria (Langston and Irwin, 1986; Kinemuchi et al., 1987). As would be expected in vivo damage to nigrostriatal DA neurons can be prevented by pretreatment with DA uptake inhibitors such as nomifensine, amfonelic acid, cocaine and GBR 13098 (Fig. 1) (reviewed by Kinemuchi et al., 1987). In contrast no protection was afforded by selective serotonin or noradrenaline uptake inhibitors (Melamed et al., 1985). The effects of MPTP on DA uptake sites have also been studied using [3H]mazindol as a radioligand (Donnan et al., 1986). Baldessarini et al. (1986) have investigated a series of phenylpiperidines structurally related to MPTP for their abilities to inhibit [3H]DA uptake. Although certain analogues were more potent than MPTP itself, the compound with the highest affinity was MPP +. In a study in two rodent species, Jossan et aL (1989) were unable to find any correlation beween MAO-B activities or DA uptake rates and the neurotoxic effect of MPTP. 2.3. PSYCHOTOMIMETICS In vivo administration of 0.1 and 0.5 mg/kg of LSD to rats induced an increase in the in vitro uptake of [14C]DA into synaptosomal fractions from the nucleus accumbens (Hetey and Quiring, 1980). Pbencyclidine is known to inhibit DA uptake into synaptosomes (Garey and Heath, 1976; Smith et al., 1977) and Ary and Komiskey (1980) have shown that high concentrations (ICs0 -- 33/ZM) also inhibit the vesicular accumulation of [3H]DA. Replacement of the phenyl ring in PCP by a benzothiophenyl group leads to a more potent inhibitor of DA uptake (ICso -- 7 riM) (Vignon et al., 1988). From a group of 37 phencyclidine analogues, a new compound BTCP, was found which is a very potent and selective inhibitor of [3H]DA uptake. In contrast,

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it has a much lower affinity for the PCP receptor (Chandieu et al., 1989). Although ketamine is not normally classed as a psychotomimetic it can produce hallucinatory disturbances in man and it is related structurally to PCP. The ( + ) isomer of ketamine has been shown to be a more potent inhibitor of DA uptake than the ( - ) enantiomer (Smith et al., 1981). A series of 13 hallucinogens were investigated in vitro by Whipple et al. (1983) who found that the indolealkylamines were generally more potent than the phenylethylamines. LSD and 2-bromoLSD were inactive. LSD, DMT and mescaline have been reported to enhance DA uptake/n vitro (Hetey et al., 1985). 2.4. ANTIDEPRF..S$ANTS A very useful comparative survey of the inhibition of uptake selectivity of various antidepressants (and in addition neuroleptics) is provided by Bogeso et aL (1986). This complements an earlier study from the same source (Hyttel, 1978). Other compounds of this class which have been investigated included FS32 and FS97 (Koide and Uyemura, 1980), Lu 19-005, which is a very potent inhibitor of DA, NA and 5-HT uptake (DA, ICs0--0.99nM) (Hyttel and Larsen, 1985) and nomifensine (Broch, 1987). In the case of the latter compound it was pointed out that in contrast to most antidepressants currently in clinical use, it is a good inhibitor of DA uptake. Using a double labelling technique Lemasson et aL (1984) have concluded that it appears that various antidepressants in the range l0 -~ to l0 -5 M exert an apparent inhibition of DA uptake which in fact corresponds to a DA release. The latter effect is probably due to a transient physicochemical mechanism and does not involve the DA carrier or exocytosis. 2.5. MISCELLANEOUSDRUGS Various other compounds have been examined recently for their effects on [3H]DA uptake/n vitro. Estrogen agonists and antagonists (Michel et aL, 1987), branched chain amino acids (Cobert et aL, 1985) tetrahydro-~-carbolines (Komulainen et aL, 1980), polyamines (Law et aL, 1984), amfonelic acid (Schmidt and Gibb, 1985), monofluoramphetamine (Benderly et aL, 1981), rigid DA analogues (Tuomisto and Tuomisto, 1987), tetrahydroisoquinolines (ZaraKaczian et al., 1986), aminepetine (Bonnet et al., 1987), phenylethanolamine N-methyl-transferase inhibitors (Liang et al., 1985), p-chlorophenylalanine (Pazdernik and Tuomisto, 1983), antihistamines (Brown and Vernikos, 1980; Gupta et al., 1982), local anesthetics (Bradford and Marinetti, 1982), and last but not least a peptide-containing fraction from the urine of schizophrenics (Hole et al., 1979). 2.6. UP-REGULATION OF THE D A TRANSPORTER BY DRUGS

It has been shown by Hanbauer et al. (1984) that chronic adminstration of cocaine induces increased [3H]cocaine binding to the striatal DA uptake system.

