|Molecular Vision 2002;
Received 12 January 2000 | Accepted 3 July 2002 | Published 9 July 2002
The mineralocorticoid receptor in rodent retina: Ontogeny and molecular identity
Nady Golestaneh,1 Serge Picaud,2
1INSERM E9912, Faculté de Médecine Paris VI, Paris, France; 2INSERM E9918, Universite Louis Pasteur, Medicale A, Strasbourg, France
Correspondence to: Dr. M. Mirshahi, INSERM E9912, Faculté de Médecine de Paris VI, 15 Rue de l'Ecole de Médecine, 75006 Paris, France; FAX: 33 1 43 26 26 73; email: email@example.com
Purpose: Mineralocorticoid hormones contribute to ion-water balance in all cell types. In this study, we investigated the presence of mineralocorticoid receptors in rat and bovine ocular tissues and during retinal development.
Methods: Isolated photoreceptors and/or intact retina, retinal pigment epithelium (RPE) cells, and ciliary body were analyzed for the expression of MR (Mineralocorticoid Receptor) using the polymerase chain reaction (PCR) technique. Since aldosterone can stimulate the expression of epithelial Na+ channels (ENaC), expression of this gene in RPE was measured under basal and aldosterone-induced level.
Results: MR was expressed in rat photoreceptors and in the inner retina (inner nuclear layer and ganglion cell layer) even one day after birth, almost hundred percent identity was observed between rat retina and kidney MR gene products. The expression was also present in the RPE and in the ciliary body. ENaC gene was expressed in RPE and generated a predicted band at 520 bp following RT-PCR amplification which was 95% homology to that of ENaC mRNA from bovine kidney. The incubation of RPE cells in vitro with aldosterone increased the mRNA level of ENaC.
Conclusions: MR expression in the mammalian retina, RPE, and ciliary body extends the potential field of action for mineralocorticoid hormones. Results on RPE cells are consistent with the idea that steroid hormones may regulate the physiology of these tissues by modulating ENaC expression. This study provides new light on the potential effect of mineralocorticoid in this area of the nervous system.
Steroid hormones influence the development, differentiation, and homeostasis of a large number of mammalian tissues by binding to receptors that are members of a superfamily of proteins involved in the transcription regulation of cell-specific genes [1,2]. Mineralocorticoid hormones regulate the ion-water balance in many cell types of mesodermal and ectodermal origins [3,4] via appropriate modulation of ion channels and pumps in cell membranes. Both the epithelial Na+ channel (ENaC) and the Na+/K+/ATPase pump were for instance found to be modulated by mineralocorticoids [4,5].
In ocular tissues, ionic and fluid transport mechanisms play a prominent role in the maintenance of several physiological processes that include aqueous humor secretion and intraocular pressure , hydration and transparency of the cornea  and the lens , adhesion of the retina, and photoreceptor function . Intracellular and extracellular ionic environment is regulated by the Na+/K+/ATPase pump in several cell types such as the non pigmented ciliary epithelium , retinal pigment epithelium , lens epithelium , and corneal endothelium . Na+ flux are also very important in retinal neurons especially photoreceptors to generate their light responses.
In a previous study, we found evidence of both MR [12,13] and ENaC  in ocular and retinal tissues with immunocytochemical methods. To further analyze MR molecular composition, we isolated mRNA from different tissues and processed them for RT-PCR. Regulation of ENaC expression was used to assess the presence of functional MR in RPE cells.
RPE cells were isolated from bovine enucleated eyes, obtained from the local slaughter house. Eyes were stored for 24 h at 4 °C to reduce adhesion between the neural retina and the pigmented epithelium, and bisected posterior to the ora serrata. After removal of the vitreous body and of the retina, interior of the eyecup was rinsed with RPMI-1640 (Gibco BRL, Paisley, Scotland, UK) and incubated with 1 ml trypsin (0.25%, Gibco BRL) for 1 h at 37 °C. RPE cells were pipetted out of the Bruch's membrane, washed with RPMI by centrifugation (10 min, 1500x g) and finally resuspended in RPMI 1640 supplemented with 5 mM glutamine, 100 mg/ml penicillin, 100 mg/ml streptomycin and 20% fetal bovine serum. The cells were seeded at 5x104 cells/cm2 in tissue culture flasks and incubated at 37 °C in 5% CO2 atmosphere. The influence of aldosterone 10 mM (dissolved in ethanol 1% final) and ethanol (1%) was assessed on RPE plated at 30,000 cells/ml in tissue culture and cultured for 12, 24, and 36 h in 10% FCS until confluent.
