Gestational changes in Ca2+ transport across rat placenta and mRNA for calbindingK and Ca2+-ATPase J. D. GLAZIER, P. T. SHARPE,

D. E. ATKINSON, K. L. THORNBURG, D. EDWARDS, R. D. H. BOYD, AND C. P. SIBLEY

Departments of Child Health and Physiological Sciences, The Epithelial Membrane Research and the Molecular Embryology Laboratory, Department of Cell and Structural Biology, The University of Manchester, St. Mary’s Hospital, Manchester Ml3 OJH, United Kingdom Glazier, J. D., D. E. Atkinson, K. L. Thornburg, P. T. Sharpe, D. Edwards, R. D. H. Boyd, and C. P. Sibley. Gestational changes in Ca2+ transport across rat placenta and mRNA for calbindingK and Ca2+-ATPase. Am. J. Physiol. 263 (Regulatory Integrative Comp. Physiol. 32): R930R935, 1992.-The unidirectional maternofetal clearance (K,J of 45Cawasmeasuredacrossthe rat placenta over the last onethird of gestation. J& for 45Canormalized to its diffusion coefficient in water (K,&J increased72-fold between days 15 and 22 of gestationfrom 3.5 t 0.3 to 253.1t 22.0cm/g placenta, respectively. At 15 and 18 days of gestation, K,,lD, for 45Ca was similar to K,JD, for the paracellular marker [14C]mannitol, but at 21 and 22 days of gestation, K,,lD, for 45Cawas significantly higher than K,JD, for [14C]mannitol, indicating that an additional route of transfer, other than diffusion, becomesavailable to calcium during this period. Northern hybridization analysis demonstrated that rat placental calbindingKto-p-actin mRNA ratio increased135-fold between 15 and 22 days of gestation and was temporally associatedwith the gestational increasein K,JDw for 45Ca.In contrast, rat placental Ca2+-ATPase-to-P-actin mRNA ratio increased only two- to threefold over the samegestational period and did not mirror the gestationalchangesin calcium clearance.These trends suggestthat the expressionof placental calbindingK, but not Ca2+ATPase, may be rate limiting to placental calcium transport in the rat. calcium clearance; calcium binding protein; calcium pump; pregnancy CALCIUM ACCRETION in the rat increases markedly over the last one-third of gestation (ll), reflecting an increased net transplacental flux of calcium. At term, placental calcium transport in the rat is by an active mechanism, with the maternofetal calcium flux almost equalling net flux (27). Active transport underlies the higher concentrations of total and free calcium in fetal plasma compared with maternal plasma in the rat as well as in other species (reviewed in Ref. 22). In the intestine, transcellular transport of calcium probably involves three steps (5): 1) passive Ca2+ entry into the enterocyte; 2) translocation of Ca2+ through its cytosol, probably bound to the calcium binding protein calbindingK; and 3) active extrusion of Ca2+ across the basolateral membrane. The presence of calbindingK in placenta (7) and the localization of Ca2+-ATPase at the fetal facing trophoblast membrane of human and rat placenta (3) lends support to the hypothesis that transplacental Ca 2+ flux is mediated in a similar way (22). The calbindin gK of rat chorioallantoic placenta is identical to that from the rat intestine (19). This protein is also found in the rat yolk sac and uterus (20). The concentrations of calbindingK in all of these tissues show gestation-dependent changes, with placental levels increasing significantly over the last one-third of gestaFETAL

