CLCA protein and chloride transport in canine retinal pigment epithelium

Matthew E. Loewen,1 Nicola K. Smith,1 Don L. Hamilton,1 Bruce H. Grahn,2 and George W. Forsyth1

Departments of 1Veterinary Biomedical Sciences and 2Small Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B4

Submitted 20 May 2003 ; accepted in final form 8 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Problems in ion and fluid transfer across the retinal pigment epithelium (RPE) are a probable cause of inappropriate accumulations of fluid between the photoreceptors of the retina and the RPE. The activities of Cl- transporters involved in basal fluid transfer across the RPE have been compared to determine whether Ca2+- or cAMP-dependent channels may be responsible for basal housekeeping levels of secretory activity in this tissue. The role of a candidate Ca2+-dependent CLCA protein in the basal RPE transport of Cl- has been investigated. Low concentrations of the Cl- conductance inhibitors glibenclamide and 5-nitro-2-(3-phenylpropylamino)benzoate reduced the short-circuit current in dog RPE preparations mounted in Ussing chambers and decreased the Ca2+-dependent Cl- efflux from fibroblasts expressing the pCLCA1 Cl- conductance regulator. However, these same agents did not inhibit the rate of Cl- release from cultured fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive Cl- channel. Addition of ionomycin to primary cultures of canine RPE cells or to fibroblasts expressing the pCLCA1 channel regulator increased the rate of release of Cl- from both types of cultured cells. However, the presence of pCLCA1 also increased cAMP-dependent Cl- release from fibroblasts expressing CFTR. We conclude that Ca2+-dependent Cl- transport may be more important than cAMP-dependent Cl- transport for normal fluid secretion across the RPE. Furthermore, CLCA proteins expressed in the RPE appear to regulate the activity of other Cl- transporters, rather than functioning as primary ion transport proteins.

chloride channel; cystic fibrosis transmembrane conductance regulator, calcium; fluid secretion


ION TRANSPORT ACTIVITY in isolated retinal pigment epithelium (RPE) choroid preparations has been characterized by Tsuboi and coworkers (20, 21) and by others (1). Their findings are consistent with a model where Na+-K+-ATPase activity in the apical RPE membrane, adjacent to the rod and cone photoreceptors, generates a Na+ gradient across this membrane. High extracellular levels of Na+ are harnessed to drive uptake of Cl- and K+ by the RPE via a coupled Na+-K+-2Cl- cotransporter localized to the same apical membrane. Activity of this cotransporter maintains RPE intracellular K+ and Cl- concentrations above their respective equilibrium potentials. Net "absorptive" fluid movement (from the vitreous to the choroid) occurs with increased opening of basal Cl- conductance channels on the choroidal side of the RPE, permitting Cl- release down its electrochemical potential. Na+ ions follow the released Cl- to maintain electroneutrality, and osmotic forces draw water after the electrolytes to cause fluid to be "secreted" from the vitreous to the choriocapillaris. Disruptions in the direction or magnitude of this ion and fluid transport are one possible cause of conditions such as serous retinopathy (7).

The activity of the basal Cl- conductance channels that regulate this ion and fluid transport process is controlled by changes in the concentration of intracellular second messenger molecules including adenosine 3',5'-cyclic monophosphate (cAMP) (7, 16) and intracellular Ca2+ (10, 22). However, there seems to be a significant degree of uncertainty about the relative importance of cAMP-dependent vs. Ca2+-dependent Cl- conductance in this tissue. Recent studies on human fetal RPE indicate the presence of an apical adrenergic receptor in these cells (18). Transepithelial potentials in the human fetal RPE cells responded positively to both cAMP and ionomycin, indicating activation of Cl- conductance by A-kinase and by a Ca2+-dependent mechanism. Expression of the cystic fibrosis transmembrane regulator protein (CFTR) and variants of the ClC chloride channel, including ClC-3, has been reported in human RPE (15, 24). Although there is much evidence for CFTR expression in this tissue, there are no reports of serous retinopathy in cystic fibrosis patients. On the basis of this evidence, it seems likely that Ca2+-dependent Cl- channels may be responsible for most of the basal housekeeping levels of secretory activity in RPE.

