Molecular Vision 2015; 21:939-947
Received 18 December 2014 | Accepted 22 August 2015 | Published 25 August 2015
#The first two authors contributed equally to this work.
1Tianjin Eye Hospital, Clinical College of Ophthalmology, Tianjin Medical University, 4 Gansu Road, Heping District, Tianjin, China; 2Department of Ophthalmology, Qilu Hospital, Shandong University, Jinan, Shandong, China; 3The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital, Shandong University, Jinan, Shandong, China; 4School of Optometry and Vision Science, Faculty of Health, and Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia; 5Department of Ophthalmology, The Second People's Hospital of Jinan, Jinan, Shandong, China
Correspondence to: Xin Tang, Tianjin Eye Hospital, Tianjin Medical University, 4, Gansu Road, Heping District, Tianjin 300020, China; Phone: +86-022-27313336; FAX: +86-022-27313133; email: firstname.lastname@example.org
Purpose: Gamma-aminobutyric acidA (GABAA) receptors (GABAARs), which are ionotropic receptors involving chloride channels, have been identified in various neural (e.g., mouse retinal ganglion cells) and nonneural cells (e.g., mouse lens epithelial cells) regulating the intracellular calcium concentration ([Ca2+]i). GABAAR β-subunit protein has been isolated in the cultured human and rat RPE, and GABAAα1 and GABAAρ1 mRNAs and proteins are present in the chick RPE. The purpose of this study was to investigate the expression of GABAAα1 and GABAAρ1, two important subunits in forming functional GABAARs, in the cultured human RPE, and further to explore whether altering receptor activation modifies [Ca2+]i.
Methods: Human RPE cells were separately cultured from five donor eye cups. Real-time PCR, western blots, and immunofluorescence were used to test for GABAAα1 and GABAAρ1 mRNAs and proteins. The effects of the GABAAR agonist muscimol, antagonist picrotoxin, or the specific GABAAρ antagonist 1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) on [Ca2+]i in cultured human RPE were demonstrated using Fluo3-AM.
Results: Both GABAAα1 and GABAAρ1 mRNAs and proteins were identified in cultured human RPE cells; antibody staining was mainly localized to the cell membrane and was also present in the cytoplasm but not in the nucleus. Muscimol (100 μM) caused a transient increase of the [Ca2+]i in RPE cells regardless of whether Ca2+ was added to the buffer. Muscimol-induced increases in the [Ca2+]i were inhibited by pretreatment with picrotoxin (300 μM) or TPMPA (500 μM).
Conclusions: GABAAα1 and GABAAρ1 are expressed in cultured human RPE cells, and GABAA agents can modify [Ca2+]i.
Gamma-aminobutyric acidA (GABAA) receptors (GABAARs), which form a subclass of receptors of the inhibitory neurotransmitter GABA, are ionotropic receptors involving chloride channels that mediate fast synaptic inhibition when activated by GABA . GABAARs include 19 subunits (alpha 1–6, beta 1–3, gamma 1–3, delta, epsilon, theta, pi, and rho 1–3) . Most native GABAARs are thought to consist of two alpha, two beta, and one gamma or delta subunits, and some GABAARs can be formed from homo- or heteropentamers composed of rho subunits . The GABAARs being formed from rho subunits are also called GABAAOr receptors (previously termed GABAC receptors) [2,3].
GABAARs are mainly located in the neural system and retina [3,4], but have also been detected in many nonneural cells and tissues, for example, in human peripheral blood mononuclear cells , human hepatic cells and carcinomas , the human prostate , the human thyroid , murine enteroendocrine cell line STC-1 , cat chemosensory glomus cells , and the rat taste bud  and kidney . In the eye, GABAAR B-chain protein has been detected in human corneal stem cells  and the GABAAR β-subunit (GABAAβ) protein in the cultured human RPE . In animal models, the GABAAR beta 3 subunit protein has been identified in cultured mouse lens epithelial cells , GABAAβ protein isolated from the cultured rat RPE , and GABAAR alpha 1 (GABAAα1) and rho 1 subunit (GABAAρ1) mRNAs and proteins present in the chick RPE ; GABAAρ1 has also been visualized in the chick sclera .