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tion. However, any functional implication of this change for placental Ca 2+ transport has not been investigated. There has also been no investigation into any gestational changes in Ca 2+-ATPase expression. In this study, our aim was to determine the temporal relationship between the unidirectional maternofetal clearance of calcium across the rat placenta over the last one-third of gestation and the expression of placental calbindin gK and Ca2+-ATPase. METHODS Measurement of Unidirectional Maternofetal Clearance Animals. FemaleSprague-Dawleyrats were mated overnight

a.ndcheckedeach morning for vaginal plugs.The day that plugs were found was designatedas day 1 of gestation. Protocol. The method used to determine unidirectional maternofetal clearance(K,J wassimilar to that of Flexner and Pohl (16) and has been previously described(2, 21). Rats were studied on days 15, 18,21, and 22 of gestation (term is 23 days). The animalswere anesthetizedusing I IO mg/kg intraperitoneal thiobutabarbital sodium (Inactin, BYK Gulden, Hamburg, FRG), and cannulaswere placed in the trachea, carotid artery, and jugular vein. Radiolabeled tracers, either 45Caor [‘“Clmannitol (Amersham International, Aylesbury, Bucks, UK), were injected into the maternal circulation via the jugular vein cannula. Fetuseswere excised, and at least four maternal blood samples(0.5 ml from the carotid cannula after removing dead space)were subsequentlytaken at known times after administration of tracer. After removal, the fetuseswere gently blotted, weighed,and the radioisotopecontent of these, and of the maternal plasmasamples,determined (2). Placentaswerealsocarefully blotted and weighed. For measurementof unidirectional clearance, it is critical that fetusesbe taken before fetal plasmaradioactivity becomes high enough for flux from fetus to mother to be quantitatively important (22). Thesetimes weredetermined for both tracers at each gestational age.At 15 and 18 days of gestation, it was not technically possibleto collect fetal blood samples,so fetuses were taken at several time points after injection of tracer into the maternal circulation. Fetal radioactivity was then normalized to the prevailing maternal plasma radioactive concentration and plotted againsttime for each tracer. Normalized fetal radioisotope concentration increasedinitially as a linear function of time but then more slowly, presumably as backflux becamequantitatively important. Fetuseswere taken for analysis at times during which the plot was linear. At 21 and 22 days of gestation, blood sampleswere taken from fetusesat severaltime points after tracer administration to the maternal circulation (blood from 3 fetuses was pooled at each time point), and the time at which fetal plasmaconcentration reached at maximum 12% of maternal plasma concentration was determined. Fetuseswere subsequentlyexcised before this time point. Recovery of radioisotope from fetal tissueswas checked for both tracers at each gestation by injecting a known amount of

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radioisotope into fetuses at each gestational age and then processing them as outlined above. Recoveries varied from 70 to 83% for both tracers over the gestational period studied, and fetal radioactive content was corrected appropriately. K,f was calculated as follows

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separated by chromatography on a Sephadex G-50 microcolumn. RNA Northern

Hybridization

Total RNA was electrophoresedon 1.2% agarosegel under denaturing conditions (18) overnight (N 18 h) at 25 V in 1X 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (20 mM MOPS, 5 mM sodiumacetate, and 1 mM Na,EDTA, pH 7.0). where Nx is the total radioactivity accumulated by a fetus (corTotal RNA wasthen transferred from the agarosegelto a nylon rected for recovery) taken at x min after injection of tracer into membrane (Hybond N; Amersham International) using 20x the maternal circulation, Wis the wet weight of the placenta for SSC (1X SSC is 0.15 M NaCl and 0.015 M sodium citrate) as that fetus, and S; Cm(t) dt is the time integral of radioactivity describedby Thomas (28). The nylon filters were exposedto concentration in maternal plasma from 0 to x min. ultraviolet illumination to fix RNA to the filters. The Kmf value obtained for eachtracer wasnormalized to its Filters were prehybridized in buffer [50% formamide, 5~ diffusion coefficient in water (D,) to give Kmf/Dw. D, values SSC, 5~ Denhardt’s solution, 50 mM Na,HPO, (pH 6.8), 0.2% usedwere 9.9 X 10s6cm2/s for [14C]mannitol (26) and 10.6 X sodium dodecyl sulfate (SDS), 10% dextran sulfate, and 300 lOA6cm2/s for 45Ca(1). pg/ml heat-denatured tRNA] for 18 h at 42°C. The hybridizaThe data for [14C]mannitol, obtained at the sametime asthat tion buffer consistedof the above buffer except only 100pg/ml for 45Ca,have already beenreported (2) but are included in this tRNA was included plus 32P-cDNA probe. report for comparisonpurposes,asan exampleof a paracellular After hybridization (18 h at 42”(Z), the filters were washed diffusion-limited marker (22). twice at room temperature in 2~ SSC and 0.1% SDS for ~30 min, followedby a final washin the samesolution at 60°C (65°C for P-actin) for 20 min. Statistics The blots were autoradiographedat -8OOCwith Fuji RX100 A three-way analysis of variance using a nested random de- film for ~6 h. The autoradiogramswereanalyzed by spectrodensign model with data from placentas obtained from each adult sitometry (Shimadzu Scientific Instruments, Kyoto, Japan), rat nested within tracer by gestation was used to assessthe and transcript density peak area wasquantified. statistical significance of differences between K,,lD, values. Preliminary studiesshowedthat under these conditions the This modeltakes into account any possibledifferencesbetween hybridization signalswere within the linear range of the film data obtained from each individual adult rat. The appropriate n used. value was the number of placentas; at least three litters were studied for each data point. Data are presentedas means~frSE. NC Klnf = WJ;; Cm(t) dt