Retina and ciliary body were obtained by dissection of rat eyes cup posterior to the ora serrata after removal of the vitreous body under a binocular microscope. The photoreceptor layer was isolated as described previously  by horizontal sectioning of the retina with a vibratome series 1000 (Technical product international, Saint Louis, MO). Briefly, the retina was flattened by putting the scleral surface down on a 20% gelatin block using 4% gelatin warmed to 40 °C in CO2 free DMEM. When the razor blade just touched the vitreal surface of the retinal tissue, it was lowered by 150 mm in order to remove the inner half of the retina (preliminary microscopic studies enabled the determination of the cut depths required to remove cleanly the inner nuclear and ganglion cell layers). The remaining photoreceptor layer was then retrieved with excess attached gelatin.
Analysis by PCR
Cell RNA extract was prepared using TrizolTM Reagent (Gibco BRL). Briefly, the cells (5x106) were lysed with 1 ml of TrizolTM and the lysate was passed several times through a pipette. After addition of 200 ml of chloroform, the mixture was shaken vigorously and centrifuged at 12000x g for 15 min at 4 °C. The aqueous phase was transferred into a fresh tube, 500 ml of isopropyl alcohol was added, and the mixture was centrifuged at 12000x g for 10 min at 4 °C. The RNA precipitate in the pellet was washed with 1 ml 75% ethanol, mixed, and centrifuged at 7500x g for 5 min at 4 °C. Total RNA was dissolved in 30 ml RNAse free water and quantified by UV absorption. The ratio OD 260 nm/OD 280 nm was 1.6-2.0 in all cases and the RNA yield varied from 4-8 mg. RNA samples were kept frozen at -80 °C until use [16-18].
Reverse transcription into the complementary DNA (cDNA) was done using 1 μg of total RNA. RNA was denatured for 10 min at 70 °C and incubated for 60 min at 42 °C in a final volume of 20 ml in the presence of 0.5 mM dNTP (Pharmacia, Uppsala, Sweden), 0.01 M dithiothreitol (Promega, Madison, WI), 10 pmol oligo(dT) (Promega), 200 units SuperscriptTM II RNAse H- reverse transcriptase (Gibco BRL), and 1 unit RNAsin (Promega) in 50 mM Tris-HCl, pH 8.3, 75 mM KCl, and 3 mM MgCl2. The mixture was heated to 95 °C for 5 min at the end of the reaction .
The Polymerase Chain Reaction (PCR) for the β-actin was performed with the primers 5'-CTGGAGAAGAGCTATGAGCTG-3' (sense) and 5'-AATCTCCTTCTGCATCCTGTC-3' (antisense) synthesized by Genosys (UK). The MR was similarly analyzed with the primers 5'-AGGCTACCA CAGTCTCCCTG-3' (sense) and 5'-GCAGTGTAAAATCTCCAGTC-3' (antisense). The reaction mixture consisted of 6 ml (15 ng) of cDNA, 2.5 units Taq DNA polymerase (Gibco BRL), 200 mM dNTP, 0.2 mM of the respective oligonucleotide primers, 10 mM Tris-HCl, pH 8.3, 50 mM KCl and 1.5 mM MgCl2 in a final volume of 50 ml. The mixture was overlaid with mineral oil (Sigma, St. Louis, MO) and amplified in a thermal cycler (Crocodile III, Appligene, France). Denaturation was carried out at 94 °C for 1 min (3 min in the first cycle), followed by an annealing step at either 53 ° C or 55 ° C (depending upon the primer) for 1 min, and an extension step at 72 °C for 1 min (10 min in the last cycle) to a total of 25 cycles for β-actin and 35 cycles for the MR and the ENaC. The PCR products (10 ml) were electrophoresed on 1.2% agarose gels (Gibco BRL) in 0.45 mM Tris-borate containing 0.2% ethidium bromide and 1 mM EDTA. A negative H2O control was similarly amplified in parallel to check the efficiency of the PCR technique . Several leukemic cells used as negative control. Another control were performed by using of total RNA from each tissue. The gels were photographed along with the molecular weight markers run in parallel.
