Bret A Hughes

University of Michigan, Ann Arbor, Michigan, United States

Are you Bret A Hughes?

Claim your profile

Publications (31)112.46 Total impact

  • Xiaoming Zhang, Bret A Hughes
    [Show abstract] [Hide abstract]
    ABSTRACT: Human, monkey, and bovine retinal pigment epithelial (RPE) cells exhibit an M-type K+ current, which in many other cell types is mediated by channels composed of KCNQ α-subunits and KCNE auxiliary subunits. Recently, we demonstrated the expression of KCNQ1, KCNQ4, and KCNQ5 in the monkey RPE. Here, we investigated the expression of KCNQ and KCNE subunits in native bovine RPE. RT-PCR analysis revealed the expression of KCNQ1, KCNQ4, and KCNQ5 transcripts in the RPE, but, in Western blot analysis of RPE plasma membranes, only KCNQ5 was detected. Among the five members of the KCNE gene family, transcripts for KCNE1, KCNE2, KCNE3, and KCNE4 were detected in bovine RPE, but only KCNE1 and KCNE2 proteins were detected. Immunohistochemistry of frozen bovine retinal sections revealed KCNE1 expression near the apical and basal membranes of the RPE, in cone outer segments, in the outer nuclear layer, and throughout the inner retina. The localization of KCNE1 in the RPE basal membrane, where KCNQ5 was previously found to be present, suggests that this β-subunit may contribute to M-type K(+) channels in this membrane.
    Experimental Eye Research 11/2013; 116:424-32. · 3.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: KCNJ13 encodes Kir7.1, an inwardly rectifying K(+) channel that is expressed in multiple ion-transporting epithelia. A mutation in KCNJ13 resulting in an arginine to tryptophan change at residue 162 (R162W) of Kir7.1 was associated with Snowflake vitreoretinal degeneration (SVD), an inherited autosomal dominant disease with vitreous degeneration and mild retinal degeneration. We used the Xenopus laevis oocyte expression system to assess the functional properties of the R162W (mutant) Kir7.1 channel and determine how wild-type (WT) Kir7.1 is affected by the presence of the mutant subunit. Recordings via the two-electrode voltage-clamp technique revealed that oocytes injected with mutant Kir7.1 cRNA resulted in currents and cation selectivity that were indistinguishable from those in water-injected oocytes, suggesting that the mutant protein does not form functional channels in the plasma membrane. Co-injection of oocytes with equal amounts of mutant and WT Kir7.1 cRNAs resulted in inward K(+) and Rb(+) currents with amplitudes that were about 17% of those in oocytes injected with WT Kir7.1 cRNA alone, demonstrating a dominant-negative effect of the mutant subunit. Similar to oocytes injected with WT Kir7.1 cRNA alone, co-injected oocytes exhibited inwardly-rectifying Rb(+) currents that were more than 7 times larger than K(+) currents, indicating that mutant subunits did not alter Kir7.1 channel selectivity. Immunostaining of Xenopus oocytes or MDCK cells expressing mutant or WT Kir7.1 demonstrated distribution of both proteins primarily in the plasma membrane. Our data suggest that the R162W mutation suppresses Kir7.1 channel activity, possibly by negatively impacting gating by membrane phosphadidylinositol 4,5-bisphosphate.
