Dopamine (DA) is a catecholamine that has a variety of roles in both the CNS and periphery, including behavior, emotion, cognition, locomotor functions, hormone production, renal functions, blood pressure, and motility of the GI-tract. Dopamine is also involved in many diseases, including schizophrenia, Parkinson's disease, attention deficit/ hyperactivity disorder (ADHD), and hyperprolactinemia. Catecholamines, which contain a catechol ring and an amine side chain, are a class of neurotransmitters that include DA, norepinephrine, and epinephrine. The catecholamines are small, water-soluble molecules derived from the amino acid tyrosine. Catecholamines are related to another group of neurotransmitters called indolamines, which include serotonin and melatonin, and both catecholamines and indolamines are classified as monoamines. Like other monoamines, DA is packaged into secretory granules, which prevent degradation from metabolic enzymes and allow for regulated, rapid release. In the brain, catecholamines act as classical neurotransmitters by facilitating communication between neurons through their secretion into the synaptic cleft between neurons. However, outside of the brain catecholamines can also function as hormones, and in the pituitary DA is the primary inhibitor of PRL (103).
A. Synthesis and Metabolism
DA is synthesized from the amino acid tyrosine by a sequential series of enzymatic reactions. The primary source of tyrosine in the circulation is from the diet, although tyrosine can also be produced by the hydroxylation of dietary phenylalanine in the liver by the enzyme phenylalanine hydroxylase. Tyrosine from the circulation is taken up by the neuron by an energy-dependent amino acid transport mechanism. In the rate-limiting step of DA biosynthesis, the enzyme tyrosine hydroxylase causes the hydroxylation of tyrosine into L-dihydroxyphenylalanine (L-DOPA). Then L-DOPA is converted to DA by DOPA decarboxylase (DDC, or L-aromatic amino acid decarboxylase) (224). L-DOPA, but not DA, can cross the blood-brain barrier. In noradrenergic neurons, DA can be converted into norepinephrine (nor-adrenaline) by dopamine β-hydroxylase (DBH), and in adrenergic neurons, phenylethanolamine N-methyl transferase can convert norepinephrine into epinephrine (adrenaline) (Fig. 9)-. The localization of noradrenergic and adrenergic neurons differs greatly from that of dopaminergic neurons. After synthesis, DA in neurons is stored in secretory vesicles at high concentrations of about 0.5- 0.6 M. An action potential causes the influx of calcium ions, allowing the secretory vesicles to fuse with the plasma membrane to release DA into the synapse between neurons (225).
DA can be deactivated both by reuptake into the nerve terminal and by conversion into inactive forms (Fig. 10). The pre-synaptic neurons contain DA transporters (DAT) in their membranes. DAT can recycle DA that has been secreted into the synaptic cleft by active transport back into the original neuron. Multiple pathways are involved in DA catabolism. Monoamine oxidase (MAO) causes oxidative deamination of DA into 3,4-dihydroxyphenylacetic acid (DOPAC), while catechol-O-methyltransferase (COMT) converts DA into 3-methoxytyramine (3-MT) by O-methylation. These products, DOPAC or 3-MT, can then be converted into homovanillic acid (HVA) by the other enzyme, either COMT or MAO, respectively (224). UDP-glucuronosyltransferases (UGTs) can conjugate DA into DA-glucuronide, although this process is more common in rodents (226). DA can also be conjugated with a sulfate group by sulfotransferases. The amount of each metabolite depends on the concentration and activity of the various enzymes, which can differ between regions of the brain, cell types, and species. In the brains of primates, HVA is the primary metabolite of DA, while in brains of rats the major product is DOPAC (224).
