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J Bioenerg Biomembr (2009) 41:85–94DOI 10.1007/s10863-009-9199-5 Purinergic signalling in rat GFSHR-17 granulosa cells:an in vitro model of granulosa cells in maturing follicles Willem Bintig & Judith Baumgart & Wilhelm J. Walter &Alexander Heisterkamp & Holger Lubatschowski &Anaclet Ngezahayo Received: 18 October 2008 / Accepted: 21 January 2009 / Published online: 4 February 2009 # Springer Science + Business Media, LLC 2009 Abstract Purinergic signalling in rat GFSHR-17 granulosa Keywords Granulosa cells . Purinergic receptors .
cells was characterised by Ca2+-imaging and perforated Cl− channels . Follicle maturation . Perforated patch-clamp .
patch-clamp. We observed a resting intracellular Ca2+- concentration ([Ca2+]i) of 100 nM and a membranepotential of −40 mV. This was consistent with high K+−and Cl− permeability and a high intracellular Cl− concen- tration of 40 mM. Application of ATP for 5–15 s every 3min induced repeated [Ca2+]i increases and a 30 mV Granulosa cells form a monolayer that surrounds oocytes in hyperpolarization. The phospholipase C inhibitor U73122 primary follicles. Upon external stimulation, granulosa cells or the IP3-receptor antagonist 2-aminoethoethyl diphenyl begin to proliferate simultaneously with an increase in borate suppressed ATP responses. Further biochemical and oocyte volume. Proliferating granulosa cells express FSH pharmacological experiments revealed that ATP responses receptors as this hormone begins to be secreted by the were related to stimulation of P2Y2 and P2Y4 receptors and pituitary gland. The proliferation of the granulosa cells and that the [Ca2+]i increase was a prerequisite for hyperpolar- the volume increase in the oocyte correlate with the ization. Inhibitors of Ca2+-activated channels or K+ channels reorganization of the follicle. An antral cavity is formed, did not affect the ATP-evoked responses. Conversely, which contains a fluid consisting of water, ions and many inhibitors of Cl− channels hyperpolarized cells to −70 mV other components such as peptides. The antral fluid is and suppressed further ATP-evoked hyperpolarization. We secreted by the granulosa cells via a mechanism that remains propose that P2Y2 and P2Y4 receptors in granulosa cells to be elucidated. The granulosa cells of the maturing follicles modulate Cl− permeability by regulating Ca2+-release.
express receptors for pituitary hormones and other physio-logical ligands such as ATP. It has been shown that ATP, atphysiologically relevant concentrations, stimulates an in-crease in the intracellular free Ca2+-concentration ([Ca2+]i) W. Bintig A. Ngezahayo (*) (Tai et al. ) and that ATP modulates K+ as well as Institute of Biophysics, Leibniz University Hannover, Cl− channels in cumulus cell-enclosed oocytes mainly Herrenhäuser-Str. 2, composed of granulosa cells (Arellano et al. These D-30419 Hannover, Germanye-mail: [email protected] findings demonstrate the expression of purinergic receptorsby granulosa cells.
J. Baumgart : A. Heisterkamp : H. Lubatschowski Two families of purinergic receptors are known. The P2X Laser Zentrum Hannover e.V., Hollerithallee 8, 1–7), which are not sensitive to UTP, form a D-30419 Hannover, Germany family of related ionotropic receptors (King and Townsend-Nicholson The binding of ATP to P2X receptors opens the channels and allows the flow of cations through Molecular and Cell Physiology, Hannover Medical School, the membrane, which can lead to increases in [Ca2+] Carl-Neuberg-Str. 1, D-30625 Hannover, Germany P2Y receptor family consists of eight subtypes (P2Y1, J Bioenerg Biomembr (2009) 41:85–94 P2Y2, P2Y4, P2Y6 and P2Y11–14) and belongs to the class permeability of the cells to K+ and Cl−, which could be of seven-transmembrane G-protein-coupled receptors involved in the secretion of antral fluid. We also found that (7TM-GPCRs). These receptors share a common membrane Cl− permeability is modulated by P2Y2 and P2Y4 receptors topology and the ability to stimulate various G-proteins.
via regulation of Ca2+-release from intracellular stores.
Like other metabotropic 7TM-GPCRs, the P2Y purinergicreceptors stimulate various intracellular signalling pathways(Abbracchio et al. ; King and Townsend-Nicholson Materials and methods von Kügelgen For instance, P2Y12–14 recep-tors block the synthesis of cAMP by inhibiting adenylyl cyclase via the activation of Gi proteins. The P2Y1, P2Y2,P2Y4 and P2Y6 receptors act via the activation of Gq If not otherwise stated, all chemicals and cell culture media proteins, stimulate phospholipase C (PLC), and can be were obtained from Sigma–Aldrich (Taufkirchen, Germany).
distinguished by their different agonists and inhibitors(Abbracchio et al. ; von Kügelgen , ). P2Y1 is not sensitive to UTP (Abbracchio et al. ; vonKügelgen P2Y11 is sensitive to UTP and The rat GFSHR-17 granulosa cells (Keren et al. were inhibited by reactive blue or suramin (Abbracchio et al.