Studies on an inherited multifocal retinopathy (5) and on the distribution of a member of the CLCA Cl- conductance channel family (2) cloned recently in our laboratory (3) have led us to use inhibitor sensitivity profiles to examine the relative roles of cAMP-dependent CFTR vs. Ca2+-dependent Cl- conductance proteins in the basal housekeeping levels of secretory activity occurring in this tissue. Basal and activated short-circuit current has been measured across RPE-choroid preparations. Extension of these studies to primary cultures of canine RPE cells has permitted comparisons of the importance of CFTR and Ca2+-dependent Cl- channels in this process. The results of this study suggest a novel role for the canine isoform of pCLCA1 in RPE cells.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ussing chamber electrophysiology. Guidelines of the Canadian Council on Animal Care were followed in harvesting eyes from healthy dogs that had been euthanized by others for nonmedical reasons. The posterior portion of the eye was bisected to separate tapetal and nontapetal areas. After the sclera and the retina were removed, the RPE-choroid preparations were mounted in Costar 5-mm-diameter vertical diffusion chambers. The bathing solution was Krebs-Ringer bicarbonate buffer, pH 7.5, containing 5.5 mM glucose and 5.0 mM K+. Tissues were maintained at 37° and oxygenated with 95% O2-5% CO2. Electrical measurements were made with glass barreled microelectrodes (Navicyte) by using an EVC 400 Precision voltage-current clamp (World Precision Instruments). Measurements were made after stable transepithelial potentials were achieved (20-30 min after tissues were mounted). Cl- conductance inhibitors were added to tissue bathing solutions from stock solutions in dimethyl sulfoxide (maximum final DMSO concentration of 0.1%.)

Conditions for primary culture of dog RPE. Enucleation was carried out under sterile conditions. Eyes were rinsed with Betadine and dissected in a sterile environment. The anterior segment and the vitreous were removed, and a small area of retina was peeled away, creating a well that was filled with 0.5% trypsin (GIBCO). After 5 min, RPE cells were dislodged by pipetting, collected by centrifugation, washed, and suspended in DMEM (GIBCO) buffered with 20 mM HEPES, pH 7.4, supplemented with 10% fetal bovine serum (GIBCO), 10 ng/ml basic fibroblast growth factor (GIBCO), and 50 µg/ml Pen-Strep. Cells were grown to confluence on 35-mm Primera plates coated with mouse laminin (2.5 µg/cm2). Identity of the RPE cells was confirmed by immunohistochemical staining for cytokeratin and vimentin (14).

RT-PCR conditions. RT-PCR was carried out on total RNA from confluent cultures of RPE cells at passage 3. Cells were frozen in 0.15 M NaCl, 5 mM dithiothreitol (DTT), and 10 mM Tris, pH 8.0, containing 10% (vol/vol) RNase inhibitor (RNA Guard; GIBCO). Total RNA from 12.5 cells was reverse transcribed with antisense primer specific to a unique 3'-untranslated sequence in pCLCA1 (5'-GAGAAAGCTTGCGGCCGCTCGTGCAGAAAGTCTAAAATG-3') or to a conserved region of CFTR (5'-GCACTGGGTTCATCAAGCAG-3'). The cDNA from the reverse transcription reaction was used as template in a PCR reaction containing 200 µM dNTPs, 25 pmol of primers, 5% DMSO, and 1.25 units of Taq and 1.25 units of Pfu DNA polymerases. Pfu was omitted from PCR reactions intended for TA cloning. Antisense primers were the same as that used in the reverse transcription, whereas sense primers were CFTR 3578 (5'-CCAGCATAGATGTGGATAG-3') and pCLCA1 33 (5'-ACGATGCAAATGGTCGATACAG-3'). PCR was run for 30 cycles with annealing for 45 s at 52°, extension for 1 min at 72°, and denaturation for 45 s at 94°. The identity of the amplified cDNA in the PCR of pCLCA1 was determined by TA cloning and sequencing of three independent clones (Core DNA Services, University of Calgary, Alberta, Canada).

cRNA templates were produced from CFTR cDNA in Bluescript (clone T16-4.5; American Type Culture Collection) and pCLCA1 cDNA cloned into the NotI site of pcDNA3 (3). DNA (10 µg) from each vector with insert was linearized by a single cut at the 3'-end of the insert, using XbaI for pCLCA1 and SpeI for CFTR, run through a Qiagen PCR clean-up column, and used as template for cRNA synthesis. The synthesis system also contained 0.1 M DTT, 250 µM dNTPs, 1 µl of RNA Guard (GIBCO), and 100 units of T7 RNA polymerase in a final volume of 100 µl. The reaction was allowed to proceed for 2 h at 37°. The cRNA product was incubated with 10 units of pancreatic DNase for 30 min at 37° and separated from DNA digestion products with the use of a Qiagen RNeasy column. The cRNA products were quantitated by optical absorbance at 260 and 280 nm, and known amounts of the cRNA were used in parallel RT-PCR experiments to quantitate mRNA in cell extracts.

