Dopamine Receptors

CLASSIFICATION

The original discovery and classification of DA receptors was based on the results of three distinct studies:

(1) Stimulation of adenylate cyclase

(2) Ligand binding

(3) Inhibition of prolactin release

The adenylate cyclase discovered originally in bovine superior cervical ganglia, and then found in homogenates of rat striatum, was specific to DA, in that it was activated by other DA agonists like ADTN, but not greatly by NA or 5-HT. Some other drugs with established DA-like effects proved, however, to be either partial agonists (apomorphine) or ineffective (bromocriptine). Also while some neuroleptic (anti-psychotic) drugs that are DA antagonists in behavioural studies, such as the thioxanthenes and phenothiazines, antagonised this effect with a relative potency that compared with their antipsychotic activity, other potent neuroleptics like the butyrophenones were relatively ineffective. Overall there was a poor correlation between antipsychotic activity and DA antagonism as measured by blockade of DA-induced cAMP production.

Ligand-binding studies, originally with [3H] dopamine and [3H] haloperidol but subsequently using [3H] spiperone, demonstrated the existence of a specific binding site for them in membrane preparations from mammalian striatum. Displacement studies with a whole range of neuroleptic drugs also showed that not only was the rank order different from that for blocking the adenylate cyclase but also correlated much better with antipsychotic activity. Additionally DA agonists like bromocriptine, which were ineffective in increasing cAMP production, showed appropriate binding.

When tested on prolactin release in isolated mammatrophs of bovine anterior pituitary, apomorphine appeared a full agonist (inhibiting release) while antagonism of the inhibition of prolactin release by the neuroleptics showed a potency more similar to that for binding than for blocking cAMP production. Also the inhibition of prolactin release by DA was not accompanied by any change in intracellular cAMP and therefore was not linked to it.

Thus the establishment of two clear dopamine effects, one directly linked to stimulation of adenylate cyclase and the other inhibition of prolactin release, which was independent of adenylate cyclase stimulation but associated with distinct binding sites led to the concept, formulated by Kebabian and Calne (1979), that DA effects were mediated through two distinct receptors. One was linked to stimulation of adenylate cyclase (D1) while the other (D2) did not appear to be associated with the enzyme but had distinct binding sites. The justification for this classification was subsequently enhanced by the synthesis of two compounds, SKF 38393 and SCH 23390. The former activated the DA adenylate cyclase without affecting prolactin release or spiperone binding, i.e. it was a D1 agonist, while the latter blocked the stimulation of adenylate cyclase, again without affecting prolactin release or binding. It was a D1 antagonist. The basis for this early classification is shown in Table 7.1.

Although some subsequent pharmacological studies suggested that perhaps there could be a subdivision of both the D1 and D2 receptors, the paucity of appropriate agonists and antagonists (and indeed of test responses) precluded its justification until molecular biology took over. Cloning studies show that structurally there are two distinct groups of DA receptors, D1 and D2. There is a D5 variant of D1 as well as D3 and D4 forms of D2. The D1 and D5 receptors are linked to activation of adenylate cyclase and the D2 group to its inhibition, although this is not its main effect on neurons (see later).

Despite this profusion of receptors the D1 and D2 predominate (over 90% of total) and most known effects of DA, its agonists and antagonists are mediated through the D2 receptor. Although the above nomenclature is now accepted it might have been better, as suggested by Sibley and Monsma (1992), to retain D1 and D2 to represent the two families and then subdivide them as D1A for (D1), D1B for (D5), then D2A for (D2), D2B for (D3) and D2C for (D4), even though variants of all five have been found. Their

Table 7.1 Evidence for the initial basic classification of D1 and D2 dopamine receptors

(a) Discovery of specific DA stimulated striatal adenylate cyclase

But (i) Effect only antagonised by some neuroleptics, phenothiazines-YES thioxanthenes-YES butyrophenones-NO (metoclopromide inactive) (ii) Effect not reproduced by DA agonists like bromocriptine.

(b) Binding studies: 3H dopamine 3H haloperidol

Displacement studies with a wide range of neuroleptics showed good correlation with their clinical potency in schizophrenia.

DA agonists such as bromocriptine show binding.

(c) Prolactin release from isolated mammatrophs from anterior pituitary

DA agonists — bromocriptine, apomorphine and ADTN decrease release.

Blocked by neuroleptics — similar in effectiveness to their binding affinities (b).

Not linked to stimulation of adenyl cyclase.