seeded (2–5×105 cells/ml) on cover slips in petri dishes King and Townsend-Nicholson ; von Kügelgen containing Dulbecco's Modified Eagle's Medium (DMEM) White et al. ). However, P2Y11 receptor supplemented with 5% foetal calf serum, penicillin and gene is absent in murine and rat genome (Abbracchio et al.
streptomycin. Cells were cultivated at 37°C in a humidified In rat cells such as GFSHR-17 granulosa cells, P2Y2 atmosphere containing 5% CO2. The culture medium was and P2Y4 can be distinguished from other Gq coupled P2Y renewed every 2–3 days. Cells were used for the experi- receptors by their sensitivity to UTP and insensitivity to UDP.
ments 2–5 days after plating. The GFSHR-17 cells were Conversely, P2Y6 can be stimulated by UDP and inhibited used up to a total of 25 passages.
by reactive blue as well as by pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS) (Abbracchio et al.
; Burnstock von Kügelgen , Atlow ATP concentration (<10 µM), the stimulation of P2Y2 A cover slip with cells was transferred to a superfusion and P2Y4 receptors can be antagonized by reactive blue chamber containing 0.5 ml of a bath solution composed of (Wildman et al. ). P2Y2 and P2Y4 can be distinguished (in mM) 121 NaCl, 5 KCl, 6 NaHCO3, 5.5 glucose, 0.8 by the P2Y4 sensitivity to Zn2+. Applied together with ATP, MgCl2, 1.8 CaCl2, and 25 HEPES. The pH was adjusted to Zn2+ blocked P2Y4 but does not affect P2Y2 (Wildman et al.
7.4 by addition of 10–15 mM NaOH. The chamber was ). The P2Y receptors that activate Gq stimulate PLC and mounted on a Zeiss inverted microscope (Oberkochen, thereby induce hydrolysis of the membrane phosphoinositol- Germany). Cells were washed with 10 ml (2 ml/min) of the 4,5-bisphosphate (PIP2) to yield inositol-1,4,5-trisphosphate bath solution and allowed to adapt to room temperature (IP3) and 1,2-diacylglycerol (DAG). As an intracellular (20–24°C) for at least 30 min. Perforated patch-clamp second messenger, IP3 activates the IP3 receptor, a ligand- configuration was established on single cells using a patch- gated Ca2+ channel expressed in the ER membrane that clamp amplifier EPC 7 (List Medical, Darmstadt, Germany).
releases Ca2+ into the cytosol when activated. The increased A stock solution of 50 mg/ml amphothericin B in DMSO [Ca2+]i is involved in stimulating various signal transduction was diluted to 250 µg/ml in the pipette filling solution pathways and, along with DAG, activates protein kinase C composed of (in mM) 140 KCl, 5 NaCl, 1 MgCl2, 0.25 (PKC), which in turn stimulates various cellular activities CaCl2, 0.5 EGTA, 1 glucose, and 10 HEPES (pH 7.4). A such as proliferation. Ca2+ can also activate or inhibit stable perforated patch-clamp configuration was achieved Ca2+-sensitive ion channels and thus alter the membrane within 3–5 min of the establishment of the cell-attached potential. These alterations can be registered as hyperpolar- patch-clamp configuration. The membrane potential was ization or depolarization of the cell membrane.
registered in current-clamp mode. The data were filtered at We used the perforated patch-clamp technique coupled 3 kHz and digitised at 10 kHz via an interface ITC 16 with imaging of [Ca2+]i by the Fura 2/AM ratiometric (Instrutech, Minnesota, USA). Data acquisition and off- method (Grynkiewicz et al. ) in rat GFSHR-17 line analyses were performed using the software Pulse granulosa cells expressing FSH receptors. These cell line Pulsefit (HEKA Electronics, Lamprecht, Germany), Excel represents an in vitro model for rat granulosa cells in (Microsoft, USA) and Origin (Microcal Software, Inc, maturing follicle (Keren et al. ). We observed a high Northampton, USA).
J Bioenerg Biomembr (2009) 41:85–94 Measurement of [Ca2+]i containing (in mM): 137 NaCl, 20 Tris-HCl, 0.1% Tween(pH 7.5) and then incubated for 1–2 h with goat-anti-rabbit Measurements of [Ca2+]i concentration were performed as IgG secondary antibodies conjugated with alkaline phos- described previously (Grynkiewicz et al. Ngezahayo phatase and diluted to 1:500. Proteins were visualised et al. ). Cells were loaded with Fura 2/AM (Calbiochem- using Sigma Fast BCIP/NBT (5-Bromo-4-chloro-3-indolyl Novabiochem, Schwalbach am Taunus, Germany) for 20– phosphate/Nitro blue tetrazolium) followed by a final 30 min at room temperature. The Fura 2/AM loaded cells washing step in H2O. During all washing steps and the were then transferred to the superfusion chamber mounted on incubations with primary and secondary antibody, milk an inverted microscope (see above). Cells were then washed (3%) was used to neutralize non-specific binding.
