Department of Obstetrics, Gynecology, and Women's Health, New Jersey Medical School, Newark, New Jersey
Submitted 10 March 2004 ; accepted in final form 4 December 2004
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ABSTRACT |
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sodium/hydrogen antiporter; pH regulation; fluorescence; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
Na+/H+ antiporter activity has been described in the human syncytiotrophoblast and in carcinoma cells derived from trophoblasts (1, 11, 15, 22, 26), with different isoforms of the antiporter present on the microvillous and basal membranes (14, 19, 26). A Cl/HCO3 exchanger has been described on both microvillous and basal membranes of the syncytiotrophoblast (10, 23). Most of the information gathered regarding the activity of the placental Na+/H+ antiporter and the Cl/HCO3 exchanger has been collected from experiments using purified plasma membrane vesicles. Although this model is useful for the study of individual transporter mechanisms in isolation, it cannot be used to investigate the interaction between cellular acid-base transporters and the pathways that regulate them. The aim of this investigation was to define systematically the homeostatic mechanisms by which the syncytiotrophoblast cell maintains pHi and specifically to determine the transporter(s) that function(s) to allow the cell to recover from an intracellular acid load.
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METHODS |
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For fluorescence microscopy, 22-mm sterile circular glass coverslips were placed in 35-mm wells containing 2 ml of medium (keratinocyte growth medium, KGM; see below), and 0.3 x 106 cells (in 0.10.2 ml) were carefully dropped into the center of the coverslips. The coverslip-containing plates were kept stationary for 15 min to permit the cells to settle and begin initial attachment. Subsequently, the cells were transferred to a humidified CO2 incubator and cultured for 72 h at 37°C in a medium composed of keratinocyte basal medium (KBM) fortified with bovine pituitary extract, epidermal growth factor, insulin, and hydrocortisone (KGM) plus 10% FBS (KGM-FBS) as described by Douglas and King (7). The medium was exchanged after the initial 24-h period. In all of the experiments described here, the cell preparations were used after a total of 72 h in culture. Cultures grown in parallel with those used for measurements of pHi were stained with propidium iodide and antidesmosomal protein to verify the syncytial nature of the cultures, as described previously (8).
Solutions and dye loading. The content of the HCO3-free and HCO3-containing solutions used in these experiments is given in Table 1. Dye loading buffer was of a composition identical to that of solution A but adjusted to pH 7.38 at room temperature. On the day of the experiment, cells were loaded with the pH-sensitive probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The cells were loaded by incubating the coverslip in the dark for 30 min at room temperature in dye loading solution containing 1 µM BCECF-AM, 0.25% BSA, and 0.02% Pluronic F-127. After loading, the coverslip was washed twice with dye-free loading buffer, placed in a temperature-controlled well on the stage of an epifluorescence microscope, and equilibrated with the appropriate buffer for 15 min.
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Experimental protocols.
All experiments were performed at 32°C. The coverslips were placed in a chamber on the microscope stage and filled with buffer (either a HEPES-buffered or a HCO3-buffered balanced salt solution). In the HCO3-buffered experiments, all solutions were continuously gassed before use with 5% CO2, resulting in a HCO3 concentration of 20 mM and a pH of
7.4. A cover was set up over the tissue chamber to allow for the continuous gassing of the bathing solution with 5% CO2-95% air during the experiment.
The first experimental protocol consisted of the measurement of the rate of change of pHi (pHi) at resting or basal pHi over 5 min, followed by measurement after ion substitution or inhibitor addition. In the second protocol, after measurement of basal pHi, cells were acid loaded and the effects of ion substitution or inhibitors on recovery were measured. Acidification was achieved in HCO3-free (HEPES buffered) experiments by switching from 140 mM Na+-containing buffer (solution A) to one in which the Na+ was replaced isotonically with N-methyl-D-glucamine (NMG+; solution B) and which contained the K+/H+ ionophore nigericin at a concentration of 0.5 µM. In HCO3-buffered experiments, acidification was achieved in a similar manner except that NaHCO3 was replaced with choline bicarbonate (solution F). Nigericin was removed through binding to BSA; removal was accomplished by switching to a Na+-free buffer (solution B) containing 0.5% BSA.
Data analysis.
Data are presented as means ± SE, with n representing the number of experiments (coverslips used). The number of individual primary trophoblast preparations used in each experiment is presented in parentheses after n values. Statistical comparisons were performed with Student's t-test, paired t-test, or ANOVA (post hoc tests: Student-Newman-Keuls or Dunnett). Recovery rates were measured as the initial rate of pHi toward basal pHi after acid loading. Linear regression analysis was used to assess the relationship between 495/440 and pH and between
pHi/min and pHi.