    AJP Cell Physiology 12/2012; · 3.71 Impact Factor
  • Bikash R Pattnaik, Bret A Hughes
    [Show abstract] [Hide abstract]
    ABSTRACT: Recently, we demonstrated the expression of KCNQ1, KCNQ4, and KCNQ5 transcripts in monkey retinal pigment epithelium (RPE) and showed that the M-type current in RPE cells is blocked by the specific KCNQ channel blocker XE991. Using patch-clamp electrophysiology, we investigated the pharmacological sensitivity of the M-type current in isolated monkey RPE cells to elucidate the subunit composition of the channel. Most RPE cells exhibited an M-type current with a voltage for half-maximal activation of approximately -35 mV. The M-type current activation followed a double-exponential time course and was essentially complete within 1 s. The M-type current was inhibited by micromolar concentrations of the nonselective KCNQ channel blockers linopirdine and XE991 but was relatively insensitive to block by 10 μM chromanol 293B or 135 mM tetraethylammonium (TEA), two KCNQ1 channel blockers. The M-type current was activated by 1) 10 μM retigabine, an opener of all KCNQ channels except KCNQ1, 2) 10 μM zinc pyrithione, which augments all KCNQ channels except KCNQ3, and 3) 50 μM N-ethylmaleimide, which activates KCNQ2, KCNQ4, and KCNQ5, but not KCNQ1 or KCNQ3, channels. Application of cAMP, which activates KCNQ1 and KCNQ4 channels, had no significant effect on the M-type current. Finally, diclofenac, which activates KCNQ2/3 and KCNQ4 channels but inhibits KCNQ5 channels, inhibited the M-type current in the majority of RPE cells but activated it in others. The results indicate that the M-type current in monkey RPE is likely mediated by channels encoded by KCNQ4 and KCNQ5 subunits.
    AJP Cell Physiology 11/2011; 302(5):C821-33. · 3.71 Impact Factor
  • Xiaoming Zhang, Dongli Yang, Bret A Hughes
    [Show abstract] [Hide abstract]
    ABSTRACT: Previous studies identified in retinal pigment epithelial (RPE) cells an M-type K(+) current, which in many other cell types is mediated by channels encoded by KCNQ genes. The aim of this study was to assess the expression of KCNQ genes in the monkey RPE and neural retina. Application of the specific KCNQ channel blocker XE991 eliminated the M-type current in freshly isolated monkey RPE cells, indicating that KCNQ subunits contribute to the underlying channels. RT-PCR analysis revealed the expression of KCNQ1, KCNQ4, and KCNQ5 transcripts in the RPE and all five KCNQ transcripts in the neural retina. At the protein level, KCNQ5 was detected in the RPE, whereas both KCNQ4 and KCNQ5 were found in neural retina. In situ hybridization in frozen monkey retinal sections revealed KCNQ5 gene expression in the ganglion cell layer and the inner and outer nuclear layers of the neural retina, but results in the RPE were inconclusive due to the presence of melanin. Immunohistochemistry revealed KCNQ5 in the inner and outer plexiform layers, in cone and rod photoreceptor inner segments, and near the basal membrane of the RPE. The data suggest that KCNQ5 channels contribute to the RPE basal membrane K(+) conductance and, thus, likely play an important role in active K(+) absorption. The distribution of KCNQ5 in neural retina suggests that these channels may function in the shaping of the photoresponses of cone and rod photoreceptors and the processing of visual information by retinal neurons.
    AJP Cell Physiology 07/2011; 301(5):C1017-26. · 3.71 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The retinal pigment epithelium (RPE) is considered a primary site of pathology in age-related macular degeneration (AMD), which is the most prevalent form of irreversible blindness worldwide in the elderly population. Extracellular adenosine triphosphate (ATP) acts as a key signaling molecule in numerous cellular processes, including cell death. The purpose of this study was to determine whether extracellular ATP induces apoptosis in cultured human RPE. RPE apoptosis was evaluated by caspase-3 activation, Hoechst staining, and DNA fragmentation. Intracellular Ca(2+) levels were determined by both a cell-based fluorometric Ca(2+) assay and a ratiometric Ca(2+) imaging technique. P2X(7) mRNA and protein expression were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confocal microscopy, respectively. The authors found that both the endogenous P2X(7) agonist ATP and the synthetic, selective P2X(7) agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) induced RPE apoptosis, which was significantly inhibited by P2X(7) antagonist oxidized ATP (oATP) but not by the P2 receptor antagonist suramin; both ATP and BzATP increase intracellular Ca(2+) via extracellular Ca(2+) influx; both ATP- and BzATP-induced Ca(2+) responses were significantly inhibited by oATP but not by suramin; ATP-induced apoptosis was significantly inhibited or blocked by BAPTA-AM or by low or no extracellular Ca(2+); and P2X(7) receptor mRNA and protein were expressed in RPE cells. These findings suggest that P2X receptors, especially P2X(7) receptors, contribute to ATP- and BzATP-induced Ca(2+) signaling and apoptosis in the RPE. Abnormal Ca(2+) homeostasis through the activation of P2X receptors could cause the dysfunction and apoptosis of RPE that underlie AMD.