with the bath solution (2 ml/min) for at least 5 min to removeexternal Fura 2/AM. The dye in the cells was excited at340 nm and 380 nm using a monochromator polychrome II (T.I.L.L. Photonics GmbH, Planegg, Germany) equipped witha 75 W XBO xenon lamp. The fluorescent images and Stimulation of rat GFSHR-17 granulosa cells with ATP intensities at 510 nm were registered with a digital CCDcamera (C4742-95, Hamamatsu Photonics K.K.; Japan) and Repeated pulse applications of ATP (10–50 µM) to rat used to calculate the fluorescence ratio (F340/F380). [Ca2+]i GFSHR-17 granulosa cells every 3 min for 3–15 s was estimated from F340/F380 ratio as described by stimulated a repetitive increase in [Ca2+]i (Fig. a). This Grynkiewicz et al. (using the program Aquacosmos Ca2+ signal was characterized by an increase from a non- (Hamamatsu Photonics K.K.; Japan). Agonists or inhib- stimulated [Ca2+]i of about 100 nM to a maximum itors were applied during electrophysiological as well as concentration of approximately 200 nM within 5–10 s Ca2+-imaging experiments using a Small Volume Perfusion (Fig. The increase was followed by a decrease to the System setup (Bioscience Tools, San Diego, USA).
initial concentration within 30 s. The ATP-related stimula-tion of [Ca2+]i increase could be repeated every 3 min by applying ATP for less than 15 s (Fig. SimultaneousCa2+-imaging and membrane potential measurement using To isolate the proteins, cells were collected from culture the perforated patch-clamp technique revealed a resting dishes in ice cold phosphate buffered solution (PBS) membrane potential of approximately −40 mV. Application containing (in mM): 137 NaCl, 2.7 KCl, 10 Na2HPO4, of ATP induced a 10 mV depolarization of the cells and 1.8 KH2PO4 (pH 7.4). After centrifugation at 500 g at (Fig. ) followed by a hyperpolarization of approximately 4°C for 5 min, the supernatant was discarded and the cells 30 mV (Fig. c, Table The depolarization and hyper- were diluted in a lysis buffer containing (in mM): 10 NaCl, polarization corresponded to inward and outward current 25 HEPES, 2 EDTA, and protease inhibitors (aprotinin and (results not shown). The GFSHR-17 are strongly coupled phenylmethylsulphonyl fluoride), (pH 7.5). Cells were then via gap junctions (Ngezahayo et al. which renders sonicated at 4°C for 10 min followed by a centrifugation difficult to adequately space-clamp in order to measure the step at 15,000 g at 4°C for 30 min. The supernatant was currents through the single cell membrane. We therefore again discarded and the pellet was dissolved in 30–50 µl of measured the membrane potential in the current clamp a solubilization buffer containing (in mM): 200 NaCl, 50 mode. The depolarization could also be induced by pressure HEPES, protease inhibitors (pH 7.5). An equal volume of a (superfusion with control bath solution). This was not 2% Chaps solution was added to the solubilization buffer, observed in all experiments, and was most likely related to and a centrifugation step was performed at 6,500 g at 4°C pressure-dependent opening of gap junction hemichannels for 10 min. The protein concentration in the supernatant was estimated using the Bradford technique. For each Extending the duration of the presence of ATP or reducing experiment, samples containing 5–10 µg of protein were the intervals between successive ATP applications compro- applied to the SDS polyacrylamide gel and separated by mised the ability to stimulate the cells, indicating a desensi- electrophoresis. The separated proteins were transferred to a tization of the receptors (Fig. For a ATP presence longer nitrocellulose membrane using 1.2 mA/cm2 for 120 min.
than 1 min, the desensitization was characterised by a Staining the nitrocellulose membrane was performed by continuous reduction of the Ca2+ signal. The decline of the overnight incubation at 4°C with the corresponding primary signal began even when ATP was still present. The removal anti-P2Y receptor antibodies (Alomone Labs Ltd., Jerusalem, of ATP was not followed by a spontaneous recovery of Israel) diluted to 1:1000 (P2Y2, 0.8 mg/ml) or 1:500 (P2Y4, purinergic sensitivity. The recovery from the desensitization 0.3 mg/ml). The membrane was washed with TBST took a long time of 30–60 min (Fig. d).
J Bioenerg Biomembr (2009) 41:85–94 Fig. 1 Intracellular Ca2+homeostasis in rat GFSHR-17granulosa cells. a Repeatedapplication of 25 µM ATP(bars) for 5–15 s every 3 mininduced a repetitive Ca2+ signal.
b Applying 25 µM ATP inducedan increase in [Ca2+]i and c adepolarization of approximately10 mV followed by a hyperpo-larization of approximately30 mV. A detailed analysisshowed that the depolarizationwas most likely unrelated toATP. The results in b and c areaverages, the error bars repre-sent the SEM for n=25 experi-ments, respectively. The resultin a is an average for n=42. Forclarity, the errors are not indi-cated but are comparable tothose shown in b. d Ca2+ signalsevoked by consecutive ATP ap-plication for 5 min each.