The nonradioactive DIG-11-dUTP system, containing the steroid hapten digoxigenin, was used for the quantification of ENaC message by luminescence, according to the instructions of the manufacturer (Enzo Diagnostics, Boehringer Mannheim). To this end, the PCR was carried out in the presence of DIG-11-dUTP for a total of 25 cycles for both β-actin and ENaC. The reaction mixture was transferred to nitrocellulose membranes, that were hybridized with anti-digoxigenin Fab-alkaline phosphatase conjugated. The chemiluminescence signal was developed with CSPD which was dephosphorylated by alkaline phosphatase for light emission at 477 nm and recorded on X-ray film.
The PCR products were purified by electrophoresis on 1.5% agarose gels followed by the electroelution (2 h at 400 V, 15 mA) of the DNA fragment in an Amicon centrilutor apparatus (Millipore Corporation, Bedford, MA). The products were concentrated by ultrafiltration in a Centricon-100 microconcentrator (Amicon, Beverly, MA) according to the recommendations of the manufacturer. For sequencing, 1 pmol of purified DNA was incubated with 2.5 U of Klenow enzyme in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and 10 mM NaCl, for 45 min at 37 °C in the presence of 200 mM dATP and fluorescence-labeled terminators, 16 nM ddGTP, 1.2 nM ddTTP, 60 nM ddATP and 600 nM ddCTP. The 3'-end-labeled DNA was separated from the free nucleotide triphosphates by gel filtration using C-100 spin columns (Clontech, East Meadow Circle, CA) and vacuum dried. The purified 3'-end labeled fragments were thereafter degraded by 10% (v/v) aqueous piperidine (30 min at 95 °C), followed by 80% (v/v) formamide (10 min at 110 °C), and finally quantified in an ABI 377 automatic sequencer (Applied Biosystems, Foster City, CA) [19,20].
Figure 1A shows the expression of MR at the level of mRNA following RT-PCR analysis of total RNA isolated from rat retina (Lane A), bovine RPE cells (Lane B), and rat ciliary body (Lane C). With our set of specific primers, PCR products were resolved as a predicted band of 843 bp for MR. In our laboratory, for PCR analysis of MR and ENaC, we have several leukemic cells that were used as negative controls (data not shown). Another control were performed by using total RNA from each ocular tissue (Lane E for retina, Lane F for RPE, and Lane G for ciliary body). To localize MR expression at a cellular level, the photoreceptor layer was isolated from the inner retina by vibratome sectioning (Figure 1B). The 843 bp product was then recovered in both the photoreceptor layer (Lane A) and in the inner retina (inner nuclear and ganglion cell layers, Lane B). Amplification of β-actin was used in these experiments as a control for mRNA expression 460 bp (Lanes C and D).
The nucleotide sequences of the PCR products were determined for the photoreceptor layer using four different chain terminators in a single electrophoretic run . The sequences of reverse and forward strands were aligned to correct undetermined N bases. The complete sequence was then compared with available nucleotide sequences for ENaC and MR using the BLAST software on the EMBL/GenBank database. The sequence of MR in the retina was identical to those from rat kidney (98%). Moreover, 691 thymidine, 762 guanine, and 1146 adenine in the previously cloned MR are replaced by cytosine, cytosine, and guanine respectively, in the PCR product using retina RNA; residue 550 could not be identified under these conditions.
To analyze the pattern of MR expression during retinal development, total RNA was isolated from rat retina from birth to adulthood. Figure 2 illustrates the expression of MR gene from 1, 3, 6, 8, and 11 day old rat retina and indicates that the MR gene was already present at 1 day postnatal and during the development of the animal. These results suggest however that steroid hormones may regulate photoreceptors function immediately after birth through a receptor-mediated mechanism.
