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Am J Physiol Regul Integr Comp Physiol 282: R1077-R1085, 2002; doi:10.1152/ajpregu.00564.2001
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Vol. 282, Issue 4, R1077-R1085, April 2002

ATP-stimulated Ca2+-activated K+ efflux pathway and differentiation of human placental cytotrophoblast cells

L. H. Clarson1, V. H. J. Roberts1, S. L. Greenwood1, and A. C. Elliott2

1 Academic Unit of Child Health, University of Manchester, St. Mary's Hospital, Manchester M13 0JH and 2 School of Biological Sciences, University of Manchester, Manchester M13 9PL, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of this study was to determine whether extracellular ATP ([ATP]o) stimulated a Ca2+-activated K+ efflux in trophoblast cells that was dependent on extracellular Ca2+ ([Ca2+]o). Cytotrophoblast cells, isolated from human placenta, were examined following 18 h (relatively undifferentiated) and 66 h (multinucleate cells) of culture. Potassium efflux was measured using 86Rb as a trace marker. Intracellular Ca2+ ([Ca2+]i) was examined by microfluorometry using fura 2. [ATP]o significantly increased 86Rb efflux to a peak that declined to control (18-h cells) or an elevated plateau (66-h cells) and was inhibited by 100 nM charybdotoxin. Removing [Ca2+]o significantly reduced 86Rb efflux in both groups as did application of 150 µM GdCl3. [ATP]o significantly increased [Ca2+]i in both groups of cells. The response was reduced by removing [Ca2+]o and applying 150 µM GdCl3. For both 86Rb efflux and microfluorometry experiments, the response to [ATP]o was more dependent on [Ca2+]o in 66-h cells compared with 18-h cells (~70% greater). Cytotrophoblast cells exhibit an [ATP]o-stimulated Ca2+-activated K+ efflux. The dependency of this pathway on [Ca2+]o is greater in the 66-h multinucleate syncytiotrophoblast-like cells, suggesting that the mechanism for Ca2+ entry may be altered during differentiation of trophoblast cells.

intermediate calcium-activated potassium channel; calcium entry; human placenta


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE TRANSPORTING EPITHELIUM of the human placenta, the syncytiotrophoblast, is formed by differentiation of stem cytotrophoblast cells, such that these mononuclear cells fuse to form the multinucleate syncytiotrophoblast layer. This process, which is ongoing throughout gestation, can be reproduced in vitro using cytotrophoblast cells isolated from human term placenta (7, 18). Ion exchange across the syncytiotrophoblast is essential for normal fetal homeostasis; however, little is known about the expression and function of ion conductances in the syncytiotrophoblast or during its formation by differentiation, or how these conductances may be regulated by agonists. Information concerning such regulation can be summarized as follows.

Cronier et al. (12) showed that Cl- currents in cultured cytotrophoblast cells could be regulated by the placental specific hormone human chorionic gonadotrophin as well as by intracellular cAMP. A Ca2+-activated Cl- current and a volume-activated Cl- current were also identified in these cells (28), as well as a volume-stimulated Ca2+-activated Cl- efflux pathway (50). Regulation of cation conductances is less well defined. However, there is preliminary evidence showing activation of a K+ efflux pathway following exposure of cytotrophoblast cells to the Ca2+ ionophore A-23187 (46), as well as a volume-stimulated Ca2+-activated K+ efflux pathway (11, 19).

The placenta is unique among the organs of the human body, in that it is not innervated. The role of humoral and autocrine/paracrine factors is, consequently, of great importance in the regulation of both tissue homeostasis and nutrient transfer to the fetus. In other tissues, such as the nervous system and epithelia, extracellular ATP is important as a paracrine/autocrine regulator of cell function (17, 40, 49) and is involved in activation of both K+ and Cl- conductances (4, 13, 15, 31, 41, 43). It is thought that ATP is released from cells following shear stress/cell activation, in response to hyposmotic challenge and/or via ATP-binding cassette (ABC) transporters (1, 5, 13, 40, 44). In the human placenta there is evidence for the ABC transporters cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance (MDR) on the microvillous membrane of the syncytiotrophoblast, which might provide a pathway for cellular ATP exit (2, 14). There are also data demonstrating increased intracellular Ca2+ ([Ca2+]i) in isolated cytotrophoblast cells following exposure to extracellular ATP (26, 39). Thus ATP may be an important autocrine/paracrine regulator in the human placenta.

Regulation of cation channels following stimulation by agonists has not hitherto been examined in the syncytiotrophoblast. Thus the overall aim of our work is to determine mechanisms of regulation of ion transport by the trophoblast, particularly in regard to the role of [Ca2+]i. In the present study we tested the hypothesis that ATP stimulates a K+ efflux pathway in cytotrophoblast cells via elevation of [Ca2+]i. We determined 1) whether extracellular ATP stimulated directly a Ca2+-activated K+ efflux pathway in cultured cytotrophoblast cells, 2) how extracellular ATP raised [Ca2+]i in these cultured cytotrophoblast cells, and 3) the effect of cytotrophoblast cell differentiation on these processes. A preliminary account of this work has been presented previously (9-11).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

Na2ATP, apamin, iberiotoxin, and charybdotoxin were purchased from Sigma-Aldrich (Poole, UK), SKF-96365 from Calbiochem (Nottingham, UK), and fura 2-AM from Molecular Probes Europe (Leiden, The Netherlands). All other reagents were analytical grade from standard suppliers.

Cell Isolation and Culture

Cytotrophoblast cells were isolated using an adaptation of the method of Kliman et al. (30) as previously described in detail (7, 18). Briefly, human placentas were collected within 30 min of delivery, and the villous tissue was removed, roughly minced, and washed thoroughly with saline. The dissected tissue was digested in trypsin and DNase, and the isolated cells were separated by centrifuging through a discontinuous Percoll gradient. The isolated cytotrophoblast cells were plated into 35-mm culture dishes (~4 × 106 cells per 35-mm dish) for efflux experiments or onto flamed 16-mm glass coverslips in 12-well plates for microfluorometry experiments. All cells were maintained at 37°C in a humidified incubator gassed with 5% CO2-95% air for up to 3 days of culture. We have previously shown that during this time cytotrophoblast cells isolated from human term placenta undergo both morphological and biochemical differentiation to form multinuclear syncytiotrophoblast-like cells (7, 18). Cells were studied at two time points in culture: after 18 h, when the majority of cells are mononuclear, and after 66 h, when the majority of cells are multinucleate syncytiotrophoblast-like cells.

