Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
The dopamine receptors fall into two families called
D1-like and D2-like receptors, based on their structural and
pharmacological features[1]. The D1 family includes D1- and
D5-receptor subtypes, and the D2 family consists of D2-, D3-
and D4-receptor subtypes. Dopamine receptors are mainly
expressed in the central nervous system and control motor
function, emotional state and endocrine
physiology[2,3].
Many central nervous system (CNS), cardiovascular and
renal diseases have been shown to be associated with
alterations in dopamine receptors. These diseases include
Parkinson disease, schizophrenia, migraine, drug dependence,
depression and Gilles de la Tourette
syndrome[4]. Dopamine is one of the principal neurotransmitters in the basal ganglia,
and plays a critical role in motor control and cognitive
function through interactions with dopamine receptors. The
possible role of dopamine D1-like receptors in brain function,
especially in learning and memory, has recently been
studied extensively[5-7]. Indeed, D1-like receptors play essential
roles in working memory[8] and other forms of cognition
activity[9]. The abnormality of these receptors also contribute
to Parkinson disease[10,11]. In addition, the D5 receptor
subtype is involved in modulating the release of hippocampal
acetylcholine, a neurotransmitter implicated in a variety of
cognitive processes[12].
Much evidence has been accumulated to indicate that
dopamine receptor agonists or antagonists can be
developed into therapeutic drugs for the treatment of CNS
diseases. It has been reported that D1-like receptor agonists
improve learning and memory in different animal
models[13,14]. Furthermore, D1-like receptor agonists or antagonists are
potential therapeutic drugs for addiction and Parkinson
disease[15-18]. Receptor agonists and antagonists also provide
essential tools for pharmacological and functional
characterization of the receptors.
Several high throughput screening methods to screen
agonists and antagonists of G-protein coupled receptors
(GPCR) have been developed[19-22]. Recently, we developed
a universal functional assay for
GPCR[23]. In the present report, we further modified this functional assay for human
D1-like agonist screening. A number of natural compounds
were identified that can specifically activate both human D1
and D5 receptors. Detailed pharmacological analysis
demonstrated that one of the agonists had distinct
pharmacological properties for these 2 receptors.
Materials and methods
Plasmid construction Human D1 and D5 receptors were
cloned by polymerase chain reaction (PCR). The primers
used for the PCR were: D1R5กฏ, 5กฏ-GCT GGA
TCC GTG CCC AAG ACA GTG ACC T-3กฏ; D1R3กฏ, 5กฏ-GGGAG CTC CGA GGG
GTA CAA ACA TCA-3กฏ; D5R5กฏ, 5กฏ-GGC GAA TTC GCG TGT
GTG TGC GTG CTT GTC AGT GT-3กฏ; and D5R3กฏ, 5กฏ-GGG AAG
CTT CTG AAG TTG GGA CCG CGC ACA GAC CG-3กฏ. PCR products were digested with
EcoRI and HindIII and subcloned into the
EcoRI/HindIII-digested mammalian
expression vector pCDNA3.1 (Invitrogen) to
generate pCDNA3.1/D1 and pCDNA3.1/D5. The 6×CRE and a mini promoter with
49 bps, containing a TATA box, were synthesized and
inserted into the pGL3 basic vector (Promega) to produce
pCRE/TA/Luci as a reporter gene. All plasmids were
confirmed by DNA sequencing.
Cell culture, transfection and stable cell line
generation CHO cells were maintained in RPMI-1640
medium containing 10% fetal calf serum at 37 °C. Cells were
transfected with dopamine D1 and D5 receptors and the
reporter construct using Lipofectin (Invitrogen). Stably
transfected cells were generated in the presence of 0.8 mg/mL
G418.
Natural product extracts Traditional Chinese Medicines
(TCM) were purchased from a local pharmacy in Chongqing,
China. Fifty grams of each TCM were extracted twice with
500 mL water at 80 °C for 2 h. The extracts were
concentrated to 100 mL by evaporation under low pressure at 80 °C.
Using this method, we prepared more than 300 samples for
human dopamine receptor agonist screening.
Luciferase assay Aliquots of 90 µL
cells (3×105 cells/mL) were seeded into each well of 96-well plates and
incubated overnight at 37 °C. A natural product sample
(10 µL) was added to each well and incubated at 37 °C for 6 h-10 h.
Bright-GloTM Lucifease assay reagent (100 µL; Promega) was
then added to each well, and the luciferase activity was
measured using AnalystTM HT (Molecular Device).
