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Cell Calcium 50 (2011) 381– 392

Contents lists available at ScienceDirect

Cell Calcium

j ourna l ho me page: www.elsev ier .com/ locate /ceca

ABAergic signaling in primary lens epithelial and lentoid cells and itsnvolvement in intracellular Ca2+ modulation

arija Schwirtlicha,1, Andrea Kwakowskya,d,1, Zsuzsa Emrib, Károly Antalb,sombor Laczac, Attila Cselenyákc, Zoya Katarovaa,2, Gábor Szabóa,∗,2

Laboratory of Molecular Biology and Genetics, Department of Gene Technology and Developmental Neurobiology, Institute of Experimental Medicine,ungarian Academy of Sciences, Budapest, HungaryDepartment of Zoology, Eszterházy College, Eger, HungaryDepartment of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest, HungaryCentre for Neuroendocrinology and Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin 9054, New Zealand

r t i c l e i n f o

rticle history:eceived 7 January 2011eceived in revised form 6 July 2011ccepted 7 July 2011vailable online 5 August 2011

eywords:ADABA receptors

ntracellular calciumenseurotransmitters

a b s t r a c t

Primary lens epithelial cell (LEC) cultures derived from newborn (P0) and one-month-old (P30) mouselenses were used to study GABA (gamma-aminobutyric acid) signaling expression and its effect on theintracellular Ca2+ ([Ca2+]i) level. We have found that these cultures express specific cellular markers forlens epithelial and fiber cells, all components of the functional GABA signaling pathway and GABA, thusrecapitulating the developmental program of the ocular lens. Activation of both GABA-A and GABA-Breceptors (GABAAR and GABABR) with the specific agonists muscimol and baclofen, respectively induces[Ca2+]i transients that could be blocked by the specific antagonists bicuculline and CGP55845 and weredependent on extracellular Ca2+. Bicuculline did not change the GABA-evoked Ca2+ responses in Ca2-containing buffers, but suppressed them significantly in Ca2+-free buffers suggesting the two receptorscouple to convergent Ca2+ mobilization mechanisms with different extracellular Ca2+ sensitivity. Pro-longed activation of GABABR induced wave propagation of the Ca2+ signal and persistent oscillations.

ABA signaling in developmentECell differentiation

The number of cells reacting to GABA or GABA + bicuculline in P30 mouse LEC cultures expressing pre-dominantly the synaptic type GABAAR did not differ significantly from the number of reacting cellsin P0 mouse LEC cultures. The GABA-induced Ca2+ transients in P30 (but not P0) mouse LEC could beentirely suppressed by co-application of bicuculline and CGP55845. The GABA-mediated Ca2+ signalingmay be involved in a variety of Ca2+-dependent cellular processes during lens growth and epithelial cell

differentiation.

. Introduction

GABA (gamma-aminobutyric acid) is a versatile molecule serv-ng as an inhibitory neurotransmitter in the central nervous system

Abbreviations: CNS, central nervous system; GABA, gamma-aminobutyric acid;AD65, glutamic acid decarboxylase, 65 kDa form; GAD67, glutamic acid decarboxy-

ase, 67 kDa form; GAT, GABA transporter (membrane); GABAR, GABA receptor(s);ABAAR, GABA-A receptor(s); GABAA�3, GABA-A receptor �3 subunit (GABA-inding); GABABR, GABA-B receptor; GABABR2, GABA-B receptor R2 subunit; VGCC,oltage-gated calcium channel(s); VGAT, vesicular GABA transporter; [Ca2+]i, intra-ellular Ca2+ concentration; P0, postnatal day 0; P30, postnatal day 30; AAH, artificialqueous humor; DAPI, 4′ ,6-diamidino-2-phenylindole; LEC, lens epithelial cell(s);-cad, N-cadherin; E-cad, E-cadherin; Cx43, connexin 43.∗ Corresponding author at: Institute of Experimental Medicine, Hungariancademy of Sciences, POB 67, H-1450 Budapest, Hungary. Tel.: +36 1 210 9980;

ax: +36 1 323 3011.E-mail address: szabog@koki.hu (G. Szabó).

1 These authors have contributed equally to this work.2 These authors share senior authorship.

143-4160/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.ceca.2011.07.002

© 2011 Elsevier Ltd. All rights reserved.

(CNS) of vertebrates, a paracrine/autocrine signaling molecule andmetabolite in numerous cells of the nervous system and periphery[1–5]. GABA is synthesized by GAD (glutamic acid decarboxylase)in GABAergic neurons and non-neuronal cells and is thereafterreleased by either a vesicular (VGAT) or membrane (GAT) GABAtransporters into the extracellular space activating two types ofGABA receptors: the ionotropic GABAAR and metabotropic GABABR.In contrast to the adult CNS where GABARs mediate fast synap-tic or tonic inhibitory neurotransmission, activation of GABAARin neuronal progenitors triggers a rise in the intracellular Ca2+

([Ca2+]i) which in turn regulates a variety of key cellular processes,including cell proliferation, migration and differentiation [6–8].The G protein-coupled metabotropic GABABR is generally thoughtto inhibit the [Ca2+]i elevation through presynaptic inhibition ofVGCCs (voltage-gated calcium channels) or post-synaptic stimu-

lation of GIRK (G-protein coupled inwardly rectifying potassium)channels [9,10]. However, it has been revealed that GABABR canalso induce [Ca2+]i rise in hippocampal astroglia [11–13] as well asneurons [14,15].

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Recently we have shown that all components of the GABAignaling machinery are expressed in two non-neuronal cellularystems: mouse embryonic stem cells (mESC) and the developingens [16–18] both affording valuable in vitro and in vivo models totudy the GABA signaling and the effect of GABAR activation onhe [Ca2+]i level during embryonic development and cellular dif-erentiation. We have demonstrated that undifferentiated mousembryonic stem cells (mESC) express molecular components ofhe GABA and Ca2+ signaling indistinguishable from their nervousystem counterparts, but in a contrasting way both GABARs evokeynergistic [Ca2+]i rise, albeit acting through different mechanisms18].