390

A. S, Hot~x

In a similar manner Wiener et al. (1989) have found that chronic L-deprenyl treatment of mice (10 mg/kg i.p. twice weekly for 4 weeks) induced an up-regulation of the [3H]maxindol binding site associated with the striatal DA carrier. 3. EFFECTS OF METALLIC IONS ON [3H]DA UPTAKE 3.1. LEAD Due to its well documented effects on behaviour and brain neurochemistry (Singhal and Thomas, 1980) there has been considerable interest in the effects of lead on DA uptake. Chronic exposure of mice to inorganic lead from birth produced hyperactive animals which displayed significant decreases in in vitro high affinity DA transport (Silbergeld and Goldberg, 1975). Similar effects were found following in vitro exposure of striatal synaptosomes to high concentrations of lead (Silbergeld, 1977). Analogous studies in rats were unable to confirm the above results, i.e. no significant differences were found in DA uptake following chronic lead exposure (Ramsay et al., 1980). The latter authors suggested that their more strignent kinetic criteria or species differences might account for this discrepancy. These negative results were also supported by in vitro work reported by Komutainen and Tuomisto (1981) who found that 100/~m Pb -'+ did not inhibit [3H]DA uptake. There was a tendency to increased uptake of [~H]DA under these conditions. In a subsequent study Komulainen et al. (1983) showed that acute in vivo administration of toxic doses of lead acetate, triethyl lead or tetraethyl lead resulted in an increased in vitro uptake of [3H]DA into striatal synaptosomes. The mechanism of this effect was unclear. 3.2. MISCELLANEOUS METALS Lai et al. (1981) showed that Cd"*, Mn :+ and AP + were all able to inhibit the in vitro uptake of [3H]DA into striatal synaptosomes; Mn:* was the least effective. Komulainen and Tuomisto (1981) found that a concentration of 10 ././M, C d 2+, C o 2+ , Hg 2+, Mn 2÷ and Sn 2+ had no influence on the uptake of [3H]DA. However, Cu '+ at this concentration produced almost complete inhibition. Leung et al. (1982) found that chronic manganese treatment of rats from conception onward resulted in an increase in striatal uptake of [3H]DA. Lithium treatment (2mequiv/kg/day) of rats resulted in an increase in the uptake of DA and the authors suggest that the therapeutic effect of lithium may be related to alterations in the uptake processes for monoamines (Ahluwalia and Singhal, 1981). 4. EFFECTS OF HYPOXIA AND ISCHEMIA ON I3HIDA UPTAKE Due to the sensitivity of the nervous system to even short periods of hypoxia and ischemia, several groups have investigated the effects of these treatments on DA uptake.

Hypoxic incubations (O: tension 30 mmHg instead of 530mmHg) of guinea pig striatal slices with [3H]DA resulted in a lowering of the V~, by one-third and an unchanged Km value (Saijoh et aL. 1985). The authors suggested that the change in the V~, value could be due to a change in the number of transport sites or a change in the turnover rate of transport. Odarjuk et al. (1987) found that a single exposure of adult rats to hypoxia leads 'to short-term changes in DA uptake. The affinity was reduced (269% of control) whilst the Vm~~ increased up to 301%, resulting in an increase of total uptake of [~HIDA into striatal synaptosomes. One week of recovery resulted in a normalization of these parameters. In a gerbil model of ischemic stroke no change in [3H]DA uptake was seen for up to 8 hr (Weinberger and Cohen, 1982). However, at 16 hr marked impairments of DA uptake were found, i.e. to 15.2% of control values. The dopaminergic system was found to be more sensitive in this model than neurons containing glutamate.

5. RADIOLIGAND BINDING STUDIES AND DA UPTAKE SITES It is interesting that although the striatal DA uptake system was characterized in vitro at the end of the 1960s by Snyder's group using [3H]DA, it was not until the beginning of the 1980s that attention was focused on radioligands and the DA transporter. There are at least two reasons for this. First, in the intervening years attention was directed to the characterization of monoamine receptor sites in the CNS. Secondly, as in other areas, suitable ligands had to be developed and then prepared in a radioactive form.