DA is mainly produced in the CNS. There are multiple dopaminergic systems that start within the midbrain, including the mesolimbic, nigrostriatal, and mesocorticol neuronal systems, and they project into the limbic system, striatum, and cortex, respectively. These systems regulate cognition, emotions, and locomotion but are not directly involved in control of the pituitary. The hypothalamus contains dopamine perikarya in several sites, including the posterior hypothalamus (A11), arcuate nucleus (A12), zona incerta (A13), periventricular nucleus (A14), and the lateral and ventral hypothalamus (A15). The tuberoinfundibular dopaminergic (TIDA) and tuberohypophysial dopaminergic (THDA)/ periventricular-hypophysial dopaminergic (PHDA) neurons originate in the arcuate nucleus and periventricular nucleus, respectively. The TIDA neurons are the major source of DA for the anterior pituitary. TIDA neurons end in the median eminence, where they release DA that is carried to the pituitary by hypophysial portal blood vessels to the anterior pituitary. As another source of DA for the pituitary, THDA/PHDA neurons extend into the neural and intermediate lobes of the pituitary (103).
Outside of the CNS and pituitary, small amounts of DA are also synthesized in the adrenal medulla, which also secretes epinephrine and norepinephrine. In addition, DA has been detected in other peripheral tissues and cells, including the pancreas, anterior pituitary, and macrophages (227). DA is released into circulation, and serum DA levels are about 0.1 nM, while the other catecholamines norepinephrine and epinephrine circulate at 1 nM and 0.2 nM, respectively (228).
Dopamine Sulfate and Arylsulfatase
Catecholamines can be deactivated by reuptake by neurons, deamination, O-methylation, glucuronidation, or sulfoconjugation. In humans, DA-sulfate (DA-S) is the primary form of DA in serum. The half-life of DA-S, at 3-4 hours, is much longer than that of free DA, which is only several minutes in circulation. Fasting DA-S concentrations in serum are approximately 10 nM, therefore, over 95% of circulating DA is in the form of DA-S in humans. After a meal, the concentration of DA-S in serum increases by over 50-fold, with much smaller increases in DA (228).
Sulfoconjugation is carried out by the monoamine-preferring SULT1A3 sulfotransferase, which is responsible for the sulfoconjugation of catecholamines and most of their metabolites. In humans, SULT1A3 has greater affinity for DA than other catecholamines due to a single amino acid substitution. The GI tissues contain large amounts of SULT1A3, and thus most sulfoconjugation occurs in the GI-tract (229). However, unlike these other types of deactivation, sulfoconjugation can be reversed by arylsulfatase A (ARSA), a lysosomal enzyme that is secreted by cells. Outside of the cell, ARSA can convert DA-S back into DA by removing the sulfate group, thus making DA bioactive again (230). Arylsulfatase C (ARSC), which de-conjugates steroid sulfates, is the subject of investigation for its potential role in increasing estrogen levels in breast cancer. Although ARSC is highly expressed in adipose tissue, it is unknown whether ARSA is also present in adipose tissue (231).
In addition to DA and other catecholamines, galactosyl and lactosyl sulfatides are substrates of ARSA. ARSA deficiency results in metachromatic leukodystrophy (MLD), a lysosomal storage disease. This rare condition is characterized by accumulation of these sulfatides in myelin forming cells, macrophages, and other peripheral tissues, which result in motor, cognitive, and psychiatric problems, and eventually leads to death. The human ARSA gene is located on chromosome 22 and is 3.2 kb long, and includes eight exons. There are more than 100 mutations in the ARSA gene, with varying impact on enzyme function (232).
Six catechols are readily detectable in human plasma, i.e. DA, epinephrine, norepinephrine, L-DOPA, DOPAC, and dihydroxyphenylglycol (DHPG), a metabolite of norepinephrine. However, DA-S is not detectable by rountine methods and thus has not received much attention until recently. The increase in DA-S after a meal may be the result of consumption of L-DOPA, DA, or DA-S, the conversion of dietary tyramine to DA, greater secretion and metabolism of endogenous DA in cells lining the GI-tract, or by generation of L-DOPA by tyrosinase in the GI lumen. Also, tyrosine derived from dietary protein could be converted to L-DOPA in sympathetic nerves or other cells that contain tyrosine hydroxylase. This L-DOPA could then be decarboxylated to DA in the bloodstream, then sulfoconjugated to DA-S in the GI-tract (228). DA-S is also derived from non-dietary sources, as demonstrated by low DA-S levels in patients with deficient in the enzyme DDC. In these patients, DA infusion results in increased plasma DA-S, indicating that DA-S in circulation is partially from DA in serum. In normal patients, more than 90% of dopamine sulfoconjugation occurs before DA goes into circulation. Other studies have indicated that DA-S in the bloodstream is not derived from DA in sympathetic nerves (233).
