Sheldon S. Miller

MicroRNA-204/211 alters epithelial physiology

MicroRNA (miRNA) expression in fetal human retinal pigment epithelium (hfRPE), retina, and choroid were pairwise compared to determine those miRNAs that are enriched by 10‐fold or more in each tissue compared with both of its neighbors. miRs‐184, 187, 200a/200b, 204/211, and 221/222 are enriched in hfRPE by 10‐ to 754‐fold compared with neuroretina or choroid (P<0.05). Five of these miRNAs are enriched in RPE compared with 20 tissues throughout the body and are 10‐ to 20,000‐fold more highly expressed (P<0.005). miR‐204 and 211 are the most highly expressed in the RPE. In addition, expression of miR‐204/211 is significantly lower in the NCI60 tumor cell line panel compared with that in 13 normal tissues, suggesting the progressive disruption of epithelial barriers and increased proliferation. We demonstrated that TGF‐β receptor 2 (TGF‐βR2) and SNAIL2 are direct targets of miR‐204 and that a reduction in miR‐204 expression leads to reduced expression of claudins 10, 16, and 19 (message/protein) consistent with our observation that anti‐miR‐204/211 decreased transepithelial resistance by 80% and reduced cell membrane voltage and conductance. The anti‐miR‐204‐induced decrease in Kir7.1 protein levels suggests a signaling pathway that connects TGF‐βR2 and maintenance of potassium homeostasis. Overall, these data indicate a critical role for miR‐204/211 in maintaining epithelial barrier function and cell physiology.—Wang, F. E., Zhang, C., Maminishkis, A., Dong, L., Zhi, C., Li, R., Zhao, J., Majerciak, V., Gaur, A. B., Chen, S., Miller, S. S. MicroRNA‐204/211 alters epithelial physiology. FASEB J. 24, 1552–1571 (2010). www.fasebj.org

Retinal pigment epithelial transport mechanisms and their contributions to the electroretinogram

Abstract The translocation of ions, fluid and macromolecules across epithelia is made possible by the asymmetric distribution of transport proteins, enzymes and receptors in two physically distinct plasma membrane domains that form the apical and basolateral sides of the cell. Each side faces a different extracellular environment. In the back of the vertebrate eye, the retinal pigment epithelium (RPE) apical membrane receives a continuous stream of paracrine signals that are generated by a variety of retinal neurons in the light and dark. These signals help regulate RPE function, and conversely, alterations in RPE function can modify the activity of retinal neurons. At the basolateral surface, there is a continual exchange of nutrients and waste products, along with a flow of hormonal signals from the choroidal blood supply, all of which serve to maintain the health and integrity of the distal retina and in particular, the photoreceptors. This review provides an integrated summary of the apical and basolateral membrane and intracellular signaling mechanisms that mediate the vectorial traffic of ions and fluid across the RPE. These same mechanisms help regulate the chemical milieu within the cell and in the extracellular spaces that surround the cell. They also generate specific components of the electrical signals that are recorded clinically across the intact human eye, the electroretinogram (ERG) and the electrooculogram (EOG). The last part of this review is focused on the light-induced photoreceptor-dependent decrease in subretinal potassium concentration ([K] 0 ) that occurs in the intact eye and serves as a paracrine signal for the RPE. This signal plays a central role in regulating RPE physiology and in mediating retina/RPE interactions, following transitions between light and dark; it is mimicked in vitro by a small (3 m m ) change in [K] 0 on the apical side of the epithelium. The clinical implications are discussed in terms of the transport mechanisms that regulate hydration of the subretinal space and that potentially mediate fluid absorption out of the retina.

Dopamine Induces Light-Adaptive Retinomotor Movements in Bullfrog Cones via D2 Receptors and in Retinal Pigment Epithelium via D1 Receptors

