J. Phyeiol. (1977), 265, pp. 691- 703 Printed in Great Britain
691
PLACENTAL TRANSPORT OF SODIUM IN THE GUINEA-PIG
BY J. ATULC AND J. SVIHOVEC From the Department of Pharmacology, Faculty of Pediatrics, Charles University, Prague, Czechoslovakia
(Received 16 June 1976) SUMMARY
1. The mechanism of placental transport of Na was studied in guineapigs in placentae with intact umbilical blood circulation or in the preparation of the placenta perfused in situ. 2. A constant level of 22Na was maintained in maternal plasma for 60 min, and from the quantity of 22Na recovered from the foetus at the end of this period the influx of Na from mother to foetus was calculated. Ligation of the omphalomesenteric vessels (supplying the everted yolk sac with blood) had no effect on the influx, the corresponding values of influx in the control and treated foetuses being 0-235 + 0-020 and 0-247 + 0*029 ,u-mole/min . g foetal weight (n = 6, the limits are S.E. of mean). The specific activity of Na in amniotic fluid was below that of the maternal or foetal plasma Na by two orders of magnitude. These observations indicate that the extraplacental transport of Na into the foetus is negligibly low. 3. The electrical potential difference (p.d.) and unidirectional fluxes of Na across the placenta perfused in situ were measured by means of 22Na and 24Na administered to the opposite sides of the placental barrier. The fluxes varied with the weight of the foetuses whose placentae were perfused. The flux from the maternal to the foetal side was 0-270 + 0-017 ,/mole/min . g foetal weight, the flux from the foetal to the maternal side was 0 340 + 0-018 ,umole/min. g foetal weight (n = 38). The corresponding p.d. was - 20*7 + 1-2 mV (foetal side negative). 4. The active component of Na transport across the placenta was calculated from the unidirectional fluxes and the p.d. The active transport was directed from the foetal to the maternal side, and its rate was 0*211 + 0*0155umole/min. g foetal weight (n = 38). During perfusion ofthe placenta with KCN (10-3 M) the active transport decreased by approximately one third. 5. The flux of Na from the foetal to the maternal side of the perfused placenta was higher than the flux from the maternal to the foetal side. A similar asymmetry of Na fluxes was observed in the non-perfused placenta,
J. vTULC AND J. 9VIHOVEC 692 the flux from mother to foetus being 0 180 + 0-013,mole/min.g foetal weight and the flux from foetus to mother 0*235+00024 #smole/min.g foetal weight (n = 12). This indicates that the asymmetry of Na fluxes is caused by the anaesthesia and/or by the trauma of the operation rather than by the perfusion of the placenta. 6. The permeabilities ofthe perfused placenta to Na and sucrose measured simultaneously from the maternal to the foetal side were 0-0767 + 00183 and 0-0324 + 0 0094 cm3/min (n = 7), respectively. The permeability values bear the same relation to each other as the respective coefficients of free diffusion in water, suggesting that the passive transport of Na across the placenta takes place as simple diffusion through wide aqueous channels. 6. The observations of this work are consistent with two different mechanisms of Na transport across the placenta: (1) simple diffusion (presumably bidirectional); and (2) unidirectional active transport from the foetal to the maternal side of the barrier. INTRODUCTION
The accumulation of inorganic ions in the foetus during gestation has been relatively thoroughly studied both in humans and in experimental animals, yet very little is known about the mechanism of their transfer across the placenta. The most simple mechanism of ion transport across the placenta would be simple diffusion. However, diffusion cannot be the only mechanism. In a variety of species an electrical potential difference (p.d.) across the placenta has been observed (Meschia, Wolkoff& Barron, 1958; Widdas, 1961; Mellor, 1969, 1970). If the ion transport across the placenta were solely diffusional the distribution of ions between maternal and foetal plasma at equilibrium would approach that predicted by the Nerust equation. This, however, is not the case. In spite of sometimes very high transplacental p.d.s (as high as 133 mV in the goat; Meschia et al. 1958) the maternal and foetal plasma concentrations of most ions are similar. From this it follows that the ions on the two sides of the placental barrier are not in equilibrium. Under these conditions there would be a net passive movement of ions across the placenta down the respective electrical potential gradient. For some ions this net passive movement may be directed to the foetal side and its rate be just equal to the rate of accumulation of the ion by the foetal tissues. In such a case no mechanism of placental transport other than simple diffusion need be assumed. For all other ions, however, active transport across the placenta must be postulated to explain their distribution between the maternal and foetal plasma. On such grounds active transport of Na from the foetal to the maternal
693 PLACENTAL TRANSPORT OF Na side of the placenta has been postulated and examined experimentally in the guinea-pig (8tulc & 8vihovec, 1973). In this species the foetal side is approximately 20 mV negative with respect to the maternal side (Mellor, 1969; 8tulc, Rietveld, Soeteman & Versprille, 1972). The p.d. recorded across the placenta perfused in situ decreased when Na was withdrawn from the perfusion fluid or when the placenta was treated with KCN. These observations were consistent with the postulated active transport. In the present work the study of the mechanism of Na transport across the guinea-pig placenta has been continued. Unidirectional fluxes of Na across the placental barrier have been measured simultaneously with the p.d., and from the values obtained the passive and active components of transport have been calculated. In this system the effects of KCN on the active component have been tested. METHODS
Materials and techniques Animals. Pregnant guinea-pigs between 40 days and full term were used. Placental perfuwion and recording of the p.d. The techniques were described in detail previously (Stulc & 8vihovec, 1973). The animals were anaesthetized with pentobarbitone (Spofa) injected through a thin polyethylene cannula into the cubital vein under local anaesthesia with 1 % procaine (Spofa). The carotid artery was cannulated, the abdomen opened, and cannulae inserted into the umbilical vessels through a small incision in the uterine wall and foetal membranes. The umbilical vascular bed of the placenta was perfused with Krebs fluid (Gaddum, 1959) containing dextran (mol. wt. 70,000, Spofa) 6 gl 100 ml., gassed with 95 % 02 and 5 % C02. The composition of the fluid (m-mole/l.) was NaCl 118-0, KCl 4 7, CaCl2 2.5, MgC12 1.2, NaHCO3 25-0, NaH2PO4 1.0, glucose 5B5. The perfusion was carried out at a constant perfusion rate of 1 ml./min (the corresponding perfusion pressure was between 8 and 35 mmHg). Effluent from the umbilical venous cannula was drained into graded test tubes and sampled at 10 min intervals. When the rate of flow from the placenta was less than 95 % of the perfusion rate, the samples were discarded and the perfusion was discontinued. The exposed uterus and the lower part of the animal were maintained at 37-38° C by immersion in a thermostabilized liquid paraffin-bath. The perfusion pressure was recorded with a Bell-Howell pressure transducer (type 4-327-L221) connected to a Schwartzer PEE-4 polygraph. The electrical potential difference was recorded between the maternal blood in the cubital vein and perfusion fluid in the umbilical venous cannula with a microvoltmeter (Tesla BM 483). The output of the microvoltmeter was integrated, and from the integral a mean value of the p.d. during the given interval was calculated. Radioactive 8ubatance8. 22NaCl and 24NaCl were supplied by the Radiochemical Centre, Amersham, and by Zentralinstitut fur Kernforschung, Dresden, respectively. Sucrose uniformly labelled with "IC was supplied by IYVVVR, Prague. Flame photometry and radioactivity counting. The concentrations of Na in the maternal plasma, foetal plasma, amniotic fluid, and perfusion fluid were measured with the Zeiss flame photometer after appropriate dilution with deionized water. Gamma radioactivity was counted with a well-type NaI scintillation detector
