Ca -activated ATPase of the mouse chorioailantoic placenta:
developmental expression, characterization and cytohistochemical
localization
ROCKY S. TUAN and NEIL BIGIONI
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA and Department of Orthopaedic Surgery, Thomas JeffersonUniversity, Philadelphia, PA 19107, USA
Summary
A membrane-associated, Ca2+-activated, Mg2+-depen-dent ATPase activity has been identified in the mousechorioailantoic placenta. The enzyme activity is ex-pressed and increases as a function of gestation. Bio-chemical characterization shows that the enzyme ishighly specific for Ca2+ and nucleotide triphosphates,with a Km of 0.97 mM [Ca2+] and a V ^ of 1.05 nmol P,released mg"1 placental protein min"1. The mouse pla-cental Ca2+-ATPase activity has a pi of approximately6.8, and corresponds to two apparent MT values of 118and 150 xlO3, based on Ferguson analysis of non-denaturing electrophoretograms. Enzyme activity is in-
hibited by phenothiazin (suggesting a calmodulin depen-dence), vanadate, erythrosin B and quercetin, but not byouabain or levamisole. Enzyme cytohistochemistry re-vealed that the Ca2+-ATPase is localized topolyploidtrophoblastic cells of the mouse inner placenta. Theseresults suggest that the enzyme may be a functionalcomponent of transplacental calcium transport duringmouse embryonic development.
During mammalian embryonic development, the feto-placental structure transfers nutrients from the ma-ternal to the fetal circulation. Although some nutrientsare translocated passively (e.g. gases) or diffuse in afacilitated manner (e.g. sugars) by virtue of concen-tration gradients, most of the nutrients are probablytranslocated actively, i.e. in an energy-dependent man-ner, across the placental barrier (e.g. see reviews byBoyd, 1987; Munro et al. 1983; Shennan and Boyd,1987; Truman and Ford, 1984).
Our laboratory has been studying the mechanism ofcalcium transport in human and mouse placentae bycharacterizing the biochemical components that may befunctional in the transport pathway. Our studies haveidentified high-Mr, Ca -binding proteins (CaBPs) inhuman (Tuan, 1982 and 1985) and mouse (Tuan andCavanaugh, 1986) placentae, and a Ca2+-activatedATPase in the human placenta (Tuan and Kushner,1987). In addition, the functional involvement of theCaBP and Ca2+-ATPase in calcium transport has beendemonstrated in vitro using cell-free membrane vesiclesisolated from term human placenta (Tuan, 1985).Although human and mouse placentae are both hemo-chorial in nature (Ramsey, 1975), they differ signifi-
cantly in tissue architecture. The former is character-ized by well-defined chorionic villi enveloped in thematernal circulation, whereas the latter consists of alabyrinth of polyploidal trophoblasts intertwined withmaternal blood vessels and sinuses (Ramsey, 1975;Hogan et al. 1986). The human and mouse CaBPs arestructurally related since they are immuno-crossreac-tive (Tuan and Cavanaugh, 1986) and the mouse CaBPcDNA cross-hybridizes with RNAs from both tissues(Tuan and Kirwin, 1988). Interestingly, human andmouse placental CaBPs are both expressed as a func-tion of gestation (Tuan, 1982; Tuan and Cavanaugh,1986), corresponding to the increased fetal need forcalcium during development (Widdowson, 1968; Garel,1983). Furthermore, the distribution of both CaBPs islocalized to the trophoblasts (Tuan, 1985; Tuan andCavanaugh, 1986), the cell type believed to be respon-sible for nutrient transport in the placenta (Deardenand Ockleford, 1983). These findings thus suggest thatcalcium transport by human and mouse placentae mayindeed involve a similar, developmentally regulatedmechanism.
In this study, we have further analyzed the mechan-istic aspect of calcium transport in the mouse placentaby examining its Ca2+-ATPase activity, based on therationale that such an activity is probably also a
506 R. S. Tuan and N. Bigioni
functional component of the transport machinery, as inthe human placenta. We report here that a develop-mentally expressed Ca2+-activated ATPase activity isindeed present in the mouse placenta that shares manycommon properties with the human placental enzyme.In addition, the enzyme has been localized histochemi-cally to the trophoblastic cells of the mouse chorioallan-toic placenta, consistent with its possible functionalinvolvement in placental calcium transport.