5.1. [3H]CoCAINE The saturable binding of [3H]cocaine to mouse brain membrane fractions was first reported by Reith et ai. (1980). In 1983 Kennedy and Hanbauer investigated the relationship between [~H]cocaine binding sites and the DA uptake system in rat striatum. Several pieces of evidence suggested that the two parameters are related. Sodium ions increased the Bin,~ selectivity in the striatum. 6OHDA (but not kainic acid) lesions prevented the Na*-induced increase in [3H]cocaine binding. A strong correlation was found between the ability of various drugs to inhibit DA uptake and block pH]cocaine binding. Finally saturable binding could be demonstrated in the same ionic environment in which cocaine inhibited DA uptake. In the presence of 50 mM NaCI these authors report a B ~ x = 13+ 1.4pmol/mg protein and a KD = 0.36 + 0.03/~M. In a somewhat similar study Sehoemaker et al. (1985) confirmed the above results in rat striatum (Bronx= 5.56 pmol/mg protein, KD = 0.36 #M) and in addition were able to demonstrate that the Na*dependent, high affinity binding of [3H]cocaine to membrane fractions of human putamen showed characteristics identical to those in the rat striatum. In putamens obtained from parkinsonian subjects the binding was prominently decreased.

Dop^ua~ U~I'AK~ The work of Calligaro and Eldefrawi (1988) on [3H]cocaine binding has already been mentioned (see Section I.I). 5.2. GBR DERIVATIVES An extremely important advance in DA uptake research was the development of a new class of very potent and selective DA uptake inhibitors by van der Zee et al. (1980), i.e. the GBR series of compounds (Fig. 1). The best characterized analogue of this series to date is GBR 12909, which has been extensively investigated by Anderson (1989). He was able to show that GBR 12909 is a highly selective inhibitor of DA uptake both in vivo and in vitro. In addition he suggested that it may find use as an antidepressant. 5.2.1. [3H]GBR 12935 Paul's group (Berger et al., 1985; Janowsky et al., ! 986) showed that [3H]GBR 12935 is a suitable ligand for selective labelling of the DA transporter. Their criteria were similar to those used to study [3H]cocaine binding. In a study of [3H]GBR 12935 binding reported by Anderson (1987), a KD=5.5nM and a Bma~= 760 pmol/mg tissue were found. Two binding sites were detected, one corresponded to the DA uptake site and the other was apparently a piperazine acceptor site. There was a good correlation between DA uptake inhibitor and blockade of [3H]GBR 12935 binding. Using the technique of quantitative in vitro receptor autoradiography, Dawson et al. (1986) found that [3H]GBR 12935 binding sites were localized in several discrete areas corresponding to structures in the mesostriatal and mesolimbocortical systems of the rat brain. There was a marked correlation between the distribution of these binding sites and DA nerve terminals as well as cell bodies. In studies on human post-mortem caudate nucleus Janowsky et al. (1987) found that the specific binding of [3H]GBR 12935 had a Bm~ = 1.36 + 0.18 pmol/mg protein and a KD = 2.34_+0.18 riM. There was a good correlation between the potencies of a series of drugs in displacing pH]GBR 12935 binding to human cuadate and rat striatum as well as in inhibiting DA uptake in rat striatal synaptosomes. As in the case of [3H]cocaine binding (Schoemaker et al., 1985), [3H]GBR 12935 binding was decreased in the caudates of parkinsonian patients. The specific in vitro binding of [3H]GBR 12935 to various areas of human brain was investigated by Marcusson and Eriksson (1988). As expected DA uptake inhibitors were potent displacers but D t and D2 receptor agonists had much lower affinities. The regional distribution of the binding corresponded to the known distribution of DA in the rat brain. The highest binding densities were in the caudate nucleus and putamen (Bin,~ 1500-2000 fmol/mg protein). The KD was I-1.5 riM. 5.2.2. [3H]GBR 12783 This ligand, which has been used mainly by Costenins's group, differsfrom G B R 12935 only-in the presence of a double bond (Fig. I) Bonnet et al.

391

(1986) in similar studies to those reported for [3H]GBR 12935 in rat brain, found a KD = 1.6 nM and a Bm~ = 10.3 pmol/mg protein for this ligand. Bcnmansour et al. (1987) investigated the composition of the incubation medium used for such binding studies and found that Tris and Krebs-Ringer buffers contain ions which can exert an inhibitory effect on the binding of [3H]GBR 12783 at low sodium concentrations. The selectivity of GBR 12783 for the DA uptake system was demonstrated by Bonnet and Costenin (1986) in a series of in vivo and ex vivo experiments. In further in vivo studies in mice, Chagraoui et al. (1987) found that following an i.v. injection of [3H]GBR 12783 the difference in binding between the stdatum and the cerebellum was maximal after I hr. The accumulation of radioactivity in the striatum was unaffected by high doses of DA receptor agonists or antagonists and of 5-HT or NA uptake inhibitors. Intrastriatal injections of 6OHDA resulted in a similar decrease in both synaptosomai [3H]DA uptake and in vivo [3H]GBR 12783 binding. Leroux-Nicollet and Costenin (1988) have found that the specific in vivo labelling observed after systemic injection of [3H]GBR 12783 in mice displays essentially the same cerebral distribution as that observed in rat brain slices. The post-mortem modifications in synaptosomal [3H]DA uptake and [3H]GBR 12783 binding in the rat striatum were investigated by Thibaut et al. (1989). They found that the uptake of [3H]DA was completely abolished after 24 and 72 hr storage at 21°C and 4°C respectively, whereas the binding of [3H]GBR 12783 was only slightly decreased. The Km and KD were virtually unchanged whilst the Vma~ and Bma~ were markedly decreased. 5.3. MISCELLANEOUSRADIOLIGANDS

