P2Y Receptor-Mediated Stimulation of Müller Glial DNA Synthesis

Abstract

purpose. To determine whether activation of P2Y receptors may increase the DNA synthesis rate of cultured Müller cells and to investigate whether adenosine 5′-triphosphate (ATP)–induced Müller cell proliferation is mediated by an intracellular calcium increase.

methods. Primary cultures of Müller cells of the guinea pig were treated with test substances for 16 hours. The DNA synthesis rate was assessed by a bromodeoxyuridine immunoassay, and ATP-induced elevations of the intracellular calcium concentration were recorded by fura-2 imaging.

results. ATP or uridine triphosphate (UTP) increased the DNA synthesis rate whereas α,β-methylene-ATP, 2-methyl-thio-ATP, and adenosine were ineffective, indicating that the action of ATP was through P2Y receptors. The effect of ATP was dose dependent, with an EC₅₀ of 5.9 μM. The mitogenic effect of ATP required an elevation of the intracellular calcium and a calcium influx into Müller cells. Blockers of calcium-permeable channels (nickel ions) or of calcium-dependent potassium (BK) channels (iberiotoxin, charybdotoxin) inhibited the ATP-stimulated DNA synthesis. In calcium-imaging experiments, ATP-evoked intracellular calcium transients were significantly shortened in the presence of extracellular nickel ions or of iberiotoxin. A correlation was found between the duration of the ATP-evoked calcium transients and the basal proliferation rate of the cultures.

conclusions. The results indicate that the ATP-induced elevation of Müller glial DNA synthesis is dependent on an influx of calcium ions from the extracellular space and that the inhibiting effect of BK channel blockers on ATP-evoked DNA synthesis is caused by an inhibition of this influx. The amount of the calcium influx seems to be directly correlated to the strength of the ATP-evoked proliferation.

Diseases of the sensory retina are regularly accompanied by a reactive gliosis of retinal glial (Müller) cells. During proliferative vitreoretinopathy, for example, Müller cells re-enter the proliferation cycle, migrate out of the sensory retina and participate in the formation of periretinal cellular membranes. ^{1 2 3} Gliotic Müller cells are characterized by altered expression of various different enzymes, ion channels, and receptors. During proliferative vitreoretinopathy of the human retina, Müller cells change their expression and activity of certain ion channels. ^{4 5} Among them, the activity of calcium-activated potassium channels of big conductance (BK) was found to be increased. ⁶ Moreover, an enhanced expression of purinergic receptors has been recently suggested, as indicated by the observation that P2X₇ receptor-mediated cation currents in Müller cells from patients with proliferative vitreoretinopathy are increased compared with cells from healthy donors. ⁷ A possible role of extracellular adenosine 5′-triphosphate (ATP) in the induction or maintenance of reactive astrogliosis has been suggested. ^{8 9} Among other effects, extracellular ATP stimulates the proliferation of cultured astrocytes. ⁸  

Müller cells of the rat and human retinas express metabotropic P2Y receptors that are coupled to transient intracellular calcium release. ^{7 10 11} However, until now, it is not known whether P2Y receptor activation leads to a stimulation of Müller cell proliferation and whether an increase of the intracellular calcium concentration is necessary for a possible proliferation-stimulating effect. Therefore, the purpose of the present study was to determine whether extracellular ATP stimulates the DNA synthesis in cultured Müller glial cells and to investigate which intracellular signaling mechanisms may underlay a possible proliferation-stimulating effect of ATP. In the findings in this study, cultured Müller cells expressed P2Y receptors and activation of these receptors increased the DNA synthesis rate. The mitogenic effect of ATP depends on an elevation of the intracellular free calcium concentration, on an influx of calcium ions from the extracellular space, and on the activity of BK channels. Moreover, the duration of the ATP-induced calcium influx correlates with the proliferation rate of cultured Müller cells. 

