Limits...
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

Phenotype identification of the cultured human RPE cells using immunofluorescence (representative image; n = 5). A: All of the cultured cells were positively stained with the RPE65 antibody. B: All of the cultured cells were negatively stained with the S100 antibody. Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI). Scale bar = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4548790&req=5

f1: Phenotype identification of the cultured human RPE cells using immunofluorescence (representative image; n = 5). A: All of the cultured cells were positively stained with the RPE65 antibody. B: All of the cultured cells were negatively stained with the S100 antibody. Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI). Scale bar = 50 μm.

Mentions: The cultured primary human RPE cells reached confluence in 2–3 weeks. The third passage cells were all positively stained with the RPE 65 antibody, and were negative for the S100 antibody (Figure 1). This suggests that the cultured cells were RPE cells and were not contaminated with other cells such as glial cells [43], Müller cells [44], fibroblasts [43], or choroidal melanocytes [45].


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)

Phenotype identification of the cultured human RPE cells using immunofluorescence (representative image; n = 5). A: All of the cultured cells were positively stained with the RPE65 antibody. B: All of the cultured cells were negatively stained with the S100 antibody. Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI). Scale bar = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Phenotype identification of the cultured human RPE cells using immunofluorescence (representative image; n = 5). A: All of the cultured cells were positively stained with the RPE65 antibody. B: All of the cultured cells were negatively stained with the S100 antibody. Nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI). Scale bar = 50 μm.
Mentions: The cultured primary human RPE cells reached confluence in 2–3 weeks. The third passage cells were all positively stained with the RPE 65 antibody, and were negative for the S100 antibody (Figure 1). This suggests that the cultured cells were RPE cells and were not contaminated with other cells such as glial cells [43], Müller cells [44], fibroblasts [43], or choroidal melanocytes [45].

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