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GABAAα1 and GABAAρ1 subunits are expressed in cultured human RPE cells and GABAA receptor agents modify the intracellular calcium concentration.

Cheng ZY, Wang XP, Schmid KL, Han XG, Song H, Tang X - Mol. Vis. (2015)

Bottom Line: 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.Muscimol-induced increases in the [Ca(2+)]i were inhibited by pretreatment with picrotoxin (300 μM) or TPMPA (500 μM).GABAAα1 and GABAAρ1 are expressed in cultured human RPE cells, and GABAA agents can modify [Ca(2+)]i.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

Purpose: 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.

No MeSH data available.


Related in: MedlinePlus

Gamma-aminobutyric acidAα1 (GABAAα1) and GABAAρ1 protein expression in cultured human RPE cells detected by western blots (A) and immunofluorescence (B) (representative image; n = 5). A: Specific bands presented at the approximate location of 51 kDa (GABAAα1), 48 kDa (GABAAρ1), and 43 kDa (β-Actin) in lysates of RPE and the retina (positive control, PC). B: Immunofluorescence staining of GABAAα1 and GABAAρ1 in cultured human RPE. Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI; blue; bar = 20 μm). NC, negative control.
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f3: Gamma-aminobutyric acidAα1 (GABAAα1) and GABAAρ1 protein expression in cultured human RPE cells detected by western blots (A) and immunofluorescence (B) (representative image; n = 5). A: Specific bands presented at the approximate location of 51 kDa (GABAAα1), 48 kDa (GABAAρ1), and 43 kDa (β-Actin) in lysates of RPE and the retina (positive control, PC). B: Immunofluorescence staining of GABAAα1 and GABAAρ1 in cultured human RPE. Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI; blue; bar = 20 μm). NC, negative control.

Mentions: Western blots revealed intense bands at 51 kDa, 48 kDa, and 43 kDa for samples incubated with the anti-GABAAα1, anti-GABAAρ1, and anti-β-Actin antibody, respectively, in cultured human RPE and retinal samples (positive control; Figure 3A).


GABAAα1 and GABAAρ1 subunits are expressed in cultured human RPE cells and GABAA receptor agents modify the intracellular calcium concentration.

Cheng ZY, Wang XP, Schmid KL, Han XG, Song H, Tang X - Mol. Vis. (2015)

Gamma-aminobutyric acidAα1 (GABAAα1) and GABAAρ1 protein expression in cultured human RPE cells detected by western blots (A) and immunofluorescence (B) (representative image; n = 5). A: Specific bands presented at the approximate location of 51 kDa (GABAAα1), 48 kDa (GABAAρ1), and 43 kDa (β-Actin) in lysates of RPE and the retina (positive control, PC). B: Immunofluorescence staining of GABAAα1 and GABAAρ1 in cultured human RPE. Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI; blue; bar = 20 μm). NC, negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4548790&req=5

f3: Gamma-aminobutyric acidAα1 (GABAAα1) and GABAAρ1 protein expression in cultured human RPE cells detected by western blots (A) and immunofluorescence (B) (representative image; n = 5). A: Specific bands presented at the approximate location of 51 kDa (GABAAα1), 48 kDa (GABAAρ1), and 43 kDa (β-Actin) in lysates of RPE and the retina (positive control, PC). B: Immunofluorescence staining of GABAAα1 and GABAAρ1 in cultured human RPE. Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI; blue; bar = 20 μm). NC, negative control.
Mentions: Western blots revealed intense bands at 51 kDa, 48 kDa, and 43 kDa for samples incubated with the anti-GABAAα1, anti-GABAAρ1, and anti-β-Actin antibody, respectively, in cultured human RPE and retinal samples (positive control; Figure 3A).

Bottom Line: 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.Muscimol-induced increases in the [Ca(2+)]i were inhibited by pretreatment with picrotoxin (300 μM) or TPMPA (500 μM).GABAAα1 and GABAAρ1 are expressed in cultured human RPE cells, and GABAA agents can modify [Ca(2+)]i.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

Purpose: 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.

No MeSH data available.


Related in: MedlinePlus