Abstract
Inhibition of p-glycoprotein under hyperglycemic conditions has been reported in various barrier tissues including blood-brain barrier, intestine, and kidney, and has been linked to significant clinical complications. However, whether this is also true for the outer blood-retinal barrier constituted by retinal pigment epithelium, or has a role in pathogenesis of diabetic retinopathy is not yet clear. In this study, using cultured human retinal pigment epithelium cell line D407, we found that high glucose exposure induced a significant decrease in p-glycoprotein expression both at mRNA and at protein levels, accompanied by an attenuated p-glycoprotein activity determined by intracellular rhodamine 123 retention. In marked contrast, the expressions of both mRNA and protein levels of inducible nitrate oxide synthase (iNOS) increased, and were accompanied by increased extracellular nitrate/nitrite production by Griess reaction. In addition, mRNA levels of nuclear receptors revealed a decreased expression of pregnane X receptor after the exposure of high glucose. However, the subsequent alterations in production of nitrate/nitrite, functional expression of p-glycoprotein, and mRNA levels of pregnane X receptor were partially blocked when pretreated with S,S′-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea•2HBr (PBITU), a selective iNOS inhibitor. Moreover, the effects of PBITU were antagonized with the addition of L-arginine, a substrate for NO synthesis. Our in vitro results suggest for the first time that iNOS induction plays a novel role in decreased p-glycoprotein expression and transport function at the human outer blood-retinal barrier under hyperglycemic conditions and further support the concept of inhibiting iNOS pathway as a therapeutic strategy for diabetic retinopathy.
Highlights
The retinal pigment epithelium (RPE) is a monolayer of pigmented cells located between the neural retina and the choroid, and constitutes the outer blood-retinal barrier (BRB, for a review see ref. 3)
We show that inducible nitrate oxide synthase signaling pathway is induced in the cultured human RPE cells by the exposure of high glucose, and high glucose-induced iNOS pathway results in the inhibition of functional expression of P-gp and transcriptional expression of pregnane X receptor (PXR), a nuclear receptor that regulates expression of drug metabolizing enzymes and efflux transporters [13,21]
To examine the changes of P-gp expression across time at the outer BRB under hyperglycemic conditions, we first examined the MDR1 mRNA level in the RPE cells incubated for 6–144 h in intermediate (12.5 mM) or high glucose (25 or 50 mM)
Summary
The retinal pigment epithelium (RPE) is a monolayer of pigmented cells located between the neural retina and the choroid, and constitutes the outer blood-retinal barrier (BRB, for a review see ref. 3). The retinal pigment epithelium (RPE) is a monolayer of pigmented cells located between the neural retina and the choroid, and constitutes the outer blood-retinal barrier Because the RPE forms the outer BRB and is essential for removal of waste products and entry of nutrients into the retina, any disturbance in normal transport function of these cells necessarily has detrimental consequences for the retina. Numerous studies on pathogenesis of DR have been focused on the impairment of the neural retina and the inner BRB [4,7]. We suspected that the abnormal elevated blood glucose levels during the development of DR may potentially disturb the barrier function at the outer BRB. The direct data for the effects of high glucose or hyperglycemia on the transport functions at the outer BRB are not yet available
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