Journal of Phy8iology (1990), 420, pp. 295-311 With 8 figures Printed in Great Britain

295

TRANSPORT OF CALCIUM ACROSS THE DUALLY PERFUSED PLACENTA OF THE RAT

BY J. STULC, B. STULCOVA AND J. SVIHOVEC From the Department of Pharmacology, Faculty of Pediatrics and Department of Physiology, Faculty of Medicine, Charles University, Albertov 4, 128 00 Prague 2, Czechoslovakia (Received 21 September 1988) SUMMARY

1. A rat placenta was dually, perfused in situ with modified Krebs fluid. Perfusion was carried out through the femoral artery on the maternal side and through the umbilical artery on the fetal side. 2. Transfer of 46Ca2+ and [3H]L-glucose across the placenta was measured in the maternal-fetal direction. The transcellular component of the maternal-fetal transport of Ca2+, Jmf,tc, was estimated from transfer rates of the two tracers and from Ca2+ concentration in maternal perfusate, [Ca2+]m. 3. At [Ca2+]m of 1.1 mm (physiological concentration of Ca2+ in plasma) Jmf, tc was 92-4 + 13-7 nmol min-1 (mean + S.D.), which is about 90% of the transport expected in an intact placenta. The permeability-surface area product (PS) of the placenta to [3H]L-glucose was 13-8 + 3.9 ul min-', about 4 times higher than that expected in intact placenta. 4. Transport of 45Ca2+ changed rapidly when [Ca2+]m was varied. Kinetic constants of the transcellular transport of Ca2+ are the Michaelis constant, Km, = 045 mM and the maximum rate of transport, Vmax, = 116 nmol min-'. It follows from this that at physiological levels of Ca2 , transport of Ca2+ to the fetus is relatively independent of changes in [Ca2+]m. 5. Strontium and barium (SrCl2 and BaC12, 1 mM) decreased Jmf, tc; the response was prompt and reversible. Magnesium (2 mM) had no effect. Maternal-fetal transport of 85Sr2+ and l33Ba2+ was decreased rapidly and reversibly by elevating [Ca2+]m from 0 35 to 2 mm. These observations suggest that Sr2+ and Ba2+ are transported across the placenta by the Ca2+ transport system. This means that the transport is not substrate specific. 6. Cadmium (1 mM-CdCl2) decreased Jmf, tc irreversibly with some latency. The slowness of the response suggests a non-competitive inhibition. Cadmium (0-02 mMCdCl2) was without effect on Jmf, tc. 7. A Ca2+ channel blocker, nifedipine (10 /M), administered to the maternal side had no effect on Jmf,tc,

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J. STULC, B. STULCOVA AND J. SVIHOVEC INTRODUCTION

Movement of Ca2+ across the haemochorial placenta has paracellular and transcellular components. The paracellular component is represented by bidirectional diffusion through wide aqueous channels in the placental barrier. The transcellular transport is active. It is highly directional, generating net flux of Ca2+ from mother to fetus and a concentration gradient of Ca2+ between maternal and fetal plasma (for evidence and reasoning, see Stulc & Stulcova, 1986). It is believed that the general pattern of transcellular transport of Ca2+ across the placenta is similar to that of transcellular Ca2+ transport across the intestinal epithelium, that is, the transport is a sequence of three steps: passive Ca2+ entry into the placental trophoblast across the maternal border from the maternal side; movement of Ca2+ through the trophoblast cytosol to the fetal border; and active extrusion of Ca2+ from the trophoblast across the fetal border to the fetal side (Van Dijk, 1981). The last step is effected by an ATP-driven Ca2+ pump which has a high affinity for Ca2+ and which is stimulated by calmodulin (Fisher, Kelly & Smith, 1987). The mechanism of the first two steps is not known. In this study we have estimated the kinetic parameters of the transcellular maternal-fetal transport of Ca2+ and have examined its substrate specificity and its sensitivity to nifedipine and Cd2+, substances which are known to block Ca2+ channels in excitable tissues. For the reasons explained in the Discussion we believe that our observations describe the properties of the Ca2+ entry into the trophoblast across the maternal border. We artificially perfused both fetal and maternal vascular beds of the rat placenta and measured maternal-fetal transfer of 45Ca2+ and [3H]L-glucose (a substance with an extracellular distribution moving across the placenta through paracellular routes) at a steady state. From the transfer rates of the two tracers we estimated the transcellular component of the maternal-fetal transport of Ca2+, Jmf, tc Along with the transfer of the two radioactive substances we measured transfer of antipyrine as an indicator of the perfusion conditions of the preparation. METHODS

Drugs, chemicals and radioactive substances. The following chemicals and radiochemicals were used in the study: antipyrine, pentobarbitone and heparin (Spofa, Czechoslovakia); nifedipine (Bayer, FGR); bovine serum and lyophilized proteins of bovine serum (Sera and Vaccines, Czechoslovakia); bovine serum albumin V-powder (Sigma); [1-3H]L-glucose (UVVVR, Czechoslovakia); 45Ca-labelled calcium chloride (IZINTA, Hungary); 855r-labelled strontium chloride (Institute of Nuclear Research, Swierk Otwock, Poland); 99mTc-labelled-pertechnetate, 133Balabelled barium chloride (Amersham International); albumin microspheres (3M, Minnesota, USA). The microspheres were labelled with radioactive pertechnetate according to the instructions of the manufacturer. Perfusion system. The perfusion systems on the maternal and on the fetal side of the placenta consisted of a reservoir containing perfusion fluid thermostabilized at 38 °C, a roller pump, air bubble trap, arterial cannula and, on the fetal side, a venous cannula (no venous cannula was used on the maternal side). The arterial cannula on the maternal side was made from polyethylene tubing, and on the fetal side from a hypodermic needle 06 mm o.d. with blunted tip. The umbilical venous cannula was pulled from polyethylene tubing to an o.d. of about 1 mm. The perfusion pressures were recorded using mercury manometers.