86Rb Efflux

It has previously been demonstrated that 86Rb can be used as a congener of K+ in human placenta (3) and that Ca2+-activated K+ channels are permeable to Rb+ (22); therefore, for the purposes of this study, 86Rb was used as a trace isotope for K+. Cytotrophoblast cells at either 18 or 66 h in culture were loaded with 5 µCi/ml 86Rb in control Tyrode buffer (composition in mM: 135 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2, 5.6 glucose, 10 HEPES pH 7.4 with 10 M NaOH) for 2 h at room temperature. Cells were washed with 50 ml control buffer over 3 min to remove extracellular isotope. Sequential 1-ml samples were collected over 10 min at 1-min intervals by replacing the incubation medium with fresh buffer at the beginning of each 1-min period. At the end of the experimental period, cells were lysed in 0.3 M NaOH for 1-2 h to determine the amount of 86Rb remaining in the cells. All samples were counted in a Packard gamma counter to determine 86Rb activity. For experiments examining the effect of removing extracellular Ca2+, following loading with isotope, the cells were washed in a Ca2+-free Tyrode solution (as described above with no added CaCl2 and containing 0.5 mM EGTA), and thereafter the experiment was carried out in Ca2+-free buffer. Experiments were carried out with an agonist (100 µM ATP) in the presence and absence of extracellular Ca2+ and inhibitors of Ca2+-activated K+ channels (apamin, charybdotoxin, iberiotoxin) or of Ca2+ entry (GdCl3 and SKF-96365). The agonist ATP was applied after the first 5 min of the efflux time course and throughout the remainder of the experiment. Channel blockers were added 1 min before application of ATP.

Efflux of 86Rb was expressed as percent per minute and was calculated as
<FR><NU><SUP>86</SUP>Rb in 1-min efflux sample cell</NU><DE><SUP>86</SUP>Rb at start of 1-min collection</DE></FR><IT>×</IT>100

Microfluorometry

Cells were loaded with 1 µM fura 2-AM in control Tyrode buffer containing 1% BSA for 30-45 min at 37°C. The coverslip was transferred to a small superfusion chamber (150-µl vol; Warner Instruments, Hamden, CT) on the stage of a Nikon Diaphot inverted microscope. The cells were superfused with control Tyrode solution from a gravity-fed perfusion system at a rate of 3 ml/min. Fluorescence was excited and observed through a Nikon ×40 oil-immersion objective (numerical aperture 1.3). Excitation light at 340 and 380 nm was supplied via a filter wheel driven by a stepper motor (Newcastle Photometric Systems). A diaphragm in the emitted light path was used to limit light collection to a small cluster of cells (~2-3 cells), and emitted fluorescence was measured by a photomultiplier tube and a Newcastle Photometric Systems photon-counting system. Fura 2 signals were not calibrated in terms of absolute values of [Ca2+]i, since the accuracy of such estimates is debatable (52) and since calibration was not necessary for the analysis of the data.

Cells were stimulated by superfusion of extracellular ATP, which was applied for 2 min. To assess the role of extracellular Ca2+, experiments were repeated in Ca2+-free Tyrode buffer (composition as above), which was present for 8 min before application of ATP (comparable with 86Rb efflux experiments) and for the duration of ATP exposure. The Ca2+ entry blocker GdCl3 was added 1 min before ATP.

Data Analysis and Statistics

All data are expressed as means ± SE, where n is the number of placentas from which cells were isolated.

Efflux time courses were assessed statistically using a repeated-measures ANOVA to show overall change with time and an ANOVA with Bonferroni correction to determine differences at individual time points. Rate constants of 86Rb efflux from 18- and 66-h cytotrophoblast cells were calculated over 5-10 min for each experimental condition (control, Ca2+-free, ATP ± channel blockers). The rate constants were calculated from plots of ln[Rbi(t)/Rbi(t = 0)] as a function of time, where Rbi(t = 0) were the counts associated with the cells at the start of the time course and Rbi(t) were the counts remaining at time t. Least-squares linear regression was used to analyze the data, and the negative slopes of the graph were taken as a measure of the unidirectional efflux rate constants (min-1). The area under the curve was calculated from 5 min to 10 min of the time course to determine total efflux over the experimental period and expressed as percent increase above control. Differences in efflux between 18- and 66-h cytotrophoblast cells were examined using the nonparametric Mann-Whitney U-test.

For microfluorometry data, the change in fura 2 fluorescence ratio above control (Delta R) was measured at the peak response to ATP and following 2 min of ATP exposure. Data were analyzed statistically using an ANOVA with Bonferroni correction.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

86Rb Efflux

Rate constants, taken as the slopes of the regression lines fitted over the experimental period (5-10 min), were calculated for all conditions tested, and values for 18- and 66-h cytotrophoblast cells are given in Table 1. In every case the data could be fitted by a single exponential, consistent with loss from an intracellular source. For the purposes of this study, we routinely plotted the data as the time course of percent 86Rb efflux per minute.

                              
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  		Table 1.   Rate constant data for 86Rb efflux from 18- and 66-h cytotrophoblast cells

ATP stimulation. Basal (unstimulated) 86Rb efflux from both 18- and 66-h cytotrophoblast cells was stable over 3-10 min (see Figs. 1 and 2). Addition of 100 µM ATP at 5 min caused a rapid, significant increase in 86Rb efflux that reached a peak at 6 min. In 18-h cytotrophoblast cells, 86Rb efflux subsequently declined back to control values. However, in 66-h cytotrophoblast cells, 86Rb efflux declined to a plateau (from 8 to 10 min), which was significantly elevated above control (see Figs. 1 and 2).