    Investigative ophthalmology & visual science 11/2010; 52(3):1522-30. · 3.43 Impact Factor
  • Bikash R Pattnaik, Bret A Hughes
    [Show abstract] [Hide abstract]
    ABSTRACT: The inwardly rectifying K+ (Kir) current in mammalian retinal pigment epithelial (RPE) cells, which is largely mediated by Kir7.1 channels, is stable in cells dialyzed with MgATP but runs down when intracellular ATP is depleted. A potential mechanism for this rundown is a decrease in phosphatidylinositol 4,5-bisphosphate (PIP2) regeneration by ATP-dependent lipid kinases. Here, we used the whole cell voltage-clamp technique to investigate the membrane PIP2 dependence of Kir channels in isolated bovine RPE cells. When RPE cells were dialyzed with ATP-free solution containing PIP2 (25-50 microM), rundown persisted but was markedly reduced. Removal of Mg2+ from the pipette solution also slowed rundown, indicating that elevated intracellular Mg2+ concentration contributes to rundown. Cell dialysis with the PIP2 scavenger neomycin in MgATP solution diminished Kir current in a voltage-dependent manner, suggesting that it acted at least in part by blocking the Kir channel. Kir current in MgATP-loaded cells was partially inhibited by bath application of quercetin (100 microM), phenylarsine oxide (100 microM), or wortmannin (50 microM), inhibitors of phosphatidylinositol (PI) kinases, and was completely inhibited by cell dialysis with 2 mM adenosine, a PI4 kinase inhibitor. Both LY-294002 (100 microM), an inhibitor of PI3 kinases, and its inactive analog LY-303511 (100 microM) rapidly and reversibly inhibited Kir current, suggesting that these compounds act as direct channel blockers. We conclude that the activity of Kir channels in the RPE is critically dependent on the regeneration of membrane PIP2 by PI4 kinases and that this may explain the dependence of these channels on hydrolyzable ATP.
    AJP Cell Physiology 08/2009; 297(4):C1001-11. · 3.71 Impact Factor
  • Source
    Dongli Yang, Xiaoming Zhang, Bret A Hughes
    [Show abstract] [Hide abstract]
    ABSTRACT: Previously, we demonstrated that the inwardly rectifying K(+) (Kir) channel subunit Kir7.1 is highly expressed in bovine and human retinal pigment epithelium (RPE). The purpose of this study was to determine whether any of the 14 other members of the Kir gene family are expressed in native human RPE. Conventional reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that in addition to Kir7.1, seven other Kir channel subunits (Kir1.1, Kir2.1, Kir2.2, Kir3.1, Kir3.4, Kir4.2 and Kir6.1) are expressed in the RPE, whereas in neural retina, all 14 of the Kir channel subunits examined are expressed. The identities of RT-PCR products in the RPE were confirmed by DNA sequencing. Real-time RT-PCR analysis showed, however, that transcripts of these channels are significantly less abundant than Kir7.1 in the RPE. Western blot analysis of the Kir channel subunits detected in the RPE by RT-PCR revealed the expression of Kir2.1, Kir3.1, Kir3.4, Kir4.2, Kir6.1, and possibly Kir2.2, but not Kir1.1, in both human RPE and neural retina. Our results indicate that human RPE expresses at least five other Kir channel subtypes in addition to Kir7.1, suggesting that multiple members of the Kir channel family may function in this epithelium.