The following ligands have been reported to label the DA uptake site in rodent striatum: [3H]mazindol (Javitch et al., 1983, 1984), [3H]nomifensine (Dubocovich and Zahniser, 1985) and [3I-I]threo (+)methylphenidate (Janowsky et al., 1985). It has been suggested that these compounds lack the potency and selectivity of the GBR derivatives (Bonnet et al., 1986). The phencyclidine derivative [3H]N-[l-(2-benzo(b)thiophenyl)cyclohexyl]-piperidine ([3H]BTCP) has been found to be a useful ligand for the invitro and in vivo labelling of the DA uptake system in rodent brain (Filloux et al., 1989; Maurice et al., 1989). It is perhaps of interest to point out the fact that the in vitro binding characteristics of [SH](+)amphetamine in the rat CNS led the authors (Hauger et al., 1984) to conclude that the binding site was probably not related to a neurotransmitter uptake system.

6. THE STABILITY OF I N V I T R O PREPARATIONS USED TO STUDY [SH]DA UPTAKE Various aspects of the lability of the in vitro systems that are commonly used to study DA

392

A.S. HoRn'

transport have been investigated by the following authors: Cooper and Carlson (1983), Heikkila and Cabbat (1983), Schoemaker and Nickolson (1983b), and Bianchi and Takimoto (198o,).

7. THE UPTAKE OF IJHIDA INTO THE MEDIAN EMINENCE AND POSTERIOR PITUITARY Demarest and Moore (1979) found that homogenates of the median eminence and posterior pituitary were less able to accumulate [3H]DA than other DA-rich brain areas. They suggested that if DA released from tuberoinfundibular neurons is quickly transported away from the terminals in the blood there would be little need for an efficient uptake system. In addition they reasoned that this could also explain the small reduction of serum prolactin observed following systemic administration of DA reuptake blockers. The above results and conclusions were supported by Annunziato et al. (1980). On the basis of a differential effect of DA uptake inhibitors and releasers on plasma prolactin in the rat, Celia et al. (1982) have also concluded that DA uptake into tuberoinfundibular nerve terminals does not play a major role in inactivating DA released into the hypophyseal portal system. Sarkar et al. (1983), in an in vitro study on the uptake and release of [3H]DA in the median eminence, found that the K= for [3H]DA uptake was greater than in the rat striatum. A detailed kinetic study of [~H]DA uptake into various areas of the hypothalamus by George and van Loon (1982) showed that all the DA neurons of the hypothalamus have lower affinities for DA than do the neurons of the striatum. In contrast to the above authors they suggest that the lower affinity for DA is not an exclusive property of the tuberoinfundibular neurons and cannot be ascribed merely to their role as neurosecretory neurons.

8. STUDIES ON THE UPTAKE OF 13HIDA INTO MISCELLANEOUS TISSUES AND PREPARATIONS Dopamine uptake has also been studied in the following tissues: the periglomerutar cells of the rat olfactory bulb (Priestley et aL, 1979), chicken ultimobranchial gland (Di Lauro et al., 1980), the perfused lung and lung homogenates of the rat (Ben-Harari and Youdim, 1981), the rabbit celiac artery (Amenta and Ferrante, 1983), astroglial cells (Hansson et aL, 1984) and rabbit, goldfish and cat retina (Thomas et aL, 1978; Vijay Sarthy and Lam, 1979; Dacey, 1988). In a comparative study between crude, partially purified and purified synaptosomal preparations, Tuomisto and Tuomisto (1979) have shown that for general purposes (i.e. the determination of ICs0 values) simple crude fractions are often good enough and they offer several practical advantages such as ease of preparation. Using synaptosomes obtained from various areas of rat brain, Bonanno and Raiteri (1987) have provided evidence for the hypothesis that uptake systems

for DA and GABA may coexist on the same nerve terminal. Near et al. (1988) have done a comparative study on [3H]DA uptake into rat striatal chopped tissue, homogenates and synaptosomes. It appears that only one uptake system for DA exists. Although two uptake systems appear to exist in more intact preparations, this appears to be the result of diffusion into the intact tissue.