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  		Fig. 1.   Effect of Ca2+-activated K+ channel blockers on ATP-stimulated 86Rb efflux from 18-h cytotrophoblast cells (A) and 66-h cytotrophoblast cells (B). The solid bar indicates application of ATP, the open bar indicates application of channel blockers; , control; black-triangle, 100 µM ATP; , 100 µM ATP + 100 nM iberiotoxin (ibtx); triangle , 100 µM ATP + 100 nM apamin (ap); down-triangle, 100 µM ATP + 100 nM charybdotoxin (chtx). Values are means ± SE, n = 3. *** P < 0.001, ** P < 0.01, * P < 0.05, ATP vs. control; +++P < 0.001, ++P < 0.01, +P < 0.05, ATP + chtx vs. ATP. ATP + ap/ibtx was not significantly different from ATP.



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  		Fig. 2.   ATP-stimulated 86Rb efflux in Ca2+-free buffer from 18-h cytotrophoblast cells (A) and 66-h cytotrophoblast cells (B). , Control; open circle , Ca2+-free buffer; black-triangle, 100 µM ATP; triangle , 100 µM ATP + Ca2+-free buffer. ATP-stimulated 86Rb efflux in the presence of GdCl3 from 18-h cytotrophoblast cells (C) and 66-h cytotrophoblast cells (D). , Control; black-triangle, 100 µM ATP; triangle , 100 µM ATP + 150 µM GdCl3. ATP-stimulated 86Rb efflux in the presence of SKF-96365 in 18-h cytotrophoblast cells (E) and 66-h cytotrophoblast cells (F). , Control; black-triangle, 100 µM ATP; triangle , 100 µM ATP + 30 µM SKF-96365. Values are means ± SE, n = 6. *** P < 0.001, ** P < 0.01, * P < 0.05, vs. control; +++P < 0.001, ++P < 0.01, +P < 0.05 vs. ATP.

Ca2+-activated K+ channel blockers. We tested whether 86Rb efflux was affected by several selective blockers of Ca2+-activated K+ channels (apamin, iberiotoxin, and charybdotoxin). Neither apamin nor iberiotoxin had any effect on ATP-stimulated 86Rb efflux from either 18- or 66-h cytotrophoblast cells. However, application of charybdotoxin to both 18- and 66-h cytotrophoblast cells largely prevented ATP-stimulated 86Rb efflux (see Fig. 1), suggesting stimulation of a Ca2+-activated K+ efflux pathway.

Ca2+ entry. We next examined the effect of removing extracellular Ca2+ on both basal and ATP-stimulated 86Rb efflux. In both 18- and 66-h cytotrophoblast cells, incubation in Ca2+-free Tyrode solution caused no significant change in basal efflux. However, the peak ATP-stimulated 86Rb efflux was reduced in both 18- and 66-h cytotrophoblast (Fig. 2, A and B). The plateau response in 66-h cytotrophoblast cells was also significantly reduced to control values (Fig. 2B). The remaining ATP-stimulated increase in 86Rb efflux in Ca2+-free buffer was abolished by 100 nM charybdotoxin (data not shown). This suggests that the residual 86Rb efflux (in Ca2+-free buffer) was Ca2+ dependent, most likely stimulated by release of Ca2+ from intracellular stores.

The effect of blockers of Ca2+ entry via Ca2+-permeable channels was also examined. Trivalent lanthanides block most Ca2+ entry pathways. In keeping with this, 150 µM GdCl3 significantly reduced the peak ATP-stimulated 86Rb efflux at 6 min in both 18- and 66-h cytotrophoblast cells (Fig. 2, C and D). In 66-h cytotrophoblast cells, GdCl3 also significantly reduced the elevated plateau in response to ATP down to control levels (see Fig. 2D). This response was similar to that seen with Ca2+-free buffer. In contrast, the organic Ca2+ entry blocker SKF-96365 had no significant effect on ATP-stimulated 86Rb efflux from 18-h cytotrophoblast cells. It did cause a small but consistent reduction in 66-h cytotrophoblast cells over the duration of the experimental period, but this reduction was only significant at the 10% level (Fig. 2, E and F).

Figure 3 shows data for total efflux, calculated as area under the curve, expressed as a proportion of control efflux. ATP-stimulated 86Rb efflux was significantly greater in 66-h compared with 18-h cytotrophoblast cells. However, charybdotoxin inhibited all the ATP-stimulated 86Rb efflux in both cell types, thus demonstrating that all 86Rb efflux occurred via the Ca2+-activated K+ efflux pathway. Stimulation of this efflux pathway in the absence of extracellular Ca2+ was considerably reduced, but the remaining efflux was not significantly different between 18- and 66-h cytotrophoblast cells. Thus the large increase in ATP-stimulated Ca2+-activated K+ efflux from 66-h cytotrophoblast cells was dependent predominantly on extracellular Ca2+. Indeed, the extracellular Ca2+- dependent component (i.e., ATP-stimulated 86Rb efflux in the absence of extracellular Ca2+ subtracted from ATP-stimulated 86Rb efflux in the presence of extracellular Ca2+) for 66-h cytotrophoblast cells was 70% greater than that of 18-h cytotrophoblast cells. Furthermore, the Gd3+- and SKF-96365-insensitive components of 86Rb efflux were also not significantly different in 18- and 66-h cytotrophoblast cells, whereas the Gd3+- and SKF-96365-sensitive components were 3 times and 4.5 times greater, respectively in 66-h cytotrophoblast cells compared with 18-h cytotrophoblast cells. These data indicate that the [Ca2+]i increase evoked by ATP is considerably more dependent on extracellular Ca2+ in 66-h than in 18-h cytotrophoblast cells, and thus suggest that the pathways for Ca2+ entry in cytotrophoblast cells may be altered with differentiation.