Depending on the delay of thesecond application, the ampli-tude and duration of the inducedCa2 signal was reduced in com-parison to the first one, indicat-ing a desensitization of thereceptors. To evoke a signalcomparable to the first one, adelay of 45–60 min wasrequested The purinergic receptors of rat GFSHR-17 granulosa cells receptors, which induce the release of Ca2+ from intracellularstores. To analyse whether the ATP-evoked [Ca2+]i-increase Two families of purinergic receptors are known. There are was related to an influx of Ca2+ from the extracellular the ionotropic P2X receptors, which allow a Ca2+ influx solution (i.e., P2X receptors) or to a Ca2+-release from from the extracellular space, and the metabotropic P2Y intracellular stores (i.e., P2Y receptors), ATP was applied in Table 1 The values of the membrane potential predicted by the Goldmann–Hodgkin equation and measured values at various [K+]o in non-stimulated cells and in ATP-stimulated cells (25 µM) Predicted Um (mV) with assumption Predicted Um (mV) with assumption that pK+ was increased (pK+=10) that pCl− was decreased (pCl−=0.01) −40.6±0.2 (n = 25) −71.1 −67.5±2.1 (n = 25) −34.8±0.4 (n = 8) −45.0±2.9 (n = 8) −35.2±0.2 (n = 12) −31.6±2.4 (n = 12) −24.2±0.1 (n = 15) −21.7±1.2 (n = 15) For the calculation of Um, pK+ = pCl− =1, pNa+ =0.025, as well as intracellular concentrations for K+ =140 mM, Na+ =10 mM, Cl− =40 mM, andexternal concentrations for K+ =5–60 mM, Na+ =140 mM and Cl− =130 mM were assumed for non-stimulated cells. The results are average ± SEM

J Bioenerg Biomembr (2009) 41:85–94 the presence of EGTA in the external bath solution. We P2X receptors, was equally efficient in stimulating the rat found that the absence of Ca2+ in the bath solution did not GFSHR-17 cells (Figs. b).
alter the ATP-evoked increase in [Ca2+]i, indicating a Ca2+- In rat cells, P2Y2, P2Y4 and to a minor extend P2Y6 can release from intracellular stores (Fig. ). This result suggests be stimulated by UTP (Abbracchio et al. von that the observed ATP-evoked response of rat GFSHR-17 Kügelgen The P2Y2, P2Y4 and P2Y6 receptors granulosa cells was mainly due to P2Y receptors and not P2X activate Gq proteins, which are linked to PLC, PIP2 receptors. Consistent with this conclusion, the application of hydrolysis and increases in [Ca2+]i. We found that the Ca2+ P2X receptor agonists such as α, β-methyleneadenosine 5′- signal could be completely suppressed by the PLC inhibitor triphosphate lithium salt (α, β-meATP) did not induce any U73122 or by the IP3 receptor blocker 2-aminoethoethyl measurable changes in the [Ca2+]i concentration of the cells diphenyl borate (2-APB; Calbiochem-Novabiochem, (results not shown). Moreover, UTP which does not stimulate Schwalbach am Taunus, Germany) (Fig. c). These results Fig. 2 Pharmacological analysis of the ATP-related Ca2+ response in suppress the ATP (5 µM) related stimulation. Zn2+ did not suppress rat GFSHR-17 granulosa cells. a EGTA in the extracellular solution the stimulation by 25 µM ATP which stimulated almost the whole cell did not alter the Ca2+ response to 25 µM ATP. b UTP (25 µM) was population. The experiments are representative of 36 cells (note the equally efficient in stimulating the Ca2+ response. c The presence of oscillating Ca2+ signal elicited by 5 µM ATP. This was observed in U73122 (10 µM), an inhibitor of PLC or 2-APB (100 µM), an IP3 some cells. Currently we do not understand how such oscillations are receptor antagonist, suppressed the Ca2+ response to ATP. The results regulated). (E) P2Y2 and P2Y4 expression was analyzed by Western are averages and the error bars represent the SEM for at least seven blot. The P2Y2 and P2Y4 antibodies stained bands at 42 kDa and experiments for each treatment. d Stimulation of the rat GFSHR-17 50 kDa, respectively (lanes 2 and 4). The bands were absent when granulosa cells by 5 µM ATP. Some cells could be stimulated while the primary antibodies were pre-absorbed with the respective antigenic others could not. Within the responding population Zn2+ could peptides (lanes 1 and 3) J Bioenerg Biomembr (2009) 41:85–94 suggest the presence of only P2Y2, 4 and 6 receptor types. An absence of any ligand induced an increase in [Ca2+]i extended pharmacological battery showed that antagonists of followed by the hyperpolarization of the cells (Fig. b, c).