Notes:

Studies with various agonists and antagonists showed that the effects on (a) differed in potency from both (b) and (c) and were thus associated with a receptor (D1) different from that (D2) linked to (b) and (c). See text for detail.

structure has been established in both rat and human brain and they are generally similar in the two. The human D2 receptor shows a protein sequence which is 96% identical to that of the rat D2 and although the similarity is only 91% between the human and rat D1 receptor, it is 96% in the transmembrane region. It is differences in the amino-acid sequences in this region that primarily justify the classification into two groups (D1 and D2) rather than their total amino-acid number. Basically the D1 (and D5) receptors differ from the D2 (D3, D4) in having a much shorter third cytoplasmic loop and a much longer intracellular C-terminus (Fig. 7.4), which appears to be a feature of receptors linked to Gs and the stimulation of adenylate cyclase. Based on amino-acid sequencing the D3 receptor is only 53% homologous with the D2 (but 75% in the transmembrane region) while with D4 it is only 41% (56%). The D5 receptor shows 50% homology with the Di rising to 80% in the transmembrane region. So-called short and long variants of the D2 receptor (D2S and D2l) have also been discovered, differing by the presence or absence of a run of 29 amino acids in the third intracellular loop. For more detail see Sibley and Monsma (1992).

DISTRIBUTION AND MECHANISMS

The potential value of the discovery, classification and subdivisions of any NT receptors rests on the knowledge that nh2

Figure 7.4 Comparative schematic representation of the D1 (• • •) and D (—) dopamine receptor. The figure attempts to highlight the major differences between extra- and intracellular loops, especially the intracellular loops between transmembrane sections 5 and 6 and the much longer C terminal of the D1 compared with the D2 receptor. It is based on the proposed topography of Sibley and Monsma (1992). The thickened length of the D2 receptor represents the amino-acid sequence missing in the short form of the receptor. No attempt has been made to show differences in amino-acid sequencing or transmembrane topography

Figure 7.4 Comparative schematic representation of the D1 (• • •) and D (—) dopamine receptor. The figure attempts to highlight the major differences between extra- and intracellular loops, especially the intracellular loops between transmembrane sections 5 and 6 and the much longer C terminal of the D1 compared with the D2 receptor. It is based on the proposed topography of Sibley and Monsma (1992). The thickened length of the D2 receptor represents the amino-acid sequence missing in the short form of the receptor. No attempt has been made to show differences in amino-acid sequencing or transmembrane topography

(1) Those receptors are linked to different cellular actions and/or are located in different brain regions or parts of the neuron so as to produce different functional effects.

(2) There are appropriate specific agonists or antagonists to establish and exploit those differences.

To some extent these requirements are cyclic since the establishment of different functions (1) depends on the availability of appropriate drugs (2). There is no shortage of drugs, especially antagonists, but since the main difference in structure between DA receptors is intracellular, rather than at the binding or recognition site, very specific drugs may be hard to produce. Since receptors can be expressed in cell lines the affinity of drugs for the different receptors can, however, be established, as can their cellular actions. Detection of appropriate mRNA also makes it possible to map the distribution of the receptors. The main characteristics of the DA receptors are summarised below and in Table 7.2.

Dj receptor family

Dj Highest expression in human striatum and nucleus accumbens and olfactory tubercle but also some in cortex and hypothalamus. In the striatum 50% of medium sized striato-nigral neurons, which also express substance P, express them. They are

Table 7.2 Dopamine receptor characteristics

Main class

Di

D2

Subclass

dIA

DiB

D2A

D2B

D2C

Named

Di

d5

D2

D3

D4

No. amino acid (human)

446

477

414 (s) 443 (l)

440

387

DA affinity

Low

Moderate

Moderate

High

High

Ki (nM) approx.

2000

200

599

20

20

Effector

Activation of adenylate cyclase

t

t

1

(I)

(I)

IP3 turnover

(t)

-

t

-

-

Ca2+ influx

(t)

-

I

I

K+ efflux

-

-

t

-

-

Agonists Antagonists Number Distribution

High

High

7-OHDPAT?

Agonists Antagonists Number Distribution

Striatum

Nuc. accumbens

Frontal cortex

Hippocampus

Hypothalamus

Substantia nigra

VTA (auto-receptors)

High

High

7-OHDPAT?

Clozapine Low

(Midbrain)

Note:

S = short, L = long versions of D2 receptor, f = main effect observed, (|) = some evidence of an effect. See Sibley and Monsma (1997), Sokoloff and Schwartz (1995) and Strange (1996).

linked primarily to stimulation of adenylate cyclase but also increase IP3 turnover. They have a low micromolar affinity for DA (K1 — 2 ^M). D5 Highest concentration in hippocampus and hypothalamus but much lower expression overall. Also linked to stimulation of adenylate cyclase but higher submicromolar affinity for DA (K — 200 nM). Also found in rat striatum and nucleus accumbens.

