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Induction of dopamine D1 and D5 receptors in R28 cells by light exposures
Accepted Manuscript Induction of dopamine D1 and D5 receptors in R28 cells by light exposures Yan Ke, Wentao Li, Zhiqun Tan, Zhikuan Yang PII:
S0006-291X(17)30565-X
DOI:
10.1016/j.bbrc.2017.03.099
Reference:
YBBRC 37486
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 1 March 2017 Accepted Date: 19 March 2017
Please cite this article as: Y. Ke, W. Li, Z. Tan, Z. Yang, Induction of dopamine D1 and D5 receptors in R28 cells by light exposures, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.03.099. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Induction of dopamine D1 and D5 receptors in R28 cells by light exposures Yan Kea, b,1, Wentao Lia, b, c,1, Zhiqun Tanb,*, Zhikuan Yanga,* Aier School of Ophthalmology, Central South University, Changsha, Hunan Province,
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a
China b
Institute for Memory Impairments and Neurological Disorders, University of California,
Huizhou No.3 People's Hospital, Guangzhou Medical University, Huizhou, Guangdong
Province, China 1
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c
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Irvine, CA 92697, USA
These two authors contributed equally.
*: corresponding authors:
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Dr. Zhiqun Tan
UCI MIND, 835 Health Science Road, 140 Irvine Hall, Irvine, CA 92697-1280 Tel: +1 949-8241669; Email: [email protected]
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Prof. Zhikuan Yang
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Aier Eye Hospital Group, 198 New Century Building, Furong Middle Road, Changsha 41000, China
Tel: +86 13380071988; Email: [email protected]
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ABSTRACT Dopamine is known to play an important role in the pathophysiological process of myopia development relevant to the ambient lighting, but it is still poorly understood
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about how lighting regulates dopamine and its interaction with dopamine receptors to mediate the pathogenic signal transduction leading to alterations of ocular globe and the pathogenesis of myopia. Many studies have highlighted changes of ocular dopamine
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amount in response to different lighting conditions, but little attention has been paid to the dopamine receptors during these processes. Here we examined the effects of
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different lighting exposures on the expression of dopamine receptors in rat R28 retinal precursor cells. R28 cells normally grown in dark were exposed to a low (10 lux) or high (500 lux) intensity of a source of LED white light (5000 K~6000 K) for 12 h and total RNA was isolated either immediately or after certain time continuous growing in dark.
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Both conventional and real-time RT-PCR were performed to determine the expression of all five different dopamine receptors in cells after treatments. While the transcripts of dopamine D2, D3, and D4 receptors were not detected in the total RNA preparations of
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all the cells, those of D1 and D5 receptors (DRD1 and DRD5) were induced by lighting in contrast to the dark control. Elevated levels of DRD1 and DRD5 mRNA returned back
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close to the original levels once the cells were maintained in dark after light exposures. Immunofluorescence microscopy using a specific antibody confirmed an increase in the immunoreactivity of DRD1 in the cells exposed to 500 lux lighting versus dark control. Notably, treatments of R28 cells with nanomolar dosages of dopamine (0~500 nM) directly downregulated expression of both DRD1 and DRD5, whereas haloperidol (0~50 nM), a DRD1 antagonist, significantly induced expression of DRD1. These results
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suggest that dopamine receptors in the retinal cells might actively respond to the environmental lighting to act as an important player in the activation of the dopaminergic system in the ocular structures relevant to the lighting-induced pathogenic development
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of myopia.
Highlights
Dopamine receptors (DRD1 and DRD5) are expressed in R28 cells.
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Expression of DRD1 and DRD5 in R28 cells is induced by ambient lighting, but not
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•
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dopamine.
Expression of DRD1 in R28 cells is also upregulated by haloperidol.
Chemical compounds
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Keywords: dopamine receptors; R28 cells; ambient lighting.
Abbreviations
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Dopamine (PubChem CID: 681); Haloperidol (PubChem CID: 3559)
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LED, light emitting diode; PCR, polymerase chain reaction; RT-PCR, reverse transcription - polymerase chain reaction; qPCR, quantitative polymerase chain reaction; DRD1, dopamine receptor D1; DRD2, dopamine receptor D2; DRD3, dopamine receptor D3; DRD4, dopamine receptor D4; DRD5, dopamine receptor D5; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CT, cycle threshold; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; PBS, phosphate-
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buffered solution; DAPI, 4',6-diamidino-2-phenylindole; WGA, wheat germ agglutinin; FITC, fluorescein isothiocyanate.
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Acknowledgements This study was supported by the Science Research Foundation of Aier Eye Hospital Group [Grant No. 201301]. We thank Dr. Gail M. Seigel for generously granting us the permission to use R28 cells, which were kindly provided by
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Dr. Ping Bu.
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1. Introduction Myopia, also called nearsightedness or short-sightedness is the most common vision
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disorder affecting approximately over two thirds of the population in most Asian countries or districts [1]. Poor lighting has been known as an important risk factor for the development of nearsightedness in juniors [2,3]. Evidence-based studies demonstrate that outdoor activities significantly reduce the prevalence of myopia and slow down its
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progression in children [4,5,6], and ambient outdoor lighting shows protective effects on
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the development of myopia in animal models [7,8]. Growing studies have highlighted a role of dopamine in the pathogenic development of myopia [9]. While levels of dopamine and related metabolites exhibit a pattern of diurnal-nocturnal changes in the retina [10,11], lighting is the major factor regulating these light/dark cycle-dependent alterations [12,13]. Changes of intraocular dopamine are further linked to the growth
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rhythm of the eye in animal studies [14,15]. Compared with the low vision light environment, appropriate outdoor lighting induces a higher level of dopamine in the retina and blocks deprivation myopia [14,15,16]. When artificial refractive errors were
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induced in animal models by lenses, high levels of bright illuminance reduced the
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compensation rate for negative lenses and enhanced the rate of positive lenses, these effects were blocked by a dopamine antagonist [17,18]. These observations further suggest that light-induced myopia development might be mediated, at least in part, by dopamine-dopamine receptor(s)-directed activities. In fact, dopamine is known to function through its receptors, a family of five different types, which are divided into two groups, the excitatory type of D1-class (DRD1 and DRD5) and the inhibitory type of D2class (DRD2, DRD3, and DRD4) [19]. Studies have found that, except for DRD3, all the
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other four different types of receptors differentially express in the mammalian retina [20]. Molecular genetic analysis and pharmacological experiments reveal that increased dopamine-induced activation of dopamine D2 receptor promotes ocular axis growth to
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contribute to the development of myopia. In contrast, activation of D1 receptor by the increase of dopamine generates a stop signal to suppress axial growth once the eye has reached emmetropization [4,21,22,23]. Nevertheless, it is still poorly known that
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how the expression of dopamine receptors coordinates with dopamine in the retinal cells in response to ambient lighting to mediate signal transduction leading to the
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pathogenic development of myopia. To determine whether the ambient lighting directly affects expression of dopamine receptors in ocular cells, we employed both conventional and real-time quantitative PCR (qPCR) to examine the expression of all five different types of dopamine receptors in rat R28 cells exposed to the different
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lighting conditions. The results suggest that ambient lighting might directly regulate expression of dopamine receptors by which contributes to the activation of the dopaminergic pathway in ocular cells.