694
J. STULC AND J.
SVIHOVEC
coupled with the Tesla NZQ-717T counter. When the samples contained both radioactive isotopes of Na, the radioactivity of 24Na was counted with the discriminator level set so that all the radioactivity due to 22Na was filtered out. The samples were recounted for the 22Na after all 24Na had died out. Radioactivity of 04C was counted in a dioxane scintillation fluid (Spolana SLD 31) with the Packard 3300 liquid scintillation counter. Evaluation and presentation of the data. The fluxes of Na across the placenta varied with the weight of the foetus. Therefore, the values of Na fluxes are expressed per gram of the foetal weight. The sign of the p.d. denotes the polarity of the foetal side of the placenta with respect to the maternal side. For the individual groups of placenta the values of Na fluxes, p.d. or permeabilities are expressed as mean values ± s.E. of mean with the number of placenta in brackets. The differences between the individual groups were evaluated using Student's t test. Na fluxce across the perfumed placenta Estimation of the unidirectional fluxem. Unidirectional fluxes of Na across the placenta were measured simultaneously by means of 22Na and 24Na administered to the opposite sides of the barrier. The isotope 22Na was added to the perfusate in a concentration of approx. 0005 &c/ml. The isotope 24Na was administered intravenously to the mother as a single initial dose followed by infusion. The rate of infusion decreased in steps according to the following schedule (the infusion rate is expressed as a fraction of the initial single dose administered per minute): 0-5 min, 0 110; 5-12 min, 0 055; 12-24 min, 0-027; from 24 min on, 0 007. In this way the level of 24Na in maternal plasma was maintained within less than ± 10 % of the mean concentration. The total quantity of 24Na administered to the animal was approx. 100l ac. Simultaneously with the initial injection of 24Na approximately 500 u. heparine (Spofa) were administered. Assuming that the movement of the radioactive Na across the barrier was in a steady state, the flow of 22Na (J*) and 2"Na (J**) were determined as the quantity of 22Na radioactivity leaving and 24Na radioactivity entering the perfusate per unit time (c.p.m./min), respectively. From the tracer flows and from the Na concentrations on the two sides of the placenta the unidirectional fluxes of Na, i.e. the flux from the foetal to the maternal side (J,) and the flux in the opposite direction (Jo), were calculated. Provided that the behaviour of the tracer and the abundant species is identical, the flow of the tracer across the barrier can be regarded as the algebraic sum of the products of the unidirectional fluxes and the respective mean specific activities of Na along the barrier on the side of their origin. The mean specific activities along the barrier cannot be measured but the placental transport of Na was sufficiently slow to assume that neither the concentrations of the tracers nor the total Na concentration changed much along the barrier and that the concentrations in the exchange vessels were approximately equal to the inflow concentrations. Since the inflow concentration of 24Na on the foetal side of the barrier (in the perfusion fluid) was zero, and the inflow concentration of 22Na on the maternal side (arterial plasma concentration) was negligibly low, unidirectional fluxes of Na could be calculated according to the following simple equations:
Jmt
=
Jtm
=
(J**/[24Na]m) [Na]m K.,[Na]., (J*/[22Na]f) [Na]f Kfn[Na]f, =
=
PLACENTAL TRANSPORT OF Na
695
where ["2Na], [24Na] and [Na] are concentrations (c.p.m./ml. and mole/ml., respectively), K., and Kf. are unidirectional transfer constants (ml./min), and m and f denote maternal and foetal side of the barrier, respectively. Calculation of active transport. It has been postulated that Jtm consists of a passive and an active component (see Introduction). The passive component of Jtf was calculated from the values of J,,f, [Na]m, [Na]1 and p.d. using the Ussing flux ratio equation (Ussing, 1949). The active component (J't) was obtained by subtracting the passive component from Jfm. Jlf. was thus calculated according to the following equation: Jft = Jfm-Jmt([Na]f/[Na]m) exp (VFIRT), where V is the p.d., and F, R and T are the Faraday constant, gas constant and absolute temperature, respectively. The equation assumes that the passive movement of Na across the placenta proceeds entirely by simple diffusion uncomplicated by solvent drag or by coupling to other possible flows across the barrier. To prove that this assumption is valid seems impossible at present. Nevertheless, it will be shown below that the passive movement of Na across the placenta is compatible with simple diffusion being the means of passive transport. The space of "Na and 24Na of the placenta. The spaces of radioactive Na in the placenta were calculated as the ratio of the respective radioactivity of 1 g placental tissue to the radioactivity of 1 ml. perfusate in the case of 22Na, or 1 ml. plasma in the case of 24Na. The space is expressed as ml./g tissue. Experimental procedures. After establishing the placental perfusion, administration of 24Na to the mother was started. The moment of the initial i.v. injection of the isotope was taken as the zero-time. Collection of the effluent was started at 10 min and continued for six 10 min collecting periods. For the first 40 min (i.e. until the end of the third collecting period) the placenta was perfused with Krebs fluid without any experimental treatment in all animals. Then either the perfusion was terminated or the placenta was perfused for another 30 min with normal Krebs fluid (control group) or with Krebs fluid containing KCN (10-3 M). In all groups the placenta was sampled at the end of perfusion for estimation of the radioactivity. Maternal bood was sampled from the carotid artery at 5 min, 10 min, and from then on at 10 min intervals. The assumption of steady-state conditions of the tracer movement across the placenta. The present calculations of Na fluxes assume that the movement of the tracers across the placental barrier was in a steady state. This condition was met in the non-treated placenta, as indicated by the following evidence. (1) If a steady state were not reached before the period of flux measurement, the calculated values of Jmf would increase and Jfm decrease during perfusion, as a steady concentration of 24Na and 22Na within the barrier would be gradually approached. The calculated fluxes actually did change in this way during the first 30 min of perfusion (Table 1). Therefore, the data obtained during this period were rejected from further evaluation. After 30 min a reasonably steady level of the tracer flows across the placenta was attained. (2) There was no significant difference between the 22Na and "4Na spaces of the placenta sampled at 30 and 70 min (see below). In the placenta treated with KCN, a steady state of Na transport was no longer maintained. The Na fluxes during the KCN treatment, therefore, could be calculated only approximately. Na fluxes across the non-perfused placenta The animals were prepared in the same way as described for the perfusion experiments. The omphalomesenteric vessels were exposed through a small incision in the
696
J. STULC AND J. SVIHOVEC
uterus and foetal membranes and cannulated with thin polyethylene cannulae in the foetal direction. The umbilical vessels were left intact. The exposed uterus and the lower half of the animal were immersed in a thermostabilized liquid paraffin-bath at 37-380 C. A known dose of 22Na (approx. 5 #ic) in 0-2 ml. saline containing approx. 200 u. heparine (Spofa) was injected into the foetus through the omphalomesenteric venous cannula simultaneously with the injection of the initial dose of 24Na into the cubital vein of the mother. Infusion of 24Na into the maternal circulation followed. Samples of maternal blood were taken at 10 min intervals from the carotid artery. Foetal blood was sampled from the omphalomesenteric arterial cannula 10 min after injection of the isotopes, in the middle and at the end of the experiment. The foetus was removed from the uterus 60-120 min (80 min in most animals) after injection of the isotopes. The blood was centrifuged and samples of maternal and foetal plasma were removed for estimation of 22Na and 24Na radioactivity and of total Na concentration. The foetus was dried in an oven at 110-1500C, weighed and pulverized. Five samples of the powder (0.3-1.5 g) were taken from each foetus for the radioactivity assay. Recovery of 22Na and 24Na radioactivity from the foetus, estimated in a separate group of animals, was 98-6 ± 0.5 % and 98*1 + I1 0% (n = 14), respectively. The transfer constants Kid and Kim (ml./min) were calculated by dividing the amount of 24Na and 22Na radioactivity, respectively, transferred across the placenta per minute (c.p.m./min) by the mean concentration difference of the radioactivity of the respective isotope (c.p.m./ml.) between maternal and foetal plasma. The values of the unidirectional fluxes were obtained by multiplying Kmf or Kfm by total Na concentration in maternal or foetal plasma, respectively.