Methods

Cultures

Primary cultures of Müller glial cells were obtained from guinea pigs (250–400 g). Animal care and handling were performed in accordance with applicable German laws and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were deeply anesthetized by urethane (2 g/kg, intraperitoneally) before decapitation and enucleation of the eyes. The excised retinas were dispersed in calcium- and magnesium-free phosphate buffer supplemented with nagarse (1 mg/mL) for 30 minutes at 37°C. After they were washed in phosphate buffer containing DNase I (200 U/mL), the dissociated cells were seeded on uncoated coverslips (diameter 15 mm; Glaswarenfabrik Hecht, Sontheim/Rhön, Germany). Retinal cells from two eyes were distributed on 54 coverslips; 100 μL of cell suspension was used per slip. Cells were cultured in minimal essential medium supplemented with 10% fetal calf serum at 37°C in 5% CO₂ in air. The medium was exchanged twice a week. Just before achieving confluence after 8 days in culture, the test substances were added to the culture medium 16 hours before the cultures were fixed. During this latter period, substances were tested in serum-free medium. The basal proliferation rate of the cultures (between 0.12 and 0.25) that was measured when no test substances were applied during the 16-hour incubation period was used as control value. A decrease of the control proliferation rate below 0.05 is accompanied by necrotic damage of cells (not shown). 

DNA Synthesis Rate

The DNA synthesis rate was determined by measuring bromodeoxyuridine (BrdU) incorporation. BrdU (10 μM) was added simultaneously with the test substances, 16 hours before fixation with 4% paraformaldehyde. BrdU incorporation was detected by a murine anti-BrdU IgG-antibody (Bu 33; Sigma, Deisenhofen, Germany) and cyanogen (Cy3)-tagged secondary antibodies. Counterlabeling of all cell nuclei was performed with acridine orange. In the peripheral (i.e., nonconfluent) regions of the cultures, six distinct areas of each coverslip (each approximately 60.000μ m², resulting in a total area of 0.42 mm² per coverslip) were studied by means of a semiautomatic image analysis system (SIS; Soft-Imaging Systems, Münster, Germany). The results from three coverslips per culture were averaged; every experiment involved at least three independent cultures. The ratio of BrdU-immunoreactive versus total cell nuclei was taken as a marker for the DNA synthesis rate. 

Calcium Imaging

For fluorescence measurements, cells were cultured for 8 days in minimal essential medium containing 10% serum and then for 16 hours in serum-free medium. The cells were loaded with 10 μM fura-2/acetoxymethylester (AM) for 30 minutes at 37°C. Measurements were performed at room temperature by using a bath solution that contained (in millimolar) 129 NaCl, 3 KCl, 1 CaCl₂, 0.2 MgCl₂, 20 glucose, and 10 HEPES (pH 7.4 adjusted with NaOH). Calcium-free bath solution was made by omitting CaCl₂. A fluorescent measurement system (Fucal 5.12B; Till-Photonics, Munich, Germany) was used. Fluorescence was excited at 340 (F₃₄₀) and 380 nm (F₃₈₀), and images were recorded every 15 or 6 seconds. Cultures were continuously perfused for at least 20 minutes before application of test substances. Substances were applied by rapidly switching the control perfusate into a perfusate containing test substances. Perfusate switches did not evoke any changes of the intracellular calcium concentration. 

Materials

Calpain inhibitors were obtained from Calbiochem (Bad Soden, Germany) and dissolved in dimethyl sulfoxide (DMSO). Vehicle alone did not affect the DNA synthesis rate. Nagarse (subtilisin) was from Serva (Heidelberg, Germany). Iberiotoxin and charybdotoxin were from Alomone Laboratories (Jerusalem, Israel). Hoechst 33258 and fura-2/AM were from Molecular Probes (Eugene, OR). To stain glial fibrillary acidic protein (GFAP), a polyclonal rabbit anti-cow GFAP serum (1:500; Dakopatts, Copenhagen, Denmark) and Cy3-tagged secondary antibodies (pig anti-rabbit; Dianova, Hamburg, Germany) were used. All other substances were obtained from Sigma (Deisenhofen, Germany). 

