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Paula E. Pecen, Padmanabhan P. Pattabiraman, Corey Morris, Vasanth Rao; MRP-4, a Multidrug Resistance-Associated Protein and its Role in Trabecular Meshwork Relaxation and Homeostasis of Intraocular Pressure. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5092.
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Multidrug resistance-associated proteins (MRP) are the subfamily of ATP-binding cassette class C (ABCC) transporters that regulate efflux of intracellular cyclic nucleotides (cAMP and cGMP), which in turn is involved in controlling cell contractile and relaxation characteristics. Here we investigated the role of MRP-4 in regulation of TM cell relaxation characteristics in cell culture and intraocular pressure (IOP) in rabbits.
Expression of different isoforms of MRPs was determined in TM cells derived from human donor eyes using RT-PCR. Both human and porcine TM cells were treated with MRP-4 specific inhibitor- MK571 and assessed for changes in cell shape, actin cytoskeletal integrity and focal adhesions by immunohistochemical analysis and myosin light chain phosphorylation (pMLC) by immunoblotting. Changes in the intracellular cAMP levels were assessed using an enzyme-linked immunoassay. Effects of MK571 on IOP were evaluated on Dutch-belted male rabbits by topical application.
RT-PCR analysis of HTM cell RNA derived from two different donors demonstrated the expression of MRP-1, -2, -3, -4 and -5 subtypes. Treatment of HTM and PTM cells with MK571 or Probenecid led to cell-cell separation and cell rounding in a dose- (10-100 μM and 0.5 to 1 mM, respectively) and time- (30 to 90 minutes) dependent manner. These alterations were completely reversible in less than one hour after drug washout. MK571 or Probenecid treatment led to a dramatic depolymerization of actin stress fibers, loss of focal adhesion and significantly decreased pMLC (p<0.005, n=4). Intracellular cAMP levels in PTM cells were significantly higher (22 fold increase, p<0.001, n=4) after treatment with MRP-4 inhibitor. MK571-induced TM cellular relaxation was found to be partially blocked by Nifedipine and Gadolinium, which are inhibitors of voltage-gated calcium ion channel activity. A single topical application of MK571 (0.2%) in rabbits showed a significant drop in IOP (16%, p<0.01, n=4) after one hour, but returned back to baseline by 4 hours post-treatment.
This study identifies the cyclic nucleotide efflux controlling protein, MRP-4, as a critical regulator of homeostasis of IOP by modulating the trabecular meshwork relaxation characteristics.
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