Immunohistochemistry. Polyclonal rabbit antiserum was raised to a KLH-conjugated 17-mer peptide sequence (CKEKNHNKEAPNDQNQK), corresponding to deduced amino acid sequence residues 250-266 in pCLCA1. Immunohistochemistry was carried out with the capillary gap method (Fisher Code-On IHC stainer) or with the Venta Benchmark flatbed IHC stainer. Formalin-fixed, paraffin-embedded sections from a normal dog eye were deparaffinized, treated to inactivate endogenous peroxidase, exposed to protease (Code-On) or heat (Benchmark) for epitope retrieval, and blocked before being exposed overnight to 1:1,000 diluted rabbit anti-pCLCA1 17-mer peptide. Secondary antibody was biotinylated anti-rabbit IgG. Avidin-peroxidase complex was added after secondary antibody, followed by NovaRED peroxidase substrate (Vector). Slides were then counterstained with hematoxylin and prepared for viewing.

Cl- release assay. Stable CFTR and pCLCA1 transfectants of mouse 3T3 fibroblasts expressed under the control of the cytomegalovirus (CMV) promoter were grown in DMEM with 10% fetal calf serum and maintained by selection on G418 (500 µg/ml). These stable transfectants or cultured canine RPE cells (P2) were grown to confluence on 35-mm plates and loaded with 36Cl by removing growth media and incubating with loading buffer containing 5 mM glucose, 10 mM HEPES, pH 7.5, and 120 mM NaCl plus 2 µCi/ml 36Cl for 90 min. Release of 36Cl from cells equilibrated with the loading buffer was measured after rapidly washing the cells four times with 1 ml of efflux buffer (loading buffer without 36Cl) to remove extracellular Cl-. The rate of release of Cl- from the cells was then determined by removing and replacing 1 ml of the efflux medium at 2-min intervals with liquid scintillation counting of 36Cl. The sensitivity of Cl- release from loaded cells to perturbations in cell second messenger systems was studied with 10 µM ionomycin in efflux buffer of cells expressing pCLCA1 or with 10 µM forskolin and 0.5 mM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) plus 2 mM 3-isobutyl-1-methylxanthine (IBMX) in cells expressing CFTR. These efflux agonists were added after the fourth wash and were present in solutions during sampling for 36Cl release. Inhibitor effects on Cl- release were determined by including inhibitor (or vehicle) in the wash solution and during the subsequent timed 36Cl efflux.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Secretory fluid transport across the RPE should transfer any accumulated serous fluid from between the retina and the RPE through the RPE epithelial layer and into the choroidal capillary drainage. The highest frequency of focal serous retinopathy lesions has been observed in tapetal areas (5). This situation could be caused by the tapetal pigments acting as a physical barrier to ion and fluid transport across tapetal regions of the retina. The short-circuit current and resistance measurements across canine RPE-choroid preparations from tapetum and from nontapetal areas mounted in Ussing chambers are shown in Fig. 1. Averaged short-circuit currents in the nontapetal area measured with the transepithelial potential reduced to zero exceeded the values in the tapetal area at each observation time. There was also a consistent trend toward larger transepithelial potential differences in nontapetal preparations (data not shown). These findings were consistent with greater ion and fluid transport activity in the RPE cell layer of the nontapetal tissue. Tissue from this nontapetal region of the canine eye was used for subsequent measurements of antagonist effects on short-circuit current across the canine RPE.



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Fig. 1. Short-circuit current (circles) and resistance values (squares) in dog retinal pigment epithelium (RPE)-choroid preparations mounted in Ussing chambers. Tissues were mounted in 5-mm-diameter aperture Ussing chambers and bathed in oxygenated Krebs-Ringer buffer. Data collection commenced after parameters stabilized. Filled symbols represent tapetum (n = 6); open symbols represent nontapetal area (n = 10). Values are means ± SE.

 

Previous reports of cAMP- and Ca2+-dependent chloride conductance in RPE indicate that two regulatory processes, and possibly two ion transport processes, may be present in these tissues (8). Increments in RPE short-circuit current after the addition of either forskolin and IBMX or the Ca2+ ionophore ionomycin to nontapetal RPE tissues mounted in Ussing chambers are shown in Fig. 2. These results reveal that agonists operating through activation of adenylate cyclase or increases in free intracellular Ca2+ are both capable of stimulating Cl- transfer from the retina to the choroidal vessels in this tissue.



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Fig. 2. Increments in short-circuit current caused by activation of Cl- conductance in RPE-choroid tissue preparations. Tissues were mounted in Ussing chambers as described in Fig. 1 legend. Activation agonists were added at time 0, and the increments in short-circuit current ({Delta}) following agonist addition are indicated. A: addition of forskolin (10 µM) and 3-isobutyl-1-methylxanthine (IBMX; 2 mM). B: addition of ionomycin (10 µM).