    Experimental Eye Research 09/2008; 87(3):176-83. · 3.03 Impact Factor
  • Bret A Hughes, Anuradha Swaminathan
    [Show abstract] [Hide abstract]
    ABSTRACT: Inwardly rectifying K(+) (K(ir)) channels in the apical membrane of the retinal pigment epithelium (RPE) contribute to extracellular K(+) homeostasis in the distal retina by mediating K(+) secretion. Multiple lines of evidence suggest that these channels are composed of Kir7.1. Previously, we showed that native K(ir) channels in bovine RPE are modulated by changes in intracellular pH in the physiological range. In the present study, we used the Xenopus laevis oocyte expression system to investigate the pH dependence of cloned human Kir7.1 channels and several point mutants involving histidine residues in the NH(2) and COOH termini. Kir7.1 channels were inhibited by strong extracellular acidification and modulated by intracellular pH in a biphasic manner, with maximal activity at about intracellular pH (pH(i)) 7.0 and inhibition by acidification or alkalinization. Replacement of histidine 26 (H26) in the NH(2) terminus with alanine eliminated the requirement of protons for channel activity and increased sensitivity to proton-induced inhibition, resulting in maximal channel activity at alkaline pH(i) and smaller whole cell currents at resting pH(i) compared with wild-type Kir7.1. When H26 was replaced with arginine, the pH(i) sensitivity profile was similar to that of the H26A mutant but with the pK(a) shifted to a more acidic value, giving rise to whole cell current amplitude at resting pH(i) that was comparable to that of wild-type Kir7.1. These results indicate that Kir7.1 channels are modulated by intracellular protons by diverse mechanisms and suggest that H26 is important for channel activation at physiological pH(i) and that it influences an unidentified proton-induced inhibitory mechanism.
    AJP Cell Physiology 03/2008; 294(2):C423-31. · 3.71 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Previous studies on bovine retinal pigment epithelium (RPE) established that Kir7.1 channels compose this epithelium's large apical membrane K+ conductance. The purpose of this study was to determine whether Kir7.1 and potential Kir7.1 splice variants are expressed in native adult human RPE and, if so, to determine their function and how they are generated. RT-PCR analysis indicated that human RPE expresses full-length Kir7.1 and a novel Kir7.1 splice variant, designated Kir7.1S. Analysis of the human Kir7.1 gene (KCNJ13) organization revealed that it contains three exons, two introns, and a novel alternative 5' splice site in exon 2. In human RPE, the alternative usage of two competing 5' splice sites in exon 2 gives rise to transcripts encoding full-length Kir7.1 and Kir7.1S, which is predicted to encode a truncated protein. Real-time PCR indicated that Kir7.1 transcript is nearly as abundant as GAPDH mRNA in human RPE whereas Kir7.1S transcript expression is 4-fold lower. Western blot analysis showed that the splice variant is translated in Xenopus oocytes injected with Kir7.1S cRNA and revealed the expression of full-length Kir7.1 but not Kir7.1S in adult human RPE. Co-expression of Kir7.1 with Kir7.1S in Xenopus oocytes had no effect on either the kinetics or amplitude of Kir7.1 currents. This study confirms the expression of Kir7.1 in human RPE, identifies a Kir7.1 splice variant resulting in predicted changes in protein sequence, and indicates that there is no functional interaction between this splice variant and full-length Kir7.1.