Materials. BCECF-AM, Pluronic-127, and dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H2DIDS) were obtained from Molecular Probes (Eugene, OR). KGM was purchased from Clonetics (San Diego, CA). Nigericin, amiloride, and all other chemicals were obtained from Sigma (St. Louis, MO).
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RESULTS |
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Experiments at basal pHi in HCO3-free buffer.
In HCO3-free buffer, the resting or basal pHi obtained after loading, washing, and equilibration of the cells in Na+-containing buffer (solution A) was 7.26 ± 0.04 [n = 16 (9)]. Initial experiments were performed to determine whether the operation of various transporters such as the Na+/H+ antiporter and the Cl/HCO3 exchanger was necessary to maintain basal pHi. In these experiments, pHi was assessed before and after ion substitution or addition of specific transport inhibitors. In some cases, a slow drift in pHi was observed under basal conditions; however, the mean rate of
pHi was not significantly different from zero (Table 2; t-test). In HCO3-free buffer, the manipulations tested were 1) removal of extracellular Na+ by substitution of NMG+ (solution B), 2) removal of extracellular Cl by substitution of gluconate (solution C), and addition of 3) the Na+/H+ antiporter inhibitor amiloride (0.5 mM) or 4) the anion exchange inhibitor H2DIDS (0.2 mM). In all cases
pHi after manipulation was not significantly different from the basal rate (Table 2; t-test). In a nominally HCO3-free buffer, in which transporters that carry HCO3 are inoperative, these results demonstrate that transporters such as the Na+/H+ antiporter, the Cl/HCO3 exchanger, the Na+-dependent Cl/HCO3 exchanger, and the Na+-HCO3 cotransporter are not functional at basal pHi.
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DISCUSSION |
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A variety of transporters have been implicated in pHi homeostasis, either separately or in combination. Those most commonly involved in the protecting against acidification are the Na+/H+ antiporter (3, 24, 28), the Na+/HCO3 cotransporter (5, 16, 18), and the vacuolar proton pump (V-ATPase; Refs. 9, 12, 29). Ion substitution and inhibitor experiments suggest that none of these transporters is active at basal pHi; transporters requiring Na+, Cl, or HCO3 are not involved in maintaining pHi at its basal level. Moreover, transporters inhibitable by amiloride or H2DIDS do not seem to be involved in basal pHi maintenance. The lack of an effect of Na+ removal or amiloride addition on basal pHi is contrary to the effects observed previously in term gestational villous fragments, where both modifications produced a significant acidification (22). In preterm fragments, amiloride had a minimal effect and there was a lesser effect of Na+ removal on pHi; however, there is no evidence to suggest that the syncytial cells used in vitro here have characteristics of preterm rather than term tissue. One obvious difference stems from the nature of the primary syncytiotrophoblast culture. These cells, although grown in the presence of 10% FBS, were not exposed to the full range of circulating maternal and fetal factors; nor did they grow in contact with or close proximity to the various other cell types that compose the placental environment. Thus it is possible that they display a complement of transporters different from the villous fragments either in type or level of expression. It is also possible, however, that differences are due to conditions extant at the time of the experiments. The acidification seen in the fragment experiments may be due to a higher rate of anaerobic metabolism (and hence proton generation) in the fragments compared with the cultured syncytial cells. In the villous fragments syncytial access to oxygen and other substrates is likely to be more limited compared with the cultured cell monolayer. Moreover, the presence of a relatively hypoxic villous core, lacking any capillary circulation, will also contribute to syncytial acidification. In addition, the experiments using cultured primary cells were performed at 32°C, potentially reducing the metabolic rate relative to that in the fragments that were maintained at 37°C. Thus a greater rate of generation of intracellular H+ in the fragments combined with inhibition of the Na+/H+ antiporter might lead to the decrease in pHi observed in the fragment experiments after Na+ removal or addition of amiloride, compared with the stable pHi observed in the cultured cells.
In syncytial cells, the degree of acidification observed after addition of nigericin in a Na+-free buffer was reduced in the presence of HCO3, demonstrating that there is an increase in intracellular buffering power due to the presence of intracellular HCO3. After acidification, the initial rate of recovery in the presence of HCO3 was only 45% of that in HEPES (HCO3-free) buffer; however, the starting point for the recovery was higher, at pH 6.53, compared with the starting point for recovery in HEPES buffer (pH 6.20). When calculated from a starting point of pH 6.53, the mean rate of recovery in HEPES buffer was not significantly different from that in HCO3 [0.0052 ± 0.007 vs. 0.0092 ± 0.0021; n = 16 (5) and 14 (8), respectively].