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2.1 Reagents
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2. Materials and methods
Dulbecco’s modified Eagle’s medium (DMEM), heat-inactivated fetal bovine serum, PenStrep, TrypLe, Trizol Reagent, High-Capacity cDNA Reverse Transcription Kit, Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody, Hoechst 33342 and fluorescent
wheat
germ
agglutinin
(WGA)
were
purchased
from
Invitrogen/ThermoFisher Scientific (Waltham, MA, USA). DNase I, dopamine (H8502),
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and haloperidol (PHR1724) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Quick-RNA MiniPrep was obtained from Zymo Research (Irvine, CA, USA). Taq 2x Master Mix was purchased from New England Biolabs (Ipswich, MA, USA). 2x SsoFast
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EvaGreen Supermix was purchased from Bio-Rad (Bio-Rad, Hercules, CA, USA). Rabbit polyclonal antibody against carboxyl terminus of DRD1 was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Vectashield mounting medium was purchased
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DNA Technologies (Coralville, IA, USA).
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from Vector Laboratories (Burlingame, CA, USA). All primers were synthesized by IDT
2.2 Cells and treatments
The R28 retinal precursor cell line that was originally immortalized from postnatal day 6 Sprague-Dawley rat retina was used for the experiments [24]. Cells were grown in high
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glucose DMEM supplemented with heat-inactivated 10% fetal bovine serum and 1% PenStrep in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. Medium was changed every other day and cells were replated after dissociation with 1xTrypLe as far
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as they reached about 95%~100% confluence (about every 3 or 4 days after plating). Cell passages within 10-20 were used for the experiments. To examine the effects of
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light exposures on cells, R28 cells in log-phase growth were first replated into 10 cm plates at about 60%~70% confluence. About 24 h after replating culture media were replaced with fresh medium and cells were placed in the same culture condition with extra lighting at either 10 lux or 500 lux intensity. Cells were either immediately harvested or continuously grow without lighting for another 12 or 24 h prior to harvest for total RNA isolation. The light was provided by a LED bulb which emits pure white light (5000K~6000K, 3W, CS Power Corp., CA, USA). The light intensity around the
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surface of cell culture medium was calibrated by a digital luxmeter (Dr. Meter DMLX1330B). Considering the potential possibility of lighting-induced changes of ingredients in the culture medium, which might directly affect expression of genes of
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interest, culture medium that was pre-exposed to 12 h corresponding lighting was used for dark control.
For drug treatments, dopamine and haloperidol were dissolved in distilled water and
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dimethyl sulfoxide (DMSO), respectively, to make micromolar scale stock solution, then
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1~2 µl stock solution was directly added into cell culture medium to obtain the final concentrations as indicated. Equal volume of the solvent was used as vehicle control. All cells were harvested for total RNA isolation 12 h after treatments. 2.3 RNA isolation and synthesis of cDNA
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Total RNA was isolated using either Invitrogen Trizol Reagent or Quick-RNA MiniPrep according to the manufacturers’ instruction or as previously described [25]. Prepared total RNA was further treated with DNase I to remove possible DNA contamination.
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Total RNA (2µg) was used for reverse transcription to make cDNA using the HighCapacity cDNA Reverse Transcription Kit following the manufacturer’s manual. The
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reaction was terminated by adding 200 µl TE buffer (10 mM Tris-HCl, 1mM EDTA, pH7.4).
2.4 PCR analysis
Conventional PCR was conducted as previously described [25]. Briefly, each PCR reaction was done with 5µl diluted cDNA mixture, 400 nM each primer, and 25µl Taq 2x Master Mix in a final volume of 50µl followed by agarose gel electrophoresis analysis.
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The detailed information for the specific primers and related PCR amplification conditions were listed in Table 1. PCR using primers for rat GAPDH was simultaneously performed and the results were used as a cDNA loading control. Rat brain (cortex)
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derived cDNA was used as a positive control for all five different receptors.
