Expression of transient receptor potential vanilloid channels TRPV5 and TRPV6 in retinal pigment epithelium.

PURPOSE: Hydration and ionic composition of the subretinal space (SRS) is modulated by the retinal pigment epithelium (RPE). In particular calcium concentration (Ca(2+)) in the SRS varies with light exposure, and although this change is regulated by RPE transport activity, the specific transport proteins involved have yet to be defined. Two members of the transient receptor potential vanilloid family, TRPV5 and TRPV6, are calcium selective ion channels and are known to be expressed in calcium-transporting epithelial tissues. The present work characterizes of TRPV5 and TRPV6 in RPE. METHODS: Reverse transcriptase PCR was used to examine the presence of TRPV5 and TRPV6 mRNA in cultured human RPE. Protein expression was assessed by western blotting using TRPV5- and TRPV6-specific antibodies. Immunocytochemistry was employed to examine subcellular localization of TRPV5 and TRPV6 in frozen, formaldehyde-fixed sections of native RPE-choroid tissue and in cultured human RPE monolayers. Finally, TRPV5/TRPV6 activity was assessed in cultured RPE, using Ca(2+) indicator dyes to follow [Ca(2+)](i) as a function of changes in [Ca(2+)](o) with and without addition of the TRPV5/TRPV6 inhibitor ruthenium red. RESULTS: Direct sequencing of PCR DNAs documented the presence of TRPV5 and TRPV6 transcripts in human RPE. Immunocytochemistry showed that TRPV5 and TRPV6 are expressed in native RPE-choroid tissue with strong immunoreactivity for both channels on the apical as well as the basal plasma membranes. Immunostaining for both channels was also positive in monolayers of cultured RPE cells. In cultured cells subcellular localization was variable with immunoreactivity present in the cytoplasmic domain as well as on the plasma membrane. Plasma membrane staining was increased with phagocytosis. The reported molecular weight of the core protein for both TRPV5 and TRPV6 is about 75 kDa, with the expected size of the glycosylated proteins in the range of 85-100 kDa. Western blot analysis of TRPV6 in RPE detected a distinct band at approximately 85 kDa, with another strong band at approximately 60 kDa. A similar pattern was seen for TRPV5, with strong bands at 82 kDa and 71 kDa. In live-cell imaging experiments, [Ca(2+)](i) was lower in the presence of the TRPV5/TRPV6 inhibitor ruthenium red. CONCLUSIONS: RPE expresses the epithelial calcium channels TRPV5 and TRPV6, the most calcium-selective channels of the TRP superfamily. Present findings suggest that these channels could function in RPE to mediate calcium influx from SRS and thus regulate changes in SRS calcium composition that accompany light/dark transitions.

Taurine suppresses the spread of cell death in electrically coupled RPE cells.

PURPOSE: To determine whether taurine exerts a protective effect on retinal pigment epithelium (RPE) cells exposed to a cytotoxic agent, cytochrome C (cyC), shown previously to induce apoptosis and produce cell death in electrically coupled neighboring cells. METHODS: Monolayer cultures of confluent human RPE (ARPE-19) cells, which express gap-junctional proteins, were incubated in culture medium with or without taurine. After scrape loading cyC into the cells, we assayed these cells for caspase 3 activity and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to determine the spread of apoptosis. RESULTS: We found that cyC, too large a molecule to traverse gap junctional channels, produced apoptosis in cells injured by the scrape as well as those distant from the site of the scrape, presumably by the intercellular transmission of a toxic agent through the gap junctions that couple these cells. Incubation in taurine, or the gap-junction blocker, octanol, before application of cyC, reduced significantly the fraction of cells undergoing apoptosis. Voltage clamp recordings from electrically coupled Xenopus oocytes transfected with Cx43 showed that junctional communication was unaffected by taurine. CONCLUSIONS: Our results indicate that taurine can serve to suppress cell death in RPE cells independent of any effect on gap junctions. We have considered various avenues by which taurine can exert its protective effect, but the precise mechanism involved under these experimental conditions has yet to be identified.

Alternative splice variants of plasma membrane calcium-ATPases in human corneal epithelium.

