Creatine Kinase in Human Retinal Pigment Epithelium

Kennedy, Brian G.; Haley, Brian E.; Mangini, Nancy J.

doi:10.1006/exer.1999.0766

Cited by 36 publications

(43 citation statements)

References 26 publications

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“…Rhodamine 123, a fluorescent substrate for efflux transporters, has been used as a typical probe to assess in vitro and in vivo P-gp function in various multidrug-resistant cells and various normal tissues including the human outer BRB. 5,9 After dexamethasone treatment (10 À5 M), we detected a significant decrease in the fluorescence intensity of intracellular rhodamine 123, indicative of an increased P-gp function. As expected, the rhodamine 123 retention was increased with coculture of RU486 when compared with dexamethasone alone, but did not fully return to the level of the control, untreated cultures (Fig.…”

Section: Dexamethasone-induced Activity Of P-gp Was Partially Reversementioning

confidence: 86%

“…Although efflux pumps such as P-gp are best known as major barriers to drug delivery in brain and gut, [11][12][13] they have also received attention for their potential roles in barrier maintenance at the outer BRB. Kennedy and Mangini 9 have demonstrated that P-gp is expressed on both the apical as well as basal membranes of the RPE cells. P-gp on the RPE cells may thus affect permeation of substrates from the vitreous humor into the systemic circulation and vice versa 3,14,15 and could be a major factor behind the inability of systemic and transscleral routes of administration to generate and maintain therapeutic concentrations of P-gp substrates in the retina.…”

mentioning

confidence: 99%

“…7 As a well-characterized efflux transporter, P-gp is strongly expressed by retinal vascular endothelial cells 8 and has recently been identified in human RPE. 9 P-gp displays broad specificity, accepting many structurally, functionally, and mechanistically unrelated compounds, 10 and its role in limiting drug penetration across biological barriers is well established. Although efflux pumps such as P-gp are best known as major barriers to drug delivery in brain and gut, [11][12][13] they have also received attention for their potential roles in barrier maintenance at the outer BRB.…”

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confidence: 99%

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Expression and Activity of p-Glycoprotein Elevated by Dexamethasone in Cultured Retinal Pigment Epithelium Involve Glucocorticoid Receptor and Pregnane X Receptor

Zhang

Lü

Sun

et al. 2012

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. To investigate whether dexamethasone has an effect on functional expression of p-glycoprotein in cultured human RPE and, if so, whether this occurs through interaction with glucocorticoid receptor (GR) and pregnane X receptor (PXR).METHODS. The human RPE D407 was treated with increasing concentrations of dexamethasone and/or RU486 for various time periods up to 24 hours. Treated cells were collected for cell viability, expressions of p-glycoprotein and PXR, and rhodamine 123 accumulation assays. GR expression plasmid and rifampicin were chosen to investigate the relationship of GR/PXR activation and p-glycoprotein expression.RESULTS. Significant increases in p-glycoprotein, as indicated by mRNA and protein levels, as well as by functional activity, were induced within 12 hours of dexamethasone treatment, persisted as long as 24 hours, and were dose-dependent and attenuable with coculture of RU486. In parallel, a dosedependent upregulation of PXR was notable at both mRNA and protein levels by 24 hours of dexamethasone treatment, and was partially reversible with RU486 coculture. Additionally, transfection of GR expression plasmid increased the transitional expressions of PXR and p-glycoprotein in untreated cells, and enhanced PXR transcriptional expression in dexamethasone-treated cells. Further, PXR silencing inhibited the dexamethasone-induced p-glycoprotein alterations; however, rifampicin had no apparent effects on the dexamethasoneinduced p-glycoprotein alterations. CONCLUSIONS.Our results suggest for the first time that expression and activation of p-glycoprotein involve GR and PXR in human RPE. (Invest Ophthalmol Vis Sci. 2012;53:3508-3515) DOI:10.1167/iovs.11-9337 R PE plays an essential role in protecting neural tissues from toxic materials and in maintaining vision and neural function in the retina by forming the outer blood-retinal barrier (BRB). It has been demonstrated that the outer BRB not only regulates the ionic environment of the subretinal space, secretes factors for structural integrity of the retina, phagocytizes shed outer segments of photoreceptors, and participates in the visual cycle, 1 but also limits vitreal penetration of drugs administered by the systemic and transscleral routes. 2-4 Efflux transport systems provide further barriers for the retina, by actively removing cytotoxic drugs and specific xenobiotic compounds from the retina and transferring them back into the systemic circulation.5,6 P-glycoprotein (P-gp), a 170-kDa protein encoded by the multiple drug resistance human MDR1 gene, is a member of the ABC superfamily of energy-dependent transport systems.7 As a well-characterized efflux transporter, P-gp is strongly expressed by retinal vascular endothelial cells 8 and has recently been identified in human RPE. 9 P-gp displays broad specificity, accepting many structurally, functionally, and mechanistically unrelated compounds, 10 and its role in limiting drug penetration across biological barriers is well established. Although efflux pumps such as P-gp are best known ...

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Section: Dexamethasone-induced Activity Of P-gp Was Partially Reversementioning

confidence: 86%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Expression and Activity of p-Glycoprotein Elevated by Dexamethasone in Cultured Retinal Pigment Epithelium Involve Glucocorticoid Receptor and Pregnane X Receptor

Zhang

Lü

Sun

et al. 2012

Invest. Ophthalmol. Vis. Sci.