Data Presentation

Statistical analysis (Mann–Whitney test, two-tailed) and curve fits were made by computer (Prism software; GraphPad Software, Inc., San Diego, CA). The fluorescence ratio F₃₄₀ to F₃₈₀ is presented to describe relative changes of the intracellular calcium concentration. An increase in the ratio indicates an increase in intracellular free calcium. ¹² Data are expressed as the mean ± SEM (BrdU incorporation levels) or as the mean ± SD (calcium imaging). Statistical significance was accepted at P < 0.05. 

Results

Müller Cell Cultures

Müller glial cells of the guinea pig formed layers of flat polygonal cells when they grew 8 days in culture (Fig. 1A) . As previously shown, ¹³ the majority of the cells (99% and 96%, respectively) expressed immunoreactivities for GFAP (Fig. 1A) and for vimentin (not shown). Because the guinea pig retina does not contain astrocytes, the majority of the cultured cells were considered to represent Müller cells. The moderate density of the cells allowed estimation of the ratio of BrdU-labeled cell nuclei versus all cell nuclei (Fig. 1B) . 

P2 Receptor-Mediated DNA Synthesis

To determine whether activation of P2 receptors affects the DNA synthesis rate of cultured Müller cells, different agonists were tested (Fig. 2A) . Addition of ATP or of uridine 5′-triphosphate (UTP), an agonist equipotent with ATP at P2Y₂ and P2Y₄ receptors, to the culture medium resulted in an increase of the BrdU incorporation in cultured Müller cells.α ,β-Methylene-ATP (α,β-meATP), an agonist for several P2X receptor subtypes, and 2-methyl-thio-ATP (2-meS-ATP), an agonist for P2Y₁ receptors, did not increase the DNA synthesis rate (Fig. 2A) . Adenosine up to 5 mM did not change the BrdU incorporation, indicating that P1 receptors were not involved in the mitogenic effect of ATP. The putative nonselective P2 receptor antagonists suramin (10 μM) and pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid (PPADS, 10 μM) prevented the increase of the DNA synthesis caused by ATP (Fig. 2B) . The results indicate that extracellular ATP stimulates the DNA synthesis through activation of P2Y receptors. The effect of ATP on the DNA synthesis was concentration dependent (Fig. 2C) , with a mean EC₅₀ of 5.9 μM. Addition of ATP (500 μM) or of fetal calf serum (5%) to the culture medium resulted in increases in DNA synthesis (Fig. 2D) . Simultaneous application of both agents stimulated the DNA synthesis additively, suggesting that ATP and serum evoke different intracellular signaling pathways. 

Calcium Dependence of ATP-Induced DNA Synthesis

To examine whether an elevation of intracellular calcium is necessary for ATP-induced stimulation of DNA synthesis, the effect of the intracellular calcium chelator 1,2-bis (o-aminophenoxy)-N,N,N′-tetraacetic acid/acetoxymethyl ester (BAPTA/AM) was tested. BAPTA/AM (20 μM) had no effect, per se, on basal BrdU incorporation but fully reversed the ATP-stimulated DNA synthesis (Fig. 3A) . To determine whether the stimulation of DNA synthesis by ATP is dependent on an influx of calcium ions from the extracellular space into the Müller cells, nickel ions (40 μM) were added to the culture medium. Nickel ions block various calcium-permeable ion channels that are expressed in the plasma membrane of cells. The sensitivity of ATP-stimulated DNA synthesis to the presence of nickel ions (Fig. 3B) indicates that the ATP-induced stimulation of the DNA synthesis is dependent on calcium influx from the extracellular space. 

The calcium influx may serve to activate intracellular calcium-dependent enzymes. This assumption was tested in respect to different enzymes: protein kinase C and the calpains. Short-term application of phorbol ester (within minutes) activates protein kinase C, whereas long-term exposure leads to a downregulation of protein kinase C activity. ^{14 15} The effect of ATP on DNA synthesis is probably mediated by activation of protein kinase C, because depletion of phorbol ester–sensitive protein kinase C by long-term exposure of phorbol-12-myristate-13-acetate (PMA; 10 nM) inhibited the mitogenic effect of ATP (Fig. 4A) . PMA per se had no effect on the BrdU labeling of our cultures. Application of vehicle (DMSO) alone did not alter the DNA synthesis (control: 0.12 ± 0.02; DMSO: 0.14 ± 0.03, n = 4). Similar results were obtained by a protein kinase C inhibitor Gö6976 (100 nM; Fig. 4B ), which selectively inhibits calcium-dependent isoforms of protein kinase C. 