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  		Fig. 3.   Total 86Rb effluxed from cells over the experimental period (5-10 min of efflux time course) expressed as a percent of control. A, 100 µM ATP (n = 16, 18-h cytotrophoblast cells; n = 15, 66-h cytotrophoblast cells); A+0C, 100 µM ATP in Ca2+-free buffer (n = 6 both groups); A+G, 100 µM ATP + 150 µM GdCl3 (n = 6 both groups); A+S, 100 µM ATP + 30 µM SKF-96365 (n = 6 both groups); A+Ch, 100 µM ATP + 100 nM charybdotoxin (n = 3 both groups). Values are means ± SE. +++P < 0.001 18-h vs. 66-h cytotrophoblast cells (nonparametric Mann-Whitney test).

Microfluorometry

ATP stimulation. There was no significant difference in resting fluorescence ratio between 18- and 66-h cytotrophoblast cells (means ± SE: 0.52 ± 0.01, n = 60, and 0.55 ± 0.02, n = 52, respectively). Application of extracellular ATP to both 18- and 66-h cytotrophoblast cells caused a rapid increase in [Ca2+]i (as indicated by the change in fura 2 fluorescence ratio). This increase in [Ca2+]i then declined, in the continuous presence of ATP, toward a sustained plateau (see Fig. 4) in ~85% of cells. The sustained plateau in 66-h cytotrophoblast cells was noticeably more prominent than that seen in 18-h cytotrophoblast cells. Cells responded to increasing concentrations of ATP (from 1 to 100 µM) with changes in the fura 2 ratio of increasing magnitude (Fig. 5). Both groups of cells demonstrated receptor desensitization with both 10 and 100 µM ATP (i.e., there was a decrease in magnitude of response to ATP following second and third applications), which was most marked with 100 µM ATP (data not shown).


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  		Fig. 4.   ATP-stimulated increase in intracellular Ca2+ ([Ca2+]i), as reflected by the change in fluorescence ratio in 18-h cytotrophoblast cells (A) and 66-h cytotrophoblast cells (B). Arrows indicate points for measurement. C, control (measurement is made prior to ATP application); +Ca2+, bath contains 1.8 mM Ca2+; 0Ca, recording in the absence of extracellular Ca2+; peak, maximum increase in fluorescence ratio in response to ATP; plat, measurement after application of ATP for 2 min. The increase above control (Delta R) for the peak response and after 2 min of ATP is used for statistical analysis.



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  		Fig. 5.   Change in [Ca2+]i in response to increasing concentrations of ATP in 18-h cytotrophoblast cells (A) and 66-h cytotrophoblast cells (B). Delta R, change in ratio above control measured at the peak response to ATP (peak) and after 2 min application (plat); open bars, 1 µM ATP; hatched bars, 10 µM ATP; solid bars, 100 µM ATP. Values are means ± SE, n = 5. * P < 0.05 vs. peak 1 µM ATP; +P < 0.05 vs. plat 1 µM ATP.

Ca2+ entry. We also examined ATP-stimulated changes in [Ca2+]i in Ca2+-free buffer to determine whether entry of extracellular Ca2+ was important for this response. For this series of experiments, 10 µM ATP was used to stimulate cells, since desensitization was less marked with this concentration of ATP than with 100 µM ATP. In addition, the duration of ATP stimulation was limited to 2 min. Although this was a shorter exposure than that used for the efflux experiments, this was found to be necessary to strike a balance between allowing us to obtain a second response to ATP (albeit reduced) and maintaining the exposure to ATP for some time beyond the peak of the 86Rb efflux (~1 min after exposure to ATP). Thus, following the response in Ca2+-free buffer, stimulation with ATP was repeated in the presence of extracellular Ca2+ to ensure that cells could respond with an increase in [Ca2+]i. Cells were exposed to Ca2+-free buffer for 8 min before application of ATP, so that the duration of exposure to Ca2+-free conditions was comparable to that in 86Rb efflux experiments. In both 18- and 66-h cytotrophoblast cells, exposure to Ca2+-free buffer significantly reduced the peak response to ATP (Figs. 4 and 6). This reduction was considerably greater in 66-h cytotrophoblast cells than in 18-h cytotrophoblast cells (>90% and 65%, respectively). Incubation in Ca2+-free buffer also reduced the [Ca2+]i increase after 2 min of stimulation, although this was not significant at the 5% level in 18-h cytotrophoblast cells. Once again the reduction was greater in 66-h compared with 18-h cytotrophoblast cells (90% and 65%, respectively). These data suggest that extracellular Ca2+ makes a greater contribution to ATP-stimulated changes in [Ca2+]i in 66-h cytotrophoblast cells compared with 18-h cytotrophoblast cells.


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  		Fig. 6.   Effect of Ca2+-free buffer on ATP-stimulated increase in [Ca2+]i. Delta R, change in fluorescence ratio above control; open bars, 10 µM ATP peak (control); solid bars, 10 µM ATP after 2 min (control); hatched bars, 10 µM ATP peak response (Ca2+-free); cross-hatched bars, 10 µM ATP after 2 min (Ca2+-free). Values are means ± SE, n = 5. *** P < 0.001, ** P < 0.01, * P < 0.05 vs. peak control ATP; +P < 0.05 vs. control ATP after 2 min.

We tested the effect of Gd3+ on ATP-stimulated changes in [Ca2+]i in both groups of cells (Fig. 7). We added GdCl3 1 min before addition of 100 µM ATP, again corresponding to the 86Rb efflux protocol. Addition of GdCl3 significantly reduced both the peak [Ca2+]i response and the [Ca2+]i increase after 2 min of ATP exposure in 18- and 66-h cytotrophoblast cells. The GdCl3-sensitive component (response in the presence of GdCl3 subtracted from the control response to ATP) was similar for the peak increase in [Ca2+]i in both 18- and 66-h cytotrophoblast cells (~75%). However, the GdCl3-sensitive component of [Ca2+]i increase after 2 min of ATP exposure was greater in 66-h compared with 18-h cytotrophoblast cells (65% and 45%, respectively). These data, taken together, once again support the hypothesis that ATP-stimulated changes in [Ca2+]i show a greater dependence on extracellular Ca2+ in 66-h cytotrophoblast cells.