P2Y6 receptors such as reactive blue or PPADS (Abbracchioet al. ; von Kügelgen did not affect ATP-evoked Hyperpolarization of rat GFSHR-17 granulosa cells and ion responses. Furthermore, UDP, the agonist of the P2Y6 receptor, was able to stimulate the cells, but only at a highconcentration of 200 µM (results not shown). These In the perforated patch-clamp configuration, rat GFSHR-17 pharmacological experiments indicate that the observed granulosa cells showed a resting membrane potential (Um) ATP-dependent stimulation of the rat GFSHR-17 granulosa of approximately −40 mV (Table We estimated the Um cells was mainly related to the P2Y2 and P2Y4 receptors.
using the Goldmann–Hodgkin equation: Working with low ATP concentration (5–10 µM), we observed that the likelihood to stimulate the cells with ATP m ¼ F ln pKþ Kþ was reduced. Some cells responded while others did not.
Within the responding cell population, Zn2+ which is known where p is the coefficient of membrane permeability to the to inhibit P2Y4 receptors (Wildman et al. blocked the respective ion and indices i and o indicate intracellular response of some cells to stimulation with 5–10 µM ATP. At and extracellular spaces, respectively. The gas constant 25 µM however, ATP was able to stimulate almost all cells (8.314 J mol−1 K−1), the temperature in Kelvin (room even in presence of 300 µM Zn2+ (Fig. d). We also temperature: 295°K) and the Faraday constant (96,485 C analyzed the expression of both molecules with western blot mol−1) are represented by R, T, and F, respectively. In our experiments. Monoclonal antibodies against P2Y2 and P2Y4 experiments, the extracellular bath solution contained 5 mM receptors recognized molecules of about 42 kDa and 50 kDa, K+, 140 mM Na+, and 130 mM Cl−. In the intracellular respectively (Fig. e).
space, the values of 140 mM K+ and 10 mM Na+ were Simultaneous [Ca2+]i imaging and electrophysiological assumed. To achieve the observed resting potential of measurements showed that the suppression of the [Ca2+]i approximately −40 mV, the values pK+ = pCl− = 1 and signal using a PLC inhibitor or an IP 3 receptor antagonist = 0.025 as well as 40 mM Cl− in the intracellular space (Fig. correlated with the suppression of hyperpolarization were estimated.
(Fig. a). Additionally, it was found that applying the The ATP-stimulated increase of [Ca2+]i was followed by SERCA pump inhibitor cyclopiazonic acid (CPA) in the a hyperpolarization to −67.5 mV (Fig. c, Table ). The Fig. 3 The relationship betweenthe increase in [Ca2+]i and hy-perpolarization. a Thepresence of U73122 (10 µM) or2-APB (100 µM) suppressedboth the ATP-induced Ca2+response and hyperpolarization.
b Applying CPA (50 µM) inducedan increase in [Ca2+]i that wasfollowed by c hyperpolarizationof the membrane potential. Theresults are averages and the errorbars represent the SEM for atleast five experiments for eachtreatment. It is noteworthy thatCPA induced an increase in[Ca2+]i and a hyperpolarizationwith amplitudes similar to thosestimulated by 25 µM ATP J Bioenerg Biomembr (2009) 41:85–94 ATP-induced hyperpolarization was suppressed by U73122 apamine, clotrimazole (CLT) or iberiotoxin (IbTx), the (an inhibitor of PLC) as well as by 2-APB (IP3-receptor blockers of Ca2+-activated K+-channels with low conduc- antagonist) (Fig. a). Furthermore, inhibition of SERCA tance (SK-channels), intermediate conductance (IK-channels) pumps using CPA stimulated an increase in [Ca2+]i and was or high conductance (BK-channels), respectively, did not able to induce hyperpolarization of the cells (Fig. b, c), alter ATP-related stimulation individually or as a three-drug indicating that the [Ca2+]i-increase was a prerequisite for cocktail (Fig. Other inhibitors of K+ channels such as the hyperpolarization. The hyperpolarization could be tetraethylammonium chloride (TEA) also failed to affect the achieved by opening K+ channels or by inhibiting Cl− ATP-stimulated hyperpolarization (results not shown). Phar- channels. To achieve the observed Um of −67.5 mV by ATP macological inhibition of Cl− channels with DIDS, mibefradil application, the Goldmann–Hodgkin equation would predict or diphenylamine-2-carboxylic acid (DPC) hyperpolarized that ATP induces a increase of pK+ to 10 or a decrease of the cells to a level comparable to that achieved by ATP pCl− to 0.01.
application under control conditions and suppressed further Changes in the external concentrations of K+ ([K+]o) or ATP-induced hyperpolarization (Fig. c, Table the substitution of Cl− with gluconate in the extracellularsolution affected the Um as predicted by the Goldmann–Hodgkin equation (Tables if we assume a permeability of gluconate of 0.3 (Kim et al. ). It was not possible todistinguish whether the ATP-related hyperpolarization was This report characterises the link between purinergic due to the activation of K+ channels or the inhibition of Cl− receptors and the regulation of membrane potential in permeability by application of ATP in presence of various granulosa cells of maturing follicles using the rat GFSHR- [K+]o concentrations (Fig. a, Table ). When NaCl was 17 granulosa cell line. The GFSHR-17 granulosa cells replaced by Na-gluconate in external solution, however, the express the FSH receptor and are therefore a suitable in application of ATP hyperpolarized the cell to −80 mV. If vitro model for this purpose (Keren et al. ). The aim of ATP were increasing the pK+ to 10 or reducing the pCl− to this study was to elucidate the role of purinergic receptors 0.01, the Goldmann–Hodgkin equation would predict a Um in the granulosa cells during follicular maturation.