 

Basal levels of ion transport processes are presumed to be responsible for housekeeping transfer of fluid from the vitreous to the choroid. The issue of which type of Cl- transporter was responsible for this basal transport was investigated by the following studies, employing inhibitors of conductive Cl- transport. The nonspecific Cl- channel antagonist 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) has been reported to inhibit active Cl- transport in RPE (8). Those findings were confirmed in the nontapetal RPE-choroid preparations used in this study by a reduction of short-circuit current to ~50% of the normal value on addition of 1 mM SITS (Fig. 3). The inhibitory action of SITS occurred immediately upon addition, but tissue integrity is shown in the constant tissue resistance. The rapid drop in short-circuit current was consistent with other observations suggesting a role for anion conductance in the short-circuit currents seen in this tissue. The oral hypoglycemic sulfonylurea compound glibenclamide is reported to be a relatively specific inhibitor of Cl- transport occurring through the CFTR Cl- conductance channel (9). Addition of 50 µM glibenclamide to RPE-choroid preparations had no effect on transepithelial resistance, but the short-circuit current measured across the tissue was reduced by ~40% (Fig. 4). These are the results that would be expected if canine CFTR or some other glibenclamide-sensitive Cl- conductance activity contributes to the ion and fluid transport across the canine RPE.



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Fig. 3. Effect of addition of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) on the short-circuit current measured in canine RPE. SITS (1.0 mM) was added at time 0 to stabilized canine RPE-choroid preparations mounted in Ussing chambers. {bullet}, Control short-circuit current (n = 8); {circ}, short-circuit current with SITS addition; {square}, tissue resistance with SITS addition (n = 4). Values are means ± SE.

 


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Fig. 4. Effect of glibenclamide on short-circuit current in dog RPE-choroid preparations in Ussing chambers. Vehicle or glibenclamide (50 µM) was added at time 0. {bullet}, Control short-circuit current; {circ}, short-circuit current in preparations treated with glibenclamide; {square}, tissue resistance in preparations treated with glibenclamide. Values are means ± SE (n = 7).

 

5-Nitro-2-(3-phenylpropylamino)benzoate (NPPB) is a relatively potent inhibitor of Cl- conductance activity. Addition of 10 µM NPPB to the RPE-choroid preparations caused a significant inhibition of the short-circuit current (Fig. 5). Earlier investigations with this compound have indicated that 10 µM concentrations were not inhibitory for the Cl- conductance activity of CFTR (6). Recently, Walsh and coworkers (23) reported a Kd of 166 µM for the interaction of NPPB with CFTR.



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Fig. 5. Effect of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) on short-circuit current in dog RPE-choroid preparations in Ussing chambers. Vehicle or NPPB (10 µM) was added at time 0. {bullet}, Control short-circuit current; {circ}, short-circuit current in preparations with NPPB; {square}, tissue resistance in preparations treated with NPPB. Values are means ± SE (n = 8).

 

Short-circuit current in secretory epithelium is believed to be to a large extent a measurement of Cl- release through basal or apical Cl- conductance channels in polarized epithelial cells. Because the concentration of NPPB that inhibited the short-circuit current in RPE-choroid was significantly less than the levels reported to inhibit the CFTR Cl- conductance, we extended these studies to compare the activity of CFTR and a Ca2+-activated Cl- conductance channel in a cell culture system. Mouse fibroblasts expressing CFTR under the control of the CMV promotor have a significant cAMP-dependent Cl- efflux when loaded with 36Cl. This cAMP-dependent Cl- efflux is much less prominent in control mouse fibroblasts transfected with the vector alone, without the CFTR cDNA insert (data not shown). The concentrations of glibenclamide (50 µM) and NPPB (10 µM) used in the short-circuit current experiments with canine RPE-choroid preparations had no inhibitory effect on the efflux of 36Cl from mouse fibroblasts transfected with and expressing the CFTR Cl- conductance channel (Fig. 6).



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Fig. 6. Effect of glibenclamide or NPPB on Cl- efflux from mouse fibroblasts expressing CFTR or pCLCA1. Permanently transfected fibroblasts grown to confluence in 35-mm dishes and loaded with 36Cl for 2 h were removed from loading medium, washed rapidly 4 times in the presence of the indicated inhibitors, and then used to measure the rate of Cl- release. Efflux medium (1 ml) was added to plates and replaced at 2-min intervals for quantifying 36Cl release. Cl- conductance was activated by time 0 additions of IBMX (2 mM), 5-chlorophenyl-cAMP (50 µM), and forskolin (10 µM) to fibroblasts expressing CFTR or 10 µM ionomycin to fibroblasts expressing pCLCA1. Changes in the rate of Cl- efflux caused by inhibitors are shown 2 min after agonist addition to cells loaded with 36Cl. Values are means ± SE (n = 6). Glib, glibenclamide.