    Experimental Eye Research 02/2008; 86(1):81-91. · 3.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mutations in the membrane frizzled-related protein (MFRP) gene cause nanophthalmos in humans, and a splice site mutation causes recessive retinal degeneration in the rd6 mouse. In human and mouse genomes, the MFRP gene lies adjoining to the complement 1q tumor necrosis factor-related protein 5 (CTRP5/C1QTNF5) gene involved in causing retinal degeneration and abnormal lens zonules in human. The purpose of this study was to characterize the spatial and temporal expression of the mouse Mfrp gene, determine tissue and subcellular localization of MFRP protein, and study its interaction with CTRP5. Expression of the Mfrp gene in the mouse was studied by quantitative (q)RT-PCR. MFRP protein expression and distribution were studied by Western blot analysis, immunohistochemistry, and immunoelectron microscopy. Interaction with CTRP5 was studied by immunoprecipitation and immunoblot analysis, using mouse eye and human retinal pigmented epithelium (RPE) choroid extracts and by expressing full-length CTRP5 and MFRP in a heterologous system. The Mfrp gene is specifically expressed in RPE and ciliary body (CB), and its expression starts during early stages of embryogenesis. In the albino mouse eye, MFRP is localized to the apical and basal membranes of RPE and ciliary epithelium (CE). In addition, MFRP and CTRP5 were found to colocalize in RPE, CE, and MDCK cells, a general model of polarized epithelia. These proteins interact with each other in ocular tissues and also in a heterologous system. MFRP is localized to the plasma membrane of CE and RPE, and colocalizes and interacts with CTRP5 indicating a functional relationship between these two proteins.
    Investigative Ophthalmology &amp Visual Science 01/2007; 47(12):5514-21. · 3.44 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In a prior study, a S163R mutation in the complement-1q tumor necrosis factor-related protein 5 (CTRP5/ C1QTNF5) was reported to be associated with early-onset long anterior zonules (LAZ) and late-onset retinal degeneration (L-ORD). The ocular tissues involved in the phenotype are the retinal pigment epithelium (RPE) in the posterior segment and ciliary epithelium (CE) and lens in the anterior segment. The purpose of this study was to characterize the spatial and temporal expression of the mouse Ctrp5 gene, determine tissue and subcellular localization, and study the effect of the S163R mutation. The expression of the Ctrp5 gene in the mouse was studied by quantitative (q)RT-PCR and in situ hybridization. CTRP5 protein expression and distribution were studied by Western blot analysis, immunohistochemistry, and immunoelectron microscopy. Cellular location of wild-type and mutant CTRP5 in MDCK and COS-7 cells was determined by immunofluorescence and immunoblot analysis. A significant level of Ctrp5 expression was detected in the adult mouse in the ciliary body (CB) and RPE, and expression started at a very early stage of embryogenesis. Immunohistochemical analysis showed CTRP5 protein in the apical processes of the RPE and forming a hexagonal lattice associated with the RPE lateral membranes. In the ciliary body, CTRP5 was localized to the apical aspects of the CE, the region between the bilayered ciliary epithelial cells. The membrane association of CTRP5 in the RPE and CE was further confirmed by immunoelectron microscopy. Furthermore, cultured cells were used to show that the CTRP5 is a secretory protein and that its secretion is impaired by the S163R mutation. CTRP5, a secretory and membrane-associated protein, is localized to the lateral and apical membranes of the RPE and CB. Impaired secretion of the mutant protein may underlie the pathophysiology of L-ORD and LAZ.
    Investigative Ophthalmology &amp Visual Science 01/2007; 47(12):5505-13. · 3.44 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Reactive oxygen metabolites (ROMs) may contribute to several eye diseases, such as age-related macular degeneration, although the underlying mechanisms are unclear. The present study shows that human photoreceptor outer segments (POS) prime human retinal pigment epithelial (RPE) cells for massive ROM release in response to lipopolysaccharide (LPS) and interferon-gamma. However, no ROM priming of human RPE cells is observed for bovine POS. ROM production appears to be linked with underlying metabolic oscillations involving the hexose monophosphate shunt.