Real-time qPCR was performed as previously described with a minor modification [26]. Each qPCR reaction (20 µl) included diluted cDNA (about 50 ng), 2x SsoFast EvaGreen
(antisense)
TGTGTATCATCAGCGTGGACCGTT-3’
ATTGAGTTGGACCGGGATGAAGGA-3’ AGGTGACCGCATCTTCTTGT-3’
for
DRD1
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GGGCAGAGTCTGTAGCATCC-3’
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Supermix (10 µl), and primers: 5’-CCTTCGATGTGTTTGTGTGG-3’ (sense) and 5’-
(sense)
(antisense)
(sense)
and
for
[27],
or
and DRD5
[28],
5’5’-
or
5’-
5’-CTTGACTGTGCCGTTGAACT-3’
(antisense) for GAPDH [29]. The PCR reactions were subjected to 40 cycles (95℃ × 5
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s, 60℃ × 5 s) in a C1000 Thermal Cycler (Bio-Rad), and results were analyzed by CFX384 Real-Time System (Bio-Rad, USA). Each sample was run in triplicate. The specificity of qPCR reaction was validated by both melting curve analysis and agarose
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gel resolution of PCR products as described [26]. Once the number of cycle threshold
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(CT) was determined for each gene by the qPCR system, the relative abundance of the interest genes versus the internal control of GAPDH was depicted by ∆CT, which is calculated from CTinterest - CTGAPDH [26,30]. 2.5 Immunofluorescence microscopy For immunofluorescence, cells were grown on laminin-coated coverslips and treated as same as these grown in the plates. After the treatments (light exposures) both treated
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and control cells were rinsed with pre-chilled 1x phosphate-buffered solution (PBS, pH7.4) once and fixed in 4% paraformaldehyde 1xPBS for 10 min on ice. Prior to antibody incubation, fixed cells on coverslips were rinsed with 1xPBS and blocked with
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a blocking buffer as previously described [31]. Then cells were incubated with a specific rabbit polyclonal antibody against carboxyl terminus of DRD1 (H-109, 1:20 in 1xPBS) over night at 4 ℃ followed by 1xPBS wash, and further staining with fluorescent wheat
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germ agglutinin (Alexa Fluor 488-conjugated, 1:500 in 1xPBS) Alexa Fluor 594conjugated goat anti-rabbit secondary antibody (Invitrogen, 1:1000 in 1xPBS), and
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Hoechst 33342 (1:1000 in 1xPBS) for 40 min. Stained cells were coverslipped with Vectashield mounting medium after wash with 1xPBS (5 min x 3) for fluorescence microscopic examination under a Leica DM6B fluorescence microscope attached with
2.6 Data analysis
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SPOT Insight CCD camera.
All experiments were repeated independently at least 3 times. PCR gel pixel intensity
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was quantified by GelQuantNet program (www.gelquant-net.com). The data are depicted as the mean ± S.E.M. from three to six independent experiments. Statistical
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analysis was performed by one-way ANOVA analysis followed by Bonferroni test on SPSS Statistics 24. A p-value < 0.05 was considered to be statistically significant. 3. Results
3.1 Light exposures induce expression of DRD1 and DRD5 in R28 cells To examine possible effects of ambient lighting on the expression of dopamine receptors in R28 cells, we first conducted conventional RT-PCR to screen the
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transcripts of all the five receptors using specific primer sets (Table 1). The schematic diagrams in Fig.1A (left) show the relative target positions of each primer on the sequences of genes or cDNA. The results demonstrate no signal detected for all five
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receptors in all the cells which were grown in dark without lighting during the experiment, while the positive controls and GAPDH show all predicted band(s) for each gene and no genomic DNA signal, respectively. In contrast, increased expression of
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both DRD1 and DRD5 is noticeable in cells directly related to light intensity after 12 h lighting, whereas there is still no change with that of the D2-class receptors (DRD2,
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DRD3, and DRD4) in response to lighting. Detection of DRD1 and DRD5 signal was further confirmed by qPCR melting curves followed by agarose gel electrophoresis and quantification as shown in Fig. 1B and Fig. 3B, respectively. Moreover, the induction of DRD1 transcript in R28 cells by lighting was also corroborated by the increased
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immunoreactivity of DRD1 protein in both membrane and cytoplasmic compartments in contrast to DAPI-stained nuclei and FITC-WGA-labeled membrane structures as visualized by immunofluorescence microscopy following immunostaining using DRD1
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specific H-109 antibody and counterstaining (Fig.2).
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Following detection of DRD1 and DRD5 induction in R28 cells by lighting, we next examined whether elevated expression of both transcripts returned to the basal levels in dark after lighting exposures. Accordingly, cells were maintained in another 12 h or 24 h in dark prior to RNA isolation after 12 h lighting as shown in Fig. 3A. RT-qPCR results revealed that elevated expression of DRD1 and DRD5 by 12 h lighting gradually declined and returned to the point very close to their original basal levels within a 24 h period (Fig. 3B).
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3.2 Dopamine and its antagonist differentially affects expression of dopamine receptors As ambient lighting is associated with elevation of dopamine in the eyes, we next
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sought to determine whether dopamine or its antagonist directly modifies expression of DRD1 and DRD5 in R28 cells. R28 cells grown in a dark environment in a regular culture incubator were treated with nanomolar scale dopamine or its antagonist haloperidol for 12 h followed by RNA isolation for qRT-PCR analysis. The results
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demonstrate a significant decrease in abundance of both DRD1 and DRD5 transcripts
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in R28 cells treated with 50nM~500nM dopamine (Fig. 4A). In contrast, 10nM~50nM haloperidol treatments enhanced DRD1 expression about 20% (Fig. 4B). 4. Discussion
As a part of the central nervous system, the retina is known to express all the dopamine
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receptors except for DRD3 in mammals [20]. Although R28 cells were originally derived from the retina of an immature rat, there is only one report about the detection of DRD3 mRNA from a microarray-based gene expression profiling study [24]. Thus, it is still
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unclear whether R28 cells express any dopamine receptor(s). The results shown here indicate a low-level expression of the D1-class receptors, i.e., DRD1 and DRD5, in
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these cells. Furthermore, the ambient lighting directly regulates the expression of these two receptors in these cells and the exchange of darkness and light in the environment mediates corresponding cyclic changes in the abundance of expression. A large body of experimental evidence supports an important role of the dopaminergic activation in the control of eye growth and the pathogenic development of myopia [6,9,23]. Changes in expression of dopamine receptors in cells could be part of the accommodative response
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to the ambient lighting conditions thereby to regulate the dopaminergic activity. Interestingly, when treated with nanomolar scale dopamine, the expression of both DRD1 and DRD5 was inhibited perhaps due to the saturation of occupancy of the
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receptors by added dopamine. These dosages are close to the range of physiological concentrations of dopamine in the extracellular environment in the retina [13,32]. Thus, further studies are warranted to examine whether the dopamine receptors are still
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upregulated in the retina when bright light induces an increase in intraocular dopamine
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amount in animal models.