In the eyes of lower vertebrates, retinal photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) exhibit characteristic retinomotor movements in response to changes in ambient illumination and to signals from an endogenous circadian clock. We previously reported that 3,4‐dihydroxyphenylethylamine (dopamine) mimicked the effect of light on these movements in photoreceptors and RPE cells of green sunfish, Lepomis cyanellus, by interacting with D2 dopaminergic receptors. Here, we report that dopamine also mimics the effect of light on cone and RPE retinomotor movements in bullfrogs, Rana catesbeiana, i.e., dopamine induces cone contraction and RPE pigment dispersion. Dopamine induced cone contraction in isolated dark‐adapted bullfrog retinas incubated in constant darkness in the presence of the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX). This effect of dopamine was inhibited by a D2 but not a D1 antagonist and mimicked by a D2 but not a D1 agonist. These results suggest that induction of cone contraction by dopamine is mediated by D2 dopaminergic receptors and that cone adenylate cyclase activity is inhibited. Thus, dopamine acts via the same type of receptor in both bullfrog and green sunfish retinas to induce cone contraction. In contrast, dopamine influences RPE retinomotor movement via different receptors in fish and bullfrog. Dopamine induced light‐adaptive pigment dispersion in isolated dark‐adapted bullfrog RPE‐eyecups incubated in constant darkness in normal Ringers solution. Because the retina was not present, these experiments demonstrate a direct effect of dopamine on bullfrog RPE. This effect of dopamine on bullfrog RPE was inhibited by a D1 but not a D2 antagonist and mimicked by a D1 but not a D2 agonist. Furthermore, agents that increase the concentration of intracellular cyclic AMP also induced pigment dispersion in dark‐adapted bullfrog RPE‐eyecups incubated in the dark. These results suggest that dopamine induces pigment dispersion in bullfrog RPE via D1 dopaminergic receptors. Thus, dopamine acts via different receptors on bullfrog (D1) versus green sunfish (D2) RPE to induce pigment dispersion. In addition, inhibitor studies indicate that pigment dispersion is actin dependent in teleost but not in bullfrog RPE. Dopamine‐induced pigment dispersion was inhibited by cytochalasin D in isolated RPE sheets of green sunfish but not in RPE‐eyecups of bullfrogs. Together, these observations indicate that dopamine mimics the effect of light on cone and RPE retinomotor movements in both fish and bullfrogs. However, in the RPE, different receptors mediate the effect of dopamine, and different cytoskeletal mechanisms are used to affect pigment transport. These findings suggest that the association of dopamine release with light onset is of earlier evolutionary origin than the appearance of retinomotor movements and that retinomotor movements may have evolved separately in teleosts and amphibians.

CO2-induced ion and fluid transport in human retinal pigment epithelium.

In the intact eye, the transition from light to dark alters pH, [Ca2+], and [K] in the subretinal space (SRS) separating the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE). In addition to these changes, oxygen consumption in the retina increases with a concomitant release of CO2 and H2O into the SRS. The RPE maintains SRS pH and volume homeostasis by transporting these metabolic byproducts to the choroidal blood supply. In vitro, we mimicked the transition from light to dark by increasing apical bath CO2 from 5 to 13%; this maneuver decreased cell pH from 7.37 ± 0.05 to 7.14 ± 0.06 (n = 13). Our analysis of native and cultured fetal human RPE shows that the apical membrane is significantly more permeable (≈10-fold; n = 7) to CO2 than the basolateral membrane, perhaps due to its larger exposed surface area. The limited CO2 diffusion at the basolateral membrane promotes carbonic anhydrase–mediated HCO3 transport by a basolateral membrane Na/nHCO3 cotransporter. The activity of this transporter was increased by elevating apical bath CO2 and was reduced by dorzolamide. Increasing apical bath CO2 also increased intracellular Na from 15.7 ± 3.3 to 24.0 ± 5.3 mM (n = 6; P < 0.05) by increasing apical membrane Na uptake. The CO2-induced acidification also inhibited the basolateral membrane Cl/HCO3 exchanger and increased net steady-state fluid absorption from 2.8 ± 1.6 to 6.7 ± 2.3 µl × cm−2 × hr−1 (n = 5; P < 0.05). The present experiments show how the RPE can accommodate the increased retinal production of CO2 and H2O in the dark, thus preventing acidosis in the SRS. This homeostatic process would preserve the close anatomical relationship between photoreceptor outer segments and RPE in the dark and light, thus protecting the health of the photoreceptors.

Retinal pigment epithelial function: a role for CFTR?

In the vertebrate eye, the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE) are separated by a small extracellular (subretinal) space whose volume and chemical composition varies in the light and dark. Light onset triggers relatively fast (ms) retinal responses and much slower voltage and resistance changes (s to min) at the apical and basolateral membranes of the RPE. Two of these slow RPE responses, the fast oscillation (FO) and the light peak, are measured clinically as part of the electrooculogram (EOG). Both EOG responses are mediated in part by apical and basolateral membranes proteins that form a pathway for the movement of salt and osmotically obliged fluid across the RPE, from retina to choroid. This transport pathway serves to control the volume and chemical composition of the subretinal and choroidal extracellular spaces. In human fetal RPE, we have identified one of these proteins, the cystic fibrosis transmembrane conductance regulator (CFTR) by RT-PCR, immunolocalization, and electrophysiological techniques. Evidence is presented to suggest that the FO component of the EOG is mediated directly or indirectly by CFTR.

Potassium transport across the frog retinal pigment epithelium.