Results
Characterization of the CRE/TA/Luci reporter gene in
CHO cells We generated a reporter gene construct for
human dopamine D1 and D5 agonist screening. The construct
contained 6 copies of CRE and a TATA box linked to the
luciferase gene. The reporter gene construct was stably
transfected into CHO cells to generate the
CRE/TA/Luci/CHO cell line. This cell line was treated with forskolin (an
adenyl cyclase activator) at different concentrations, and
luciferase activity was measured. Our results showed that
forskolin stimulated reporter gene luciferase expression in a
dose-dependent manner (Figure 1). This result indicated that
increasing intracellular cAMP levels led to the activation of
CRE and TATA promoter and suggested that this assay could
be used for Gs-coupled receptor agonist screening. To
examine whether there was any endogenous dopamine
receptor in the CHO cells, we tested several dopamine receptor
agonists in the stable reporter gene cell line. Our results
demonstrated that none of the dopamine receptor agonists
had any effect on the cell line, indicating that no dopamine
receptor was expressed in CHO cells.
Development of reporter gene assay for D1-like
receptor agonist screening Mammalian expression vectors
containing human dopamine D1 or D5 receptors were transfected
into the reporter gene cell line. Activation of the receptors
leads to the elevation of cAMP and activates the cAMP
response element, and therefore induces the expression of
the reporter gene luciferase. We tested the natural ligand
dopamine and two other D1-like receptor agonists, dihydrexidine and chloro-APB, in the stable cell lines
expressing both dopamine receptors and the reporter gene.
The results are shown in Figure 2. The rank order of potency,
dopamine>dihydrexidine>chloro-APB, agreed with the
ligand-receptor binding analysis[24,25].
Optimization of the reporter gene assay
conditions We carried out a series of experiments to optimize the reporter
gene assay for drug screening. First, we examined the
incubation time for the D1-like receptor agonist in the assay.
Different concentrations of dihydrexidine were used for the
experiment. Our results showed that approximately 8 h
incubation with the agonist gave the highest response at all
concentrations (Figure 3). Therefore, we used an 8-h incubation
time for all of our experiments, unless otherwise indicated.
Second, because the compounds were in dimethylsulphoxide
(Me2SO) solution, we tested the effects of
Me2SO at different concentrations in the assay.
Me2SO concentrations of 1% or less had no effect on the signal (data not shown). In our
compound screen assays, the final concentration of
Me2SO was adjusted at 1% or less. Finally, we found that the
number of cells in each well may influence the reporter gene
assay. Figure 4 shows that although increasing cell number
gave a better signal after agonist stimulation, the background
was also higher. The best number of cells to use was
between 2×105 cells/mL and
4×105 cells/mL. Therefore, we
used 3×105 cells/mL in all compound screens using the
reporter gene assay. In addition, we calculated the coefficient
of variation (CV) for the assay in a standard 96-well plate.
The CV values were 6.5% for non-activated cells and 5.8%
for dopamine-stimulated cells. The Zกฏ
factor[26] was 0.58. These results suggest that the reporter gene assay system
was suitable for dopamine receptor agonist screening.
Identification of extracts active for the D1-like
receptor More than 300 herb extract samples were used for
the D1-like receptor agonist screening. From our previous
experiences for other G-protein coupled receptor agonist or
antagonist screens, we found that it was necessary to use
different concentrations of the raw extracts for the screen.
Therefore, in the D1-like receptor agonist screen, the extract
samples were screened twice at different concentrations. One
was at the original concentration and the other was a 5-fold
dilution of the sample. The samples that gave signals larger
than the mean value +3SD were selected as agonist
candidates (Figure 5). To eliminate the possibility that the agonist
candidates activated the reporter gene expression through
intracellular pathways other than the D1-like receptor, we
tested the samples in a cell line expressing the reporter gene
alone, without the D1-like receptor. We found that some of
the agonist candidates, such as Chinese ester pillar fungus,
could stimulate the reporter gene luciferase expression in
the reporter gene cell line (Figure 6). In contrast, sample
SBG492 activated the expression of the reporter gene in the
D1-like receptor expressing cell line, but had no effect in the
reporter gene cell line. This result suggested that SBG492
activated the reporter gene expression through human
D1-like receptor (Figure 6). Furthermore, we tested the sample
in more than 20 different GPCR using the same reporter gene
assay system (data not shown). Our results demonstrated
SBG492 could not activate other GPCR, suggesting that the
sample contained specific human D1-like receptor agonist.
We further analyzed the pharmacological properties of
SBG492 for human D1 and D5 receptors. The
EC50 values of SBG492 were 342.7 µg/mL for the D1 receptor and
31.7 µg/mL for the D5 receptor (Figure 7).
Discussion
Dopamine receptors are a subclass of the superfamily of
GPCR. Within the dopamine receptor family, both D1 and D5
are Gs-coupled receptors. Interaction of the receptors with
the natural ligand dopamine or other agonists leads to the
activation of adenyl cyclase and increases the intracellular
concentration of cAMP. The cAMP second messenger
system ultimately activates CRE and induces gene expression.