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Perfusion fluids. The perfusion fluids were based on the bicarbonate Krebs fluid of the following composition (mM): NaCl, 118&0; KCl, 4-7; NaHCO3, 25-0; MgCl2, 1P2; Na2HPO4, 1P0; glucose, 5-5. Calcium was added to the fluid as the chloride; the concentration of Ca2+ in the standard perfusion fluid was 1 1 mM. The maternal perfusion fluid was a 4: 1 mixture of Krebs fluid and calf serum. The fetal perfusion fluid was Krebs fluid with 10 g 1-1 of lyophilized proteins of bovine serum (RPBoS) in the first part of the study (the concentration-dependence experiments) or Krebs fluid with 10 g 1-1 of bovine serum albumin in the rest of the study. Heparin was added to both perfusion fluids to a concentration of 2500 units 1-1. The fluids were equilibrated with 95% 02 and 5 % C02 at 38 0C. Antipyrine, [3H]L-glucose and 45Ca2+ were added to the maternal perfusate to approximate concentrations of 80 mg 1-1 and 0 5 MBq 1-1, respectively. The perfusion rates were 2 ml min-' on the maternal side and 1 ml min-' on the fetal side. Dual perfusion of the placenta. The experiments were performed on rats of our own breed on day 21 of pregnancy. The animals were anaesthetized with pentobarbitone administered into the tail vein in a dose of approximately 40 mg kg-'. Additional small doses of the anaesthetic were injected at short time intervals until the analgesia was achieved (complete lack of response to algic stimuli). The total dose of pentobarbitone administered before the onset of the surgical procedures was about 60 mg (kg body weight)-'. The skin was cut from the left inguina to the mid-line of the lower abdomen and a similar incision was made in the abdominal muscles. The ligamentum inguinale was cut through. All accessible branches of the left external iliac artery and vein, except the superior vesical vessels, were ligated. The urinary bladder was ligated at its base to tie off its vascular supply. A loop was placed around the left common iliac artery and vein just below the bifurcation of the aorta and tied. The femoral artery was cannulated retrogradely and infusion of the maternal perfusate was started at a rate of about 0-2 ml min-'. (The perfusion was thus carried out via the femoral, iliac, superior vesical and uterine arteries.) The animal was then transferred into a Ringer fluid bath thermostabilized at 37-38 'C and placed on its left side. The left horn of the uterus was exposed and the fetus, the placenta of which was to be perfused, was separated from its neighbours by loops of thread tied round the uterus. Patches of tissue paper were glued to the upper and lower face of the selected segment of the uterus with cyanoacrylic adhesive to prevent contraction of the uterine muscle after removing the fetus. All vessels supplying the uterus and placentae between the selected placenta and vagina were ligated. Umbilical perfusion was established as described previously (Stulc & Stulcova', 1986). The perfusion rate on the maternal side was increased to 2 ml min-', the uterine vessels were ligated just next to the perfused placenta in the ovarian direction and the uterine vein was opened. Maternal effluent was not collected, it was let out from the opened uterine vein into the bath. The animal was killed with an overdose of

pentobarbitone. Transport measurements and calculations. After dual perfusion had been established and the placenta perfused for at least 6 min, the collection of the umbilical effluent was started (this time is taken as time zero). The effluent was collected at 2 min intervals into pre-weighed vessels for 36-40 min. Each sample of effluent was deproteined with 50 ,ul of concentrated perchloric acid and centrifuged. Aliquots of 0 5 ml of supernatant fluid were taken for radioactivity counting and for estimation of antipyrine concentration. Transplacental clearance, Kmf, of antipyrine, 4SCa2+ and [3H]L-glucose was calculated as the amount of antipyrine or of the 45Ca2+ and 3H+radioactivity transferred into the umbilical perfusate per minute (mg min-' and c.p.m. min-', respectively) divided by the corresponding concentration or radioactivity in the maternal perfusate (mg ml-' or c.p.m. ml-', respectively). In the case of 45Ca2+ the ratio was further divided by the fraction of ionized calcium in the maternal perfusate. The Kmf of [3H]L-glucose is taken to represent the permeability-surface area product, PS, of the placenta to [3H]L-glucose. The rate of paracellular diffusion of small hydrophilic molecules across the rat placenta is approximately proportional to the respective diffusion coefficients in water (Stulc & Stulcova, 1986). On the basis of this observation the diffusion component of the transplacental clearance of 45Ca2+ was estimated by multiplying the PS of the placenta to [3H]Lglucose by the Ca2+/L-glucose diffusion coefficient ratio (estimated below). Transcellular clearance of 4SCa2+, Kmr, tc was calculated as the difference between Kmf and the diffusion component of the transplacental clearance. The transcellular transport of Ca2+, Jmf, tc, was obtained by multiplying Kmft, te by the concentration of ionized calcium in the maternal perfusate, [Ca2+]m. Using transplacental clearance to calculate the flux in the way described here rests on the