Materials and methods
Animals and placental tissuesMice (females, C57BL; males SJL) were obtained from theJackson Laboratory (Bar Harbor, ME, USA). Mating wascarried out randomly, and the first presence of a vaginal plugin the female was counted as day 1 of gestation. Animals werekilled by cervical dislocation, and the chorioallantoic placentawas dissected and cleared from the amniotic sac and uterinewall, rinsed thoroughly in cold 0.9% NaCl, and used immedi-ately for membrane preparation or extraction. Occasionally,the tissues were kept frozen at — 80°C and then extracted.
Tissue extraction and membrane preparationPlacental tissues were either extracted directly or processedfor microsomal membranes as follows. For direct extraction,the placenta was homogenized (1:4, w/v) in a Triton-Trisbuffer (13.7 mM Tris-HCl, pH 7.4, 0.12 M NaCl, 4.74 mM KC1,98.5/iM glucose, 0.02% NaN3, and 1% Triton X-100; Tuanand Knowles, 1984). The soluble placental extract was de-fined as the supernate after centrifugation of the homogenateat 31000 g for 30 min. Microsomal membranes were preparedas described previously for human term placenta (Tuan, 1985;Tuan and Kushner, 1987). Briefly, a freshly prepared, iso-osmotic homogenate of mouse placentae was fractionated bydifferential centrifugation, and microsomes were obtained bycentrifugation (80000g, 80 min) of the post-mitochondrialfraction. Microsomal membrane vesicles were suspended in10mM imidazole-HCl, pH7.0, containing 5min MgCl2 and0.1M KC1 to a concentration of 5-10 mg of protein m l .
Assay of Co2*-ATPase activityThe ATPase enzyme activity was determined using tissueextract or solubilized microsomal membranes at pH8.0 asdescribed previously (Tuan and Knowles, 1984) using thephosphomolybdate-Malachite Green colorimetric reaction,and based on differential activities in the presence of Ca2+
and ethylene glycol bis(amino-ethyl ether)tetra-acetate(EGTA). Levamisole (lmM) was included in most assays toinhibit placental alkaline phosphatase activity (van Belle,1972). Enzyme activity was expressed as moles of phosphatereleased per minute.
Protein determinationProtein was estimated by the method of Lowry et al. (1951)with bovine serum albumin as standard.
Co2*-ATPase cytohistochemistryThe procedure used was as described previously (Tuan andKnowles, 1984; Tuan and Kushner, 1987), using 10^m cryo-sections of frozen-embedded mouse placenta. The incubationmixture contained ATP, lead citrate, CaQ2 and levamisole toinhibit alkaline phosphatase. The reaction product of leadphosphate was visualized by incubation with (NH^S. Con-
trols included omission (or substitution) of ATP (ADP),CaCl2 (EGTA), or lead citrate from the incubation mixture.All stained sections were mounted in glycerine for micro-scopic observation.
Co2*-ATPase zymogramSolubilized samples were subjected to non-denaturing poly-acrylamide gel electrophoresis (Tuan and Knowles, 1984), orisoelectric focusing in polyacrylamide gel using the LKBMultiphor unit as described previously (Ono and Tuan, 1986).For isoelectric focusing, ampholytes (IsoLytes, IsoLab)covering the range of pH3-10 were used. After electrophor-etic or isoelectric fractionation, the gels were rinsed andreacted histochemically for Ca2+-ATPase as described pre-viously (Tuan and Knowles, 1984). The pH gradient of thefocusing gel was determined using a flat-surface pH electrodeacross a full-length portion of the gel.
Placental membrane calcium uptakeThe procedure used was as described previously (Tuan, 1985:Tuan et al. 1986). Microsomal membranes were isolated froman isosmotic homogenate of day-14 mouse placenta by differ-ential centrifugation (80000^, 80 min) of the post-mitochon-drial fraction. Membrane vesicles were obtained by suspen-sion in 10mM imidazole-HCl, pH7.0, containing 5mMMgCl2, and 0.1M KC1 to a concentration of 3—4 mg ofprotein ml"1. The calcium uptake assay conditions were:imidazole buffer, 0.1 mM CaCl2 (containing approximately0.5/iCi of ^CaClzmT1), 5mM ATP, and incubation at 37°C.At the end of incubation, membranes were separated fromthe solution phase by centrifugation through a layer of siliconeoil (specific gravity=1.041). Uptake was measured as the rateof Ca incorporation from 30 s to 120 s.
