title: MRP4-mediated regulation of intracellular cAMP and cGMP levels in trabecular meshwork cells and homeostasis of intraocular pressure.
abstract: OBJECTIVE
  Multidrug, resistance-associated protein-4 (MRP4) is a membrane transporter that regulates the cellular efflux of cyclic nucleotides (cAMP and cGMP) involved in various physiologic responses. This study examined the expression and distribution of MRP4 in the trabecular meshwork (TM) cells and its role in homeostasis of IOP.
  
  
  METHODS
  Expression and distribution of MRP4 in human TM (HTM) cells and aqueous humor (AH) outflow pathway was determined by RT-PCR, immunoblotting, and immunofluorescence. Effects of inhibiting MRP4 activity and suppression of MRP4 expression on cAMP and cGMP levels, myosin light chain (MLC) phosphorylation, actin filament organization and activity of protein kinase G (PKG), protein kinase A (PKA), Rho guanosine triphosphatase (GTPase), and MLC phosphatase was monitored in HTM cells using ELISA, siRNA, biochemical, and immunofluorescence analyses. Topical application of the MRP4 inhibitor MK571 was tested to assess changes in IOP in rabbits.
  
  
  RESULTS
  RT-PCR, immunoblot, and immunofluorescence analyses confirmed the expression of MRP4 in HTM cells and distribution in human AH outflow pathway. Inhibition of MRP4 in HTM cells by MK571 or probenecid resulted in cell shape changes and decreases in actin stress fibers and MLC phosphorylation. Levels of intracellular cAMP and cGMP in HTM cells were increased significantly under these conditions. MK571-induced HTM cell relaxation appeared to be mediated predominantly via activation of the cGMP-dependent PKG signaling pathway. Topical application of MK571 significantly decreased IOP in Dutch-Belted rabbits.
  
  
  CONCLUSIONS
  These observations reveal that cyclic nucleotide efflux controlling transporter-MRP4 plays a significant role in IOP homeostasis potentially by regulating the relaxation characteristics of AH outflow pathway cells.
authors: Department of Ophthalmology, Duke University School of Medicine, Durham, North Carolina 27710, USA.
authors: Padmanabhan P
authors: PP
authors: Pattabiraman
authors: Paula E
authors: PE
authors: Pecen
authors: Ponugoti Vasantha
authors: PV
authors: Rao
mesh_terms: D000199:Actins / Q000502:physiology / Q000648:ultrastructure; D000328:Adult; D000368:Aged; D000818:Animals; D001082:Aqueous Humor / Q000166:cytology / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D006152:Cyclic GMP / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology; D006706:Homeostasis / Q000502:physiology*; D006801:Humans; D007429:Intraocular Pressure / Q000502:physiology*; D020024:Leukotriene Antagonists / Q000494:pharmacology; D008875:Middle Aged; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018994:Myosin Light Chains / Q000502:physiology; D010766:Phosphorylation / Q000502:physiology; D011339:Probenecid / Q000494:pharmacology; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000502:physiology; D011817:Rabbits; D015398:Signal Transduction / Q000502:physiology; D014129:Trabecular Meshwork / Q000166:cytology* / Q000378:metabolism
chemical_list: D000199:Actins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D006152:Cyclic GMP; D017869:Cyclic GMP-Dependent Protein Kinases; D020024:Leukotriene Antagonists; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D018994:Myosin Light Chains; D011339:Probenecid; D011422:Propionates; D011804:Quinolines; D034741:RNA, Small Interfering; C073492:ABCC4 protein, human; C059141:verlukast
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