D2 receptor family

D2 Mostly in striatum, nucleus accumbens and olfactory tubercle but also on neuron cell bodies in substantia nigra and ventral tegmentum where they are the auto-receptors for locally (dendritic) released DA. The loss of specific D2 antagonist binding in the striatum after lesions of the afferent nigro-striatal tract indicates their presynaptic autoreceptor role on terminals there. Other lesion studies have also established D2 receptors on other inputs such as the cortico striatal tract.

As with D1 receptors some 50% of striatal medium-sized cells contain them but they are different neurons as they co-express enkephalin rather than substance P. The importance of this difference in the therapy of Parkinsonism is taken up later (Chapter 15). D2 receptors are also expressed on larger cells — probably cholinegic. Although linked to inhibition of adenylate cyclase (and IP3 turnover) this is not their primary action. They increase K+ conductance (hyperpolarise neurons) but also inhibit Ca2+ entry through voltage-sensitive channels, probably directly. When functioning as autoreceptors, these effects would also reduce DA release. The affinity for DA is slightly higher for the D2 (K1 — 400 nM) than for D1 receptors. No pharmacological differences have been established between the long or short forms of the D2 receptor.

D3 Much less abundant than D2. Mainly in limbic regions (nucleus accumbens and olfactory tubercle) but also in hypothalamus. Some in caudate and cortex and also expressed on DA neurons in substantia nigra, presumably as autoreceptors. No effect on adenylate cyclase but inhibits Ca2+ entry (autoreceptor role). High affinity for DA (K1 - 25 nM).

D4 Again very few in number compared with D2 but located in frontal cortex, midbrain and amygdala. High affinity for DA (K1 — 20 nM) and a number of variants in humans.

Comparison of the K1 values of various agonists and antagonists for the different receptors (Table 7.3) shows that whereas there are a number of drugs that readily distinguish between the D1 and D2 families and can be used to study their function, none distinguish between D1 and D5 and there is little to choose between D2, D3 and D4 activities. Some differences that have been exploited are the low affinity of raclopride for D4 receptors (compared with D2 and D3), the high affinity of clozapine and the benzamide derivative YM 43611 for the D4 (cf. D2, D3) and that of 7-OH-DPAT for D3. Since only the latter is an agonist, however, their value in establishing the roles of the D3 and D4 receptors is limited, although the high affinity of clozapine for D4 receptors and their location in the frontal cortex has been considered, somewhat controversially, to be of significance in the aetiology and therapy of schizophrenia (see Chapter 17).

Table 7.3 (a) Dissociation constants for various agonists and antagonists at the different dopamine receptors, (b) Indication of specificity for D2 compared with Dj receptors and between D2 (i.e. D2, D3 and D4) receptors (a)

Di

D5

D2

D3

D4

Agonist

Bromocriptine

440*

440*

8*

5*

290*

Quinpirole

1900

-

5

24*

30*

7-OH-DPAT

5000*

-

10

1*

650

SKF 38393

1

0.5*

150*

5000*

1000*

Apomorphine

0.7

-

0.7*

32*

4*

Antagonist

Chloropromazine

90*

130*

3

4

35

Haloperidol

80*

100*

1

7*

2

Clozapine

170*

330*

230*

170*

21

Raclopride

18 000

1.8

3.5

2400

Spiperone

350*

3500*

0.06

0.6

0.08

S-Sulpiride

45 000

77 000

15*

13*

1000

YM 43611

10000+

10 000+

43

11

2

SCH 23390

0.2*

0.3

1100*

800*

3000*

(b)

Specific for D2 family

Ratio to D1

activity

Agonist

D2 = D3>D4

Bromocriptine

50

D2>D3 = D4

Quinpirol

<400

d3>d2>> d4

7-OH-DPAT

<5000

Antagonist

d2 = d3>> d4

Raclopride

10 000

D2=D4 > D3

Spiperone

6000

D2=D3 > D4

S-Sulpiride

3000

d4>d3 = d7

YM 43611

250

Specific for D1 family

Ratio to D2

activity

Agonist

D1 = D5

SKF 38393

150+

Antagonist

D1 = D5

SCH 23390

1000

All values shown are taken from Seeman and Van Tol (1994) except for those for YM 43611 (Hidaka et al. 1996). Asterisked values are considered approximate.

Note:

All values shown are taken from Seeman and Van Tol (1994) except for those for YM 43611 (Hidaka et al. 1996). Asterisked values are considered approximate.

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