Structure and Variants
DA receptors (DAR) belong to the seven transmembrane (TM) domain GPCR superfamily. The DAR are divided into five subtypes: D1R, D2R, D3R, D4R and D5R. DAR consist of 387 to 475 amino acid residues in a single polypeptide chain, with the seven TM helices arranged in a ring to form a hydrophobic pocket encompassed by three extracellular and three intracellular loops. The sequence of the TM domains is highly conserved between receptors in the GPCR family. In the late 1970s, physiological, biochemical, and pharmacological evidence suggested that there were two opposing types of dopamine receptors, called D1R and D2R (234). Subsequently, it was determined that the D1R interacts with G-stimulatory (Gs) proteins to stimulate intracellular cAMP accumulation, while the D2R couples to G-inhibitory (Gi) proteins to decrease intracellular cAMP levels (Fig. 11). Three additional dopamine receptors were later discovered, and the DAR are now categorized as D1-like (D1R and D5R) and D2-like (D2R, D3R, and D4R).
In 1988, the rat gene for the D2R was cloned by utilizing sequence homology to other GPCRs (235). Shortly thereafter several groups cloned the D1R (236-238), and now all five DAR have been cloned and characterized. D1R, D2R, D3R, D4R, and D5R are located on chromosomes 5q, 11q, 3q, 11p, and 4p, respectively (239). The five DAR contain structural similarities and differences, with substantial sequence homology between receptors of the same family. The TM domain of D1R shares 80% homology with the TM domain of D5R, and the genes for these receptors lack introns. However, the genes for the D2-like receptors contain introns (6, 5, and 3 introns in the D2R, D3R, and D4R, respectively). The D2R shares 75% homology with D3R and 53% homology with D4R in their TM domains. The D1-like and D2-like receptors may have diverged from two gene subfamilies that defined by the absence or presence of introns, respectively (240).
In all five receptor subtypes, the N-terminus has a similar amount of amino acid residues, but possesses a varying number of consensus N-glycosylation sites. D1-like receptors contain one N-glycosylation site in their amino-terminus and one in their second extracellular loop (Fig. 11). There are four N-glycosylation sites in the D2R, three in the D3R and one in the D4R. The carboxyl-terminal tail is about seven times longer in D1-like receptors than in D2-like receptors. The COOH-terminal tail is rich in threonine and serine residues and includes a conserved cysteine residue that is palmitoylated to anchor the cytoplasmic tail to the plasma membrane (241;242). This cysteine residue is located at the end of the carboxy terminus in D2-like receptors, while in D1-like receptors, the cysteine residue is near the beginning of the carboxyl-terminal tail.
Like many GPCRs that interact with Gs proteins, the D1-like receptors have a short third intracellular loop, while the D2-like receptors have a longer third intracellular loop, which is characteristic of receptors that couple to Gi proteins. The receptor structure is stabilized by a disulfide bond formed between two cysteine residues on the second and third extracellular loops. There are also multiple phosphorylation sites on intracellular loop 3 and the carboxyl-terminal tail that are involved in desensitization of the receptor (243).
Studies utilizing protein modeling and site-directed mutagenesis suggest that agonist binding probably occurs within a narrow pocket of hydrophobic TM domains and involves several conserved amino acids. The amine group on the side chain of the catecholamine likely interacts with an aspartate residue in TM 3, while the hydroxyl groups of the catechol ring interact with two serine residues in TM 5. An aspartate residue in TM 2 and a phenylalanine residue in TM 6 are also involved in stabilization of ligand binding and receptor activation (244).
There are two main variants of the D2R, a long and a short isoform. These isoforms are formed by alternative splicing of an 87 bp exon, which creates a difference of 29 amino acid residues in the third cytoplasmic loop of the receptors. The long isoform has 443 amino acids, while the short isoform has 414 amino acids. The long and short D2R isoforms are similar in their pharmacological properties and distribution, although there is typically lower gene expression of the short D2R (245). However, they differ in their functional characteristics, since the third cytoplasmic loop is involved in intracellular signaling, and in the regulation of receptor internalization. While both isoforms inhibit adenylate cyclase, the long D2R has less effect than the short D2R (246).