Other calcium-dependent enzymes are the calpains, a family of cysteine proteases that has been shown to be involved in gliosis-related upregulation of intermediate filament immunoreactivity. ¹⁶ To examine whether calpain activity is involved in the maintenance of Müller cell proliferation, calpain inhibitor I (500 nM) was tested. This inhibitor had, per se, no effect on basal BrdU incorporation but inhibited ATP’s effect (Fig. 4B) . Calpain inhibitor II, although structurally similar to calpain inhibitor I, did not attenuate the ATP-stimulated DNA synthesis (Fig. 4C) . The results indicate that the calcium ions that enter Müller cells after ATP stimulation may intracellularly activate several distinct calcium-dependent enzymes that are necessary for the maintenance of Müller cell proliferation. 

BK Channel Involvement in the Mitogenic Action of ATP

During proliferative gliosis of Müller cells, the activity of calcium-activated big-conductance potassium (BK) channels has been observed to be enhanced. ⁶ To determine whether activation of BK channels is necessary for the mitogenic effect of ATP, two blockers of BK channels were tested: iberiotoxin and charybdotoxin. Although iberiotoxin (70 nM) did not alter basal BrdU incorporation (control: 0.17 ± 0.04; iberiotoxin: 0.17 ± 0.05, n= 5), it fully inhibited the effect of ATP (500 μM) on the DNA synthesis (Fig. 5A) . Similar results were obtained with charybdotoxin (100 nM; Fig. 5B ). Tetrodotoxin (10 μM), a blocker of voltage-gated sodium channels, displayed no effect on the mitogenic effect of ATP (Fig. 5C) , suggesting that the effects of other channel blockers tested were not unspecific. The effect of serum (5%) on DNA synthesis was not inhibited by simultaneous application of iberiotoxin (100 nM; Fig. 5D ), which further support the view that ATP and serum evoke different intracellular signaling pathways in Müller cells. 

P2Y Receptor-Evoked Intracellular Calcium Transients

To explore whether the ion channel blockers tested exert their effects during the activation of P2Y receptors or after a time delay (i.e., downstream in the signaling pathway), fluorometric calcium imaging was performed on cultured cells. Extracellular application of ATP (50 μM) induced rapid increases in intracellular free calcium that slowly returned to the basal level within 3 to 10 minutes after the beginning of drug exposure (Fig. 6A) . When cells were preincubated for 5 minutes with the P2 receptor antagonist PPADS (200 μM), the ATP-evoked increase in intracellular calcium was largely depressed (Fig. 6A) . The experiments were performed on sister cultures with identical treatment to rule out effects of rapid receptor desensitization during multiple applications of ATP. Preincubation for 4 minutes with the phospholipase C inhibitor U73122 (4 μM) blocked the ATP (50 μM)-induced intracellular calcium transient by 95% (Fig. 6B) , suggesting the view that the increase of intracellular calcium was largely mediated by metabotropic P2Y receptors and that functional ionotropic P2X receptors were not present. Extracellular ATP releases intracellular calcium stores as indicated by the block of the ATP effect after preincubation of the cells with cyclopiazonic acid (5 μM; Fig. 6C ). Cyclopiazonic acid is an inhibitor of the endoplasmic reticulum calcium-ATPase, thus causing a slow release of intracellular calcium stores. 