GABAARs have been reported to regulate intracellular calcium concentration ([Ca2+]i) in a variety of cells. The GABAAR agonist muscimol increases [Ca2+]i in rat astrocytes , as well as in embryonic and early postnatal neocortical cells , embryonic rat ventral spinal cord neurons , embryonic rat striatal neurons , and rat cerebellar Purkinje neurons . It also alters [Ca2+]i in rat pituitary lactotrophs , immortalized gonadotropin -releasing hormone neurons , and alphaT3–1 gonadotropes . Within ocular tissues, muscimol increases [Ca2+]i in postnatal mouse retinal ganglion cells  and mouse lens epithelial cells ; these increases have been prevented by GABAAR antagonists bicuculline and picrotoxin.
The RPE is a single layer of predominantly hexagonal, pigmented cells that interact apically with the interphotoreceptor matrix and the photoreceptor outer segments and basally with the Bruch’s membrane of the vascular choriocapillaris (reviewed in ). Ca2+ signals play essential roles in the function of the RPE [28,29], and a normal [Ca2+]i appears to be essential if the RPE is to conduct its normal retinal maintenance functions . Abnormal [Ca2+]i levels in the RPE have been reported to be associated with high lipofuscin formation , retinal dystrophy , and cell death . The [Ca2+]i in RPE can be modified by various neural transmitter receptors, for example, acetylcholine muscarinic receptors , alpha7 nicotinic acetylcholine receptors , adrenergic receptors [36,37], and GABAB receptors . Whether stimulation of GABAARs modifies the [Ca2+]i in RPE is unknown. The purpose of this study was to investigate the expression of GABAAα1 and GABAAρ1—two important subunits in forming functional GABAARs —and the effects of the GABAAR agonist muscimol, antagonist picrotoxin, and specific GABAAρ antagonist TPMPA  on the [Ca2+]i in the cultured human RPE.
The RPE cell cultures were established from five donor eyecups from one eye of each of five previously healthy adults after the corneas were removed for donor cornea transplantation surgery. Donors included three males (aged 38, 73, and 75 years) and two females (aged 66 and 70 years) of Han nationality. Eyecups were received within 24 h of death. The study was approved by the Institutional Review Board of Shandong University’s Qilu Hospital and was performed in accordance with “The Code of Ethics of the World Medical Association (Declaration of Helsinki)” for experiments involving humans.
Primary cultures of human RPE cells were produced as previously described [40,41]. After the anterior segments and vitreous were removed, the retinal tissue was separated from the RPE and was retained for use as the positive control. After being washed three times in PBS (1X; 120 mM NaCl, 20 mM KCl, 10 mM NaPO4, 5 mM KPO4, pH 7.4; Gibco, Rockville, MD), The posterior eyecups were incubated with 0.25% trypsin-EDTA (Gibco) and the RPE cells were collected using Dulbecco’s modified Eagle’s medium (DMEM; manufacturer product number: 11965-092, Gibco, Rockville, MD) with 10% fetal bovine serum (Gibco, Rockville, MD) and seeded in a culture flask; cells from each donor were kept separate (n = 5). When reaching confluence, the cells were digested using 0.25% trypsin-EDTA, and were passaged; the experiment used the third passage cells. The cell phenotype was identified using immunofluorescence with an RPE-specific marker, namely the RPE 65 antibody (Millipore, Billerica, MA) . To determine whether the cultured cells were contaminated by other cells such as glial cells , Müller cells , fibroblasts , or choroidal melanocytes , the cultured cells were also stained with S100 antibody (Zhongshan Jinqiao Company, Beijing, China).