In addition to the 400 amino acid D3R, two variants of the D3R have also been identified. These shorter variants are the result of alternative splicing and appear to be nonfunctional (247). In the mouse, the D3R is alternatively spliced into two variants that differ by 21 amino acids, but have similar pharmacological properties and distribution (248).
The D4R contains a variable number of tandem repeats (VNTR) polymorphism in the third intracellular loop that links the TM 5 and 6. This VNTR is 48 bp and may have 2 to 11 direct repeats. The 2, 4 and 7 repeats are the most common with a global frequency of 8%, 64% and 21%, respectively (249). However, there is considerable variability in allele frequency between different populations (i.e. the D4R containing a 7 repeat VNTR is found in less than 1% of Asian populations). While the seven-repeat allele is estimated to be five to ten times more recent than the four-repeat allele, positive selection has increased the frequency of the seven-repeat allele (250). The seven-repeat allele has been linked to ADHD, novelty seeking, addiction, and sexual behavior (249;251). One study using cells transfected with the three most common D4R isoforms found no difference between antagonist or agonist binding to these isoforms, or between the ability of these isoforms to couple to Gi proteins (252). However, another study has shown that the seven-repeat allele has lower potency for adenylate cyclase than the two- and four-allele repeats (249). In addition to the VNTR, the promoter for D4R contains three single-nucleotide polymorphisms (SNPs) at C-521T, C-616G, and A-809G, and a 120 bp tandem duplication, which appear to be related to sexual behavior (251).
The DAR are located in most areas of the CNS, the pituitary, and peripheral tissues, including the gut, kidneys, cardiovascular system, immune cells, and adrenal glands. The D1R and D2R are generally the most highly expressed of the DAR. The expression of the five DAR varies throughout the regions of the brain and is tissue-specific in peripheral tissues. The hippocampus, substantia nigra, and ventral tegmental area contain high amounts of D2R. The amygdala mainly expresses D1R, with little expression of D2R mRNA. Both D1R and D2R are highly expressed in the nucleus accumbens, the olfactory tubercle, and caudate putamen. In the hypothalamus, D1R and D2R are expressed at moderate levels, while D4R and D5R are expressed at lower levels. The DAR are also expressed in the pituitary, where they regulate the production of hormones, including PRL. The D2R is the most abundantly expressed DAR in the anterior pituitary, especially in lactotrophs (103).
In the kidneys, proximal tubular cells express all five DAR and produce DA through decarboxylation of L-DOPA taken up from circulation. Exogenous DA stimulates sodium excretion and glomerular filtration as well as dilating renal blood vessels and acting as a diuretic (228). The D4R are also expressed in the heart where DA increases cardiac contractility, and D2R and D1R have been found in blood vessels, where they are involved in vasodilation (244). Both D1R and D2R are also present in the GI-tract. Activation of D2R decreases acetylcholine release, thus D2R antagonists such as domperidone are used to treat gastric stasis, emesis, and functional dyspepsia (253). DAR are also expressed in immune cells. Four of the DAR (D2R, D3R, D4R, and D5R) are expressed in leukocytes, with the highest expression in B cells and Natural Killer cells (254).
Both the long and short isoforms of D2R are also expressed in the adrenal gland as well as several types of both benign and malignant adrenal tumors. Dopamine has been shown to regulate the secretion of catecholamines and aldosterone from the adrenal gland. In addition, benign hormone-secreting adenomas and cortisol-secreting carcinomas express D4R, while pheochromocytomas express both D4R and D5R. However, DAR expression has not been detected in androgen- and aldosterone-secreting carcinomas (255).
There are substantial differences between the pharmacological profiles of the D1-like and D2-like receptor subfamilies. Most agonists and antagonists preferentially bind to certain DAR, and there is less variation in the affinity of compounds to the receptors within each subfamily (Tables 1 and 2). However, binding affinities, usually represented by the dissociation constant (Kd), can differ significantly depending on the method of measurement. The Kd of a compound for each DAR varies with the radioligand utilized to determine the Kd. Greater concentrations of the compound must be used to compete with highly fat-soluble radioligands (e.g. [3H]spiperone) than with other radioligands (e.g. [3H]raclopride), and the lowest Kd value appears to be the most accurate (256).