J. STULC, B. STULCOVA AND J. SVIHOVEC 298 following assumptions: (1) back flux of 45Ca2+ from the fetal to the maternal side is negligible; (2) the 45Ca2+ radioactivity as well as [Ca2+]m do not change significantly during the passage of the perfusate along the exchange barrier in the placenta. The first assumption is nearly correct. At Kmf for 45Ca2+ of about 85 ,d min-' (the average Kmf at [Ca2+]m of 1.1 mM) and the fetal perfusion rate oft ml min-' the mean 45Ca2+ radioactivity on the fetal side of the placental barrier will be about 4% of that on the maternal side. The movement of Ca2+ across the rat placenta is highly asymmetrical, the fetal-maternal/maternal-fetal flux ratio in the umbilically perfused preparation is about 0-2 (Stulc & Stulcova, 1986). Assuming that the flux ratio in the dually perfused placenta is similar, only less than 1% of 45Ca2+ transferred to the fetal side returns back to the maternal side. The second assumption is not correct as the 45Ca2+ radioactivity, and presumably also the Ca2+ concentration, decreases during the passage of the maternal perfusate through the placenta. The resulting error, however, is unlikely to be large because the decrease can only be small. For instance, at a Kmf of 85 #sl min-' and the average flow rate through the maternal vascular bed of the placenta of about 07 ml min-' (see below) the mean 45Ca2+ radioactivity on the maternal side of the placenta will be only about 6 % lower than the inflow radioactivity. Estimation of the kinetic parameters of the Ca2+ transport. All experiments of this type followed the same schedule (Fig. 1). For the first 8 min (four 2 min collecting periods) the placenta was perfused with the standard perfusate ([Ca2+]m of 1.1 mM). Then [Ca2+]m was changed. In each experiment two different concentrations of Ca2+ were tested successively, each for 10 min. Then the placenta was perfused with the standard perfusate again for 10 min. The concentrations of Ca2+ tested were (in mM): 0 35 (n = 5), 0 7 (n = 4), 0 95 (n = 3), 1.1 (n = 15), 1-6 (n = 6), 2-1 (n = 7) and 3-1 (n = 4). To minimize the effect of the spontaneous decrease of the Ca2+ transport during perfusion on the mean values of Jmf, tc at different Ca2+ concentrations the order ofthe fluids with different Ca2+ content was changed from experiment to experiment. The concentration of Ca2+ in the fetal perfusate was 1 1 mm throughout perfusion. An inverse value plot of Jmf, tc versus [Ca2+]m fitted a straight line indicating that the transport conformed to the Michaelis-Menten kinetics. Estimating the kinetic parameters from the linearizing plots has statistical dangers (Dowd & Riggs, 1965), in particular when the number of observations is small and not equal at all concentrations tested, as was the case in our study. To estimate the kinetic parameters from our data, therefore, we have used a numerical equivalent of the graphical method of Eisenthal & Cornish-Bowden (1974) which is statistically sound. In each individual experiment the corresponding values of Jmf,tc and [Ca2+]m were inserted in the Michaelis-Menten equation and the equations were paired and solved for the Michaelis constant, Km, and the maximum rate of transport, Vmax. The median value of each parameter was taken as being representative of the experiment. Effects ofMg2+, Sr2+, Ba2+, Cd2+ and nifedipine on the transport of 45Ca2+. All experiments followed the same schedule: 12 min perfusion with the control fluid (control period), 12 min exposure to the substance under test added to the maternal perfusate (experimental period), and 12 min perfusion with the control perfusate again (recovery period). In the group of control placentae transport of Ca2+ decreased spontaneously during perfusion (see below). This spontaneous decrease had to be taken into account when evaluating the effects of experimental treatment. This was done in the following way. All values of Kmf, tc in the control group were normalized with respect to the mean value during the first six collecting intervals, and the normalized values were fitted by a regression line (time being the independent variable). In the experimental groups the control values were extrapolated beyond the control period by multiplying the regression equation by the mean Kmf, tc during the control period. The effects of the experimental treatment were evaluated by comparing the observed Kmf, tc with the corresponding extrapolated control values. Estimation of the flow rate through the maternal vascular bed of the perfused placenta. The flow rate through the maternal side of the preparation was estimated using albumin microspheres labelled with 9omTc. Microspheres, suspended in 0-2 ml of saline, were injected into the maternal perfusion system close to the arterial cannula; the dose of radioactivity administered was about 5 kBq. After 10 min, placenta. adjacent uterine muscle, the rest of the left uterine horn, right uterine horn, vagina, abdominal viscera, and carcass were sampled and their radioactivity counted. The fraction of flow to the placenta was estimated as the radioactivity of the placenta divided by the total radioactivity recovered. The flow rate was obtained as a product of the fraction of flow to the placenta and the pump rate.