The ion substitution and inhibitor experiments performed during acidification and recovery cycles, both in HCO3-free and HCO3-containing buffers, show that recovery from acidification is Na+ dependent and inhibited by amiloride. The demonstration that absence of HCO3 had no effect on recovery, and the lack of an effect of H2DIDS, suggest that recovery does not involve the Na+/HCO3 cotransporter or HCO3 conductances (17, 21). The inhibition of recovery in the absence of Na+ or in the presence of amiloride shows that there is little or no contribution of a V-ATPase or a proton conductance to recovery, despite the previous identification of the latter in the syncytiotrophoblast (4, 20). The lack of effect of Cl removal on recovery suggests that transporters such as the putative Cl-dependent Na+/H+ exchanger (2) do not function to relieve acidosis.
We conclude that syncytiotrophoblast recovery from acidification is a Na+-dependent, amiloride-inhibitable process that terminates at basal pHi. These data strongly suggest that a Na+/H+ antiporter is the moiety responsible for the recovery from acidification. After recovery from acidification and stabilization of pHi, the addition of monensin provoked a further increase in pHi, showing that a driving force for Na+/H+ exchange is still present, despite recovery to basal pHi. This suggests that it is the inactivation of the antiporter rather than loss of the driving force that terminates the recovery of pHi from acidification. These results are consistent with the operation of the Na+/H+ antiporter in other cells and tissues, in which it has been determined that the antiporter has two intracellular H+ binding sites, one that operates as the transport site and the other that, when occupied, activates the transporter. Thus as pHi decreases, the antiporter is activated, and as recovery proceeds and pHi increases, this second site is deprotonated, leading to progressive deactivation of the antiporter until its activity finally terminates at basal pHi.
Little has been reported thus far on the pathways by which pHi homeostasis is maintained in human placental syncytiotrophoblast cells, the main barrier layer between mother and fetus. A previous report from this laboratory (22) demonstrated the functional presence of a Na+/H+ antiporter in the syncytium of villous tissue fragments. The basal pHi reported here is similar to that observed previously in the term villous fragments (7.31 ± 0.03, 7.30 ± 0.03; Ref. 22). The Na+/H+ antiporter has been demonstrated to be present in both microvillous and basal membranes of the human syncytiotrophoblast cell in vivo (11, 14). Western blotting and immunohistochemical data suggest that the Na+/H+ exchanger (NHE)1 isoform of the Na+/H+ antiporter is the predominant syncytial form, with greater expression and activity on the microvillous than on the basal membrane (14, 19, 25). The distribution of expression and activity of the other isoforms is less clear; NHE3 appears to be predominantly associated with the basal membrane, although some small quantity may be found on the microvillous membrane (19). If the enlargement factor introduced by the involution of the microvillous membrane is taken into account (27), it is clear that the overwhelming majority of the Na+/H+ antiporter is NHE1 on the microvillous membrane of the syncytium. Western blotting of the cultured syncytial cells used in these experiments revealed the presence of NHE1 and NHE3 (data not shown), as predicted from the isolated plasma membrane data. These measurements were performed on whole cell extracts because methods do not exist currently for the preparation of microvillous and basal membranes from cultured cells; thus the relative quantities and microvillous-basal distribution cannot be determined.
The movement of H+ generated by fetal metabolic activity from the fetal circulation and placenta to the maternal circulation has been postulated as a means of fetal pH homeostasis. With respect to intrasyncytial pH homeostasis in vivo, it will be important to determine whether, under the conditions of reduced oxygenation extant in vivo, there is increased generation of lactic acid and a decreased pHi, compared with cultured syncytial cells, and thus a more permanent activation of the antiporter. The activation of antiporter activity at a pHi below basal would ensure that the system responds to increases in syncytial production of H+ or uptake from the fetal circulation. It should be noted that all the experiments described here were performed with cells both grown and measured in conditions that are relatively hyperoxic compared with those present in the intervillous space in vivo.
In summary, we have shown that maintenance of resting pHi in cultured human placental syncytiotrophoblast cells does not require the active operation of commonly expressed H+/OH transporters. We have shown for the first time that the syncytial cell is protected from acidification solely by means of an Na+/H+ antiporter, which is activated only when pHi falls below its basal or resting value. No other transporters appear to be necessary or functional in defending intrasyncytial pH from an acid load. The functioning of the antiporter is consistent with activation through an allosteric, intracellular H+ binding site, as observed in other cell types.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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