Results
Identification and developmental expression ofplacental Co2*-ATPaseThe initial enzyme assay was carried out using totalextract from day-18 mouse placental tissues. The ex-tract exhibited Ca2+-activated ATP hydrolysis in a time-and substrate-dependent manner, with a mean specificactivity of 14-18 nmol phosphate mg"1 protein min"1.The membranous nature of the enzyme activity wasindicated, as the specific activity was increased 5- to 10-fold when microsomal membrane preparations wereassayed.
The total Ca2+-ATPase activity of the mouse pla-centa was measured as a function of embryonic devel-opment. As shown in Fig. 1, the specific activity level ofthe enzyme increased steadily during gestation, particu-larly during the late stages, perhaps reflecting theincreased calcium need for skeletal mineralization ofthe rapidly growing rodent embryo (Bruns et al., 1978;Garel, 1983). The developmental profile of the mouseplacental Ca2+-ATPase is thus consistent with a poss-ible functional role for the enzyme in placental calciumtransport.
Characterization of placental Ca2+-ATPaseThe enzyme activity was next characterized with respectto its ion dependence and specificity, apparent MT, pi,kinetic parameters, substrate specificity, temperatureand pH optima, and thermal stability.
Mouse placental Ca2+-ATPase 507
20
18
ivit
o
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:ein
o
1°CL"
"o
1
16
14
12
10
h3 ^
on
hi
13 14 15 16 17 18 19Embryonic age (days)
Fig. 1. Developmental profile of mouse placental Ca2+-ATPase activity. Placental tissues were dissected from miceat different stages of gestation and were extracted andassayed for Ca -ATPase activity as described in Materialsand methods. The activities (closed circles) are themean±s.D. from 2 experiments and are expressed asspecific activities (nmol Pi mg"1 protein min"1). Forcomparison, the age profile of embryo wet weight (opencircles) is also presented based on previously publishedvalues (Brans et al. 1978; Garel, 1983).
10 100Calcium (//M)
1000
Fig. 2. Ca2+ and Mg2"1" dependence of mouse placentalATPase. Mouse placental extract was assayed in varying[Ca2+] and [Mg2"1"] as indicated. All activities are the meanof triplicates and expressed as a percentage of the highestvalue.
(1) Ion dependence and specificityIonic activation of the placental enzyme was examinedin varying [Ca2+] and [Mg2"1"], and the results are shownin Fig. 2. The data clearly showed a Ca2+-activated,Mg2+-dependent ATPase activity. Thus, at all Mg2"1"concentrations, Ca2+ served to activate the enzyme,with the activation being optimal at 1CT5 M [Mg2"1"]. It isnoteworthy that although Mg2"1" at 1CT5 M was requiredfor the Ca-activated ATPase activity, higher [Mg2"1"]actually appeared to inhibit the Ca2+-activated enzyme,particularly at low [Ca2+], except at [Ca2+] 3^500/iM,where some Mg2+-activated ATPase activity was ob-served. This latter finding suggested the presence in thetotal placental extract of another enzyme that operatedat high [Mg24"] and [Ca2+]. However, this activity wasnot observed when placental microsomal membraneswere assayed.
- . 1 0 0
i^ 80
« 60
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, Cd
\ Mg\
\
1 1
/
/
1 Sr
/
v / minus Ca
Ba
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0.06 0.08 0.10 0.12Ionic radius (nm)
0.14
Fig. 3. Ion specificity of mouse placental Ca2+-ATPaseactivity. Mouse placental extract was assayed for ATPaseactivity as described in Materials and methods in theabsence of Ca2+ (2mM EGTA; minus Ca2 +) , in thepresence of Ca2+ (100 HM), or equimolar concentrations ofCa2+ and the indicated divalent cations (100/iM total). Allactivities are the mean of triplicates, and are expressed as apercentage of the activity in the presence of Ca alone andplotted against the ionic radii of the respective ions.