299 PLACENTAL TRANSPORT OF CALCIUM Estimation of diffusion coefficients. The diffusion coefficients of Ca2+ and L-glucose were estimated by the method of diffusion measurements in agar gel (Schantz & Lauffer, 1962) as described by Hedley & Bradbury (1980), using 45Ca2+ and [3H]L-glucose. The diffusion coefficients of the two substances were measured simultaneously in a single diffusion experiment at 38 'C. The values obtained (10-5 cm2 S-1) were 0 79 in the case of Ca2+ and 0-98 in the case of [3H]L-glucose. The diffusion coefficient of L-glucose estimated here agrees well with the value of 0-99 x 10-5 cm2 s-1 given by Schneider, Sodha, Progler & Young (1985) and is similar to that of D-glucose (0.9 x 10-5 cm2 s-1, Landis & Pappenheimer, 1963). The corresponding Ca2+/[3H]L-glucose diffusion coefficient ratio is 0-8. Chemical and radioactivity measurements. fl-Radioactivity of 3H and 45Ca was measured by liquid scintillation. y-Radioactivity of 85Sr, 99mTc and '33Ba was measured in a well scintillation detector. Concentration of Ca2+ was measured using the Radiometer equipment (Copenhagen, Denmark), total concentration of Ca2+ by a colorimetric method (KX-kit, Lachema, Czechoslovakia), concentration of antipyrine by ultraviolet spectrophotometry after extraction in chloroform (Stulc & Stulcova, 1986). Criteriafor rejection. The data were rejected if the PS of the placenta to [3H]L-glucose was higher

than 25 1 min-' or if the umbilical outflow rate decreased below 90 % of the pump rate. Of the 122 successfully started perfusions six ended in trivial mishaps, six were stopped because of a decreasing umbilical outflow rate (none of the placentae perfused with albumin fluid on the fetal side) and in the course of four perfusions the placenta separated from the uterus. The results of nine experiments were discarded because of a high PS for [3H]L-glucose, the results of four experiments were discarded because of a low Kmf of antipyrine. The observations in the remaining ninety-three placentae are presented below. Presentation of the data. The data are presented as means, the limits are standard deviations. Statistical significance of the observed differences was tested using the Wilcoxon signed ranks test. A difference is taken to be significant at P < 0.05. RESULTS

GOeneral observations The average fetal and placental weights were 3*57 + 0-42 g and 0-53 + 0-12 g (n = 93), respectively. The mean initial values of the variables recorded (n = 93) were: Kmf of antipyrine, 0-57 + 0-15 ml min-'; PS to [3H]L-glucose, 13-8 + 3.9,1t min-'; Jmf, tc, 92-4 + 13-7 nmol min-'; perfusion pressure on the maternal side, 51 + 17 mmHg; perfusion pressure on the fetal side, 10-7 + 5.3 mmHg. The PS to [3H]Lglucose and fetal perfusion pressure did not change significantly during perfusion, Kmf of antipyrine decreased by 8 % on average and maternal perfusion pressure increased by 12 %. In a group of six control placentae Jmf, tc decreased to 85 + 9 % during 40 min. The values of Kmf, tc normalized with respect to the mean Kmf, tc during the first 12 min (six collecting intervals), were fitted by a regression line Kmf,tc (normalized) = 1-025-0s046 t, where t is time (min); r = 0-90. The equation was used in the experimental groups to extrapolate the values of Kmf, tc beyond the control period (see Methods). The spontaneous decrease of Jmf, tc seems to be due to a decrease in Vmax (see below). The flow fraction to the maternal side of the placenta was 34-5 +11-2 % (n = 5). At a pump rate of 2 ml min-' the corresponding flow rate is about 0 7 ml min-', which is equal to the maternal placental blood flow in the rat near to term (0-7 ml min-'; Bruce, 1976). Of the other tissues analysed most of the flow was to the lower part of the carcass (24%), the myometrium of the perfused uterine segment (20%) and the

vagina (16%).

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Estimation of the kinetic parameters of the Ca2+ transport Changing [Ca2+]m had a rapid and great effect on Kmf, te (Fig. 1). After a change in the Ca2+ concentration in the reservoir a new steady level ofKKmf, te was attained within about 6 min. Much of this delay, however, seems to be caused by a delay in the [Ca2+]. (MM) 150

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20 30 Time (min) Fig. 1. The dependence ofthe transcellular clearance of 45Ca2+, Kmf, t, on the concentration of Ca2+ in the maternal perfusate [Ca2+]m. The line is the clearance observed. 0 represents the clearance predicted if a new steady state of transport of 45Ca2+ across the trophoblast was attained instantaneously. The predicted clearance was calculated from the response of the outflow concentration from the maternal arterial cannula to a step change in the concentration of a test substance in the reservoir (recorded in a separate experiment) according to the equation Kmt,tc (calculated) = Vmax/(Km+C0), where CO is the mean concentration of Ca2+ in the fluid infused during collection of the sample n. The constants Vmax and Km were estimated from the data obtained in the same experiment. For each sample the value of Vmax was corrected for the spontaneous decrease in the transport during perfusion assuming a linear time course. Results from a single experiment.