Compared to a poor ambient lighting environment, the bright natural outdoor lighting results in higher levels of intraocular dopamine and/or is associated with a lower risk for the development of myopia [4,9]. Our results from R28 cells suggest that ambient lighting directly regulate the expression dopamine receptors in addition to the alteration
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of local dopamine levels. Recent findings from molecular biology analyses reveal that activation of DRD2 is critical for the development of myopia, as genetic or pharmacological silencing of DRD2 attenuates the development of form-deprivation
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myopia in mouse [21], whereas pharmacological manipulation of DRD1 activity results
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in opposite effects on the eye growth in guinea pig [22]. Although D2-class dopamine receptors were not detected in these cells, cells treated with haloperidol, a D2-class receptor antagonist, appeared slightly increased expression of DRD1. In addition, a previous study reported expression of DRD3 in R28 cells [24], but we failed to detect DRD3 transcripts in these cells. Our observations appear consistent with the results from the retina [20]. This discrepancy might result from the variation of PCR primers
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and/or the presence of alternative splicing of DRD3 transcripts in R28 cells leading to the observation in the previous study [24].
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In summary, our findings indicate expression of dopamine D1 and D5 receptors in the rat R28 retinal precursor cell line. Ambient lighting conditions significantly regulate levels of expression of dopamine receptors in R28 cells. These observations suggest that changes in expression of dopamine receptors might also occur in the retina in
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response to the environmental lighting in vivo to contribute to the dopamine-related
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signal transduction in the eye. Nevertheless, further studies are needed to extend these findings. Disclosure Statement
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The authors declare no conflict of interest.
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Figure legends Figure 1. Light exposures induce expression of dopamine D1 and D5 receptor transcripts in R28 cells. A). Conventional PCR shows differential induction of both
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DRD1 and DRD5 mRNA in R28 cells exposed to 10 lux or 500 lux light for 12 h. Schematic on the left shows the approximate positions of specific primers (arrows) listed in Table 1 and predicted sizes of PCR products. PCR from rat brain cDNA was
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used as a positive control for each receptor. Black arrows indicate the two predicted bands (317 bp and 404 bp) detected in cDNA from rat brain but not R28 cells. Empty
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arrow indicates no signal of GAPDH PCR product from genomic DNA in all samples. B). Melting curves of qPCR and agarose gel images of the qPCR products for DRD1 and DRD5.
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Figure 2. Increased DRD1 immunoreactivity in R28 cells by light exposure. Immunofluorescence microscopy demonstrates weak DRD1 (red) staining in control cells (in dark) and markedly increased immunoactivity in cells after 12 h exposure to
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500 lux cool white light. Cell membrane is counterstained by wheat germ aggrelutinin
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(WGA, green), while nuclei are stained with Hoechst 33342 (blue).
Figure 3. Changes in expression of both DRD1 and DRD5 transcripts in response to ambient lighting conditions. A). A diagram showing the different schedules for R28 cells exposed to 500 lux lighting and/or maintained in dark. B). qPCR results showing the relative abundance of DRD1 and DRD5 transcripts in R28 cells grown under
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different ambient lighting conditions. Bars are depicted as mean ± S.E.M.; *denotes p< 0.05.
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Figure 4. Dopamine and its antagonist differentially affect expression of DRD5 and/or DRD1 in R28 cells. qPCR analysis demonstrates that nanomolar scale
dopamine (A) inhibits expression of DRD1 and DRD5 and haloperidol (B) increases
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expression of DRD1 in R28 cells. Bars are depicted as mean ± S.E.M.; *denotes p<
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0.05.
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Table 1. Primers and PCR conditions GenBank accession
Primer
number (target)
Forward:
NC_005116 (gDNA)
5’-TGACATCATGTGCTCTACGGC-3’ Reverse:
Forward: 5’-GCAGTCGAGCTTTCAGAGCC-3’
M35077 (cDNA)
303-323(F); 590-570 (R)
AC_000076 (gDNA)
56468-56487 (F); 59297-59277 (R)
M36831 (cDNA)
Reverse: 5’-TCTGCGGCTCATCGTCTTAAG-3’
X53278 (cDNA)
Forward:
Reverse: 5’-AGGAGTTCCGAGTCCTTTCCACG-3’ Forward:
Reverse: 5’-AGCCCAGCCAGGTGACAGCA-3’ Forward: DRD5
5’-GGAGGAAGGCTGGGAGCTAGAA-3’ Reverse: 5’-GCTGACACAAGGGAAGCCAGTC-3’ Forward:
GAPDH
54713-54735 (F); 58669-58647 (R)
×45''→ 57
size (bp)
94
×5'→ (94
×30'' →
72
×2') ×35 →72℃ ×5'→ 4℃
M84009 (cDNA)
717-740 (F); 1196-1177 (R)
594-615 (F); 996-975 (R)
NM_012768 (cDNA)
594-615 (F); 996-975 (R)
NC_005103 (gDNA)
3047-3066 (F); 3697-3678 (R)
2830
94℃×5'→ (94℃×30'' → 57℃×30'' → 404
72℃ ×1') ×35 → 72℃ ×5' → 4℃
317
95
×30''→ (95
→ 68
×20''→ 60.5
×20''
461
94 ×5' → (94 72
×30''→ 60
×30'' →
480
94
×5' → (94
×30'' → 60
×30'' →
72
×35'') ×35 →72℃ × 5'→ 4℃
→72℃ ×45'') ×35 → 72℃ ×5'→4℃
F, forward; R, reverse; bp, base pair.
403
403
Reverse: 520-539 (F); 971-952 (R)
672
×45'') ×35 →72℃ ×5'→ 4℃
94
AF106860 (cDNA)
3957
×30'') ×35→68℃×5'→ 4℃
5’-ACCACAGTCCATGCCATCAC-3’
5’-TCCACCACCCTGTTGCTGTA-3’
288
288
616-638 (F); 1075-1053 (R)
2303-2326 (F); 2974-2955 (R)
NC_005113 (gDNA)
Product
PCR condition
810-829 (F); 1126-1106 (R)
NC_005100 (gDNA)
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5’-TGCCCTGTCCGCTCATGCTACTGC-3’
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DRD4
NM_017140 (cDNA)
809-828 (F); 1212-1192 (R)
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DRD3
NC_005110 (gDNA)
5’-AGCATCTGCTCCATCTCCAACCC-3’
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5’-GAAATGGCATACGTCCTGCTC-3’
DRD2
1977-1997 (F); 2264-2244 (R)
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DRD1
Primer position
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Gene
×5' → (94
× 45'' → 56
× 45''
651
452
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S0006-291X(17)30565-X
DOI:
10.1016/j.bbrc.2017.03.099
Reference:
YBBRC 37486
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 1 March 2017 Accepted Date: 19 March 2017
Please cite this article as: Y. Ke, W. Li, Z. Tan, Z. Yang, Induction of dopamine D1 and D5 receptors in R28 cells by light exposures, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.03.099. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Induction of dopamine D1 and D5 receptors in R28 cells by light exposures Yan Kea, b,1, Wentao Lia, b, c,1, Zhiqun Tanb,*, Zhikuan Yanga,* Aier School of Ophthalmology, Central South University, Changsha, Hunan Province,
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a
China b
Institute for Memory Impairments and Neurological Disorders, University of California,
Huizhou No.3 People's Hospital, Guangzhou Medical University, Huizhou, Guangdong
Province, China 1
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c
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Irvine, CA 92697, USA
These two authors contributed equally.
