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P. A. Knepper, T. Koga, M. J. Nolan, B. Y. J. T. Yue, J. R. Samples, A. Sheppard, A. F. Clark; Multidrug Resistance Proteins and the Human Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2056.
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© ARVO (1962-2015); The Authors (2016-present)
To determine whether trabecular meshwork (TM) cells express functionally active ABC transporters. Recent evidence indicates that adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily members respond to stressors such as hypoxia, cytokine signaling, increased pressure, mechanical stretch, and aging. The ABCB1 transporter (multidrug resistance protein (MDR) p-glycoprotein), expression and function are controlled by hyaluronic acid (HA) and the HA receptor CD44, both of which are altered in primary open angle glaucoma (POAG). Dysregulation of HA and CD44 interaction could result in decreased MDR activity and consequently increased cell vulnerability to stress.
The expression profile of ABCB1 transporter in ocular cells was determined using Affymetrix U133A chip gene array. A functional assay of MDR activity was developed in TM cells in culture that used calcein-AM, an excellent substrate for targeted exclusion by multi-drug transporters. If ABC transporters are active, the hydrophobic calcein-AM will be removed intact before it can be hydrolyzed. If these transporters are less active, calcein-AM is enzymatic cleaved by esterase, yielding a fluorescent calcein which was measured by confocal microscopy. In vitro, normal human TM cells were compared for their ability to accumulate and to release fluorescent calcein in the presence and absence of verapamil (calcium channel blocker) as a positive control and metabolic stress using 1 and 10 mM lactate.
ABC transporter genes ABCB1, ABCA6, ABCA8, ABCA2, ABCF2, and ABCA5 were all flagged as present in TM tissue by Affymterix microarray analysis. ABCF2 maps to 7q36 which is in the published glaucoma loci GLC1F (7q35-36). In the MDR functional assay, both verapamil and lactate treatment reduced calcein release indicating that MDR activity in TM cells is responsive to stress.
Several ABC transporter gene transcripts are expressed in TM tissue, suggesting that MDR activity may be present. Calcein-AM functional assay of MDR showed that TM cells incorporate calcein-AM, and MDR activity is inhibited by metabolic stress. These results support our hypothesis that MDR is expressed in cells relevant to the POAG disease process and abnormal stress may lead to decreased MDR activity and TM cell dysfunction in POAG. Expression of ABCF2 in the TM and localization of the gene to the GLC1F locus suggests that ABCF2 may be a glaucoma candidate gene.
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