Plasma membrane calcium-ATPases (PMCAs) play a critical role in regulating intracellular calcium concentration. Four genes encode PMCA proteins with alternative splicing of transcripts at three sites (A, B and C) serving to increase isoform diversity. Our previous work shows that all four PMCAs are expressed and have specific locations in human corneal epithelium (hCE). The present work examined which splice variants of PMCAs are expressed in hCE. Total RNA was extracted from hCE scraped from cadaver corneas of five different donors (two females and three males, age range 55-76 years). RT-PCR was performed using PMCA isoform-specific primers designed to amplify transcripts that included either splice site A or splice sites B and C. PMCA cDNAs were sequenced or cloned, and then sequenced. There was uniformity in the PMCA1 and PMCA4 expression profile among the five donors. Specifically, every donor expressed PMCA4 transcripts (4x at site A and 4b at site B/C). Every donor also expressed PMCA1 transcripts at sites B/C, specifically PMCA1b and PMCA1kb. In contrast, PMCA2 and PMCA3 expression varied; PCR DNAs were detected in two of five donors. One donor expressed PMCA2a and a novel PMCA2 variant we termed PMCA2((i)). PMCA3a transcript was demonstrated in a different donor. Finally, for all the donors, bands encoding site A transcripts for PMCA4 were obtained but no PCR transcripts were detected at site A for PMCA1, PMCA2 and PMCA3. This investigation showed that hCE expressed multiple splice variants of PMCA isoforms. Furthermore, this study documented the expression of the PMCA1k variant (PMCA1kb) previously only described in intestine and pancreatic beta cells and describes a novel PMCA2((i)) variant. Finally, this study suggests that the molecular configuration of PMCA1, PMCA2 and PMCA3 in the region of splice site A in hCE must be different than in other tissues since the same primers that produced site A transcripts in several other tissues were ineffective in priming PCR in hCE.

Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes.

Melanin, which is responsible for virtually all visible skin, hair, and eye pigmentation in humans, is synthesized, deposited, and distributed in subcellular organelles termed melanosomes. A comprehensive determination of the protein composition of this organelle has been obstructed by the melanin present. Here, we report a novel method of removing melanin that includes in-solution digestion and immobilized metal affinity chromatography (IMAC). Together with in-gel digestion, this method has allowed us to characterize melanosome proteomes at various developmental stages by tandem mass spectrometry. Comparative profiling and functional characterization of the melanosome proteomes identified approximately 1500 proteins in melanosomes of all stages, with approximately 600 in any given stage. These proteins include 16 homologous to mouse coat color genes and many associated with human pigmentary diseases. Approximately 100 proteins shared by melanosomes from pigmented and nonpigmented melanocytes define the essential melanosome proteome. Proteins validated by confirming their intracellular localization include PEDF (pigment-epithelium derived factor) and SLC24A5 (sodium/potassium/calcium exchanger 5, NCKX5). The sharing of proteins between melanosomes and other lysosome-related organelles suggests a common evolutionary origin. This work represents a model for the study of the biogenesis of lysosome-related organelles.

SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans.

Lighter variations of pigmentation in humans are associated with diminished number, size, and density of melanosomes, the pigmented organelles of melanocytes. Here we show that zebrafish golden mutants share these melanosomal changes and that golden encodes a putative cation exchanger slc24a5 (nckx5) that localizes to an intracellular membrane, likely the melanosome or its precursor. The human ortholog is highly similar in sequence and functional in zebrafish. The evolutionarily conserved ancestral allele of a human coding polymorphism predominates in African and East Asian populations. In contrast, the variant allele is nearly fixed in European populations, is associated with a substantial reduction in regional heterozygosity, and correlates with lighter skin pigmentation in admixed populations, suggesting a key role for the SLC24A5 gene in human pigmentation.

Expression and immunolocalization of plasma membrane calcium ATPase isoforms in human corneal epithelium.