SummaryPrevious experiments indicate that the apical membrane of the frog retinal pigment epithelium contains electrogenic Na∶K pumps. In the pressent experiments net potassium and rubidium transport across the epithelium was measured as a function of extracellular potassium (rubidium) concentration, [K]o ([Rb]o). The net rate of retina-to-choroid42K(86Rb) transport increased monotonically as [K]o ([Rb]o), increased from approximately 0.2 to 5mm on both sides of the tissue or on the apical (neural retinal) side of the tissue. No further increase was observed when [K]o ([Rb]o) was elevated to 10mm. Net sodium transport was also stimulated by elevating [K]o. The net K transport was completely inhibited by 10−4m ouabain in the solution bathing the apical membrane. Ouabain inhibited the unidirectional K flux in the direction of net flux but had not effect on the back-flux in the choroid-to-retina direction. The magnitude of the ouabain-inhibitable42K(86Rb) flux increased with [K]o ([Rb]o). These results show that the apical membrane Na∶K pumps play an important role in the net active transport of potassium (rubidium) across the epithelium. The [K]o changes that modulate potassium transport coincide with the light-induced [K]o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium.

CAMP-dependent absorption of chloride across airway epithelium

Elevated levels of Na and Cl in airway surface liquid may play a major role in the airway pathology of cystic fibrosis (CF) (J. J. Smith, S. M. Travis, E. P. Greenberg, and M. J. Welsh. Cell85: 229-236, 1996) and could be caused by block of transcellular Cl absorption due to lack of a functional CF transmembrane conductance regulator (CFTR). To test for transcellular absorption of Cl across non-CF epithelium, we studied how fluid absorption was affected by the opening and closing of Cl channels. Forskolin (an activator of CFTR) tripled fluid absorption across primary cultures of bovine tracheal epithelium but had no effect on human cells. However, in both species, fluid absorption was markedly inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, a blocker of CFTR. Microelectrode studies suggested that the magnitude of the absorptive response to forskolin in bovine cells depended on the size of an inwardly directed electrochemical driving force for Cl movement across the apical membrane. Patch-clamp measurements of bovine cells revealed CFTR in the apical membrane and a cAMP-activated, inwardly rectifying Cl channel in the basolateral membrane. We conclude that a significant fraction of absorbed Cl passes transcellularly in bovine tracheal epithelial cultures, with CFTR as the path of entry in the apical membrane and a novel cAMP-activated Cl channel as the exit route in the basolateral membrane. Our data further indicate that a similar pathway may exist in non-CF human tracheal epithelium.

IFNγ regulates retinal pigment epithelial fluid transport

The present experiments show that IFNgamma receptors are mainly localized to the basolateral membrane of human retinal pigment epithelium (RPE). Activation of these receptors in primary cultures of human fetal RPE inhibited cell proliferation and migration, decreased RPE mitochondrial membrane potential, altered transepithelial potential and resistance, and significantly increased transepithelial fluid absorption. These effects are mediated through JAK-STAT and p38 MAPK signaling pathways. Second messenger signaling through cAMP-PKA pathway- and interferon regulatory factor-1-dependent production of nitric oxide/cGMP stimulated the CFTR at the basolateral membrane and increased transepithelial fluid absorption. In vivo experiments using a rat model of retinal reattachment showed that IFNgamma applied to the anterior surface of the eye can remove extra fluid deposited in the extracellular or subretinal space between the retinal photoreceptors and RPE. Removal of this extra fluid was blocked by a combination of PKA and JAK-STAT pathway inhibitors injected into the subretinal space. These results demonstrate a protective role for IFNgamma in regulating retinal hydration across the outer blood-retinal barrier in inflammatory disease processes and provide the basis for possible therapeutic interventions.

Pseudomonas aeruginosa induces changes in fluid transport across airway surface epithelia.