of −69 mV and −77 mV, respectively (Table The Purinergic receptors are classified in two groups: comparison between the measured and the estimated values ionotropic P2X receptors and metabotropic P2Y receptors.
for Um suggests that an inhibition of Cl− permeability likely The P2X receptors are mainly permeable to cations when produced the ATP-evoked hyperpolarization. Accordingly, activated by purines and can induce depolarization as well Fig. 4 a Increasing [K+]o from5 mM to 35 mM shifts themembrane potential as predictedby the Goldmann–Hodgkin equa-tion (Table and suppressedATP-induced hyperpolarization.
b The continuous presence of acocktail containing the inhibitorsof Ca2+-activated K+ channels(0.1 µM iberiotoxin, 2 µMclotrimazole, 1 µM apamin) didnot alter the ATP induced Ca2+release (not shown) or hyperpo-larization. c The inhibition of Cl−channels by DPC (2.5 mM)induced hyperpolarization. Simi-lar results were obtained usingDIDS (500 µM) or mibefradil(30 µM). The results are averagesand the error bars represent theSEM for at least five experiments J Bioenerg Biomembr (2009) 41:85–94 Table 2 The values of the membrane potential predicted by the Goldmann–Hodgkin equation and measured values under ATP (25 µM) and non-ATP-induced stimulation of the cells when NaCl in external solution was replaced by Na-Gluconate (pGluconate=0.3; [Cl−]o=10 mM) Substitution of NaCl by Na-Gluconate + 25 µM ATP Predicted Um (mV) with assumption Predicted Um (mV) with assumption that pK+ was increased (pK+=10) that pCl− was decreased (pCl−=0.01) −36.9±0.3 (n = 5) −81.5±1.9 (n = 5) The results are average ± SEM as increases in [Ca2+]i due to the influx of cations, primarily follicle are characterized by a strong proliferation of the Na+ and Ca2+ from the external space. It is shown that granulosa cells. Whether purinergic stimulation is involved applying ATP stimulates depolarization of the rat GFSHR- in regulation of the proliferative activity of the granulosa 17 granulosa cells (Fig. c). As proposed by Bintig et al.
cells is at moment matter of speculation. We estimated the ), however, it seems that this depolarization is not doubling time of the rat GFSHR-17 granulosa cells. For cell related to the activation of P2X receptors but to opening cultivated under control conditions and in presence of ATP gap junction hemichannels. This is consistent with observa- or the non hydrolysable ATP-γ-S, doubling times of 25.8 h, tions by other authors. It was recently shown that granulosa 24.5 h and 24.2 h were respectively found. These values are cells express gap junction hemichannels (Tong et al. ).
not significantly different. Because of desensitization, a Additionally, the hemichannels are mechanosensitive and long lasting presence of ATP induced a single Ca2+ signal function as ATP-release channels (Bintig et al. Gomes (Fig. d). It could therefore be expected that cultivation of et al. Romanello et al. ). Moreover, it is shown the cells with ATP would not affect the proliferation that the ATP-related Ca2+ signal could be elicited even in the activity. However, the doubling times show a tendency that presence of EGTA in the extracellular solution (Fig.
the purinergic stimulation could increase the proliferative These results strongly suggest that the ATP-related increase activity of granulosa cells. A careful study combining in [Ca2+]i does not depend on the stimulation of P2X analysis of frequency of ATP application and the whole receptors, but on the activation of P2Y receptors.
duration of stimulation is needed for a definitive conclusion.