Our studies in the mouse lens have revealed that the GABAignaling machinery is present from the earliest stages of lensevelopment in the epithelial sheet forming the lens placode16,17]. Later, the placode cells give rise to the ocular lens, aolarized organ, composed of a monolayer of cubical epitheliumt the anterior pole, central differentiated primary lens fibersnd differentiating secondary fiber cells. The latter arise fromontinuously proliferating epithelial cells, which undergo termi-al differentiation characterized by extensive fiber elongation,oncomitant loss of all cellular organelles and upregulation ofhe lens crystallins [19,20]. During development multiple GABAeceptor subunits and other components of the GABA signal-ng show characteristic spatio-temporal patterns of expressionhat overlap in the lens epithelium, but segregate to primary orecondary lens fibers along with the two GAD isoforms GAD65nd GAD67, respectively [16,17]. The two GABARs exhibit a clearmbryonic-to-postnatal shift in subunit composition characterizedy an upregulation of the fast synaptic type GABAAR and down-egulation of the GABABR2 receptor subunit during postnatal stages16]. The functionality of the lens-specific GABAR was demon-trated by GABA administration to acutely isolated newborn mouseenses, which resulted in the elevation of [Ca2+]i level in elongat-ng fiber cells at the equatorial region. We have proposed thathe responding cells may correspond to the population of chickennnular pad cells that also respond to mAchR (muscarinic acetyl-holine receptor) stimulation with a [Ca2+]i rise [16,21]. However,sing the above approach only superficial fibers could be visu-lized with a poor resolution at the cellular level (discussed in16]).

Here we have expanded these studies in primary mouse LECultures, which represent a faithful in vitro model system of lenspithelial cell differentiation [22,23] and have been successfullysed before to study different aspects of the lens Ca2+ signaling24–29]. We show that primary LEC cultures prepared from new-orn and one-month-old mouse lenses express GAD, GABA, GABAeceptors and transporters. Activation of both GABARs induced

rise in [Ca2+]i that was dependent on the presence of extra-ellular Ca2+ and could be blocked by specific antagonists inoth P0 and P30 mouse LEC cultures. This synergistic elevationf [Ca2+]i following activation of GABAAR and GABABR may benvolved in the maintenance of Ca2+ homeostasis, which is aey regulator of a variety of cellular processes involved in lensevelopment.

. Materials and methods

.1. Animal use

Mice of strain FVB/Ant [30] housed in the Medical Gene Techno-ogical Unit of the Institute of Experimental Medicine were used. Allxperiments with animals were conducted in compliance with NIHNIH Publication #85-23, 1985) and EC (86/609/EEC/2) guidelinesnd approved by in-house and national committees.

ium 50 (2011) 381– 392

2.2. Primary lens epithelial cell (LEC) culture andimmunocytochemistry

Lenses dissected from newborn (P0) mice were cleaned of ciliaryepithelium and retina with fine forceps and collected in ice-coldcell-culture grade Tyrode’ salts (Sigma–Aldrich, St. Louis, MO, USA).Subsequently the lenses were incubated in 0.02% EDTA solution in0.5 mM Dulbecco’s Phosphate Buffered Saline (Sigma–Aldrich) at37 ◦C for 10 min, transferred to pre-warmed Medium 199 (Invit-rogen, Carlsbad, CA, USA) supplemented with 10% fetal bovineserum (FBS, EuroClone, Pero-Milano, Italy) triturated 10 times andcentrifuged at 200 × g for 2 min. Cells were resuspended in freshMedium 199 + 10% FBS and plated at the equivalent of one lens/cm2

on glass coverslips pre-coated with BD MatrigelTM (BD Biosciences,NJ, USA; 0.4 mg/ml in PBS). In addition, primary LEC were alsocultured from lens capsules isolated from one-month-old (P30)mice and plated on matrigel-coated coverslips at 1 capsule/cm2

in Medium 199 + 10% FBS. Primary LEC cultures were grown at37 ◦C at 5%CO2/95%O2 with medium changed every two days.Two to three-week-old cultures containing single lens epithelialcells, epithelial monolayers and lentoids of different sizes wereused for immunocytochemistry and Ca2+ imaging. Cultures werefixed in 4% (w/v) paraformaldehyde (PFA) in PBS (phosphate-buffered saline) for 20 min at room temperature (RT) then washed3× 5 min in PBS. For GABA detection fixation was done in 4%PFA–0.1% glutaraldehyde–PBS for 30 min at RT. Cells were per-meabilized in 0.2% Triton X-100 (Sigma–Aldrich)–PBS for 5 min atRT and blocked in 0.5% blocking reagent (Roche Applied Sciences,Penzberg, Germany)–PBS for 1 h at RT. Primary antibodies wereapplied in 1% BSA–PBT (PBS + 0.05% TWEEN® 20, Sigma–Aldrich)overnight at 4 ◦C as follows: goat anti-�A-crystallin (Santa CruzBiotechnology, Santa Cruz, CA, USA, 1:500), rabbit anti-GABAA�3(1:1000) and rabbit anti-�B-crystallin (1:500) both from NovusBiologicals, Littleton, CO, USA, mouse anti-uvomorulin (E-cadherin,Sigma–Aldrich, 1:1000), mouse anti-N-cadherin (1:1000), mouseanti-GABABR2 (1:500) and mouse anti-Cx43 (1:1000) all fromBD Biosciences; rabbit anti-GABA (Sigma–Aldrich, 1:5000); rab-bit anti-GAD serum #6799 [17] at 1:500 or mouse anti-GAD67(EMD Millipore-Chemicon, Billerica, MA, USA) mouse anti-GAD65developed by David Gottlieb and obtained through the Develop-mental Studies Hybridoma Bank (Iowa City, IA, USA, 1:250); guineapig anti-VGAT (Calbiochem, Merck KGaA, Darmstadt, Germany;1:5000), guinea pig anti-mouse GAT-1 (gift from N. Nelson, Tel AvivUniversity), rabbit anti-GAT-3 (Abcam, Cambridge, UK, 1:1000).Coverslips were washed 3× 10 min in PBT and incubated withanti-rabbit, mouse, goat or guinea-pig secondary antibodies con-jugated either with Alexa Fluor® 594 or with Alexa Fluor® 488(Invitrogen, diluted 1:500 in 1% BSA-PBT) for 1 h at RT. The anti-GABA antibody was reacted first with biotinylated goat anti-rabbitIgG (Vector, Burlingame, CA, USA) for 90 min at RT in PBT–1%BSA, followed by Cy3-streptavidin (Jackson ImmunoResearch, WestGrove, PA, USA, 1:5000) in PBS for 1 h at RT. The actin cytoskele-ton was stained with rhodamin-phalloidin (Invitrogen, 1:100) for30 min at RT in PBS. Cells were washed 3× 10 min in PBT, 1× PBSand incubated for 5 min at RT with DAPI (Invitrogen) as recom-mended by the manufacturer. Coverslips were mounted in Mowiol(Calbiochem) containing DABCO (Sigma–Aldrich, 0.1 mg/ml) andexamined under Zeiss Axioscop-2 equipped with AxioCam HRc dig-ital camera using AxioVision 4.6 software or Zeiss LSM 510 META.