*: corresponding authors:
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Dr. Zhiqun Tan
UCI MIND, 835 Health Science Road, 140 Irvine Hall, Irvine, CA 92697-1280 Tel: +1 949-8241669; Email: [email protected]
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Prof. Zhikuan Yang
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Aier Eye Hospital Group, 198 New Century Building, Furong Middle Road, Changsha 41000, China
Tel: +86 13380071988; Email: [email protected]
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ABSTRACT Dopamine is known to play an important role in the pathophysiological process of myopia development relevant to the ambient lighting, but it is still poorly understood
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about how lighting regulates dopamine and its interaction with dopamine receptors to mediate the pathogenic signal transduction leading to alterations of ocular globe and the pathogenesis of myopia. Many studies have highlighted changes of ocular dopamine
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amount in response to different lighting conditions, but little attention has been paid to the dopamine receptors during these processes. Here we examined the effects of
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different lighting exposures on the expression of dopamine receptors in rat R28 retinal precursor cells. R28 cells normally grown in dark were exposed to a low (10 lux) or high (500 lux) intensity of a source of LED white light (5000 K~6000 K) for 12 h and total RNA was isolated either immediately or after certain time continuous growing in dark.
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Both conventional and real-time RT-PCR were performed to determine the expression of all five different dopamine receptors in cells after treatments. While the transcripts of dopamine D2, D3, and D4 receptors were not detected in the total RNA preparations of
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all the cells, those of D1 and D5 receptors (DRD1 and DRD5) were induced by lighting in contrast to the dark control. Elevated levels of DRD1 and DRD5 mRNA returned back
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close to the original levels once the cells were maintained in dark after light exposures. Immunofluorescence microscopy using a specific antibody confirmed an increase in the immunoreactivity of DRD1 in the cells exposed to 500 lux lighting versus dark control. Notably, treatments of R28 cells with nanomolar dosages of dopamine (0~500 nM) directly downregulated expression of both DRD1 and DRD5, whereas haloperidol (0~50 nM), a DRD1 antagonist, significantly induced expression of DRD1. These results
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suggest that dopamine receptors in the retinal cells might actively respond to the environmental lighting to act as an important player in the activation of the dopaminergic system in the ocular structures relevant to the lighting-induced pathogenic development
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of myopia.
Highlights
Dopamine receptors (DRD1 and DRD5) are expressed in R28 cells.
•
Expression of DRD1 and DRD5 in R28 cells is induced by ambient lighting, but not
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•
•
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dopamine.
Expression of DRD1 in R28 cells is also upregulated by haloperidol.
Chemical compounds
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Keywords: dopamine receptors; R28 cells; ambient lighting.
Abbreviations
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Dopamine (PubChem CID: 681); Haloperidol (PubChem CID: 3559)
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LED, light emitting diode; PCR, polymerase chain reaction; RT-PCR, reverse transcription - polymerase chain reaction; qPCR, quantitative polymerase chain reaction; DRD1, dopamine receptor D1; DRD2, dopamine receptor D2; DRD3, dopamine receptor D3; DRD4, dopamine receptor D4; DRD5, dopamine receptor D5; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CT, cycle threshold; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; PBS, phosphate-
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buffered solution; DAPI, 4',6-diamidino-2-phenylindole; WGA, wheat germ agglutinin; FITC, fluorescein isothiocyanate.
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Acknowledgements This study was supported by the Science Research Foundation of Aier Eye Hospital Group [Grant No. 201301]. We thank Dr. Gail M. Seigel for generously granting us the permission to use R28 cells, which were kindly provided by
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Dr. Ping Bu.
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1. Introduction Myopia, also called nearsightedness or short-sightedness is the most common vision
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disorder affecting approximately over two thirds of the population in most Asian countries or districts [1]. Poor lighting has been known as an important risk factor for the development of nearsightedness in juniors [2,3]. Evidence-based studies demonstrate that outdoor activities significantly reduce the prevalence of myopia and slow down its
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progression in children [4,5,6], and ambient outdoor lighting shows protective effects on
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the development of myopia in animal models [7,8]. Growing studies have highlighted a role of dopamine in the pathogenic development of myopia [9]. While levels of dopamine and related metabolites exhibit a pattern of diurnal-nocturnal changes in the retina [10,11], lighting is the major factor regulating these light/dark cycle-dependent alterations [12,13]. Changes of intraocular dopamine are further linked to the growth
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rhythm of the eye in animal studies [14,15]. Compared with the low vision light environment, appropriate outdoor lighting induces a higher level of dopamine in the retina and blocks deprivation myopia [14,15,16]. When artificial refractive errors were
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induced in animal models by lenses, high levels of bright illuminance reduced the
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compensation rate for negative lenses and enhanced the rate of positive lenses, these effects were blocked by a dopamine antagonist [17,18]. These observations further suggest that light-induced myopia development might be mediated, at least in part, by dopamine-dopamine receptor(s)-directed activities. In fact, dopamine is known to function through its receptors, a family of five different types, which are divided into two groups, the excitatory type of D1-class (DRD1 and DRD5) and the inhibitory type of D2class (DRD2, DRD3, and DRD4) [19]. Studies have found that, except for DRD3, all the
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other four different types of receptors differentially express in the mammalian retina [20]. Molecular genetic analysis and pharmacological experiments reveal that increased dopamine-induced activation of dopamine D2 receptor promotes ocular axis growth to
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contribute to the development of myopia. In contrast, activation of D1 receptor by the increase of dopamine generates a stop signal to suppress axial growth once the eye has reached emmetropization [4,21,22,23]. Nevertheless, it is still poorly known that
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how the expression of dopamine receptors coordinates with dopamine in the retinal cells in response to ambient lighting to mediate signal transduction leading to the
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pathogenic development of myopia. To determine whether the ambient lighting directly affects expression of dopamine receptors in ocular cells, we employed both conventional and real-time quantitative PCR (qPCR) to examine the expression of all five different types of dopamine receptors in rat R28 cells exposed to the different
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lighting conditions. The results suggest that ambient lighting might directly regulate expression of dopamine receptors by which contributes to the activation of the dopaminergic pathway in ocular cells.