RESULTS

Measurement of CalbindingK mRNA and Ca2+-ATPase mRNA Levels

Unidirectional

Rats, mated as describedabove, were taken on either 13, 15, 18, 21, or 22 days of gestation.The animalswere anesthetizedby ether inhalation, and the placentaswere excisedand immediately frozen in liquid nitrogen and stored at -80°C. Tissuewasthawed at room temperature in guanidinium thiocyanate buffer (4 M guanidinium thiocyanate, 0.5% sarkosyl, 0.1% Antifoam A emulsion,25 mM sodium citrate, pH 7, and 0.1 M 2-mercaptoethanol), and total RNA was extracted as describedby Chomczynski and Sacchi (9). The final RNA pellet was suspendedin water, quantified by measuring the absorbance at 260 nm, and stored at -80°C. Preparation of 32P-cDNA probes. A 460-bp cDNA for calbindingKwasthe generousgift of Dr. M. Thomasset(Institut National de la Sante et de la Recherche Medicale, Paris, France). This probe is complementary to a mRNA coding for the rat intestinal calbindingK (13). Ca2+-ATPasecDNA wasthe generousgift of Dr. E. Strehler (ETH, Zurich, Switzerland), coding for a human isoform of the calcium pump (29). The EcoR I-BaZ I fragment corresponding to positions 809-2,278 in the sequenceof Verma et al. (29) was usedasprobe. ,&Actin cDNA wasusedas a control to correct for variations in loading between samples.Initial studies suggested@actin was constitutively expressedin rat placenta basedon the following observations:equal loadingsof rat placental RNA from 13 to 22 daysof gestation hybridized with @actin cDNA showed little variation in transcript abundance,and the useof an alternative control probe, cDNA to cyclophilin, a protein which is constitutively expressedin other tissues,gave identical results to those obtained with @-actin. All probeswere labeledwith 32Pbv random priming (15) and Tissue preparation

and extraction

of

RNA.

Clearance Measurements

The &/I&, value increased with gestation by 7- and 72-fold for [ 14C] mannitol and 45Ca, respectively, between day 15 and day 22 of gestation (Fig. 1). The analysis of variance showed that the two tracers behaved differently over the period of gestation studied (P = 0.03, F = 3.45 with 3 degrees of freedom). Further analysis, by simple contrasts, at individual times showed a significant difference in I&/D, values for the two tracers at days 21 and 22 (P = 0.002 and P = 0.02, respectively). This suggests that between days 18 and 21 of gestation an additional route of transfer, inaccessible to mannitol, becomes available to calcium.

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Fig. 1. Unidirectional maternofetal clearance normalized to diffusion coefficient in water (I&&&) values for 45Ca (A) and [14C]mannitol (m) over last one-third of gestation in the rat. Means t SE are shown (unless SE falls within size of symbol) with n (number of placentas) in Parentheses.