    Experimental Eye Research 10/2004; 79(3):431-5. · 3.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Retinal pigment epithelial (RPE) cells mediate the recognition and clearance of effete photoreceptor outer segments (POS), a process central to the maintenance of normal vision. Given the emerging importance of Toll-like receptors (TLRs) in transmembrane signaling in response to invading pathogens as well as endogenous substances, we hypothesized that TLRs are associated with RPE cell management of POS. TLR4 clusters on human RPE cells in response to human, but not bovine, POS. However, TLR4 clustering could be inhibited by saturating concentrations of an inhibitory anti-TLR4 mAb. Furthermore, human POS binding to human RPE cells elicited transmembrane metabolic and calcium signals within RPE cells, which could be blocked by saturating doses of an inhibitory anti-TLR4 mAb. However, the heterologous combination of bovine POS and human RPE did not trigger these signals. The pattern recognition receptor CD36 collected at the POS-RPE cell interface for both homologous and heterologous samples, but human TLR4 only collected at the human POS-human RPE cell interface. Kinetic experiments of human POS binding to human RPE cells revealed that CD36 arrives at the POS-RPE interface followed by TLR4 accumulation within 2 min. Metabolic and calcium signals immediately follow. Similarly, the production of reactive oxygen metabolites (ROMs) was observed for the homologous human system, but not the heterologous bovine POS-human RPE cell system. As (a) the bovine POS/human RPE combination did not elicit TLR4 accumulation, RPE signaling, or ROM release, (b) TLR4 arrives at the POS-RPE cell interface just before signaling, (c) TLR4 blockade with an inhibitory anti-TLR4 mAb inhibited TLR4 clustering, signaling, and ROM release in the human POS-human RPE system, and (d) TLR4 demonstrates similar clustering and signaling responses to POS in confluent RPE monolayers, we suggest that TLR4 of RPE cells participates in transmembrane signaling events that contribute to the management of human POS.
    The Journal of General Physiology 09/2004; 124(2):139-49. · 4.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To determine the presence of Kir2.1 channels in native bovine corneal endothelial (BCE) cells and assess their contribution to the resting membrane potential. RT-PCR and Western blot analysis were used to detect the expression of Kir2.1 mRNA and protein in native BCE cells. Whole-cell patch-clamp recording was used to characterize Kir2.1 currents in freshly isolated, single BCE cells, as well as in BCE cell clusters. The contribution of Kir2.1 channels to the membrane potential (V(m)) was assessed by whole-cell recording in the zero-current clamp mode in the absence and presence of Ba(2+). RT-PCR analysis confirmed that Kir2.1 was expressed in the native BCE cells. Western blot analysis with native BCE cell protein and a polyclonal anti-Kir2.1 antibody revealed a approximately 60-kDa band that was blocked by the corresponding synthetic Kir2.1 peptide. Both single BCE cells and BCE cell clusters exhibited an inwardly rectifying K(+) (Kir) current that was dependent on the extracellular K(+) concentration. The Kir current was blocked by external Ba(2+) or Cs(+) in a voltage- and concentration-dependent manner. In 5 mM K(+) Ringer's, the V(m) of cell clusters averaged -40.0 +/- 4.1 mV (n = 14) and in 140 mM K(+) Ringer's it depolarized to -7.4 +/- 1.8 mV. Application of Ba(2+) in 5 mM K(+) Ringer's produced a concentration-dependent depolarization of V(m), with 10 mM Ba(2+) depolarizing V(m) from -53.4 +/- 4.8 mV to -27.8 +/- 6.3 mV (n = 6). Native BCE cells express functional Kir2.1 channels that help determine the membrane potential and probably also play a role in transendothelial transport.
    Investigative Ophthalmology &amp Visual Science 09/2003; 44(8):3511-9. · 3.44 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: To determine the expression and functional activity of proton-coupled oligopeptide transporters (POT) in retinal pigment epithelial (RPE) cells. RT-PCR was used to probe the presence of POT mRNA in freshly isolated bovine RPE (BRPE) and human RPE (HRPE) cells, a human RPE cell line (ARPE-19), and human and bovine neural retina. [14C]GlySar uptake was used to characterize POT activity in cultured ARPE-19 cells and freshly isolated BRPE cell sheet suspensions. PHT1 mRNA was expressed in BRPE, HRPE, ARPE-19, and bovine and human neural retina. In contrast, PEPT2 and PHT2 were expressed only in bovine and human retina, and PEPT1 could not be detected. GlySar exhibited a linear uptake over 6 h at pH values of 6.0 and 7.4, with greater uptake at pH 7.4 (p < 0.01). GlySar uptake did not exhibit saturability (5-2000 microM) and was unchanged when studied in the presence of 1 mM L-histidine. In contrast, GlySar uptake was significantly decreased when studied at 4 degrees C or in the presence of endocytic inhibitors at 37 degrees C (p < 0.01). Studies in BRPE cell sheet suspensions validated the results obtained in ARPE-19 cells and strongly suggested the absence of POT on the apical and basolateral membranes of RPE. PHT1 mRNA is present in native bovine and human RPE and a human RPE cell line. However, the data argue against PHT1 being expressed on plasma membranes of RPE. Overall, GlySar appears to be taken up by RPE cells via a low-affinity, endocytic process.