9. POSSIBLE RELATIONSHIPS BETWEEN DA UPTAKE AND RELEASE It has been suggested that the DA transport system could also be the means whereby DA is released into the synaptic gap (Fischer and Cho. 1979; Raiteri et al., 1979). The latter authors showed, in fact, that in vitro DA can be released by two different mechanisms, i.e. exocytosis and via the amine pump operating in an outward direction. They were able to do this by using nomifensine which is a compound that has virtually no releasing action, i.e. a pure uptake inhibitor. They also found that the carrier system is involved in the mode of action of certain phenylethylamines which bring about DA release. Cubeddu et al. (1983) investigated the effects of various DA uptake inhibitors on the in vitro release of DA and ACh in the rabbit striatum. The effects were variable and dependent on the frequency of stimulation (0.3 or 30 Hz). However, in the presence of the presynaptic D 2 antagonist sulpiride these DA uptake inhibitors produced a large enhancement of DA overflow. Similar results were reported subsequently in in vivo studies (Westerink et al., 1987a). Cubeddu et al. (1983) suggest that the net effect of DA uptake inhibitors on DA overflow depends on the balance of two factors, i.e. the degree of blockade of DA reuptake, which enhances overflow and the degree of autoreceptor activation, which inhibits overflow. Various authors have suggested the possible existence of a functional coupling between mechanisms of transmitter uptake and presynaptic receptors controlling release (Galzin et al., 1982; Langer and Motet, 1982; G6thert et aL, 1983; LaBeUa, 1985). However, Raiteri et al. (1984), using superfused synaptosomal preparations, was unable to detect any effect of reuptake inhibitors on the actions of presynaptic receptor agonists and transmitter release. Hoffman et al. (1986) have provided further evidence for their hypothesis that the interaction between neuronal uptake blockers and agonists acting on release-modulatory receptors is mediated by endogenous DA. Using synaptosomes and field-stimulated brain slices, Hunt et at. (1979) were able to differentiate between drugs causing an inhibition of uptake or release. They confirmed earlier findings that benztropine and nomifensine are uptake inhibitors whereas amphetamine causes a release of [3H]DA (see Horn, 1979). Nicolaysen and Justice (1988) have proposed a mathematical model for the effects of cocaine on DA release and uptake. By the use of [3H]DA and [J*C]DA, Bonnet et al. 0984) were able to evaluate simultaneously the drug

DOPAMI~ UPTAKE

induced inhibition of uptake and release. They also showed that nomifensine and benztropine are potent inhibitors of uptake and that cocaine is less active. In addition neither nomifensine nor cocaine caused a significant release of DA. At concentrations of benztropine above 10-5 M it was able to induce a release of DA. However, inhibition of uptake was its most noticeable effect.

10. I N V I V O STUDIES ON DA REUPTAKE IN ANIMALS Intracerebral microdialysis is a new technique for investigating the in vivo release of DA and certain other neurotransmitters (for a review see Westerink et al., 1987b). In a study of two reuptake blockers, GBR 12909 and amfonelic acid, the former compound was found to induce a 3-fold increase in the output of DA from the striatum whilst surprisingly amfonelic acid was without effect (Westerink et al., 1987b). In a similar study, Church et al. (1987) showed that nomifensine, cocaine and benztropine produced dose-dependent elevations in extracellular dopamine. Hurd and Ungerstedt (1989a) examined various DA uptake inhibitors and found that they induced dosedependent accumulations of extracellular DA. These authors (Hurd and Ungerstedt, 1989b) also studied the calcium dependence of DA release under the influence of amphetamine, nomifensine and Lu19005. Using a push-pull perfusion technique Sirinathsinghji et al. (1988) found that benztropine (10 -6 M) caused a 1-7 fold increase in DA levels in the rat striatum. An additional technique which has been employed to study DA uptake in rico is fast cyclic voltammetry (FCV) in which carbon fibre microelectrodes are used. The advantage with this technique, in comparison with microdialysis, is that it allows sub-second time resolution of changes in extracellular DA and enables one to obtain kinetic data about uptake and release processes. Stamford et al. (1984) found that electrical stimulation of the median forebrain bundle in anesthetized rats evoked DA release which could be monitored by FCV. The extracellular levels of DA fell to sub-detectable levels in 15 sec. The authors suggested that this was due to a low affinity uptake system that could be inhibited by nomifensine. The same group (Stamford et al., 1986) reported in subsequent studies that the decline in DA levels was biphasic, showing zero and first-order components. The uptake system could be blocked by d-amphetamine, methylphenidate and nomifensine but not by benztropine, amfonelic acid or mazindol. In a third paper in this series (Stamford et al., 1988), a comparison was made between DA uptake in the rat caudate and nucleus accumbens. It was found that DA uptake was significantly slower in the accumbens than in the caudate. Using similar technology Wightman et al. (1988) were also able to determine kinetic parameters for DA uptake and found that the system could be inhibited by nomifensine. In the above context it is perhaps worth mentioning that in vitro studies, which have examined DA