 

The recently cloned Cl- conductance regulator pCLCA1 is a member of a family of proteins named for a Ca2+-dependent Cl- channel activity (3, 12, 13). We investigated the sensitivity of Cl- transport activated by pCLCA1 expression to the concentrations of inhibitors shown to reduce short-circuit current in canine RPE-choroid preparations. Efflux of 36Cl from mouse fibroblasts transfected with the pCLCA1 channel increased significantly upon exposure of these cells to the Ca2+ ionophore ionomycin (3, 13). There was no significant Cl- efflux response in these cells to treatment with the cAMP agonists that activated the CFTR channel. Ionomycin- or cAMP-dependent stimulation of 36Cl efflux in control, vector-transfected cells was also insignificant (results not shown). 36Cl efflux from the pCLCA1-transfected mouse fibroblast cell line was inhibited significantly by 50 µM glibenclamide and by 10 µM NPPB, concentrations that reduced short-circuit current in the intact canine RPE-choroid preparations (Fig. 6).

The possibility that the pCLCA1 Cl- channel regulator was expressed in canine RPE cells was investigated by RT-PCR. With primary cultures of canine RPE cells as a source of RNA and with the RT reaction primed with an antisense primer located in the 3'-untranslated region of the pCLCA1 cDNA sequence, the resulting cDNA was tested in PCR for the presence of pCLCA1. An 861-base pair cDNA fragment was generated in these PCR reactions. Template contamination by genomic DNA is ruled out by alignments of pCLCA1 (GenBank accession no. AF095584 [GenBank] ) that indicate a minimum of two introns in the genomic sequence between the locations of the PCR primer pair. The 861-base pair PCR product was ligated directly from the PCR reaction into Invitrogen's TA cloning vector. Three independent clones isolated from the resulting transformation had 99% sequence identity to the reported pCLCA1 sequence (7 base changes out of 830 bases in the clones). These sequence changes give one conservative amino acid replacement (I845-> V) and three nonconservative replacements (Fig. 7). In contrast to these minor cross-species differences for canine and pCLCA1, there are 52 different amino acids in this 252-amino acid region between hCLCA1 and pCLCA1, confirming that canine pCLCA1 is not a species isoform of hCLCA1. The 3'-untranslated sequence contained in three dog pCLCA1 clones was identical to the reported sequence of pCLCA1.



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Fig. 7. Alignment of predicted amino acid sequence from a partial clone of canine CLCA with related CLCA proteins. The predicted sequence of a partial clone of the canine isoform of pCLCA1 (c1), the porcine isoform (p1), and h1 (hCLCA1), the closest currently identified human homologue, are shown.

 

Semiquantitative RT-PCR gave an estimate of 37 ± 7 copies of the pCLCA1 mRNA expressed in each canine RPE cell growing in tissue culture (Fig. 8A). CFTR mRNA was expressed in the same cells at ~5 copies per cell (Fig. 8B). Expression of cCLCA1 antigen in the formalin-fixed canine eye was prominent in RPE cells, with the use of both heat- and protease-induced epitope retrieval (Fig. 9). Antibody dilutions from 1:100 to 1:10,000 produced positive identification of the RPE (data not shown). Staining indicated that the pCLCA1 antigen was present at highest concentrations on the choroidal (basal) side of the RPE epithelial layer. There was relatively less pCLCA1 epitope evident on the apical membrane processes that interdigitate with and feed the photoreceptor cells (note the reverse nomenclature for apical and basal relative to other secretory epithelial tissues). This polarized localization of pCLCA1 is consistent with the known direction of net fluid transport by the RPE from the vitreous to the choroid.



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Fig. 8. RT-PCR quantitation of RPE mRNA species. DNA bands produced by PCR, separated by agarose gel electrophoresis, and stained with ethidium bromide were quantitated by image analysis software (Bio-Rad Discovery). A: fluorescence intensity (adjusted volume, with background subtracted) of an 861-base pair (bp) product amplified with primers specific to pCLCA1 cDNA template. cDNA template was produced by reverse transcribing 50 ({bullet}), 250 ({blacksquare}), or 500 ({blacktriangleup}) molecules of pCLCA1 cRNA or total RNA from 12 RPE cells ({diamond}). The cDNA product of the RT reaction was used as a template for PCR for the indicated number of cycles. Numbers on plots indicate the calculated number of mRNA copies per RPE cell at 22, 24, and 26 cycles. Inset: DNA banding at PCR cycle 22. Lane 1: 100-bp standards. Lanes 2-5: PCR from cDNA templates produced by reverse transcribing 50 (lane 2), 250 (lane 3), or 500 (lane 4) molecules of pCLCA1 cRNA or RNA from 12 RPE cells (lane 5). Lane 6: positive control; lane 7: negative (no template) control. B: DNA banding patterns of a 650-bp product amplified with primers specific to canine CFTR cDNA template. Lane 1: 100-bp standards. Lanes 2-6: PCR from cDNA templates produced by reverse transcribing total RNA from 12 RPE cells (lane 2) or 10 (lane 3), 100 (lane 4), 1,000 (lane 5), and 10,000 molecules (lane 6) of CFTR cRNA. Lane 7: positive control; lane 8: negative (no template) control.

