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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

Evidence that the gamma-aminobutyric acidA receptor (GABAAR) agonist muscimol, the antagonist picrotoxin and the GABAAρ antagonist 1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) can modify the intracellular calcium concentration ([Ca2+]i) in the cultured human RPE. A: Muscimol (100 μM) increased the [Ca2+]i in cultured human RPE cells in buffer with Ca2+ (paired t-test, p<0.05). B: Pretreatment with picrotoxin (300 μM) completely inhibited the muscimol (100 μM) induced increase of [Ca2+]i in buffer with Ca2+. C: Pretreatment with TPMPA (500 μM) completely blocked the muscimol (100 μM) induced increase of [Ca2+]i in buffer with Ca2+. D: Muscimol (100 μM) increased the [Ca2+]i in cultured human RPE cells in buffer without Ca2+ (paired t-test, p<0.05). E: Pretreatment with picrotoxin (300 μM) completely inhibited the muscimol (100 μM) induced increase of [Ca2+]i in buffer without Ca2+. F: Pretreatment with TPMPA (500 μM) completely blocked the muscimol (100 μM) induced increase of [Ca2+]i in buffer without Ca2+. Left, representative images (1, 8, and 15 images were acquired, respectively). Right, fluorescent intensity in the images acquired before and after muscimol application. The red bar represents the time when muscimol was added. FI represents the fluorescence intensity color scale, with the direction of the arrow indicating higher intensity. *indicates a p<0.05, and ** indicates a p<0.01, compared to the baseline, paired t-test, n=5 for each group.
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f4: Evidence that the gamma-aminobutyric acidA receptor (GABAAR) agonist muscimol, the antagonist picrotoxin and the GABAAρ antagonist 1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) can modify the intracellular calcium concentration ([Ca2+]i) in the cultured human RPE. A: Muscimol (100 μM) increased the [Ca2+]i in cultured human RPE cells in buffer with Ca2+ (paired t-test, p<0.05). B: Pretreatment with picrotoxin (300 μM) completely inhibited the muscimol (100 μM) induced increase of [Ca2+]i in buffer with Ca2+. C: Pretreatment with TPMPA (500 μM) completely blocked the muscimol (100 μM) induced increase of [Ca2+]i in buffer with Ca2+. D: Muscimol (100 μM) increased the [Ca2+]i in cultured human RPE cells in buffer without Ca2+ (paired t-test, p<0.05). E: Pretreatment with picrotoxin (300 μM) completely inhibited the muscimol (100 μM) induced increase of [Ca2+]i in buffer without Ca2+. F: Pretreatment with TPMPA (500 μM) completely blocked the muscimol (100 μM) induced increase of [Ca2+]i in buffer without Ca2+. Left, representative images (1, 8, and 15 images were acquired, respectively). Right, fluorescent intensity in the images acquired before and after muscimol application. The red bar represents the time when muscimol was added. FI represents the fluorescence intensity color scale, with the direction of the arrow indicating higher intensity. *indicates a p<0.05, and ** indicates a p<0.01, compared to the baseline, paired t-test, n=5 for each group.

Mentions: There were no significant differences in baseline fluorescent intensity across treatment groups in the buffer either with Ca2+ (one-way ANOVA, p>0.05, n=5), or without Ca2+ (one-way ANOVA, p>0.05, n=5). The GABAAR agonist muscimol (100 μM) induced a rapid and significant [Ca2+]i increase in the cultured human RPE cells in the buffer either with Ca2+ (paired t-test, p<0.05, n=5; Figure 4A) or without Ca2+ (paired t-test, p<0.05, n=5; Figure 4D). The [Ca2+]i reached its peak in 20–40 s, and then gradually declined (Figure 4A,D). When the cultured human RPE cells were preincubated with either the GABAAR antagonist picrotoxin (300 μM; Figure 4B,E) or the GABAAρ antagonist TPMPA (500 μM; Figure 4C,F), the muscimol (100 μM) induced [Ca2+]i increase was completely blocked (both when the buffer contained Ca2+, Figure 4B,C, and when it did not, Figure 4E,F). The addition of the control agent, PBS, did not alter the [Ca2+]i of the cultured human RPE cells (paired t-test, p>0.05, n=5).