To examine the ion specificity of the Ca2+ activationof the enzyme, a number of divalent cations were testedfor their ability to compete with Ca2+. The data inFig. 3 clearly demonstrated the Ca2+ specificity ofenzyme activation, and furthermore indicated that theability of other cations to displace Ca2+showed someapparent relationship to the similarity of their ionicradii to that of Ca2+, suggesting some degree of stericspecificity for Ca2+ in the binding site of the enzyme.
(2) Apparent Mr
To determine the apparent native MT of the placentalCa2+-ATPase, we carried out Ferguson analysis of non-denaturing gel electrophoresis (Chrambach, 1980), fol-lowed by enzyme histochemistry as described pre-viously (Tuan and Knowles, 1984; Tuan and Kushner,1987). The histochemical electrophoretogram revealedtwo dark brown bands of enzyme activity, bands I andII (Fig. 4A). The two activity bands, which wereobserved in both whole placental extract as well asplacental microsomal membranes (total staining shownin Fig. 4B), behaved as distinct moieties with differentpatterns of electrophoretic mobility in acrylamide gelsof varying %T (acrylamide+bis-acrylamide). It shouldbe noted that band II was consistently the moreprominent activity species. By plotting the R{ valuesversus varying %T, the retardation coefficients, KT, ofthe two activity bands were calculated (Chrambach,1980) and compared to those of standard proteins ofknown Mx (Fig. 4C). This comparison yielded apparentMr values of 118xlO3 and 150X103 for bands I and II,respectively.
(3) Isoelectric point (pi)The isoelectric zymogram of the placental Ca2+-ATPase revealed only one species of enzyme (Fig. 5),which corresponded to a pi of 6.8-7.0.
508 R. S. Tuan and N. Bigioni
P MM P M
30 40 50 60 70- K r (xlOOO)
80 90 100
Fig. 4. Electrophoretic analysis of mouse Ca2+-ATPase.Total mouse placental extract and solubilized microsomeswere fractionated by non-denaturing Triton X-100polyacrylamide gel electrophoresis, and Ca2+-ATPaseactivity was visualized by enzyme histochemistry asdescribed in Materials and methods. (A) Enzyme histogramof Ca2+-ATPase showing two activity bands (arrows, BandsI and II). Lane P, total placenta. Lanes M, microsomes.(B) Coomassie blue staining of proteins on the denaturinggel. Lanes P, total placenta; Lanes M, microsomes. (C) Sizeestimation of Ca2+-ATPase activity by means of Fergusonanalysis of the enzyme histogram compared to standardproteins of known molecular weights. Bands I and IIcorrespond to apparent Mv values of 118xlO3 and 150X103,respectively.
(4) Kinetic parametersA Lineweaver-Burke reciprocal plot of l /v versusl / [Ca 2 +] of the placental Ca2+-ATPase activity (Fig. 6)yielded an apparent Km of 0.97 ^M [Ca2+] and a Vmax of1.05 nmol phosphate released mg" 1 placental protein
mm- i
(5) Substrate specificityThe Ca2+-activated enzyme activity was tested usingvarious substrates. The relative activities (%) based onphosphate released were: ATP, 100; ADP, 52; AMP, 5;/5-glycerophosphate, 3; and GTP, 100. These data
A
B
1
i
3.77 5.42 6.78 7.39 7.82 8.20 8.77 9.50 10.96pH
Fig. 5. Isoelectric focusing profile of mouse Ca2+-ATPaseactivity. Mouse placental extract was electrofocussed inpolyacrylamide gel containing Triton X-100 as described inMaterials and methods. Lanes A and B are two separatepreparations of placental extract; the enzyme activity(arrow) corresponded to a pi of 6.8-7.0.
• § 2 4S 23
2 21
- 2000
!
| 1 8 o 50 100 150 200 250
Fig. 6. Lineweaver-Burke kinetics plot of mouse placentalCa^-ATPase activity. The plot yielded a Km of 0.97 UM[Ca2+] and a Vmax of 1.05 nmol Pi mg"1 protein min .
indicated that the enzyme activity was highly specific fornucleotide triphosphates, and did not result from non-specific phosphatases.