Currently, it is difficult to distinguish the D1R and D5R pharmacologically, since their ability to bind to specific compounds, except DA itself, is nearly identical. DA, which binds to all five DAR, has a ten-fold greater affinity for D5R than D1R (244). The three D2-like receptors can be differentiated from one another due to the preferential binding of select agonists and antagonists. DA has 20 times higher affinity for D3R than D2R, and dopamine also has more affinity for D4R than D2R. The D2R agonists bromocriptine and cabergoline preferentially bind to D2-like receptors, but can also bind to D1-like receptors. However, cabergoline has a higher affinity for D2R, D3R, and D4R than bromocriptine (257). Therefore, cabergoline, which is more selective for D2-like receptors than bromocriptine, is becoming the preferred treatment for hyperprolactinemia due to its greater effectiveness and fewer side-effects (258). While compounds can distinguish between members of the D2-like receptor subfamily, no agonist or antagonist is selective for either the long and short isoforms of D2R. However, two antagonists of D2-like receptors, raclopride and sulpiride, show small differences in their binding affinities for the two D2R isoforms (259). The atypical anti-psychotic, clozapine, is about 10 times more specific for the D4R than other D2-like receptors. Some compounds do not differentiate between the DAR, such as apomorphine, a pan-agonist that has similar affinity for all five DAR. Haloperidol, a classical antipsychotic, and spiperone are antagonists of DAR that block all five receptors, but with greater affinity for D2-like receptors than D1-like receptors (244).
DA binds to its receptors to activate multiple signal transduction pathways. The most important of these is the cAMP/PKA pathway. When DA activates the D1-like receptors (D1R and D5R), the receptor couples to Gs proteins and activates AC. AC then stimulates an increase in cAMP levels, which leads to phosphorylation of PKA and transcription factors such as CREB or C/EBP and subsequent regulation of gene transcription (Fig. 12). Binding of DA to D2-like receptors (D2R, D3R, and D4R) causes the receptor to couple to Gi proteins and inhibit the cAMP/PKA pathway. DA also regulates potassium and calcium channels as well as the activity of phospholipase C (PLC) and the release of arachidonic acid. Na-K ATPase and Na/H exchangers are also controlled by DA. The short isoform of D2R also mediates stimulation of phospholipase D, which, together with DAG, is involved in cell metabolism, proliferation, and differentiation. Both D2R isoforms can couple with Gβγ subunits and protein kinase C (PKC) to induce the MAPK signaling pathway (103).
In rat pituitary GH3 cells, activation of D2R and the MAPK pathway results in apoptosis, while in other cells DA can also bind to D1R to activate the MAPK pathway (260;261). In GH3 cells, DA can also increase apoptosis through DAT and oxidative stress (260). In addition, DA can stimulate the cGMP/PKG pathway through activation of the D1R (262). Similar to classical GPCRs, the DARs can be desensitized by phosphorylation of the receptor, which results in the binding of an arrestin-like protein. The receptor is subsequently uncoupled from its G protein and the receptor is internalized into an endosomal compartment. Similar to other GPCRs, the DAR can form homo-oligomers (groups of the same receptor) or hetero-oligomers (groups of different receptors), although the function of this is not clear (245). In addition, DA can activate the PI3K/Akt pathway through D2R and c-Src/ epidermal growth factor receptor (EGFR), which results in protection against apoptosis in PC12 cells (263).
Dopamine Regulation of PRL in the Pituitary
In lactotrophs, DA binds to D2R, which rapidly stimulates membrane hyperpolarization and subsequent inactivation of voltage-gated calcium channels within seconds. This decreases calcium influx and intracellular free calcium and stimulates intracellular potassium (Fig. 12). This occurs either through direct or indirect coupling of D2R to Gα0 and potassium channels, which causes calcium influx to drop and immediate suppression of PRL secretion. Intracellular calcium is also reduced due to decreased mobilization of calcium from the endoplasmic reticulum, which is caused by inhibition of PKC and PLC. Finally, the D2R couples to Gi proteins, decreasing intracellular cAMP accumulation and PKA activation and results in the inhibition of PRL gene expression. DA also suppresses lactotrophs proliferation and treatment with DA agonists reduces the size of prolactinomas (103).