change in Ca2+ concentration in the fluid infused into the placenta. Figure 1 compares the time course of Kmf, tc observed and Kmf, tc predicted from the response of the outflow concentration at the tip of the maternal arterial cannula to a step change in the concentration of a test substance in the reservoir (recorded in a separate experiment), assuming that the transport responds to a change in [Ca2+]m without delay. The relatively small difference between Kmf, tc predicted and observed suggests that the change in transcellular Ca2+ transport is practically instantaneous. This observation indicates that the Ca2+ transport pool within the trophoblast is very small. The dependence of the transcellular transport of Ca2+ on [Ca2+]m is shown in

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Fig. 2. Above [Ca2+]m of about 1 mm the transport increases with increasing concentration of Ca2+ only very slowly. An inverse value plot of Jmf, tc versus [Ca2+]m fitted a straight line indicating that the kinetics of the transcellular transport of Caa2+ is consistent with the Michaelis-Menten 150-

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[Ca2+]L (mM) Fig. 2. The dependence of the transcellular transport of Ca2+, t,f t on the concentration of Ca2+ in the maternal perfusate. *, the observed values of Jmf, tc, the limits are S.D. The curve is the dependence predicted by the Michaelis-Menten equation.

model (Fig. 3). Kinetic parameters of the transport were estimated from the data as described in Methods. Km ranged from 0-08 to 1-13 mm, with a median value of 0 45 mM; Vmax ranged from 76 to 172 nmol min-', with a median value of 116 nmol min-1. We take the median values to be the best estimates. They are closely similar to the values of Km = 0'45 mm and Vmax = 117 nmol min-1, estimated from the inverse value plot in Fig. 3.

Effects of Mg2+, Sr2+ and Ba2+ The divalent ions of the second Mendeleev group of elements were tested to examine the specificity of the Ca2+ transport system in the placenta. If the transport system did not discriminate Ca24 and Mg2+, Sr2+ and Ba2+, the transport of Ca2+ would be inhibited competitively by the ions tested. The first group of experiments was performed at [Ca2+]m reduced to 0'85 mm. Strontium chloride and barium chloride were added to the perfusate to a concentration of 1 mm, the concentration of magnesium chloride was increased to 2 mM from 1 mm in the control perfusate. Adding Mg2+ had no effect on Kmf, tc (n = 5, the results are not shown). Sr2+ and Ba2+ decreased Kmf, tc by 9 + 4% (n = 7) and 21+26% (n = 13), respectively, both effects being statistically significant. The

J. S6TULC, B. STULCOVA AND J. SVIHOVEC response was prompt and readily reversible (the effect of Ba2+ is shown in Fig. 4, the effects of Sr2+ are not shown). Both foreign ions increased perfusion pressure reversibly on the maternal side. It can be calculated from the kinetic parameters of the Ca2+ transport that adding 1 mM of CaCl2 to the maternal perfusate would decrease Kmf, te by 37 %. If the inhibition of the Ca2+ transport by Sr2+ and Ba2+ was competitive, the much smaller effect of these two ions would indicate that the affinity of the Ca2+ transport system for Sr2+ and Ba2+ is much lower than that for Ca2+. 302

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Fig. 3. The inverse value plot of Jmf, tc and Ca2+ concentration in maternal perfusate. *, the observed values of Jmf, tc, the limits are S.D. The equation of the regression line is y = 00085 + 00039x, r = 0974, the corresponding kinetic constants are Vmax = 117 nmol min-' and Km = 045 mm.

To examine the nature of the inhibition of the Ca2+ transport by Sr2+ and Ba2+ we tested the effect of the two ions on the kinetic parameters of the transport. (If the inhibition was competitive Km would increase.) In addition to [Ca2+]m of 0-85 mm the transport was measured at [Ca2+]m (in mm) of 0 35 (n = 8), 1-1 (n = 7) and 2X3 (n = 8) in the case of Sr2 , and 0 35 (n = 8), 1-4 (n = 6) and 2-1 (n = 9) in the case of Ba2 . The inverse value plot of Jmf, tc versus [Ca2+]m in the barium experiments is shown in Fig. 5. The control values and recovery values could be fitted by straight lines. The upward shift of the recovery values indicates a decrease in Vmax, the magnitude of which corresponds roughly to the spontaneous decrease of transport during perfusion observed in control placentae. In the presence of Ba2+, however, the values could not be fitted by a straight line. The plot was curvilinear, concave upwards, suggesting that the transport no longer conformed to the Michaelis-Menten kinetics. The change in the kinetics of Ca2+ transport during perfusion with Sr2+ was qualitatively similar,

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only it was less pronounced (the results are not shown). Estimation of the kinetic parameters of the transport under such conditions did not seem possible. The possible mechanisms of the effects of Sr2+ and Ba2+ on the transport kinetics are discussed below.

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20 30 Time (min) Fig. 4. The effects of Ba21 on Kmf, te of 45Ca2+. The presence of Ba2+ is indicated by filled symbols. Mean values of nine experiments, the limits are S.D. The line is the expected course if the placenta was not exposed to Ba2+.