    Pharmaceutical Research 09/2003; 20(9):1364-72. · 4.74 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The purpose of this study was to identify the molecular basis of the apical membrane K(+) conductance in native bovine retinal pigment epithelium (RPE). RT-PCR, Northern blot, and Western blot analyses were used to detect the expression of the inwardly rectifying K(+) (Kir) channel subunits Kir7.1 and Kir4.1 in native bovine RPE and neural retina. The distribution of Kir7.1 protein was determined in frozen sections of bovine retina-RPE-choroid by indirect immunofluorescence analysis. RT-PCR analysis revealed Kir7.1 transcript in both RPE and neural retina, but Kir4.1 transcript only in the neural retina. In Northern blot analysis, Kir4.1 probe hybridized to an appropriately sized-transcript in neural retina but not in RPE. Kir7.1 probe hybridized to a major transcript of approximately 1.5 kb in both RPE and neural retina, but with greater expression in RPE. In Western blot analysis, Kir7.1 antibody recognized a major monomer of approximately 53 kDa in RPE, whereas Kir4.1 antibody recognized a monomer of approximately 60 kDa in neural retina but not in RPE. Intense Kir7.1 immunolabeling was present on the apical surface of all RPE cells and appeared to extend over the length of the apical processes. Na(+),K(+)-ATPase expression varied among RPE cells, but in highly expressing cells, it colocalized with Kir7.1. These results indicate that the Kir7.1 channel subunit, but not Kir4.1, is a major component of the apical K(+) conductance in bovine RPE. Kir7.1 is distributed over the length of apical processes, where it probably functions in the regulation of K(+) transport and the electrical response of the RPE to light-evoked changes in subretinal K(+) concentration.
    Investigative Ophthalmology &amp Visual Science 08/2003; 44(7):3178-85. · 3.44 Impact Factor
  • Source
    Yukun Yuan, Masahiko Shimura, Bret A Hughes
    [Show abstract] [Hide abstract]
    ABSTRACT: Inwardly rectifying K+ (Kir) channels in the apical membrane of the retinal pigment epithelium (RPE) play a key role in the transport of K+ into and out of the subretinal space (SRS), a small extracellular compartment surrounding photoreceptor outer segments. Recent molecular and functional evidence indicates that these channels comprise Kir7.1 channel subunits. The purpose of this study was to determine whether Kir channels in the RPE are modulated by extracellular (pHo) or intracellular pH (pHi), both of which change upon illumination of the dark-adapted retina. The Kir current (IKir) in acutely dissociated bovine RPE cells was recorded in the whole-cell configuration while altering pHo or pHi. In cells dialysed with pipette solution buffered to pH 7.2, step changes in pHo from 7.4 to 8.0, 7.0 or 6.5 had little effect on IKir. Acidification to pHo 6.0, however, caused a transient activation of IKir followed by a slower inhibition. To determine the dependence of IKir on pHi, we altered pHi within individual RPE cells at constant pHo by imposing transmembrane acetate concentration gradients. These experiments revealed a biphasic relationship between IKir and pHi: IKir was maximal at about pHi 7.1, but decreased sharply at more acidic or alkaline levels. To evaluate the role of Kir7.1 channels in the pHi-dependent changes in IKir, we tested the effect of transmembrane acetate concentration gradients on Rb+ currents, which are 10-fold larger than K+ currents for this channel subtype. Inwardly rectifying Rb+ currents were maximal at about pHi 7.0 and were inhibited by intracellular alkalinization or acidification. We conclude that the Kir conductance in the RPE is modulated by intracellular pH in the physiological range and that this reflects the behaviour of Kir7.1 channels. This sensitivity to pHi may provide an important mechanism linking photoreceptor activity and RPE function.