To determine whether the ATP-induced intracellular calcium release evoked secondarily a calcium influx from the extracellular space into the Müller cells, calcium responses were evoked in regular (calcium-containing) extracellular solution and in calcium-free extracellular solution. As shown in Figure 7A , the ATP-evoked calcium transients were significantly shorter in calcium-free extracellular solution, indicating that addition of ATP also evoked a transient calcium influx from the extracellular space into the Müller cells. The mean decay time of the calcium transients (i.e., the duration from the beginning of the transients to the time point at which the transients decayed to 50% of their maximal amplitudes) was 188 ± 104 seconds (n = 39) in calcium-containing and 130 ± 64 seconds (n = 32) in calcium-free extracellular solution (P < 0.05). 

The presence of a calcium entry pathway that is activated by intracellular calcium release was determined by application of cyclopiazonic acid (5 μM) in calcium-free extracellular solution (Fig. 7B) . After readdition of calcium-containing solution, a prolonged and increased steady state increase in intracellular free calcium was observed, caused by calcium entry from the extracellular space. The data indicate that cultured Müller cells express calcium release-activated calcium (CRAC) channels in their plasma membranes that serve to refill released intracellular calcium stores. A similar increased steady state increase in intracellular free calcium was observed after readdition of calcium-containing solution when the internal calcium stores had been depleted by extracellular ATP (500μ M) in calcium-free extracellular solution (Fig. 7C) . The calcium influx from the extracellular space was decreased by 59% ± 31% (n = 16; P < 0.05) by 2-aminoethoxydiphenylborane (2-APB; 75 μM), a blocker of inositol 1′,4′,5′-triphosphate (IP₃) receptors and of CRAC channels. The data may indicate that extracellular ATP evokes an intracellular calcium release from IP₃ receptor-gated intracellular stores, which is followed by a transient calcium influx from the extracellular space, probably mediated by CRAC channels. 

Ion Channel Modulation of the ATP-Evoked Calcium Transients

We next examined whether ion channels contribute to the ATP-evoked calcium transients in cultured Müller cells. The experiments were performed on sister cultures with identical treatment. Coapplication of nickel ions (40 μM) and of ATP decreased the ATP-induced increase in intracellular calcium—that is, the intracellular calcium concentration returned to the basal level at a significantly shorter time than with the application of ATP alone (Fig. 8A) . Similarly, addition of iberiotoxin (100 nM; Fig. 8B ) to the bath solution significantly shortened the ATP-induced increase in intracellular calcium. The half-inactivation latency was 347 ± 136 seconds in the case of the ATP application and 165 ± 81 seconds in the case of the coapplication of ATP and iberiotoxin (P < 0.05; Fig. 8C ). To investigate the effect of blockers on the calcium entry more directly, prolonged CRAC entry was induced. Extracellular application of nickel ions (Fig. 8D) or of iberiotoxin (Fig. 8E) decreased the mean amplitude of this prolonged calcium influx by 35% ± 23% (n = 9; P < 0.05) and 87% ± 10% (n = 35; P < 0.05), respectively. The present results indicate that both calcium-permeable ion channels and BK channels are necessary to maintain the calcium influx into Müller cells after P2Y receptor stimulation and implicate calcium influx regulation as a site for BK channel action on Müller cell proliferation. 

Duration of ATP-Evoked Calcium Transients and DNA Synthesis Rate

Both the ATP-induced DNA synthesis rate (Figs. 3B 5A) and the duration of the ATP-evoked calcium transients (Fig. 8) were found to be dependent on calcium influx from the extracellular space and on the activity of BK channels in cultured Müller cells. Therefore, we determined whether the duration of the ATP-evoked calcium transient is related to the DNA synthesis rate in different independent cultures. As shown in Figure 9 , there is a small but significant correlation between both parameters. The longer the mean calcium transient of a culture, the higher the basal rate of DNA synthesis in this culture (r = 0.59, n = 16 cultures, P < 0.01). 

Discussion

In the present study, for first time, cultured Müller cells were shown to express P2Y receptors, and activation of these receptors resulted in a stimulation of DNA synthesis. The mitogenic action of ATP was dependent on an increase of the intracellular calcium concentration, on calcium influx into Müller cells, and on the activity of calcium-dependent potassium (BK) channels. The calcium influx may serve to stimulate the activity of different calcium-dependent enzymes that must be activated to maintain Müller cell proliferation (e.g., protein kinase C and calpains). BK channels may be involved in the regulation of the strength of the calcium influx into Müller cells and therefore in the regulation of Müller cell proliferation, ¹³ as indicated by the correlation between the duration of the ATP-evoked calcium transients and the DNA synthesis rate. 