393

uptake by striatal synaptosomes during the fast phase of depolarization-induced DA release, suggest that presynaptic neuronal uptake may be unable to immediately remove the relatively large amount of DA released following depolarization (Woodward et al., 1986). These authors argue that presynaptic uptake may be more important in the long-term regulation of transmitter release by maintaining storage pools which are linked to synthesis and release.

11. DA UPTAKE INHIBITORS AND BEHAVIOUR Various inhibitors of DA uptake have a stimulating influence on behaviour. Ross (1979) has suggested that not all indirectly acting central stimulants act via an inhibition of DA uptake. He has suggested that they can be divided into a methylphenidate-like group (methylphenidate, amfonelic acid, mazindoi and nomifensine) and an amphetamine-like group ((+)-amphetamine, N-methylamphetamine). In reserpinized mice only the latter group was active in causing hypermotility. In addition the former group was able to antagonize the hyperactivity induced by amphetamine in reserpinized mice. On this basis, Ross suggested that the methyiphendiate group acts via an inhibition of DA uptake whilst amphetamine causes a release of DA. These results were confirmed by Heikkila (1981) who also studied the very potent and selective DA uptake inhibitors GBR 13069 and GBR 13098. Heikkila et al. (1979) also examined two structural analogues of cocaine, Win 35065 and Win 35140. The former compound was a more potent inhibitor of [3H]DA uptake and inducer of motor activity in mice and rotational behaviour in 60HDA rats, than cocaine. Fung and Uretsky (1980) found that mazindol, nomifensine, benztropine and cocaine were all able to inhibit amphetamine-induced turning in 6OHDA lesioned rats. Heikkila et al. (1981) examined several structural analogues of mazindol and showed that they caused large increases in motor activity in normal mice but not in reserpinized animals. Heikkila and Manzino (1984) showed that GBR 12909, 13069 and 13098 are very potent and specific DA uptake inhibitors. They caused ipsilateral turning in 6OHDA-lesioned rats and increased locomotor activity in mice. Gratton et at. 0988) found that unilateral infusions of morphine into the ventral tegmental area induced contraverse circling in rats. Administration of either nomifensine or GBR 13069 resulted in a 3-fold increase in the rate of turning. The authors suggested that the data were consistent with the hypothesis that morphine causes contraverse circling through local activation or disinhibition of dopaminergic cell firing. Marshall and Altar (1986) have reported that administration of nomifensine or bupropion to senescent rats produced an improvement in their swimming performance to levels comparable with young adult animals. As the two groups showed identical K= and V=,~values for the uptake of [3H]DA into striatal homogenates it was suggested that amine reuptake may limit the synaptic effectiveness of DA released in the aged striatum.

394

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In studies on schedule-controlled behaviour in squirrel monkeys, McKearney (1982) found that benztropine, bupropion, mazindol and nomifensine markedly increased responding under a fixed-interval schedule but not under a fixed-ratio schedule. With the exception of benztropine the potency differences seen in these experiments were similar to those reported for in vitro inhibition of DA uptake. In studies on isolation-induced fighting in mice, Hadfield (198 I) found that after such a conflict there was a specific increase in both Kr, and I/m~ values in the prefrontal cortex but not in the caudate-putamen. Neonatal rats have ultradian rather than circadian rhythms. In a study by Barber et al. (1989) of the effects of selective uptake inhibitors on the activity rhythms of developing rats, it was found that GBR 12909 had little effect on ultradian rhythms throughout the prominent 7-16 cycle per day domain.

of various tetrahydro-/3-carbolines and tryptamines on the uptake of [3H]DA and 5-HT into human blood platelets, Airaksinen et al. (1980) showed that there were clear differences in the rank order of potencies as compared to rat brain synaptosomes. In a preliminary study Szekely et al. (1980) reported that the uptake of DA into platetets from autistic children is about 15-20% higher than in psychotic children. However, the authors were cautious regarding their conclusions due to the limitations of this model as demonstrated by Omenn and Smith (1978). McLean and Nihei (1981) found that patients at risk to develop Huntington's chorea have a significantly higher uptake of DA into blood platelets than controls. In studies with platelets obtained from schizophrenics, Shah et aL (1982) found that compared with controls, DA uptake in 12 patients with active symptoms was significantly increased.