This is the basis of our reporter gene assay, which contains
6×CRE linked to the reporter gene luciferase.
The D1 and D5 dopamine receptors are genetically
distinct, sharing over 80% sequence homology within the
highly conserved 7 transmembrane-spanning domains, but
display only 50% overall homology at the amino acid
level[27]. The D1-like dopamine receptors, including D1 and D5, have
similar pharmacological properties[28]. Indeed, it is often difficult to pharmacologically distinguish between dopamine
D1 and D5 receptors using the same ligands. Therefore,
identification of agonists that have distinct pharmacological
properties for these 2 receptors should provide a useful tool
for their functional studies.
In our previous agonist or antagonist screening for GPCR
and other targets, we have successfully isolated active
components from crude extracts of
herbs[29-31]. Subsequent purification of the active components lead to the identification
of a single effective compound. The advantage of using the
crude extract is that the samples are easy to prepare and a
large amount of herbs can be screened in a short period of
time. On the other hand, since the crude extracts contain
hundreds of different compounds, some of them may have
negative effects on the targets or may even be harmful to
cells. Indeed, we found that in some cases, high
concentrations of the extracts in our cell-based assay had no effects,
while after dilution the samples showed agonist or
antagonist activity. Therefore, we used 2 concentrations of each
extract for the screening to increase the chances of
identifying D1-like receptor agonists. A number of extracts were
isolated as potential receptor agonists using the reporter
gene assay.
There are several other possible ways in which that the
extracts can activate the reporter gene expression other than
as human D1-like receptor agonists. For example, the
extracts may activate or inhibit other components in the cAMP
signal pathway, such as adenyl cyclase, protein kinase A,
cAMP-dependent phosphodiesterase or CRE binding protein, and eventually lead to the activation of the reporter
gene. It is also possible that the extracts may activate other
endogenous GPCR in the cell and lead to the induction of
reporter gene expression. To examine these possibilities, we
tested the activity of the herb extracts in a reporter gene cell
line that did not express human D1-like receptors. We found
that SBG492 did not induce the expression of the reporter
gene in the cell line, indicating that the extract activated the
reporter gene through the receptor. It is interesting to note
that SBG492 is approximately 10 times more potent for
human D1 receptor than for D5 receptor. Further
characterization of the agonist could provide important information for
pharmacological and functional studies on human D1-like
receptors.
Acknowledgements
We thank Hong GAO, Lu WANG, and Li-ping WANG for
their excellent technical assistance.
References
1 Braun AR, Laruelle M, Mouradian MM. Interactions between D1
and D2 dopamine receptor family agonists and antagonists: the
effects of chronic exposure on behavior and receptor binding in
rats and their clinical implications. J Neural Transm 1997; 104:
341-62.
2 Vallone D, Picetti R, Borrelli E. Structure and function of dopamine
receptors. Neurosci Biobehav Rev 2000; 24: 125-32.
3 Meyer ME, Cottrell GA, Van Hartesveldt C, Potter TJ. Effects
of dopamine D1 antagonists SCH23390 and SK&F83566 on
locomotor activities in rats. Pharmacol Biochem Behav 1993; 44:
429-32.
4 Emilien G, Maloteaux JM, Geurts M, Hoogenberg K, Cragg S.
Dopamine receptors-physiological understanding to therapeutic
intervention potential. Pharmacol Ther 1999; 84: 133-56.
5 Xu M, Moratalla R, Gold LH, Hiroi N, Koob GF, Graybiel AM,
et al. Dopamine D1 receptor mutant mice are deficient in
striatal expression of dynorphin and in dopamine-mediated
behavioral responses. Cell 1994; 79: 729-42.
6 El-Ghundi M, Fletcher PJ, Drago J, Sibley DR, O'Dowd BF, George
SR. Spatial learning deficit in dopamine D(1) receptor knockout
mice. Eur J Pharmacol 1999; 383: 95-106.
7 Centonze D, Grande C, Saulle E, Martin AB, Gubellini P, Pavon
N, et al. Distinct roles of D1 and D5 dopamine receptors in
motor activity and striatal synaptic plasticity. J Neurosci 2003;
23: 8506-12.
8 Lidow MS, Koh PO, Arnsten AF. D1 dopamine receptors in the
mouse prefrontal cortex: immunocytochemical and cognitive
neuropharmacological analyses. Synapse 2003; 47: 101-8.
9 Williams GV, Goldman-Rakic PS. Modulation of memory fields
by dopamine D1 receptors in prefrontal cortex. Nature 1995;
376: 572-5.
10 Hurley MJ, Mash DC, Jenner P. Dopamine D(1) receptor
expression in human basal ganglia and changes in Parkinson's disease.