In cultured Müller cells of the guinea pig, we found no evidence for functional P2X receptors, based on the following two findings:α ,β-Methylene-ATP, an agonist for several P2X receptor subtypes, did not increase the DNA synthesis rate (Fig. 2A) , and after preincubation of the cells with a phospholipase C inhibitor, the ATP-evoked calcium transients were largely depressed (Fig. 5B) . It is unclear whether guinea pig Müller cells do not express P2X receptor protein or whether these receptors were not active at the conditions used. The present results are in agreement with patch–clamp studies on freshly isolated Müller cells from the guinea pig, in which we were unable to detect ATP-induced cationic currents that would indicate the presence of P2X receptors (Pannicke T, unpublished data, 2001). Similarly, freshly isolated Müller cells of the rat displayed no cationic currents in response to different P2X receptor agonists, indicating that these cells do not express functional P2X receptors. ¹⁷  

Both BK channels and calcium-permeable ion channels have been implicated in the regulation of growth factor–induced proliferation of Müller cells. ^{13 18 19} The present results extend these findings and show that the activity of BK channels is also necessary for the ATP-induced proliferation of Müller cells. Although it cannot be ruled out that secondary steps after primary P2Y receptor activation may be modulated by BK channels, the present results support the view that they are already involved in the primary signaling transduction process just after binding of external ATP to the P2Y receptors, causing an elevation of intracellular free calcium through activation of phospholipase C and release of IP₃-gated intracellular calcium stores, which is followed by a transient influx of calcium ions from the extracellular space. BK channels may have a crucial role in supporting calcium influx from the extracellular space after P2Y receptor activation, probably through membrane hyperpolarization that results in an enhanced electrochemical driving force for calcium influx through open calcium-permeable channels. ²⁰ Blocking BK channels inhibits the ATP-induced calcium influx into Müller cells (Figs. 8B 8C 8E) as well as the mitogenic effect of ATP (Figs. 5A 5B) . 

The type of channel that mediates the P2Y receptor-mediated calcium influx in Müller cells is unclear and difficult to determine, because specific blockers are not available. The ATP-induced intracellular calcium release may secondarily cause an opening of CRAC channels in the Müller cell membranes that serve to refill the intracellular calcium stores. Indeed, the calcium-imaging experiments shown in Figure 7 indicate that cultured Müller cells express CRAC channels in their plasma membranes. Because proliferating Müller cells, at least from the human ^{5 6} and rabbit retinas, ²¹ have no inwardly rectifying potassium channels, they show a relatively low membrane potential. Opening of BK channels should hyperpolarize the membrane around the calcium-permeable CRAC channel to levels near the potassium equilibrium potential. This should strongly facilitate the influx of calcium ions from the extracellular space. Calcium-activated, calcium-permeable cation channels have been described to be present in cultured human Müller cells and to be activated by bFGF. ²² A relation between an enhanced capacitative calcium entry and an increased proliferation rate was recently established in arterial myocytes. ²³ A similar correlation between the DNA synthesis rate and the duration of the ATP-evoked calcium transients could be established for cultured Müller cells (Fig. 9) . 

An involvement of purinergic receptors in induction or maintenance of gliosis has been proposed, based on the observation that P2 receptor agonists, when infused into the brain, induce astrogliosis, leading to hypertrophy and proliferation of astrocytes. ^{24 25} ATP is a mitogen for multipotent precursor cells during retinal development, ²⁶ and the P2 receptor-evoked responses of Müller cells were found to be enhanced during proliferative Müller cell gliosis. ⁷ Both the increased activity of BK channels ⁶ and the enhanced expression of P2 receptors ⁷ may support Müller cell proliferation during gliosis, probably through an enhanced calcium influx. 