Total RNA extraction and reverse transcription were performed as previously described . Based on the sequences reported in the GenBank database, primers for human GABAAα1 and GABAAρ1 were designed and ordered from Shanghai Biosune Biotechnology Company (Shanghai, China). The primer sequences of human GABAAα1 were F: 5’- ACT TTT CAG CTG CTC CAG CCC G-3’, R 5’- CTC CCA ATC CTG GTC TCA GGC GA-3’. The sequences of human GABAAρ1 were F: 5’- GGC TGG TAC AAC CGT CTC TA-3’, R: 5’- CAC AAA GCT GAC CCA GAG GT-3’. RNA concentration and purity were determined at an optical density ratio of 260:280 using a spectrophotometer. SYBR Green real-time PCR was accomplished according to the manufacturer’s protocol. Briefly, denaturation was performed for 10 s at 95 °C, annealing for 10 s at 60 °C, and extension for 10 s at 72 °C. β-Actin was used as the housekeeping gene. The real-time PCR products were sent to the Shanghai Biosune Biotechnology Company for sequence analysis. Correct product size was confirmed by DNA agarose gel and lack of primer dimer formation was verified by melt curve analysis. The human retina was used as the positive control, and the samples without cDNA were used as the negative control.
Western blots were performed as previously described . Total protein was extracted separately from each RPE sample and human retina sample (which served as the positive control). Proteins were separated by 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes. After being blocked in 5% fat-free milk diluted in Tris-buffered saline with Tween (TBST; 0.1% Tween-20, 150 mM NaCl, 50 mM Tris, pH 7.5) for 1 h, the membrane was incubated with the polyclonal goat anti-human GABAAα1 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal goat anti-human GABAAρ1 antibody (1:100, Santa Cruz, CA), or monoclonal mouse anti-human β-Actin antibody (Zhongshan Jinqiao Company, Beijing, China) overnight at 4 °C, and then was followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000) for 1 h at 37 °C. The bands developed by use of HRP-conjugated secondary antibody detection kits (Jingmei, Shenzhen, China) were scanned and analyzed with FluorchemTM 9900 Gel Imaging System (Alpha Innotech, San Leandro, CA).
Immunofluorescence was performed as previously described [16,46,47]. Briefly, after being fixed with 4% paraformaldehyde for 15 min, cells were blocked with 10% normal donkey serum for 30 min at room temperature and then were incubated with monoclonal mouse anti-human RPE 65 antibody (1:300), monoclonal mouse anti-human S100 antibody (1:200), polyclonal goat anti-human GABAAα1 antibody (1:50), and polyclonal goat anti-human GABAAρ1 antibody (1:50) overnight at 4 °C. Subsequently, the cells were incubated with donkey anti-mouse secondary antibody (Alexa Fluor 568 conjugated; 1:1,000; Invitrogen, CA) and donkey anti-goat secondary antibody (Alexa Fluor 488 conjugated; 1:1,000; Invitrogen) for 30 min at 37 °C. Cells incubated with PBS instead of primary antibodies served as negative control. A drop of Prolong Gold anti-fade reagent with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen) was added before cell images were acquired using an LSM 710 laser confocal microscope (EC Plan-Neofluar 40×/1.30 Oil objective, N.A. 0.55) equipped with ZEN 2009 Light Edition software (Zeiss, Germany).
[Ca2+]i was measured as previously described ; Ca2+-dependent RPE functions and the use of RPE cell cultures to study these have been recently reviewed . RPE cells were seeded onto specialized cell culture dishes 35 mm in diameter with a glass disc of 20 mm in diameter inserted in the middle of the base (NEST, Wuxi, China). When cultures were confluent, the cells were incubated with 5 μM fluo3-acetoxymethyl ester (Fluo3-AM) calcium indicator for 20 min in the dark at 37 °C in normal physiological saline solution (N-PSS; 140 mM NaCl, 1 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 5 mM HEPES, pH 7.4). After being rinsed twice with N-PSS, cells were kept in N-PSS for another 10 min; the cell culture dishes with RPE attached were placed on the viewing stage of a confocal microscope (LSM 710, Zeiss). While images were being captured, the GABAAR agonist muscimol (100 μM; Sigma, MO) was added to the assay at set time points. To determine the impact of the GABAAR antagonist, cells were preincubated with either the GABAAR antagonist picrotoxin (300 μM; Sigma) or the GABAAρ antagonist TPMPA (500 μM; Sigma) for 10 min before muscimol (100 μM) was added. To determine the effects of muscimol on [Ca2+]i in buffer without Ca2+, cells were rinsed and kept in PSS without Ca2+ added. PBS was added instead of agents as the control. Sequences of images were acquired using the laser confocal microscope (LSM 710, Zeiss) equipped with a 488 nm laser at 5 s intervals.