2.3. Ca2+ imaging studies

Primary LEC were loaded with 5 �M Fluo-4/AM in the pres-ence of 0.1% pluronic acid F-127 (both from Invitrogen) in growthmedium at 37 ◦C for 45 min, in a humidified atmosphere of 5%CO2/95%/O2. Subsequently the medium was changed and de-

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sterification of the dye was allowed to proceed for another5–30 min. The glass coverslips with attached cells were placed in aOC-R type submerged chamber (PeCon GmbH, Erbach, Germany).n each experiment cells were perfused for 2 min at 2 ml/min withrtificial aqueous humor (AAH) of the following composition (inM): NaCl, 130; KCl, 5; CaCl2, 1; MgCl2, 0.5; d-glucose, 5; NaHCO3,

; HEPES, 10; pH 7.25 to obtain a steady baseline. GABA (1 mM),uscimol (100 �M) or baclofen (20 �M), all from Sigma–Aldrichere applied for 2 min, followed by a 10 min perfusion with AAH. In

eneral, the visual field selected under phase contrast and fluores-ence microscopy for recordings included single cells, monolayersnd multilayered lentoid-like structures.

Whenever GABAR agonists in combination with antagonistsere used, cells were initially perfused with the antagonist: 20 �M

icuculline (Sigma–Aldrich; a GABAAR antagonist) for 2 min or0 �M of CGP55845 (Tocris Bioscience, Bristol, UK; a GABABRntagonist) for 2–4 min. This was followed by the co-application ofgonist and antagonist: 100 �M muscimol + 20 �M bicuculline or0 �M baclofen + 20 �M CGP55845, respectively, for 10 min withAH. Cells were then perfused for 2 min with AAH containing

he agonist alone (muscimol or baclofen, respectively). WheneverABA was applied as agonist cells were initially perfused withAH (with or without Ca2+) until steady baseline was achieved,

hen 50 mM GABA drop was added (to final concentration 1 mM)ollowed by AAH (4 min), AAH + bicuculline (2 min), GABA drop,AH + bicuculline + CGP55845 (7 min), GABA drop, AAH (10 min).ABA (50 mM) and KCl (1 M) drops were added to verify thexcitability of the cells at the end of the recording. Cultures werebserved under a Zeiss LSM 510 META (Carl Zeiss) laser scan-ing confocal microscope and scanned every second. All settingsf the laser, optical filter and microscope as well as data acquisi-ion were controlled by LSM 510 META software. Excitation was at88 nm and emitted light was read at 515 nm. Fluorescence inten-ity within regions of interest covering a single cell was followed

ver time and expressed in arbitrary units. Data are presented asepresentative single experiments or the mean ± S.E.M. of pooledata from a number of experiments, as indicated. Statistical signifi-ance was estimated by Mann–Whitney unpaired test, with p < 0.05

ig. 1. Features of primary LEC cultures prepared from newborn mouse lenses. Images ofith large intact nuclei and strong staining for actin stress fibers (arrowheads in A, E and

nd F), flat sheets of tightly packed cells with small pyknotic nuclei (empty asterisks in Bmall pyknotic nuclei (B–D, F and G, solid asterisks). At later stages of lentoid differentianuclear bulges (solid asterisks in D). Cells were visualized by phase contrast (C), DIC micrhodamin-phalloidin (E–G). Cell nuclei were counterstained with DAPI (blue color in A, Bnd G) using AxioVersion 4.6 software. Scale bars: 50 �m (A, D and E); 100 �m (B–C, F, ans referred to the web version of the article.)

ium 50 (2011) 381– 392 383

considered statistically significant compared to control if not statedotherwise.

3. Results

3.1. Primary LEC cultures – features of the in vitro differentiationof lens epithelial cells

Primary LEC cultures were derived primarily from newbornmouse lenses, as they express high levels of GABA signaling com-ponents [17] as well as provide an ample supply of easy-to-dissectepithelial and fiber cells. In some experiments primary LEC werederived from P21 to P30 (postnatal days 21–30) mouse lens cap-sules containing the attached lens epithelial cells. Under the cultureconditions used here, the lens epithelial cells continuously prolifer-ate, migrate and subsequently differentiate. After two weeks in vitrothe primary LEC cultures were comprised of single epithelial cellsof flattened irregular shape (Fig. 1A and E), epithelial monolayers(Fig. 1B and C) and randomly distributed lentoids–multilayered cellaggregates of various sizes and shapes that appear to bulge from thesurrounding epithelial cells (Fig. 1B–D and G). Based on morphol-ogy and expression profiling lentoids are considered an equivalentof aggregates of elongating fiber cells [23,31–33]. The transitionbetween epithelial cells, monolayers and lentoids involves a grad-ual change in cell compaction and nuclear morphology that couldbe traced by staining of the actin cytoskeleton and nuclear DAPIstaining (Fig. 1E–G). Single cells are clearly distinguished by theirlarge nuclei and the presence of stress fibers – bundles of actinfilaments (Fig. 1E). Cells with large nuclei initially form monolay-ers with strong staining of cortical actin fibers (Fig. 1F). Furthercompaction of cells with concomitant reduction of nuclear sizeand chromatin condensation produces compact monolayers andmultilayered bulging lentoids that could be distinguished morpho-logically (Fig. 1B, C and F) and by staining with cellular markers

(see below). Lentoids may contain transluscent anuclear parts cor-responding to terminally differentiated fiber cells (Fig. 1D). Cellborders of cells forming lentoids could not be distinguished underphase contrast.