To determine whether MR were physiologically active in ocular tissues, their induction of ENaC expression [21,22] was evaluated in RPE cells. Cultured RPE cells were incubated in the presence of aldosterone (10 mM) during 12, 24, and 36 h. When mRNA were processed for RT-PCR with primers for ENaC (Figure 3A), the treatment greatly increased ENaC expression after 12 h as compared to that of the control RPE cells (Figure 3B). The β-actin was used as a control for mRNA expression 460 bp (Figure 3B). Sequences analysis of the residues 14-208 in the PCR products revealed 95% homology with the residues located between 1939 (3' end) and 2135 (5' end) of bovine kidney MR mRNA (data not shown). Moreover, thymidine 1944, guanine 1962, and cytosines 1963, 1966, 2045 are replaced by adenine and thymidine residues 2064 and 2134 are replaced by adenine and cytosine, respectively, in RT-PCR product using RPE cell RNA. Assuming aldosterone at the concentrations tested has effect only through MR activation, MR thus appears to be physiologically active in RPE cells and could stimulate ENaC expression as previously observed in other epithelial cells [23,24].
Steroids can regulate cellular functions in many tissues via paracrine and/or autocrine actions . Despite demonstration of their local production in ocular tissues , their targets have generally escaped analysis in these tissues. Earlier studies have just demonstrated the presence of glucocorticoid receptors in the lens using radioligand binding [27,28] and the induction of glutamine synthetase by such hormonal receptors [29,30]. We here demonstrated MR expression in photoreceptors and more widely in the retina, in pigment epithelium and ciliary body.
Using an antibody directed against MR, we had previously found evidence of MR in the human and rat retina [12,13]. The immunostaining was localized in the retina and the ciliary body. The present data using RT-PCR analysis further confirmed MR expression in the mammalian retina. Isolating photoreceptors enabled us to demonstrate the presence of MR in these neurons as well as in the other inner layers of the retina. The sequence of the photoreceptor PCR-products revealed almost hundred percent homology to that in rat kidney . To our knowledge, this is the first demonstration of the presence of MR genes in a non-epithelial ocular cell. This pattern of expression in the retina was therefore consistent with the immunostaining in the outer nuclear layer and ganglion cell layer . Surprisingly, MR expression was detected in the retina just after birth, suggesting that mineralocorticoids may play an important role in modulating vision throughout life. These results further demonstrate our earlier immunological staining indicating the presence of the MR protein in photoreceptor cells [12,13]. The primers used here also indicate that the initiation sites for these genes in rat retina are similar to those in other tissues . This may be of further help in the analysis of MR genes in epithelial as well as non-epithelial cells of diverse origins.
In epithelial cells, MR were found in the ciliary body by binding sites for aldosterone [33,34] or specific antireceptor antibody [12,13]. Expression of such MR was confirmed in our study at the mRNA level. The expression was also detected in retinal pigment epithelium where ENaC had been reported previously. Using a culture of RPE cells, we could demonstrate that these cells expressed functional MR that could stimulate ENaC expression in the presence of aldosterone as previously demonstrated [21,22]. Sequences analysis of the PCR products in RPE cells revealed 95% homology with the alpha subunit of bovine renal epithelial sodium channel mRNA. Despite the substituted residues, whose functional significance remains unknown, it is clear that the cell contains the ENaC mRNA.
Since photoreceptors , other retinal neurons , and epithelial cells of the ciliary body [14,35] also display ENaC, the MR may regulate their cell function similarly by modulating Na+ flux through ENaC. MR activation may therefore affect eye functions where Na+ plays a prominent role such as phototransduction, visual information processing in retinal neurons, aqueous humor secretion, and regulation of the intraocular pressure, hydration, and transparency of the cornea and lens [11,36-39]. In photoreceptors, for instance, light responses might be greatly affected by a modulation of ENaC since dark currents are generated by cyclic GMP-dependent channels that also generate a Na+ conductance. In RPE cells, activating ENaC channels may alter photoreceptor outer segment shedding, or visual pigment recycling.
In conclusion, our demonstration here of the presence of the MR in ocular tissues identifies new targets to delineate the mechanism of action of mineralocorticoid hormones and thereby adding an important new dimension in the understanding of various ocular functions.
Thanks are due to Association Française Retinitis Pigmentosa for financial assistance and to Valerie Forster for her technical assistance in producing the vibratome section of the retina.
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