    The Journal of Physiology 07/2003; 549(Pt 2):429-38. · 4.38 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Purpose . To determine the expression and functional activity of proton-coupled oligopeptide transporters (POT) in retinal pigment epithelial (RPE) cells. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/41501/1/11095_2004_Article_471009.pdf
    Pharmaceutical Research 01/2003; · 4.74 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To generate a profile of genes expressed in the native human retinal pigment epithelium and identify candidate genes for retinal and macular diseases. Two cDNA libraries (one amplified, the other unamplified) were constructed using RNA isolated from native human RPE sheets. The sequence from the 5' end was obtained for randomly selected clones from the two libraries. Of these, more than 2000 expressed sequence tags (ESTs) were analyzed for similarity to sequences and gene clusters in public databases. EST analysis revealed several known RPE-expressed genes and more than 500 genes that have been characterized previously but were not known to be expressed in the RPE. Transthyretin and 90-kDa heat shock protein represent the most abundant transcripts identified in these RPE libraries. More than 200 novel ESTs and putative proteins were identified. An additional 344 sequences matched only the human genomic sequence. High-complexity cDNA libraries were generated from native human RPE. Analysis of ESTs generated from these libraries has yielded a profile of genes expressed in the native RPE. Several of the identified genes are known to play a significant role in the RPE. Novel ESTs, putative proteins, and genomic hits may represent as yet unidentified RPE-expressed genes and many of these, mapping in the region of retinal disease loci, may serve as candidate genes. In addition, the nonredundant set of more than 1100 genes and ESTs described herein will be a valuable resource for generating gene microarrays, which can assist in delineating RPE expression profiles during human disease pathogenesis.
    Investigative Ophthalmology &amp Visual Science 04/2002; 43(3):603-7. · 3.44 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We report a novel 3-dimensional model for visualizing tumor cell migration across a nylon mesh-supported gelatin matrix. To visualize migration across these model barriers, cell proteolytic activity of the pericellular matrix was detected using Bodipy-BSA (fluorescent upon proteolysis) and DQ collagen (fluorescent upon collagenase activity). For 3-dimensional image reconstruction, multiple optical images at sequential z axis positions were deconvoluted by computer analysis. Specificity was indicated using well-known inhibitors. Using these fluorescent proteolysis markers and imaging methods, we have directly demonstrated proteolytic and collagenolytic activity during tumor cell invasion. Moreover, it is possible to visualize migratory pathways followed by tumor cells during matrix invasion. Using cells of differing invasive potentials (uPAR-negative T-47D wild-type and uPAR-positive T-47D A2--1 cells), we show that the presence of the T-47D-A2--1 cells facilitates the entry of T-47D wild-type cells into the matrix. In some cases, wild-type cells follow T-47D A2--1 cells into the matrix whereas other T-47D-wild-type cells appear to enter without the direct intervention of T-47D A2--1 cells. Thus, we have developed a new 3-dimensional model of tumor cell invasion, demonstrated protein and collagen disruption, mapped the pathways followed by tumor cells during migration through an extracellular matrix, and illustrated cross-talk among tumor cell populations during invasion.
    The FASEB Journal 05/2001; 15(6):932-9. · 5.70 Impact Factor

Publication Stats

433 Citations
112.46 Total Impact Points

Institutions

  • 1993–2012
    • University of Michigan
      • Department of Ophthalmology and Visual Sciences
      Ann Arbor, Michigan, United States
  • 2004
    • Concordia University–Ann Arbor
      Ann Arbor, Michigan, United States