LEC cultured grown for two weeks in vitro. These cultures comprise epithelial cells G), flat cell monolayers with large nuclei and cortical actin filaments (arrows in A, C and F) and bulging multilayered lentoids made of highly compacted cells withtion cells around lentoids become sparse with some lentoid parts developing intooscopy (A, B and D) or by fluorescence following staining of filamentous actin with, D and E–G). Fluorescent images were superimposed on the DIC images in (A, B, Dd G). (For interpretation of the references to color in this figure legend, the reader

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In addition to morphological criteria, we also used cellulararkers to distinguish between epithelial and differentiated fiber

ells (Fig. 2). The molecular chaperone �A-crystallin, one of theost abundant proteins of the lens epithelium and fibers and

he small �B-crystallin, more abundant in fiber cells [34] wereetected at high levels in multilayered lentoids and were onlyeakly expressed in epithelial cells and monolayers (Fig. 2A, D,

and K). Lentoids, but not epithelial cells stained strongly for the

ig. 2. Newborn mouse primary LEC cultures express different markers for lens epitheliaells were used: �A- and �B-crystallin, N- and E-cadherin (N-cad and E-cad, respectively)isualization was by indirect immunofluorescence using Alexa 488 (A, D, G and K) or Alexaith DAPI (A–C, F, I, J and M). (A) The fluorescent image is superimposed on the phase-co

n A) as well as larger multilayered lentoids (A) staining also for N-cad (B). N-cad is detC, arrowhead). (D) �B-crystallin strongly stains lentoids (solid asterisk), also displaying aabeled with a �B-crystallin-specific antibody (arrowheads in D and F) and negative for

sterisks in G and H) stains weakly for �B-crystallin (G) and strongly for the epithelial mn a complementary manner. (K–M) Flat region (K and L – empty asterisks) and bulgingppositely for Cx43 and �B-crystallin, respectively. Scale bars: 50 �m.

ium 50 (2011) 381– 392

adhesion protein N (neural) cadherin typically expressed on thebroad face of fiber cells (Fig. 2B, C and E), while E-cad (epithelial-cadherin) only stained flat, presumably epithelial, but not bulginglentoid regions (Fig. 2H and J and [35]) distinguished by the promi-

nent �B-crystallin staining (Fig. 2G and J). Similarly, Cx43 (connexin43), a gap junction channel protein known to be expressed abun-dantly in lens epithelium [36] was localized to flat monolayersincluding those found at the periphery of �B-crystallin stained

l and differentiating fiber cells. Antibodies staining differentially epithelial or fiber and connexin-43 (Cx43, a gap juction protein channel specific for lens epithelium).

594 (B, C, E, H and L) conjugated secondary antibodies. Nuclei were counterstainedntrast image. �A-crystallin is strongly expressed in small cellular aggregates (insetected in multicellular lentoids (C, arrows) while epithelial cells remain unstained

prominent membrane staining for N-cad (arrows in E). Epithelial cells are weaklyN-cad (E and F). (K–L) Flat monolayer area at the periphery of the lentoid (empty

arker E-cad (H), while the multilayered regions (solid asterisk in G and J) stains regions (solid asterisks in K and M) of a multicellular lentoid-like structure stain

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ultilayered lentoid-like regions (Fig. 2K–M). DAPI staining ofuclei clearly demonstrated that compact monolayers composedf closely packed cells expressing epithelial markers and bulgingentoids both possess similar sized “pyknotic” nuclei (Fig. 2I, J, Mnd data not shown).

.2. Expression of GABA signaling components in primary LECultures

Similar to intact lenses [16,17] primary LEC cultures preparedrom newborn mouse lenses expressed both forms of the GABA-ynthesizing enzyme GAD (i.e. GAD65 and GAD67), as well asesicular (VGAT) and membrane GABA transporters (Fig. 3A–I). Ineneral, the bulging multilayered lentoids clearly identifiable underhase contrast (Fig. 3A, D and G) showed the strongest expres-ion for GABA signaling components, while epithelial cells wereess prominently stained (Fig. 3B, C, E, F, H and I). GAD65 and theesicular GABA transporter VGAT showed a uniform distributionithin multilayered lentoid-like aggregates (Fig. 3B and C), while

he membrane GABA transporter GAT-1 and especially GAD67 werenriched in the most differentiated lentoid thickenings devoid ofuclear DAPI staining (Fig. 3D–F) that also stained strongly forABA (see below). GAT-3 was found in virtually all types of cells:

n both single or monolayer epithelial cells as well as multilayerentoid-line structures (Fig. 3G–I and data not shown). GAD67

as also expressed in a subpopulation of previously describedmall spindle-like cells of epithelial origin (data not shown and31]).

Prominent labeling of the GABA-binding GABAA�3 subunit wasetected over the cell borders of flattened (putative monolayer)egions found at the periphery of bulging lentoid-like structuresnd was stronger in the multilayer region (Fig. 4A and C). GABABR2

ig. 3. Primary LEC cultures from newborn mouse lenses express different members of the

(C); GAD67-rabbit #6799 (F) or mouse GAD67 (I); VGAT (B); GAT-1 (E) and GAT-3 (H). (n (B, C), (E, F) and (H, I), respectively. (B, F and H) Nuclei were counterstained for DAPI.o-localization of GAT-1 and GAD67: The flat area of the lentoid-like structure (empty astAD67 (F), while the bulging multilayered regions (E, solid asterisks) are strongly labeled

entoid structure shown in (G); the surrounding flat monolayer is weakly labeled. Scale b

ium 50 (2011) 381– 392 385

staining displayed a uniform staining over the entire lentoid sur-face (Fig. 4G and I). An almost identical staining pattern of thetwo receptors was observed in epithelial monolayers with typi-cal large nuclei (Fig. 4D–F and J–L). The staining product was of apunctate appearance outlining cell borders at the periphery of themuticellular assembly (Fig. 4D, F, J and L) and highly accumulatedat presumptive intercellular interfaces and underlying perinuclearspace of internally positioned cells, as demonstrated in confocalimages (Fig. 4D–F and J–L). Only a subpopulation of single cellsstained for GABAR (data not shown).

In conclusion, some single epithelial cells, most if not all cellsof epithelial monolayers and multilayered lentoid-like structuresexpressed all components required for a functional GABA signalingpathway.

3.3. Primary newborn mouse LEC cultures produce GABA

Using a highly specific antibody, we could detect strong GABAstaining in newborn mouse primary LEC cultures. Multilayeredlentoid structures showed high accumulation of GABA especiallyin their more differentiated anuclear regions, but also in theadjacent flatter regions (Fig. 5A and A′-boxed area in A), whilesingle cells remained unstained (Fig. 5C and C′-boxed area inC; fluorescence in C′ is digitally enhanced to demonstrate thepresence of cells that are NOT stained). This expression gradientwas typically found in the vicinity of all lentoid-like structurescontaining epithelial cells at different stages of differentiation

condensation normally accompanying fiber maturation (Fig. 5B,B′-boxed area in B and [37]). Rare neurons could be also identi-fied by their typical morphology and high content of GABA (Fig. 5Aand A′).