 


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Fig. 9. Immunohistochemical staining of pCLCA1 antigen in canine RPE. The RPE cell bodies on the choroidal side of the polarized epithelium are indicated by filled arrows. The "apical" membrane processes that interdigitate with the (missing) photoreceptors are identified by open arrows. Counterstaining was with hematoxylin. Antibody binding was identified by NovaRED peroxidase substrate. A: heat-induced epitope retrieval; B and C: enzymatic (protease) epitope retrieval. In A and C, a 1:1,000 dilution of anti-pCLCA1 immune serum primary antibody was used; in B, a preimmune serum primary antibody was used. Bars, 10 µm.

 

Evidence for the coexistence of the canine isoforms of pCLCA1 and CFTR in RPE raises the possibility that these proteins could be responsible for the Ca2+-dependent and cAMP-dependent increases in short-circuit current in the RPE reported in Fig. 2. This point was investigated indirectly by producing dual transfectants of NIH/3T3 fibroblasts with both CFTR and pCLCA1. When 36Cl efflux from NIH/3T3 cells expressing both pCLCA1 and CFTR was examined in the presence of forskolin and IBMX, the cells expressing both proteins had a significant increase in the rate of Cl- efflux relative to the parental cell line only expressing CFTR (Fig. 10). This finding resembles results of a previous report that pCLCA1 stimulates the cAMP-dependent Cl- transport occurring when CFTR is expressed (12). Because other Ca2+-dependent Cl- conductance proteins have been reported in the canine RPE, it is possible that pCLCA1 may play a regulatory role for Cl- channels, rather than functioning as a channel itself.



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Fig. 10. A-kinase activation in NIH/3T3 fibroblasts cotransfected with pCLCA1 and CFTR. A 3T3 cell line transfected with CFTR (pcDNA3, G418 selection) was subsequently transfected with pCLCA1 (pcDNA3.1, hygromycin selection). 36Cl release from loaded cells is reported. All cells were treated with 10 µM forskolin and 2 mM IBMX from time 0. {circ}, Cells expressing pCLCA1 and CFTR; {triangledown}, cells expressing only CFTR; {bullet}, cells expressing only pCLCA1; {blacktriangledown}, control cells transfected with pcDNA3 vector. Values are means ± SE (n = 8). *P < 0.05.

 

The Cl- efflux system in cultured canine RPE cells was investigated by the same system used to assess threshold inhibitor concentrations for transfected mouse fibroblasts. The pattern of ionomycin (Ca2+)-dependent 36Cl efflux from the cultured RPE cells was similar to that observed with pCLCA1-transfected mouse fibroblasts, although the maximum efflux rate was greater in RPE cells. This ionomycin-dependent increase in the rate of 36Cl efflux was completely blocked by addition of 10 µM NPPB. Unlike the situation observed with in situ RPE in Ussing chambers (Fig. 2), there was no stimulation of 36Cl release from primary cultures of RPE cells in response to treatment with A-kinase agonists plus IBMX (Fig. 11).



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Fig. 11. 36Cl efflux from cultured canine RPE cells. RPE cells grown to confluence on laminin-coated 35-mm dishes were loaded with 36Cl and washed rapidly 4 times, and the release of 36Cl was measured in media containing the following additions: 10 µM ionomycin ({bullet}); 10 µM ionomycin plus 10 µM NPPB ({triangledown}); 10 µM forskolin, 2 mM IBMX, and 500 µM CPT-cAMP ({blacktriangledown}); and control ({circ}). Values are means ± SE (n = 6); missing error bars are smaller than symbols.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of the tapetum on tissue electrical properties, including slight reductions in short-circuit current and the reduced trans-RPE-choroid potential difference between tapetal and nontapetal fundic regions have not been previously described. The significance of the tapetal pigments as a barrier to ion transport across the RPE is an interesting question, given that the difference in driving force for transport observed in this study correlates with reports of a distinct tapetal sparing effect on retinal degeneration secondary to glaucoma that have been reported in the dog and cat (13, 18) and observed in the horse (Grahn B, unpublished observation). We have also noted an increased incidence/severity of RPE dysplasias in the tapetal region (4). The association of tapetal structure with localized differences in ion transport raises significant questions concerning a causal relationship between regional RPE transport differences and the manifestations and pathogenesis of retinal disease.