12. IJH]DA UPTAKE INTO HUMAN BLOOD PLATELETS The characteristics of [3H]DA uptake into blood platelets have been discussed in a previous review article (Horn, 1979). Omenn and Smith (1978), in a comparative study on the kinetics of [JH]DA and [~H]5-HT uptake into human platelets, reached the conclusion that the uptake of DA appears to be mediated inefficiently via the specific 5-HT carrier system. These authors are of the opinion that reports on abnormal DA uptake into platelets in Huntington's disease (Aminoff et aL, 1976) and parkinsonism (Barbeau et al., 1975) should be re-assessed. Shah et aL (1980) report studies from which they conclude that approximately 50% of DA uptake into platelets is mediated through a 5-HT carrier mechanism. The other 50% is possibly due to passive diffusion and low affinity uptake. This view was supported by Lingjaerde and Kildemo (1981) who showed that DA uptake into human platelets encompassed two different saturable components, one chloride-dependent and one non-chloride-dependent. The former is probably identical with the 5-HT uptake mechanism and the latter appears to be facilitated diffusion. These authors argue against the view of Stahl and Meltzer (1978) that all D A uptake into human platelets is non-saturable and due to passive diffusion. Airaksinen (1979) found that in patients suffering from progressive myoclonus epilepsy the uptake of DA, G A B A and 5-HT into platelets was not significantly different from controls. However, the uptake of taurine was lower. In an SAR study on the effects

13. THE DA UPTAKE SYSTEM IN THE HUMAN BRAIN The regional distribution of DA uptake and the concentrations of DA in the human brain have been studied by Hardy et al. (1987). Post-mortem material was obtained within 24 hr from individuals who had died of myocardial infarctions and without any psychiatric or neurological disorders. Twelve areas of the brain were dissected for investigation: neuropathological examination revealed no abnormalities. DA uptake was demonstrated in seven areas (Table 2), the highest being the caudate, but it could not be detected in the remaining five (Table 2, legend). Zelnik et aL (1986), in in vitro studies on the binding of [~H]GBR 12935 to post-mortem membrane preparations of caudate nuclei, have found substantial alterations with age. The density of binding sites (B=~,) was age-dependent and decreased from 2.76 + 0.34 pmol/mg protein in children (0-10 years) to 1.36 _+0.18 pmol/mg protein in adults (ages 21-82 years). No significant differences in the Ko were observed between the age groups. Using the above radioligand, Maloteaux et al. (1988) found decreased in vitro binding in the putamen and caudate nucleus of parkinsonian patients (33 and 46% of control values, respectively) and subjects with supranuclear palsy (38 and 57% control values, respectively). The problems associated with storage and cryopreservation of post-mortem samples of human brain in relation to/n vitro uptake studies were investigated by Haberland et al. (1985) and Haberland and Hetey

TABLE 2. CONCENTRATIONOF DOPAMINEAND DISTRIBUTIONOF DOPAMINE UPTAKE IN H~M.~.'¢ BRAIN

Caudate 12.6 I00

Putamen 18.6 62.1

Globus pallidus 1.6 3.5

Frontal Hippocampus Hypothalamus cortex 0.2 1.8 0.2 2.8 11.0 2.8

Cingulate cortex 0.2 1.5

DA (nmol/g) DA uptake (pmol/g rain) K= (/aM) 0.14 0.26 0.02 -0.22 0.22 0.22 Experiments were carded out in vitro with homogenates. NO [~H]DA uptake could be detected in the following brain regions; substantia nigra, olfactory tubercle, thalamus, pons and medulla. For further details see Hardy et al. (1987).