The effect of Ca2+ on the transport of 85Sr2+ and l33Ba2+ The above experiments did not answer the question of whether Sr2+ and Ba2+ compete with Ca2+ for the Ca2+ transport system. Therefore, we approached the problem in the opposite way -we tested the effect of changing [Ca2+]m on the maternal-fetal transport of 85Sr2+ and l33Ba2+ (the foreign ions were in maternal perfusate at a concentration of 0-02 mM). If Sr2+ and Ba2+ competed with Ca2+ for the transport system, the transport of 85Sr2+ and 13Ba2+ across the placenta would be inhibited reversibly by an increased [Ca2+]m. This indeed has been found (Fig. 6). The Kmf of each of S5Sr2+ and l33Ba2+ was much above the PS of the placenta to [3H]L-glucose, indicating that most of the transport was transcellular. The inhibition of the transport by increased [Ca2+]m suggests that the transport of Sr2+ and Ba2+ across the trophoblast is effected by the Ca2+ transport system. It follows from this that none of the processes involved in the transcellular transport of Ca2+ is entirely substrate specific. The effects of Cd2+ on the transport of Ca2+ The effects of Cd2+ on the transport of Ca2+ have been tested for three reasons: (1) Cd2+ in low concentrations (0-01-0-1 mM) blocks Ca2+ channels competitively in excitable tissues (see for example Tsien, Hess, McCleskey & Rosenberg, 1987; Reuter

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& Porzing, 1988), (2) Cd21 is a highly toxic general pollutant and (3) Cd2+ in nanomolar concentrations inhibits the Ca2+ pump (Verbost, Senden & van Os, 1987; Verbost, Flik, Lock & Wendelaar Bonga, 1988). In our experiments 0 1 mM-CdCl2 inhibited transport of 45Ca2+ (Fig. 7). Unlike the 0-05

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1/[Ca2+]", (MM-,) Fig. 5. The effects of Ba2+ on the kinetics of the transcellular transport of Ca2+. Inverse value plot of Jmf, tc versus [Ca2+]m. 0, control period; *, perfusion with Ba2+; E], recovery

period. The points are mean values in the individual groups of experiments. Straight lines are the regression lines fitted to the data. The interrupted line has been fitted to the data by eye. The kinetic parameters estimated from the regression lines are Vmax = 124 nmol min-' and Km = 0-38 mm in the control period, and Vmax = 104 nmol min-' and Km = 0 33 mm in the recovery period.

effects of Ba2+ and Sr2+, however, the response of Kmf, tc to Cd2+ was sluggish and not reversible within 12 min. We believe, therefore, that inhibition of the transport of 4Ca2+ was not caused by competition between Cd2+ and Ca2+ for the transporter in the maternal surface of the trophoblast. The inhibition may be due rather to an effect of Cd2+ on the processes beyond the entry step, presumably on the Ca2+ pump in the fetal border. The delay of the response may reflect the slow build-up of the concentration of free Cd2+ in the trophoblast cytosol due to binding of Cd2+ to cytosolic proteins (Wier & Miller, 1987). If these conclusions are correct, the mechanism of Ca2+ entry into the trophoblast is not sensitive to Cd2+, unlike the Ca2+ channels in excitable tissues.

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Cadmium chloride, at a concentration of 0-02 mm, had no effect on the transport of 45Ca2+ (tested in four experiments, the results are not shown). This concentration is about 100 times higher than the concentration of Cd2+ in the blood of people professionally exposed to Cd2+ (Lauwerijs, Buchet & Roels, 1976). However, the observed lack of effect of Cd2+ (0-02 mM) on transplacental transport of Ca2+ does not indicate that the Ca2+ transport across the placenta may not be impaired by Cd2+ in chronically exposed persons, since in our experiments only very rapid effects could be detected. [Ce2]. (mM) 2.0

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Time (min) Fig. 6. The effects of Ca2+ on Kmf of 85Sr2+ and l33Ba2+. The concentration of Ca2+ in the maternal perfusate is indicated across the top of the graph. 0, Kmf of 85Sr2+ (n = 3); O, Kmf of 133Ba2+ (n = 6); A, PS of the placenta to [3H]L-glucose (measured only in the lS3Ba2+ transport experiments). Mean values, the limits are S.D.

Effects of nifedipine A calcium channel blocker, nifedipine, in a concentration of 0-02 mm had no effect at all on Jmf, tc (tested in seven experiments, the results are not shown). The concentration of nifedipine in these experiments was several orders higher than the concentrations blocking Ca2+ channels (L-channels) in excitable tissues (see reviews by Schramm, Bechem, Franckowiak, Thomas & Towart, 1986; Schwartz, 1988). We do not think that the lack of effect of nifedipine on Jmf, tc could be explained by a lack of access of the drug to the relevant surface of the trophoblast. Both morphological and physiological observations suggest that the extracellular spaces in the placental barrier of the rat are wide enough to allow passage of even large molecules (Tillack, 1966; Stulc & Stulcovat, 1986). Such pathways could hardly discriminate Ca2+ and nifedipine on the basis of molecular (ionic) size. Since no other

306

J. STULC, B. STULCOVA AND J. SVIHOVEC explanation of our observations seems available we conclude that the Ca2+ entry into the trophoblast from the maternal side is not sensitive to dihydropyridine Ca2+ antagonists.