GABA signaling pathway. The following specific antibodies were used: GAD65–GAD-A, D and G) Phase contrast images corresponding to the fluorescent images shown

(A–C) VGAT co-localizes with GAD65 in bulging lentoid-like structures. (D and E)erisks in D–F) exhibits a weak membrane staining for GAT-1 (E) and no staining for

for both (E and F). GAT-3 (H) and GAD67 (I) antibodies both stain the multilayeredar: 100 �m.

386 M. Schwirtlich et al. / Cell Calcium 50 (2011) 381– 392

Fig. 4. Expression of GABAA and GABAB receptors in primary LEC cultures from newborn mice. Primary LEC cultures were stained with rabbit anti-GABAA�3 (A–F) or mouseanti-GABABR2 (G–L) antibodies and visualized by epifluorescence (A–C and G–I) or laser scanning microscopy (D–F and J–L). Nuclei were counterstained with DAPI (B, C,E, F, H, I, K and L). Staining shows patchy vesicular appearance outlining the presumed cellular membranes at the lentoid periphery (arrows in A, D, F and J). Considerablee n D anG

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nrichment is observed at intercellular interfaces of LEC monolayers (arrowheads i–I).

.4. GABA receptor-mediated Ca2+ responses

The functional assembly and activity of GABAA and GABABeceptors expressed by primary LEC was studied by measuringCa2+]i changes in cultures loaded with Fluo-4/AM upon applica-ion of selective agonists/antagonists. Most of the cells of the visualeld selected for recordings were successfully loaded, includingpithelial cells, either singly or in monolayers and multicellularentoid-like aggregates distinguished under phase contrast anduorescence (Supplementary Fig. 1; Supplementary movies 1–6nd data not shown). Larger lentoids, likely to represent a latertage of lens cell differentiation [31] remained unlabeled. Theabeled cells did not show spontaneous Ca2+ transients in AAHolution.

.4.1. Modulation of [Ca 2+]i levels by selective GABAAR orABABR ligands

The GABAAR agonist muscimol (100 �M) and GABABR agonistaclofen (20 �M), applied in AAH induced a simultaneous increase

f fluorescence (Fig. 6A1–4 and B1–2) mostly in small lentoid-liketructures (Fig. 6A1 and B1; Supplementary movie 1), but also inpithelial cells in flat monolayers; Fig. 6A3), which were identifiedn initially obtained phase contrast images. The average number of

d J) and lentoid surfaces (A and G). Bar 20 �m in (D–F and J–L); 50 �m in (A–C and

cells per observation field responding to muscimol was 4.2 ± 1.02,p < 0.05, n = 10 fields (Fig. 6C) with a mean amplitude of fluores-cence change �F = 46.5 ± 5, p < 0.05. In comparison, Ca2+ transientswere almost fully blocked in the presence of the GABAAR antago-nist bicuculline (cell number 0.5 ± 0.3, n = 6, p < 0.05; amplitude offluorescence change �F = 19.5 ± 5.84; Fig. 6C) demonstrating thespecificity of the response.

Baclofen induced relatively small Ca2+ transients in a por-tion of single cells (data not shown), which could be explainedby their low number of receptors or the relative insensitivityof the latter to baclofen. The response to baclofen (average cellnumber 3.4 ± 0.5, n = 8 fields, p < 0.05; mean amplitude change�F = 37.0 ± 14.5, n = 25, p < 0.05; Fig. 6C) was typically completelyblocked in the presence of the GABABR antagonist CGP55845(0.3 ± 0.3; n = 3 fields, p < 0.05; mean amplitude change �F = 93.6,n = 1; Fig. 6C). The large-amplitude transients exhibited by rarecells responding to baclofen in the presence of CGP55845 mayresult from release of Ca2+ from the intracellular Ca2+ stores [18,38].Continuous application of baclofen sometimes induced persistent

oscillations and a secondary wave of signal propagation within themultilayered cell aggregates and adjacent epithelial monolayers(Fig. 6B1; Supplementary movie 2), which were sustained evenafter complete washout of baclofen (Fig. 6B2).

M. Schwirtlich et al. / Cell Calcium 50 (2011) 381– 392 387

Fig. 5. GABA detection in primary LEC cultures derived from newborn mice with anti-GABA antibody. (A, A′ , C and C′) Visualization was with streptavidin-Cy3. (B, B′) Nucleiwere revealed by DAPI staining. Boxed regions in A, B and C are enlarged in A′ , B′ and C′ , respectively. A large multicellular structure (A–C) is highly positive for GABA inits bulging anucleated lentoid part (solid asterisk in A, A′ and C) and less pronouncedly stained at the flat, presumably epithelial monolayer regions (empty asterisk in A′).Rare neurons express similar levels of GABA (arrows in A, A′). Cells outside the multicellular structure with large (C′ arrowheads) or partially disintegrated (numbered 1–3in B′) nuclei remained unstained (B, B′ , C and C′; boxed area in C devoid of staining was digitally enhanced in C′ to reveal the presence of unstained cells in this area). Cellsbelonging to the lentoid structure with strongly stained small nuclei (#4 in B′) are GABA+ (yellow asterisk in B and C). (B′) Circled nuclei of different shapes and level ofchromatin compaction (numbered 1–4) correlating with different stages of lens epithelial-to-fiber cell differentiation proceeding from less differentiated (#1) [5] to moredifferentiated (# 4) stages [37]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 6. Changes of [Ca2+]i in primary newborn mouse LEC cultures in response to muscimol (a GABAAR agonist) and baclofen (a GABABR agonist). Cultures grown for twoweeks in vitro and loaded with Fluo-4/AM were used. (A1 and A2) Perfusion was with AAH buffer followed by AAH containing 100 �M muscimol. (A3 and A4) Perfusion wasdone initially with AAH containing 100 �M muscimol in the presence of 20 �M of the GABAAR antagonist bicuculline then by 100 �M muscimol alone, followed by 1 mMGABA and a drop addition of 1 mM KCl (A4). Note the complete blockade of muscimol action by bicuculline (A3 and A4). (B1 and B2) representative recordings of baclofen-evoked fluorescence changes. Cells were initially perfused with AAH containing 20 �M CGP55845 (CGP, a GABABR antagonist) then AAH containing 20 �M CGP55845 + 20 �Mbaclofen followed by baclofen alone. Baclofen induces a simultaneous rise of fluorescence (*) in closely apposed cell stacks followed by oscillations (**). Note that CGP55845 at20 �M completely blocks the baclofen-evoked Ca2+ responses (B1, B2). (A1, A3 and B1) Cells manifesting Ca2+ transients are circled on the confocal images taken at differenttime points of the recording using fluorescent images taken at the peak of fluorescence. (A2, A4, and B2): traces of fluorescence intensity changes in arbitrary units (F) overtime in cells circled on the corresponding images shown in (A1, A3 and B1). (C) Bar chart illustrating the average number of cells (upper panel) and amplitude of fluorescencechanges (�F, lower panel) responding to muscimol (Musc), muscimol in the presence of bicuculline (Musc + Bic), baclofen (Bacl) and baclofen in the presence of CGP55845(Bacl + CGP). Data represent the means ± S.E.M.