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Fig. 2. Northern hybridization of rat placental RNA with 32P-cDNA probes. Single Northern blot probed with calbindinan (A) and /3-actin (B). Gestational age in days is given above each lane with 70,45,15,5, and 7 fig total RNA/lane at 13,15,18, 21, and 22 days gestation, respectively. Position of RNA size markers (in kb) (GIBCO, Uxbridge, Middlesex, UK) is shown on right. Arrows indicate major RNA species at 0.6 kb (A) and 2.4 kb (B). Transcript density peak area values (arbitrary units) are given beneath each lane. Data are representative of 4 independent analyses.

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mRNA

Northern hybridization of rat placental RNA with calbindinsk cDNA revealed a single transcript of 0.6 kb at all gestational ages. It was necessary to load relatively more RNA at the earlier gestational ages to enable spectrodensitometric quantitation from an autoradiograph of the same exposure, since the abundance of this transcript increased markedly with gestation (Fig. 2A). Reprobing with Ca2+-ATPase cDNA demonstrated a major transcript of 5.5 kb at all gestational ages (Fig. 3). When RNA loadings were standardized to P-actin mRNA levels (Fig. 2B), a 135fold increase in placental calbindinsK-to-/3-actin mRNA ratio occurred between 15 and 22 days of gestation. The increase in the abundance of the placental calbindinex transcript between days 15 and 22 of gestation showed a close temporal relationship

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with the increase in maternofetal 45Ca clearance during this period (Fig 4). In contrast, Ca2+-ATPase-to-P-actin mRNA ratio in rat placenta increased only two- to threefold over the same gestational period and did not parallel the increase in maternofetal 45Ca clearance (Fig. 4). DISCUSSION

Gestational

Changes

in Placental

Calcium

Transport

Over the last 7 days of gestation, the rat fetus accumulates >99% of its body calcium (11). The increase in fetal calcium accretion during this period closely matches the observed gestational increase in maternofetal calcium clearance (Fig. l), suggesting that the unidirectional maternofetal calcium flux becomes increasingly predominant and approaches net flux, with the fetomaternal flux being relatively small. This supports the earlier finding of Stulc and Stulcova (27) in the near-term rat placenta.

Fig. 3. Northern hybridization of rat placental RNA with 32P-cDNA for Ca2+-ATPase. Same Northern blot as in Fig. 2 probed with cDNA for Ca2+-ATPase is shown. Gestational age in days is shown above each lane. Position of RNA size markers (in kb) is shown on right. Arrow indicates major RNA species at 5.5 kb. Transcrint densitv neak area values (arbitrary units) are given beneath each lane. Data are representative of 4 independent analyses.

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latter is more compatible with the dramatic increase in placental calbindingK transcription as placental calcium transport rises.

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Gestational Changes in Ca2+-ATPase mRNA Levels

160 CI)

v 80 e Y 2 40 0 16

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20

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Fig. 4. Relationship between &/& for 45Ca (A) and mRNA levels for calbindingK (w) and Ca 2+-ATPase (0) in rat placenta during gestation. K,flD, for 45Ca is taken from Fig. 1, and mRNA levels are shown as density values for calbindingk or Ca2+-ATPase mRNA normalized to @actin mRNA density.

The induction of a higher maternofetal clearance of calcium across the rat pl .acenta between 18 and 21 days of gesta tion, relative to the paracellular marke lr [ 14C] mannitol, suggests an additional route of transfer is available to calcium from this time, which coincides with the onset of rat fetal ossification (25). Because calcium is transported across the term rat placenta by an active mechanism (27), it is likely that this additional route is the transcellular pathway. The gestational increase in rat placental calcium transport could be attributable to an increase in the concentration, and/or the functional activity, of the transport that mRNA for proteins involved. The demonstration calbindingK increased concomitantly with J&J&, for 45Ca between days 15 and 22 of gestation (Fig. 4) suggests this protein is stoichiometri tally involved in transplacental calcium transport. Gestational Changes in CalbindingK mRNA Levels