The fluorescent intensity over the cultured human RPE cell body was measured before and after agent application, and was calculated and analyzed using Zen 2009 Light Edition software (Zeiss).
Data were acquired from the five different cell samples and each was repeated at least in duplicate. Numerical data were analyzed using SPSS software (SPSS Inc., version 16.0, Chicago, IL) and were expressed as mean ± standard deviation (SD). The differences in the baseline fluorescent intensity across treatment groups in the buffer either with Ca2+ or without Ca2+ were analyzed using one-way ANOVA and the Dunnett’s post-hoc test. The differences of fluorescent intensity between the baseline and that after either muscimol or PBS was added in each group were analyzed using paired t-test. Differences were defined as significant at p<0.05.
The cultured primary human RPE cells reached confluence in 2–3 weeks. The third passage cells were all positively stained with the RPE 65 antibody, and were negative for the S100 antibody (Figure 1). This suggests that the cultured cells were RPE cells and were not contaminated with other cells such as glial cells , Müller cells , fibroblasts , or choroidal melanocytes .
Real-time PCR showed that GABAAα1 and GABAAρ1 mRNAs were detected in cultured human RPE and in the retina (positive control), but not in the negative control. Ethidium bromide–stained agarose gels of real-time PCR products showed specific bands present at the positions of 211 bp (GABAAα1), 263 bp (GABAAρ1), and 302 bp (β-Actin) in cultured human RPE and retina samples, but not in the negative control (Figure 2). The sequence analysis revealed that the sequence of the products corresponded to the targeted mRNA sequence of the GABAAα1 and GABAAρ1.
Western blots revealed intense bands at 51 kDa, 48 kDa, and 43 kDa for samples incubated with the anti-GABAAα1, anti-GABAAρ1, and anti-β-Actin antibody, respectively, in cultured human RPE and retinal samples (positive control; Figure 3A).
Immunofluorescence revealed immunoreactivity to antibodies for GABAAα1 and GABAAρ1 in the cultured human RPE cells but not in the negative control. Immunofluorescence was mainly observed in the cell membrane and was also identified in the cytoplasm, but not in the nucleus (Figure 3B).
There were no significant differences in baseline fluorescent intensity across treatment groups in the buffer either with Ca2+ (one-way ANOVA, p>0.05, n=5), or without Ca2+ (one-way ANOVA, p>0.05, n=5). The GABAAR agonist muscimol (100 μM) induced a rapid and significant [Ca2+]i increase in the cultured human RPE cells in the buffer either with Ca2+ (paired t-test, p<0.05, n=5; Figure 4A) or without Ca2+ (paired t-test, p<0.05, n=5; Figure 4D). The [Ca2+]i reached its peak in 20–40 s, and then gradually declined (Figure 4A,D). When the cultured human RPE cells were preincubated with either the GABAAR antagonist picrotoxin (300 μM; Figure 4B,E) or the GABAAρ antagonist TPMPA (500 μM; Figure 4C,F), the muscimol (100 μM) induced [Ca2+]i increase was completely blocked (both when the buffer contained Ca2+, Figure 4B,C, and when it did not, Figure 4E,F). The addition of the control agent, PBS, did not alter the [Ca2+]i of the cultured human RPE cells (paired t-test, p>0.05, n=5).
GABAAα1 and GABAAρ1 mRNAs and proteins were expressed in the cultured human RPE cells; the protein was primarily located in the cell membrane, and was also present in the cytoplasm, but was not observed in the nucleus. The GABAAR agonist muscimol induced a [Ca2+]i rise in the cultured human RPE cells irrespective of whether the buffer contained Ca2+ or not, and the [Ca2+]i increase was completely blocked by the GABAAR antagonist picrotoxin and the GABAAρ antagonist TPMPA. This suggests that both GABAAα1 and GABAAρ1 occur in cultured human RPE cells and that in these cells, GABAAR stimulation can modify [Ca2+]i. This means that GABAAβ , GABAB1 and GABAB2 , and now GABAAα1 and GABAAρ1 have been identified in cultured human RPE cells, collectively showing that human RPE cells possess various GABA receptors, and thus that the GABAergic pathways in the RPE are likely complex.