388 M. Schwirtlich et al. / Cell Calcium 50 (2011) 381– 392

Fig. 7. GABA induces [Ca2+]i rise in primary LEC cultures from newborn mouse lenses through synergistic activation of GABAAR and GABABR. (A1–A6) Representativerecordings of GABA-evoked changes in fluorescence of Fluo-4/AM loaded LEC cultures grown in vitro for two weeks. Perfusion was initially with AAH followed by AAH withbicuculline (Bic; 20 �M) and finally with AAH containing 20 �M bicuculline + 20 �M CGP55845 (Bic + CGP). (A1–A6) Drop application of GABA (final concentration 1 mM)induced a similar Ca2+ rise in cells perfused with AAH or AAH + bicuculline, but not in AAH containing both GABAR antagonists bicuculline and CGP (Bic + CGP). (A1, A3 and A5):Cells manifesting [Ca2+]i transients are circled on the confocal images taken at different time points of the recording. (A2, A4 and A6) Traces of fluorescence intensity changes(F, in arbitrary units) measured over time in cells circled in the corresponding images in (A1, A3 and A5). (B) GABA-evoked responses in cells perfused in Ca2+-free AAH buffer( ucullin 2) resC the m

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0 Ca2+), followed by AAH (0 Ca2+) containing either 20 �M bicuculline or 20 �M bicumber of cells (C1) and amplitude of fluorescence change �F in arbitrary units (CaCl2 (closed bars) or AAH buffer without CaCl2 (0 Ca2+, open bars). Data represent

.4.2. Responses to GABA in newborn mouse LEC culturesIn general, both cells of larger lentoid surfaces and adjacent

pithelial cells of primary newborn mouse LEC responded to GABAith a quick rise in fluorescence (Fig. 7A1–4; Supplementaryovies 3–5). The average cell number was 5.0 ± 1.4; p < 0.05;

= 58.9 ± 9.5, p < 0.05, n = 10 fields (Fig. 7C1–2). Addition ofABA in the presence of the GABAAR antagonist bicucullineroduced similar responses compared to GABA added in AAHFig. 7A1–2 and A5–6; Supplementary movie 6) with regard to

oth cell number and amplitude of reaction (average cell num-er 5.6 ± 2.6; �F = 68.4 ± 10.6, p > 0.05 for differences comparedo GABA responses, n = 3 fields; Fig. 7C1–2). Some cells clearlyhowed a delayed response first at this stage, as shown in

ne (Bic) and 20 �M CGP55845 (CGP). (C1 and C2) Bar charts illustrating the averageponding to GABA. Cultures were perfused with either AAH buffer containing 1 mMean ± S.E.M.

Fig. 7A5–6. Blockade of both GABAR by applying bicuculline andCGP55845 generally suppressed all GABA-evoked Ca2+ responses(Fig. 7A1–6) and only occasional single Ca2+ transients wererecorded under these conditions (average cell number 0.8 ± 0.6,n = 6 fields, p < 0.05 for calculated differences compared to GABAresponses; �F = 97.3 ± 56.6, n = 6 fields; Fig. 7C1–2).

While wave propagation of the signal was clearly observed(Supplementary movies 3–5) it was rarely followed by oscilla-tions. Drop application of KCl induced a simultaneous Ca2+ rise

in most, if not all cells of a visual field (Supplementary movie3; Supplementary Fig. 1), which demonstrated that the late Ca2+

transients and waves do not result from a slow diffusion ofGABA.

M. Schwirtlich et al. / Cell Calcium 50 (2011) 381– 392 389

Fig. 8. GABA induces Ca2+ transients through activation of both GABAA and GABAB receptors in LEC cultures derived from lens capsules of one-month-old (P30) mice:similarities with newborn (P0) mouse LEC cultures. LEC cultures grown for 2–3 weeks in vitro were used. (A1 and A2) Bar charts illustrating the average cell number (A2)and the amplitude of fluorescence change expressed in arbitrary units �F (A2) of primary LEC cultures derived from newborn (solid black bars) or P30 (open bars) micedisplaying Ca2+ transients in response to GABA. (B) Traces of fluorescence intensity (F) changes induced by GABA over time recorded in individual cells of P30 mouse LECc 0 �M;G culturw BA in

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ulture. Initial perfusion was with AAH followed by AAH containing bicuculline (2ABA as added in a drop (1 mM final concentration) induced Ca2+ transients in LECith AAH or AAH containing bicuculline, but only newborn cultures respond to GA

Using Ca2+-free buffer we still observed Ca2+ transients inesponse to GABA (Fig. 7B), although with considerably reducedverage cell number (2.8 ± 0.8, n = 4, p < 0.05) and mean amplitudef the responses (�F = 33.5 ± 16.4, n = 4 fields, p > 0.05; Fig. 7C1–2)ompared to GABA responses in the presence of Ca2+. The num-er of cells reacting to GABA with Ca2+ transients in the presencef bicuculline or bicuculline + CGP55845 in Ca2+-free buffer waseduced even further (GABA + bicuculline: cell number: 1 ± 0, n = 3elds; �F = 27.0 ± 4.6, n = 3 fields; GABA + bicuculline + CGP55845:ell number 1 ± 0.6, n = 3 fields; �F = 30.4 ± 5.0, n = 3 fields).