Extraplacental transport of Na into the foetus The animals were anaesthetized with pentobarbitone, the abdomen was opened and the foetal vessels supplying the yolk sac (omphalomesenteric vessels) were ligated through a small incision in the uterus without opening the foetal membranes. The abdomen was closed again, the animal covered with a cellulose tissue and heated with an infra-red lamp. A steady level of 22Na was maintained in the maternal plasma (as described above for 24Na) for 60 min. At the end of this period the abdomen was reopened and the amniotic fluid and umbilical venous blood from the treated foetus and from a non. treated foetus in the opposite horn of the uterus (control foetus) were simultaneously sampled. The influx of Na into the foetus was calculated from the 22Na recovered from the foetus and from the 22Na and total Na concentrations in maternal and foetal plasma, as already described. In these calculations the concentration of 22Na in foetal plasma was assumed to have followed a linear course. This simplification is unlikely to cause a great error in estimation of Na influx since the radioactivity of the foetal plasma at the end of the experiment did not exceed 25 % of that in the maternal plasma. Estimation of the placental permeability to Na and sucrose The placental permeabilities from the maternal to the foetal side to Na and sucrose were measured simultaneously. The animals were nephrectomized before the perfusion. The placenta was perfused, and a constant level of 24Na in maternal plasma was maintained as described above. Approximately 10 Atc [14C]sucrose were injected i.v. into the mother together with the initial dose of 24Na. The perfusion liquid contained no 22Na. The placenta was
PLACENTAL TRANSPORT OF Na
697
perfumed for 50 min, and effluent from the placenta and maternal blood were sampled during the last 10 min. Placental permeability to sucrose was calculated as the quantity of [14C]sucrose transferred into the perfusate per unit time and unit concentration difference. The 14C-material recovered from the effluent was shown to be [14C]sucrose for each sample of effluent by paper chromatography in a system butanol:ethanol:water (4:1:5). The permeability of the placenta to Na was calculated by dividing the constant Kmt by a factor that represents the effect of the p.d. on the diffusion of ions across the membrane. This factor is VF/RT exp (VF/RT)-I' where V is the p.d. (Schultz & Zalusky, 1964, eqn. (6)). The permeability refers to the whole exchange area of the placental barrier and its dimension is therefore
cm3/min. RESULTS
Transport across the perfumed placenta. Control values and general observations. The values of Na fluxes across the placenta together with the values of the p.d. during a 70 min perfusion in a group of control animals are in Table 1. The initial increase of Jmf and decrease Of *fm are probably not real. They may result from an incorrect estimation of their values due to the non-steady-state conditions after the introduction of the tracers into the system (see Methods). Using 24Na, Flexner & Pohl (1941) measured the flux of Na from mother to foetus in guinea-pigs. The animals were exposed to no stress except light ether anaesthesia at the time of isotope injection and at the end of the experiment. The values of flux into the foetus can therefore be taken to be closely representative of those in normal unstressed animals. The values of 'corrected' transfer of radioactive Na in Fig. 7 of their publication are equivalent to Kmf in this work, except that an hour is used as the unit of time. Therefore, Jmf per gram of foetal weight in intact guinea-pigs can be estimated by multiplying the values in Fig. 7 of Flexner & Pohl by [Na]m and dividing them by 60 x (foetal weight). Taking the value of 137 m-mole/l. obtained in this work for [Na]m (136-8 + 0-8 m-mole/l.) the flux of Na from mother to foetus calculated from the data of Flexner & Pohl is 0*219 + 0.015 ,mole/min.g (n = 18). (The calculation was performed only for the foetuses with a body weight above 25 g, so that the range of foetal weight would be comparable with that in the present work.) The values of Jmf across the perfused placenta (Table 1) are in good agreement with the flux into the foetus estimated for intact animals. Since Jmf occurs down the electrochemical potential gradient and therefore can be taken as passive this indicates that no gross changes of placental permeability to Na were caused by the perfusion.
J. STULC AND J. 9VIHOVEC 698 The 22Na and 24Na spaces of the placenta at 30 min were 0 337 + 0-031 ml./g and 0-227 + 0 030 ml./g (n = 6), respectively. The radioactive Na spaces did not change during the next 40 min, the corresponding values at 70 min being 0 345 + 0*017 ml./g and 0-225 + 0016 ml./g (n = 7), respectively. The weight of the placenta at the end of a 70 min perfusion was 3-496 + 0 300 g (n = 7). TABLE 1. Na fluxes and the electrical potential difference across the control perfused placentae Time (min)
J., (pmole/min.g) J,., (.umole/min.g) PAd (mV) J't
(,umole/min.g)
10-20 0.240 + 0*021 0 357 ± 0.036 -210 +2.0 *
20-30 0.250 ± 0.024 0 355 + 0 035 -20X5 +2.0 *
30-40 0.252 + 0.024 0.338 ± 0.034 -19 9
±20
0f206
+ 0*032
40-50 0.258 ± 0.025 0-334 + 0 035 -20X6 +2-1
0f215
+ 0 033
50-60 0.257 ± 0.027 0-321 + 0 035 -19 9 +2.1 0 199 + 0*032
60-70 0.260 + 0-027 0'334 + 0-034 -19 1 +2.2
0f204
+ 0.030
Mean + S.E. of mean, number of placenta = 13. The fluxes are expressed per gram foetal weight, mean foetal weight is 50-2 + 3-7 g
(n
=
13).