Transient changes in the rate of fluid absorption across the RPE could set up pressure gradients that increase the probability of retinal detachment from the RPE or RPE separation from the choroid. Information about the identity of the protein responsible for Cl- conductance across the RPE could be an important contribution to understanding the pathology of serous retinopathies. The cystic fibrosis transmembrane regulator protein is expressed in human, canine, and bovine RPE (15, 24), and the expression site has been localized to the basal membrane (17). However, uncertainties remain concerning the contribution of the CFTR channel to Cl- conductance in the RPE. There are no reports of increased risk of serous retinopathy or retinal detachments in cystic fibrosis patients. Hence it is possible that Cl- transport through other conductance channels coexpressed with CFTR could account for the general housekeeping levels of tissue short-circuit current and intracellular Cl- release from the RPE. The comparative inhibition strategy employed in this study provides evidence that, under the conditions employed, CFTR may be less important than other Cl- conductance channels as a mediator of basal RPE-choroid Cl- transport. However, it is clear that there is a significant cAMP-dependent Cl- conductance in RPE-choroid preparations. Signaling cascades that might activate A-kinase in vivo in response to increased requirements for trans-RPE fluid movement have not been reported.

The loss of cAMP-dependent Cl- conductance in cultured RPE cells has been reported previously in chick and bovine RPE (11, 19). The A-kinase agonist mixture that failed to activate Cl- conductance activity in cultured canine RPE cells was able to increase the rate of 36Cl release from transfected mouse fibroblasts expressing CFTR under the control of the CMV promoter (see Fig. 7A). Because we have demonstrated the presence of CFTR mRNA in cultured RPE cells, there may be a problem with the activation of cAMP-dependent protein kinase in these cells.

Our findings of a significant Ca2+-dependent Cl- release from cultured canine RPE cells are in agreement with a role for intracellular Ca2+ in controlling Cl- conductance, as reported by Strauss et al. (19) in cultured rat RPE cells. Expression of pCLCA1 mRNA in canine RPE cells was considered to be a probable source of a Ca2+-dependent Cl- conductance in this tissue. Protein expression and basal membrane localization was confirmed by immunohistochemistry. The basal side of the RPE that locates against the choriocapillaris is functionally equivalent to the apical membrane of secretory epithelial cells in the trachea or small intestine. Hence the basal membrane of the RPE is the site of Cl- conductance proteins involved in active ion transport. Concentration of the pCLCA1 antigen on the basal side of RPE cells was consistent with a role in either Cl- transport or in the regulation of Cl- transport proteins (12). However, the regulatory effect of pCLCA1 on cAMP-dependent Cl- transport that we have reported previously in Caco-2 cells has also been observed in the fibroblast cell model used in this study. These results suggest that the primary function of the CLCA protein may involve regulation of the activity of other proteins that mediate Cl- conductance.

These studies indicate that cAMP-dependent ion channels are less important than Ca2+-dependent channels to basal housekeeping levels of conductive Cl- transport in canine RPE. The extension from pCLCA1 expression in RPE cell membranes to regulatory effects on Cl- transport activity in 3T3 fibroblasts raises questions about the function of this protein as Ca2+-dependent Cl- channel in canine RPE. However, the localization of the CLCA protein at the basal RPE cell membrane is consistent with the regulatory effects of this protein on Ca2+-and cAMP-dependent Cl- conductance reported previously in the Caco-2 human colon carcinoma cell line (12).


    DISCLOSURES
 
We gratefully acknowledge research support by the Canadian Cystic Fibrosis Foundation.


    ACKNOWLEDGMENTS
 
We thank Darlene Hall, who provided excellent technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. Forsyth, Veterinary Biomedical Sciences, Univ. of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5B4 (E-mail: george.forsyth{at}usask.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Bialek S and Miller SS. K+ and Cl- transport mechanisms in bovine pigment epithelium that could modulate subretinal space volume and composition. J Physiol 475: 401-417, 1994.[Abstract]

2. Fuller CM and Benos DJ. Ca2+-activated Cl- channels: a newly emerging anion transport family. News Physiol Sci 15: 165-171, 2000.[Abstract/Free Full Text]

3. Gaspar KJ, Racette KJ, Gordon JR, Lowen ME, and Forsyth GW. Cloning a chloride conductance mediator from the apical membrane of porcine ileal enterocytes. Physiol Genomics 3: 101-111, 2000.[Abstract/Free Full Text]

4. Grahn BH and Cullen CL. Retinopathy of the Great Pyrenees dog: fluorescence angiography, light microscopy and transmission and scanning electron microscopy. Vet Opthalmol 4: 191-199, 2001.