D O P A M I ~ Ulq'/~rdE

(1987a). They found that slow freezing to -25~C followed by immersion in liquid nitrogen of tissue preincubated with 5% DMSO produced the best results. This procedure was not found to affect the kinetic paraineters of DA uptake. In a follow-up study these authors (Haberland and Hetey, 1987b) studied DA uptake kinetics in vitro in post-mortem brain regions of schizophrenics. The Km and Vm~ were significantly elevated in the nucleus accumbens and in the nucleus caudatus of schizophrenics in comparison with controls. DA uptake in the frontal cortex was unchanged. The authors stated that these changes did not seem to be primarily dependent on neuroleptic medication. In a comparative post-mortem study between controls and schizophrenics, Hetey et al. (1987) found that in the latter group DA uptake inhibition by nomifensine was significantly weaker than in the corresponding control brain regions. As part of a preparatory investigation for human in vivo studies, Aquilonius et al. (1987) examined striatal DA uptake sites in the Rhesus monkey using UC-nomifensine and positron emission tomography (PET). As expected the highest binding was found in the striatum and the lowest in the cerebellum. This difference was markedly reduced after nomifensine and mazindol but not after desipramine and spiperone. In a human volunteer the regional uptake of radioactivity was similar to that in the monkey. The striatal/cerebellar ratio was 1.6. In a hemiparkinsonian patient this ratio was 1.1 contralaterally and 1.3 ipsilaterally to the affected side. In an additional study from the above group (Tedroff et al., 1988) they investigated 6 parkinsonian patients and 7 controls using the same methodology. In the control group there was a marked age-related decline in ItC-nomifensine binding in all brain regions investigated relative to the cerebellum. In the group of parkinsonian patients with mainly unilateral involvement, the contralateral putamen exhibited the most pronounced decrease. Only 3 of the parkinsonian patients aged 63 and younger showed markedly lower ~C-nomifensine binding in striatal areas as compared to age-matched controls. 14. THE MOLECULAR NATURE OF THE DA TRANSPORT SYSTEM Due to recent developments in the isolation and structure elucidation of various membrane-bound receptors for the biogenic amines (Dohlman et al., 1987) and the availability of several potent and selective radioligands for the DA uptake site, the stage is set for the isolation and molecular characterization of the DA transporter. At the current rate of progress in the area of the molecular biology of receptors it is reasonable to predict that this goal should be achieved within a short period from now. One reason for this optimism is the fact that the system has already been solubilized in an active form by two groups (Kuhar et al,, 1988; Sa11¢¢et al., 1988) using the detergent digitonin and characterized by [3H]GBR 12935 as the radioligand. A full paper on this work has recently been published by Lew et al. (1989). In addition the same group has developed a photoaffinity ligand ~:3I-DEEP (Fig. 3) which is able

395

,Is/

N~

28 I . D f : E p

FxG. 3. A photoaffinity label for the DA transporter.

to selectively label the DA transporter (Grigoriadis et al., 1989). The ligand was found to incorporate into a protein with a molecular weight of 50 kDa. Compounds containing an isothiocyanate group also appear to be useful affinity labels for the DA transporter (Zimanyi et al., 1989). Sallee et al. (1988) found that the solubilized material can be absorbed by and specifically eluted from, a WGA-lectin column suggesting that the material is a transmembrane glycoprotein. In this respect it is of interest that Zaleska and Erecinska (1987) have shown that a sialic acid moiety is involved in DA transport across the synaptosomal membrane. The membrane cholesterol/phospholipid ratio has also been shown to influence the uptake of [~H]DA (Maguir¢ and Druse, 1988). At the moment the neurotransmitter transport system which is the most well characterized is the one for GABA (Radian et al., 1986; Keynan and Kanner, 1988). It has been shown to be a glycoprotein with a molecular weight of 80 kDa. Experiments with antibodies have shown that there is very little homology between the GABA transporter and the GABA receptor. 15. CONCLUSIONS AND FUTURE PROSPECTIVES In the last review, published in 1979, a plea was made for the development of a more selective inhibitor of DA uptake (Horn, 1979); this goal has been achieved with the GBR series of compounds. The need to obtain more information about the biochemical composition of the DA transporter was stressed and some progress in this area has also been achieved. The neuropharmacological actions of several drugs which are currently in the public and academic spotlights, i.e. cocaine and MPTP, have been linked directly to an interaction with the DA transporter, The influence of DA uptake inhibitors on animal behaviour has also been the focus of increasing attention. The impact of in vitro radioligand binding studies in this field is one of the most important developments in recent years. These studies have been extremely useful in their own right and also in preparing the ground for the in vivo PET scan work which is the most exciting clinical development in this area. In the near future we can expect a full molecular characterization of the DA transporter as well as other biogenic amine uptake systems. Will they all belong to a large "transporter family" as is the case with certain receptors for these amines? Further refinement of radioactive ligands for PET scanning which can be used on a more routine basis can also be expected. Hopefully, research on the DA transporter in the next 10 years will be as interesting as it has been during the past two decades.

396

A.S. HORN REFERENCES

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