* Jt was not calculated for the first two collecting periods. It took approx. 30min for the movement of radioactive Na across the placenta toattain a steadystate (see Methods). Consequently the value of unidirectional fluxes obtained during this period are in error, and the values of active transport calculated from these data would be meaningless.
Extraplacental transport. In the perfusion experiments Jfm was higher than Jmf (Table 1) which means that there was a net flux of Na from the foetal to the maternal side of the placenta. Existence of this net flux follows independently from the positive arteriovenous concentration difference of Na in the perfused umbilical vascular bed of the placenta (the average value in seventeen placentae was 5*3 + 0-6 m-mole/l.). A net flux of Na from the foetal to the maternal side of the placenta could exist in intact animals only if it were made good by a net flux of Na into the foetus by extraplacental transport via foetal membranes. In rodents the foetal membranes are represented by the everted yolk sac and amnion. The everted yolk sac is the outer membrane and is richly supplied with blood by the omphalomesenteric vessels. Material passing through the foetal membranes could therefore reach the foetus by two pathways: (1) through the vitelline blood circulation; or (2) through the amniotic fluid.
PLACENTAL TRANSPORT OF Na 699 Ligation of the omphalomesenteric vessels had no effect on the Na transport into the foetus, the influx into the foetus in control and treated animals being 0-235 + 0-020 /tmole/min. g and 0*247 + 0*029 #smole/min . g (n = 6), respectively. Specific activity of Na in the amniotic fluid was less than 2 % of that in foetal plasma in both groups. From this it follows that no significant quantity of Na can reach the foetus by extraplacental pathways. The net flux of Na across the perfused placenta, therefore, must be caused by the perfusion and/or by the stress of the operation and anaesthesia. To decide between these two possibilities Na fluxes were measured across the non-perfused placenta under conditions similar to those of the perfusion experiments. Transport across the non-perfused placenta. The flux of Na from mother to foetus was 0 180 + 0 013 Ismole/min .g, the flux in the opposite direction was 0-240 + 0'024 #smole/min. g (n = 12). These values are lower than those observed in the present perfusion experiments (Table 1) or those obtained by Flexner & Pohl (1941) in intact guinea-pigs. The reason for this difference is not clear. The concentration of Na in foetal plasma was 133-9 + 0-9 m-mole/l. (n = 12). The ratio Of Jfm/Jmf in the perfused placenta is 1-39 + 0-08 (n = 13) which is close to the corresponding flux ratio 1-35 ± 0 09 (n = 12) in the nonperfused placenta. (The former value was calculated from the fluxes across the perfused placenta during the collecting period 30-40 min. The mean values of the fluxes are given in Table 1.) This suggests that the asymmetry of Na fluxes in the present experiments bears no relation to the perfusion procedure and that it is probably due to the stress of the animal under conditions of acute experiment (i.e. to the trauma of operation and/or anaesthesia). Further search for the factors causing the asymmetry of Na fluxes in these experiments was given up at this point since it would require control measurements of Na fluxes and the p.d. in non-stressed, non-anaesthetized animals. Such measurements would be technically impossible in the guinea-pig. The mechanism of passive transport. Calculation of the passive and active components of Na transport from the unidirectional fluxes and the p.d. requires that the passive movement of Na across the placenta proceeds entirely by simple diffusion. There is, however, no evidence that this condition was met. Usually, the flux of a solute across a membrane is taken to be diffusional when its magnitude is directly proportional to the electrochemical potential of the solute on the side from which the penetration is occurring. However, such an approach could not be used to test the nature of passive movement of Na across the placenta since neither the Na concentration in the maternal
700 J. STULC AND J. SVIHOVEC plasma nor the p.d. could be varied. The test performed here was less general: a single mode of passive transfer, which seemed a prior most likely, was assumed and verified experimentally. Since nothing was known about the permeability properties of the guinea-pig placenta, observations from the morphologically related placenta of the rabbit were used as a
guide. It has been observed in the rabbit that the values of placental permeability to lipid insoluble substances are approximately proportional to their coefficients of free diffusion in water (Faber & Hart, 1967; 9tulc, Friedrich & Jificika, 1969; Faber, Green & Long, 1970). This suggests that the lipid insoluble molecules move across the rabbit placenta by unrestricted diffusion through wide aqueous channels. In this work the permeability properties of the guinea-pig placenta were assumed to be similar, and the assumption was tested by measuring the simultaneous values of placental permeability to Na and sucrose. It was expected that if the assumption were correct the values of placental permeability would bear the same quantitative relation to each other as the respective diffusion coefficients in water. The placental permeability was measured from the maternal to the foetal side, i.e. in the direction in which the movement of Na can be taken as entirely passive. The values of placental permeability to Na and sucrose obtained are 0-0767 + 0*0183 cm3/min, and 0'0324 + 0-0094 cm3/min (n = 7), respectively. The diffusion coefficients of NaCl and sucrose are 1-7 x 10-5 cm2/sec (Schultz & Zalusky, 1964) and 0 7 x 10-5 cm2/sec (Landis & Pappenheimer, 1963), respectively. The Na: sucrose ratios of the placental permeability and the diffusion coefficients calculated from the above data are 2*57 + 0-20 and 2-40, respectively. This is consistent with simple diffusion in aqueous medium being the mechanism of passive transport of Na across the placental barrier in the guinea-pig. Effects of KCN on Na transport across the perfumed placenta. At the end of a 30 min perfusion with KCN the values of the 22Na and 24Na space and of the weight of the placenta were, respectively, 0-616 + 0-071 ml./g, 0X175 + 0.041 ml./g and 3-836 + 0183 g (n = 7). The space of 22Na is significantly above the corresponding value in the control placentae, the other two variables are not significantly changed. The increase in the 22Na space indicates that part of the 22Na which left the perfusion fluid during the KCN treatment did not reach the maternal side of the barrier. To obtain a correct value of J*, therefore, the quantity of 22Na retained by the placenta should be subtracted from the quantity of 22Na cleared from the perfusate during the same period. The rate of 22Na accumulation in the placenta could not be exactly assessed since it was impossible to obtain two successive samples from one placenta, but it was estimated in the following way. From the weight of the placenta perfused
PLACENTAL TRANSPORT OF Na 701 with KCN and from the average value of the 22Na space in the control placentae the quantity of 22Na expected in the placenta if the perfusion fluid contained no KCN was calculated. This value, subtracted from the actual 22Na content of the placenta at the end of perfusion with KCN, was taken to represent the approximate quantity of 22Na retained in the placenta due to the KGN treatment. TABLE 2. Effects of KCN (10-3 M) in the perfusion fluid on the Na fluxes and the p.d. across the perfused placenta Time (min)
J,. (ismolelmin . g) P.d. (mV)
30-40 (Control period) 0.322 + 0.014 0-402 + 0.033 -18.7+2.2
40-70 (KCN treatment) 0-394 + 0 055 0 393 + 0 050
J'f~t (#mole/min.g)
0-228+0.014
0.1444+0-021
J,,, (#smole/min g) .