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  		Fig. 7.   Effect of GdCl3 on ATP-stimulated increase in [Ca2+]i. Delta R, change in fluorescence ratio above control; open bars, 100 µM ATP peak; solid bars, 100 µM ATP after 2 min; hatched bars, 100 µM ATP peak + 150 µM GdCl3; cross-hatched bars, 10 µM ATP after 2 min + 150 µM GdCl3. Values are means ± SE, n = 5. *** P < 0.001 vs. ATP peak; +P < 0.05 vs. ATP after 2 min.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have identified an ATP-stimulated K+ (86Rb) efflux pathway in cultured cytotrophoblast cells, which is sensitive to charybdotoxin and is largely dependent on extracellular Ca2+. This is the first demonstration of such a pathway in human placental cytotrophoblast cells. Interestingly, the requirement for extracellular Ca2+ for stimulation of this pathway by ATP appears to be more marked in 66-h cytotrophoblast cells compared with 18-h cytotrophoblast cells. This suggests that the mechanism by which ATP raises [Ca2+]i in these cells alters with differentiation.

Characterization of the K+ Channel Underlying the 86Rb Efflux Pathway in Cytotrophoblast Cells

We used selective peptide blockers to characterize the K+ channel underlying the ATP-stimulated K+ (86Rb) efflux pathway identified in this study. Broadly, Ca2+-activated K+ channels can be distinguished into three families: BK, large-conductance channels that are strongly inhibited by iberiotoxin and charybdotoxin; SK, small-conductance channels that are inhibited by apamin but are insensitive to charybdotoxin and iberiotoxin; and IK, intermediate-conductance channels that are strongly inhibited by charybdotoxin, sensitive to iberiotoxin at very high concentrations, and insensitive to apamin. BK channels are voltage- and Ca2+ dependent, whereas both IK and SK only require Ca2+ for activation (34). The pharmacological profile of the ATP-stimulated 86Rb efflux in cultured cytotrophoblast cells suggests that activation of IK underlies this pathway, because 86Rb efflux was sensitive to charybdotoxin but insensitive to both apamin and iberiotoxin. In other epithelia there is good evidence for ATP-stimulated Ca2+-activated K+ conductances from electrophysiological or efflux studies [airway epithelia (42); human sweat gland (53); exocrine cells (33); rat parotid acinar cells (47)]. Several IK channels have also been cloned from a human placental cDNA library (22, 24, 25). Preliminary PCR data have also provided evidence for mRNA encoding IK in term human placental tissue (A. Y. Al-Taher, unpublished observations); in the same study the authors were unable to detect BK mRNA in this tissue. However, the present study provides the first functional evidence for the IK channel in cytotrophoblast cells of human placenta.

In 66-h cytotrophoblast cells, removal of extracellular Ca2+ completely abolished the ATP-stimulated increase in [Ca2+]i (Fig. 6). However, efflux experiments under parallel conditions showed that substantial 86Rb efflux, inhibited by charybdotoxin, still occurred. One possible explanation of this paradox arises from the fact that the EC50 for Ca2+ activation of IK is very low [95-320 nM (21, 24)]. Both studies proposed that activity of this channel would be expected to occur in unstimulated cells, and thus even a marginal increase in [Ca2+]i, which might be barely detectable, may be sufficient to activate such an efflux pathway. Alternatively, this anomaly might reflect efflux from a mixed population of cells. In the 66-h cytotrophoblast cell group, although the majority of cells are multinucleate and syncytiotrophoblast-like, some mononuclear cytotrophoblast cells are also present (7, 8, 18). In contrast, only multinucleate cells were selected for microfluorometry studies in the 66-h cytotrophoblast cell group.

Physiological Role of IK in the Human Placenta

Unlike BK and SK, which predominate in excitable tissue, IK is the main Ca2+-activated K+ channel in nonexcitable tissues such as epithelia, endothelia, and T lymphocytes (15, 22, 32, 37, 38). Activation of this channel has been associated with several functions including growth and differentiation (32, 38) and regulation of Na+ and Cl- secretion in epithelia via recycling of K+, rather than K+ secretion per se (22, 24, 29). Recycling of K+ via activation of IK may also be important in the human placenta. In the present study 86Rb efflux is from the media-facing membrane, which, from electron microscopy studies, is microvillous in structure (7). Indeed, ATP-stimulated 86Rb efflux has also been measured across the microvillous membrane of placental villous fragments (45). It is likely, therefore, that IK is located on the microvillous membrane, although expression of the channel on the basal plasma membrane cannot be ruled out. Expression of Na+-K+-ATPase has been demonstrated on both microvillous and basal plasma membranes of the syncytiotrophoblast (23), and there is also evidence for an inwardly rectifying K+ channel (8, 36). It is probable, therefore, that following stimulation of IK, which would result in efflux of K+, both the Na+-K+-ATPase and the inwardly rectifying K+ channel could act as pathways for K+ uptake into the syncytiotrophoblast, thus providing a mechanism for K+ recycling. Recycling of K+ could maintain a driving force for Na+-dependent cotransport of, e.g., amino acids, toward the fetus.

ATP Receptors

It has been demonstrated in other tissues that ATP can exit the cell following shear stress or via ABC transporters after a hyposmotic challenge, although this is still controversial (1, 5, 13, 40, 44). Although the mechanism for ATP exit in human placenta has not been determined directly, the components for this are in place, and there is evidence for expression of ABC transporters in the syncytiotrophoblast of the human placenta (2, 14). After ATP exits the cell, it is thought to act in a paracrine/autocrine manner as a regulator of cell function (17, 40, 49).

Extracellular ATP exerts its effect by acting on P2 purinergic receptors. This family of receptors can be broadly subdivided into two groups: P2X (ionotropic) and P2Y (metabotropic) (40, 49). Stimulation of P2X and P2Y receptors results in elevated [Ca2+]i via direct Ca2+ influx or via release of Ca2+ from inositol-1,4,5- trisphosphate-sensitive stores, respectively (40, 49), which, in epithelia, can result in activation of K+ and/or Cl- conductance pathways (4, 13, 15, 41, 43).