(6) Thermal properties, pH optimum, and stabilityThe mouse placental Ca2+-ATPase was thermolabile;more than 60% of activity was lost by a 15 minincubation at temperatures >65°C, and boiling for5 min completely destroyed enzyme activity. The tem-perature optimum of the enzyme activity itself was 45 °C(Fig. 7A). The pH profile of the placental Ca2+-ATPaseactivity was also measured. As shown in Fig. 7B, theenzyme activity was maximal at pH values (8.0-8.5)slightly above physiological, and was thus unlikely to berelated to placental alkaline phosphatase (also see dataon inhibitors below). With respect to stability, enzymeactivity was preserved in whole placental tissues orpelleted microsomal membranes kept at —80°C for atleast 6 months; however, detergent-solubilized enzymelost activity after >24h upon storage at 4°C or -20°C.
Inhibitors of mouse placental Ca2+-ATPaseVarious pharmacochemical reagents that were reported
Mouse placental Ca2+-ATPase 509
100r (A)
•a 80
60
40
20 40 60Temperature (°C)
80
100 r (B)
I"« 60
40
20
10
Fig. 7. Thermal (A) and pH (B) optima of mouse placentalQr+-ATPase. The enzyme assays were carried out at theindicated temperatures (A), or in Hepes-Pipes buffers atthe indicated pH (B). The activities are the mean oftriplicates and expressed as a percentage of the highestvalue.
to affect ATPase or phosphatase activities were testedfor their ability to interfere with the mouse placentalCa2+-ATPase activity. These compounds included anti-calmodulin compounds (phenothiazin: Gietzen et al.1980; Vincenzi, 1982), erythrosin B (Morris etal. 1982),ouabain, vanadate (Caroni and Carafoli, 1981), quer-cetin (Barzilari and Rahamimoff, 1983; Bambauer et al.1984), and levamisole (van Belle, 1972), varying dosesof which were used to treat the placental extract prior toand during the Ca2+-ATPase assay. The results in Fig. 8showed that whereas ouabain and levamisole did notaffect the activity at all doses, the other compoundswere effective inhibitors of the placental Ca2+-ATPaseactivity. The relative inhibitory strength based oneffective dose appeared to be: phenothiazin>sodiumvanadate, erythrosin B^quercetin (Fig. 8). The highsensitivity to phenothiazin suggested a possible func-tional association between calmodulin and the enzyme.
Cytohistochemical localization of placental Ca2+-ATPaseThe cellular location of the Ca2+-ATPase in the mouseplacenta was detected histochemically as describedpreviously for the human term placenta (Tuan andKushner, 1987) and the chick embryonic chorioallantoicmembrane (Tuan and Knowles, 1984). As shown inFig. 9(A-D), Ca2+-ATPase histochemistry on cryosec-tions of day-16 mouse placenta revealed the presence ofenzyme activity in the inner placenta region; further-
Control
Phenothiazin
Erythrosin B
Quercetin 1 m M
0.01 mM
Ouabain
Levamisole
1 mM0 01 mM
1 mM
20 40 60 80 100
Relativ tnzyme actMty (%)
Fig. 8. Inhibition of mouse placental Ca2+-ATPase activity.Mouse placental extract was incubated with the respectiveagents at the indicated concentrations for 15 min and thenassayed for enzyme activity under the same conditions. Allactivities are the mean of 2-3 experiments and areexpressed as a percentage of the activity in the absence ofany pharmacochemical agents (control).
more, the reaction product was associated with aggre-gates of polyploid trophoblastic cells. Occasionally,reaction product could also be seen associated withcellular components of the fetal vasculature (notshown), perhaps a result of the Ca2+-ATPase activity ofthe vascular smooth muscle (Githens, 1983; Godfraind-De Becker and Godfraind, 1980). The specificity of theenzyme histochemistry was demonstrated, as in pre-vious studies (Tuan and Knowles, 1984; Tuan andKushner, 1987), by the lack of reaction product in theabsence of Ca2+ (Fig. 9E and F) or ATP (not shown).Levamisole (1 mM) was routinely included in the incu-bation mixture to eliminate interference from alkalinephosphatase (van Belle, 1972).