.4.3. Responses to GABA in one-month-old mouse LEC culturesWe also studied the GABA-induced Ca2+ signaling in LEC cul-

ures derived from one-month-old (P30) mouse lens capsulesith attached epithelium (Fig. 8A1–2 and B). As described forewborn mouse LEC, the P30 mouse LEC cultures contain sin-le epithelial cells, epithelial sheets and lentoids that derive fromens epithelial cells migrating out of the lens capsules (data nothown).

Similar to P0, the P30 mouse LEC responded to GABA with Ca2+

ransients (4 and 8 cells, n = 2 fields compared to LEC from P0ouse lenses: average cell number 5.2 ± 0.9, n = 26 fields, respec-

ively; Fig. 8A1). Furthermore, the responses observed for GABAdded in the presence of bicuculline did not differ significantlyetween the two types of culture (P0: GABA + bic: 5.7 ± 2.2, n = 7elds vs. P30: 8.0 ± 2.2 cells, n = 3 fields). However, the mean ampli-ude of Ca2+ transients in response to GABA in P30 mouse LECultures differed compared to P0 cultures (P30: �F = 31.8 ± 5.7,

= 2 vs. P0: �F = 58.3 ± 3.9, n = 10 fields, respectively; Fig. 8A2).his difference between P0 and P30 mouse LEC cultures waseduced when GABA was applied in the presence of bicucullineFig. 8A2; P0: �F = 69.4 ± 10.6, n = 3 fields; P30: �F = 68.4 ± 10.8,

= 2 fields, p > 0.05). Blockade of both GABAAR and GABABR withicuculline + CGP55845 abolished all responses to GABA in P30ouse LEC cultures (0, n = 2). In comparison, low numbers of P0ouse LEC still produced Ca2+ transients (0.8 ± 0.6, p < 0.05 n = 6;

ig. 8A1) with higher amplitude (97 ± 56.0, n = 2; Fig. 8A2) underdentical conditions.

. Discussion

.1. Primary LEC cultures as a model system for studying GABA

ignaling during development

GABA signaling has been drawing continuous attention due tots widespread expression and versatile role in variety of neuronal

Bic) and AAH containing both bicuculline and CGP55845 (20 �M each; Bic + CGP).es derived from either newborn or one-month-old mouse lenses during perfusion

the presence of both bicuculline and CGP55845 (A1, A2 and B).

and non-neuronal cell types. Its hyperpolarizing inhibitory effectmediated by the two classes of GABARs is indispensable for thefunctionality of neuronal circuitry and control of excitability ofthe vertebrate CNS. In recent years it has become apparent thatthe GABAARs exert a predominantly Ca2+-elevating activity in neu-ronal progenitors prior to synaptic engagement [5], whereas muchless is known about the effect of GABABR, the possible interactionsbetween the two receptors and downstream effects in differenti-ating neural precursors due to the complexity of the developingnervous system. We chose to use primary LEC cultures derivedfrom postnatal mouse lenses since they recapitulate the develop-ment of the mammalian lens [23], which expresses high levels ofGABA signaling pathway components [16,17,39,40]. In addition,primary LEC cultures have been extensively used before for highresolution Ca2+-imaging [24,29,41–43]. Two weeks after platingthe mouse LEC cultures from both newborn and one-month-oldlenses contain cells at virtually all stages of differentiation, whichcan be easily identified by size, nuclear shape, chromatin con-densation and staining for different cellular markers like �A- and�B-crystallin (epithelial cells and lentoids), Cx43 and E-cadherin(epithelial monolayers) or N-cadherin (lentoids). The transitionbetween different differentiated stages, however, is gradual andcells at intermediate stages may share common morphological andphysiological features.

The highest expression of GABA receptors and transporters wasdetected in multilayer lentoid structures composed of nucleatedcells corresponding to the elongating equatorial lens fibers [24],while epithelial monolayers and a population of single epithe-lial cells showed lower level of expression. Interestingly, GADand GABA were highly enriched in anucleated lentoid regions,corresponding to terminally differentiated lens fiber cells. Fur-thermore, GABA and VGAT were never detected in single cellsindicating GABA synthesis and VGAT-coupled release increasesin more differentiated cells. This expression pattern correspondswell to that described for the intact mouse lens [17] and clearlydemonstrates that primary LEC cultures faithfully recapitulatethe GABAergic signaling expression pattern during lens develop-ment.

4.2. Both GABAAR and GABABR induce synergistic elevation of[Ca2+]i in primary LEC cultures from newborn or one-month-oldmouse lenses

This is the first ever study dealing with the GABA-evoked Ca2+

signaling in primary lens epithelial cultures and one of few that hasaddressed the role of both GABARs in conveying the GABA signal

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o downstream Ca2+ signaling. Therefore, here we have addressedasic issues like the nature of responses of primary LEC cultureserived from newborn (P0) or P30 mice to specific GABA recep-or agonists or GABA, and sensitivity of the responses to selectiventagonists in the presence/absence of extracellular Ca2+. Since theast majority if not all cells exhibiting Ca2+ transients throughouthese experiments were nucleated cells belonging to monolayers ormall lentoid-like structures that did not differ substantially in theiresponses, the recorded fluorescent intensity values were pooledor statistical analysis. Anucleated lentoid cells presumably alsoacking intracellular Ca2+ stores [24] did not load under conditionsescribed here and were excluded from this analysis.

.2.1. Studies with GABAR-selective agonistsBoth muscimol (GABAAR agonist) and baclofen (GABABR ago-

ist) evoked Ca2+ transients in all responding cells that could belocked by the selective antagonists bicuculline and CGP55845,espectively. While the GABAAR-mediated Ca2+ rise in primary LECs entirely consistent with the channel properties in immature neu-ons [1,5,44] and/or non-neuronal cells [3,45], the GABABR-evokeda2+ transients were unusual, as GABABR is widely considered toe inhibitory in both embryos and in the adult [3,44,46,47]. Suchransients were also observed in the rare occasionally survivingretinal) neurons in the LEC cultures indicating that this propertyf GABABR might be a much wider phenomenon during develop-ent. We have also found a greater variability in the P0 mouse LEC

esponses to baclofen compared to muscimol. This phenomenonas been previously described for hippocampal astroglia and ishought to be an intrinsic feature of the GABABR [11]. This vari-bility may arise from the superimposed inhibitory and excitatoryffects of GABABR existing in different cells as described for mESells [18]. However, the inhibitory component is probably smallompared to the excitatory one, since application of the antago-ist CGP55845 alone usually had no effect, but occasionally evoked

ong-lasting small-amplitude oscillations, the mechanism of whichs obscure at present.