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2.1 Reagents
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2. Materials and methods
Dulbecco’s modified Eagle’s medium (DMEM), heat-inactivated fetal bovine serum, PenStrep, TrypLe, Trizol Reagent, High-Capacity cDNA Reverse Transcription Kit, Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody, Hoechst 33342 and fluorescent
wheat
germ
agglutinin
(WGA)
were
purchased
from
Invitrogen/ThermoFisher Scientific (Waltham, MA, USA). DNase I, dopamine (H8502),
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and haloperidol (PHR1724) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Quick-RNA MiniPrep was obtained from Zymo Research (Irvine, CA, USA). Taq 2x Master Mix was purchased from New England Biolabs (Ipswich, MA, USA). 2x SsoFast
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EvaGreen Supermix was purchased from Bio-Rad (Bio-Rad, Hercules, CA, USA). Rabbit polyclonal antibody against carboxyl terminus of DRD1 was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Vectashield mounting medium was purchased
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DNA Technologies (Coralville, IA, USA).
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from Vector Laboratories (Burlingame, CA, USA). All primers were synthesized by IDT
2.2 Cells and treatments
The R28 retinal precursor cell line that was originally immortalized from postnatal day 6 Sprague-Dawley rat retina was used for the experiments [24]. Cells were grown in high
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glucose DMEM supplemented with heat-inactivated 10% fetal bovine serum and 1% PenStrep in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. Medium was changed every other day and cells were replated after dissociation with 1xTrypLe as far
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as they reached about 95%~100% confluence (about every 3 or 4 days after plating). Cell passages within 10-20 were used for the experiments. To examine the effects of
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light exposures on cells, R28 cells in log-phase growth were first replated into 10 cm plates at about 60%~70% confluence. About 24 h after replating culture media were replaced with fresh medium and cells were placed in the same culture condition with extra lighting at either 10 lux or 500 lux intensity. Cells were either immediately harvested or continuously grow without lighting for another 12 or 24 h prior to harvest for total RNA isolation. The light was provided by a LED bulb which emits pure white light (5000K~6000K, 3W, CS Power Corp., CA, USA). The light intensity around the
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surface of cell culture medium was calibrated by a digital luxmeter (Dr. Meter DMLX1330B). Considering the potential possibility of lighting-induced changes of ingredients in the culture medium, which might directly affect expression of genes of
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interest, culture medium that was pre-exposed to 12 h corresponding lighting was used for dark control.
For drug treatments, dopamine and haloperidol were dissolved in distilled water and
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dimethyl sulfoxide (DMSO), respectively, to make micromolar scale stock solution, then
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1~2 µl stock solution was directly added into cell culture medium to obtain the final concentrations as indicated. Equal volume of the solvent was used as vehicle control. All cells were harvested for total RNA isolation 12 h after treatments. 2.3 RNA isolation and synthesis of cDNA
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Total RNA was isolated using either Invitrogen Trizol Reagent or Quick-RNA MiniPrep according to the manufacturers’ instruction or as previously described [25]. Prepared total RNA was further treated with DNase I to remove possible DNA contamination.
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Total RNA (2µg) was used for reverse transcription to make cDNA using the HighCapacity cDNA Reverse Transcription Kit following the manufacturer’s manual. The
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reaction was terminated by adding 200 µl TE buffer (10 mM Tris-HCl, 1mM EDTA, pH7.4).
2.4 PCR analysis
Conventional PCR was conducted as previously described [25]. Briefly, each PCR reaction was done with 5µl diluted cDNA mixture, 400 nM each primer, and 25µl Taq 2x Master Mix in a final volume of 50µl followed by agarose gel electrophoresis analysis.
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The detailed information for the specific primers and related PCR amplification conditions were listed in Table 1. PCR using primers for rat GAPDH was simultaneously performed and the results were used as a cDNA loading control. Rat brain (cortex)
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derived cDNA was used as a positive control for all five different receptors.
Real-time qPCR was performed as previously described with a minor modification [26]. Each qPCR reaction (20 µl) included diluted cDNA (about 50 ng), 2x SsoFast EvaGreen
(antisense)
TGTGTATCATCAGCGTGGACCGTT-3’
ATTGAGTTGGACCGGGATGAAGGA-3’ AGGTGACCGCATCTTCTTGT-3’
for
DRD1
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GGGCAGAGTCTGTAGCATCC-3’
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Supermix (10 µl), and primers: 5’-CCTTCGATGTGTTTGTGTGG-3’ (sense) and 5’-
(sense)
(antisense)
(sense)
and
for
[27],
or
and DRD5
[28],
5’5’-
or
5’-
5’-CTTGACTGTGCCGTTGAACT-3’
(antisense) for GAPDH [29]. The PCR reactions were subjected to 40 cycles (95℃ × 5
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s, 60℃ × 5 s) in a C1000 Thermal Cycler (Bio-Rad), and results were analyzed by CFX384 Real-Time System (Bio-Rad, USA). Each sample was run in triplicate. The specificity of qPCR reaction was validated by both melting curve analysis and agarose
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gel resolution of PCR products as described [26]. Once the number of cycle threshold
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(CT) was determined for each gene by the qPCR system, the relative abundance of the interest genes versus the internal control of GAPDH was depicted by ∆CT, which is calculated from CTinterest - CTGAPDH [26,30]. 2.5 Immunofluorescence microscopy For immunofluorescence, cells were grown on laminin-coated coverslips and treated as same as these grown in the plates. After the treatments (light exposures) both treated
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and control cells were rinsed with pre-chilled 1x phosphate-buffered solution (PBS, pH7.4) once and fixed in 4% paraformaldehyde 1xPBS for 10 min on ice. Prior to antibody incubation, fixed cells on coverslips were rinsed with 1xPBS and blocked with
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a blocking buffer as previously described [31]. Then cells were incubated with a specific rabbit polyclonal antibody against carboxyl terminus of DRD1 (H-109, 1:20 in 1xPBS) over night at 4 ℃ followed by 1xPBS wash, and further staining with fluorescent wheat
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germ agglutinin (Alexa Fluor 488-conjugated, 1:500 in 1xPBS) Alexa Fluor 594conjugated goat anti-rabbit secondary antibody (Invitrogen, 1:1000 in 1xPBS), and
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Hoechst 33342 (1:1000 in 1xPBS) for 40 min. Stained cells were coverslipped with Vectashield mounting medium after wash with 1xPBS (5 min x 3) for fluorescence microscopic examination under a Leica DM6B fluorescence microscope attached with
2.6 Data analysis
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SPOT Insight CCD camera.