The developmental profile of Ca2+-ATPase ex-pression was also examined by cytohistochemistry.Fig. 10 illustrates the results obtained using day-11 and-14 placenta samples. In agreement with the biochemi-cal activity data shown in Fig. 1, day-11 placenta did notexhibit any enzyme staining (Fig. 10A and B), whereasday-14 placenta was clearly reactive (Fig. 10C-F).Interestingly, the first appearance of Ca2+-ATPase wasseen in the inner placenta (Fig. 10C-E), also associatedwith aggregates of polyploid trophoblastic cells(Fig. 10F). The outer placenta remained negative forCa -ATPase histochemical activity, at least up to day16.
Calcium uptake by cell-free mouse placentalmembranes and functional involvement of Ca2+-ATPaseCell-free membranes isolated by differential centrifu-gation fractionation of mouse placenta exhibited ATP-dependent 45Ca uptake (Fig. 11) in a manner analogousto similar membranes isolated from the human termplacenta (Tuan, 1985), although the apparent specific
510 R. S. Tuan and N. Bigioni
Fig. 9. Histochemical localization of Ca2+-ATPase activity in mouse placenta. Cryosections (8/an) of mouse placenta werestained for Ca2+-ATPase as described in Materials and methods. (A and B) Positive reaction for Ca2+-ATPase activityobserved using phase contrast (A) and bright-field (B) optics. Note association of reaction product with large polyploid cells(arrows). (C and D) Similar to A and B, respectively. Some reactivity was also observed in the erythrocytes located in afetal capillary (open arrow). (E and F) Similar to A and B, respectively, except that Ca2+ was omitted from the incubationmedium. Bar=10/an.
activity was lower in the mouse placenta. Functionalinvolvement of the Ca2+-ATPase was also suggestedsince pre-incubation with phenothiazin, a potent inhibi-tor of its activity (Fig. 8), also significantly inhibitedmembrane calcium uptake (Fig. 11).
Discussion
We have reported here the identification, characteriz-ation and cellular localization of a specific, Ca2+-activated, Mg2+-dependent ATPase in mouse chorioal-lantoic placenta. Enzyme activity is expressed as afunction of fetal gestation, and is localized principally inthe large, polyploid fetal trophoblasts of the innerplacenta. The enzyme has a pi of 6.8-7.0, and corre-sponds to two Mr species of 118 and 150X103. The
activity is highly sensitive to phenothiazin, suggesting acalmodulin dependence, and is also inhibited by vana-date, erythrosin B and quercetin. Phenothiazin alsostrongly inhibits calcium uptake by placental mem-branes in vitro.
The biochemical properties of the mouse placentalCa2+-ATPase suggest that it is a membrane-boundenzyme that functions at physiological pH. Althoughtwo electrophoretic forms of the enzyme are observedon non-denaturing gels (Fig. 4A), they are isoelectric(see Fig. 5) and probably differ only in apparent MT. Bycomparison with the enzyme of the human placenta(Treinin and Kulkarni, 1986; Tuan and Kushner, 1987),and the known properties of other Ca2+-ATPases (seereviews by Sarkadi; 1980; Penniston, 1982; Schatz-mann, 1983; Carafoli, 1987), we suggest that the118X103 form is derived from the vasculature, whereas
Mouse placental Ca2* -ATPase 511
Fig. 10. Developmental profile of Ca2+-ATPase activity in mouse placenta revealed by enzyme histochemistry. Cryosectionsof mouse placentae (day-11, A and B; day-14, C-F) were stained as described in the legend to Fig. 9. (A,C,F) Phasecontrast optics; (B,D,E) bright-field optics; op, outer placenta; ip, inner placenta. (A and B) Day-11 placenta showing nodetectable enzyme activity; opposing arrowheads demarcate the apparent border between the inner (ip) and outer (op)placenta. (C and D) Similar regions in a day-14 placenta, showing distinct activity staining in the inner placenta, whereas theouter placenta remained negative for enzyme activity. (E) Another region of the placenta with a staining pattern similar toD. (F) Higher magnification of stained regions, showing association of enzyme activity (arrowheads) with the peripheral areaof large, polyploid cells. Bar=40^m in A-E, and 10/an in F.