In human placental cytotrophoblast cells there is previous evidence for an ATP-stimulated increase in [Ca2+]i (26, 39). The authors demonstrated that the increase in [Ca2+]i was dependent on both release of Ca2+ from stores and entry of extracellular Ca2+. Both sets of authors observed a similar response with UTP and therefore proposed that ATP was acting via P2Y receptors. Subsequently, Somers et al. (48) demonstrated expression of mRNA for the P2Y6 receptor, which is stimulated by both ATP and UTP, in cytotrophoblast cells and the syncytiotrophoblast layer of both term and first-trimester placenta. The authors therefore suggested that this receptor may be important in regulation of [Ca2+]i in the human placenta.

P2Y receptors are presumed to activate depletion-activated Ca2+ entry. A putative blocker of such store-depletion-operated Ca2+ channels is SKF-96365 (51). In our study, SKF-96365 had a small inhibitory effect on ATP-stimulated 86Rb efflux from 66-h cytotrophoblast cells. There is also both a small ATP-stimulated increase in [Ca2+]i and residual 86Rb efflux in both 18- and 66-h cytotrophoblast cells in the absence of extracellular Ca2+, thus suggesting some release of Ca2+ from intracellular stores in response to ATP. These data together suggest that Ca2+ is released from intracellular stores. This Ca2+ release may in turn stimulate a depletion-activated Ca2+ influx pathway.

In our study, entry of Ca2+ appeared to be of increasing importance during differentiation, given its greater contribution to increase in [Ca2+]i evoked by ATP in the more differentiated 66-h cytotrophoblast cells. The difference between our study and previous reports may be due to the stage of differentiation of the cells examined. Karl et al. (26) used cytotrophoblast cells after 2 days of culture, when they would be less well differentiated, with fewer multinucleate syncytiotrophoblast-like cells. In fact, the form of the response to ATP that they reported was similar to that which we observed in the less differentiated 18-h cytotrophoblast cells, with a rapid increase in [Ca2+]i to a peak, followed by a relatively small plateau.

What does the change in size of the ATP response during cytotrophoblast differentiation represent? First, it could be attributed to increased expression of P2Y receptors in 66-h cytotrophoblast cells. However, Ca2+ release from intracellular stores (in the absence of extracellular Ca2+) was similar in both 18- and 66-h cytotrophoblast cells, suggesting that the changes in the ATP response largely reflected increased Ca2+ influx specifically. We cannot, therefore, exclude the possibility that ionotropic P2X receptors are involved. Certainly, influx of Ca2+ via either P2X or P2Y receptors can be inhibited by Gd3+ (27). Preliminary data from PCR experiments demonstrate that both placental tissue and cultured cytotrophoblast cells express mRNA for P2X4 (6), and it is therefore possible that, in this study, ATP stimulates Ca2+ entry via activation of P2X receptors, especially in more differentiated 66-h cytotrophoblast cells. Alternatively, there might be upregulation of some other agonist-dependent entry with differentiation. A number of putative Ca2+ channels have been identified in human placental tissue [ECaC (20); PKD2 (16, 35); Cat-L (54)], but their functional role remains wholly unexplored. Clearly, the exact involvement of intracellular Ca2+ stores and the specific Ca2+ influx pathways present in human placental trophoblast cells will be explored in future studies.

Summary

In conclusion, we have identified a Ca2+-activated K+ efflux pathway that is stimulated by extracellular ATP in human placenta. Pharmacological evidence implies that the channel underlying this pathway is the intermediate-conductance Ca2+-activated K+ channel (IK). It is likely that the main function of this channel in the human placenta is in recycling K+, thereby providing a driving force for Na+-driven cotransport. ATP controls IK in cytotrophoblast cells by raising [Ca2+]i following activation of P2Y and, possibly, P2X receptors. Influx of Ca2+ in response to ATP is of increasing importance during cytotrophoblast cell differentiation, because its contribution to the ATP-evoked increase in [Ca2+]i is greater in differentiated 66-h cytotrophoblast cells compared with 18-h cytotrophoblast cells. This study provides a basis for future work on the regulation and nature of Ca2+ influx pathways activated by purinergic receptors in the syncytiotrophoblast and their functional role in placental physiology.


    ACKNOWLEDGEMENTS

We thank the staff of the Central Delivery Unit at St. Mary's Hospital, Manchester, UK, for their assistance in collection of the tissue. We are grateful to Professor Colin Sibley for his encouragement throughout the work and for comments on the manuscript.


    FOOTNOTES

This work was funded by the Medical Research Council, UK.

Address for reprint requests and other correspondence: L. H. Clarson, Academic Unit of Child Health, Univ. of Manchester, St. Mary's Hospital, Hathersage Rd., Manchester M13 0JH, UK (E-mail: lorraine.clarson{at}man.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpregu.00564.2001

Received 17 September 2001; accepted in final form 30 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Al-awqati, Q. Regulation of ion channels by ABC transporters that secrete ATP. Science 269: 805-806, 1995[Free Full Text].

2.   Atkinson, DE, Glazier JD, Greenwood SL, and Sibley CP. Expression and localisation of MDR1 and MRP1 in the human placenta (Abstract). J Physiol (Lond) 528: 34P, 2000.

3.   Boyd, CA. Cotransport systems in the brush border membrane of the human placenta. Ciba Found Symp 95: 300-326, 1983[Medline].

4.   Brodin, B, and Nielsen R. Electrophysiological evidence for an ATP-gated ion channel in the principal cells of the frog skin epithelium. Pflügers Arch 439: 227-233, 2000[ISI][Medline].

5.   Cantiello, HF. Nucleotide transport through the cystic fibrosis transmembrane conductance regulator. Biosci Rep 17: 147-171, 1997[ISI][Medline].

6.   Clarson, LH, and Glazier JD. Expression of mRNA for Ca2+-permeable channels in human placenta and cultured trophoblast cells (Abstract). J Soc Gynecol Invest 7, Suppl.: 140A, 2000.

7.   Clarson, LH, Glazier JD, Greenwood SL, Jones CJ, Sides MK, and Sibley CP. Activity and expression of Na+-K+-ATPase in human placental cytotrophoblast cells in culture. J Physiol (Lond) 497: 735-743, 1996[ISI][Medline].