-13.1±+18
P < 0-05
> 0 05
691
PLACENTAL TRANSPORT OF SODIUM IN THE GUINEA-PIG
BY J. ATULC AND J. SVIHOVEC From the Department of Pharmacology, Faculty of Pediatrics, Charles University, Prague, Czechoslovakia
(Received 16 June 1976) SUMMARY
1. The mechanism of placental transport of Na was studied in guineapigs in placentae with intact umbilical blood circulation or in the preparation of the placenta perfused in situ. 2. A constant level of 22Na was maintained in maternal plasma for 60 min, and from the quantity of 22Na recovered from the foetus at the end of this period the influx of Na from mother to foetus was calculated. Ligation of the omphalomesenteric vessels (supplying the everted yolk sac with blood) had no effect on the influx, the corresponding values of influx in the control and treated foetuses being 0-235 + 0-020 and 0-247 + 0*029 ,u-mole/min . g foetal weight (n = 6, the limits are S.E. of mean). The specific activity of Na in amniotic fluid was below that of the maternal or foetal plasma Na by two orders of magnitude. These observations indicate that the extraplacental transport of Na into the foetus is negligibly low. 3. The electrical potential difference (p.d.) and unidirectional fluxes of Na across the placenta perfused in situ were measured by means of 22Na and 24Na administered to the opposite sides of the placental barrier. The fluxes varied with the weight of the foetuses whose placentae were perfused. The flux from the maternal to the foetal side was 0-270 + 0-017 ,/mole/min . g foetal weight, the flux from the foetal to the maternal side was 0 340 + 0-018 ,umole/min. g foetal weight (n = 38). The corresponding p.d. was - 20*7 + 1-2 mV (foetal side negative). 4. The active component of Na transport across the placenta was calculated from the unidirectional fluxes and the p.d. The active transport was directed from the foetal to the maternal side, and its rate was 0*211 + 0*0155umole/min. g foetal weight (n = 38). During perfusion ofthe placenta with KCN (10-3 M) the active transport decreased by approximately one third. 5. The flux of Na from the foetal to the maternal side of the perfused placenta was higher than the flux from the maternal to the foetal side. A similar asymmetry of Na fluxes was observed in the non-perfused placenta,
J. vTULC AND J. 9VIHOVEC 692 the flux from mother to foetus being 0 180 + 0-013,mole/min.g foetal weight and the flux from foetus to mother 0*235+00024 #smole/min.g foetal weight (n = 12). This indicates that the asymmetry of Na fluxes is caused by the anaesthesia and/or by the trauma of the operation rather than by the perfusion of the placenta. 6. The permeabilities ofthe perfused placenta to Na and sucrose measured simultaneously from the maternal to the foetal side were 0-0767 + 00183 and 0-0324 + 0 0094 cm3/min (n = 7), respectively. The permeability values bear the same relation to each other as the respective coefficients of free diffusion in water, suggesting that the passive transport of Na across the placenta takes place as simple diffusion through wide aqueous channels. 6. The observations of this work are consistent with two different mechanisms of Na transport across the placenta: (1) simple diffusion (presumably bidirectional); and (2) unidirectional active transport from the foetal to the maternal side of the barrier. INTRODUCTION
The accumulation of inorganic ions in the foetus during gestation has been relatively thoroughly studied both in humans and in experimental animals, yet very little is known about the mechanism of their transfer across the placenta. The most simple mechanism of ion transport across the placenta would be simple diffusion. However, diffusion cannot be the only mechanism. In a variety of species an electrical potential difference (p.d.) across the placenta has been observed (Meschia, Wolkoff& Barron, 1958; Widdas, 1961; Mellor, 1969, 1970). If the ion transport across the placenta were solely diffusional the distribution of ions between maternal and foetal plasma at equilibrium would approach that predicted by the Nerust equation. This, however, is not the case. In spite of sometimes very high transplacental p.d.s (as high as 133 mV in the goat; Meschia et al. 1958) the maternal and foetal plasma concentrations of most ions are similar. From this it follows that the ions on the two sides of the placental barrier are not in equilibrium. Under these conditions there would be a net passive movement of ions across the placenta down the respective electrical potential gradient. For some ions this net passive movement may be directed to the foetal side and its rate be just equal to the rate of accumulation of the ion by the foetal tissues. In such a case no mechanism of placental transport other than simple diffusion need be assumed. For all other ions, however, active transport across the placenta must be postulated to explain their distribution between the maternal and foetal plasma. On such grounds active transport of Na from the foetal to the maternal
693 PLACENTAL TRANSPORT OF Na side of the placenta has been postulated and examined experimentally in the guinea-pig (8tulc & 8vihovec, 1973). In this species the foetal side is approximately 20 mV negative with respect to the maternal side (Mellor, 1969; 8tulc, Rietveld, Soeteman & Versprille, 1972). The p.d. recorded across the placenta perfused in situ decreased when Na was withdrawn from the perfusion fluid or when the placenta was treated with KCN. These observations were consistent with the postulated active transport. In the present work the study of the mechanism of Na transport across the guinea-pig placenta has been continued. Unidirectional fluxes of Na across the placental barrier have been measured simultaneously with the p.d., and from the values obtained the passive and active components of transport have been calculated. In this system the effects of KCN on the active component have been tested. METHODS
Materials and techniques Animals. Pregnant guinea-pigs between 40 days and full term were used. Placental perfuwion and recording of the p.d. The techniques were described in detail previously (Stulc & 8vihovec, 1973). The animals were anaesthetized with pentobarbitone (Spofa) injected through a thin polyethylene cannula into the cubital vein under local anaesthesia with 1 % procaine (Spofa). The carotid artery was cannulated, the abdomen opened, and cannulae inserted into the umbilical vessels through a small incision in the uterine wall and foetal membranes. The umbilical vascular bed of the placenta was perfused with Krebs fluid (Gaddum, 1959) containing dextran (mol. wt. 70,000, Spofa) 6 gl 100 ml., gassed with 95 % 02 and 5 % C02. The composition of the fluid (m-mole/l.) was NaCl 118-0, KCl 4 7, CaCl2 2.5, MgC12 1.2, NaHCO3 25-0, NaH2PO4 1.0, glucose 5B5. The perfusion was carried out at a constant perfusion rate of 1 ml./min (the corresponding perfusion pressure was between 8 and 35 mmHg). Effluent from the umbilical venous cannula was drained into graded test tubes and sampled at 10 min intervals. When the rate of flow from the placenta was less than 95 % of the perfusion rate, the samples were discarded and the perfusion was discontinued. The exposed uterus and the lower part of the animal were maintained at 37-38° C by immersion in a thermostabilized liquid paraffin-bath. The perfusion pressure was recorded with a Bell-Howell pressure transducer (type 4-327-L221) connected to a Schwartzer PEE-4 polygraph. The electrical potential difference was recorded between the maternal blood in the cubital vein and perfusion fluid in the umbilical venous cannula with a microvoltmeter (Tesla BM 483). The output of the microvoltmeter was integrated, and from the integral a mean value of the p.d. during the given interval was calculated. Radioactive 8ubatance8. 22NaCl and 24NaCl were supplied by the Radiochemical Centre, Amersham, and by Zentralinstitut fur Kernforschung, Dresden, respectively. Sucrose uniformly labelled with "IC was supplied by IYVVVR, Prague. Flame photometry and radioactivity counting. The concentrations of Na in the maternal plasma, foetal plasma, amniotic fluid, and perfusion fluid were measured with the Zeiss flame photometer after appropriate dilution with deionized water. Gamma radioactivity was counted with a well-type NaI scintillation detector