Fluid transport across cultures of bovine tracheal epithelium was measured with a capacitance probe technique. Baseline fluid absorption ( J v) across bovine cells of 3.2 μl ⋅ cm-2 ⋅ h-1was inhibited by ∼78% after 1 h of exposure to suspensions of Pseudomonas aeruginosa, with a concomitant decrease in transepithelial potential (TEP) and increase in transepithelial resistance ( R t). Effects of P. aeruginosa were blocked by amiloride, which decreased J v by 112% from baseline of 2.35 ± 1.25 μl ⋅ cm-2 ⋅ h-1, increased R t by 101% from baseline of 610 ± 257 Ω ⋅ cm2, and decreased TEP by 91% from baseline of -55 ± 18.5 mV. Microelectrode studies suggested that effects of P. aeruginosa on amiloride-sensitive Na absorption were due in part to a block of basolateral membrane K channels. In the presence of Cl transport inhibitors [5-nitro-2-(3-phenylpropylamino)-benzoic acid, H2-DIDS, and bumetanide], P. aeruginosa induced a fluid secretion of ∼2.5 ± 0.4 μl ⋅ cm-2 ⋅ h-1and decreased R twithout changing TEP. However, these changes were abolished when the transport inhibitors were used in a medium in which Cl was replaced by an impermeant organic anion. Filtrates of P. aeruginosa suspensions had no effect on J v, TEP, or R t. Mutants lacking exotoxin A or rhamnolipids or with defective lipopolysaccharide still inhibited fluid absorption and altered bioelectrical properties. By contrast, mutations in the rpoN gene encoding a ς factor of RNA polymerase abolished actions of P. aeruginosa. In vivo, changes in transepithelial salt and water transport induced by P. aeruginosa may alter viscosity and ionic composition of airway secretions so as to foster further bacterial colonization.Fluid transport across cultures of bovine tracheal epithelium was measured with a capacitance probe technique. Baseline fluid absorption (Jv) across bovine cells of 3.2 microliter. cm-2. h-1 was inhibited by approximately 78% after 1 h of exposure to suspensions of Pseudomonas aeruginosa, with a concomitant decrease in transepithelial potential (TEP) and increase in transepithelial resistance (Rt). Effects of P. aeruginosa were blocked by amiloride, which decreased Jv by 112% from baseline of 2.35 +/- 1.25 microliter. cm-2. h-1, increased Rt by 101% from baseline of 610 +/- 257 Omega. cm2, and decreased TEP by 91% from baseline of -55 +/- 18.5 mV. Microelectrode studies suggested that effects of P. aeruginosa on amiloride-sensitive Na absorption were due in part to a block of basolateral membrane K channels. In the presence of Cl transport inhibitors [5-nitro-2-(3-phenylpropylamino)-benzoic acid, H2-DIDS, and bumetanide], P. aeruginosa induced a fluid secretion of approximately 2.5 +/- 0.4 microliter. cm-2. h-1 and decreased Rt without changing TEP. However, these changes were abolished when the transport inhibitors were used in a medium in which Cl was replaced by an impermeant organic anion. Filtrates of P. aeruginosa suspensions had no effect on Jv, TEP, or Rt. Mutants lacking exotoxin A or rhamnolipids or with defective lipopolysaccharide still inhibited fluid absorption and altered bioelectrical properties. By contrast, mutations in the rpoN gene encoding a sigma factor of RNA polymerase abolished actions of P. aeruginosa. In vivo, changes in transepithelial salt and water transport induced by P. aeruginosa may alter viscosity and ionic composition of airway secretions so as to foster further bacterial colonization.

Flagellin-stimulated Cl− secretion and innate immune responses in airway epithelia: role for p38

Activation of an innate immune response in airway epithelia by the human pathogen Pseudomonas aeruginosa requires bacterial expression of flagellin. Addition of flagellin (10(-7) M) to airway epithelial cell monolayers (Calu-3, airway serous cell-like) increased Cl(-) secretion (I(Cl)) beginning after 3-10 min, reaching a plateau after 20-45 min at DeltaI(Cl) = 15-50 microA/cm(2). Similar, although 10-fold smaller, responses were observed in well-differentiated bronchial epithelial cultures. Flagellin stimulated I(Cl) in the presence of maximally stimulating doses of the purinergic agonist ATP, but had no effects following forskolin. IL-1beta (produced by both epithelia and neutrophils during infections) stimulated I(Cl) similar to flagellin. Flagellin-, IL-1beta-, ATP-, and forskolin-stimulated I(Cl) were inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) blockers GlyH101, CFTRinh172, and glibenclamide. Neither flagellin nor IL-1beta altered transepithelial fluxes of membrane-impermeant dextran (10 kDa) or lucifer yellow (mol wt = 457), but both activated p38, NF-kappaB, and IL-8 secretion. Blockers of p38 (SB-202190 and SB-203580) reduced flagellin- and IL-1beta-stimulated I(Cl) by 33-50% but had smaller effects on IL-8 and NF-kappaB. It is concluded that: 1) flagellin and IL-1beta activated p38, NF-kappaB, IL-8, and CFTR-dependent anion secretion without altering tight junction permeability; 2) p38 played a role in regulating I(Cl) and IL-8 but not NF-kappaB; and 3) p38 was more important in flagellin- than IL-1beta-stimulated responses. During P. aeruginosa infections, flagellin and IL-1beta are expected to increase CFTR-dependent ion and fluid flow into and bacterial clearance from the airways. In cystic fibrosis, the secretory response would be absent, but activation of p38, NF-kappaB, and IL-8 would persist.