8.   Clarson, LH, Greenwood SL, Mylona P, and Sibley CP. Inwardly rectifying K+ current and differentiation of human placental cytotrophoblast cells in culture. Placenta 22: 328-336, 2001[ISI][Medline].

9.   Clarson, LH, Greenwood SL, and Sibley CP. The effect of a non-selective cation (NSCC) channel blocker on ATP-induced 86Rb efflux from cytotrophoblast cells (CYTS) (Abstract). Placenta 20: A18, 1999.

10.   Clarson, LH, Roberts VHJ, and Elliott AC. Role of extracellular calcium ([Ca2+]o) in ATP-stimulated K+ (86Rb) efflux from human placental cytotrophoblast cells (CYTS) in culture (Abstract). J Physiol (Lond). 535: 38P, 2001.

11.   Clarson, LH, Roberts VHJ, and Greenwood SL. Charybdotoxin-sensitive 86Rb efflux from human placental cytotrophoblast cells (CYTS) (Abstract). J Physiol (Lond) 528: 20P, 2000.

12.   Cronier, L, Bois P, Herve JC, and Malassiné A. Effect of human chorionic gonadotrophin on chloride current in human syncytiotrophoblasts in culture. Placenta 16: 599-609, 1995[ISI][Medline].

13.   Dezaki, K, Tsumara T, Maeno E, and Okada Y. Receptor-mediated facilitation of cell volume regulation by swelling-induced ATP release in human epithelial cells. Jpn J Physiol 50: 235-241, 2000[ISI][Medline].

14.   Faller, DP, Egan DA, and Ryan MP. Evidence for location of the CFTR in human placental apical membrane vesicles. Am J Physiol Cell Physiol 269: C148-C155, 1995[Abstract/Free Full Text].

15.   Gerlach, AC, Gangopadhyay NN, and Devor DC. Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1. J Biol Chem 275: 585-598, 2000[Abstract/Free Full Text].

16.   Gonzalez-Perrett, S, Kim K, Damiano AE, Zotta E, Batelli M, Harris PC, Reisin IL, Arnout MA, and Cantiello HF. Polycistin-2, the protein mutated in autosomal dominant polycystic kidney disease (APKD), is a Ca2+-permeable nonselective cation channel. Proc Natl Acad Sci USA 28: 1182-1187, 2001.

17.   Gordon, JL. Extracellular ATP: effects, sources and fate. Biochem J 233: 309-319, 1986[ISI][Medline].

18.   Greenwood, SL, Clarson LH, Sides MK, and Sibley CP. Membrane potential difference and intracellular cation concentrations in human placental trophoblast cells in culture. J Physiol (Lond) 492: 629-640, 1996[ISI][Medline].

19.   Greenwood, SL, Sides MK, and Sibley CP. Ca2+ sensitivity of volume regulatory 86Rb (K+) efflux from human term placental cytotrophoblast cells in culture (Abstract). J Physiol (Lond) 493: 32P, 1996.

20.   Hoenderop, JGJ, Van der Kemp AWCM, Hartog A, Van de Graaf SFJ, Van Os CH, Willems PHGM, and Bindels RJM Molecular identification of the apical Ca2+ channel in 1,25-dihydroxyvitamin D3 responsive epithelia. J Biol Chem 274: 8375-8378, 1999[Abstract/Free Full Text].

21.   Ishii, TM, Silvia C, Hirschberg B, Bond CT, Adelman JP, and Maylie J. A human intermediate conductance calcium-activated potassium channel. Proc Natl Acad Sci USA 94: 11651-11656, 1997[Abstract/Free Full Text].

22.   Jensen, BS, Strøbæk D, Christophersen P, Jørgensen TD, Hansen C, Silahtaroglu A, Olesen S, and Ahring PK. Characterisation of the cloned human intermediate-conductance Ca2+-activated K+ channel. Am J Physiol Cell Physiol 275: C848-C856, 1998[Abstract/Free Full Text].

23.   Johansson, M, Jansson T, and Powell TL. Na+-K+-ATPase is distributed to microvillous and basal membrane of the syncytiotrophoblast in human placenta. Am J Physiol Regulatory Integrative Comp Physiol 279: R287-R297, 2000[Abstract/Free Full Text].

24.   Joiner, WJ, Wang L, Tang MD, and Kaczmarek LK. hSK4, a member of a novel subfamily of calcium-activated potassium channels. Proc Natl Acad Sci USA 94: 11013-11018, 1997[Abstract/Free Full Text].

25.   Jørgensen, TD, Jensen BS, Strøbæk D, Christophersen P, Olesen S, and Ahring PK. Functional characterisation of a cloned human intermediate-conductance Ca2+-activated K+ channel. Ann NY Acad Sci 868: 423-426, 1999[Free Full Text].

26.   Karl, PI, Chusid J, Tagoe C, and Fisher SE. Ca2+ flux in human placental trophoblasts. Am J Physiol Cell Physiol 272: C1776-C1780, 1997[Abstract/Free Full Text].

27.   Kerst, G, Fischer K, Normann C, Kramer A, Leipziger J, and Greger R. Ca2+ influx induced by store release and cytosolic Ca2+ chelation in HT29 colonic carcinoma cells. Pflügers Arch 430: 653-665, 1995[ISI][Medline].

28.   Kibble, JD, Greenwood SL, Clarson LH, and Sibley CP. A Ca2+-activated whole cell Cl- conductance in human placental cytotrophoblast cells activated via a G protein. J Membr Biol 151: 131-138, 1996[ISI][Medline].

29.   Klaerke, DA. Purification and characterisation of epithelial Ca2+-activated K+ channels. Kidney Int 48: 1047-1056, 1995[ISI][Medline].

30.   Kliman, HJ, Nestler JE, Senmasi E, Sanger JM, and Strauss JF, III. Purification, characterisation and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118: 1567-1582, 1986[Abstract].

31.   Kong, ID, Koh SD, and Sanders KM. Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes. Am J Physiol Cell Physiol 278: C352-C362, 2000[Abstract/Free Full Text].