694
J. STULC AND J.
SVIHOVEC
coupled with the Tesla NZQ-717T counter. When the samples contained both radioactive isotopes of Na, the radioactivity of 24Na was counted with the discriminator level set so that all the radioactivity due to 22Na was filtered out. The samples were recounted for the 22Na after all 24Na had died out. Radioactivity of 04C was counted in a dioxane scintillation fluid (Spolana SLD 31) with the Packard 3300 liquid scintillation counter. Evaluation and presentation of the data. The fluxes of Na across the placenta varied with the weight of the foetus. Therefore, the values of Na fluxes are expressed per gram of the foetal weight. The sign of the p.d. denotes the polarity of the foetal side of the placenta with respect to the maternal side. For the individual groups of placenta the values of Na fluxes, p.d. or permeabilities are expressed as mean values ± s.E. of mean with the number of placenta in brackets. The differences between the individual groups were evaluated using Student's t test. Na fluxce across the perfumed placenta Estimation of the unidirectional fluxem. Unidirectional fluxes of Na across the placenta were measured simultaneously by means of 22Na and 24Na administered to the opposite sides of the barrier. The isotope 22Na was added to the perfusate in a concentration of approx. 0005 &c/ml. The isotope 24Na was administered intravenously to the mother as a single initial dose followed by infusion. The rate of infusion decreased in steps according to the following schedule (the infusion rate is expressed as a fraction of the initial single dose administered per minute): 0-5 min, 0 110; 5-12 min, 0 055; 12-24 min, 0-027; from 24 min on, 0 007. In this way the level of 24Na in maternal plasma was maintained within less than ± 10 % of the mean concentration. The total quantity of 24Na administered to the animal was approx. 100l ac. Simultaneously with the initial injection of 24Na approximately 500 u. heparine (Spofa) were administered. Assuming that the movement of the radioactive Na across the barrier was in a steady state, the flow of 22Na (J*) and 2"Na (J**) were determined as the quantity of 22Na radioactivity leaving and 24Na radioactivity entering the perfusate per unit time (c.p.m./min), respectively. From the tracer flows and from the Na concentrations on the two sides of the placenta the unidirectional fluxes of Na, i.e. the flux from the foetal to the maternal side (J,) and the flux in the opposite direction (Jo), were calculated. Provided that the behaviour of the tracer and the abundant species is identical, the flow of the tracer across the barrier can be regarded as the algebraic sum of the products of the unidirectional fluxes and the respective mean specific activities of Na along the barrier on the side of their origin. The mean specific activities along the barrier cannot be measured but the placental transport of Na was sufficiently slow to assume that neither the concentrations of the tracers nor the total Na concentration changed much along the barrier and that the concentrations in the exchange vessels were approximately equal to the inflow concentrations. Since the inflow concentration of 24Na on the foetal side of the barrier (in the perfusion fluid) was zero, and the inflow concentration of 22Na on the maternal side (arterial plasma concentration) was negligibly low, unidirectional fluxes of Na could be calculated according to the following simple equations:
Jmt
=
Jtm
=
(J**/[24Na]m) [Na]m K.,[Na]., (J*/[22Na]f) [Na]f Kfn[Na]f, =
=
PLACENTAL TRANSPORT OF Na
695
where ["2Na], [24Na] and [Na] are concentrations (c.p.m./ml. and mole/ml., respectively), K., and Kf. are unidirectional transfer constants (ml./min), and m and f denote maternal and foetal side of the barrier, respectively. Calculation of active transport. It has been postulated that Jtm consists of a passive and an active component (see Introduction). The passive component of Jtf was calculated from the values of J,,f, [Na]m, [Na]1 and p.d. using the Ussing flux ratio equation (Ussing, 1949). The active component (J't) was obtained by subtracting the passive component from Jfm. Jlf. was thus calculated according to the following equation: Jft = Jfm-Jmt([Na]f/[Na]m) exp (VFIRT), where V is the p.d., and F, R and T are the Faraday constant, gas constant and absolute temperature, respectively. The equation assumes that the passive movement of Na across the placenta proceeds entirely by simple diffusion uncomplicated by solvent drag or by coupling to other possible flows across the barrier. To prove that this assumption is valid seems impossible at present. Nevertheless, it will be shown below that the passive movement of Na across the placenta is compatible with simple diffusion being the means of passive transport. The space of "Na and 24Na of the placenta. The spaces of radioactive Na in the placenta were calculated as the ratio of the respective radioactivity of 1 g placental tissue to the radioactivity of 1 ml. perfusate in the case of 22Na, or 1 ml. plasma in the case of 24Na. The space is expressed as ml./g tissue. Experimental procedures. After establishing the placental perfusion, administration of 24Na to the mother was started. The moment of the initial i.v. injection of the isotope was taken as the zero-time. Collection of the effluent was started at 10 min and continued for six 10 min collecting periods. For the first 40 min (i.e. until the end of the third collecting period) the placenta was perfused with Krebs fluid without any experimental treatment in all animals. Then either the perfusion was terminated or the placenta was perfused for another 30 min with normal Krebs fluid (control group) or with Krebs fluid containing KCN (10-3 M). In all groups the placenta was sampled at the end of perfusion for estimation of the radioactivity. Maternal bood was sampled from the carotid artery at 5 min, 10 min, and from then on at 10 min intervals. The assumption of steady-state conditions of the tracer movement across the placenta. The present calculations of Na fluxes assume that the movement of the tracers across the placental barrier was in a steady state. This condition was met in the non-treated placenta, as indicated by the following evidence. (1) If a steady state were not reached before the period of flux measurement, the calculated values of Jmf would increase and Jfm decrease during perfusion, as a steady concentration of 24Na and 22Na within the barrier would be gradually approached. The calculated fluxes actually did change in this way during the first 30 min of perfusion (Table 1). Therefore, the data obtained during this period were rejected from further evaluation. After 30 min a reasonably steady level of the tracer flows across the placenta was attained. (2) There was no significant difference between the 22Na and "4Na spaces of the placenta sampled at 30 and 70 min (see below). In the placenta treated with KCN, a steady state of Na transport was no longer maintained. The Na fluxes during the KCN treatment, therefore, could be calculated only approximately. Na fluxes across the non-perfused placenta The animals were prepared in the same way as described for the perfusion experiments. The omphalomesenteric vessels were exposed through a small incision in the
696
J. STULC AND J. SVIHOVEC
uterus and foetal membranes and cannulated with thin polyethylene cannulae in the foetal direction. The umbilical vessels were left intact. The exposed uterus and the lower half of the animal were immersed in a thermostabilized liquid paraffin-bath at 37-380 C. A known dose of 22Na (approx. 5 #ic) in 0-2 ml. saline containing approx. 200 u. heparine (Spofa) was injected into the foetus through the omphalomesenteric venous cannula simultaneously with the injection of the initial dose of 24Na into the cubital vein of the mother. Infusion of 24Na into the maternal circulation followed. Samples of maternal blood were taken at 10 min intervals from the carotid artery. Foetal blood was sampled from the omphalomesenteric arterial cannula 10 min after injection of the isotopes, in the middle and at the end of the experiment. The foetus was removed from the uterus 60-120 min (80 min in most animals) after injection of the isotopes. The blood was centrifuged and samples of maternal and foetal plasma were removed for estimation of 22Na and 24Na radioactivity and of total Na concentration. The foetus was dried in an oven at 110-1500C, weighed and pulverized. Five samples of the powder (0.3-1.5 g) were taken from each foetus for the radioactivity assay. Recovery of 22Na and 24Na radioactivity from the foetus, estimated in a separate group of animals, was 98-6 ± 0.5 % and 98*1 + I1 0% (n = 14), respectively. The transfer constants Kid and Kim (ml./min) were calculated by dividing the amount of 24Na and 22Na radioactivity, respectively, transferred across the placenta per minute (c.p.m./min) by the mean concentration difference of the radioactivity of the respective isotope (c.p.m./ml.) between maternal and foetal plasma. The values of the unidirectional fluxes were obtained by multiplying Kmf or Kfm by total Na concentration in maternal or foetal plasma, respectively.