D. Dopamine Associated Pathologies
Parkinson's disease is a neurodegenerative condition that results in tremor, muscle rigidity, impaired speech, bradykinesia (slowing of movement), gait and posture abnormalities, cognitive dysfunction, and sometimes akinesia (loss of movement). This is a chronic and progressive disease that is the result of reduced function or death of dopaminergic neurons in the substantia nigra and striatum, which decreases stimulation of the basal ganglia. Parkinson's disease is primarily treated by administration of L-DOPA, which relieves symptoms by increasing basal ganglia function. Certain mutations in the dopamine system are related to Parkinson's, including a Taq1A polymorphism in the D2R (264).
Schizophrenia is a complex psychiatric disease characterized by altered cognitive and emotional functions. This disorder has both positive symptoms (e.g. hallucinations, psychosis, delusions and paranoia) and negative symptoms (e.g. slowed speech, low motivation, and decreased energy). Both genetic and environmental factors have been implicated as causes for schizophrenia. The effectiveness of classical antipsychotics, (i.e. DAR antagonists such as haloperidol), in treating schizophrenia suggest the involvement of dopamine in the pathogenesis of the disease. In addition, the second generation of antipsychotics, called atypical antipsychotics (including aripiprazole and olanzapine), bind to both DAR and serotonin receptors. Many mutations in the DAR or enzymes related to dopamine metabolism have been correlated with schizophrenia. One study found a single-nucleotide polymorphism of COMT (rs6267) that is associated with decreased risk of schizophrenia (265). A functional polymorphism in the D2R promoter, (-141C Ins/Del), and a polymorphism in the DRD2 gene (Ser311Cys) are also both associated with schizophrenia (266;267). However, other studies have only shown more moderate associations between mutations in the DAR and schizophrenia. Both classical and atypical anti-psychotics often have severe side effects, including significant weight gain and the metabolic syndrome as well as Parkinsonism (268). Life expectancy is greatly reduced in schizophrenic patients due to both obesity and an increase in cardiovascular risk factors associated with the use of anti-psychotic medications (269). One mechanism by which antipsychotics induce weight gain is through increased appetite via dopamine, serotonin, and histamine receptors. Hyperprolactinemia caused by antipsychotics may also be related to increased BMI (188). Most studies have focused on the actions of antipsychotics in the brain as the mechanism for weight gain, thus it is unknown whether antipsychotics can also directly affect adipocyte function.
Treatment of hyperprolactinemia patients with D2R agonists often results in the loss of the excess weight associated with this condition. Long-term treatment with bromocriptine or cabergoline causes weight loss in obese patients without hyperprolactinemia. Furthermore, in healthy obese women, short-term bromocriptine therapy improves symptoms of the metabolic syndrome, including reducing blood glucose and insulin levels while increasing the resting energy expenditure and serum FFA levels, indicating increased lipolysis (270). This suggests that blockade of the D2R may be a suitable target for weight-loss drugs, although the mechanism of action, whether in the brain or periphery, remains unclear. In addition, this is further evidence that the D2R is involved in the weight gain caused by neuroleptics.
Mutations in the D4R gene are associated with ADHD. ADHD is a disease that typically begins in childhood and is characterized by inattention, hyperactivity, distraction, and impulsivity. ADHD is correlated with the 7-repeat allele for the VNTR of the D4R (271). This mutation in the D4R is also associated with novelty seeking, impulsivity, addiction, and sexual behavior (251). A polymorphism in the 5' UTR of the DRD1 gene (DRD1-48A>G) is associated with severity of alcoholism, as well as novelty seeking, harm avoidance, and persistence (272). ADHD is commonly treated with psycho-therapy and drugs such as amphetamines, including methylphenidate (Ritalin), which increase extracellular dopamine and norepinephrine by blocking reuptake (249).