100

0

0

10

20 30 Time (min) Fig. 7. The effects of Cd2+ on Kmf, te of 45Ca2+. The presence of Cd2+ is indicated by filled symbols. Mean values of five experiments, the limits are S.D. The line is the expected course if the placenta was not exposed to Cd2+. The total concentration of Cd was 01 mM.

DISCUSSION

The preparation Three variables measured in our experiments can be taken to characterize the conditions of the preparation: the PS of the placenta to [3H]L-glucose, Jmf, tc and Kmf of antipyrine. The PS of the placenta to [3H]L-glucose indicates integrity of the placental barrier. In intact rats the PS of the placenta to radioactive mannitol, a substance of a comparable molecular size, is 2 9 ,u min-' (Stulc & Stulcova, 1986). The PS to [3H]L-glucose of the dually perfused placenta is about 4 times higher than the expected PS of an intact placenta. The increase in the paracellular permeability of the placenta is a common problem in placental perfusion studies (discussed for example in Stulc, 1985). The reasons are not known. At the approximately physiological [Ca2+]m of 1-1 mm, Jmf, tc was 92-4 nmol min-1, which was only slightly lower than the physiological value (about 100 nmol min-', Stulc & Stulcova, 1986). This suggests that the Ca2+ transport systems were not damaged by the perfusion. The reason for the spontaneous decrease in the transport during perfusion is not clear. It may reflect a slow deterioration of the conditions of the preparation or a lack of some substance which is normally present in plasma and which is necessary for the transport to be fully active. Antipyrine diffuses rapidly across the placenta. The rate of placental transport of

307 PLACENTAL TRANSPORT OF CALCIUM antipyrine is not limited by the transplacental diffusion but by the rate of convective transport to and from the exchange barrier, that is, by the flow rate through two vascular beds of the placenta (Meschia, Battaglia & Bruns, 1967). The transport decreases when the flow rate(s) decreases or when the flows become maldistributed (Faber, 1969). In this study Kmf of antipyrine was used to indicate changes in perfusion of the maternal side of the placenta which could not be monitored directly. The Kmf of antipyrine was high and relatively stable during perfusion suggesting that the hydrodynamic conditions of the preparation were reasonably good. 2

x

-_E 0)

~

1

0

-0.5 0.5

log [Ca2+Jm Fig. 8. The Hill plot of Jmf, tc in the presence of Sr2+ and Ba21. @, perfusion with Sr2+; 2, perfusion with Ba2 . Straight lines are regression lines fitted to the data (r > 099 in both instances). The Hill numbers estimated from the slopes of the regression lines are 2-3 in the case of Sr2+ and 2-0 in the case of Ba2+, suggesting that the Ca2+ transporter may contain at least two binding sites for Ca2+.

As in the umbilically perfused rat placenta (Stulc & Stulcova', 1986) the initial Kmf of antipyrine in the present preparation was nearly equal to the estimated flow rate through the maternal side. Such a high transfer rate is compatible with a crosscurrent or countercurrent arrangement of flows in the two vascular beds of the placenta (Faber, 1969). The properties of Ca2+ transport The transport of Ca2+ across the trophoblast consists of at least three steps in series: entry into the trophoblast from the maternal side, movement through the trophoblast cytosol and extrusion to the fetal side. The slowest step will be rate limiting to the whole process of transcellular transport. It can be assumed that the response of Jmf,tc to the experimental treatment represents the response of the rate-limiting step. The dependence of Jmf, tc on [Ca2+]m

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was consistent with the simple Michaelis-Menten model. This suggests that over the whole range of [Ca2+]m tested the transport was limited by the same rate-limiting process. (This may not have been the case in the presence of Sr2+ and Ba2+, as will

be discussed below.) The observed Km indicates that the rate-limiting step is represented by the Ca2` entry into the trophoblast. A Km of about 0 5 mm can hardly characterize any other part of the Ca2+ transport since Km of the Ca2+ pump in the fetal border is in submicromolar range (Fisher et al. 1987) and the same presumably applies to the dissociation constant of the Ca2+-binding proteins in the cytosol (Carafoli, 1987). Thus all other parts of the transcellular transport would have Km several orders of magnitude lower. The entry of Ca2+ into the trophoblast being the slowest part of the transcellular transport may be an important factor in maintaining the Ca2+ homeostasis of the trophoblast cytosol. Under conditions of the large transcellular movement of Ca2+ it will protect the trophoblast from being flooded by Ca2 . On the basis of the above considerations we assume that most of our observations relate to the Ca2+ entry into the trophoblast from the maternal side. If this assumption is correct, the entry mechanism has the following properties: (1) it has a high affinity for Ca2+. At physiological levels of Ca2+ in plasma most of the transport will be saturated. This will make the Ca2+ transport to the fetus relatively insensitive to fluctuations of Ca2+ in maternal plasma, which may be important for maintaining fetal Ca2+ homeostasis (in this respect the Ca2+ transport system is similar to that of inorganic phosphate, see Brunette & Alard, 1985); (2) the transport is not substrate specific (it can also transport Sr2+ and Ba2+); (3) the transport is not sensitive to dihydropyridine Ca2+ channel blockers (nifedipine). Sweiry & Yudilevich (1984) estimated the kinetic parameters of the Ca2+ uptake by the maternal and fetal borders of the trophoblast of the dually perfused guineapig placenta using the single-injection paired-tracer dilution method. They also demonstrated inhibition of the uptake of 45Ca2+ by Sr2+, Ba2+ and other divalent ions. The values of Km for the entry from the maternal side, presented by those authors, range from 0-18 to 1-15 mm. Our own values are almost exactly in the same range. However, it is not clear how far the observations by Sweiry & Yudilevich and those of this study relate to the same phenomena. The paired-tracer dilution technique cannot distinguish between uptake of radioactive Ca2+ into cells and its binding to the binding sites on cell surfaces and extracellular matrix. (The problems in measuring cellular uptake of Ca2+, arising from the extracellular binding of radioactive Ca2+, are discussed in detail, for example, by van Breemen, Farinas, Casteels, Gerba, Wuytack & Deth, 1973.) The kinetic parameters estimated by Sweiry & Yudilevich are therefore likely to represent some overall dissociation constant of the interaction of Ca2+ with the extracellular binding sites (including the