Extraplacental transport of Na into the foetus The animals were anaesthetized with pentobarbitone, the abdomen was opened and the foetal vessels supplying the yolk sac (omphalomesenteric vessels) were ligated through a small incision in the uterus without opening the foetal membranes. The abdomen was closed again, the animal covered with a cellulose tissue and heated with an infra-red lamp. A steady level of 22Na was maintained in the maternal plasma (as described above for 24Na) for 60 min. At the end of this period the abdomen was reopened and the amniotic fluid and umbilical venous blood from the treated foetus and from a non. treated foetus in the opposite horn of the uterus (control foetus) were simultaneously sampled. The influx of Na into the foetus was calculated from the 22Na recovered from the foetus and from the 22Na and total Na concentrations in maternal and foetal plasma, as already described. In these calculations the concentration of 22Na in foetal plasma was assumed to have followed a linear course. This simplification is unlikely to cause a great error in estimation of Na influx since the radioactivity of the foetal plasma at the end of the experiment did not exceed 25 % of that in the maternal plasma. Estimation of the placental permeability to Na and sucrose The placental permeabilities from the maternal to the foetal side to Na and sucrose were measured simultaneously. The animals were nephrectomized before the perfusion. The placenta was perfused, and a constant level of 24Na in maternal plasma was maintained as described above. Approximately 10 Atc [14C]sucrose were injected i.v. into the mother together with the initial dose of 24Na. The perfusion liquid contained no 22Na. The placenta was
PLACENTAL TRANSPORT OF Na
697
perfumed for 50 min, and effluent from the placenta and maternal blood were sampled during the last 10 min. Placental permeability to sucrose was calculated as the quantity of [14C]sucrose transferred into the perfusate per unit time and unit concentration difference. The 14C-material recovered from the effluent was shown to be [14C]sucrose for each sample of effluent by paper chromatography in a system butanol:ethanol:water (4:1:5). The permeability of the placenta to Na was calculated by dividing the constant Kmt by a factor that represents the effect of the p.d. on the diffusion of ions across the membrane. This factor is VF/RT exp (VF/RT)-I' where V is the p.d. (Schultz & Zalusky, 1964, eqn. (6)). The permeability refers to the whole exchange area of the placental barrier and its dimension is therefore
cm3/min. RESULTS
Transport across the perfumed placenta. Control values and general observations. The values of Na fluxes across the placenta together with the values of the p.d. during a 70 min perfusion in a group of control animals are in Table 1. The initial increase of Jmf and decrease Of *fm are probably not real. They may result from an incorrect estimation of their values due to the non-steady-state conditions after the introduction of the tracers into the system (see Methods). Using 24Na, Flexner & Pohl (1941) measured the flux of Na from mother to foetus in guinea-pigs. The animals were exposed to no stress except light ether anaesthesia at the time of isotope injection and at the end of the experiment. The values of flux into the foetus can therefore be taken to be closely representative of those in normal unstressed animals. The values of 'corrected' transfer of radioactive Na in Fig. 7 of their publication are equivalent to Kmf in this work, except that an hour is used as the unit of time. Therefore, Jmf per gram of foetal weight in intact guinea-pigs can be estimated by multiplying the values in Fig. 7 of Flexner & Pohl by [Na]m and dividing them by 60 x (foetal weight). Taking the value of 137 m-mole/l. obtained in this work for [Na]m (136-8 + 0-8 m-mole/l.) the flux of Na from mother to foetus calculated from the data of Flexner & Pohl is 0*219 + 0.015 ,mole/min.g (n = 18). (The calculation was performed only for the foetuses with a body weight above 25 g, so that the range of foetal weight would be comparable with that in the present work.) The values of Jmf across the perfused placenta (Table 1) are in good agreement with the flux into the foetus estimated for intact animals. Since Jmf occurs down the electrochemical potential gradient and therefore can be taken as passive this indicates that no gross changes of placental permeability to Na were caused by the perfusion.
J. STULC AND J. 9VIHOVEC 698 The 22Na and 24Na spaces of the placenta at 30 min were 0 337 + 0-031 ml./g and 0-227 + 0 030 ml./g (n = 6), respectively. The radioactive Na spaces did not change during the next 40 min, the corresponding values at 70 min being 0 345 + 0*017 ml./g and 0-225 + 0016 ml./g (n = 7), respectively. The weight of the placenta at the end of a 70 min perfusion was 3-496 + 0 300 g (n = 7). TABLE 1. Na fluxes and the electrical potential difference across the control perfused placentae Time (min)
J., (pmole/min.g) J,., (.umole/min.g) PAd (mV) J't
(,umole/min.g)
10-20 0.240 + 0*021 0 357 ± 0.036 -210 +2.0 *
20-30 0.250 ± 0.024 0 355 + 0 035 -20X5 +2.0 *
30-40 0.252 + 0.024 0.338 ± 0.034 -19 9
±20
0f206
+ 0*032
40-50 0.258 ± 0.025 0-334 + 0 035 -20X6 +2-1
0f215
+ 0 033
50-60 0.257 ± 0.027 0-321 + 0 035 -19 9 +2.1 0 199 + 0*032
60-70 0.260 + 0-027 0'334 + 0-034 -19 1 +2.2
0f204
+ 0.030
Mean + S.E. of mean, number of placenta = 13. The fluxes are expressed per gram foetal weight, mean foetal weight is 50-2 + 3-7 g
(n
=
13).
