GABA Maintains the Proliferation of Progenitors in the Developing Chick Ciliary Marginal Zone and Non-Pigmented Ciliary Epithelium

GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABAA receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABAA receptor system. To quantify the effects on proliferation by GABAA receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABAA receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABAA receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl–transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABAA receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABAA receptors. This supported the depolarising role for the GABAA receptors. Inhibition of L-type voltage-gated Ca2+ channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABAA receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27KIP1, along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27KIP1 after inhibition of either the GABAA receptors or the L-type VGCCs suggests a link between the GABAA receptors, membrane potential, and intracellular Ca2+ in regulating the cell cycle

GABA-A channel subunits immunolabeled in the cytoplasm and in the plasma membrane.

<p>(A) Rat CD4⁺ T cell, β3 GABA-A channel subunit immunolabeling was observed both in the cytoplasm and in the plasma membrane (n = 41). (B) Jurkat cell, α1 GABA-A subunit immunolabeling was observed prominently in the plasma membrane as punctate pattern (n = 59). Plasma membrane labelling: DiI (red); subunit colour-labeling: β3 (green), α1 (green). The nuclei were stained with DAPI (blue). Scale bar = 2 µM.</p

Detection of GABA-A channel subunit proteins in CD4⁺ and CD8⁺ T cells from rats, mice and the Jurkat cell line.

<p>(A–C). Rat CD4⁺ and CD8⁺ T cell protein extracts immunoprecipitated with anti-α1–6 (A, n = 6), anti-α1 (B, n = 3) or anti-β3 antibody (C, n = 3) and bands at the correct molecular weight were identified. The α1 and β3 GABA-A channel subunits proteins were also identified in extracts from Jurkat cells (B and C). (D) The γ2 GABA-A channel subunit protein was detected in mouse CD4⁺ and CD8⁺ T cells (n = 4). Protein extracts from rat and mouse brains served as positive controls. In A–D, the blots with β-actin served as loading controls. (E–F) The bands specific for γ2 subunit were absent in the presence of the γ2 blocking peptide in protein extracts from mouse brain (E) and mouse CD4+ and CD8+ T cells (F). The amounts of proteins loaded were: (A) 20 µg for all lanes; (B) 60 µg for rat CD4⁺, CD8⁺ T cells and Jurkat cells, 15 µg for rat brain; (C) 30 µg for all lanes; (D, E, F) 60 µg for mouse CD4⁺ and CD8⁺ T cells; 10 µg for mouse brain. Molecular weight in kDa is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042959#pone.0042959.s001" target="_blank">Table S1</a>.</p

GABA-A channel subunit mRNA expression in rat, mouse and human T cells (both CD4⁺ and CD8⁺) and Jurkat cells.

<p>++: Abundant expression; +: Modest expression; (+): Low expression: −: No expression.</p

Immunolabeling of GABA-A channel subunits in rats, mice and human T cells and the Jurkat cell line.

<p>(A) Rat CD4⁺ (n = 85) and CD8⁺ (n = 186) T cells, β3 GABA-A channel subunit immunolabeling is observed in most cells. Insert in (A) represent the cell identified with the arrowhead. (B) Rat CD4⁺ and CD8⁺ T cells, α1 (n = 44; n = 43), α2 (n = 53; n = 55) GABA-A subunit immunolabeled. The representative images show the punctate labeling pattern (C) Human CD4⁺ (n = 34) and CD8⁺ (n = 43) T cells, α1 GABA-A subunit immunolabeled. (D) Jurkat cells, α1 (n = 104) or β3 (n = 65) GABA-A subunit immunolabeled. (E) Mouse CD4⁺ and CD8⁺ T cells, α2 (n = 42; n = 53) or γ2 (n = 36; n = 54) GABA-A subunit immunolabeled. (F) In mouse CD4⁺ T cells, the γ2 GABA-A subunit immunolabeling was absent in the presence of the blocking peptide (n = 35). Subunit colour-labeling: α1 green; α2 green, β3 red; γ2 green. The nuclei were stained with DAPI (blue). Scale bars in A and F = 5 µm, in insert in A and in B–E = 2 µm.</p

Human, rat and mouse primers list for quantitative real-time RT-PCR.

<p><i>B2M</i>: beta-2 microglobulin; <i>Hprt</i>: hypoxanthine guanine phosphoribosyl transferase.</p>*<p>Primer sequences were extracted from Alam S, et al. (2006) Mol Immunol, 43(9): 1432-42.</p

GABA activates GABA-A currents in T cells.

<p>Whole-cells currents were evoked by application of 1 µM or 1 mM GABA to rat CD4⁺ T cells (A, B, F), rat CD8⁺ T cells (C) or Jurkat E6. 1 cells (D, E, G). In symmetrical chloride solutions the currents were inward at negative potentials (A, B, C, D; −80 mV and in G, −60 mV) and outward at positive potential (E, F; +40 mV). Picrotoxin (PTX) inhibits the GABA-activated transient current. (F, G) Tonic currents were activated by GABA (1 µM, 1 mM) and inhibited by 100 µM SR95531 (F) or 100 µM bicuculline (G), GABA-A channel antagonists. The difference between the dotted lines shows the amplitude of the tonic current (51 pA). Applications of drugs are indicated by the bars located above the current traces.</p

Expression of GABA-A channel subunit mRNAs in CD4⁺ or CD8⁺ T cells from rats, mice and human donors and the Jurkat cell line.

<p>Expression of all 19 GABA-A receptor channel subunit isoforms was examined using RT-qPCR in T cells. Subunit isoforms that were detected are shown in A–D. In CD4⁺ (open bar) and CD8⁺ (grey bar) T cells from mesenteric lymph nodes of Wistar rats (A, n = 4) or C57BL/6J mice (B, n = 3), 13 and 8 different GABA-A channel subunit mRNAs were detected, respectively. The mRNA expression level for each subunit did not differ between the CD4⁺ and the CD8⁺ T cells. (C) In both CD4⁺ (open bar) and CD8⁺ (grey bar) T cells isolated from human pancreatic lymph nodes (4 different donors) 5 different GABA-A channel subunit mRNAs were detected. (D) In Jukart cells, 9 different GABA-A channel subunit mRNAs were detected. The mRNA level of each subunit was normalized to reference genes, calculated as 2^−ΔCt and presented as mean with SEM. The reference genes were hypoxanthine phophoribosyltransferase (<i>Hprt</i>) for rat T cells, β-actin (<i>Actb)</i> for mouse T cells, and β2-microglobin (<i>B2M</i>) for human T cells and Jurkat cells.</p

Revaluation of magnetic properties of Magneto

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