Brain Res Mol Brain Res 2001; 87: 271-9.
11 Mailman R, Huang X, Nichols DE. Parkinson's disease and D1
dopamine receptors. Curr Opin Invest Drugs 2001; 2: 1582-91.
12 Hersi AI, Kitaichi K, Srivastava LK, Gaudreau P, Quirion R.
Dopamine D-5 receptor modulates hippocampal acetylcholine
release. Brain Res Mol Brain Res 2000; 76: 336-40.
13 Castellano C, Cestari V, Cabib S, Puglisi-Allegra S. Post-training
dopamine receptor agonists and antagonists affect memory
storage in mice irrespective of their selectivity for D1 or D2 receptors.
Behav Neural Biol 1991; 56: 283-91.
14 Castner SA, Goldman-Rakic PS. Enhancement of working
memory in aged monkeys by a sensitizing regimen of dopamine
D1 receptor stimulation. J Neurosci 2004; 24: 1446-50.
15 Linazasoro G. Conversion from dopamine agonists to
prami-pexole. An open-label trial in 227 patients with advanced
Parkinson's disease. J Neurol 2004; 251: 335-9.
16 Jenner P. Dopamine agonists, receptor selectivity and
dyskinesia induction in Parkinson's disease. Curr Opin Neurol 2003; 16
Suppl 1: 3-7.
17 Ruggieri S, Stocchi F, Baronti F, Viselli F, Horowski R, Lucarelli
C, et al. Antagonist effect of terguride in Parkinson's disease.
Clin Neuropharmacol 1991; 14: 450-6.
18 Meyer ME, Van Hartesveldt C, Potter TJ. Locomotor activity
following intra-accumbens microinjections of dopamine D1
agonist SK&F 38393 in rats. Synapse 1993; 13: 310-4.
19 Kariv II, Stevens ME, Behrens DL, Oldenburg KR. High
throughput quantitation of camp production mediated by activation of
seven transmembrane domain receptors. J Biomol Screen 1999;
4: 27-32.
20 Fitzgerald LR, Mannan IJ, Dytko GM, Wu HL, Nambi P.
Measurement of responses from Gi-, Gs-, or Gq-coupled receptors by
a multiple response element/cAMP response element-directed
reporter assay. Anal Biochem 1999; 275: 54-61.
21 George SE, Bungay PJ, Naylor LH. Functional analysis of the
D2L dopamine receptor expressed in a cAMP-responsive
luciferase reporter cell line. Biochem Pharmacol 1998; 56:
25-30.
22 Hertzberg RP, Pope AJ. High-throughput screening: new
technology for the 21st century. Curr Opin Chem Biol 2000; 4:
445-51.
23 Jiang C, Chen G, Zeng X, Ouyang K, Hu Y. Generation of a
bioactive neuropeptide in a cell-free system. Anal Biochem 2003;
316: 34-40.
24 Gilmore JH, Watts VJ, Lawler CP, Noll EP, Nichols DE,
Mailman RB. "Full" dopamine D1 agonists in human caudate:
biochemical properties and therapeutic implications.
Neuropharmacology 1995; 34: 481-8.
25 Grandy DK, Zhang YA, Bouvier C, Zhou QY, Johnson RA, Allen
L, et al. Multiple human D5 dopamine receptor genes: a
functional receptor and two pseudogenes. Proc Natl Acad Sci USA
1991; 88: 9175-9.
26 Zhang JH, Chung TD, Oldenburg KR. A simple statistical
parameter for use in evaluation and validation of high throughput
screening assays. J Biomol Screen 1999; 4: 67-73.
27 Sidhu A. Coupling of D1 and D5 dopamine receptors to multiple
G proteins: Implications for understanding the diversity in
receptor-G protein coupling. Mol Neurobiol 1998; 16: 125-34.
28 Seeman P, van Tol HH. Dopamine receptor pharmacology.
Curr Opin Neurol Neurosurg 1993; 6: 602-8.
29 Xu ZL, Gao H, Ou-Yang KQ, Cai SX, Hu YH. Establishment of a
cell-based assay to screen regulators for Klotho gene promoter.
Acta Pharmacol Sin 2004; 25: 1165-70.
30 Zheng XX, Ou-Yang KQ, Gao H, Xu ZL, Hu YH, Cai SX. Study
on high throughput screening method of identifying agonist for
neuromedin U2 receptor. Chin Pharm J 2004; 9: 185-8. Chinese.
31 Gao H, Ou-Yang KQ, Zheng XX, Xu ZL, Hu YH, Cai SX. High
throughput screening method of identifying agonist for
muscarinic receptor. Chin Pharmacol Bull 2003; 19: 776-9. Chinese.
|