512 R. S. Tuan and N. Bigioni
3 100
§ • _ 80
1 ^ - 60
S;E 40
S o Control -ATP +PHE
Fig. 11. Calcium uptake by cell-free placental membranesin vitro. The membrane preparation and calcium uptakeprocedures were as described in Materials and methods.Calcium uptake activity was expressed as pmolmg"1
protein min"1. Samples were treated as follows: Control,standard assay conditions; —ATP, in the absence of ATP inthe incubation mixture; and +PHE, membranes pre-treatedwith 100 HM phenothiazin and assayed in the presence of theinhibitor. Values represent the mean of triplicates, and areexpressed as percentage of control (10.1pmolmg~1min~1).
the more predominant, higher Mr, 150X103 form islikely to be a plasma membrane component of theplacental trophoblasts, which stain prominently forCa2+-ATPase activity in the histochemical reaction.Indeed, Borke et al. (1989) have recently detected byimmunohistochemistry epitopes of the human erythro-cyte plasma membrane 140 xlO3 Ca2+-ATPase in thebasal membranes of human and rat placental tropho-blasts. It is highly likely that the Ca -ATPase activitydescribed here is similar to that reported by theseworkers.
Calcium transport is a major function of the placentalstructure, and serves to provide the necessary calciumto the developing embryo. In vivo and in vitro calciumuptake studies (e.g. Fisher et al., 1987; Miller andBerndt, 1975; Shami et al. 1975; Sweiry et al., 1986;Tuan, 1985; Twardock, 1967; van Kreel and van Dijk,1983; Whitsett and Tsang, 1980), using whole animal orperfused placenta or membrane vesicles derived fromhuman and other animal species, have clearly estab-lished a bioenergetic requirement for calcium transport.Since the active nature of placental calcium transportwould suggest that it is coupled to ATP hydrolysis, theCa2+-ATPase identified and characterized in this studymay indeed be functionally involved in the transportprocess, as suggested by the ATP dependence ofcalcium uptake by mouse placental membranes asshown here (Fig. 11). Similar association of placentalmembrane calcium uptake transport with ATP hydroly-sis has also been reported by other workers (Fisher etal., 1987; Shami et al., 1975; Treinen and Kulkarni,1987). In support of this hypothesis, the mouse enzymeshares many common properties with the human pla-cental Ca2+-ATPase, which has been clearly implicatedin placental membrane calcium uptake in vitro (Tuanand Kushner, 1987). These common properties includeMr, substrate specificity and, most importantly, sensi-tivity to various pharmacochemical compounds, whichalso inhibit calcium uptake by placental membranevesicles (Fig. 11). However, it should be pointed outthat since these compounds are not specific inhibitors of
Ca -ATPase activities (as there are no 'ouabain-equivalent' specific inhibitors of plasma membraneCa2+-ATPases), the similarities between the two pla-cental Ca2+-ATPases thus only serve as circumstantialcorrelations. Additional support for a transport role forthe mouse placental Ca2+-ATPase is provided by itscellular distribution within the mouse placenta, i.e.association with the fetal trophoblast cells. It is thus ofinterest to note that the human (Tuan, 1985) and mouse(Tuan and Cavanaugh, 1986) CaBPs and their mRNA(Tuan et al. 1988), as well as the human Ca2+-ATPase(Tuan and Kushner, 1987), are also specifically local-ized within the trophoblastic cells, which are generallybelieved to the transporting cell type of the hemochor-ial placenta (Dearden and Ockleford, 1983; Sideri et al.1983; Truman and Ford, 1984).
At present, the mechanisms of trans-placental trans-port processes are poorly understood (Shennan andBoyd, 1987). With respect to calcium transport, thework in our laboratory has established the high MrCaBPs as functional components, and much progresshas been made on their biochemical and molecularcharacteristics (Tuan, 1982 and 1985). Specific reagentsfor the CaBPs, such as antibodies (Borke et al. 1989;Tuan and Cavanaugh, 1986) and cDNAs (Tuan andKirwin, 1988), have also been prepared. Work iscurrently in progress to extend these studies to theplacental Ca -ATPases to gain further understandingof the basic mechanism and regulation of placentalcalcium transport.
This research was supported in part by grants from theNational Institutes of Health (HD 15822, HD 21355), Marchof Dimes Birth Defects Foundation (1-1146), and U. S.Department of Agriculture (88-37200-3746). The authorsalso acknowledge the assistance of Robert Akins and JamesKirwin in the microsomal membrane study.
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