title: GABAAα1 and GABAAρ1 subunits are expressed in cultured human RPE cells and GABAA receptor agents modify the intracellular calcium concentration.
abstract: OBJECTIVE
  Gamma-aminobutyric acid A (GABAA) receptors (GABAARs), which are ionotropic receptors involving chloride channels, have been identified in various neural (e.g., mouse retinal ganglion cells) and nonneural cells (e.g., mouse lens epithelial cells) regulating the intracellular calcium concentration ([Ca(2+)]i). GABAAR β-subunit protein has been isolated in the cultured human and rat RPE, and GABAAα1 and GABAAρ1 mRNAs and proteins are present in the chick RPE. The purpose of this study was to investigate the expression of GABAAα1 and GABAAρ1, two important subunits in forming functional GABAARs, in the cultured human RPE, and further to explore whether altering receptor activation modifies [Ca(2+)]i.
  
  
  METHODS
  Human RPE cells were separately cultured from five donor eye cups. Real-time PCR, western blots, and immunofluorescence were used to test for GABAAα1 and GABAAρ1 mRNAs and proteins. The effects of the GABAAR agonist muscimol, antagonist picrotoxin, or the specific GABAAρ antagonist 1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo3-AM.
  
  
  RESULTS
  Both GABAAα1 and GABAAρ1 mRNAs and proteins were identified in cultured human RPE cells; antibody staining was mainly localized to the cell membrane and was also present in the cytoplasm but not in the nucleus. Muscimol (100 μM) caused a transient increase of the [Ca(2+)]i in RPE cells regardless of whether Ca(2+) was added to the buffer. Muscimol-induced increases in the [Ca(2+)]i were inhibited by pretreatment with picrotoxin (300 μM) or TPMPA (500 μM).
  
  
  CONCLUSIONS
  GABAAα1 and GABAAρ1 are expressed in cultured human RPE cells, and GABAA agents can modify [Ca(2+)]i.
authors: Tianjin Eye Hospital, Clinical College of Ophthalmology, Tianjin Medical University, 4 Gansu Road, Heping District, Tianjin, China ; Department of Ophthalmology, Qilu Hospital, Shandong University, Jinan, Shandong, China.
authors: Zhen-Ying
authors: ZY
authors: Cheng
authors: The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital, Shandong University, Jinan, Shandong, China.
authors: Xu-Ping
authors: XP
authors: Wang
authors: School of Optometry and Vision Science, Faculty of Health, and Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia.
authors: Katrina L
authors: KL
authors: Schmid
authors: Department of Ophthalmology, The Second People's Hospital of Jinan, Jinan, Shandong, China.
authors: Xu-Guang
authors: XG
authors: Han
authors: Tianjin Eye Hospital, Clinical College of Ophthalmology, Tianjin Medical University, 4 Gansu Road, Heping District, Tianjin, China.
authors: Hui
authors: H
authors: Song
authors: Tianjin Eye Hospital, Clinical College of Ophthalmology, Tianjin Medical University, 4 Gansu Road, Heping District, Tianjin, China.
authors: Xin
authors: X
authors: Tang
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D058785:GABA-A Receptor Agonists / Q000494:pharmacology; D058787:GABA-A Receptor Antagonists / Q000494:pharmacology; D006801:Humans; D051379:Mice; D009118:Muscimol / Q000494:pharmacology; D010852:Picrotoxin / Q000494:pharmacology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics* / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
chemical_list: D002118:Calcium; D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; D009118:Muscimol; D010852:Picrotoxin; D021122:Protein Subunits; D012333:RNA, Messenger; D011963:Receptors, GABA-A