In summary, activation of P2Y receptors in cultured Müller glial cells resulted in a stimulation of DNA synthesis. Activation of P2Y receptors led to an increase of intracellular free calcium and to an influx of extracellular calcium. The increase of the intracellular calcium activated BK channels. BK channel activity may support the calcium influx and therefore the ATP-induced stimulation of DNA synthesis. 

²  These authors contributed equally to the work.

 Supported by Grant 01KS9504, Project C5, from the Bundesministerium für Bildung und Forschung (BMBF; Federal Ministry of Education, Science, Research, and Technology) to the Interdisciplinary Center for Clinical Research (Interdisziplinäre Zentrum klinische Forschung; IZKF) at the University of Leipzig; and by Grant Re 849/8-1 from the Deutsche Forschungsgemeinschaft (DFG).

 Submitted for publication June 7, 2001; revised October 26, 2001; accepted November 13, 2001.

 Commercial relationships policy: N.

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked“ advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Cultured Müller cells of the guinea pig. (A) The majority of cultured cells expressed GFAP immunoreactivity (red). The cell nuclei were counterstained with Hoechst 33258 (blue). (B) Cell nuclei that showed DNA synthesis were stained using BrdU immunocytochemistry (red). All cell nuclei were counterstained with acridine orange (green). BrdU-positive cell nuclei are shown by yellow-orange double labeling. Arrows: sites of chromosome accumulations in a cell that is in the telophase of cell cycling. Scale bars, 20 μm.

Figure 2.

 

The mitogenic effect of extracellular nucleotides was mediated by P2Y receptors. (A) Effects of different nucleotides and of adenosine, respectively, on BrdU incorporation. (B) The ATP-induced elevation of the DNA synthesis rate was blocked by suramin (10 μM) or by PPADS (10 μM). (C) Concentration–response curve for ATP. Extracellular ATP was tested at concentrations of 0.1, 1, 10, 50, 100, 250, and 500 μM. The curve was fitted with f = (b · x)/(x + a), with a = 5.9 μM and b = 0.435. (D) The effects of ATP (500 μM) and fetal calf serum (5%) on DNA synthesis were additive. Data are the mean of three to six independent experiments. (•) Significantly different from control levels (P < 0.05); (○) significant effects of the blockers (P < 0.05).

Figure 3.

 

The mitogenic effect of ATP was dependent on an influx of calcium from the extracellular space. Buffering of intracellular free calcium by (A) BAPTA/AM (20 μM) or exposure to (B) nickel ions (40 μM) inhibited the DNA synthesis induced by ATP (500 μM). Data are the mean of four independent experiments. (•) Significantly different from control levels (P < 0.05); (○) significant effects of the blockers (P < 0.05).

Figure 4.

 

Activation of calcium-dependent enzymes was necessary for ATP-induced Müller cell proliferation. (A) Downregulation of protein kinase C by long-term exposure to PMA (10 nM) inhibited the DNA synthesis induced by ATP. (B) Similarly, the mitogenic effect of ATP was blocked by Gö6976 (100 nM), which selectively inhibits calcium-dependent isoforms of protein kinase C. (C) Calpain inhibitor I (Ci-I, 500 nM) inhibited the mitogenic effect of ATP. (D) However, Ci-II (500 nM) displayed no significant effect on ATP-induced DNA synthesis. ATP was added to the culture medium at a concentration of 500 μM. Means of three to five independent experiments. (•) Significantly different from control levels (P < 0.05); (○) significant effects of the blockers (P < 0.05).

Figure 5.

 

ATP (500 μM)-induced DNA synthesis was inhibited by blockers of BK channels. (A) Iberiotoxin (70 nM) reversed the mitogenic effect of ATP. (B) Charybdotoxin (100 nM) inhibited the ATP effect. (C) Simultaneous application of tetrodotoxin (10μ M), a blocker of voltage-gated sodium channels, did not inhibit ATP’s effect. (D) Iberiotoxin (100 nM) did not prevent the increase of the DNA synthesis evoked by fetal calf serum (5%). Data are the mean of 3 to 11 independent experiments. (•) Significantly different from control values (P < 0.05); (○) significant effects of the blockers (P < 0.05).