* Jt was not calculated for the first two collecting periods. It took approx. 30min for the movement of radioactive Na across the placenta toattain a steadystate (see Methods). Consequently the value of unidirectional fluxes obtained during this period are in error, and the values of active transport calculated from these data would be meaningless.
Extraplacental transport. In the perfusion experiments Jfm was higher than Jmf (Table 1) which means that there was a net flux of Na from the foetal to the maternal side of the placenta. Existence of this net flux follows independently from the positive arteriovenous concentration difference of Na in the perfused umbilical vascular bed of the placenta (the average value in seventeen placentae was 5*3 + 0-6 m-mole/l.). A net flux of Na from the foetal to the maternal side of the placenta could exist in intact animals only if it were made good by a net flux of Na into the foetus by extraplacental transport via foetal membranes. In rodents the foetal membranes are represented by the everted yolk sac and amnion. The everted yolk sac is the outer membrane and is richly supplied with blood by the omphalomesenteric vessels. Material passing through the foetal membranes could therefore reach the foetus by two pathways: (1) through the vitelline blood circulation; or (2) through the amniotic fluid.
PLACENTAL TRANSPORT OF Na 699 Ligation of the omphalomesenteric vessels had no effect on the Na transport into the foetus, the influx into the foetus in control and treated animals being 0-235 + 0-020 /tmole/min. g and 0*247 + 0*029 #smole/min . g (n = 6), respectively. Specific activity of Na in the amniotic fluid was less than 2 % of that in foetal plasma in both groups. From this it follows that no significant quantity of Na can reach the foetus by extraplacental pathways. The net flux of Na across the perfused placenta, therefore, must be caused by the perfusion and/or by the stress of the operation and anaesthesia. To decide between these two possibilities Na fluxes were measured across the non-perfused placenta under conditions similar to those of the perfusion experiments. Transport across the non-perfused placenta. The flux of Na from mother to foetus was 0 180 + 0 013 Ismole/min .g, the flux in the opposite direction was 0-240 + 0'024 #smole/min. g (n = 12). These values are lower than those observed in the present perfusion experiments (Table 1) or those obtained by Flexner & Pohl (1941) in intact guinea-pigs. The reason for this difference is not clear. The concentration of Na in foetal plasma was 133-9 + 0-9 m-mole/l. (n = 12). The ratio Of Jfm/Jmf in the perfused placenta is 1-39 + 0-08 (n = 13) which is close to the corresponding flux ratio 1-35 ± 0 09 (n = 12) in the nonperfused placenta. (The former value was calculated from the fluxes across the perfused placenta during the collecting period 30-40 min. The mean values of the fluxes are given in Table 1.) This suggests that the asymmetry of Na fluxes in the present experiments bears no relation to the perfusion procedure and that it is probably due to the stress of the animal under conditions of acute experiment (i.e. to the trauma of operation and/or anaesthesia). Further search for the factors causing the asymmetry of Na fluxes in these experiments was given up at this point since it would require control measurements of Na fluxes and the p.d. in non-stressed, non-anaesthetized animals. Such measurements would be technically impossible in the guinea-pig. The mechanism of passive transport. Calculation of the passive and active components of Na transport from the unidirectional fluxes and the p.d. requires that the passive movement of Na across the placenta proceeds entirely by simple diffusion. There is, however, no evidence that this condition was met. Usually, the flux of a solute across a membrane is taken to be diffusional when its magnitude is directly proportional to the electrochemical potential of the solute on the side from which the penetration is occurring. However, such an approach could not be used to test the nature of passive movement of Na across the placenta since neither the Na concentration in the maternal
700 J. STULC AND J. SVIHOVEC plasma nor the p.d. could be varied. The test performed here was less general: a single mode of passive transfer, which seemed a prior most likely, was assumed and verified experimentally. Since nothing was known about the permeability properties of the guinea-pig placenta, observations from the morphologically related placenta of the rabbit were used as a
guide. It has been observed in the rabbit that the values of placental permeability to lipid insoluble substances are approximately proportional to their coefficients of free diffusion in water (Faber & Hart, 1967; 9tulc, Friedrich & Jificika, 1969; Faber, Green & Long, 1970). This suggests that the lipid insoluble molecules move across the rabbit placenta by unrestricted diffusion through wide aqueous channels. In this work the permeability properties of the guinea-pig placenta were assumed to be similar, and the assumption was tested by measuring the simultaneous values of placental permeability to Na and sucrose. It was expected that if the assumption were correct the values of placental permeability would bear the same quantitative relation to each other as the respective diffusion coefficients in water. The placental permeability was measured from the maternal to the foetal side, i.e. in the direction in which the movement of Na can be taken as entirely passive. The values of placental permeability to Na and sucrose obtained are 0-0767 + 0*0183 cm3/min, and 0'0324 + 0-0094 cm3/min (n = 7), respectively. The diffusion coefficients of NaCl and sucrose are 1-7 x 10-5 cm2/sec (Schultz & Zalusky, 1964) and 0 7 x 10-5 cm2/sec (Landis & Pappenheimer, 1963), respectively. The Na: sucrose ratios of the placental permeability and the diffusion coefficients calculated from the above data are 2*57 + 0-20 and 2-40, respectively. This is consistent with simple diffusion in aqueous medium being the mechanism of passive transport of Na across the placental barrier in the guinea-pig. Effects of KCN on Na transport across the perfumed placenta. At the end of a 30 min perfusion with KCN the values of the 22Na and 24Na space and of the weight of the placenta were, respectively, 0-616 + 0-071 ml./g, 0X175 + 0.041 ml./g and 3-836 + 0183 g (n = 7). The space of 22Na is significantly above the corresponding value in the control placentae, the other two variables are not significantly changed. The increase in the 22Na space indicates that part of the 22Na which left the perfusion fluid during the KCN treatment did not reach the maternal side of the barrier. To obtain a correct value of J*, therefore, the quantity of 22Na retained by the placenta should be subtracted from the quantity of 22Na cleared from the perfusate during the same period. The rate of 22Na accumulation in the placenta could not be exactly assessed since it was impossible to obtain two successive samples from one placenta, but it was estimated in the following way. From the weight of the placenta perfused
PLACENTAL TRANSPORT OF Na 701 with KCN and from the average value of the 22Na space in the control placentae the quantity of 22Na expected in the placenta if the perfusion fluid contained no KCN was calculated. This value, subtracted from the actual 22Na content of the placenta at the end of perfusion with KCN, was taken to represent the approximate quantity of 22Na retained in the placenta due to the KGN treatment. TABLE 2. Effects of KCN (10-3 M) in the perfusion fluid on the Na fluxes and the p.d. across the perfused placenta Time (min)
J,. (ismolelmin . g) P.d. (mV)
30-40 (Control period) 0.322 + 0.014 0-402 + 0.033 -18.7+2.2
40-70 (KCN treatment) 0-394 + 0 055 0 393 + 0 050
J'f~t (#mole/min.g)
0-228+0.014
0.1444+0-021
J,,, (#smole/min g) .
-13.1±+18
P < 0-05
> 0 05