Previous studies using in situ hybridization have demonstrated the cytoplasmic localization of calbindingK mRNA in the syncytial cells of the rat trophoblast (30). The single calbindin gK transcript of 0.6 kb in rat placenta found here (Fig. 2A) is in agreement with previous observations (12, 30). The increase in placental calbindingK mRNA levels over the last one-third of gestation (Fig. 4) is also in broad agreement with the earlier observations of de Maintenant et al. (12). In contrast to the marked gestational increase in placental calbindingK concentration found in rat (7, 20) and mouse (8)) mRNA levels for calbindingK in the human placenta remain fairly constant throughout gestation (6). This different pattern in gestational expression of placental calbindingK is probably related to the comparatively more acute fetal demand for calcium in rodents (11). The role of calbindingK in epithelial calcium transport is not fully understood, although it is present in highest con .centration in calcium transporting epithelia such as the intestine, kidney, yolk sac, and placenta (10) and is induced, perhaps hormonally, in rat uterus, yolk sac, and placenta at the same stage of gestation (20). The distribution of calbindingK in transporting epithelia has been suggested to imply a calcium buffering role (lo), or an ability to facilitate the diffusion of calcium through the cytosol of these epithelia (4,14). Perhaps the

Northern analysis of rat placental RNA (Fig. 3) with a cDNA coding for a human isoform of the calcium pump (29) demonstrated, for the first time, the expression of this calcium pump isoform in rat placenta. To date, this is the only isoform that has been clearly identified in rat and human (23). The size of the rat placental calcium pump transcript, -5.5 kb (Fig. 3), agrees well with that found in other rat tissues (17) and in the human erythrocyte (24). Two calcium pump isoforms have so far been identified in the human erythrocyte, although the functional significance of this remains uncertain (24). It has yet to be established if placenta also expresses more than one isoform. Localization of a high-affinity calcium pump to the innermost fetal facing membrane of the rat trophoblast and to the basal (fetal facing) membrane of human trophoblast using a monoclonal antibody raised against the human erythrocyte (3), and the demonstration that ATPdependent calcium transport in isolated basal membranes from the human placenta is inhibited by this antibody (3), highlights the similarity between the placental and erythrocyte calcium pump and promotes the concept of a calcium transport role for this enzyme. However, the small (2- to 3-fold) increase in placental Ca2+-ATPase mRNA levels during E-22 days of gestation in the face of the 72-fold rise in calcium clearance (Fig. 4) infers that the concentration of this protein is unlikely to be rate limiting to rat placental calcium transport. We are grateful to Dr. V. Hillier for statistical advice and to Vera Green for skilled secretarial assistance. We would also like to thank Drs. M. Thomasset and E. Strehler for donating the cDNA probes used in this study. This work was supported by grants from the Medical Research Council and by the National Fund for Research into Crippling Diseases Endowment. K. L. Thornburg was a Fulbright Scholar on sabbatical from the Dept. of Physiology, Oregon Health Sciences University, Portland, OR. Address for reprint requests: J. D. Glazier, Dept. of Child Health, St. Mary’s Hospital, Hathersage Rd., Manchester, Ml3 OJH, UK. Received 7 October 1991; accepted in final form 6 May 1992. REFERENCES 1. American Institute of Physics. American Institute of Physics Hczndbooicz. New York: McGraw-Hill, 1957. 2. Atkinson, D. E., N. R. Robinson, and C. P. Sibley. Development of the passive permeability characteristics of the rat placenta during the last third of gestation. Am. J. Physiol. 261 (Regulatory

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Gestational changes in Ca2+ transport across rat placenta and mRNA for calbindin9K and Ca(2+)-ATPase.

The unidirectional maternofetal clearance (Kmf) of 45Ca was measured across the rat placenta over the last one-third of gestation. Kmf for 45Ca normal...
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