.2.2. GABA responsesResponses to GABA alone or in the presence of GABAR antag-

nists revealed the nature of the interactions between the twoeceptors. The elevation of the intracellular Ca2+ level by the twoABAR in Ca2+-containing buffers was not additive since GABAvoked similar responses alone or in the presence of bicuculline.his indicates that convergent intracellular Ca2+ mobilizationechanisms are activated by the two receptors as has been previ-

usly shown for histamine1R and histamine2R in splenocytes [48],r P2XR and P2YR in retinal cells [49]. Another explanation maye a GABAAR-independent potentiation effect of bicuculline on thea2+ transients described previously in rat cerebellar granule cells50]. Finally, this effect may be caused by the increased GABABRctivity due an elevated level of ambient GABA [51] followinglockade of GABAAR. Even application of both GABAR antagonistsannot suppress all GABA-evoked Ca2+ transients, which may resultrom a non-canonical GABA-evoked Ca2+ signaling, like the recentlyescribed GABA transporter (GAT)-mediated Ca2+ rise in olfactoryulb astrocytes [52]. A full blockade of the two GABAR was effec-ively achieved in P30 mouse LEC cultures (see below).

Compared to P0, Ca2+ responses to GABA in LEC cultures derivedrom P30 lenses were characterized by smaller amplitudes andreater sensitivity to CGP55845 and bicuculline. These changes areartly attributable to the switch of expression to a predominantly

ast synaptic type GABAAR since GABABR or other GABA signalingomponents do not change significantly between the two stages16]. Interestingly, this switch is not paralleled by a reversal of theeceptor properties from excitatory to inhibitory as would be pre-

ium 50 (2011) 381– 392

dicted from studies in the CNS. It remains to be seen whether anexcitatory-inhibitory shift of GABAAR-evoked responses occurs atlater developmental stages.

4.2.3. Cell-to-cell wave propagation and oscillationsFollowing prolonged applications of baclofen or GABA (but not

muscimol) we have also been able to record the synchronous reac-tion of cell groups followed by wave-propagation of the signal,delayed Ca2+ transients and, in the case of baclofen long-lastingoscillations. The cell-to-cell signal propagation is reminiscent ofresponses involving mobilization of intracellular Ca2+ in lens cul-tures mediated by ATP receptors and strongly suggests that it maybe propagated through intercellular gap junctions and/or puriner-gic receptors [25,28,29,41]. Whereas GABA, GABAA or GABABreceptor agonists evoked Ca2+ increases with similar amplitudes,only the activation of GABABR by baclofen lead to sustained [Ca2+]iresponses, similarly to cerebellar mGluRs where both mGluR1 andmGluR5 induced [Ca2+]i mobilization but only the activation ofmGluR5 results in intracellular Ca2+ oscillation [53].

4.2.4. Dependence of GABA-evoked Ca2+ responses onextracellular Ca2+

The primary mouse LEC did not elicit responses to baclofen inthe absence of extracellular Ca2+ (data not shown), but reactedto GABA under the same conditions, although the number andamplitudes of responses decreased compared to Ca2+-containingbuffers. The GABA responses observed in Ca2+-free buffers are prob-ably due to release of Ca2+ from the intracellular Ca2+ stores. Thedecreased responsiveness in Ca2+-free solution and the robust Ca2+

transients evoked by KCl application in AAH implicate VGCCs asmediators of Ca2+ influx. The presence of L-type VGCCs in primaryLEC has previously been documented [43]. Bicuculline reduced sig-nificantly the responses to GABA in Ca2+-free conditions, in contrastto Ca2+-containing buffers, where it did not exert a significanteffect (Section 4.2.2) highlighting the differential (extracellular)Ca2+ sensitivity of GABAAR and GABABR-activated Ca2+-mobilizingmechanisms.

Following extensive washes with Ca2+-free buffer lens epithe-lial and fiber cells reacted to addition of 1mM Ca2+ with a sustainedand robust capacitive Ca2+ rise (data not shown), which is usuallyattributed to Ca2+ influx through the store-operated channel (SOC)mediating the refilling of the internal stores [24,25,41,43]. There-fore, the GABA-induced changes in [Ca2+]i in lens epithelial andfiber cells of primary mouse LEC cultures are mediated throughmultiple mechanisms employing both extracellular Ca2+ influx andCa2+ efflux from (IP3-sensitive) internal stores, which is also con-sistent with expression data.

In comparison, dependence of the Ca2+ rise exclusively onintracellular (mainly IP3-dependent) Ca2+ store release coupled tostore-operated Ca2+ influx has been described for primary humanlens cells in culture after activation of G-protein coupled recep-tors [18,41] and also in astroglia and mouse ES cells after GABABRactivation [11,18]. In this respect, GABABR of primary LEC culturesderived from P0 or P30 mouse lenses show similar sensitivity toexternally added Ca2+ as CNS neurons [54].

4.3. Possible role of GABA signaling in the developing lens andimplications for developing CNS and lens pathologies

GABA is a novel neurotransmitter for the lens in addition toacetylcholine [55], which is known to increase [Ca2+]i rise in lensepithelial and fiber cells through muscarinic receptors [21,55]. The

lens expresses a fully functional GABA signaling machinery whichis remarkably similar to that of the developing nervous system asdemonstrated by expression profiling [16,17]. Hence, it affords anideal model system to study the mechanisms of GABA-induced Ca2+

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ignaling, the interplay between the two GABA receptors, possi-le involvement of other GABA signaling components and couplingo downstream second messenger pathways. Finally, although theens cells possess a remarkably high resting [Ca2+]i [24] a sustaineda2+ overload is the prime reason for lens cataract [43], underliesany pathological conditions and triggers apoptosis [56,57]. LEC

ultures of human origin could be potentially useful in studies onhe interplay between GABA, Ach and Ca2+ signaling in patholog-cal conditions associated with cataract, like IDDM or Alzheimer51,58].

onflict of interest

The authors of this paper declare no conflict of interest for theast three years with regard to the content of this paper.

cknowledgements

We acknowledged the excellent work of the members of theene Technology Unit, Institute of Experimental Medicine. Thisork was partially supported by Hungarian National Grant Agency

OTKA) grant # T038098 (to Z.K.).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.ceca.2011.07.002.

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