Figure 6.

 

Extracellular ATP induced intracellular calcium transients through activation of P2Y receptors in cultured Müller cells. The intracellular calcium concentration was recorded by fura-2 fluorometry. (A) The extracellular ATP (50 μM)-induced calcium transient was largely inhibited when PPADS (200 μM) was preincubated for 5 minutes and was coapplied with ATP. Mean curves of 64 cells (ATP) and of 63 cells (ATP + PPADS), respectively, from sister cultures with identical treatment. (B) The extracellular ATP (50μ M)-induced transient elevation of intracellular calcium was blocked in the presence of the phospholipase C inhibitor U73122 (4 μM). The blocker was preincubated for 4 minutes before ATP application and was applied simultaneously with ATP. Mean curves of 85 cells (ATP) and of 76 cells (ATP + U73122), respectively, from sister cultures with identical treatment. (C) After preincubation of the cells with cyclopiazonic acid (5 μM), ATP’s effect at 500 μM on intracellular calcium concentration was blocked. Mean (±SD) curve of 73 cells.

Figure 7.

 

The ATP-evoked calcium transients were caused by intracellular calcium release and subsequent transient activation of a calcium entry pathway in the plasma membrane. The intracellular calcium responses were recorded by fura-2 fluorometry. (A) Comparison of calcium transients that were evoked by extracellular ATP (500 μM) in the presence and absence of calcium in the extracellular solution. Mean curves are shown of 32 cells (presence of calcium) and of 39 cells (absence of calcium) from sister cultures with identical treatment. (B) Extracellular application of cyclopiazonic acid (5 μM) in calcium-free extracellular solution induced a slow increase in intracellular calcium-free concentration. After a changing was made to a calcium-containing extracellular solution, there was a prolonged steady state increase in intracellular free calcium (mean ± SD curve of 40 cells). (C) A similar effect was observed when ATP (500 μM) was applied in calcium-free extracellular solution. After a change to calcium-containing extracellular solution, an increase in intracellular free calcium was observed (mean ± SD curve of 32 cells). (D) The ATP-induced calcium influx from the extracellular space is inhibited by 2-aminoethoxydiphenylborane (2-APB; 75 μM), a blocker of IP₃ receptors and of calcium release-activated calcium channels. Example of record in one cell.

Figure 8.

 

Blockers of BK or of calcium channels reduced the calcium influx from the extracellular space in response to external ATP. The intracellular calcium response was recorded by fura-2 fluorometry. (A) Coapplication of nickel ions (40 μM) shortened the ATP-induced increase in intracellular free calcium. Mean curves of 27 and 17 cells, respectively, are shown from sister cultures with identical treatment. (B) Coapplication of iberiotoxin (100 nM) caused a shortening of the ATP-induced calcium response. Mean curves of 57 and 61 cells, respectively, are shown from sister cultures with identical treatment. (C) Mean ± SD decay times of the calcium transients (i.e., the duration from the beginning of the calcium transient to the time point at which the transients decayed to 50% of maximal amplitudes). Blockers were applied 2 minutes before and during ATP application. (D) Application of nickel ions (40 μM) decreased the calcium influx that was evoked by readdition of calcium-containing extracellular solution after ATP application in calcium-free solution. Example of recordings in three cells. (E) Application of iberiotoxin (100 nM) had a similar effect on the calcium influx. Example of recordings in three cells. ATP was applied at 500 μM. (•), P < 0.05.

Figure 9.

 

Dependence of the mean basal DNA synthesis rates of 16 independent cultures on the mean decay times of the ATP (500 μM)-evoked calcium responses. The decay time indicates the duration from the beginning of the calcium transient to the time point at which the transients decayed to 50% of their maximal amplitudes. The BrdU labeling data are averages of three coverslips per culture. The calcium imaging data represent the mean of 24 to 89 cells from sister cultures with identical treatment.

The authors thank Jana Krenzlin for preparation of the cell cultures.