32.   Logsdon, NJ, Kang J, Togo JA, Christian EP, and Aiyar J. A novel gene, hKCa4, encodes the calcium-activated potassium channel in human T lymphocytes. J Biol Chem 272: 32723-32726, 1997[Abstract/Free Full Text].

33.   Martin, SC, and Shuttleworth TJ. Activation by ATP of a P2U "nucleotide" receptor in an exocrine gland. Br J Pharmacol 115: 321-329, 1995[ISI][Medline].

34.   McManus, OB. Calcium-activated potassium channels: regulation by calcium. J Bioenerg Biomembr 23: 537-560, 1991[ISI][Medline].

35.   Mochizuki, T, Wu G, Hayashi T, Xenophontus SL, Veldhuisen B, Saris JJ, Reynolds DM, Cai Y, Gabow PA, Pierides A, Kimberling WJ, Breuning MH, Deltas CC, Peters DJM, and Somlo S. PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science 272: 1339-1342, 1996[Abstract].

36.   Mylona, P, Clarson LH, Greenwwood SL, and Sibley CP. Expression of the Kir2.1 (inwardly rectifying potassium channel) gene in the human placenta and in cultured cytotrophoblast cells at different stages of differentiation. Mol Hum Reprod 4: 195-200, 1998[Abstract/Free Full Text].

37.   Pedersen, KA, Jørgensen NK, Jensen BS, and Olesen S. Inhibition of the human intermediate-conductance, Ca2+-activated K+ channel by intracellular acidification. Pflügers Arch 440: 153-156, 2000[ISI][Medline].

38.   Peña, TL, and Rane SG. The fibroblast intermediate conductance KCa channel, FIK, as a prototype for the cell growth regulatory function of the IK channel family. J Membr Biol 172: 249-257, 1999[ISI][Medline].

39.   Petit, A, and Belisle S. Stimulation of intracellular calcium concentration by adenosine triphosphate and uridine 5'-triphosphate in human term placental cells: evidence for purinergic receptors. J Clin Endocrinol Metab 80: 1809-1815, 1995[Abstract].

40.   Rathbone, MP, Middlemiss PJ, Gysbers JW, Andrew C, Herman MAR, Reed JK, Ciccarelli R, Iorio PI, and Caciagli F. Trophic effects of purines in neurons and glial cells. Prog Neurobiol 59: 663-690, 1999[ISI][Medline].

41.   Rubera, I, Tauc M, Bidet M, Verheecke-Mauze C, De Renzis G, Poujeol C, Cuiller B, and Poujeol P. Extracellular ATP increases [Ca2+]i in distal tubule cells. II. Activation of a Ca2+-dependent Cl- conductance. Am J Physiol Renal Physiol 279: F102-F111, 2000[Abstract/Free Full Text].

42.  Rugolo M, Mastrocola T, Whorle C, Rasola A, Gruenert DC, Romeo G, and Galietta LJ. ATP and A1 adenosine receptor agonists mobilize intracellular calcium and activate K+ and Cl- currents in normal and cystic fibrosis airway epithelial cells. J Biol Chem 268: 24779-24784.

43.   Ryan, JS, Baldridge WH, and Kelly MEM Purinergic regulation of cation conductances and intracellular Ca2+ in cultured rat retinal pigment epithelial cells. J Physiol (Lond) 520: 745-759, 1999[Abstract/Free Full Text].

44.   Schwiebert, EM. ABC transporter-facilitated ATP conductive transport. Am J Physiol Cell Physiol 276: C1-C8, 1999[Abstract/Free Full Text].

45.   Sibley, CP, Greenwood SL, Glazier JD, Sides MK, and Turner M. Regulation of Cl- and K+ transport by the human placenta (Abstract). Placenta 19: A8, 1998.

46.   Sides, MK, Greenwood SL, and Sibley CP. Effect of a calcium ionophore (A23187) on 86Rb (K+) efflux from human placental trophoblast cells in culture (Abstract). Placenta 16: A10, 1995.

47.   Soltoff, SP, Mcmillian MK, Cragoe EJ, Jr, Cantley LC, and Talamo BR. Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. J Gen Physiol 95: 319-346, 1990[Abstract/Free Full Text].

48.   Somers, GR, Bradbury R, Trute L, Conigrave A, and Venter DJ. Expression of the human P2Y6 nucleotide receptor in normal placenta and gestational trophoblastic disease. Lab Invest 79: 131-139, 1999[ISI][Medline].

49.   Turner, JT, Landon LA, Gibbons SJ, and Talamo BR. Salivary gland P2 nucleotide receptors. Crit Rev Oral Biol Med 10: 210-224, 1999[Abstract/Free Full Text].

50.   Turner, MA, Sides MK, Sibley CP, and Greenwood SL. Anion efflux from cytotrophoblast cells derived from normal term human placenta is stimulated by hyposmotic challenge and extracellular A23187 but not by membrane-soluble cAMP. Exp Physiol 84: 27-40, 1999[Abstract].

51.   Wayman, CP, Wallace P, Gibson A, and McFadzean I. Correlation between store-operated cation current and capacitative Ca2+ influx in smooth muscle cells from mouse anococcygeus. Eur J Pharmacol 376: 325-329, 1999[ISI][Medline].

52.   Williams, DA, and Fay FS. Intracellular calibration of the fluorescent calcium indicator Fura-2. Cell Calcium 14: 145-159, 1990.

53.   Wilson, SM, Whiteford ML, Bovell DL, Pediani JD, Ko WH, Smith GL, Lee CM, and Elder HY. The regulation of membrane 125I- and 86Rb+ permeability in a virally transformed cell line (NCL-SG3) derived from the human sweat gland epithelium. Exp Physiol 79: 445-459, 1994[Abstract].

54.   Wissenbach, U, Niemeyer BA, Fixemer T, Schneidewind A, Tros C, Cavalie A, Reus K, Meese E, Bonkhoff H, and Flockerzi V. Expression of CAT-Like, a novel calcium selective channel, correlates with the malignancy of prostate cancer. J Biol Chem 276: 19461-19468, 2001[Abstract/Free Full Text].

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