PURPOSE: Plasma membrane Ca2+-ATPases (PMCAs) are integral membrane proteins essential to the control of intracellular Ca2+ ([Ca2+]i) concentration. Four genes encode PMCA proteins termed PMCA1-PMCA4. Little is known about the expression of these isoforms in corneal epithelium (CE). The purpose of this investigation is to characterize the expression and distribution of PMCAs in human CE (hCE). METHODS: PMCA mRNA expression was examined by RT-PCR analysis of total RNA from native hCE using PMCA gene specific primers. PMCA isoform expression at the protein level in native hCE was examined by immunoblotting using isoform specific antibodies (Abs) and a panPMCA Ab that recognizes all PMCAs. Distribution of PMCAs in postmortem and surgical sections of hCE was determined by immunohistochemistry with the same Abs. RESULTS: Immunoblot analysis with the panPMCA Ab yielded an intense band of approximately 135 kDa and several faintly staining bands above and below this major band. The isoform specific Abs labeled one or more bands that corresponded to bands detected with the panPMCA Ab. RT-PCR analysis of total RNA from hCE yielded PCR DNAs that were identified by sequencing as products of PMCA1, PMCA2, PMCA3, and PMCA4, thus confirming the immunoblot data. Immunohistochemistry demonstrated localization of PMCAs in all layers of hCE. PMCA4 was the predominant isoform, and was expressed along the plasma membrane of cells in all layers of CE, except with a notable absence along the basal cell membranes adjacent to the stroma. PMCA1 and PMCA2 were found mainly on basal and wing cells. In contrast to PMCA4, PMCA1 immunoreactivity (IR) was located on portions of basal cell plasma membranes adjacent to the stroma. PMCA2 IR was detected cytoplasmically within basal and wing cells in both central cornea and limbus. PMCA3 IR was located in basal cell nuclei in central cornea, but in a perinuclear location in the limbal, basal, and wing cells. CONCLUSIONS: Human CE expresses multiple PMCA isoforms that are differentially expressed and localized among the layers and cells that comprise the CE. We propose that the differential expression of multiple PMCA isoforms affords CE the requisite flexibility to respond to the demands for Ca2+ regulation required during renewal and regeneration of its multiple cell types.

P-glycoprotein expression in human retinal pigment epithelium.

PURPOSE: The retinal pigment epithelium (RPE) is a transporting epithelial monolayer that controls hydration and composition of the subretinal space. P-glycoprotein is an ATP-binding cassette transport protein known to transport a wide range of hydrophobic compounds. The expression of P-glycoprotein in barrier epithelial cells suggests that it could serve a normal protective function, possibly clearing potentially harmful substances from sensitive compartments, like the subretinal space. The present study is designed to determine the expression and activity of P-glycoprotein in normal human RPE. METHODS: RT-PCR and direct sequencing were employed to examine the presence of mdr1 mRNA in cultured human RPE. P-glycoprotein-specific antibodies were employed in Western blotting to identify P-glycoprotein in cultured human RPE and in an established RPE cell line (D407). Anti-P-glycoprotein antibodies were also used to localize the protein in frozen, formaldehyde-fixed sections of native human RPE/choroid by immunohistochemistry. Finally, rhodamine uptake was performed in cultured human RPE monolayers to assess P-glycoprotein activity. The inhibitory antibody 4E3 and reversins 121 and 205 were used to block transport activity. RESULTS: P-glycoprotein is expressed, and is active, in human RPE tissue not exposed to any known inducers of P-glycoprotein. RT-PCR yielded a 546 bp product that was 100% identical in sequence to published data for the mdr1 isoform of human P-glycoprotein. Western blotting demonstrated expression at the protein level, with specific bands observed at about 220 and 165 kD. In native tissue, P-glycoprotein immunoreactivity was predominantly membrane associated, with localization to both apical and basolateral cell membranes. Finally, P-glycoprotein expressed in human RPE is active. Steady-state rhodamine accumulation was increased in the presence of compounds reported to block P-glycoprotein mediated rhodamine efflux. CONCLUSIONS: Human RPE, not exposed to inducer treatment, expresses P-glycoprotein with localization to both apical and basal cell surfaces. Basolateral P-glycoprotein could serve a protective function for the neural retina helping to clear unwanted substances from subretinal space. The finding that P-glycoprotein is also on the apical surface suggests possible additional roles for P-glycoprotein in the RPE.