Two physiological processes can be evoked that explain the rise of ATP in the extracellular space of the maturing P2Y2 and P2Y4 receptors are expressed in rat GFSHR-17 follicle: paracrine secretion and nervous co-stimulation (Aguado Burnstock , ; Tai et al. As for co-stimulation, the survival and maturation of the The different subtypes of P2Y receptors can be distin- follicle are controlled by neurotransmitters such as acetyl- guished by their pharmacology as well as by their choline and norepinephrine, as well as neuropeptides intracellular signalling cascades. We observed that P2Y secreted by the ovarian nerve (Aguado ). It can receptors of the rat GFSHR-17 granulosa cells could be therefore be assumed that ATP appears in the extracellular stimulated by UTP (Fig. b) but not by ADP (results not space of the maturing follicle as a co-transmitter with other shown). We also found that blocking the IP3 pathway neurotransmitters, as suggested by Tai et al. (). Upon inhibits ATP stimulation in the cells (Figs. c and a). It can arriving in the follicular interstitial space by either paracrine therefore be assumed that rat GFSHR-17 granulosa cells secretion or co-stimulation, we suggest that ATP binds to express P2Y receptor subtypes that are sensitive to UTP the P2Y receptors and induces the cascade of reactions to and are linked to the IP3 pathway. Only P2Y2, P2Y4 and increase [Ca2+]i and stimulate hyperpolarization. Maturing P2Y6 are UTP-sensitive and are linked to the activation of Table 3 The values of the membrane potential predicted by the Goldmann–Hodgkin equation and measured values under ATP (25 µM) and non-ATP-induced stimulation of the cells, when Cl− permeability was inhibited with DPC (2.5 mM) Predicted Um (mV) with assumption Predicted Um (mV) with assumption that pK+ was increased (pK+=10) that pCl− was decreased (pCl−=0.01) −68.2±0.5 (n = 8) −72.9±1.1 (n = 8) The results are average ± SEM. Similar results were obtained with Mibefradil (30 µM) or DIDS (500 µM) J Bioenerg Biomembr (2009) 41:85–94 the IP3-Ca2+-cascade at the intracellular site (Burnstock and following experimental observations: (i) Gradual increase Williams ; King and Townsend-Nicholson ). The of [K+]o from 5 mM to 20 mM, 35 mM and 60 mM and agonist of P2Y6 UDP (Abbracchio et al. King and substitution of NaCl in the external solution with Na-gluconate Townsend-Nicholson ; von Kügelgen ), depolarized the cells as predicted by the Goldmann-Hodgkin however, could stimulate the rat GFSHR-17 granulosa cells equation from −40 mV to −34.8 mV, −35.2 mV, −24.2 mVand only at concentrations of 200 µM. Moreover, reactive blue, −37 mV, respectively (with assumed pGluconate = 0.3 an inhibitor of P2Y6 (Abbracchio et al. von Kügelgen suggested by Kim et al. ) (Fig. Tables , ). (ii) ) did not affect the ATP evoked response (results Inhibitors of Cl− channels such as DPC, DIDS or mibefradil not shown). These results suggest a minor role of P2Y6 induced hyperpolarization of the cells to approximately receptor and a major role of P2Y 2 and P2Y4 receptors in 70 mV (Fig. Table (iii) [Cl−]i in the range of ATP-induced activity in rat GFSHR-17 granulosa cells.
40 mM has been measured in various non-excitable cells This assumption is supported by Western blot experiments, such as astrocytes and in rat GFSHR-17 granulosa cells under which showed that the rat GFSHR-17 granulosa cells the whole-cell patch-clamp configuration using a pipette expressed molecules specifically recognized by anti-P2Y2 solution containing 140 mM K+, 10 mM Na+ and 30–40 mM and anti-P2Y4 antibodies (Fig. e). The anti-P2Y2 antibody Cl− (Ngezahayo et al. ). Since the theoretically predicted stained a molecule with 42 kDa (Fig. e, lane 2), which Um under various conditions and the measured Um are very correlates to the predicted size of 42.0 kDa for the P2Y2 close, it can be assumed that non-stimulated rat GFSHR-17 receptor Sage and Marcus A size of 40.7 kDa was granulosa cells are equally permeable to K+ and Cl−. This predicted for P2Y4 receptor. The anti-P2Y4 antibody, permeability to ions must be compensated by a large however, recognized a molecule of approximately 50 kDa permeability to water. Since rat GFSHR-17 granulosa cells (Fig. e, lane 4). These bands disappeared when the are an in vitro model for the granulosa cells of maturing antibodies were blocked with the corresponding antigenic follicle, we propose that this mechanism is involved in the peptides (Fig. lane 1 and 3). The discrepancy between secretion of the antral fluid during follicle maturation.
the predicted band at 40.7 kDa for P2Y4 and the The ATP-induced hyperpolarization can be associated specifically stained band at 50 kDa can be explained by with an increase in membrane permeability to K+ or a posttranslational modifications such as glycosylation (Sage decrease in membrane permeability to Cl−. To achieve the and Marcus It is therefore tempting to assume that observed Um of −67.5 mV by ATP application, the the ATP-evoked Ca2+ and electrical responses in the ratGFSHR-17 granulosa cells are related to the activation ofP2Y2 and P2Y4 receptors. Further pharmacological experi- ments revealed that the density of each receptor type couldbe variable. Using low ATP concentration (5–10 µM), there were cells which could not be stimulated. Within theresponding cell population, Zn2+ (300 µM) which inhibited 4 receptors was able to suppress the Ca2+ evoked response by 5–10 µM ATP in some cells, while other cellswere not affected (Fig. At 25 µM ATP the inhibitory Inhibition by U73122 effect of Zn2+ could not be observed (Fig. d). These resultscould be related to a various density of the P2Y2 and P2Y4 receptors in the membrane of the rat GFSHR-17 granulosacells. How both receptor subtypes participate to the regulation of the ATP-evoked response in granulosa cellsremains an interesting question. A combination of bio- Blockage by 2-APB chemical analysis and molecular biological dissection isneeded to determine the contribution of each receptor to the observed ATP-evoked response in granulosa cells.