All experiments were repeated independently at least 3 times. PCR gel pixel intensity
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was quantified by GelQuantNet program (www.gelquant-net.com). The data are depicted as the mean ± S.E.M. from three to six independent experiments. Statistical
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analysis was performed by one-way ANOVA analysis followed by Bonferroni test on SPSS Statistics 24. A p-value < 0.05 was considered to be statistically significant. 3. Results
3.1 Light exposures induce expression of DRD1 and DRD5 in R28 cells To examine possible effects of ambient lighting on the expression of dopamine receptors in R28 cells, we first conducted conventional RT-PCR to screen the
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transcripts of all the five receptors using specific primer sets (Table 1). The schematic diagrams in Fig.1A (left) show the relative target positions of each primer on the sequences of genes or cDNA. The results demonstrate no signal detected for all five
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receptors in all the cells which were grown in dark without lighting during the experiment, while the positive controls and GAPDH show all predicted band(s) for each gene and no genomic DNA signal, respectively. In contrast, increased expression of
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both DRD1 and DRD5 is noticeable in cells directly related to light intensity after 12 h lighting, whereas there is still no change with that of the D2-class receptors (DRD2,
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DRD3, and DRD4) in response to lighting. Detection of DRD1 and DRD5 signal was further confirmed by qPCR melting curves followed by agarose gel electrophoresis and quantification as shown in Fig. 1B and Fig. 3B, respectively. Moreover, the induction of DRD1 transcript in R28 cells by lighting was also corroborated by the increased
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immunoreactivity of DRD1 protein in both membrane and cytoplasmic compartments in contrast to DAPI-stained nuclei and FITC-WGA-labeled membrane structures as visualized by immunofluorescence microscopy following immunostaining using DRD1
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specific H-109 antibody and counterstaining (Fig.2).
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Following detection of DRD1 and DRD5 induction in R28 cells by lighting, we next examined whether elevated expression of both transcripts returned to the basal levels in dark after lighting exposures. Accordingly, cells were maintained in another 12 h or 24 h in dark prior to RNA isolation after 12 h lighting as shown in Fig. 3A. RT-qPCR results revealed that elevated expression of DRD1 and DRD5 by 12 h lighting gradually declined and returned to the point very close to their original basal levels within a 24 h period (Fig. 3B).
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3.2 Dopamine and its antagonist differentially affects expression of dopamine receptors As ambient lighting is associated with elevation of dopamine in the eyes, we next
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sought to determine whether dopamine or its antagonist directly modifies expression of DRD1 and DRD5 in R28 cells. R28 cells grown in a dark environment in a regular culture incubator were treated with nanomolar scale dopamine or its antagonist haloperidol for 12 h followed by RNA isolation for qRT-PCR analysis. The results
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demonstrate a significant decrease in abundance of both DRD1 and DRD5 transcripts
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in R28 cells treated with 50nM~500nM dopamine (Fig. 4A). In contrast, 10nM~50nM haloperidol treatments enhanced DRD1 expression about 20% (Fig. 4B). 4. Discussion
As a part of the central nervous system, the retina is known to express all the dopamine
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receptors except for DRD3 in mammals [20]. Although R28 cells were originally derived from the retina of an immature rat, there is only one report about the detection of DRD3 mRNA from a microarray-based gene expression profiling study [24]. Thus, it is still
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unclear whether R28 cells express any dopamine receptor(s). The results shown here indicate a low-level expression of the D1-class receptors, i.e., DRD1 and DRD5, in
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these cells. Furthermore, the ambient lighting directly regulates the expression of these two receptors in these cells and the exchange of darkness and light in the environment mediates corresponding cyclic changes in the abundance of expression. A large body of experimental evidence supports an important role of the dopaminergic activation in the control of eye growth and the pathogenic development of myopia [6,9,23]. Changes in expression of dopamine receptors in cells could be part of the accommodative response
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to the ambient lighting conditions thereby to regulate the dopaminergic activity. Interestingly, when treated with nanomolar scale dopamine, the expression of both DRD1 and DRD5 was inhibited perhaps due to the saturation of occupancy of the
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receptors by added dopamine. These dosages are close to the range of physiological concentrations of dopamine in the extracellular environment in the retina [13,32]. Thus, further studies are warranted to examine whether the dopamine receptors are still
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upregulated in the retina when bright light induces an increase in intraocular dopamine
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amount in animal models.
Compared to a poor ambient lighting environment, the bright natural outdoor lighting results in higher levels of intraocular dopamine and/or is associated with a lower risk for the development of myopia [4,9]. Our results from R28 cells suggest that ambient lighting directly regulate the expression dopamine receptors in addition to the alteration
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of local dopamine levels. Recent findings from molecular biology analyses reveal that activation of DRD2 is critical for the development of myopia, as genetic or pharmacological silencing of DRD2 attenuates the development of form-deprivation
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myopia in mouse [21], whereas pharmacological manipulation of DRD1 activity results
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in opposite effects on the eye growth in guinea pig [22]. Although D2-class dopamine receptors were not detected in these cells, cells treated with haloperidol, a D2-class receptor antagonist, appeared slightly increased expression of DRD1. In addition, a previous study reported expression of DRD3 in R28 cells [24], but we failed to detect DRD3 transcripts in these cells. Our observations appear consistent with the results from the retina [20]. This discrepancy might result from the variation of PCR primers
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and/or the presence of alternative splicing of DRD3 transcripts in R28 cells leading to the observation in the previous study [24].