Ca2+ transport sites on the trophoblast surface) rather than the kinetic parameters of the Ca2+ transport system. A similar reasoning may apply to the inhibition of uptake of 45Ca2+ by foreign divalent ions. In this study the transport of 45Ca2+ was measured at a steady state of transport of 45Ca2+ when the Ca2+ binding sites not involved in the transplacental transport were saturated by the tracer. The close agreement between Km estimated by the two different methods suggests that the

309 PLACENTAL TRANSPORT OF CALCIUM dissociation constants and other properties of all extracellular Ca2+ binding sites on the maternal side of the placenta (whether involved in Ca2+ transport or not) are similar, or that there is no significant extracellular binding of Ca2+ on the maternal side of the placenta. The observation of the above authors that most of the 45Ca2+ which had been taken up by the placenta returned rapidly to the perfusate and was recovered in the effluent suggests that the former is the case. Interaction of Sr2+ and Ba2+ with the transport of Ca2+ The kinetics of Ca2+ transport was changed qualitatively by Ba2+ (Fig. 5) and Sr2+. We see three possible explanations for this. Two of them are based on models of enzyme kinetics. There are two situations in enzyme kinetics which lead to a concave inverse value plot of velocity versus concentration (as found in Fig. 5): stimulation of the process by substrate and co-operativity of the process. The stimulation by substrate in this study would mean that the activity of the system mediating the entry of Ca2+ into the trophoblast is stimulated by the binding of Ca2+ to some allosteric (non-transport) site of the transporter. If the dissociation constant of this allosteric binding of Ca2+ was sufficiently below the range of [Ca2+]m tested in our experiments no stimulation of the transport by Ca2+ would be detected under control conditions. Competitive binding of Sr2+ and Ba2+ to these sites would increase the apparent dissociation constant of Ca2+ and stimulation might become apparent. (It is obvious that such a mechanism could not control the Ca2+ entry into the trophoblast because at physiological concentrations of Ca2+ it would be fully saturated. The binding of Ca2+, however, is a general property of cell membranes, and it is possible that Ca2+ occupation of some of the binding sites is a necessary physiological condition for the Ca2+ transporter to be normally functional.) Co-operativity of the Ca2+ transport into the trophoblast would mean that there is more than one Ca2+ transport site on each transporter and that binding of Ca2+ to one site facilitates Ca2+ binding to the other site(s). In co-operative systems, increase in co-operativity by binding of inhibitors to allosteric sites is predicted by theoretical models (see for example Dixon & Webb, 1979), which might explain why co-operativity was observed only in the presence of Sr2+ and Ba2+. The nearly perfect fit of the data to a straight line in the Hill plot (Fig. 8), the Hill number being about 2 (suggesting co-operativity between at least two binding sites) and the good agreement between the Hill numbers in the Ba2+ and Sr2+ experiments, seem to support this hypothesis. The last hypothesis takes into account the fact that the transcellular transport of Ca2+ consists of several steps in series. It is based on the observation that Sr2+ and Ba2+ can be transferred across the placenta by the Ca2+ transport system. If the two foreign ions enter the trophoblast by the Ca2+ transporter, Ca2+ will inhibit their transfer into the trophoblast. With the [Ca2+]m decreasing, the influx of Sr2+ and Ba2+ into the trophoblast will increase rapidly. It is therefore possible that at low [Ca2+]m the two foreign ions reach a concentration in the trophoblast high enough to inhibit some other step of the Ca2+ transport (for instance the Ca2+ extrusion across the fetal border by competition with Ca2+ for the Ca2+ pump) which then becomes rate limiting for the whole process of transcellular transport. These effects may thus

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cause Jmf, tc to decrease with [Ca2+]m more rapidly than could be accounted for by competition between Ca2± and the foreign ions for the transporter at the maternal

border of the trophoblast alone. Our evidence does not allow us to decide which, if any, of the above hypotheses is right. Intuitively we lean to the last one because it is the most simple. Moreover, the first two hypotheses assume that Sr2+ and Ba2+ do not inhibit Ca2+ transport by binding to the Ca2+ transport sites but by binding to allosteric sites of the transporter; that two foreign ions similar in many important respects to Ca2+ should show preferential binding to allosteric sites does not seem very likely. We thank Mrs Jana Kopeeki for her able assistance. REFERENCES

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