(Inhibition of Cl- permeability) ATP stimulates hyperpolarization in rat GFSHR-17granulosa cells Hyperpolarization The measured resting potential suggests a high pCl− andpK+ Fig. 5 Schematic representation of ATP signalling in granulosa cells (≌1) and an elevated [Cl−]i of about 40 mM under of maturing follicle. The question mark shows possible pathways that control conditions. This suggestion is supported by the remain to be elucidated J Bioenerg Biomembr (2009) 41:85–94 Goldmann–Hodgkin equation would predict that ATP tion of P2Y2 and P2Y4 receptors in rat GFSHR-17 induces an increase of pK+ to 10 or a decrease of pCl− to granulosa cells induces a Ca2+-dependent inhibition of Cl− 0.01. Accordingly, we predicted the Um that would be channels and thereby promotes hyperpolarization (Fig. achieved by application of ATP under various conditions We propose that an ATP-dependent inhibition of Cl− with the assumption that ATP affects the permeability of permeability in granulosa cells of the maturing follicle is either K+ or Cl− (Tables and ). At various [K+]o, the a key mechanism in regulating the secretion of antral fluid.
Goldmann–Hodgkin equation predicted values that werevery close when we assumed that ATP affected either K+ The work was partly supported by the NANOTOME project; Biophotonik III.
or Cl− permeability. The measured values agree with theprediction of the Goldmann–Hodgkin equation; however,these experiments cannot decipher which ion is affected byATP-application. After substitution of external NaCl with Na-gluconate, ATP-application hyperpolarized the cells to amembrane potential of −81 mV (Table If we assume that Abbracchio MP, Burnstock G, Boeynaems JM, Barnard EA, Boyer JL, Kennedy C, Knight GE, Fumagalli M, Gachet C, Jacobson KA, ATP was activating K+-channels, a Um of −69 mV would be Weisman GA (2006) Pharmacol Rev 58:281–341 predicted by the Goldmann–Hodgkin equation. Conversely, Aguado LI (2002) Microsc Res Tech 59:462–473 if we assume that ATP reduced pCl−, the Goldmann– Arellano RO, Martinez-Torres A, Garay E (2002) Biol Reprod Hodgkin equation would predict a U m of −77 mV. The Bintig W, Buchholz V, Schlie S, Baumgart J, Heisterkamp A, results therefore suggest that ATP induced an inhibition of Ngezahayo A (2007) Acta Physiologica 188(Suppl. 653):P10 Cl− permeability. Furthermore, the inhibition of Ca2+- Burnstock G (2007a) Physiol Rev 87:659–797 release with inhibitors of PLC or IP3-receptors blocked Burnstock G (2007b) Cell Mol Life Sci 64:1471–1483 the hyperpolarization (Fig. The SERCA pump inhibitor Burnstock G, Williams MJ (2002) Pharmacol Exp Ther 295:862–869Gomes P, Srinivas SP, Van Driessche W, Vereecke J, Himpens B CPA yielded an increase in [Ca2+]i (Fig. and thereby (2005) Invest Ophthalmol Vis Sci 46:1208–1216 induced hyperpolarization (Fig. c). If we assume that the Grynkiewicz G, Poenie M, Tsien RY (1985) J Biol Chem 260:3440–3450 ATP-evoked hyperpolarization was related to the activation Keren TI, Dantes Sprengel AR, Amsterdam A (1993) Mol Cell of K+ channels, it would be correct to assume that these Endocrinol 95:R1–R10 Kim SJ, Shin SY, Lee JE, Kim JH, Uhm DY (2003) Prostate 55:118–127 channels are Ca2+-activated. Pharmacological inhibition of King BF, Townsend-Nicholson A (2003) Tocris Reviews 23:1–12 the putative Ca2+-activated BK, IK and SK channels by King BF, Townsend-Nicholson A (2008) J Pharmacol Exp Ther IbTx, CLT, apamin or a cocktail containing all these inhibitors (Fig. did not alter the ATP-induced hyperpo- Ngezahayo A, Altmann B, Kolb HA (2003) J Membr Biol 194:165–176Romanello M, Pani B, Bicego M, D'Andrea P (2001) Biochem larization, nor did inhibitors of K+ channels such as TEA.
Biophys Res Commun 289:1275–1281 The Cl− channel inhibitors DPC, DIDS or mibefradil Sage CL, Marcus DC (2002) J Membr Biol 185:103–115 hyperpolarized the cells to −70 mV as predicted by the Tai CJ, Kang SK, Cheng KW, Choi KC, Nathwani PS, Leung PCK Goldmann–Hodgkin equation (Table ). An additional (2000) J Clin Endocrinol Metab 85:591–597 Tong D, Li TY, Naus KE, Bai D, Kidder GM (2007) J Cell Sci application of ATP was not able to provoke a further hyperpolarization, even though the Goldmann–Hodgkin von Kügelgen I (2000) Naunyn Schmiedebergs Arch Pharmacol equation predicted a reinforcement of hyperpolarization to −83 mV under the assumption that the permeability for K+ von Kügelgen I (2006) Pharmacol Ther 110:415–432White PJ, Webb TE, Boarder MR (2003) Mol Pharmacol 63:1356– would be increased by ATP (pK+=10) (Fig. Table ).
Biophysical analysis, combined with pharmacological Wildman SS, Unwin RJ, King BF (2003) Br J Pharmacol 140:1177– dissection, allow us to postulate that ATP-induced stimula-

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