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“…Ref. 16) to reduce the significant autofluorescence of lipofuscin and Bruch's membrane that is characteristic of native human RPE tissue (10). Sections were incubated in commercially available polyclonal primary antibody raised against rat ClC-3 (Alomone Labs, Jerusalem, Israel), ClC-5 (C1 antibody raised against residues 570-677 of rat ClC-5; Ref.…”

Section: Methodsmentioning

confidence: 99%

Oxidant and antioxidant modulation of chloride channels expressed in human retinal pigment epithelium

Weng

Godley

Jin

et al. 2002

American Journal of Physiology-Cell Physiology

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little is known about the molecular nature of chloride channels in human adult RPE (haRPE) or the effects of oxidative stress on membrane conductance properties. In the present study, we assessed ClC channel and cystic fibrosis transmembrane conductance regulator (CFTR) expression and membrane chloride conductance properties in haRPE cells. ClC-5, ClC-3, ClC-2, and CFTR mRNA expression was confirmed with RT-PCR analysis, and protein expression was detected with Western blot analysis and immunofluorescence microscopy. Whole cell recordings of primary cultures of haRPE showed an outwardly rectifying chloride current that was inhibited by the oxidant H2O2. The inhibitory effects of H2O2 were reduced in cultured human RPE cells that were incubated with precursors of glutathione synthesis or that were stably transfected to overexpress glutathione S-transferase. These findings indicate a possible role for ClC channels in haRPE cells and suggest possible redox modulation of human RPE chloride conductances.immunocytochemistry; patch clamp; glutathione; glutathione S-transferase RETINAL PIGMENT EPITHELIUM (RPE) comprises part of the blood-retina barrier and functions in several processes that are vital for the preservation of sight. Among the crucial roles of this epithelium is the regulation of the volume and electrolyte composition of the subretinal space. This function is achieved largely by regulated transepithelial transport of chloride from the subretinal space to the choroid with the obligatory movement of water. Disruption of RPE chloride transport can result in the accumulation of fluid in the subretinal space and subsequent retinal detachment (3, 26).Chloride transport across the RPE is mediated in part by chloride channels that are stimulated by calcium and cAMP (Ref. 28; cf. Refs. 13,14). In recent whole cell patch-clamp recordings in SV40-transformed cultured human fetal RPE (hfRPE) cells, we (39) identified an outwardly rectifying chloride current that was stimulated by cAMP but was inhibited by the chloride channel blocker DIDS, acidic bathing solutions, or low concentrations of the oxidative agent H 2 O 2 . The molecular identity of the chloride channel(s) responsible for this current was not determined. However, these cells expressed several candidate chloride channels including cystic fibrosis transmembrane conductance receptor (CFTR) and members of the ClC chloride channel family Ref. 39). At present, little is known about the expression of ClC chloride channels in the intact human RPE.Recent evidence suggests that the ClC family of voltage-gated chloride channels may be crucial for retinal function. At least three members of this family are associated with retinal degenerations in mice. Specifically, transgenic mice that were deficient for ClC-3 (34), ClC-2 (6), or ClC-7 (17) were found to develop retinal degenerations and blindness within weeks after birth. The basis of these degenerations is not presently understood.The finding of ClC channel expression in human fetal cells and the consequences of ...

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“…Both pumps have a broad substrate specificity, with P-gp pumps generally expelling large neutral or cationic compounds and MRPs removing large neutral or anionic compounds [97] . P-gp and MRP expression have been reported in human RPE with efflux directed towards the choroid [98][99][100] , preventing drugs from entering the retina.…”

Section: Rpe Transporter Proteinsmentioning

confidence: 99%

Transport Barriers in Transscleral Drug Delivery for Retinal Diseases

et al. 2007

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Transscleral delivery has emerged as an attractive method for treating retinal disorders because it offers localized delivery of drugs as a less invasive method compared to intravitreal administration. Numerous novel transscleral drug delivery systems ranging from microparticles to implants have been reported. However, transscleral delivery is currently not as clinically effective as intravitreal delivery in the treatment of retinal diseases. Transscleral drug delivery systems require drugs to permeate through several layers of ocular tissue (sclera, Bruch’s membrane-choroid, retinal pigment epithelium) to reach the neuroretina. As a result, a steep drug concentration gradient from the sclera to the retina is established, and very low concentrations of drug are detected in the retina. This steep gradient is created by the barriers to transport that hinder drug molecules from successfully reaching the retina. A review of the literature reveals 3 types of barriers hindering transscleral drug delivery: static, dynamic and metabolic. While static barriers have been examined in detail, the literature on dynamic and metabolic barriers is lacking. These barriers must be investigated further to gain a more complete understanding of the transport barriers involved in transscleral drug delivery.

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Creatine Kinase in Human Retinal Pigment Epithelium

Cited by 36 publications

References 26 publications

Expression and Activity of p-Glycoprotein Elevated by Dexamethasone in Cultured Retinal Pigment Epithelium Involve Glucocorticoid Receptor and Pregnane X Receptor

Expression and Activity of p-Glycoprotein Elevated by Dexamethasone in Cultured Retinal Pigment Epithelium Involve Glucocorticoid Receptor and Pregnane X Receptor

Oxidant and antioxidant modulation of chloride channels expressed in human retinal pigment epithelium

Transport Barriers in Transscleral Drug Delivery for Retinal Diseases

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