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In summary, our findings indicate expression of dopamine D1 and D5 receptors in the rat R28 retinal precursor cell line. Ambient lighting conditions significantly regulate levels of expression of dopamine receptors in R28 cells. These observations suggest that changes in expression of dopamine receptors might also occur in the retina in
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response to the environmental lighting in vivo to contribute to the dopamine-related
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signal transduction in the eye. Nevertheless, further studies are needed to extend these findings. Disclosure Statement
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The authors declare no conflict of interest.
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[31] Z. Tan, W. Qu, W. Tu, W. Liu, M. Baudry, S.S. Schreiber, p53 accumulation due to down-regulation of ubiquitin: relevance for neuronal apoptosis, Cell Death Differ 7 (2000) 675-681.
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[32] S. Ohngemach, G. Hagel, F. Schaeffel, Concentrations of biogenic amines in fundal layers in chickens with normal visual experience, deprivation, and after reserpine
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application, Vis Neurosci 14 (1997) 493-505.
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Figure legends Figure 1. Light exposures induce expression of dopamine D1 and D5 receptor transcripts in R28 cells. A). Conventional PCR shows differential induction of both
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DRD1 and DRD5 mRNA in R28 cells exposed to 10 lux or 500 lux light for 12 h. Schematic on the left shows the approximate positions of specific primers (arrows) listed in Table 1 and predicted sizes of PCR products. PCR from rat brain cDNA was
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used as a positive control for each receptor. Black arrows indicate the two predicted bands (317 bp and 404 bp) detected in cDNA from rat brain but not R28 cells. Empty
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arrow indicates no signal of GAPDH PCR product from genomic DNA in all samples. B). Melting curves of qPCR and agarose gel images of the qPCR products for DRD1 and DRD5.
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Figure 2. Increased DRD1 immunoreactivity in R28 cells by light exposure. Immunofluorescence microscopy demonstrates weak DRD1 (red) staining in control cells (in dark) and markedly increased immunoactivity in cells after 12 h exposure to
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500 lux cool white light. Cell membrane is counterstained by wheat germ aggrelutinin
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(WGA, green), while nuclei are stained with Hoechst 33342 (blue).
Figure 3. Changes in expression of both DRD1 and DRD5 transcripts in response to ambient lighting conditions. A). A diagram showing the different schedules for R28 cells exposed to 500 lux lighting and/or maintained in dark. B). qPCR results showing the relative abundance of DRD1 and DRD5 transcripts in R28 cells grown under
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different ambient lighting conditions. Bars are depicted as mean ± S.E.M.; *denotes p< 0.05.
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Figure 4. Dopamine and its antagonist differentially affect expression of DRD5 and/or DRD1 in R28 cells. qPCR analysis demonstrates that nanomolar scale
dopamine (A) inhibits expression of DRD1 and DRD5 and haloperidol (B) increases
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expression of DRD1 in R28 cells. Bars are depicted as mean ± S.E.M.; *denotes p<
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0.05.
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Table 1. Primers and PCR conditions GenBank accession
Primer
number (target)
Forward:
NC_005116 (gDNA)
5’-TGACATCATGTGCTCTACGGC-3’ Reverse:
Forward: 5’-GCAGTCGAGCTTTCAGAGCC-3’
M35077 (cDNA)
303-323(F); 590-570 (R)
AC_000076 (gDNA)
56468-56487 (F); 59297-59277 (R)
M36831 (cDNA)
Reverse: 5’-TCTGCGGCTCATCGTCTTAAG-3’
X53278 (cDNA)
Forward:
Reverse: 5’-AGGAGTTCCGAGTCCTTTCCACG-3’ Forward:
Reverse: 5’-AGCCCAGCCAGGTGACAGCA-3’ Forward: DRD5
5’-GGAGGAAGGCTGGGAGCTAGAA-3’ Reverse: 5’-GCTGACACAAGGGAAGCCAGTC-3’ Forward:
GAPDH
54713-54735 (F); 58669-58647 (R)
×45''→ 57
size (bp)
94
×5'→ (94
×30'' →
72
×2') ×35 →72℃ ×5'→ 4℃
M84009 (cDNA)
717-740 (F); 1196-1177 (R)
594-615 (F); 996-975 (R)
NM_012768 (cDNA)
594-615 (F); 996-975 (R)
NC_005103 (gDNA)
3047-3066 (F); 3697-3678 (R)
2830
94℃×5'→ (94℃×30'' → 57℃×30'' → 404
72℃ ×1') ×35 → 72℃ ×5' → 4℃
317
95
×30''→ (95
→ 68
×20''→ 60.5
×20''
461
94 ×5' → (94 72
×30''→ 60
×30'' →
480
94
×5' → (94
×30'' → 60
×30'' →
72
×35'') ×35 →72℃ × 5'→ 4℃
→72℃ ×45'') ×35 → 72℃ ×5'→4℃
F, forward; R, reverse; bp, base pair.
403
403
Reverse: 520-539 (F); 971-952 (R)
672
×45'') ×35 →72℃ ×5'→ 4℃
94
AF106860 (cDNA)
3957
×30'') ×35→68℃×5'→ 4℃
5’-ACCACAGTCCATGCCATCAC-3’
5’-TCCACCACCCTGTTGCTGTA-3’
288
288
616-638 (F); 1075-1053 (R)
2303-2326 (F); 2974-2955 (R)
NC_005113 (gDNA)
Product
PCR condition
810-829 (F); 1126-1106 (R)
NC_005100 (gDNA)
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5’-TGCCCTGTCCGCTCATGCTACTGC-3’
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DRD4
NM_017140 (cDNA)
809-828 (F); 1212-1192 (R)
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DRD3
NC_005110 (gDNA)
5’-AGCATCTGCTCCATCTCCAACCC-3’
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5’-GAAATGGCATACGTCCTGCTC-3’
DRD2
1977-1997 (F); 2264-2244 (R)
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DRD1
Primer position
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Gene
×5' → (94
× 45'' → 56
× 45''
651
452
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