In the retina, the GABAAR agonist muscimol increase the [Ca2+]i in the early embryonic chick retina  and in postnatal mouse retinal ganglion cells . The GABAAR antagonist picrotoxin has been shown to attenuate the muscimol-induced [Ca2+]i increase in postnatal mouse retinal ganglion cells . In this study, we observed that the GABAAR agonist muscimol increase the [Ca2+]i in cultured human RPE cells and that the antagonist picrotoxin blocked this rise. This suggests that activation of the GABAAR modifies the [Ca2+]i in RPE cells as it does in retinal ganglion cells .
In the retina, the GABAAρ antagonist TPMPA has been shown to suppress both the GABA-induced current and the light-evoked feedback inhibition observed in ON-cone bipolar cells and to enhance the light-evoked excitatory postsynaptic currents of ON-transient amacrine cells . TPMPA reduces the stimulation thresholds of ON-center retinal ganglion cells , and increases the light responsiveness of retinal ganglion cells in a rat model of retinitis pigmentosa . Here, we found that TPMPA inhibited the muscimol induced [Ca2+]i increase in cultured human RPE cells; suggesting that the GABAAρ receptor functions are not unique to the retina.
GABAARs have been shown to be involved in eye growth and myopia development in animal models [53-58], and levels of GABA transporter 1 (GAT-1) increased in the myopic mouse retina . The GABAAR agonist muscimol induced myopia development in chicks  and prevented the myopia-reducing effects of normal vision , whereas the GABAAρ antagonist TPMPA inhibited form-deprivation myopia in both chicks [53,54] and guinea pigs [56,58]. The pathway, targets, and mechanisms for the eye growth effects of these GABA agents are unclear. Previously, as GABAARs are expressed in retina , and the GABAAR agonist muscimol and the GABAAρ antagonist TPMPA modify the functions of many retinal cells [26,50,51], the retina was considered the most plausible target. In this study, we found that GABAAα1 and GABAAρ1 are expressed in the human RPE, and that muscimol and TPMPA modulate the [Ca2+]i. Thus, the RPE is a potential additional site for the actions of muscimol and TPMPA. The sclera ultimately determines ocular size , and thus, how activation of the GABAARs in the RPE might influence scleral physiology requires further investigation.
Additional roles of GABA receptors and their impacts on RPE physiology require investigation. Here, we have shown that GABA agents can modify the [Ca2+]i of RPE cells. Ca2+ acts as a second messenger controlling many cellular processes, including secretion, cell differentiation, and signal transmission (reviewed in ). For example, in cultures of RPE cells, calcium antagonists have been shown to reduce RPE cell proliferation and increase pigmentation . The Ca2+ balance is critical for preventing the accumulation of lipofuscin in RPE cells during phagocytosis . [Ca2+]i overload causes lipofuscin accumulation, and this can be prevented by a Ca2+ antagonist that suppresses Ca2+ influx . In cultured mouse lens epithelial cells  GABA agents can modify the [Ca2+]i, and the authors  speculate that GABA-mediated Ca2+ signaling may be used to prevent sustained Ca2+ overload, which can both cause cataract (reviewed in ) and trigger apoptosis . In the RPE, GABA-mediated Ca2+ signaling is thus likely to have similarly critical cellular roles. In summary, GABAAα1 and GABAAρ1 mRNAs and proteins were expressed in cultured human RPE cells, and the GABAAR agonist muscimol, antagonist picrotoxin, and GABAAρ antagonist TPMPA were shown to modify [Ca2+]i.
This work was supported by grants from the National Nature Science Foundation of China (81271038 to ZYC, 80200212 to XPW, and 81270984 to XT) and grants from the Postdoctoral Science Foundation of China (2013M530879 and 2014T70221 to ZYC). The authors thank the Red Cross for providing postmortem human donor eyes. The co-corresponding authors: Xin Tang, email: email@example.com; Or Zhen-Ying Cheng, email: firstname.lastname@example.org.