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)

Evidence that the gamma-aminobutyric acidA receptor (GABAAR) agonist muscimol, the antagonist picrotoxin and the GABAAρ antagonist 1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) can modify the intracellular calcium concentration ([Ca2+]i) in the cultured human RPE. A: Muscimol (100 μM) increased the [Ca2+]i in cultured human RPE cells in buffer with Ca2+ (paired t-test, p<0.05). B: Pretreatment with picrotoxin (300 μM) completely inhibited the muscimol (100 μM) induced increase of [Ca2+]i in buffer with Ca2+. C: Pretreatment with TPMPA (500 μM) completely blocked the muscimol (100 μM) induced increase of [Ca2+]i in buffer with Ca2+. D: Muscimol (100 μM) increased the [Ca2+]i in cultured human RPE cells in buffer without Ca2+ (paired t-test, p<0.05). E: Pretreatment with picrotoxin (300 μM) completely inhibited the muscimol (100 μM) induced increase of [Ca2+]i in buffer without Ca2+. F: Pretreatment with TPMPA (500 μM) completely blocked the muscimol (100 μM) induced increase of [Ca2+]i in buffer without Ca2+. Left, representative images (1, 8, and 15 images were acquired, respectively). Right, fluorescent intensity in the images acquired before and after muscimol application. The red bar represents the time when muscimol was added. FI represents the fluorescence intensity color scale, with the direction of the arrow indicating higher intensity. *indicates a p<0.05, and ** indicates a p<0.01, compared to the baseline, paired t-test, n=5 for each group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Evidence that the gamma-aminobutyric acidA receptor (GABAAR) agonist muscimol, the antagonist picrotoxin and the GABAAρ antagonist 1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) can modify the intracellular calcium concentration ([Ca2+]i) in the cultured human RPE. A: Muscimol (100 μM) increased the [Ca2+]i in cultured human RPE cells in buffer with Ca2+ (paired t-test, p<0.05). B: Pretreatment with picrotoxin (300 μM) completely inhibited the muscimol (100 μM) induced increase of [Ca2+]i in buffer with Ca2+. C: Pretreatment with TPMPA (500 μM) completely blocked the muscimol (100 μM) induced increase of [Ca2+]i in buffer with Ca2+. D: Muscimol (100 μM) increased the [Ca2+]i in cultured human RPE cells in buffer without Ca2+ (paired t-test, p<0.05). E: Pretreatment with picrotoxin (300 μM) completely inhibited the muscimol (100 μM) induced increase of [Ca2+]i in buffer without Ca2+. F: Pretreatment with TPMPA (500 μM) completely blocked the muscimol (100 μM) induced increase of [Ca2+]i in buffer without Ca2+. Left, representative images (1, 8, and 15 images were acquired, respectively). Right, fluorescent intensity in the images acquired before and after muscimol application. The red bar represents the time when muscimol was added. FI represents the fluorescence intensity color scale, with the direction of the arrow indicating higher intensity. *indicates a p<0.05, and ** indicates a p<0.01, compared to the baseline, paired t-test, n=5 for each group.
Mentions: There were no significant differences in baseline fluorescent intensity across treatment groups in the buffer either with Ca2+ (one-way ANOVA, p>0.05, n=5), or without Ca2+ (one-way ANOVA, p>0.05, n=5). The GABAAR agonist muscimol (100 μM) induced a rapid and significant [Ca2+]i increase in the cultured human RPE cells in the buffer either with Ca2+ (paired t-test, p<0.05, n=5; Figure 4A) or without Ca2+ (paired t-test, p<0.05, n=5; Figure 4D). The [Ca2+]i reached its peak in 20–40 s, and then gradually declined (Figure 4A,D). When the cultured human RPE cells were preincubated with either the GABAAR antagonist picrotoxin (300 μM; Figure 4B,E) or the GABAAρ antagonist TPMPA (500 μM; Figure 4C,F), the muscimol (100 μM) induced [Ca2+]i increase was completely blocked (both when the buffer contained Ca2+, Figure 4B,C, and when it did not, Figure 4E,F). The addition of the control agent, PBS, did not alter the [Ca2+]i of the cultured human RPE cells (paired t-test, p>0.05, n=5).

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