5. Grahn BH, Philibert H, Cullen CL, Houston DM, Semple HA, and Schmutz SM. Multifocal retinopathy of Great Pyrenees dogs. Vet Opthalmol 1: 211-221, 1998.

6. Hipper A, Mall M, Greger R, and Kunzelmann K. Mutations in the putative pore-forming domain of CFTR do not change anion selectivity of the cAMP-activated Cl- conductance. FEBS Lett 374: 312-316, 1995.[ISI][Medline]

7. Hughes BA, Gallemore RP, and Miller SS. Transport Mechanisms in the Retinal Pigment Epithelium. New York: Oxford, 1998.

8. Hughes BA and Segawa Y. cAMP-activated chloride currents in amphibian retinal pigment epithelial cells. J Physiol 466: 749-766, 1993.[Abstract]

9. Ilek B, Yankaskas JR, and Machen TE. cAMP and genestein stimulate HCO3- conductance through CFTR in human airway epithelia. Am J Physiol Lung Cell Mol Physiol 272: L752-L761, 1997.[Abstract/Free Full Text]

10. Joseph DP and Miller SS. Alpha-1-adrenergic modulation of K and Cl transport in bovine retinal pigment epithelium. J Gen Physiol 99: 263-290, 1992.[Abstract]

11. Kuntz CA, Crook RB, Dimitriev A, and Steinberg RH. Modification by cAMP of basolateral membrane chloride conductance in chick retinal pigment epithelium. Invest Opthalmol Vis Sci 35: 422-433, 1994.[Abstract]

12. Loewen ME, Bekar LK, Gabriel SE, Walz W, and Forsyth GW. pCLCA1 becomes a cAMP-dependent chloride conductance mediator in Caco-2 cells. Biochem Biophys Res Commun 298: 531-536, 2002.[ISI][Medline]

13. Loewen ME, Gabriel SE, and Forsyth GW. The calcium-dependent chloride conductance mediator pCLCA1. Am J Physiol Cell Physiol 283: C412-C421, 2002.[Abstract/Free Full Text]

14. McLellan GJ and Bedford PG. The cytoskeletal intermediate filaments of canine retinal pigment epithelial cells in vivo and in vitro. Res Vet Sci 63: 245-251, 1997.[ISI][Medline]

15. Miller SS, Rabin J, Strong T, Ianuzzi M, Adams AJ, Collins F, Reenstra W, and McCray P Jr. Cystic fibrosis gene product is expressed in retina and retinal pigment epithelium (Abstract). Invest Opthalmol Vis Sci 33: 1009, 1992.

16. Peterson WM and Miller SS. Elevation of intracellular cAMP levels in bovine retinal pigment epithelium closes basolateral Cl channels (Abstract). Invest Opthalmol Vis Sci 36: S216, 1995.

17. Peterson WM, Quong JN, and Miller SS. Mechanisms of fluid transport in retinal pigment epithelium. Proc Third Great Basin Visual Sci Symp Retinal Res 3: 34-42, 1998.

18. Quinn RH, Quong JN, and Miller SS. Adrenergic receptor activated ion transport in human fetal retinal pigment epithelium. Invest Opthalmol Vis Sci 42: 255-264, 2001.[Abstract/Free Full Text]

19. Strauss O, Steinhausen K, Mergler S, Stumpff F, and Wiederholt M. Involvement of protein tyrosine kinase in the InsP3-induced activation of Ca2+-dependent Cl- currents in cultured cells of rat retinal pigment epithelium. J Membr Biol 169: 141-153, 1999.[ISI][Medline]

20. Tsuboi S. Measurement of the volume flow and hydraulic conductivity across the isolated dog retinal pigment epithelium. Invest Opthalmol Vis Sci 28: 1776-1782, 1987.[Abstract]

21. Tsuboi S, Manabe R, and Iizuka S. Aspects of electrolyte transport across isolated dog retinal pigment epithelium. Am J Physiol Renal Fluid Electrolyte Physiol 250: F781-F784, 1986.[Abstract/Free Full Text]

22. Ueda Y and Steinberg RH. Chloride currents in freshly isolated rat retinal pigment epithelial cells. Exp Eye Res 58: 331-342, 1994.[ISI][Medline]

23. Walsh KB, Long KJ, and Shen X. Structural and ionic determinants of 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid block of the CFTR chloride channel. Br J Pharmacol 127: 369-376, 1999.[Abstract/Free Full Text]

24. Wills NK, Weng T, Mo L, Hellmich HL, Yu A, Wang T, Buchheit S, and Godley BF. Chloride channel expression in cultured human fetal RPE cells: response to oxidative stress. Invest Opthalmol Vis Sci 41: 4247-4255, 2000.[Abstract/Free Full Text]